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  • 1990-1994  (14,398)
  • 1980-1984  (6,630)
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  • Life and Medical Sciences  (17,575)
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 221 (1994), S. 309-320 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Gastrocnemius tendons of 10 White Leghorn chickens at 6, 8, and 12 weeks of age were divided into proximal, middle, and distal portions to assess regional variability in composition and growth. Body weight increases ∼ 150% during the period examined, whereas the lateral gastrocnemius muscle and tendon increase ∼ 193% and 227%, respectively. No significant changes in cellularity (DNA concentration) or hydroxypyridinium (OHP) crosslinks occur with increasing age. Hydroxyproline (HYP) concentration increases by 12 weeks of age, as hexuronate, glucosamine, and galactosamine decrease. Composition shows some regional variation: the distal region of the tendon has a lower HYP concentration, and increased GAGs and OHP crosslinks compared to either the proximal or middle regions, which do not differ from each other. The mean collagen fibril diameter increases with age, but the oldest tendons also contain more small diameter fibrils (〈40 nm). There is a unimodal fibril distribution at all three ages, although this has broadened by 12 weeks. The data from this study suggest that rapid tendon growth occurs throughout the time period examined and that changes characteristic of mature tendon, such as increased OHP crosslink concentration, have not yet developed in hatchlings because of the large amount of new tissue being produced. Whereas all three regions of the tendon are similar in size, composition of the distal region differs from that of the proximal and middle regions, suggesting that this portion of the tendon should be avoided when sampling a tendon. © 1994 Wiley-Liss, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 33-48 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The integument of Pycnogonida (Arthropoda) consists of an epicuticle decorated with tubercles and a filamentous coat, an exocuticle with a small number of ill-defined layers, and an endocuticle whose numerous layers are composed of conspicuously cross-banded fibrils. This cuticular periodicity, attributable to cross-linked chitin, has been observed previously in uncalcified and untanned cuticle of many lower crustaceans, especially branchiopods and copepods, and in scattered examples of thin respiratory or excretory cuticles of other arthropods. It is uniformly present in all representatives of all nine pycnogonid families examined to date. Stomodeal, proctodeal, and arthrodial cuticles are devoid of the endocuticular periodicity. The cuticle is decorated with sensory filaments and setae, but is more noteworthy for a dense coverage by glands, up to 1,400/mm2. Myocuticular junctions have desmosomal fine structure previously found only in chelicerates. Muscle fine structure is that of slow fibers with long sarcomeres and a high actin to myosin filament ratio, except for cardiac muscle, which has short sarcomeres. Among the arthropods, only merostomates resemble the pycnogonids in the lack of fast somatic muscle fibers. Pycnogonids display a hybrid array of fine structural features that variously serve to relate them to some arthropod subphyla and distance them from others. © 1994 Wiley-Liss, Inc.
    Additional Material: 15 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 111-111 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 73-89 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The chimaeroid holocephalian fishes are distinguished among extant chondrichthyans by the possession of three pairs of tooth plates, evergrowing and partially hypermineralized, that are not shed and replaced like the teeth of living elasmobranchs. Although derivation of the chimaeroid tooth plate from the fusion of members of a plesiomorphic chondrichthyan tooth family has been proposed, evidence for this hypothesis has been lacking. A new analysis of the development and structure of the tooth plates in Callorhinchus milii (Holocephali, Chimaeriformes) reveals the compound nature of the tooth plates in a chimaeroid fish. Each tooth plate consists of an oral and aboral territory that form independently in the embryo and maintain separate growth surfaces through life. The descending lamina on the aboral surface of the tooth plate demarcates the growth surface of the aboral territory. Comparison with the tooth plates of Chimaera monstrosa indicates that compound tooth plates may be a feature of all chimaeroids in which a descending lamina is present. The tooth plates in these fishes represent the fusion of two members of a reduced tooth family. The condition of the tooth plates in C. milii is plesiomorphic for chimaeroids and is of evolutionary significance in that it provides further evidence to support a lyodont dentition in chimaeroid fishes similar to that found in other chondrichthyans. © 1994 Wiley-Liss, Inc.
    Additional Material: 14 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 219 (1994), S. 1-6 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The process of morphological and functional regeneration was followed on a tilapid fish, a cross of Oreochromis aureus × Oreochromis niloticus, by observations on movements and the use of X-rays. A four-year-old adult fish that lost its tail as post larva, including ten vertebrae, was able to reconstruct a novel and shorter central skeleton, including a specially modified urostyle. The enlarged and strengthened pterygiophores and their junctions with the dorsal and anal spine formed a fast-holding base for the fins, the posterior part of which largely performed the functions of the missing caudal fin. Although the fish was much shorter than usual, this male behaved and functioned normally. © 1994 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 219 (1994), S. 7-13 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A study of the ultrastructure and function of the paraphysis in Bufo bufo larvae was carried out. The structure is a tubular-ramified gland made up of numerous tubules with monolayered epithelial walls surrounded by connective tissue and sinusoids. The epithelial cells secrete glycoprotein to contribute to production of the cephalorachidian fluid. The role of the paraphysis in the transport of fluids and electrolytes from the blood to the cephalorachidian fluid in regulation of ionic and osmotic homeostasis is discussed. © 1994 Wiley-Liss, Inc.
    Additional Material: 14 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 219 (1994), S. 15-20 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Spermatozoa from representatives of the five insect orders in superorder Neuropteroidea were examined by electron microscopy following a new fixation method that includes tannic acid in the primary fixative but has uranyl acetate rather than osmium tetroxide as the secondary fixative. The sperm axoneme was found to be similar in the four orders Megaloptera, Raphidioptera, Neuroptera, and Coleoptera, and is characterized above all by its so-called intertubular material being divided into two portions, one located outside, but in contact with the doublet, and the other projecting from the accessory tubule and having a beak-like shape. These features have not been seen in insects from other orders and may be a synapomorphy for these neuropteroid orders. The accessory tubules in these four orders have 16 protofilaments. The shape of the accessory bodies adjacent to the mitochondrial derivatives is nearly the same in insects from the more primitive neuropteroid orders and in Coleoptera. The sperm tail of the examined strepsipteran deviates in several respects from that of other neuropteroids: the particle row in the wall of accessory tubules is incomplete, an intertubular material is missing, and the mitochondria contain no crystal. © 1994 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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  • 8
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In the embryo of Haliotis tuberculata spiral cleavage induces size differences between the quadrants in the 4-cell embryo. These size differences, together with the formation of compact cell configurations, induce asymmetrical positions of equivalent cells in the 8- and 16-cell embryo. The asymmetries in size and position influence the final specification of the dorsoventral asymmetry in the 32-cell embryo, as well as formation of the mesentoblast. © 1994 Wiley-Liss, Inc.
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  • 9
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The anatomy and histology of the abdominal eversible vesicles and the male reproductive tract of the spoonwing lacewing Palmipenna (Neuroptera: Nemopteridae) have been examined. The eversible vesicles open as a pair of large bulbous sacs between tergites five and six, each folding into halves during retraction. They consist of highly pleated cuticle, beneath which are typical gland cells, each having a circular or oval end apparatus surrounded by closely packed microvilli. These communicate to the surface via cuticularized channels. In spite of considerable behavioral observations, male Palmipenna were never noted with everted vesicles. Even during mating trials, where females were presented to males in the field, the vesicles were never everted during the attempted copulation that ensued. Our observations indicate that mate attraction is mediated by the release of a female pheromone. The function of the eversible vesicles and their associated gland cells remains unknown, and their structure appears to be unique to the Nemopteridae. The reproductive tract is similar to that of other Neuroptera, consisting of a pair of five-lobed testes, a medium-to-large pair of seminal vesicles, and three pairs of accessory glands. The major accessory glands are surrounded by circular and longitudinal muscle, and are lined by an epithelium, the cells of which presumably secrete the amorphous rods of material always present in this pair of glands. The sperm in the seminal vesicles are elongate, with a pointed head and a 9 + 9 + 2 configuration in the flagellum. A single spermatophore, similar in shape to that described for other Neuroptera, was found occluding the bursa copulatrix of a teneral female. © 1994 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 219 (1994), S. 35-46 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Embryos of viviparous goodeid fishes undergo a 10 to 150 × increase in dry weight during gestation. Maternal nutrients are transferred across a trophotaenial placenta comprised of the ovarian lumenal epithelium and the trophotaeniae of the embryo. Trophotaeniae are externalized projections of the embryonic hindgut. Epithelial cells of the ribbon trophotaenia (Ameca splendens) resemble intestinal absorptive cells of suckling mammals and endocytose macromolecules. They possess an apical brush border, endocytotic complex, endosomal-lysosomal system, and apical and basal clusters of mitochondria. Cells of the rosette trophotaenia (Goodea atripinnis) lack an endocytotic apparatus, have small lysosomes, two mitochondrial clusters, and transport small molecules. Organelle-specific fluorescent probes were employed to characterize the functional organization of the two types of trophotaenial cells. In A. splendens, Lucifer Yellow, a membrane-impermeable tracer of vesicular transport, first appears in peripheral vesicles (15-45 sec), then passes into elongated tubular endosomes (1-3 min) and later appears in large central vacuoles (10-15 min). These vacuoles accumulate Acridine Orange, a classical probe for lysosomes, and have been shown to contain lysosomal enzymes. Endosomelysosome fusion was observed. In both A. splendens and G. atripinnis, Rhodamine 123 fluorescence was localized in two clusters of fine spots that corresponded to mitochondria. 4′,6-diaminido-2-phenyl-indole (DAPI) staining of nuclei established the positional relationships of cell organelles with respect to the nuclei. 3,3′-dihexyloxacarbo-cyanine iodide (DiOC6) revealed the perinuclear distribution of the endoplasmic reticulum. In order to compare in vivo fluorescence of Lucifer Yellow with previous ultrastructural observations, we employed fluorescence photoconversion and electron microscopy. © 1994 Wiley-Liss, Inc.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 219 (1994), S. 59-71 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The ontogenesis and structural characteristics of the seminal vesicles in Clarias gariepinus (sharptooth catfish) were studied by light and electron microscopy and are described in detail. The seminal vesicles, beginning as simple protrusions from the vas efferentia, becomes more complex with age. Their distal ends become fingerlike and the bases form palm-like extensions. Juvenile male organs do not reveal any signs of seminal vesicles although spermatogenic tissue is already well delineated. The developing gonads contain clusters of large cells, close to the sperm duct and cysts of the testis, from which seminal vesicles are formed. Secretory epithelium lines the tubules of the seminal vesicles and becomes columnar as the tissue matures. Electron micro-graphs of these epithelial cells reveal two types of cells: opaque cells and cells with very vacuolized cytoplasm. Dense pinocytotic vesicles are present between the membranes of neighbouring seminal tubules and apical cell membranes facing the lumen. Maturation and onset of secretion by the secretory cells is accompanied by morphological changes. Protruding cylindrical cells become shortened, modified to cuboidal, rounded cells that send tubular extensions into the lumen. In the final stage of differentiation, only connective tissue membranes supporting the tubule walls remain intact. At the points of contact between the testis, seminal vesicles, and sperm duct, the epithelia of these organs often become confluent. The distal parts of the seminal vesicles, rarely contain sperm; during spawning sperm accumulated in the proximal tubules of the vesicles. © 1994 Wiley-Liss, Inc.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 219 (1994) 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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  • 13
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 11-18 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The long (49-93 mm) antennae of two species of Australian gryllacridids have high total numbers of sensilla consisting of five sensillar types. Ametrus sp. 7 has 22,300 (♀) and 26,250 (♂) sensilla; although the antennae of males are 33% longer than those of females, their sensillar density was 11% less. Bothriogryllacris pinguipes has 26,700 (♂) and 31,900 (♀) sensilla; antennae of females are 55% longer than those of males but sensillar density is 23% less. Aporous sensilla chaetica form 94.5 to 99.5% of all sensilla; they are presumably mechanoreceptors. Uniporous trichoid contact chemoreceptors range from 75-900 in number. Olfactory, multiporous, basiconic sensilla range from 22-440 and olfactory, coeloconic sensilla from 16-235. Two to five multiporous lenticular organs occur on all but female A. sp. 7. Differences in sensillar abundance between males and females are discussed as well as are the relationships between sensillar diversity on gryllacridid mouthparts and antennae. © 1994 Wiley-Liss, Inc.
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  • 14
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 19-32 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Homozygous recessive cardiac mutant gene c in the axolotl, Ambystoma mexicanum, results in a failure of the embryonic heart to initiate beating. Previous studies show that mutant axolotl hearts fail to form sarcomeric myofibrils even though hearts from their normal siblings exhibit organized myofibrils beginning at stage 34-35. In the present study, the proteins titin and myosin are studied using normal (+/+) axolotl embryonic hearts at stages 26-35. Additionally, titin is examined in normal (+/c) and cardiac mutant (c/c) embryonic axolotl hearts using immunofluorescent microscopy at stages 35-42. At tailbud stage-26, the ventromedially migrating sheets of precardiac mesoderm appear as two-cell-layers. Myosin shows periodic staining at the cell peripheries of the presumptive heart cells at this stage, whereas titin is not yet detectable by immunofluorescent microscopy. At preheartbeat stages 32-33, a myocardial tube begins to form around the endocardial tube. In some areas, periodic myosin staining is found to be separated from the titin staining; other areas in the heart at this stage show a co-localization of the two proteins. Both titin and myosin begin to incorporate into myofibrils at stage 35, when normal hearts initiate beating. Additionally, areas with amorphous staining for both proteins are observed at this stage. These observations indicate that titin and myosin accumulate independently at very early premyofibril stages; the two proteins then appear to associate closely just before assembly into myofibrils. Staining for titin in freshly frozen and paraffin-embedded tissues of normal embryonic hearts at stages 35, 39, and 41 reveals an increased organization of the protein into sarcomeres as development progresses. The mutant siblings, however, first show titin staining only limited to the peripheries of yolk platelets. Although substantial quantities of titin accumulate in mutant hearts at later stages of development (39 and 41), it does not become organized into myofibrils as in normal cells at these stages. © 1994 Wiley-Liss, Inc.
