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Notes: Each statocyst in Apylsia californica contains 13 neurons. The statocyst nerve, which connects each statocyst to the cerebral ganglion, contains only the 13 axons of the statocyst neurons. The size of the statocyst, the number of neurons in the statocyst, and the average axonal diameter does not change even though the statocyst nerve lengthens greatly as the animal enlarges. A description of the statoconia and the supporting cells in this organ has been given. This prepazation may be useful for microelectrode studies designed to test whether the gap and cytoplasmic specializations that are used to identify active synapses, are necessary for all types of chemical synaptic transmission.

Notes: The venom system of Nasonia vitripennis is well-developed and composed of an unbranched acid gland and associated reservoir. Fine-structural, histochemical and electrophoretic studies indicate that the venom is produced by two protein-secreting epithelia. The bulk of the venom is synthesised in the columnar cells of the acid gland and discharged via “vesicular organelles” and the efferent ductular system into the lumen of the reservoir. The acid gland also contains squamous chitogenous cells, situated either around the central lumen or interposed between the bases of the columnar cells. Once within the reservoir, the venom is probably activated by enzymatic secretions from the reservoir secretory cells. Each of these cells has a “vesicular organelle” but, in contrast to the columnar cells of the acid gland, the cytoplasm contains a preponderance of free ribosomes, and protein segregation apparently occurs outside the Golgi complexes.The venom is expelled through the efferent discharge duct by muscular contractions, which open the duct lumen and bring it into contact with the funnel of the ovipositor. Excessive distortion of the duct is prevented by a massive ventral ligament.

Notes: The developmental cytology of the apical tissue of the testis of Tenebrio molitor and Zophobas rugipes was studied with light and electron microscopy. In the early larvae of both species the tisue was found to be a thickened protrusion of nongerminal cells appearing at the apical end of each testis follicle following gonadal differentiation. The cells persist through pupal and adult stages in both species, becoming more prominent at these stages in Z. rugipes, despite tracheal invasion in both species. In older adults the apical tissue regresses and ultimately distintegrates.Ultrastructurally the apical cells are distinguished from adjacent germinal cells by their (1) small, rounded or oval nuclei, (2) highly convoluted plasma membrane, (3) electron-opaque cytoplasm, (4) profuse concentrically-stacked, granular endoplasmic reticulum, (5) large aggregates of glycogen-like granules, (6) numerous small, tubular mitochondria, (7) well-developed Golgi centers and (8) striking arrays of microtubules. These cells have many cytological features in common with the androgenic gland cells of crustaceans, but not with the steroidogenic cells of vertebrates. Evidence for the formation of protein granules is also lacking. As yet, experimental procedures have not indicated an endocrine function for these cells in tenebrionids. However, their cytology is consistent with secretory activity of some kind.

Notes: The rectum of Periplaneta americana L. is lined with cuticle and has six radially arranged cushion-shaped thickenings, the rectal pads, composed of columnar cells. Narrow strips of simple rectal cells lie between the pads. Tall junctional cells form a thin but continuous collar around the pads where they join the rectal cells. The epithelium is surrounded by a layer composed of circular and longitudinal muscles and connective tissue. This layer of muscle and connective tissue is innervated and tracheated, and is separated from the pad surface by a subepithelial sinus. Fluid flowing through the sinus enters the haemolymph through openings in the muscle layer whre large tracheae penetrate. These openings can be sealed by muscle contractions that appress the muscle around the openings against the pad surface. The tracheae pass on into the pads, following basement membrne-lined indentations of the pad surface. Within the pad tracheolar cells send fine branches between the cells. Near the apical and basal surfaces the lateral membranes of pad cells are bridged by septate desmosomes that form a continuous band around the cells. Between apical and basal septate desmosomes is an interconnected labyrinthine system of intercellular spaces. There are three kinds of space, dilations and apical sinuses, both of variable size, and narrow communicating channels about 200 Å wide. The membranes of the latter have mitochondria closely associated with them. Continuity between the system of spaces and the subepithelial sinus is established by the basement membrane-lined invaginations of the basal surface where tracheae penetrate between pad cells. Apical surfaces of the pad cells are highly infolded and are also associated with mitochondria. However, unlike the lateral membranes facing the narrow channels, the apical membranes have a cytoplasmic coating of particles. Both associations of mitochondria with membranes constitute discrete structural entities that are found in many transporting epithelia, and we have termed them “plasmalemma-mitochondrial complexes.” As the rectal pads are organized into systems of spaces that ultimately open in the direction of fluid movement, existing models of solute-coupled water transport can be applied. However, the rectal pads are structurally more complex than fluid-transporting tissues of vertebrates. This complexity may be related to the ability of the rectum to withdraw water from ion-free solutions in the lumen. We present a structural model involving solute recycling to explain the physiological characteristics of rectal reabsorption.

Notes: The ultrastructure of mouse tracheal epithelium was examined. The three cell types, basal cells, ciliated cells and goblet cells, described for other mammalian trachea were found to be present although goblet cells occurred only rarely. A cell type, termed the nonciliated cell, not described in other mammalian trachea was frequently found in mouse tracheal epithelium. These cells contained abundant smooth and rough endoplasmic reticulum, free ribosomes, a large Golgi complex, and many mitochondria. There were many vesciles containing an electron dense material near the luminal surface of these cells; these cells were positive for PAS. These features suggested a secretory function for the cells. This, along with the scarcity of goblet cells, suggested that the nonciliated cells of mouse tracheal epithelium fulfill the function of the goblet cells found in other mammalian trachea.

Notes: Cytodifferentiation during spermiogenesis in Hydra littoralis was studied at the fine structural level. Concentration of nuclear material as well as specific orientation of granular and filamentous nuclear elements are apparent in two regions of the early spermatid: where the nuclear envelope is in contact with mitochondrial membranes at one pole of the cell and at an opposite region where the nucleus is closely apposed to the plasma membrane. Ultimately the mass of condensed nuclear material becomes concentrated at the mitochondrial pole of the cell. Additional electron-dense material is extruded from the nucleus into a large vacuole which is in continuity with the nuclear membrane as well as associated with Golgi lamellae and vesicles. Eventually all residual cytoplasm is sloughed, leaving the nucleus, mitochondria, and flagellum. These observations are suggestive of nucleocytoplasmic interactions during development, especially influences of mitochondria and plasma membranes on chromatin condensation.

Notes: The development of the eye in embryos of Rana pipiens raised at two different temperatures was studied in detail from Shumway stage 16 through Shumway stage 25. One clutch of eggs from each of ten different female frogs was divided into two groups, one of which was raised at a temperature of 14°C and the other at 19°C. The 14° to 19° difference falls in the middle of the temperature range for normal development of Rana pipiens as established by Atlas ('35), Moore ('39), and Ryan ('41). Two hundred embryos, one embryo from each of the ten clutches, raised to each of ten stages at 14° or at 19°, were sectioned for microscopic study of the eye region. Descriptions of the morphology and histology of the developing eye are illustrated by photographs and provide a reference to which development of experimental embryos may be compared. A synoptic checklist is provided which specifies the changes occurring at each stage studied.

Notes: The fine structure of the mid-gut epithelium and regenerative cells of larvae of a moth (Ephestia kühniella) is described. Particular attention is paid to the absorptive and goblet cells and their lateral junctions; these features are discussed in terms of the digestive and regulatory functions of the epithelium. One digestive pathway has been investigated with the aid of ingested ferritin; intake of this marker by endocytosis and the evident involvement of Golgi vesicles in the transformation of endocytic vesicles into multivesicular bodies, together with the fate of the latter, are discussed in terms of the digestive function of this part of the alimentary tract and of the lysosome concept.

Notes: A fine structural study of the ventricular muscle of Venus mercenaria has revealed that it is an invertebrate smooth muscle. In the relaxed state induced by acetylcholine, both thick (350 Å) and thin (80 Å) myofilaments are observed. These are loosely distributed in bundles in the periphery of the mononucleated myocytes. The central core of the cell contains an ovoid nucleus, α-glycogen rosettes, round mitochondria and numerous smooth surfaced vesicles of the endoplasmic reticulum. After exposure to serotonin, all myofilaments are compacted in the peripheral cytoplasm and become oriented parallel to the longitudinal cellular axis. This produces contraction bands visible in phase contrast microscopy. Because these myofilaments attach to the cell membrane at sites of attachment plaques, contraction of the cell results in the serial evagination or blebbing of the cell surface. The above features are clearly demonstrable in this invertebrate smooth muscle and strongly suggest a sliding filament model as the contractile mechanism in this tissue. Moreover, the cell surface is thought to play an active and major role in that process.

Notes: Three-dimensional, histological, and x-ray techniques provide a picture of body segment and limb morphogenesis. Cell multiplication begins in the proliferation region (“meristem”) during the molt from the preceding instar. By four days post-molt, the cells that will form the new segments are well under way in their anterior, lateral, and dorsal migration. It is suggested that after the anlagen for all the new segments are estabilshed, a mitotic wave commences in the most anterior anlagen and moves posteriorly during the remainder of the instar. When cell proliferation is complete, final differentiation of the segments takes place.The process of limb formation is one of cell proliferation and perhaps migration. Each limb develops in a membranous pocket during the instar following the one in which its respective body segment was formed.