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  • 15
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994) 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 16
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 113-131 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Based on a detailed description of hatchling skeletons of the precocial buttonquail (Turnix suscitator) and the altricial budgerigar (Melopsittacus undulatus), this report presents the hypothesis that the rate of avian posthatching growth is limited by the quantitative design (i.e., relative volumes of cartilage, bone, and marrow) of the hatchling skeletons. A Jarge portion of bone in the skeletal elements and fast growth are hypothesized to be mutually exclusive. This hypothesis is tested by morphometric techniques and by statistical comparison of morphometric and growth data. All predictions are met by the data, and the design of hatchling skeletons is described as determined by a tradeoff between tissue composition of skeletal elements and maximum rates of posthatching growth. The precocial design shows large bony areas that supposedly resist mechanical stress of locomotion; however, the relatively small cartilaginous areas exclude high growth rates. The altricial design shows the reverse relationship with small bony areas and a lack of locomotion on the one side but large cartilaginous areas and fast posthatching growth on the other side. © 1994 Wiley-Liss, Inc.
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  • 17
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 175-190 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Jaw protrusion is an important component of prey capture in fishes, although the mechanics of protrusion have thus far been studied largely in teleosts. Elasmobranchs are also able to protrude their jaws (Tricas and McCosker [1984] Proc. Cal. Acad. Sci. 43: 221-238; Tricas [1985] Mem. S. Calif. Acad. Sci. 8:81-91.; Frazzetta and Prange [1987] Copeia 4:979-993). Several related features of the feeding apparatus contribute to jaw protrusion in sharks. Labial cartilages form an extendible series attached dorsally to the anterolateral face of the palatoquadrate and ventrally to the anteroventral surface of Meckel's cartilage. The labial cartilage chain swings anterolaterally as the lower jaw is depressed, thrusting the labial margins forward to form a circular oral opening and displacing the jaw apparatus towards the food; this pattern is analogous to halecomorph and primitive actinopterygian fishes in which the maxilla swings forward (Lauder [1979] J. Zool. Lond. 187:543-578). The palatoquadrate and Meckel's cartilage also project anteriorly and represent the major contribution to protrusion. These movements occur simultaneously with enlargement of the oral cavity to generate suction. The wobbegong sharks (Orectolobidae) are specialized for jaw protrusion. The spotted wobbegong protrudes its jaw by 33% of its chondrocranial length using two different mechanical systems. In the first mechanism of jaw protrusion, the intermandibularis and interhyoideus muscles medially compress the lower jaw and hyomandibulae. Compression of the lower jaw results in a more acute symphyseal angle so that the anteroposterior alignment of the lower jaw increases due to the rotation of each lower jaw towards a saggital orientation. Distal compression of the hyomandibulae at their attachments to the jaws swings the jaws forward. The second mechanism involves rotation of the ceratohyal around a posterior process of the lower jaw, pushing the hyomandibulae anteroventrally, thereby pushing the jaw articulation ventrally and anteriorly to protrude the jaws. © 1994 Wiley-Liss, Inc.
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  • 18
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 203-213 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Gap junctions in mammalian heart function to provide low-resistance channels between adjacent cells for passage of ions and small molecules. It is clear that the almost unrestricted passage of ions between cells, ionic coupling, is required for coordinate and synchronous contraction. This knowledge of gap junction function has made it important to study their properties in normal and abnormal tissues. In the present study, we analyzed gap junction distribution in normal and cardiomyopathic heart tissue utilizing immunofluorescent and electron microscopy techniques. Frozen, unfixed sections of age-matched normal and cardiomyopathic cardiac tissues were immunofiuorescently stained using an antibody directed against a specific peptide sequence of the connexin-43 gap junction protein. These studies revealed a characteristic punctate staining pattern for the intercalated discs in normal tissues. Some of the intercalated discs in cardiomyopathic hearts appeared to stain normally; however, others stained diffusely. The pixel intensity distribution of the confocal images demonstrated a marked difference of up to 90% increase in the number of pixels in cardiomyopathic myocardium (CM), yet the pixel intensity of gap junctions had a decrease of approximately 60%. This suggests the possibility that connexin-43 is present in CM cells in significant quantity; however, it does not become localized on the membranes as in normal cells. Electron-microscopic findings corroborate these observations on CM cells by showing an irregular distribution of intercalated discs relatively smaller in size with abnormal orientation and distribution. © 1994 Wiley-Liss, Inc.
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  • 19
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994) 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 20
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 223-230 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We describe some significant structures of the adult ovary in a Japanese penicillate diplopod, Eudigraphis nigricans, with respect to phylogenetic implications. The ovary is a long, saclike organ lying between the alimentary canal and the ventral nerve cord from the fourth through the ninth body segment. The ovarian wall consists of a thin ovarian epithelium and a sparse muscle covering. There are two types of oogenetic sites: a single, mound-shaped germarium sitting on the center of the ventral ovarian epithelium, and ∼ 10 pairs of patchlike vitellarial areas metamerically arranged anterior and posterior to the germarium. The germarium consists of oogonia, early previtellogenic oocytes, and some somatic interstitial cells. In contrast, the vitellarial areas are composed of more advanced oocytes, follicle cells surrounding the oocytes, and some interstitial cells, but no oogonia. A few larger previtellogenic oocytes rise up from each vitellarial area into the ovarian lumen. Each of these oocytes is still connected with its own vitellarial area by a partial extension of its follicle. Vitellogenesis takes place in these oocytes rising in the ovarian lumen. The ripe primary oocytes leave their follicles to be transported forward into the oviducts. Some phylogenetic implications of the basic characteristics in ovarian structure and oogenesis of E. nigricans are discussed. © 1994 Wiley-Liss, Inc.
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  • 21
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 103-110 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The morphology of eggshells from hatched eggs of captive Chinese alligators (Alligator sinensis) was compared with that of shells from eggs with early embryonic death and with the morphology of eggshells from the American alligator (Alligator mississipiensisis). Pieces of shells were examined in the scanning electron microscope. Parameters examined included: numbers of open pores on the outer surfaces, total shell thickness, and thickness of the outer densely calcified and inner mammillary layers. Results indicate that shells from Chinese and American alligator eggs with early embryonic death have a thicker outer densely calcified layer than do shells from hatched eggs or full-term embryos. Also, eggshells from Chinese alligator eggs with dead embryos have fewer open pores on the outer surface than do shells from hatched eggs, as has been reported earlier for the American alligator (Wink et al., '90). © 1994 Wiley-Liss, Inc.
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  • 22
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 222 (1994), S. 149-173 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Development of craniofacial muscles of Monodelphis domestica (Marsupialia, Didelphidae) is described. In a period of 4-6 days all craniofacial muscles in M. domestica progress from myoblast condensation, to striated myofibers that are aligned in the direction of adult muscles and possess multiple, lateral nuclei. This process begins 1 to 2 days before birth and continues during the first few days after birth. Compared to other aspects of cranial development, muscle development in M. domestica is rapid. This rapid and more or less simultaneous emergence of craniofacial muscles differs from the previously described pattern of development of the cranial skeleton in marsupials, which displays a mosaic of acceleration and deceleration of regions and individual elements. Unlike the skeletal system, craniofacial muscles show no evidence of regional specialization during development. M. domestica resembles eutherian mammals in the relatively rapid and more or less simultaneous differentiation of all craniofacial muscles. It differs from eutherian taxa in that most stages of myogenesis occur postnatally, following the onset of function. The timing of the development of muscular and skeletal structures is compared and it is concluded that the relatively early development of muscle is not reflected by any particular acceleration of the differentiation or growth of skeletal structures. Finally, the difficulties in accounting for complex internal arrangements of muscles such as the tongue, given current models of myogenesis are summarized. © 1994 Wiley-Liss, Inc.
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  • 23
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    Journal of Morphology 222 (1994), S. 191-201 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Study of the epithelial morphology of a stingless bee ileum from the pyloric valve to the last portion of high absorptive cells shows that although the bee ileum is an anatomically undifferentiated tube, four types of epithelial cells along the tube (in addition to the valve cells) indicate physiological differentiation. The anterior end seems to be less active in reabsorption, while the posterior region contains cells with typical morphology of an ion pump and permits conclusions about the mechanisms of absorption in the posterior end of the intestine. © 1994 Wiley-Liss, Inc.
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  • 24
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    Journal of Morphology 222 (1994), S. 215-221 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: This study examines the allometric scaling relationships of the cetacean humerus, radius, and ulna. Bone lengths and diameters were measured for 20 species of odontocete and three species of mysticete cetaceans, representing eight of the nine extant cetacean families. The scaling of individual bone proportions (bone length vs. cranio-caudal diameter, bone length vs. dorso-ventral diameter), and of individual bone dimensions against estimated body mass, are compared to models of geometric and elastic similarity. The geometric similarity model describes the scaling relationship of bone length vs. cranio-caudal diameter and body mass vs. cranio-caudal diameter for the humerus only; geometric similarity also describes the scaling relationship of body mass vs. bone length for all three bones. None of the scaling relationships fits the elastic similarity model. The scaling relationships of bone length vs. dorso-ventral diameter for all three bones, and bone length vs. cranio-caudal diameter for the radius and ulna, exhibit negative allometry, indicating that large bones are less robust than small bones. Negative allometry of structural support elements has not been previously described for terrestrial mammals or plants. The high relative swimming speeds of small delphinids may generate sufficient stresses to require more robust bones relative to those of larger whales. © 1994 Wiley-Liss, Inc.
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  • 25
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    Journal of Morphology 222 (1994), S. 287-299 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The male reproductive cycle of this paedomorphic species that occurs only in Lake Pátzcuaro, Michoacán, México, was investigated by documenting changes in germinal cells during the spermatogenic cycle. Cysts of germ cells divide synchronously to complete spermatogenesis during September through December, with the proportion of evacuated cysts or cysts containing spermatozoa increasing during this period. The chromatin changes during prophase I of meiosis reveal the usual leptotene, zygotene, pachytene, and diplotene stages. A basal body at the caudal end of the spermatozoan head connects to the flagellum. After spermiation, empty cysts contain a granular substance. Spermatogenesis in this species follows an annual cycle like other north temperate salamanders, rather than the continuous spermatogenesis of some tropical salamanders. © 1994 Wiley-Liss, Inc.
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  • 26
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    Journal of Morphology 222 (1994), S. 269-286 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: PC12 cells show a classical volume regulatory process when submitted to hypo-osmotic conditions. The present study examined the effects of such osmotic shock on the structural organization of different cytoskeletal elements. Results were obtained by use of different light and electron microscopy techniques combined with immunostaining methods. It appeared that the osmotically induced changes in cell volume were concomitant with important modifications in the organization of the microfilament network. Microfilaments concentrated in the perinuclear area, leaving only radial extensions of poorly organized structures in the cytoplasm. The latter were the only actin structures immunologically stained in the cytoplasm and seemed to anchor to the plasma membrane. Measurements of the fluorescence intensity of PC12 cells treated with FITC-labeled phalloidin indicated a progressive depolymerization, followed by a repolymerization of F-actin. This occurs in parallel with microfilament reorganization and volume regulatory processes. The appearance of microfilament reorganization was a function of both the incubation period and the amplitude of the osmolarity changes. During the first minutes of osmotic shock, a decrease was observed in the density and length of microvilli, which normally cover the PC12 cell surfaces, suggesting an early reorganization of the underlying microfilament network. Microtubules and intermediate filament networks were not affected by the hypo-osmotic conditions. © 1994 Wiley-Liss, Inc.