Notes: Bovine parotid glands exhibit outstanding structural differences when compared with those of non-ruminant mammals. The acini are tortuous, branched and lined with cells of different heights, imparting a scalloped appearance to acinar lumina. Numerous microvilli, ca. 1.5 μ in length, extend into the lumina and intercellular canaliculi. Intercellular canaliculi measure ca. 3 μ in diameter and interweave in close association with intercellular tissue spaces. Intercellular tissue spaces are separated from the extraacinar spaces across a basal lamina only, whereas junctional complexes guard canaliculi from direct continuity with tissue spaces and/or extraacinar spaces. Flattened cytoplasmic lamellae extend from adjacent acinar cells and loosely interdigitate with one another across the tissue spaces. Acinar cells contain more mitochondria and less granular endoplasmic reticulum than parotid glands of non-ruminant mammals. Two types of secretory material, in the form of inclusions which vary in size and electron density, are present in the acinar cells. Intercalated ducts connect acini with striated ducts which in turn, empty into collecting ducts located between gland lobules. In terms of frequency of “basal infoldings” and numbers of mitochondria, striated ducts of calf parotid glands are not as well developed as those of certain other salivary glands. Myoepithelial cells are most often present at junctions of acini and intercalated ducts where they may attach to both acinar and ductal epithelium. Nerve “terminals” were not observed on the epithelial side of basement membranes in relation to the secretory cells.

Notes: The endoskeletal structure supporting the gill-books of Limulus polyphemus has been investigated by means of light and electron microscopy, chemical analysis and x-ray diffraction. This tissue is a cartilage which has significant correspondences with both vertebrate cartilage and plant tissues. Morphologically, the Limulus cartilage resembles certain cellular vertebrate cartilages with relatively scant matrix, and also certain plant parenchyme, collenchyme and sclerenchyme tissues. Of particular interest, was the observation that during cytoplasmic division, a phragmasome-like structure appears between the daughter cells of the dividing gill cartilage cells. This phragmasome-like structure appears to be a precursor of new matrix (cell-wall) formation between the young chondrocytes, in much the same fashion as its counterpart in plant tissues. Perichondrial cells and underlying chondrocytes contain lipid droplets, abundant glycogen and ribosomes, as do corresponding vertebrate cartilage cells. In some of the Limulus cells, glycogen and ribosomes appear to be admixed with lipid, forming aggregates in which all three materials are in intimate intraparticulate relationship. During molting, the number of ribosomes seen in chondrocytes increases. The tissue contains both hydroxyproline and hydroxylysine, and gives a weak x-ray diffraction pattern.

Notes: A principal component analysis revealed that the two major components of mandibular shape variation among individuals within breeding groups of white-tailed deer in Canada and the United States involve contrasts between the mandible and the dentition and between the premolars and the molars. Size variation appeared to account for 34% of the total variation within the groups, and the two major shape variations accounted for 23% and 8% respectively.A canonical analysis was used to identify the major components of mandibular variation among the breeding groups and to provide measures of the proportion of the total variation accounted for by each component. Among male groups, size variation was associated with latitude, and the major shape variation was closely associated with longitude, so a bivariate plot of the first two canonical variates reflected the general geographic orientation of the populations.The mandibular size in a Tennessee population that descended from Wisconsin and Michigan ancestors appears to have not decreased appreciably in the more southerly habitat after introduction more than 20 years ago. Changes in range conditions in eastern Upper Michigan over the past 30 years have not influenced local mandibular morphology as reflected by the first two canonical variates. Regardless of general smallness of individuals, the mandibular morphology of the deer from the Cross Timbers area in Kansas appears to approximate more closely that of northern populations than that of the more southerly populations from Oklahoma and Texas.Sexual dimorphism on the first two canonical axes was observed.

Notes: Structural and functional changes have been correlated during metamorphic degeneration of a single muscle fiber, the plantar retractor of G. mellonella, its axon, and their junctions to determine which features persist as long as muscle contractility. Changes commence simultaneously in muscle and nerve near cuticular attachments, and spread towards the center. Alterations associated with the muscle, including appearance of collapsed tracheoles and mitochondria with dense bodies, begin late in the last larval instar. Within 12 hours after pupal ecdysis some tracheolar withdrawal occurs, sarcoplasmic reticulum becomes reduced, and many mitochondria have dense bodies, dense membranes, or are enlarged. By 17-19 hours primary myofilaments and striations begin to disappear, microtubules and autophagic vacuole-like bodies appear, and phagocytes invade the muscle. It remains partially contractile upon electrically stimulating its nerve, the ventral nerve, until these changes spread throughout the fiber.Neuromuscular junction changes, including appearance of dense mitochondria and isolation bodies, begin late in the last larval instar. Junctions become fewer, and none remain in those muscle areas where tracheoles, sarcoplasmic reticulum, and primary myofilaments have disappeared.Preliminary studies on nerve discharge activity to the muscle suggest that nerve silence occurs at approximately the time when the muscle loses all contractility. In some axons isolation bodies appear and neurotubules are lost, other axons remain unchanged, and new ones develop later in the pupal state. Phagocytes invade the neural lamella and it disappears in the late pupa, but it reappears in the adult.The adult ventral nerve has over three times more axons and a thinner layer of glial cells than the larval nerve.

Notes: Ampullary receptor organs of the South American weakly electric gymnotid fish Eigenmannia virescens consist of a pore at the surface of the skin, a canal through the epidermis, and the expanded basal end of the canal in the corium. The cavity of the organ contains a jelly that is filled with fine fibers. The canal wall consists of three to six layers of flattened cells that appear to be derived from the adjacent skin. Along the lumen of the organ the cells are joined by tight junctions. Usually there are four spherical receptor cells in the base of the organ. They are innervated by single neural terminals. These organs are compared to tuberous receptor organs found in the same species, and the functional significance of the fine structure observed in these cells is discussed.

Notes: There are eight retinula cells in the ommatidium of the compound eye of the toadbug (Gelastocoris oculatus), two of which are central in position. Along the axial sides of the six peripheral retinula cells expand six cytoplasmic processes from the apical crystalline cone cells. These processes, which contain longitudinally-oriented microtubules, are associated with all eight retinula cells by means of desmosomal junctions. In addition to providing structural support, the possibility is set forth that the interconnecting cone processes might also serve to functionally integrate the retinula cells of an ommatidium. The eight retinula cells possess microvillus surfaces, which are especially prominent in the six peripheral cells, where they extend into the lumen of the ommatidium. There is evidence of pinocytotic activity at the bases of microvilli. Multivesicular bodies are present in the cytoplasm of retinula cells, and the means by which these bodies might be elaborated are discussed.

Notes: Development of the adult fly foot falls into clearly defined phases of cell division, growth, cuticle secretion and cell death. The pulvillus is composed dorsally of two giant cells and ventrally of thousands of minute tenent cells; the former produce the dorsal footpad cuticle and the latter the thousands of tenent hairs. Cell divisions are still occurring in future tenent cells when increase in size of the cells and in polyteny of the chromosomes is already occurring in the two dorsal cells. Also cell death occurs considerably earlier in the tenent cells, yet the sequential secretion of some six cuticular layers takes place at comparable times in dorsal and ventral cuticles. The cuticular layers formed are, in their order of secretion: ecdysial membrane, cuticulin of the epicuticle, dense exocuticle, homogeneous exocuticle, an intermediate layer, wax of the epicuticle, and an extensive mass of endocuticle. The ecdysial membrane seems to perform an important mechanical role in maintaining the shape of the delicate cytoplasmic projections of the tenent cells, before and during cuticle secretion, and in establishing the cuticular pattern of ridges in the dorsal cuticle. Comparisons are made with trichogen cell cuticle development and with tracheal cuticle. Tracheal, trichogen and dorsal footpad cuticle patterns are compared.Details of giant cell activity provide a working basis for studies of nuclear-cytoplasmic interactions, and the whole system raises many unsolved problems in the general field of cell differentiation and pattern formation.

Notes: A detailed description of the innervation of the individual muscles of the antenna of the centipede Scolopendra morsitans is given. There are six nerves supplying the antennal muscles of each side. The nerve N I consists of 26 bundles of which two are motor, 12 sensory and 12 are mixed. It innervates the intrinsic muscles of the antenna and the antennal sense organs. The nerves N II, N III and N IV innervate the dorsal extrinsic muscles and the nerve N V and N VI the ventral extrinsic muscles.

Notes: Dense bodies in the heart muscle of Venus mercenaria exist in two forms, free and attached. Free dense bodies morphologically consist of fascicles of thin filaments in parallel array and bound together by a dense, amorphous proteinaceous material. The binding of dense bodies to the cell membrane is effected via connecting filaments of the amorphous material of the dense body which join a condensation of morphologically similar material attached to the inner osmiophilic layer of the unit membrane. This composite of dense body, connecting filaments, membrane condensation and unit cell membrane has been termed collectively the attachment plaque. The attachment plaque is part of an extensive network on the cell surface which obligates that surface to a role in the contractile process. Moreover, this set of attachment plaques imposes an organization and an orientation to most thin filaments of the cell and preserves the contractile axis of the cell.

Notes: Large quantities of colloidal particles were rapidly transported around the junctional complex into the lateral intercellular spaces by flounder renal epithelial cells. Large invaginations containing particles developed in the apical cytoplasm of cells when tracer particles were injected into the tubular lumens. Some membranebounded profiles containing particles appeared close to the lateral intercellular spaces. Particles were then found in the lateral intercellular spaces, between the basal plasmalemma and the basement membrane, and within the basement membrane. It is suggested that this transport might operate in situ and provide a morphological mechanism to explain a type of protein transport noted in the renal tubules of another flounder species by Maack and Kinter ('67). It is interesting to consider that perhaps a similar mechanism for the transport of intact proteins might also operate in mammalian nephrons as well.

Notes: A rapid method for examining rat fetuses is presented. The technique consists of fixing the fetuses in Bouin's solution, serially sectioning the head, neck and lower trunk with a razor blade and doing sagittal sections of the heart after opening the thoracic cavity. Examples of sections from normal 20 day rat fetuses are given as well as some with the following abnormalities: cleft palate produced by chlorcyclizine and eye and heart malformations resulting from anti-adult rat kidney serum.

Notes: The serigenous glands of a number of different sawfly larvae have been examined. Silk is secreted by pear-shaped cells which may be fused together in pairs or triplets, or exist simply as free, single cells. The cells are arranged in numerous groups attached to a pair of wide silk reservoirs by means of short canals. Each gland cell contains a large, irregular, ramifying nucleus and an intracellular duct which receives droplets of synthesised silk protein. Two modifications of this basic arrangement are described. It is suggested that the secretory cells are dermal gland cells, and that the intracellular duct is a rudimentary end-apparatus. A comparison is made between these and some other types of dermal gland cell found in insects.

Notes: The structure of human labial salivary gland acini was studied by light and electron microscopy. Contrary to previous reports, these glands were pure mucous in nature; no serous elements were present. The acinar cells were found in all stages of maturation. Immature cells were characterized by an extensive and highly organized rough-surfaced endoplasmic reticulum. The Golgi complex was extremely prominent, consisting of stacks of flattened cisternae and swarms of small vesicles. Mucous droplets were almost completely absent. As secretory activity progressed, the endoplasmic reticulum involuted, while the Golgi cisternae became distended and formed many vacuoles. In mature mucous cells, the apical cytoplasm was filled with membrane-bounded mucous droplets, and the nucleus was displaced basally. The droplets frequently showed great variation in density from cell to cell, and even within the same cell they sometimes were quite heterogeneous. They were liberated from the acinar cells by an apocrine process, so that droplets with intact limiting membranes were often observed in the acinar lumen. These droplets soon lysed, their contents fusing into streams of mucus. Occasionally during apocrine secretion a mucous cell failed to reconstitute its apical surface, and its entire contents spilled into the acinar lumen.Unusual cytoplasmic inclusions were present in many of the acinar cells. These inclusions, which were surrounded by a single membrane, consisted of lipid droplets closely associated with bundles of fine filaments.

Notes: Submandibular glands of the opossum have been studied by light and electron microscopy and compared with other mammalian salivary glands. The glands have four parenchymal segments which connect in the order named below to convey saliva toward the oral cavity. 1Secretory units are elongated branching tubules exhibiting mucous and special serous cell types. Mucous cells predominate and resemble those in other salivary glands. Special serous cells differ from “typical” serous cells. They contain a preponderance of tubular or vesicular endoplasmic reticulum and secretory granules which vary from electron lucent to electron opaque.2Intercalated ducts are short segments lined by nonsecretory, cuboidal cells.3Striated ducts are numerous and lie in the center of the lobule. The duct epithelium has four cell types, designated light cells, dark cells, Type I basal cells, and Type II basal cells. Light cells possess basal infoldings associated with mitochondria, but the other cell types lack this characteristic.4Excretory ducts are also lined by four cell types which bear the same names as those of striated ducts. Three of the four cell types are virtually identical to those of striated ducts, but light cells differ. They do not always contain basal infoldings and the supranuclear cytoplasm lacks distinct inner and outer zones.The glands resemble salivary glands of higher mammals in many respects while possessing certain unique cytological features which may reflect the secretory needs of the organism.

Notes: Several biological distances based on cranial and mandibular variation among breeding groups of white-tailed deer were calculated and compared with geographic distances among the groups. Distances based on epigenetic variation among ten groups were calculated using 16 non-metric variants of the cranium and mandible. Penrose's size and shape distances and Mahalanobis' D2 distance were calculated for 11 groups; the calculations were based on seven skeletal and seven dental metric variables of the mandible.The biological distances were correlated with geographic distance as follows: the epigenetic distance, 0.74; Penrose's shape distance, 0.71; Penrose's size distance, 0.45; and Mahalanobis' distance, 0.37. All correlations were significant at the 0.01 level. The epigenetic and Penrose shape correlations were significantly higher than the Mahalanobis correlation.Because of the conditions under which the breeding groups were selected, it was assumed that genetic affinites among the groups would be a function of geographic distance. The results suggest that the epigenetic distance and Penrose's shape distance reflect genetic affinities among groups better than do the Penrose size and Mahalanobis distances.

Notes: The mechanism of lung ventilation in chelonians has been much debated. Electromyographic studies show that the basic mechanism in the snapping turtle, Chelydra serpentina, is dependent on the activities of four major respiratory muscles that are capable of varying the volume of the visceral cavity. The precise mechanism utilized varies in response to environmental factors, especially the depth to which the animal is submerged. Chelydra tends to reduce muscular activity to a minimum, and hydrostatic pressure or gravity replaces muscular effort whenever possible. The response is subject to hysteresis. Both the mechanics and pattern of ventilation in Chelydra differ from those of Testudo. The differences appear to be attributable in part to Chelydra's markedly reduced plastron and more extensive respiratory musculature and in part to the different habitats occupied by the two species.

Notes: The purported “neoblasts” of the serpulid Spirorbis have been studied in Spirorbis (Paradexiospira) vitreus and Spirorbis (Laeospira) borealis at both the light and electron microscopic levels. These perivasal cells occur in greatest abundance around the ventral blood vessel of the achaetous region. In light microscope preparations, the perivasal cells are intensely basophilic, containing basally situated nuclei, and relatively large nucleoli. The fine structure of the perivasal cells reveals that they contain an abundance of rough endoplasmic reticulum, well-developed Golgi complex, heterogeneous dense bodies, and cytolysomes. The respiratory pigment chlorocruorin, which has a diameter of about 230 Å and is believed to be composed of two superimposed hexagonal components, has been localized within: cisternae of the rough endoplasmic reticulum, elements of the Golgi complex, and membrane-bounded vesicles at the base of the perivasal cells. Evidence is advanced which strongly suggests that molecules of chlorocruorin are transported from the perivasal cells into the lumen of the vessel by reverse pinocytosis. It is concluded that whatever other functional role(s) the perivasal cells of Spirorbis may have, a major function is the synthesis and secretion of chlorocruorin. Whether the perivasal cells can be considered to be pluripotent is discussed.

Notes: The concept of functional components was initially proposed by van der Klaauw ('45, '52) to indicate overlap of functional influences particularly in mammalian skulls; his analysis marked a departure from the study of single characters to that of function-modified systems. A very similar set of terms is now coming into vogue to describe the mechanically separable components of highly kinetic fish, amphibian and reptilian skulls. In these cases the term functional unit often pertains only to the musculo-skeletal system and is utilized during the process of description; it is often applied before a complete functional analysis has been carried out.Yet, any structure tends to be affected by the influence of multiple functions, and any function will almost certainly affect multiple characteristics of the animal. Since functional components overlap, the term should not be used to label an essentially topographical dissection of the animal. It cannot be expected that each loosely connected component of a kinetic skull subserves as a single “function,” and that this function does not overlap onto other units.It is suggested that the term mechanical unit be substituted as a label for the mechanical sub-divisions often utilized to organize descriptions. The concept of functional units in the original sense then remains available as an analytical tool.

Notes: The cytology of the vitellogenic stages in the development of the oocyte of Drosophila melanogaster has been studied using whole mounts and sections of plastic-embedded ovaries and single egg chambers for light microscopy and cytochemistry. The migrations, changes in morphology, and synthetic products of the follicle cells are described as a function of developmental stage. The follicle cells synthesize the egg coverings, the vitelline and chorionic membranes, and elaborate the micropyle and dorsal chorionic appendages. The changing structure of the nurse cell nucleus and changes in organelle composition of its cytoplasm are described. The nurse cells synthesize ribosomes, lipid droplets, and mitochondria. These components pass through the ring canal system into the oocyte, which increases in volume some 200,000 times during its 78 hours of development.