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  • 27
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    Journal of Morphology 222 (1994), S. 241-267 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The position and structure of the olfactory organ and its openings vary among actinopterygians. The anterior nasal opening is a simple perforation in the skin in many extant actinopterygians (e.g., acipenseriforms, lepisosteids, and primitive Recent teleosts) and represents the primitive condition. Polypterids and Amia each exhibit a derived condition, in which the anterior nasal opening extends into a tube. The olfactory organ is relatively far away from the anterior end of the elongate rostrum in acipenseriforms, whereas the olfactory organs are closer to the anterior end of the snout in extant actinopterygians (e.g., polypterids, lepisosteids, and amiids). In adults, olfactory organs are cuplike structures in most actinopterygians, but these organs are tubelike in polypterids. Among extant actinopterygians, a nasal diverticulum is present only in polypterids. Teleosts have accessory nasal sacs, but chondrosteans, polypterids, lepisosteids, and amiids lack them.The olfactory rosette is formed by primary folds or lamellae that may be placed anterior, lateral, posterior, and/or medial to the axis of the organ. Large acipenserids have 20-32 lamellae, polyodontids have 13-18 lamellae, lepisosteids have 8-10 lamellae, and Amia may have over 100. In teleosts, the number of lamellae varies from none or a few to over 200. Secondary lamellae are present in acipenseriforms, lepisosteids, and some advanced teleosts; secondary lamellae are interpreted as independently acquired in these lineages. Secondary lamellae are absent in Amia and primitive teleosts such as Elops and Hiodon. Tertiary lamellae are present in Acipenser oxyrhynchus. The arrangement of the primary lamellae in relation to the axis of the organ results in at least 11 patterns of the olfactory rosette in actinopterygians. Lamellae that are enclosed in a tubelike sac and that have an anteromedial diverticulum are specializations of polypterids. Primary lamellae anterior, lateral, and posterior to an elongate axis are characteristic of lepisosteids. The presence of primary lamellae lateral, medial, and posterior to an elongate olfactory axis is a synapomorphy of Halecomorpha (Amia plus teleosts). The absence of secondary lamellae is a synapomorphy of Halecomorpha. © 1994 Wiley-Liss, Inc.
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  • 28
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: On the ventral side of each pupal abdominal segment of the housefly, there is a pair of histoblast nests, each containing about 600 diploid cells. These cells, during adult development, divide, replace intervening polytene larval epidermal cells (LEC), and form both the median sternite and the surrounding pleura of the adult segment. Since the histoblast nests and the LEC form a contiguous layer, we examined the role of these two types of cells in regulating the mitotic potential of the histoblasts during development of the median sternite. Two experimental approaches were used: deletion of one of the nests by thermocautery; and by disturbance of the continuity of the monolayered epidermis by thermocautery of, or topical application of heptanol on, the midventral LEC. Ablation of one of the contralateral nests resulted in a mirror image duplication of the hemisternite and pleura by the surviving nest. Disturbance of the continuity of the LEC produced mirror image duplication of the hemisternal pattern by each of the contralateral nests. From these results, we propose that the contralateral ventral nests mutually downregulate their mitotic potential by secreting regulatory factor(s) to produce the normal median sternite pattern and surrounding pleura. We also suggest that these chemicals act in a paracrine fashion, possibly through gap junctions in the LEC. © 1994 Wiley-Liss, Inc.
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  • 29
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    Journal of Morphology 222 (1994), S. 309-326 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The relatively large, but superficially similar, Lerista macropisthopus, L. connivens, and L. lineopunctulata differ in bodily elongation and limb reduction, inhabit sandy areas, and move under sand. Visual analysis and computer-generated excursion and curvature graphs show that each species moves differently on smooth and rough surfaces, on surfaces with and without nails, and in channels.The reduced-limbed quadruped, Lerista macropisthopus walks frequently, using its four clawed limbs, whenever traction is available. Its undulating body curves uniformly but never generates slide-pushing curves. The biped L. connivens walks with its hindlimbs, although less frequently, and/or oscillates its tail in propelling its relatively stiff, short body. The biped L. lineopunctulata rarely uses its hindlimbs but always undulates body and. tail. It can use single nails in cam-follower progression. L. macropisthopus and L. connivens walk well in channels with rough bottoms, but only L lineopunctulata uses tunnel concertina to travel in channels with smooth bottoms.Friction of body surfaces dragged and of those transmitting propulsive forces is critical to these lizards and explains the division of movement into slow and rapid progression rates. Animals that have clawed limbs, no matter how reduced, use them. Body and tail generally are used differently. The tail may be flipped anteriorly to facilitate concertina. In nail arrays, travel is by simple, never by lateral, undulation. Apparently distinct motor coordination patterns are associated with differences in morphology, habit, and habitat. © 1994 Wiley-Liss, Inc.
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  • 30
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    Journal of Morphology 222 (1994), S. 327-335 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The appearance, cellular distribution, and changes of sugar residues during tooth development in adults of the polyphyodont, Liolaemus gravenhorsti, were investigated by using horseradish-peroxidase-conjugate lectins (lectin-HRP). With Con A (Canavalia ensiformis), the ameloblasts (late bell stage) show granular supranuclear positivity and also at the Golgi zone and on their tomes process. Reactivity also appears at the apical surface of the odontoblasts and odontoblastic process. With WGA (Triticum vulgaris), the tooth germs (late bell stage) show cytoplasmatic granular positivity in the ameloblast cells, Golgi regions, and in a lesser extent of the cytoplasm. Also, the apical surface and the odontoblastic process react. WGA reaction is depressed following sialidase treatment.The significance in tooth germs of α-D-mannose, α-D-glucose as well as β-D-N-acetylglucosamine and sialic acid is difficult to ascertain. These oligosaccharides may have some significance in odontogenesis. In fact, Con A-HRP- and WGA-HRP-binding components in ameloblasts and odontoblasts may be functionally related to molecules that are thought to contribute to odontogenesis in lizards. © 1994 Wiley-Liss, Inc.
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  • 31
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    Cell Motility and the Cytoskeleton 27 (1994), S. 26-40 
    ISSN: 0886-1544
    Keywords: cleavage furrows ; cytokinesis ; actin ; phalloidin ; myosin ; filamin ; talin ; attachment plaques ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: PtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows. © 1994 Wiley-Liss, Inc.
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  • 32
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    Cell Motility and the Cytoskeleton 27 (1994), S. 69-78 
    ISSN: 0886-1544
    Keywords: kinesin ; dynein ; MAP-motor interactions ; microtubule arrays ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Bundles of native microtubules isolated from the ovarioles of hemipteran insects are seen to shimmer when observed using dark-field microscopy. This novel form of microtubule motility becomes even more obvious when the isolated bundles are detergent-extracted and reactivated. We have studied the nucleotide-specificity and the drug-sensitivity of microtubule shimmering in order to obtain information regarding the nature of the motor protein responsible, and to compare its properties with those of previously characterised microtubule motors. The involvement of structural MAPs in the shimmering and in maintenance of microtubule bundles in this system has also been investigated. © 1994 Wiley-Liss, Inc.
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  • 33
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    Cell Motility and the Cytoskeleton 27 (1994), S. 88-96 
    ISSN: 0886-1544
    Keywords: cell movement ; speed ; persistence time ; colcemid ; alveolar macrophage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The role of microtubules in random cell migration was investigated using time-lapse videomicroscopy to record in vitro the shape and motile behavior of guinea pig alveolar macrophages before and after disrupting microtubules with colcemid. Cell migration was quantified in terms of directional persistence time and speed. Motility was also correlated with morphological polarity: cells having a single lamellipodal region (monopolar cells) migrated, whereas those lacking a lamellipod (apolar cells) or with opposing lamellipodal regions (bipolar cells) did not migrate. Within 2 hours, colcemid caused a shift in polarity from 80% monopolar cells to 40% monopolar and 40% bipolar cells and a corresponding decrease from 80% to 40% in the fraction of migrating cells. Mean persistence time and speed decreased only slightly (approximately 20%) for those cells (still monopolar) which continued to migrate in the presence of colcemid. Persistence time and speed actually increased for many individual cells, indicating that random migration did not require intact microtubules. We conclude that colcemid treatment destabilizes monopolarity, leading to the gradual loss of monopolarity and consequent inhibition of migration. While a cell remains monopolar, it will continue to migrate even in the absence of intact microtubules, but microtubules are required for the long-term maintenance of cellular monopolarity and, thus, for continued motility. © 1994 Wiley-Liss, Inc.
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  • 34
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    Cell Motility and the Cytoskeleton 27 (1994), S. 327-336 
    ISSN: 0886-1544
    Keywords: HEL cells ; cell spreading ; fibronectin ; diacyl glycerol ; phorbol myristate acetate ; protein kinase C ; staurosporine ; thymosin beta four ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Human erythroleukemia (HEL) cells grow in suspension, but after treatment with nM PMA the cells adhere and spread on glass or fibronectin [Jarvinen et al., 1987: Eur. J. Cell Biol. 44:238-246]. We observed an early (20-30 min) stage of spreading in which F-actin was organized into peripheral arcs near the spreading margin and vinculin was localised to the cell's periphery at the ends of these arcs. By 1 h the cells were well spread with straight actin bundles many of which ended at more central sites terminating on patches containing vinculin and talin; thus the cells assemble typical stress fibers but do not appear to polarize. The cells also spread on RGD polymer. DiC8 (1,2-dioctanoyl-sn-glycerol, C8:0, Sigma Chemical Co., St. Louis, MO) induced spreading but only if DAG kinase inhibitor and A-23187 were also present; in their absence cells adhered but did not spread. Spreading was ∼85% inhibited by 100 nM staurosporine. PKC-β was shown to be present in the cells by immunoblotting. In cells spread for 1 h with PMA, F-actin increased to 180% of control levels as measured by RP binding and the actin sequestering complex of G-actin-thymosin β4 decreased significantly.To determine whether the F-actin increase required adhesion, we inhibited cell attachment to the substratum by adding RGDS, by coating glass surfaces with hemoglobin, or by a combined treatment. Under these conditions PMA-treated suspended cells still increased their F-actin to 126-137% of controls, a significant increase over control levels. Staurosporine inhibited F-actin increases under all the conditions studied.Permeabilized cell suspensions, incubated with rhodamine labelled G-actin, incorporated the labelled actin along cell membranes at a low level. A few minutes preincubation with either diC8 plus DAG kinase inhibitor or with PMA strongly increased the incorporation. This increased incorporation was reduced to below control levels by either staurosporine (100 nM) or cytochalasin D (1 μM).We conclude that both suspended and spreading HEL cells can be stimulated to polymerize actin by a mechanism dependent on PKC or a PKC-like molecule. In suspended cells, the polymerization occurs along the membrane. When cells spread, F-actin increased to a significantly greater extent. This second step could involve additional polymerization, perhaps at the observed adhesion sites, decreased turnover of the actin bundles, or a combined effect of both mechanisms. © 1994 Wiley-Liss, Inc.
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  • 35
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    Cell Motility and the Cytoskeleton 28 (1994), S. 34-44 
    ISSN: 0886-1544
    Keywords: exocrine gland ; protein secretion ; microtubule-disrupting drugs ; immunofluorescence microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The role of microtubules in the exocrine secretory process is not yet well established, and their disruption by anti-microtubule drugs leads to variable effects on intracellular transit and protein secretion. We investigated the involvement of microtubules in the regulated secretory process of rat parotid glands using microscopic techniques and pulse-chase experiments. We showed that 10 μM colchicine or nocodazole destroys the microtubule network in parotid acinar cells but only weakly reduces the release of newly synthesized proteins. The half-effect was obtained with 0.22 μM colchicine. Moreover, this small reduction was found to be independent of the nature of the drug (colchicine, colcemid, or nocodazole) and of the nature of the stimulation (β-adrenergic or cholinergic pathways). Using nocodazole, we have been able to determine that the steps affected by the drug are very early events in the secretory pathway. Finally, we showed by kinetic analysis that microtubule disruption slows protein release only moderately but does not reduce the total amount of secreted protein. We conclude from this study that microtubule integrity is not essential for protein secretion in rat parotid gland. © 1994 Wiley-Liss, Inc.
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  • 36
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    Cell Motility and the Cytoskeleton 28 (1994), S. 59-68 
    ISSN: 0886-1544
    Keywords: cytoskeleton ; paracrystal ; coiled ribbons ; microtubule-associated proteins ; assembly ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Isolated microtubules from cod and cow brains were compared with respect to their response to calcium ions. The effect of Ca2+ on cod microtubules was found to be temperature dependent. In contrast to cow microtubules, cod microtubules assembled at 18°C. At this temperature the assembly was inhibited by Ca2+ concentrations of 2 mM and higher. This was also found for cow microtubules at 37°C. However, at 30°C there was no effect of 2 mM Ca2+ of the amount of assembly or disassembly of cod microtubules consisting of only tubulin or of tubulin and microtubule-associated proteins (MAPs). The morphology was affected though, since some coiled ribbons formed from tubulin and MAPs. The calcium-binding calmodulin did not alter the effect of calcium on cod microtubules markedly. At higher Ca2+ concentrations (〉4 mM), coiled ribbons were formed from cod tubulin and MAPs, but mainly amorphous aggregates and very few coiled ribbons were formed from cod tubulin alone, indicating that the Ca2+ effect is modulated by cod MAPs. The modulatory effect of cod MAPs was however not species specific, since both cod and cow MAPs had the same effect on cod microtubules, in spite of a different protein composition. A MAP-dependent effect of Ca2+ was also found for cow microtubule proteins. The assembly of pure cow tubulin, as well as that of cow tubulin and MAPs, was inhibited by 2 mM Ca2+. In the presence of 10 and 20 mM Ca2+, pure cow tubulin formed amorphous aggregates, rings, and even paracrystals, while the assembly of cow tubulin and MAPs was inhibited. Our results suggest therefore that the effect of Ca2+ can be moderated by MAPs, but depends on intrinsic properties of the different tubulins. © 1994 Wiley-Liss, Inc.