Notes: The mechanisms of development of posterior levels of neural tubes of chick embryos were analyzed by study of serial cross-sections of a continuous series of normal embryos between 40 to 72 hours of incubation. Two extirpation experiments were performed in ovo on other embryos of the same stages. Descriptive studies revealed the presence of an overlap zone in which two types of neural tube formation occurred. Open neural tube formation (by fusion of neural folds) occurred dorsally in this region; closed neural tube formation (by canalization of solid medullary cord tissue) occurred ventrally. Extirpation of the posterior end of the neural plate produced defects within the lumbosacral region, indicating that the posterior neural plate participates in the formation of the lumbosacrum, and that the overlap zone is therefore in the lumbosacral region. Extirpation of the prospective neural tissue in the anterior end of the tail bud indicated that only the most posterior levels of the neural tube originate exclusively by cavitation of the tail bud. In both extirpation experiments a neural tube formed independently within the tail bud tissue, indicating that formation of the neural tube in this region is not dependent upon direct continuity with neural tissue anteriorly.

Notes: The interstitial cells of Pennaria tiarella differentiate exclusively from the central endoderm of the planula. Shortly after their appearance, most of the interstitial cells become cnidoblasts. Subsequently, as the larva transforms into a polyp, both cnidoblasts and interstitial cells migrate from the endoderm, through endoblast and mesoglea, into the ectoderm. It is suggested that some interstitial cells remain in the endoderm and differentiate into the gland and mucous cells of the polyp gastroderm.

Notes: A series of dimensions of the shoulder girdle of primates has previously been chosen as being related to function in that anatomical region. Their examination by canonical analysis suggests that they do indeed reflect aspects of the use of the shoulder in locomotion in the different primates.Further analysis is here performed using the technique of neighborhood limited classification and this confirms the basic picture presented by the previous analysis. The new method also gives more detailed information about the grouping of the specimens; thus it endorses the reality of functional divisions that appear to exist in the data. And in addition the groupings reflect differences in the structure of the shoulder that correlate well with certain taxonomic subdivisions of the order. The method maintains contact with individual specimens throughout the analysis and is capable of placing them within groups, at the boundaries of groups, within the interfaces between groups, or as satellites to groups.The new method appears to have a part to play in the description of the relationships between biological objects that is complementary to that of canonical analysis. As the mathematical concepts upon which the two techniques are based differ totally, the risk that the results might be inherent in statistical assumptions is thus averted.

Notes: In the annelid Enchytraeus albidus the ovary is composed of packets containing eight synchronously developing oocytes. Each oocyte in the packet is connected, via a bridge, to a common cytoplasmic mass. Developmental synchrony of oocytes within individual packets is probably related to the ooplasmic continuity.The young previtellogenic oocyte contains many polysomes, a few cisternae of smooth and rough endoplasmic reticulum, small Golgi complexes, and mitochondria. Many of the mitochondria are dumbbell-shaped and may thus represent division stages.Vitellogenesis is marked by the appearance of peripherally located lipid yolk and small, densely staining granules scattered throughout the ooplasm. There is an increase of smooth endoplasmic reticulum, mitochondria, and enlarged Golgi elements. Small multivesicular-like bodies, the early stages of developing yolk, are derived from the Golgi complex. The mature yolk sphere is bipartite and consists of (a) a variable number of dense spheres, the core bodies, which are produced in the ooplasm by the Golgi complex and which become embedded in (b) a dense matrix. The electron opaque tracer, horseradish peroxidase is incorporated into the oocyte and deposited in the matrix suggesting that this component of the yolk sphere is obtained by micropinocytosis. Enzyme digestions and various cytochemical techniques suggest that the core bodies are rich in carbohydrate, probably as glyco- or mucoproteins, and that the matrix is rich in lipid.

Notes: Differentiating oocytes and associated follicle cells of two species of amphineurans (Mollusca) Mopalia muscosa and Chaetopleura apiculata have been studied by techniques of light and electron microscopy. In addition to the regularly occurring organelles, the ooplasm of young oocytes contains large, randomly situated, basophilic regions. These regions are not demonstrable in mature eggs.As oocytes differentiate, lipid, pigment and protein-carbohydrate yolk bodies accumulate within the ooplasm. Concomitant with the appearance of pigment and the protein carbohydrate containing yolk bodies, the saccules of the Golgi complex become filled with a dense material. Associated with the Golgi complex are cisternae of the rough endoplasmic reticulum which are filled with an electron opaque substance which is thought to be composed of protein synthesized by this organelle. That portion of the cisternae of the endoplasmic reticulum facing the Golgi complex shows evaginations. These evaginations are thought to finalize into protein containing vesicles that subsequently fuse with the Golgi complex. Thus, the Golgi complex in these oocytes might serve as a center for packaging and concentrating the protein used in the construction of the protein containing pigment or protein-carbohydrate yolk bodies. The suggestion is made that the Golgi complex may also synthesize the carbohydrate portion of the formentioned yolk bodies.In an adnuclear position in young oocytes are some acid mucopolysaccharide containing vacuolar bodies. In mature eggs, these structures are found within the peripheral ooplasm and we have referred to them as cortical granules. There is no alteration of these cortical granules during sperm activation.

Notes: Biochemical assay of acid phosphatase in normal and lens-regenerating eyes of the urodele Diemictylus viridescens, using p-nitrophenyl phosphate as substrate, demonstrates both soluble and lysosomal fractions of the enzyme. While the specific activity of the soluble fraction remains unchanged during lens regeneration, the lysosomal fraction shows four distinct rises in specific activity during the thirty-day regeneration period studied. These peak activities on the second, eighth, fifteenth, and twenty-second days post-lentectomy apparently correspond to lysosomal activity in the processes of wound healing, iris depigmentation, and lens differentiation which occur during urodele lens regeneration. On the basis of biochemical and histochemical studies as well as observations of morphological changes in the urodele eye as lens regeneration proceeds, it is postulated that there is a significant correlation between these morphological changes and the level and localization of the lysosomal acid hydrolases in the tissues in which the changes occur.

Notes: A sensory structure in the anterior region of the food canal of two species of aphid has been examined by light and electron microscopy. The dorsal wall is innervated by a total of 60 neurones which terminate, in groups, at 14 porous papillae on the cuticle. Paired papillae have also been detected in the ventral wall of this region. The fine structure of individual neurones and their grouping around papillae indicates a chemosensory function.The examination of moulting aphids shows that the distal portions of dendrites are shed with the exuviae.

Notes: The mechanism of respiration in the bullfrog has been analyzed by means of pressure recordings from the buccal cavity, the lungs and the abdominal cavity, by cinematography and cinefluorography, and by electromyography of buccal, laryngeal and abdominal muscles. Gas flow was investigated by putting frogs in atmospheres of changing argon and nitrogen content and monitoring the concentration of the nostril efflux.Three kinds of cyclical phenomena were found. (1) Oscillatory cycles consist of rhythmical raising and lowering of the floor of the mouth, with open nares. They have a definite respiratory function in introducing fresh air into the buccal cavity. (2) Ventilatory cycles involve opening and closing of the glottis and nares and renewal of a portion of the pulmonary gas. More muscles are involved and the pattern of muscular activity is more complex than in the oscillatory cycles. (3) Inflation cycles consist of a series of ventilation cycles, interrupted by an apneic pause. The intensity of the ventilatory cycles increases before this pause and decreases immediately thereafter. This results in a stepwise increase in pulmonary pressure, to a plateau (coincident with the pause) followed by a sudden or stepwise decrease.The respiratory mechanism depends on the activity of a buccal force pump, which determines pulmonary pressure whose level is always slightly less than the peak pressure values of the ventilation cycles. The elevated pulmonary pressure is responsible for the expulsion of pulmonary gas during the second phase of the next ventilation cycle. This pressure is maintained by the elastic fibers (and the smooth masculature) of the lungs.

Notes: The complete regeneration of a new oral-disc and tentacles has been observed and described for Aiptasia diaphana. These structures are regenerated quite rapidly: seven to ten days at 20°C. At three days post-amputation, the new primary, secondary, and tertiary tentacle buds begin to develop in direct association with the underlying primary, secondary, and tertiary septae (respectively) of the column, suggesting that the latter organize the form of the regenerating oral-disc. Two days after amputation, the zooxanthellae of the presumptive oral disc arrange themselves into a ring which quite precisely delimits the area from which the tentacle buds will form. In spite of its suggestive proximity, this accumulation of algae plays no role in the induction of tentacle buds as was shown by studying regeneration in anemones which essentially lacked large quantities of these symbiotic algae.Cuts perpendicular to the longitudinal axis of the column result in an equal rate of tentacular regeneration around the entire circumference of the presumptive oral disc. Oblique amputations foster an asynchronous regeneration: the tentacle buds of the distal-most area of the severed column are larger and regenerate much sooner than those of the proximal region. Similar results were obtained by studying anemones which were cut perpendicular to their longitudinal axes at different levels along the column. The data suggest that an oral-aboral gradient exists concerning the time required for the initiation of tentacle budding and the rate of tentacle regeneration.

Notes: The ventral lobe of the adenohypophysis of the smooth dogfish, Mustelus canis, a viviparous elasmobranch, has been found to possess distinctive cells identified as basophils on the basis of staining properties. At maximum size, such a cell consists of a distended vesicle containing PAS-positive, AF-negative material surrounded by a thin envelope of cytoplasm and an eccentric nucleus. In earlier stages of these cells, vesicles are small or absent and granules in the more abundant cytoplasms are AF or Alcian-positive.Basophil numbers are high in pre-ovulation and mid-ovulation females, decrease markedly after the end of ovulation until embryos are about 1 cm long then increase greatly during August and September while embryos grow to 8 cm in length. Early high counts, if these basophils are gonadotropes, may be correlated with stimulation of the ovary and ovulation; reduced numbers suggest inhibition, possibly by ovarian hormones for a period, while subsequent increase may indicate indirect involvement in uterine conditions in this viviparous species. Conclusion are, admittedly, tentative as specimens were available during only a fraction of the ten month gestation period.

Notes: The secretory processes in the shell gland of laying chickens were the subject of this study. Three cell types contribute secretory material to the forming egg: ciliated and non-ciliated columnar cells of the uterine surface epithelium, and cells of tubular glands in the mucosa. The ciliated cells as well as the non-ciliated cells have microvilli, which undergo changes in form and extent during the secretory cycle. At the final stages of shell formation they resemble stereocilia. It is postulated that the microvilli of both cells are active in the production of the cuticle of the shell.The ciliated cell which has both cilia and microvilli manufactures secretory granules which arise from the Golgi complex in varying amounts throughout the egg laying cycle. Granule production reaches its greatest intensity during the early stages of shell deposition. The ciliated cell probably supplies proteinaceous material to the matrix of the forming egg shell.The non-ciliated cell has only microvilli. Secretory granules, containing an acid mucopolysaccharide, arise from the Golgi complex. Some granules are extruded into the uterine lumen where they supply the egg shell with organic matrix. Others migrate towards the supranuclear zone. Here a number of them disintegrate. This is accompanied by the formation of a large membraneless space, which is termed “vacuoloid.” Subsequently the vacuoloid regresses and during regression an extensive rough endoplasmic reticulum with numerous polyribosomes of spiral configuration appears. It is suggested that material in the vacuoloid originating from the disintegrating granules is resynthesized and utilized for the formation of secretory product.The uterine tubular gland cells have irregular, frondlike microvilli. During egg shell deposition, these microvilli form large blebs and are probably related to the elaboration of a watery, calcium-containing fluid.

Notes: Hearts of the Atlantic hagfish, Myxine glutinosa were studied with the electron microscope after prefixation in phosphate buffered glutaraldehyde or buffered formalin and subsequent postifxation in phosphate buffered osmium tetroxide. Epicardial, myocardial and endocardial layers are identified; however the hearts of Myxine lack an extensive capillary system comparable to the coronary vessels of other vertebrate heart tissues. Instead, blood is supplied to cells via an elaborate system of channels which extend between numerous trabeculae that make up the cardiac wall of this organism. Fine structural features of special interest include the presence of numerous dense granules (chromaffin granules) within myofibers and also specific granular cells which lack the contractile elements that are characteristic of both skeletal and cardiac myofibers. Another prominent feature noted includes an elaborate system of tubular invaginations within the subjacent sarcoplasm. These elements appear to be specific for the myofibers. They are continuous with the plasma membrane and project into the peripheral sarcoplasmic matrix. Crystalline inclusions are also observed in the sarcoplasm of the myofibers. These are compared with similar inclusions in other cellular components. The Golgi complex is very extensive in the myofibers of Myxine, and granules of varying sizes and densities often appear in the vicinity of the Golgi saccules. The observations suggest that the numerous vesicles around the Golgi Complex represent intermediate stages in the formation of the chromaffin granules. The structure and function of the extensive tubular invaginations are compared with the transverse tubules reported in several mammalian heart tissues.

Notes: Communication organs (septulae) of cheilostome Bryozoa are more complex than perviously believed. Annuli, present only in lateral septulae, are thickenings of the intercalary cuticle. Each communication pore is filled with a ring-like “pore cincture,” through which project a pair of “special cells.” Septulae of all species examined (10 species from 6 families) can be considered modifications of the same structure, varying only in degree of calcification and number of communication pores.External walls, including basal and lateral walls, are best defined as reinforcements of the ectocyst, which is derived by intussusception from the primary cuticle of the ancestrula. The lateral ectocyst must be considered a double layer formed by invagination of the distal ectocyst. Internal walls are developed by apposition from inner parts of the ectocyst; they include pore plates and transverse walls.External walls are laid down first. Lenticular masses develop unilaterally on the uncalcified lateral ectocyst; the pore plate develops by apposition from the interior part of the ectocyst. Depending on the species, the pore plate may or may not be calcified at the time of its formation. Communication pores are formed when the developing pore plate abuts against embryonic special cells. The septular ectocyst never calcifies; it breaks down when the pore plate is complete.Some ascophorans undergo “reparative budding,” in which new zoids are formed within dead zoecia. Hollow, ectocyst-covered buds lined with blastemic epithelia are produced from septulae of live zoids; adjacent buds may fuse.These findings are consistent with the view that lateral septulae are aborted zoids and that pore plates represent transverse walls.

Notes: The development of the motor horn of the foetal mouse was investigated. A sampling technique was evolved using cresyl violet stained material.A decline in the number of motor neuroblasts occurred during the development, on each side, of four definitive motor regions from a single longitudinal column. The total number of motor horn cells fell from about 100,000 on the eleventh day after mating to about 25,000 on the fourteenth day. The early stages of this decline (between 11 and 12 days) are probably explained by the fact that not all neuroblasts in the region differentiate into motor cells; he later decrease can be entirely accounted for by the number of degenerations.Irradiation of the foetal mouse with a dose of 50 rads of cobalt-60 gamma-radiation resulted in an excess of about 20,000 differentiating motor cells on the thirteenth and fourteenth days. Irradiation increased the actual number, but not the percentage, of degenerations. This suggests that differentiation is a phase in the growth process which proceeds degeneation.

Notes: Histochemical procedures for acid phosphatase in normal and lens-regenerating eyes of the urodele Diemictylus viridescens demonstrate activity in a variety of structures. In the normal urodele eye, acid phosphatase is present in conjunctival and corneal epithelial cells and associated glands, in blood vessel endothelium and posterior epithelial cells of the iris, in the anterior lens epithelium, and in the cytoplasm of the optic nerve. Acid phosphatase in the lens-regenerating eye is localized in the same structures as in the normal eye as well as in increased amounts in the corneal epithelial cells and stromal macrophages at the lentectomy wound site and in the posterior portion of the developing lens during completion of differentiation of primary into mature lens fibers characterized by loss of many intracellular organelles. On the basis of these histochemical findings, it is proposed that hydrolytic lysosomal enzymes play an important role in the processes of cellular and intracellular destruction and synthesis which occur during Wolffian lens regeneration in the urodele.

Notes: Median cord development is uniform in six families of Hemiptera and five non-hemipterans. The median cord arises independently from the lateral cords and is histologically distinguishable from the latter throughout development. Intrasegmentally, median cord nuclei possess prominent nucleoli and many small chromatin granules surrounded by clear nuclear sap. This region forms what appear to be glial elements at the midline of the neuropile. Intersegmentally, a spherical clump of eight to twelve large nuclei develops surrounded by dark-staining granular cytoplasm. Each intersegmental clump migrates anteriorly into the preceding ganglionic region but degenerates soon after katatrepsis.

Notes: The order of ossification of bones in the skeleton of Rana pipiens during larval growth and metamorphosis has been determined from observations on specimens fixed in 70% alcohol and stained with alizarin red S. The axial skeleton ossifies in a generally cephalo-caudal sequence, beginning with the parasphenoid bone at Taylor-Kollros stages IV-IX, followed by vertebrae (V-IX) and then the urostyle (IX-XIV). Exoccipitals (VII-IX), frontoparietals (XI-XII) and prootics (XIII-XVII) are additional cranial bones which successively ossify before metamorphosis. With the onset of metamorphosis at stage XVIII jawbones and rostral bones of the skull ossify in the following succession: premaxilla, maxilla, septomaxilla, nasal, dentary, angular, squamosal, pterygoid, prevomer, mentomeckelian, quadratojugal, palatine, columella, posteromedial process of “hyoid.” The sphenethmoid does not ossify until after metamorphosis.Ossification of limbbones begins with the femur or humerus at stages X-XII and progresses proximo-distally to the phalanges by stages XIII-XV. Carpals, however, do not ossify until stage XXV or after metamorphosis. The ilium of the pelvic girdle begins to ossify at stages X-XII, but the ischium is delayed until stages XX-XXIII. Scapula and coracoid of the pectoral girdle undergo initial ossification at stages XII-XIV, suprascapula and clavicle at stages XIII-XV. The sternum does not begin to ossify until stage XXIV. The possible role of thyroid hormones in stimulating osteogenesis is discussed.