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  • 37
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    Cell Motility and the Cytoskeleton 28 (1994), S. 135-142 
    ISSN: 0886-1544
    Keywords: bidirectional swimming ; flagellar movement ; helical bends ; 9+0 axoneme ; planar bends ; viscosity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Spermatozoa of the small myzostomid worm Myzostomum cirriferum usually swim with the flagellum foremost but occasionally stop and then swim with the head foremost. The spermatozoa have axoneme of the 9+0 type; thus each lacks the central pair microtubules. The flagellum emerges in the anterior end of the cell body and attaches to it with junctions. To understand the mechanism regulating the swimming direction of the spermatozoa, we recorded the sperm and their flagellar movements using a video camera with a high-speed shutter. The effects of calcium and viscosity on these movements were also examined.The cell body with the flagellum attached to it formed a curved plate during beating, while the free portion of the flagellum beats with small helical bends. Motive force to propel a spermatozoon was mainly due to the bends in the cell body. The spermatozoa reversed the direction of their swimming as a result of a change in the direction of bend propagation. The direction of bend propagation was regulated by calcium; the bends in the cell body propagated from the end of the head toward the free portion of the flagellum at low concentrations of Ca2+, whereas the direction of bend propagation was reversed at high concentrations of this ion. High viscosity of the medium stimulated a change in the direction of bend propagation. © 1994 Wiley-Liss, Inc.
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  • 38
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    Cell Motility and the Cytoskeleton 28 (1994), S. 155-164 
    ISSN: 0886-1544
    Keywords: microfilamentous cytoskeleton ; actin binding proteins ; formyl peptides ; ionic extraction ; immunoblots ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: F-actin is a major component of the neutrophil (PMN) cytoskeleton. In basal PMNs, F-actin exists in two structurally and functionally distinct pools: Triton insoluble F-actin (TIF)-cold insensitive, not depolymerizable by dilution, and distributed in pseudopods and submembranous locations; and Triton soluble F-actin (TSF)-unstable in cold, diffusely distributed, and gelsolin enriched. The element(s) conferring these unique properties to the Triton insoluble F-actin pool are unknown, but logically include distinct actin regulatory proteins. To study the morphologic and functional determinants of the Triton insoluble F-actin pool, the distribution and quantity of three candidate regulatory proteins, α-actinin, tropomyosin (TM), and actin binding protein (ABP-280), were compared in F-actin (Triton insoluble and Triton soluble) and G-actin pools isolated from basal and chemotactic factor activated human PMNs in suspension, using immunoblots and ionic extraction. F-actin content was measured by NBDphallacidin binding and gel scans. The results show that: (1) α-actinin, actin binding protein 280, and tropomyosin are localized to TIF and excluded from TSF; (2) TM, α-actinin, and ABP 280 are required to stabilize fractions of Triton insoluble F-actin in PMNs; and (3) chemotactic factor activation results in release of a fraction of TM from the Triton insoluble F-actin pool in temporal association with F-actin polymerization in the Triton insoluble F-actin pool. Shifts in ABP 280 or α-actinin do not occur. The results suggest that TM, α-actinin, and ABP 280 provide structure to TIF and that TM release from TIF is involved in chemotactic factor induced actin polymerization in PMNs. © 1994 Wiley-Liss, Inc.
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  • 39
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    Cell Motility and the Cytoskeleton 28 (1994), S. 165-178 
    ISSN: 0886-1544
    Keywords: WISH ; Keratin ; 3-D reconstruction ; mitosis ; intermediate filaments ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Three dimensional (3-D) reconstruction of four mitotic WISH cells from ultrathin sections gave an informative representation of the spatial distribution of keratin densities in these cells. The correspondence between the densities as studied by transmission electron microscopy (TEM) and the Keratin bodies initially revealed by immunoflourescent colabeling of cultures, was confirmed by immunoelectron-microscopy. The smaller, and sometimes more elongated densities, were relatively abundant just beneath the subplasmalemmal microfilament band; and at certain levels of the mitotic cell they were observed to be connected to neighboring densities by intact intermediate filaments (IFs). The larger and more spherical densities appeared to be somewhat more discrete and randomly distributed. Other observed associations of the keratin densities included the telophase contractile ring of microfilaments, chromosomes, the reformed telophase nucleus, and desmosomal junctions with neighboring interphase cells. Cytochalasin D (CD) treatment of cells displaced the peripheral keratin densities toward the cell membrane. The density volume constituted 0.52% to 1.57% of the total cell volume, and the proportional density size was decreased in the cells that had progressed into anaphase and telophase. The observed formation and subsequent dissolution of keratin densities during mitosis may represent a dynamic mechanism of restructuring the keratin cytoskeleton in an unpolymerized form in order to allow for rapid reformation of interphase cell junctions. The physical associations observed between intact IFs and the keratin densities may provide support at certain depths of the mitotic cell, and the juxtaposition of densities with nuclear components suggests a possible source of and role for keratin IFs during nuclear events. © 1994 Wiley-Liss, Inc.
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  • 40
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    Cell Motility and the Cytoskeleton 28 (1994), S. 199-204 
    ISSN: 0886-1544
    Keywords: axoneme ; cilia ; flagella ; microtubule ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Observations that were interpreted to provide evidence for equivalent functions of all axonemal dyneins should be reinterpreted, and models based on this assumption should be abandoned. In the future, attempts to understand the mechanisms for flagellar bending, oscillation, and bend propagation should start from the assumption that each type of axonemal dynein may have a specific function. At least three distinct functions can now be identified: bend initiation, maintenance of the angle of propagating bends, and generation of power to overcome viscous resistances. Only the last of these three functions is an outer arm dynein function. © 1994 Wiley-Liss, Inc.
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  • 41
    ISSN: 0886-1544
    Keywords: cytoskeleton ; actin binding ; transgelin sequence ; gelation ; gene family ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have used degenerate oligonucleotides, derived from the amino acid sequence of transgelin peptides [Shapland et al., 1993: J. Cell Biol. 121:1065-1073], to isolate and sequence overlapping cDNA clones encoding this actin gelling protein. Primers with 5′ restriction enzyme sites directed against the N and C terminal amino acids present in these clones were then used to amplify and clone the entire transgelin coding region from reverse transcribed rat small intestine cDNA (RT-PCR). These studies have shown that transgelin is the product of a single gene which is conserved between yeast, Drosophila, molluscs, and humans. Transgelin is expressed as a single message that is regulated at the level of transcription in SV40 transformed 3T3 cells. Our data have shown that transgelin and several other proteins of unknown function, SM22α [Pearlstone et al., 1987: J. Biol. Chem. 262:5985-5991], mouse p27 [Almendral et al., 1989: Exp. Cell Res. 181:518-530], and human WS3-10 [Thweatt et al., 1992: Biochem. Biophys. Res. Commun. 187:1-7], share extensive homology. More limited regions of homology shared between transgelin and other proteins such as rat NP25 (unpublished), chicken calponins α and β [Takahashi and Nadal-Ginard, 1991: J. Biol. Chem. 266:13284-13288], and Drosophila mp20 [Ayme-Southgate et al., 1989: J. Cell Biol. 108:521-531] suggest that all of these proteins may be classified as members of a new transgelin multigene family. © 1994 Wiley-Liss, Inc.
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  • 42
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    Cell Motility and the Cytoskeleton 28 (1994), S. 279-284 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 43
    ISSN: 0886-1544
    Keywords: cytoskeleton ; cell culture ; gene expression ; Northern blot ; serum-induction ; rat ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cytochalasin D and dBcAMP cause cultured astrocytes to change from flat cells to retrated process-bearing cells. F-actin was present throughout cells stimulated with dBcAMP for 16 h, whereas cytochalasin D caused F-actin to form massive aggregates at the tips of the cell processes. The two drugs differently regulated the expression of both β-actin and tropomyosin genes in astrocytes cultured in the presence or absence of serum: dBcAMP caused down-regulation and cytochalasin D caused up-regulation. Northern blot analyses indicated that: (1) serum deprivation halved the concentration of all tropomyosin transcripts (TM-1, TM-2, TM-4, TMBr-1, TMBr-2). Serum induced TM-4 via transcriptional activation, independent of protein synthesis, (2) dBcAMP induced down-regulation of β-actin (-50%) and tropomyosin transcripts (-35 to 52%) even in the presence of serum. The concentration of profilin mRNA decreased in dBcAMP-reactive astrocytes (-46%). The decrease in β-actin mRNA concentration was not blocked by cycloheximide, whereas down-regulation of tropomyosin transcripts was completely reversed when protein synthesis was inhibited, and (3) cytochalasin D induced an increase in the concentration of tropomyosin transcripts (+ 69 to 185%) which was cumulative with serum stimulation. Cytochalasin D induction of both β-actin and TM-4 operated through transcriptional activation, independent of protein synthesis.The production of all tropomyosin transcripts examined here were strictly coordinated with β-actin expression in serum-, dBcAMP- and cytochalasin D-treated astrocytes. This indicates that the differential expression of tropomyosin isoforms occurring during astrocyte maturation is due to more complex regulation than that involved in serum- or cAMP-stimulated astrocytes. © 1994 Wiley-Liss, Inc.
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  • 44
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    Cell Motility and the Cytoskeleton 28 (1994), S. 333-345 
    ISSN: 0886-1544
    Keywords: ciliary beat frequency ; metachronal wave ; ciliary coupling ; extracellular ATP ; acetylcholine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In the present work we measured in real time the metachronism and degree of correlation between beating cilia from cultured mucociliary epithelium. The method is based on simultaneous measurement of ciliary beat frequency, phase shifts, and correlation factors in two directions: parallel and perpendicular to the effective stroke direction (ESD). From the phase shifts the lengths of wave components, and consequently the metachronal wavelength and direction, were evaluated.On active ciliary areas of cultured frog esophagus under normal conditions, a relatively high degree of correlation is observed, but cilia are more correlated in direction parallel to ESD which is also the direction of the mucus propulsion. The length of the wave component parallel to ESD is more than twice as large as that of the perpendicular component. The metachronal wavelength was found to be in the range of 5-9 μm, and the direction of the wave propagation was in the range of 90°-125° clockwise to the ESD.When ciliary beat frequency was rapidly increased by extracellular ATP or acetylcholine, only minor effects were observed on the degree of correlation between beating cilia. The length of the wave component parallel to ESD showed the most dramatic effect increasing up to tenfold. The perpendicular to ESD component was not affected by the stimulation. Consequently, the metachronism became more laeoplectic with the angle between the ESD and the wave directions decreasing by 10°-30°, and the metachronal wavelength remained unaltered. © 1994 Wiley-Liss, Inc.
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  • 45
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    Cell Motility and the Cytoskeleton 29 (1994), S. 57-71 
    ISSN: 0886-1544
    Keywords: microtubule bundling ; cytoskeleton ; tau ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22°C. The bundles, which were readily visible by video-enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6-11 μm in length and 2-5 MTs in thickness. No inter-MT cross-bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP-2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt-extractable and heat-stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were “unbundled” by subtilisin or by high salt (0.5-1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt-induced unbundling was reversible with desalting. However, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor. © 1994 Wiley-Liss, Inc.
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  • 46
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    Cell Motility and the Cytoskeleton 29 (1994), S. 72-81 
    ISSN: 0886-1544
    Keywords: spectrin ; intrinsic fluorescence ; spectrin elasticity ; fluorescence quenching ; spectrin α chain ; spectrin β chain ; membrane skeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To better understand the solution structure of spectrin, the environments of its tryptophan residues have been examined by fluorescence spectroscopy. The spectra and the extent of quenching by several quenching agents have been determined for intact spectrin and its α and β subunits. The arsenal of quenchers used in the study represented both hydrophilic and hydrophobic species including anionic, cationic and neutral compounds. Effects on spectrin fluorescence of ethanol and ionic strength, which extend and/or rigidify spectrin, and of glycerol, which is commonly used in electron microscopy of the protein, have also been assessed in the presence and absence of quenchers. Most of the tryptophans of spectrin are either internally quenched or are sequestered, hindering the approach of hydrophilic quenching agents. Both the spectral shape and the extent of quenching by acrylamide indicate that some tryptophans of the β subunit are slightly more exposed in the isolated chain than in the dimer. Similar effects on spectra and on quenching of the intact dimer and of the isolated β chain are seen when the ionic strength is reduced. Ethanol and glycerol reduce spectrin tryptophan accessibility to 2-p-toluidinyl napthalene-6-sulfonic acid (TNS). It therefore appears that low ionic strength, α-β association and neutral solute (or lowered dielectric constant) all induce a similar, but modest conformational change in the domain structure. The extent of TNS binding is not increased by lowering the ionic strength, suggesting that the expansion and/or stiffening of the molecule in low electrolyte solutions does not involve exposure of significant numbers of hydrophobic sites. © 1994 Wiley-Liss, Inc.