Notes: The campaniform sensilla on halteres of Drosophila were studied by electron microscopy in order to establish the relationships of functional elements in the sensory system. The surface of the sensillum consists of an oval cuticular cap membrane which may contain resilin, the rubberlike protein. A border of denser cuticle rings the cap membrane, and extending down around the neural process is a third type of cuticle filled with a fourth light fibrous type. The four cuticular components form a system for displacement of the neural process. The neural process is differentiated into a terminal fan-shaped structure projecting from a bulbous dilatation which tapers to a neck region ending proximally with two basal bodies. The neural process is packed with microtubules. Surrounding the dendrite is an inner enveloping cell, attached to the basal body region by septate desmosomes and by desmosomes to which microtubules of the enveloping cell are applied. An outer enveloping cell surrounds the inner one. The tip of the neural process is covered with a dense secretion which is tightly bound to the cap membrane. The dense secretion is surrounded by an extracellular fluid which might be compressed hydraulically by the cuticular system. The stimulus of cuticular distortion could thus be transmitted to the neural process which may be displaced between its fixed ends.

Notes: Frog retinal rod outer segments were detached from dark adapted retinas by (1) agitation in frog Ringer's solution or (2) by crushing between two glass surfaces. The resulting suspensions were further purified by low and high speed centrifugation procedures in Ficoll density gradients. The density of the outer segments in Ficoll solutions was found to be 1.09. The large frog outer segments, unlike bovine outer segments, are not readily separated from nuclei, which were estimated to comprise 2.6-8% of the material, based on DNA analyses. The RNA/DNA ratio was 0.4-0.5, like that of neuronal nuclei. Representative enzymes of glycolysis (lactic dehydrogenase and glyceraldehyde phosphate dehydrogenase), phosphogluconate oxidation (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), the citric acid cycle (malic dehydrogenase) and ATP degradation (ATPase) were assayed. A major part of the malic dehydrogenase activity was probably due to inner segments attached to some of the outer segments. Glyceraldehyde phosphate dehydrogenase and glucose-6-phosphate dehydrogenase (but not lactic dehydrogenase) activities were lower in detached outer segments purified on Ficoll gradients than in samples of outer segment layer microdissected from freeze-dried sections of frog retina, as well as in whole retina. The data suggest that the activities of all the enzymes studied are intrinsically low in rod outer segments.

Notes: The frog, Rana pipiens, hibernates through the winter with ovaries containing oocytes which, in size and appearance, are ready for ovulation and maturation. From November through April, the normal time of egg laying, ovulation and maturation can be induced by interrupting hibernation and administering gonadotropic hormones. In the studies reported here, it has been found that oocytes taken from hibernating animals in early winter take up amino acids from a saline medium at a relatively rapid rate. Respiratory inhibition produced by such agents as dinitrophenol (DNP) and anaerobiosis, does not completely stop uptake but slows it down markedly.In late winter, amino acid uptake tends to be slower in normal cells and when DNP or cyanide is added, a marked acceleration of the rate of uptake is observed. The uptake in poisoned cells is accumulative, producing internal concentrations higher than that of the medium. At this concentration of DNP, amino acid incorporation is almost completely stopped. Fluoride abolishes the DNP stimulation of amino acid uptake. Removal of sodium ion from the incubating medium has no effect on either uptake or incorporation.These data are interpreted to mean that a capacity for oxidative phosphorylation in oocytes diminishes during winter hibernation. In the spring either an anaerobic capacity comes into being or becomes capable of being switched on. During this time the rate of amino acid uptake, even in the downhill phase, is limited by energy availability and is not dependent on a sodium gradient.

Notes: The effect of temperature on intracellular ATP levels in cells of the ascitic Hepatoma 129 has been determined using an UV-enzymic method. ATP levels are found to vary inversely with temperature over the range (4°-37°C) studied, and this variation is found to be reversible. An indication of a possible direct correlation of ADP level with temperature was also obtained.A correlation is deduced between rise in intracellular ATP level and decrease in cytoplasmic viscosity, both from the present results, and from similar effects recently reported by Landau ('61) for increase in pressure.The significance of these observations is discussed with reference to the model for cytoplasmic viscosity proposed by Marsland ('56) and the wider biological significance of this effect is explored.

Notes: Dietary zinc deficiency has been shown to decrease the in vivo incorporation of thymidine into the nuclear DNA of liver parenchymal cells in the rat. A single injection of 100 μg of zinc was sufficient to rapidly reverse the effect of zinc deficiency on the synthesis of DNA.

Notes: It has been shown by re-cloning of colonies formed in vitro from rat bone marrow cells, that normal granulocyte colonies can originate from single cells. No mixed macrophage (M) and granulocyte (G) colonies were obtained after re-cloning either M or G colonies. The results indicate, that clones of normal granulocytes and macrophages can be obtained in vitro, and that the mixed primary M and G colonies formed after seeding hematopoietic cells from animals presumably originate from a mixture of M and G cells.

Notes: The cytotoxic effect of high, as well as low, oxygen tension of proliferation and metabolism of Low line cells in culture is reversible even after several days of exposure provided the cells are returned to 95% air + 5% CO2 environment. This suggests that the activity of certain mechanisms within the cells may have been altered or in other ways inhibited by the abnormal environments but are quite rapidly regenerated once the adverse condition is removed. The cells tolerate a low O2 exposure for at least 20 days while continuous exposure to high O2 atmosphere results in degeneration and death after 7-10 days. Both glucose utilization and lactic acid production are elevated in cultures exposed to either low or high O2 tensions, although they are markedly higher in the latter condition. When cell so exposed are returned to an air + 5% CO2 atmosphere, rate of glucose uptake and lactic acid formation soon approaches that found in control cultures.

Notes: Histologic examination of the spleens in RFM/Un mice killed 6 to 9 days after 350 to 800 R whole-body x-irradiation revealed hemopoietic colonies, the numbers of which decreased exponentially with increasing radiation dose. In such animals, myelocytic colonies were the predominant type on the sixth to the eighth day. However, they decreased in number with time, being fewer than erythropoietic colonies by the ninth day after irradiation. In C57BL mice, erythropoietic colonies were relatively more numerous, markedly predominating on both the eighth and the thirteenth days.RFM/Un mice injected with nonirradiated syngeneic bone marrow cells within 24 hours after 750 R developed colonies, predominantly of erythropoietic and undifferentiated types, the numbers of which were proportional to the numbers of marrow cells injected. The number of colonies formed from exogenous marrow cells increased slightly between the sixth and ninth days after inoculation, possibly because of a greater likelihood of counting them due to an increase in their size.

Notes: In Tetrahymena pyriformis, mating type I, variety 1, cycloheximide rapidly and completely inhibited incorporation of 14C-L-leucine into protein. Actinomycin D (25 μg per ml) inhibited incorporation of 14C-uracil into cold-TCA-insoluble material, after a 5-10 minute lag. Frequently a subsequent decline in the amount of radioactivity was observed. Protein synthesis continued in actinomycintreated cultures for a variable time after cessation of RNA synthesis.Oral development was affected by cycloheximide virtually immediately, and by actinomycin D after a 10-15 minute lag. Cells affected by either drug before the onset of oral membranelle formation were permanently arrested in the stomatogenic field phase. Cells affected in the early and middle stages of membranelle formation completed development of membranelles, but did not invariably complete cell division. Cycloheximide, when added at the beginning of membranelle formation, brought about arrest or resorption of membranelles after they were completed. Actinomycin did not elicit resorption, but sometimes brought about blockage during cell division. Cells affected by either drug after membranelles were fully formed (and cell division was just beginning) completed oral development, nuclear divisions, and cell division. These results suggest that concurrent RNA and protein synthesis are essential for the initiation but not for the completion of membranelle differentiation. The results also suggest that a specific messenger RNA(s) with a very short half-life is required for the synthesis of proteins involved in the initiation of membranelle differentiation.

Notes: Growth of KB cells was inhibited by both spermine and spermidine, but the inhibition is reduced in conditioned medium. The amount of spermine required for 50% inhibition of plating varied according to the type of serum used with medium 199 (calf, fetal bovine, and horse; 0.55, 0.9, and 24 μg/ml respectively). The spermine oxidase activity of the three sera was calf 〉 horse 〉 fetal bovine, which is not the same ordering as was obtained for the inhibition. When the concentration of sera in the media was varied, the inhibition decreased as calf and fetal bovine sera concentration increased, whereas, with horse serum, an increase in serum concentration increased the inhibition. The opposite effects of increasing concentrations of the sera on the inhibition suggest that at least two factors are involved in the inhibition. A scheme which involves three factors (spermine oxidase, another enzyme and its activator) is postulated to account for the inhibitions and reversals observed. Spermine oxidase alone cannot account for the action of polyamine on cells.

Notes: Reduction in alkaline phosphatase activity was observed when HeLa S3 cells were grown in Puck's medium containing high concentrations of human serum. This effect was not seen with the enzyme of Chang liver 8A cells. The induction of increased alkaline phosphatase in HeLa S3 by prednisolone or by osmolality changes was not prevented by serum. The concentration of serum in the culture medium had no influence on acid phosphatase activity.

Notes: Functionally active 30S ribosomes can be reconstituted in vitro from 16S RNA and a mixture of 30S ribosomal proteins under certain defined conditions. Our previous studies on the specificity and kinetics of reconstitution are summarized and discussed. The reconstitution reaction is first-order with respect to formation of active 30S ribosomes. The rate-limiting reaction is probably unimolecular, and it represents the structural rearrangement of an intermediate. Presumed reconstitution intermediates, or RI particles, have been isolated from reconstitution mixtures incubated at low temperature. It has been concluded that the reconstitution takes place in stepwise fashion: 16S ribosomes. New experimental results that show the highly cooperative nature of the assembly reaction are described; the number of sites, per RNA chain, which can bind ribosomal proteins independently from each other (i.e., without cooperativity) is at most two to three. Finally, another approach to the study of ribosome assembly in vivo is described. It utilizes cold-sensitive E. coli mutants that are defective in ribosome biosynthesis at low temperature and accumulate incomplete “intermediate” ribosomal particles.

Notes: A cell line was established in vitro from a spontaneous myeloid leukemia of SL strain mice. This cell line was cytologically identified as myeloblast, and its normal differentiation appeared to be completely blocked in mass culture. When the line cells were seeded in soft agar with a conditioned medium from normal cells, either macrophages or neutrophil granulocytes appeared from a single clone. The rate of formation of colonies containing differentiated cells always increased with an increase in the concentration of conditioned medium. The conditioned medium from this line cell was not as effective as was that from normal cells in inducing differentiation.

Notes: Considerable variability has been found in the yield of cells in batch cultures of Tetrahymena pyriformis grown axenically in 1% tryptone/0.05% yeast extract. This variability has been traced to the photolysis by visible light of the flavin mononucleotide and thiamine components of yeast extract.

Notes: Colony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7-day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.

Notes: Maturation of bacterial viruses requires the formation of mature viral nucleic acid in the form found in progeny virus particles, the “packaging” of this viral nucleic acid within a capsule, and the assembly of “packaged” nucleic acid with the accessory structures necessary for infectivity. Recognition that the replicating viral nucleic acid is frequently in a form distinct from that found in mature particles has, in some instances, led to the postulation and formal description of presently unknown processes that must participate in the synthesis of the mature form. In at least some instances, packaging of the mature nucleic acid is clearly integrated with its synthesis.Examples involving the DNA bacteriophages T4, λ, and φ X174 and the RNA bacteriophages are presented to illustrate these points.

Notes: The development of a virus is programmed by a series of negative and positive controls which determine the timing and the segment on either of the two DNA strands (l or r) to be transcribed into specific messenger RNA's. Bacteriophage λ provides one of the most deeply studied systems for following the development of lysogenic viruses. In the lysogenic repressed state, only 2-4% of the λ genome is expressed. This pc-cI-rex region is transcribed leftward to produce a repressor protein which prevents any further transcription by blocking the oL and oR operators flanking the cI-rex operon (figs. 1, 2). This negative control is relieved by destruction of the repressor, and the result is the “induction” of viral development. The earliest post-induction or postinfection events are the leftward transcription of the pLoL N region from strand l and the rightward transcription mainly of the pRoR-x segment from strand r. The N product acts as a positive control, permitting a leftward transcription beyond gene N and a rightward transcription of genes cII-O-P and also Q. The int-xis system controls the excision of the λ genome, whereas the act of rightward transcription and the products of genes O and P initiate the replication of λ DNA. The product of gene Q, still another positive control, stimulates rightward transcription of the late genes which control the synthesis and assembly of the phage heads and tails as well as cell lysis. Among other types of negative control are the possible competition between the two divergent transcriptions originating in region x, the “antirepressor” effect of the x product, and the interference between the two convergent transcriptions which collide in the central b2 region. The majority of controls are based on protein-DNA interactions and can be modified by mutations. For instance, transcription can be rendered independent of negative repressor control either by constitutive, v, mutations which decrease or abolish the affinity of the o operators for the repressor or by insertion of new promoters-e.g., c17 or ric- on the “downstream” side of the operator. The need for the positive N and Q controls may also be obviated by mutations in the N- or Q-dependent promoter or terminator elements.The specific DNA structure within the controlling sites is not known. However, a remarkable coincidence was observed; namely, the occurrence of pyrimidine-rich clusters in those segments of the individual DNA strands acting as templates for RNA synthesis. This observation, which pertains to all studied DNA's, including those of phages T2, T3, T4, T5, T6, T7, λ, and φ 80, formed the basis for a proposal that implicates pyrimidine-rich clusters in the initiation, control and/or termination of transcription, and also in the determination of the preferred strand and, consequently, the orientation of transcription. General considerations regarding the possible role of the structural singularities, especially those represented by the pyrimidine clusters, in the bipartite structure of the recognition regions in DNA are discussed.

Notes: These studies have been conducted as a first step toward investigating the molecular mechanism by which DNA-dependent RNA polymerase recognizes a specific region of DNA. The DNA moieties are either bound to or freed from RNA polymerase. Results obtained so far indicate that the DNA moieties remaining as resistant segments after deoxyribonuclease digestion should be regarded as the binding regions on DNA. The results described here were carried out by use of E. coli and lambda DNA.

Notes: Sarcoplasmic reticulum fragments, prepared from skeletal muscle homogenates, were found to consist of two major types when examined after negative staining. One type possessed 90 Å subunits and was thought to be of mitochondrial origin. The other had 35 Å subunits and ws presumably derived from the sarcoplasmic reticulum. Only the latter type accumulated visible calcium oxalate deposits inside the vesicle when they were exposed to a medium containing ATP, MgCl2, K2C2O4 and CaCl2. The calcium oxalate loaded vesicles were strikingly angular in shape and did not have tails. The calcium oxalate loaded vesicles had identical membrane subunit arrangement to inactive companion membranes and nonincubated controls; this suggested that the membrane subunits were not the critical structural requirement for calcium transport. A method was described whereby the calcium accumulating membranes could be purified 3- to 4-fold on the basis of the best previously used preparation procedures.

Notes: The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10-5 M FUdR was very low (0.0001-0.0003) whether the drug was administered during early G1, late G1, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10-7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.

Notes: The effects of cycloheximide on synchronized Tetrahymena pyriformis strain GL-C were investigated at concentrations ranging from 0.01 to 10 μg/ml. The initial inhibition of protein synthesis was nearly total (〉85%) at 1 μg/ml and above, partial (50-80%) at 0.2 to 0.05 μg/ml, and slight (〈30%) at 0.02 μg/ml. Eventual recovery of protein synthesis to a rate approaching that of the controls took place at concentrations of 1 μg/ml and less. When the drug was added before a “transition point” at 55 minutes after the end of the synchronizing treatment (EST), cell division was blocked by 10 μg/ml, and delayed at concentrations of 1 μg/ml or less. The duration of delay was related to the degree of initial inhibition, and to the time required for recovery of protein synthesis; it also depended on the time after EST at which the drug was added. At a given concentration, maximum division delay was observed just prior to the “transition point;” this maximum delay was correlated with resorption of differentiating oral primordia, followed by the appearance of new primordia. The lesser delays observed at earlier times were correlated with temporary blockage of development of primordia in the “stomatogenic field” stage. Resumption of oral primordium development was, in both cases, temporally correlated with a substantial recovery of protein synthesis. After the “transition point,” cell division, and completion of oral development, was delayed slightly at the lower concentrations, and more substantially at 1 and 10 μg/ml, with some division-arrest at the latter concentration. Except for the recovery phenomenon, the developmental responses elicited by cycloheximide were similar to those observed earlier with puromycin.The bearing of these findings on the mechanism of synchronization in Tetrahymena is considered in the Discussion.

Notes: Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.

Notes: Theoretical osmotic fragility curves were calculated and drawn by computer using the van't Hoff equation and the isotonic areas and volumes of 1000 individual erythrocytes. We studied the influence on the calculated curves of theoretically altering the fraction of the volume which was osmotically active from 50 to 70%, and of altering the permissible stretch before hemolysis from zero to 10%. With the two assumptions-that the membrane does not stretch before hemolysis, and that the osmotically active fraction of the cell volume is 0.58-it was possible to duplicate the general shape of the standard fragility curve; the exact NaCl concentration, however, at which there was 50% hemolysis was approximately 0.1 gm/100 ml higher than found in vitro. The calculated osmotic fragility curves can be made quantitatively similar to in vitro ones if the following statements are true: the osmotically active volume is 58%, the permissible stretch of the membrane without lysis is 6%, the cell membrane resists a slight osmotic pressure gradient of approximately 0.1 atmospheres, and hemolysis is an all or nothing phenomenon. This set of values for the relevant factors is sufficient but not unique in causing the superposition of the calculated and experimental curves. The frequency distribution of the cells according to the hemolytic salt concentrations (the sodium chloride concentration at which an individual cell just hemolyzes) was skewed positively and was leptokurtic for each of the seven normal subjects studied.