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  • 47
    ISSN: 0886-1544
    Keywords: intermediate filaments ; phosphorylation ; sea urchin embryos ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effects of 6-dimethylaminopurine (6-DMAP) on the length of the cell cycle and on the state of phosphorylation of a putative intermediate filament protein, p117, have been studied in sea urchin embryos. Embryos were transferred into sea water containing 600 μM 6-DMAP at 0.5, 2 or 5 min after insemination, and incubated for 30 or 90 min. The effects of 6-DMAP on cell cycle length were studied by determining the time required for completion of mitosis upon return of the embryos in normal sea water. In all instances, except for the embryos transferred 0.5 min after insemination (AI) and incubated for 30 min, the duration of the M phase was shortened compared to controls, being faster in the embryos incubated for 90 minutes compared to the 30 min incubation period. However, embryos transferred 0.5 min AI have a longer M-phase than those transferred 2 minutes or later after fertilization, suggesting that between 0.5 and 2 min after fertilization, critical phosphorylating events occur which affect the commitment of the cells to enter M-phase.To study the pattern of p117 phosphorylation during the cell cycle, the eggs were transferred 2 minutes after fertilization in presence of 600 μM 6-DMAP and with 200 μCi/ml of 32P-orthophosphate. Analyses of 32P-labelled proteins after exposure of SDS-PAGE gels and their corresponding blots suggested that phosphorylation of p117 greatly increases at the time of pronuclear fusion, and then declines slightly at prophase-metaphase. This decrease is markedly enhanced when the cells are treated with 6-DMAP during metaphase in order to induce a premature breakdown of the mitotic apparatus. A causal link is suggested between the level of phosphorylation of p117 and its state of assembly. © 1994 Wiley-Liss, Inc.
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  • 48
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    Cell Motility and the Cytoskeleton 29 (1994) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 49
    ISSN: 0886-1544
    Keywords: intermediate filaments ; cytoskeleton ; filament attachment sites ; immunogold labeling ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The substructure of assembling cytoplasmic dense bodies (CDBs) and changes in the distribution of desmin and α-actinin during development of smooth muscle were studied in gizzard samples from 10- and 16-day embryos and from 1- and 7-day post-hatch chickens. CDBs in these cells lack the density of CDBs in mature or adult smooth muscle cells and, thus, allow observations of the changes inside CDBs. The random filament orientation seen in younger embryonic cells is first modified to include relatively small patches of IFs that are somewhat straighter and are approaching a side-by-side arrangement. As development proceeds, the IFs in these arrays become straighter, are parallel over longer lengths of the IFs and later acquire the density characteristic of mature CDBs. Anti-desmin labeling in embryonic 10- and 16-day cells showed that desmin intermediate filaments (IFs) were located in the myofilament compartment but were concentrated in or near assembling CDBs. Anti-desmin labeling shifted to the perimeter of CDBs after hatching. Cross sections, longitudinal sections, and stereo pairs all show that IF profiles are present inside unlabeled assembling CDBs. Anti-α-actinin labeling was directly on CDBs and was often associated with the cross-connecting filaments (CCFs) (average diameter of 2-3nm) inside CDBs. We propose, based on these data, that desmin IFs, α-actinin-containing CCFs, and actin filaments are the principal components of the substructure of assembling CDBs. We also present a proposed model for CDB assembly. © 1994 Wiley-Liss, Inc.
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  • 50
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    Cell Motility and the Cytoskeleton 29 (1994), S. 241-249 
    ISSN: 0886-1544
    Keywords: immunofluorescence ; microinjection ; mitotic apparatus ; monoclonal antibodies ; sand dollar egg ; tubulin isotypes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect on fixation on the reactivities of mitotic microtubules with monoclonal anti-tubulin antibodies was investigated by the indirect immunofluorescence procedure. All of the seven antibodies used intensely stained mitotic microtubules in sea urchin eggs lysed and fixed with methanol at -20°C, whereas only two of them stained the stabilized microtubules in the lysed eggs before the fixation. The other five did not stain the mitotic microtubules even after microtubule components other than tubulin were removed by treating the lysed eggs with 0.4 M KCl solution containing taxol. These results exclude the possibility that the fixation affects proteins, which interact with microtubules including microtubule-associated proteins (MAPs) and interfere with the binding of monoclonal antibodies with tubulin, and strongly suggest that the fixation directly affects the three-dimensional conformation of tubulin Furthermore, microinjection of these antibodies indicated the results as follows [combining the results reported previously; Oka et al., 1990: Cell Struct. Funct. 15: 373-378]: The antibodies which stained mitotic microtubules stabilized in the lysed eggs induced disassembly of native mitotic microtubules in the living eggs, but those which did not stain the stabilized microtubules did not disassemble the native microtubules. From these results, it is suggested that the monoclonal antibodies which stain microtubules in the eggs lysed but not fixed are useful for microinjection experiments. © 1994 Wiley-Liss, Inc.
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  • 51
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    Cell Motility and the Cytoskeleton 29 (1994), S. 271-279 
    ISSN: 0886-1544
    Keywords: peptide antibodies ; protein processing ; axonemes ; microtubule associated proteins ; UV photocleavage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Dyneins are multi-subunit enzymes that transduce chemical energy into the mechanical energy that makes cilia and flagella beat and moves organelles towards the minus end of microtubules. The ATPase activity is borne by heavy chains, and recent molecular analysis indicates that dynein heavy chain genes form an ancient multigene family: the similarity between the same isoform of two distantly related species is greater than that between different isoforms of the same species. We have exploited sequence identities between a Paramecium axonemal dynein heavy chain gene cloned in our laboratory and sequences of dynein heavy chains from other species to prepare antibodies against active-site peptides capable of recognizing dynein heavy chains regardless of species or isoform. One of the antibodies is perfectly specific for the larger product of V1 photolysis (HUV1) and thus incorporates a unique property of the hydrolytic ATP binding site of all known dynein heavy chains, the capacity for photocleavage in the presence of micromolar vanadate. Our characterization of these reagents suggests that they will be useful for biochemical and in situ studies of known dyneins as well as identification of potential new members of the family. © 1994 Wiley-Liss, Inc.
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  • 52
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    Cell Motility and the Cytoskeleton 29 (1994), S. 301-311 
    ISSN: 0886-1544
    Keywords: ctenophore ; egg ; nucleus ; microtubule ; endoplasmic reticulum (ER) ; sperm aster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In the large eggs (∼1 mm) of the ctenophore Beroe ovata, female pronuclei migrate long distances to join stationary male pronuclei in the peripheral cytoplasm that surrounds the yolky interior. We have investigated the mechanism of nuclear migration using time lapse video recording, automated image analysis, visualization of microtubules by immunofluorescence and rhodamine-tubulin injection, and electron microscopy. Female pronuclei migrated at average speeds of 0.2 μm/sec, and were found to show periodic oscillations in velocity. Alternating phases of acceleration and deceleration occurred with an average periodicity of 235 seconds covering distances of 47 μm (about 3 times the nuclear diameter). Migration velocities and velocity oscillations were similar in fertilized and unfertilized eggs; however, changes in migration direction were much more frequent in unfertilized eggs. Characteristic deformations of the pronuclear membrane and occasional rotation of the nuclear contents were observed during migration. Inhibitor studies indicated that microtubules are required for nuclear migration. In fertilized eggs the top of the nucleus was found to move through the dense layer of aligned sperm aster microtubules. The frequent changes in direction of pronuclear migration in unfertilized eggs reflect the random organization of the microtubule layer in the absence of sperm derived centrosomes. Densely packed endoplasmic reticulum was found intermeshed with sperm aster microtubules and connected extensively with the nuclear membrane during migration. Most nuclear pores were grouped in an infolding of the nuclear membrane. We suggest that in fertilized eggs the female pronucleus is transported to the minus ends of sperm aster microtubules using motor molecules attached either to the outer nuclear membrane and/or to the network of connecting ER. © 1994 Wiley-Liss, Inc.
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  • 53
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    Cell Motility and the Cytoskeleton 29 (1994), S. 225-230 
    ISSN: 0886-1544
    Keywords: Weber's Law ; retinal ; retinal analogs ; photoreception ; alga ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The unicellular green alga Chlamydomonas reinhardtii maintains sensitivity of its phototaxis response (alignment of swimming direction along the axis of a light beam) over several orders of magnitude of light intensities. It is widely accepted that the rotation of the swimming cell provides temporal comparisons of light intensities via periodic contrast generated by its asymmetrically positioned refractile eyespot organelle. The cells also exhibit a second behavioral response to light called the photophobic (or stop) response, which is a brief cessation of swimming caused by a temporal change in light intensity. The cells are desensitized to photophobic stimuli by light exposure. Through comparative measurements of both responses, we explain the behavioral basis of the large dynamic range of phototaxis in terms of precise desensitization of the photophobic response. The basis of the explanation is that the flagellar beat changes which cause phototactic orientation are the residual of the photophobic response after desensitization (i.e., “mini-photophobic” reactions which cause brief reorienting motions without a full stop). This interpretation predicts quantitatively the dependence of the extent of desensitization on light intensity and the dependence of onset and maintenance of phototaxis on extent of desensitization. These predictions are tested and confirmed in this report. © 1994 Wiley-Liss, Inc.
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  • 54
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    Cell Motility and the Cytoskeleton 29 (1994), S. 259-270 
    ISSN: 0886-1544
    Keywords: calsequestrin ; calreticulin ; sarcoplasmic reticulum ; skeletal muscle ; myofibril ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A major Ca2+-storing protein in endoplasmic reticulum (ER) of non-muscle cells is calreticulin (CR), which is considered to be functionally homologous to calsequestrin. Calsequestrin is a Ca2+-binding protein in sarcoplasmic reticulum (SR) of striated muscle, which stores Ca2+ during muscle relaxation. In order to investigate the expression and distribution of calsequestrin and calreticulin during skeletal muscle differentiation, cultured chick embryonic skeletal muscles were observed by immunofluorescence using anti-calsequestrin, anti-calreticulin, antidesmin, and anti-sarcomeric myosin antibodies and rhodamine-phalloidin. Within 6 hours in culture, myoblasts started to express desmin. Desmin-positive cells demonstrated the reticular staining of calreticulin, as did desmin-negative cells. Around fusion, calsequestrin and sarcomeric myosin started to appear in desmin-positive cells. The expression of calsequestrin slightly preceded that of sarcomeric myosin. As the myotubes matured, the fluorescent dots of calsequestrin increased and spread to the cell periphery along the myofibrils, while the reticular pattern of calreticulin gradually disappeared. Double labeling showed that calsequestrin colocalized with calreticulin. In mature myotubes, anti-calsequestrin staining demonstrated many dots along myofibrils, whereas calreticulin was barely seen except at the perinuclear region. These results suggest that the expression of calsequestrin and calreticulin are switched during skeletal muscle differentiation. © 1994 Wiley-Liss, Inc.
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  • 55
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    Cell Motility and the Cytoskeleton 29 (1994), S. 321-338 
    ISSN: 0886-1544
    Keywords: protrusive activity ; adherens junctions ; stress fibers ; permeabilized cell models ; myosin light chain kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Addition of protein kinase inhibitor H-7 leads to major changes in cell structure and dynamics. In previous studies [Citi, 1992: J. Cell Biol. 117:169-178] it was demonstrated that intercellular junctions in H-7-treated epithelial cells become calcium independent. To elucidate the mechanism responsible for this effect we have examined the morphology, dynamics, and cytoskeletal organization of various cultured cells following H-7-treatment. We show here that drug treated cells display an enhanced protrusive activity. Focal contact-attached stress fibers and the associated myosin, vinculin, and talin deteriorated in such cells while actin, vinculin, and N-cadherin associated with cell-cell junctions were retained. Furthermore, we demonstrate that even before these cytoskeletal changes become apparent, H-7 suppresses cellular contractility. Thus, short pretreatment with H-7 leads to strong inhibition of the ATP-induced contraction of saponin permeabilized cells. Comparison of H-7 effects with those of other kinase inhibitors revealed that H-7-induced changes in cell shape, protrusional activity, and actin cytoskeleton structure are very similar to those induced by selective inhibitor of myosin light chain kinase, KT5926. Specific inhibitors of protein kinase C (Ro31-8220 and GF109203X), on the other hand, did not induce similar alterations. These results suggest that the primary effect of H-7 on cell morphology, motility, and junctional interactions may be attributed to the inhibition of actomyosin contraction. This effect may have multiple effects on cell behavior, including general reduction in cellular contractility, destruction of stress fibers, and an increase in lamellipodial activity. It is proposed that this reduction in tension also leads to the apparent stability of cell-cell junctions in low-calcium medium. © 1994 Wiley-Liss, Inc.
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  • 56
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    Cell Motility and the Cytoskeleton 29 (1994), S. 383-383 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 57
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    Cell Motility and the Cytoskeleton 27 (1994), S. 161-168 
    ISSN: 0886-1544
    Keywords: fluorescent nucleotide analogs ; methylanthraniloyl ATP ; anthraniloyl ATP ; Chlamydomonas ; axonemal mutants ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Substrate analogs are useful for studying the structures of active sites and for distinguishing between similar enzyme activities. Fluorescent ribose-modified ATP analogs were used to investigate the functional differences between dynein ATPases. These analogs reactivate (support the movement of) sea urchin sperm axonemes, yet they do not reactivate wild-type Chalmydomonas axonemes. Surprisingly, the analogs reactivate the axonemes of mutants completely missing the outer arm dyneins. Competition experiments using ATP and these analogs provide strong evidence that the analogs bind to all dynein active sites but fail to release a subset of dyneins from rigor. We suggest that this subset of Chlamydomonas outer arm dyneins unable to use the analogs remains in rigor in the presence of the analogs and paralyzes the axoneme. © 1994 Wiley-Liss, Inc.