Notes: The fraction of cell capable of division was determined for (1) population of the human diploid cell strains, WI38 after different numbers of subcultivations in vitro and (2) a single population of WI38 cells at intervals throughout its entire in vitro lifespan. In both cases the percentage of cells capable of division decreased with increasing age in tissue culture. The rate and the magnitude of the decrease is sufficient to account for the limited in vitro lifespan reported by other investigators. Furthermore, the decrease in the fraction of cells capable of division in similar in some respects of senescence among human populations.

Notes: All tRNA sequences so far known can be folded into a cloverleaf structure. Physical data and chemical reactions allow us to draw conclusions on secondary (cloverleaf) and tertiary structure. N-oxidation of adenosine to adenosine-1-N-oxide can be done with monoperphthalic acid in non-base-paired regions of polynucleotides and can be followed easily by changes in absorption of ultraviolet light. Thus this method can be used to determine the structure of tRNA's. A fingerprint of the N-oxidation product of tRNAyeastPhe reveals that all adenosine residues are protected except the 3′-terminal adenosine and the three adenosine residues in or adjacent to the anticodon. On this basis a conformation of tRNAyeastPhe is proposed. Similar tertiary structures can be constructed for the other tRNA's. In order to connect tertiary structure of a tRNA and recognition by its aminoacylating enzyme, the rate of aminoacylation, as a function of temperature, was measured. Neither changes in the anticodon nor specific changes at the 3′-terminal adenosine abolish aminoacylation. Single crystals of tRNAyeastPhe were obtained from aqueous solutions upon addition of various organic solvents.

Notes: Assemblies of protein molecules represent a fundamental level of biological organization. The dynamic behavior of these systems-including both the assembly process and functional rearrangements-may be accounted for by the specificity of the protein interactions, which depend on environmental conditions. Analysis of the self-assembly of virus particles has established that the design of an ordered structure can be built into the specific bonding properties of the constituent proteins. Any structure which can change its state of organization is, by definition, polymorphic. The distinctive aspect of polymorphism in protein structures, contrasted with nonliving states of matter, is that the molecular design has been selected to carry out a function and that this function is part of an integrated system. The differences in molecular conformation and arrangement in all polymorphic structures-for example, allosteric enzymes or ice crystals-depend on the intrinsic interaction properties of the molecules themselves. The structures of ice and water illustrate relations between specificity and polymorphism which are relevant to the form and function of protein assemblies.Two types of polymorphism can be distinguished: modal polymorphism, which is externally moderated, as in phase transitions between different crystals forms; and positional polymorphism, which is internally moderated, as in the different disposition of identical molecules within a single crystal lattice. Positional polymorphism, exemplified by the quasi-equivalent bonding of icosahedral virus coat proteins and the different arrangement of myosin and paramyosin at the center and polar portions of the bipolar filaments, results from specific interactions that are not compatible with a strictly equivalent packing of identical molecules. The structural rearrangements in muscle contraction and the switching between the oxy and deoxy forms of hemoglobin represent the formation of different structures in response to altered external conditions. The different structural states of many protein assemblies are characterized by conserved connections which may be regarded as providing the framework for functional rearrangements. The types of polymorphism displayed by hemoglobin, virus, and muscle proteins demonstrate the relevance of the simple view that the function of a protein is determined by the potential structures it can form.

Notes: Analysis of the equilibrium of the normal biconcave human red cell, in terms of its tension and a pressure inside, suggests a force of attraction between the opposite membranes at the dimple regions. The analogous attraction that causes rouleaux formation is mediated by long-chain molecules. Single cells hanging on edge between polarizer and analyser, almost “crossed,” were photographed at different angles to the axis of the polarizer. Enlarged prints were scanned by a photometer. For single cells the records showed non-significant fluctuations of intensity, but mean values for 32 cells showed a very significant sinusoidal variation with angle, as predicted by theory for birefringence in the cell at the dimple region. For the rim region, the averaged data showed no variation with angle. In cells moderately osmotically swollen, birefringence in the centre of the dimple region was absent, but persisted close to the inside of the membranes. The latter disappeared in cells further swollen to a biconvex shape. The data is interpreted as indicating oriented chains of molecules across the interior of the cell at the dimple region. The behaviour on swelling was what had been seen in a model with nylon fibres oriented between the charged plates of a condenser, in which the variation of attractive force with distance was adequate to explain the equilibrium of the red cell membrane.

Notes: H3-Uridine microinjected in the giant axons of the squid is incorporated in a TCA insoluble material. There is no difference between stimulated and resting axons as to the amount incorporated.The amount incorporated is increased if the stimulation precedes the microinjection of the tracer.RNA was purified and characterized from the axoplasm, axon sheaths and from a purified membrane preparation obtained from squid retinal nerve.

Notes: Diploid human fibroblasts in culture (WI 38) were allowed to reach a stationary phase and were then stimulated to reenter DNA synthesis and cell division by addition of serum to the culture medium. The rate of protein synthesis increased during the first hours after addition of serum reaching at three hours a plateau value that continued for at least 24 hours after serum addition. Inhibition of protein synthesis during the early hours after serum addition abolished the stimulation of DNA synthesis occurring 20 to 28 hours later.Increased protein synthesis was preceded by a rapid decrease in the intracellular pool size of most amino acids. These changes were independent of concomitant protein synthesis. They suggest that serum exerts an immediate effect on the function of the cell membrane.

Notes: Cellular transformation was induced with avian myeloblastosis virus strain BAI-A (standard AMV) and with a strain of AMV containing subgroup B only. Cultures of muscle tissues from either chick embryo or day old chicks were used for this study. Results were similar in C/O and C/A cells. Leukemogenic virus was continuously produced by these transformed cultures.

Notes: In the presence of 1 mM ATP in the external medium, TA3 mouse ascites tumor cells showed a dramatic loss of potassium and gain of sodium down their respective concentration gradients. The volume changes detected by size discrimination with the Coulter counter have been adequately confirmed by densimetric techniques. Further, some experiments were so designed that net losses of both ions occurred and the cell shrank in response to ATP, a response which was predictable if the volume change was produced by a loss of cell water. We believe that ATP produces a major change in the passive permeability of the membrane to these ions and the effect may be due to a response of a contractile protein in the membrane to ATP.

Notes: Growth hormone, oxytocin, parathyroid hormone, prolactin and lysine vasopressin strongly stimulate mitotic activity in rat thymocyte populations maintained in vitro. These hormones have no mitotic effect on cells maintained in calcium-free medium. It is concluded that they stimulate mitosis only indirectly by sensitising the mitotically competent segment of a thymocyte population to the action of calcium. The stimulatory action of calcium itself is opposed by low concentrations of the mucopolysaccharide chondroitin sulphate. However, the inhibitory action of chondroitin sulphate can be overcome by growth hormone.A possible common mechanism of action of these hormones on mitotically competent cells is discussed.

Notes: A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 107 cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r2/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.

Notes: The progression of rat thynocytes (maintained in vitro) into mitosis is profoundly affected by the level of magnesium in the medium. Increasing the extracellular MgC12 concentration from 0 to 1.0 mM does not affect mitotic activity, but a further increase to 1.2 and 2.5 mM rapidly and strongly stimulates the flow of cells into mitosis. The level of extracellular magnesium also governs the mitogenic effectiveness of calcium ions. The mitogenic action of magnesium stems from an ability to stimulate the initiation of deoxyribonucleic acid synthesis.

Notes: Colony-forming cells (CFU), which have the general properties of hemopoietic “stem” cells, appear to be augmented in the mouse fetal liver from 12-18 days gestation and then decrease in the newborn. This finding suggests that few, if any, hemopoietic “stem” cells remain in the adult liver, an organ which appears to be unable to function erythropoietically, even at times of severe crises. In the spleen, and active adult as well as embryonic hematopoietic organ, the total number of CFU increases from 18 days gestation until at least 7 days after birth.Spleen and liver CFU augmentation seems to occur in cojunction with an analogous expansion of non-hematopoietic cells. The data suggests, in fact, that while there is an increase in the total number of liver CFU, there is also a dilution of liver CFU in the total cell population at successively later gestational ages.

Notes: The incorporation of tritiated thymidine and deoxycytidine into DNA of x-irradiated mammalian cells was studied. Both inhibition and stimulation were found due to pool changes rather than to effects on DNA synthesis, indicating that precursor uptake can be a misleading method to measure DNA synthesis rate.