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  • 58
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    Cell Motility and the Cytoskeleton 27 (1994), S. 180-191 
    ISSN: 0886-1544
    Keywords: sliding movement ; 22S dynein ; Tetrahymena cilia ; dynein-track ; singlet microtubule ; ATP ; polarity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chlamydomonas and Tetrahymena axonemal dyneins have previously been found to bind to porcine brain microtubules to produce a microtubule-dynein complex. At appropriate microtubule:dynein concentration, microtubules in the complex became covered to saturation by dynein arms of the same polarity and at a spacing of 24 nm [Haimo et al., 1979; Haimo and Fenton, 1988; Haimo, 1989; Porter and Johnson, 1983a].In the present study, two different types of microtubule-dynein complexes (α-and β-complexes) were prepared from Tetrahymena ciliary 22S dynein and porcine brain tubulin. The characteristics of the adenosine triphosphate (ATP)-induced extrusion of microtubules from these complexes were analyzed, as a simple and direct in vitro assay for the ATP-induced extrusion of single microtubules. The α-complex prepared by adding dynein to microtubules showed an interrupted sliding movement, which would stop and start several times following the addition of ATP. In the β-complex, prepared by adding dynein bound to DEAE-tubulin to pre-assembled microtubules, microtubules became covered with dynein molecules whose orientation and binding were uniform with respect to microtubule polarity. The microtubules in the β-complex extruded at 12 μm/second following the addition of ATP. Dark-field and electron microscopy indicated that the extruded microtubules had undergone sliding on a dynein-track that had become detached from the complexes and had been absorbed onto the surface of the glass slide. At higher light intensity under a dark-field microscope, the dynein-track was seen to be composed of rows of dynein molecules arranged densely. The orientation of dynein molecules in rows appeared to be uniform considering the images of bound dynein in the β-complex under electron microscope. The higher sliding velocity of the microtubules on these dynein-tracks compared to that seen on slides coated at random with dynein [Vale and Toyoshima, 1988, 1989], may be due to more efficient force generation by this dense arrangement of dynein molecules with the same polarity on the tracks. © 1994 Wiley-Liss, Inc.
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  • 59
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    Proteins: Structure, Function, and Genetics 18 (1994), S. 205-215 
    ISSN: 0887-3585
    Keywords: peptide folding ; disulfide framework ; insect toxins ; NMR ; distance geometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An analysis of the sequences of scyllatoxin and charybdotoxin suggested that it would be possible to design a core peptide sequence which would still fold to give the β-hairpin and helix seen in the toxins, but which would eliminate one disulfide and connecting residues. The core sequence was modeled, then synthesized and purified. The cysteines oxidize in air to give the same disulfide pairings as seen in the parent toxins as the major product. The three-dimensional structure of the core sequence peptide, termed Max, was determined using proton NMR spectroscopy and found to be identical in secondary structure to the toxins. However differences were found in the relative orientation of the β-hairpin and helix. The use of this structural motif, found in many insect toxins, as a disulfide framework for exploring sequence/structure/activity relationships is discussed. © 1994 John Wiley & Sons, Inc.
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  • 60
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    Proteins: Structure, Function, and Genetics 18 (1994), S. 246-253 
    ISSN: 0887-3585
    Keywords: microcalorimetry ; heat capacity ; enthalpy ; hydrogen bonding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The energetics of ubiquitin unfolding have been studied using differential scanning microcalorimetry. For the first time it has been shown directly that the enthalpy of protein unfolding is a nonlinear function of temperature. Thermodynamic parameters of ubiquitin unfolding were correlated with the structure of the protein. The enthalpy of hydrogen bonding in ubiquitin was calculated and compared to that obtained for other proteins. It appears that the energy of hydrogen bonding correlates with the average length of the hydrogen bond in a given protein structure. © 1994 John Wiley & Sons, Inc.
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  • 61
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    Proteins: Structure, Function, and Genetics 18 (1994), S. 262-266 
    ISSN: 0887-3585
    Keywords: protein structure prediction ; protein stability ; hydrogen bond ; β-sheet ; amino acid propensity ; steric effect ; hydrogen exchange ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Amino acid side chains can enhance peptide group hydrogen bond strength in protein structures by obstructing the competing hydrogen bond to solvent in the unfolded state. Available data indicate that the steric blocking effect contributes an average of 0.5 kJ per residue to protein hydrogen bond strength and accounts for the intrinsic α-sheet propensities of the amino acids. In available data for helical models, the contribution to α-helix propensities is obscured especially by large context-dependent effects. These issues are all related by a common side chain-dependent steric clash which disfavors peptide to water H-bond formation, peptide to catalyst complexation in hydrogen exchange reactions (Bai et al., Proteins 17:75-86, 1993), and peptide to peptide H-bonding in the helical main chain conformation (Creamer and Rose, Proc. Natl. Acad. Sci. U.S.A. 89:5937-5941, 1992) but not in α-strands. © 1994 John Wiley & Sons, Inc.
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  • 62
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    Proteins: Structure, Function, and Genetics 18 (1994), S. 281-294 
    ISSN: 0887-3585
    Keywords: membrane ; protein ; structure ; prediction ; G-protein coupled receptor ; rhodopsin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Integral membrane proteins (of the α-helical class) are of central importance in a wide variety of vital cellular functions. Despite considerable effort on methods to predict the location of the helices, little attention has been directed toward developing an automatic method to pack the helices together. In principle, the prediction of membrane proteins should be easier than the prediction of globular proteins: there is only one type of secondary structure and all helices pack with a common alignment across the membrane. This allows all possible structures to be represented on a simple lattice and exhaustively enumerated. Prediction success lies not in generating many possible folds but in recognizing which corresponds to the native. Our evaluation of each fold is based on how well the exposed surface predicted from a multiple sequence alignment fits its allocated position. Just as exposure to solvent in globular proteins can be predicted from sequence variation, so exposure to lipid can be recognized by variable-hydrophobic (variphobic) positions. Application to both bacteriorhodopsin and the eukaryotic rhodopsin/opsin families revealed that the angular size of the lipid-exposed faces must be predicted accurately to allow selection of the correct fold. With the inherent uncertainties in helix prediction and parameter choice, this accuracy could not be guaranteed but the correct fold was typically found in the top six candidates. Our method provides the first completely automatic method that can proceed from a scan of the protein sequence databanks to a predicted three-dimensional structure with no intervention required from the investigator. Within the limited domain of the seven helix bundle proteins, a good chance can be given of selecting the correct structure. However, the limited number of sequences available with a corresponding known structure makes further characterization of the method difficult. © 1994 John Wiley & Sons, Inc.
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  • 63
    ISSN: 0887-3585
    Keywords: triglyceride lipase ; proenzyme ; molecular replacement ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X-ray studies. It is monoclinic (C2, a = 92.8 Å, b = 128.9 Å, c = 78.3 Å, β = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P212121, with a = 79.8 Å, b = 115.2 Å, c = 73.0 Å) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form. © 1994 John Wiley & Sons, Inc.
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  • 64
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    Proteins: Structure, Function, and Genetics 18 (1994), S. 309-317 
    ISSN: 0887-3585
    Keywords: protein structure prediction ; predicted contact maps ; correlated mutations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The maintenance of protein function and structure constrains the evolution of amino acid sequences. This fact can be exploited to interpret correlated mutations observed in a sequence family as an indication of probable physical contact in three dimensions. Here we present a simple and general method to analyze correlations in mutational behavior between different positions in a multiple sequence alignment. We then use these correlations to predict contact maps for each of 11 protein families and compare the result with the contacts determined by crystallography. For the most strongly correlated residue pairs predicted to be in contact, the prediction accuracy ranges from 37 to 68% and the improvement ratio relative to a random prediction from 1.4 to 5.1. Predicted contact maps can be used as input for the calculation of protein tertiary structure, either from sequence information alone or in combination with experimental information. © 1994 John Wiley & Sons, Inc.
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  • 65
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    Proteins: Structure, Function, and Genetics 18 (1994), S. 324-337 
    ISSN: 0887-3585
    Keywords: protein structure ; secondary structure ; peptide geometry ; Ramachandran plot ; β-turns ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The polypeptide of a protein molecule can be considered as a chain of Cα atoms linked by pseudobonds between the Cα atoms of successive amino acid residues. This paper presents an analysis of the angle and dihedral angles made by these pseudobonds in protein structures determined at high resolution by X-ray crystallography. This analysis reveals a strong correlation between Cα geometry and the protein fold. The regular features of protein secondary structure such as α-helix and α-sheet are very clearly defined. In addition, it is possible to identify with some confidence the discrete populations of particular conformations of α-turn. Comparison with the traditional Ramachandran type of plot demonstrates that an analysis of protein structure on the basis of Cα geometry provides a richer description of protein conformation. In addition, the characteristics of this geometry could be a useful guide in model building of protein structure. © 1994 John Wiley & Sons, Inc.
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  • 66
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    Keywords: X-ray crystallography ; extracellular matrix ; multiwavelength anomalous diffraction (MAD) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7-10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 Å Bragg spacings. A mutant FN7-10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis. Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed. © 1994 Wiley-Liss, Inc.
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  • 67
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    Proteins: Structure, Function, and Genetics 19 (1994), S. 55-72 
    ISSN: 0887-3585
    Keywords: secondary structure prediction ; prediction of secondary structure class ; prediction of secondary structure content ; evolutionary information ; multiple alignment profiles ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Using evolutionary information contained in multiple sequence alignments as input to neural networks, secondary structure can be predicted at significantly increased accuracy. Here, we extend our previous three-level system of neural networks by using additional input information derived from multiple alignments. Using a position-specific conservation weight as part of the input increases performance. Using the number of insertions and deletions reduces the tendency for overprediction and increases overall accuracy. Addition of the global amino acid content yields a further improvement, mainly in predicting structural class. The final network system has a sustained overall accuracy of 71.6% in a multiple cross-validation test on 126 unique protein chains. A test on a new set of 124 recently solved protein structures that have no significant sequence similarity to the learning set confirms the high level of accuracy. The average cross-validated accuracy for all 250 sequence-unique chains is above 72%. Using various data sets, the method is compared to alternative prediction methods, some of which also use multiple alignments: the performance advantage of the network system is at least 6 percentage points in three-state accuracy. In addition, the network estimates secondary structure content from multiple sequence alignments about as well as circular dichroism spectroscopy on a single protein and classifies 75% of the 250 proteins correctly into one of four protein structural classes. Of particular practical importance is the definition of a position-specific reliability index. For 40% of all residues the method has a sustained three-state accuracy of 88%, as high as the overall average for homology modelling. A further strength of the method is greatly increased accuracy in predicting the placement of secondary structure segments. © 1994 Wiley-Liss, Inc.
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  • 68
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    Keywords: endonuclease overexpression ; crystallization ; X-ray diffraction ; protein-DNA complex ; Type II restriction enzyme ; vapor diffusion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We have overexpressed the type II restriction endonuclease PvuII (R.PvuII) in E. coli, prepared large amounts of the homogeneous enzyme, and crystallized it with an oligonucleotide carrying a PvuII recognition site. The cocrystals are orthorhombic space group P212121 with cell constants a = 95.8 Å, b = 86.3 Å, c = 48.5 Å, and diffract X-rays to at least 2.7 Å. There is a complex of two protein subunits and one oligonucleotide duplex in the asymmetric unit. © 1994 Wiley-Liss, Inc.
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  • 69
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    Proteins: Structure, Function, and Genetics 19 (1994) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 70
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    Proteins: Structure, Function, and Genetics 19 (1994), S. 80-83 
    ISSN: 0887-3585
    Keywords: maize protein ; crystals ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P212121 with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (Vm) of 2.36 Å 3/Da and a solvent content of 48% by volume. The X-ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuKα and synchrotron X-rays. A set of X-ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley-Liss, Inc.
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  • 71
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    Proteins: Structure, Function, and Genetics 19 (1994), S. 85-97 
    ISSN: 0887-3585
    Keywords: protein conformation ; secondary structure ; protein folding ; helix stability ; helix formation ; conformational entropy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Much effort has been invested in seeking to understand the thermodynamic basis of helix stability in both peptides and proteins. Recently, several groups have measured the helix-forming propensities of individual residues (Lyu, P. C., Liff, M. I., Marky, L. A., Kallenbach, N. R. Science 250:669-673, 1990; O'Neil, K. T., DeGrado, W. F. Science 250:646-651, 1990; Padmanabhan, S., Marqusee, S., Ridgeway, T., Laue, T. M., Baldwin, R. L. Nature (London) 344:268-270, 1990). Using Monte Carlo computer simulations, we tested the hypothesis that these differences in measured helix-forming propensity are due primarily to loss of side chain conformational entropy upon helix formation (Creamer, T. P., Rose, G. D. Proc. Natl. Acad. Sci. U.S.A. 89:5937-5941, 1992). Our previous study employed a rigid helix backbone, which is here generalized to a completely flexible helix model in order to ensure that earlier results were not a methodological artifact. Using this flexible model, side chain rotamer distributions and entropy losses are calculated and shown to agree with those obtained earlier. We note that the side chain conformational entropy calculated for Trp in our previous study was in error; a corrected value is presented. Extending earlier work, calculated entropy losses are found to correlate strongly with recent helix propensity scales derived from substitutions made within protein helices (Horovitz, A., Matthews, J. M., Fersht, A. R. J. Mol. Biol. 227:560-568, 1992; Blaber, M., Zhang, X.-J., Matthews, B. M. Science 260:1637-1640, 1993). In contrast, little correlation is found between these helix propensity scales and the accessible surface area buried upon formation of a model polyalanyl α-helix. Taken in sum, our results indicate that loss of side chain entropy is a major determinant of the helix-forming tendency of residues in both peptide and protein helices. © 1994 Wiley-Liss, Inc.
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  • 72
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    Keywords: crystallography ; hydroxamate ; high resolution ; metalloproteinase ; zinc ; X-ray ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 Å resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded β-sheet and three α-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase. © 1994 Wiley-Liss, Inc.
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  • 73
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    Proteins: Structure, Function, and Genetics 19 (1994), S. 110-119 
    ISSN: 0887-3585
    Keywords: folding intermediate ; urea denaturation ; stopped-flow circular dichroism ; molten globule ; hemindicyanide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The refolding kinetics of horse cyanometmyoglobin induced by concentration jump of urea was investigated by five optical probe stopped-flow methods: absorption at 422 nm, tryptophyl fluorescence at around 340 nm, circular dichroism (CD) at 222 nm, CD at 260 nm, and CD at 422 nm. In the refolding process, we detected three phases with rate constants of 〉 1 × 102 s-1, (4.5-9.3) S-1, and (2-5) × 10-3 s-1. In the fastest phase, a substantial amount of secondary structure (40%) is formed within the dead time of the CD stopped-flow apparatus (10.7 ms). The kinetic intermediate populated in the fastest phase is shown to capture a hemindicyanide, suggesting that a “heme pocket precursor” recognized by hemindicyanide must be constructed within the dead time. In the middle phase, most of secondary and tertiary structures, especially around the captured hemindicyanide, have been constructed. In the slowest phase, we detected a minor structural rearrangement accompanying the ligand-exchange reaction in the fifth coordination of ferric iron. We present a possible model for the refolding process of myoglobin in the presence of the heme group. © 1994 Wiley-Liss, Inc.
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  • 74
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    Proteins: Structure, Function, and Genetics 19 (1994), S. 120-131 
    ISSN: 0887-3585
    Keywords: enzymes ; protein immobilization ; microcalorimetry ; protein melting domains ; protein DSC ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Ribonuclease A has been immobilized on silica beads through glutaraldeyde-mediated chemical coupling in order to improve the stability of the protein against thermal denaturation. The thermodynamic and binding properties of the immobilized enzyme have been studied and compared with those of the free enzyme. The parameters describing the binding of the inhibitor 3′ -CMP (Ka and ΔH) as monitored by spectrophotometry and calorimetry were not significantly affected after immobilization. Conversely both the stability and unfolding mechanism drastically changed. Thermodynamic analysis of the DSC data suggests that uncoupling of protein domains has occurred as a consequence of the immobilization. The two state approximation of the protein unfolding process is not longer valid for the immobilized RNase. Protein stability strongly depends on the hydrophobicity properties of the support surface as well as on the presence of the inhibitor and pH. For example, after immobilization on a highly hydrophobic surface, the enzyme is partially in the unfolded state. The binding of a ligand is able to reorganize the protein structure into a native-like conformation. The refolding rates are different for the two protein domains and vary as a function of pH and presence of the inhibitor 3′-CMP. © 1994 Wiley-Liss, Inc.
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  • 75
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    Proteins: Structure, Function, and Genetics 19 (1994), S. 73-76 
    ISSN: 0887-3585
    Keywords: molecular recognition ; protein assembly ; protein folding ; protein interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Recent advances in the measurement and theory of “hydration” interactions between biomolecules provide a basis on which to formulate mechanisms of biomolecular recognition. In this paper we have developed a mathematical formalism for analyzing specificity encoded in dynamic distributions of surface polar groups, a formalism that incorporates newly recognized properties of directly measured “hydration” forces. As expected, attraction between surfaces requires complementary patterns of surface polar groups. In contrast to usual expectations, thermal motion can create these complementary surface configurations. We have demonstrated that assembly can occur with an increase in conformational entropy of polar residues. Elevated temperature then facilitates recognition rather than hinders it. This mechanism might underlie some temperature-favored assembly reactions common in biological systems that are usually associated with the “hydrophobic effect” only. © 1994 Wiley-Liss, Inc.
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  • 76
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    Proteins: Structure, Function, and Genetics 20 (1994), S. 174-184 
    ISSN: 0887-3585
    Keywords: coiled-coils ; keratin ; intermediate filament proteins ; link segments ; heptad phasing ; computer modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Structural discontinuities have previously been identified in four regions of the coiled-coil rod domain structure present in intermediate filament (IF) protein molecules. These include a point at which a phase shift occurs in the heptad periodicity characteristic of the sequence of polar and apolar residues in α-helical coiled-coils, and three links that lack a heptad substructure. We have studied these regions by computer-based molecular modeling and comparative sequence analysis and conclude that the phasing discontinuity can be accommodated without significant distortion of the overall double-helical chain conformation; the L2 link has a similar conformation in all different types of IF molecules, a favorable conformation being one in which the two strands wrap tightly around each other; the L12 links vary in length between different IF types but contain important sequence similarities suggestive of a partial β structure; the L1 links show larger variations in length, a lower degree of similarity, and probably diverse structures. Variations in the overall charges of the different links suggest that ionic interactions may playa significant role in filament assembly. The results also have general significance for other α-fibrous proteins in which either the characteristic heptad phasing undergoes a discontinuity or where a short non-coiled-coil sequence occurs within a coiled-coil rod domain structure. © 1994 Wiley-Liss, Inc.
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    Proteins: Structure, Function, and Genetics 20 (1994), S. 191-196 
    ISSN: 0887-3585
    Keywords: human Clara cell 10-kDa protein ; X-ray diffraction ; phospholipase A2 inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystals of recombinant human Clara cell 10-kDa protein were grown both from ammonium sulfate and polyethylene glycol (PEG) solutions. Crystals grown from ammonium sulfate solution have been characterized by X-ray diffraction studies as monoclinic with the space group C2 and lattice constants a = 69.2 Å, b = 83.0 Å, c = 58.3 Å, and β = 99.7°. The monoclinic crystals diffract to beyond 2.5 Å. Some of the crystals grown from PEG were of a similar habit to those grown from ammonium sulfate, but others were triclinic with the space group P1 and cell constants a = 40.3 Å, b = 46.3 Å, c = 51.3 Å, α = 117.7°, β = 102.3°, and γ = 71.4°. These crystals diffract to beyond 3.2 Å. © 1994 Wiley-Liss, Inc.
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    Proteins: Structure, Function, and Genetics 20 (1994), S. 185-190 
    ISSN: 0887-3585
    Keywords: heme ; secondary structure ; conformation ; hemopexin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Hemopexin is a serum glyco-protein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptormediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% β-structure, 15% α-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme-hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the hemeprotein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis. © 1994 Wiley-Liss, Inc.
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  • 79
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    Proteins: Structure, Function, and Genetics 20 (1994) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 80
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    Proteins: Structure, Function, and Genetics 20 (1994), S. 197-201 
    ISSN: 0887-3585
    Keywords: cytochrome P450 ; erythromycin ; P450eryF ; crystallization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Cytochrome P450eryF was overexpressed in Escherichia coli and purified in high yield. Crystals of the protein in the presence of the substrate, 6-deoxyerythronolide B, have been obtained by the hanging drop vapor diffusion method, using polyethylene glycol 4000 as a precipitant. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 54.16 Å, b = 79.67 Å, and c = 99.48 Å and one molecule per asymmetric unit. A complete native data set has been collected to a resolution of 2.1 Å, and anomalous dispersion difference Patterson maps have revealed the location of the single heme iron atom. © 1994 Wiley-Liss, Inc.
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    Proteins: Structure, Function, and Genetics 20 (1994), S. 203-215 
    ISSN: 0887-3585
    Keywords: SH3 ; Abl ; molecular modeling ; homology modeling ; molecular dynamics ; protein structure prediction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A tertiary structure model of the Abl-SH3 domain is predicted by using homology modeling techniques coupled to molecular dynamics simulations. Two template proteins were used, Fyn-SH3 and Spc-SH3. The refined model was extensively checked for errors using criteria based on stereochemistry, packing, solvation free-energy, accessible surface areas, and contact analyses. The different checking methods do not totally agree, as each one evaluates a different characteristic of protein structures. Several zones of the protein are more susceptible to incorporating errors. These include residues 13, 15, 35, 39, 45, 46, 50, and 60. An interesting finding is that the measurement of the Cα chirality correlated well with the rest of the criteria, suggesting that this parameter might be a good indicator of correct local conformation. Deviations of more than 4 degrees may be indicative of poor local structure. © 1994 Wiley-Liss, Inc.
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  • 82
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    Proteins: Structure, Function, and Genetics 18 (1994), S. 81-93 
    ISSN: 0887-3585
    Keywords: serine carboxypeptidase ; protein modeling ; mutation analysis ; comparative modeling ; cathepsin A ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The deficiency of the lysosomal protective protein/carboxypeptidase L (CARB L) causes the lysosomal storage disorder, galactosialidosis, characterized by neuraminidase and β-galactosidase deficiencies in patients' cells. The three enzymes form a complex inside the lysosome, and the neuraminidase and β-galactosidase deficiencies are secondary to CARB L deficiency. Sequence similarity and common enzymological properties suggest that the protomeric tertiary structure of CARB L is conserved within a family of serine carboxypeptidases which includes the yeast carboxypeptidase Y, killer expression I gene product and several plant carboxypeptidases. We used this homology to build a model of the CARB L structure based on the recently published X-ray atomic coordinates of the wheat carboxypeptidase II (CPDW-II) which shares 32% primary structure identity with CARB L. Small insertions and deletions were accommodated into the model structure by energy minimization using the DREIDING II force field. The Cα atomic-coordinates of the final CARB L model have a RMS shift of 1.01 Å compared to the corresponding conserved residues in the CPDW-II template structure. The correct orientation of the homologous catalytic triad residues Ser150, His429 and Asp392, the potential energy calculations and the distribution of hydrophobic and hydrophillic residues in the structure all support the validity of the CARB L model. Most missense mutations identified in galactosialidosis patients were located in secondary structural elements except for the Tyr211→Asn mutation which is in a loop. The other mutant residues have their side chains deeply buried in the central β-sheet of the model structure except for the Phe412→Val mutation which is located in the dimer interface. The predicted effects of specific mutations on CARB L structural stability correlates well with recently published transient expression studies of mutant CARB L (Shimmoto, M. et al., J. Clin. Invest., 91:2393-2399, 1993). © 1994 John Wiley & Sons, Inc.
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  • 83
    ISSN: 0887-3585
    Keywords: cell multipole method ; Newton-Euler inverse mass operator ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Two new methods developed for molecular dynamics simulations of very large proteins are applied to a series of proteins ranging up to the protein capsid of tomato bushy stunt virus (TBSV).For molecular dynamics of very large proteins and polymers, it is useful to carry out the dynamics using internal coordinates (say, torsions only) rather than Cartesian coordinates. This allows larger time steps, eliminates problems with the classical description of high energy modes, and focuses on the important degrees of freedom. The resulting equation of motion has the form where for T is the vector of generalized forces, M(θ) is the moments of inertia tensor, is the vector of torsions, and C is a vector containing Coriolis forces and nonbond forces. The problem is that to calculate the acceleration vector from M, C, and Trequires inverting. M(θ), an order N3calculation. Since the number of degrees of freedom might be 300,000 for a million atom system, solving these equations every time step is impractical, restricting internal coordinate methods to small systems. The new method, Newton-Euler Inverse Mass Operator (NEIMO) dynamics, constructs the torsional accelerations vector directly by an order N process, allowing internal-coordinate dynamics to be solved for super larger (million atom) systems, The first use of the NEIMO method for molecular dynamics of proteins is presented here.A second serious difficulty for large proteins is calculation of the nonbond forces. We report here the first application to proteins of the new Cell Multipole Method (CMM) to evaluate the Coulomb and van der Waals interactions. The cost of CMM scales linearly with the number of particles while retaining an accuracy significantly better than standard non bond methods (involving cutoffs).Results for NEIMO and CMM are given for simulations of a wide range of peptide and protein systems, including the protein capsid of TBSV with 488,000 atoms. The computational times for NEIMO and CMM are demonstrated to scale linearly with size. With NEIMO the dynamics time steps can be as large as 20 fs (for small peptides), much larger than possible with standard Cartesian coordinate dynamics.For TBSV we considered both the normal form and the high pH form, in which the Ca2+ ions are removed. These calculations lead to a contraction of the protein for both forms (probably because of ignoring the RNA core not observed in the X-ray). © 1994 Wiley-Liss, Inc.
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  • 84
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    Proteins: Structure, Function, and Genetics 20 (1994), S. 68-84 
    ISSN: 0887-3585
    Keywords: side chain conformation ; protein folding ; protein binding ; helix formation ; helix stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Theoretical estimations of changes in side chain configurational entropy are essential for understanding the different contributions to the overall thermodynamic behavior of important biological processes like folding and binding. The configurational entropy of any given side chain in any particular protein can be evaluated from the complete energy profile of the side chain. Calculations of the energy profiles can be performed using the side chain single bond dihedrals as the only independent variables as long as the structures at each value of the dihedrals are allowed to relax through small changes in the valence bond angles. The probabilities of different side chain conformers obtained from these energy profiles are very similar to the conformer populations obtained by analysis of side chain preferences in the proteins of the Protein Data Bank. Also, side chain conformational entropies obtained from the energy profiles agree extremely well with those obtained from the Protein Data Bank conformer populations. Changes in side chain configurational entropy in binding and folding can be computed as differences in conformational entropy because, in most cases, the frequency of the rotational oscillation around the energy minimum of any given conformer does not appear to change significantly in the reaction. Changes of side chain conformational entropy calculated in this way were compared with experimental values. The only available experimental data-the effect of side chain substitution on the stability of α-helices-were used for this comparison. The experimental values were corrected to subtract the solvent contributions. This comparison yields an excellent agreement between calculated and experimental values, validating not only the theoretical estimates but also the separability of the entropic contributions into configurational terms and solvation related terms. © 1994 Wiley-Liss, Inc.
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  • 85
    ISSN: 0887-3585
    Keywords: X-ray diffraction ; aspartic protease ; AIDS ; recombinant protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: For therapeutically relevant targets, the evaluation of enzymes in complex with their inhibitors by cocrystallization and high resolution structural analysis has become a vital component of structure-driven drug design and development. Two approaches, hanging drop vapor diffusion and a novel microtube batch method, were utilized in parallel to grow crystals of recombinant HIV -2 protease and recombinant human renin in complex with inhibitors. In the case of HIV -2 protease in complex with a reduced amide inhibitor, crystallization was achieved only by the microbatch method. In the case of human renin, the addition of precipitant was required for crystal growth. The microbatch method described here is a useful supplementary or alternative approach for screening parameters and generating crystals suitable for high resolution structural analysis. © 1994 Wiley-Liss, Inc.
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  • 86
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    Proteins: Structure, Function, and Genetics 20 (1994), S. 124-138 
    ISSN: 0887-3585
    Keywords: leghemoglobin ; hydrophobic ; interactions ; hydrophobicity ; protein folding ; structure prediction ; protein dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The essential features of the in vitro refolding of myoglobin are expressed in a solvable physical model. Alpha helices are taken as the fundamental collective coordinates of the system, while the refolding is assumed to be mainly driven by solvent-induced hydrophobic forces. A quantitative model of these forces is developed and compared with experimental and theoretical results. The model is then tested by being employed in a simulation scheme designed to mimic solvent effects. Realistic dynamic trajectories of myoglobin are shown as it folds from an extended conformation to a close approximation of the native state. Various suggestive features of the process are discussed. The tenets of the model are further tested by folding the single-chain plant protein leghemoglobin. © 1994 Wiley-Liss, Inc.
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  • 87
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    Proteins: Structure, Function, and Genetics 18 (1994), S. 19-33 
    ISSN: 0887-3585
    Keywords: Discover program ; protein dynamics ; computer simulation ; protein motions ; counterions ; dielectric ; protein electrostatics ; aqueous simulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In this report we examine several solvent models for use in molecular dynamics simulations of protein molecules with the Discover program from Biosym Technologies. Our goal was to find a solvent system which strikes a reasonable balance among theoretical rigor, computational efficiency, and experimental reality. We chose phage T4 lysozyme as our model protein and analyzed 14 simulations using different solvent models. We tested both implicit and explicit solvent models using either a linear distance-dependent dielectric or a constant dielectric. Use of a linear distance-dependent dielectric with implicit solvent significantly diminished atomic fluctuations in the protein and kept the protein close to the starting crystal structure. In systems using a constant dielectric and explicit solvent, atomic fluctuations were much greater and the protein was able to sample a larger portion of conformational space. A series of nonbonded cutoff distances (9.0, 11.5, 15.0, 20.0 Å) using both abrupt and smooth truncation of the nonbonded cutoff distances were tested. The method of dual cutoffs was also tested. We found that a minimum nonbonded cutoff distance of 15.0 Å was needed in order to properly couple solvent and solute. Distances shorter than 15.0 Å resulted in a significant temperature gradient between the solvent and solute. In all trajectories using the proprietary Discover switching function, we found significant denaturation in the protein backbone; we were able to run successful trajectories only in those simulations that used no switching function. We were able to significantly reduce the computational burden by using dual cutoffs and still calculate a quality trajectory. In this method, we found that an outer cutoff distance of 15.0 Å and an inner cutoff distance of 11.5 worked well. While a 10 Å shell of explicit water yielded the best results, a 6 A shell of water yielded satisfactory results with nearly a 40% reduction in computational cost. © 1994 John Wiley & Sons, Inc.
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  • 88
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    Proteins: Structure, Function, and Genetics 18 (1994), S. 94-101 
    ISSN: 0887-3585
    Keywords: surface representation ; molecular recognition ; protein docking ; surface triangulation ; molecular graphics ; molecular visualization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We have defined a molecular surface representation that describes precisely and concisely the complete molecular surface. The representation consists of a limited number of critical points disposed at key locations over the surface. These points adequately represent the shape and the important characteristics of the surface, despite the fact that they are modest in number. We expect the representation to be useful in areas such as molecular recognition and visualization. In particular, using this representation, we are able to achieve accurate and efficient protein-protein and protein-small molecule docking. © 1994 John Wiley & Sons, Inc.
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  • 89
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    Cell Motility and the Cytoskeleton 27 (1994) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 90
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    Cell Motility and the Cytoskeleton 27 (1994), S. 13-25 
    ISSN: 0886-1544
    Keywords: Tβ4 ; Tβ10 ; β-thymosins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The β-thymosins are a family of small proteins originally isolated from the thymus. Recently, two of the major mammalian isoforms, thymosin β4 (Tβ4) and thymosin β10 (Tβ10), are identified as significant actin monomer sequestering proteins which may be involved in regulating actin filament assembly. To study the cellular function of β-thymosins, we have used isoform-specific antibodies to determine their concentration and intracellular distribution, and examined the effects of inducing overexpression of Tβ4 and Tβ10 on actin filament structures. Immunofluorescence labeling of peritoneal macrophages showed that both β-thymosins are uniformly distributed within the cytoplasm. cDNA-mediated overexpression of β-thymosins in CV1 fibroblasts induced extensive loss of phalloidinstained actin stress fibers. Stress fibers in the cell center were more susceptible than those at the periphery. There was a decrease in the number of focal adhesions, as evidenced by a decrease in discrete vinculin staining and an increase in diffuse vinculin fluorescence. The majority of the transfected cells had normal shape in spite of extensive loss of actin filaments. Occasionally, cells overexpressing β-thymosin were observed to divide. In these cells, β-thymosin was excluded from the midbody which contains an actin filament-rich contractile ring. Our results indicate that Tβ4 and Tβ10 are functionally very similar and both are effective regulators of a large subset of actin filaments in living cells. © 1994 Wiley-Liss, Inc.
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  • 91
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    Cell Motility and the Cytoskeleton 27 (1994), S. 101-107 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 92
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    Cell Motility and the Cytoskeleton 27 (1994), S. 133-149 
    ISSN: 0886-1544
    Keywords: MAP4 depletion ; antibody blocking ; detyrosination ; midbody ; asymmetric cell processes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Previous immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule-associated protein of Mr ∼210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802-808; DeBrabander et al. (1981) J. Cell Biol. 91:438-455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double-label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277-288] to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC-7), and those made by human neuroblastoma cells (IMR-32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC-7 cells, we also observed MAP4-depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity. © 1994 Wiley-Liss, Inc.
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  • 93
    ISSN: 0886-1544
    Keywords: spermatozoa ; centriole ; axoneme ; immunogold ; acetylated tubulin ; tubulin heterogeneity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distribution of glutamylated tubulin has been analyzed in mammalian testis using the specific mAb GT335 by immunoelectron microscopy and immunoblotting. In spermatozoa of various species, immunogold labeling showed the presence of glutamylated tubulin in all of the microtubules of axoneme and centrioles, whereas the microtubule network of the spermatid manchette was unlabeled. In earlier germ cells, centriole was the only microtubule structure to be labeled. A similar distribution was observed using the anti-acetylated tubulin antibody (6-11B-1), confirming previous results of Hermo et al. [Anat. Rec. 229:31-50, 1991]. However, among testicular somatic cells, microtubules of some Sertoli cell branches were not acetylated but glutamylated. 2-D PAGE of mouse and hamster sperm extracts showed a high level of α and β-tubulin heterogeneity, comparable to that found in brain. Immunoblotting with GT335 revealed a large amount of glutamylated tubulin resolved into numerous α as well as β-tubulin isoforms. This suggests that the major testis-specific tubulin isotypes (mα3/7 and mβ3) are also glutamylatable. These results show a subcellular sorting of posttranslationally modified tubulin isoforms in spermatids, glutamylation being associated with the most stable microtubule structures. © 1994 Wiley-Liss, Inc.
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  • 94
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    Cell Motility and the Cytoskeleton 27 (1994), S. 97-97 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 95
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    Cell Motility and the Cytoskeleton 27 (1994) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 96
    ISSN: 0886-1544
    Keywords: cleavage furrow ; cytokinesis ; intercellular bridge ; polar lobe constriction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The terminal phase of cell division involves tight constriction of the cleavage furrow contractile ring, stabilization/elongation of the intercellular bridge, and final separation of the daughter cells. At first cleavage, the fertilized eggs of the mollusk, Ilyanassa obsoleta, form two contractile rings at right angles to each other in the same cytoplasm that constrict to tight necks and partition the egg into a trefoil shape. The cleavage furrow contractile ring (CF) normally constricts around many midbody microtubules (MTs) and results in cleavage; the polar lobe constriction contractile ring (PLC) normally constricts around very few MTs and subsequently relaxes without cleavage. In the presence of Ag+ ions, the PLC 1) begins MT-dependent rapid constriction sooner than controls, 2) encircles more MTs than control egg PLCs, 3) elongates much more than control PLCs, and 4) remains tightly constricted and effectively cleaves the polar lobe from the egg. If Ag+-incubated eggs are returned to normal seawater at trefoil, tubulin fluorescence disappears from the PLC neck and the neck relaxes. If nocodazole, a drug that depolymerizes MTs, is added to Ag+-incubated eggs during early PLC constriction, the PLC is not stabilized and eventually relaxes. However, if nocodazole is added to Ag+-incubated eggs at trefoil, tubulin fluorescence disappears from the PLC neck but the neck remains constricted. These results suggest that Ag+ accelerates and gradually stabilizes the PLC constriction by a mechanism that is initially MT-dependent, but that progressively becomes MT-independent. © 1994 Wiley-Liss, Inc.
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  • 97
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    Cell Motility and the Cytoskeleton 27 (1994), S. 272-283 
    ISSN: 0886-1544
    Keywords: cell cycle ; transcription ; mRNA decay ; autoregulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The single alpha-tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha-tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha-tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha-tubulins. The single alpha-tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha-tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha-tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, alpha-tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha-tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts alpha-tubulin message amounts via message decay. However the complex control of alpha-tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates. © 1994 Wiley-Liss, Inc.
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  • 98
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    Cell Motility and the Cytoskeleton 27 (1994), S. 150-160 
    ISSN: 0886-1544
    Keywords: axoneme ; bend propagation ; computer simulation ; flagella ; microtubule sliding ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distinct damped, or attenuated, bending pattern observed when demembranated sperm flagella of the tunicate, Ciona, are reactivated in the presence of 2 mM Li+ has been analysed in detail. In these patterns, bends are initiated at the base of the flagellum, but die out after they start to propagate along the flagellum, so that little or no bending is seen in the distal half of the flagellum. A quantitative descriptive analysis shows that the distinctive feature of this attenuation of bending wave amplitude is an asymmetric interbend decay, or slippage, occuring, on average, only at the transitions between a reverse bend and the preceding principal bend. This attenuation is combined with a significant amount of synchronous sliding in the distal half of the flagellum and a decrease in propagation velocity of transitions between bends in the mid-region of the flagellum.Computer simulations demonstrate that the synchronous sliding in the distal half of these flagella can be an entirely passive consequence of the mechanical interaction between active sliding and bending in the basal third of the flagellum and viscous resistances to movement of the distal region of the flagellum through the fluid environment. The current computer models do not contain a mechanism for asymmetric interbend decay that can reproduce these attenuated bending patterns. © 1994 Wiley-Liss, Inc.
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  • 99
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    Cell Motility and the Cytoskeleton 27 (1994), S. 169-179 
    ISSN: 0886-1544
    Keywords: calcium-binding proteins ; myonemes ; centrin ; contractility ; ciliates ; protozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Myonemes are bundles of thin filaments (3-6 nm in diameter) which mediate calcium-induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calciumbinding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium-modulated contractile protein centrin. Western-blot analysis indicates that the 23 kDa protein crossreacts antigenically with anti-centrin antibodies. In addition, the 23 kDa protein displays calcium-induced changes in both electrophoretic and chromatographic behaviour, and contains calcium-binding domains that conform to the EF-hand structure, as known for centrin. Based on these observations, we conclude that a calcium-binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myonememediated retraction of the ciliature. © 1994 Wiley-Liss, Inc.
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  • 100
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    Cell Motility and the Cytoskeleton 28 (1994) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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