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Notes: Summary The basic scheme of nucleosynthesis (building of heavy elements from light ones) has held up very well since it was first proposed more than 30 years ago by E.M. Burbidge, G.R. Burbidge, A.G.W. Cameron, W.A. Fowler, and F. Hoyle. Significant advances in the intervening years include (a) observations of elemental and a few isotopic ratios in many more extrasolar-system sites, including metal-poor dwarf irregular galaxies, where very little has happened, and supernovae and their remnants, where a great deal has happened, (b) recognition of the early universe as good for making all the elements up to helium, (c) resolution of heavy element burning in stars into separate carbon, neon, oxygen, and silicon burning, with fine tuning of the resulting abundances by explosive nucleosynthesis in outgoing supernova shock waves, (d) clarification of the role of Type I supernovae, (e) concordance between elements produced in short-lived and long-lived stars with those that increased quickly and slowly over the history of the galaxy, and (f) calibration of calculations of the evolution and explosion of massive stars against the detailed observations of SN 1987A. The discussion presupposes a reader (a) with some prior knowledge of astronomy at the level of recognizing what is meant by an A star and an AGB star and (b) with at least a mild interest in how we got to where we currently are.

Notes: Many drugs are recemic and therefore much effort has to be devoted towards the stereoselective synthesis of the most effective or less harmful component of a racemic mixture. High performance liquid chromatography will play an important role in the clinical analysis of racemic drugs in anticipation of regulations that are currently being discussed and are expected to be enforced by the end of this decade. In this review a number of methods for chiral resolution are outlined. These include the formation of diastereoisomers and the use of chiral stationary phases or chiral mobile phase additives.

Notes: Medium- and long-chain 3-hydroxymonocarboxylic acids represent intermediates in the β-oxidation of fatty acids: they accumulate in the plasma of patients with an inherited deficiency of long-chain 3-hydroxyacylcoenzyme A dehydrogenase. 3-Hydroxy acids with chain lengths varying from 6 to 16 were synthesized by a Reformatzky reaction. Capillary gas chromatography of the pertrimethylsilyl derivatives was performed on a CP-Sil 19 CB column, coupled to a quadrupole mass spectrometer in the electron impact mode. Calculation of the retention indices showed that the separation of the 3-hydroxy acids from the homologous straight-chain fatty acids may be troublesome, stressing the need for mass spectrometric identification.

Notes: A new method is described for the analysis of vitamin D and its metabolites utilizing thermospray (TSP) mass spectrometry as an on-line detector for high performance liquid chromatogrpahy. Ionization conditions were optimized for use with isocratic reversed phase chromatography. TSP mass spectrometry was employed in series with a UV absorbance detector to facilitate comparisons between the two methods of detection. Positive ion TSP mass spectra were recorded for vitamin D2, vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). The spectra contained protonated molecular ions, ammonium adduct ions and fragment ions due to the loss of one or more molecules of water. A comparison of quantitative precision was made by determining UV absorbance and TSP standard curves for vitamin D3 using two different methods: (1) External standard method with post-column (post UV detector) addition of ammonium acetate. (2) As (1) but using the method of internal standards with a closely eluting internal standard (vitamin D2). In each case the quantitative precision (correlation coefficient) for UV absorbance detection was superior owing to intrinsic instability of the TSP ion beam. A stable isotopically labelled internal standard was employed in the development of an assay for 1,25(OH)2D3. The assay was used to quantify in vitro enzymic conversion of 25(OH)D3 to 1,25(OH)2D3 in guinea pig and sheep renal mitochondrial incubations. TSP LC/MS was also applied to analysis of an extract of human blood plasma in which D3 and each of its principal metabolites were identified in a single analysis.

Notes: Chromatography has played a pivotal role in the advances made during the last 30 years in our knowledge of inborn errors of metabolism. This review discusses the application of some of these techniques to the analysis of organic acids and acylcarnitines. The separation of organic acids needed a comprehensive approach that would permit all of the many organic acids present in urine or other complex mixtures to be extracted, analysed and identified in a single run. This required analytical methods of great resolving power, wide linear range and universal detectors such as gas chromatography (GC), or GC coupled with mass spectrometry. Sample preparation was another problem that has been tackled by a variety of approaches. Organic solvents have been employed widely for the extraction of organic acids from physiological fluids. Unfortunately, recoveries of the different organic acids by this method are sometimes less than quantitative and variable depending on the compound. Other methods, such as the use of DEAE-Sephadex columns, have the advantage of resulting in close to 100% recoveries, but are more tedious. Liquid partition chromatography on short silicic acid columns has also been recommended as a useful clean-up step prior to GC, permitting both the identification and quantitation of organic acids in urine, plasma or amniotic fluid. Although many derivatization procedures have been used to prepare organic acids for gas chromatography, the most common is trimethylsilylation. Oxo acids are usually reacted with one of several commonly used reagents to form oximes. GC analysis of organic acids was initially done using packed columns with methylsilicone-based, non-polar stationary phases. In recent years most laboratories have switched to the commercially available, bonded-phase capillary columns. Some authors have recommended the use of two columns of slightly differing polarities to improve the reliability of identification without mass spectrometry. Carnitine and acylcarnitines are substances whose measurement may yield useful diagnostic information about inborn errors and about the basic biochemical mechanisms of disease. While free carnitine is usually measured by a radioenzymatic assay, the separation of acylcarnitines requires paper chromatography or high performance liquid chromatography. The identification of various acylcarnitines following separation has been accomplished by mass spectrometric methods. Another technique relies on gas chromatographic identification of the acyl groups liberated following alkaline hydrolysis. Recently, liquid chromatography/mass spectrometry has also been utilized to separate and identify the various acylcarnitines present in urine and other biological samples.

Notes: A sensitive and selective, reasonably fast method for the determination of glucose content has been developed. A glucose oxidase immobilized column was coupled to a small-size anion exchange column/borate buffer chromatograph. The hydrogen peroxide produced in the enzyme reaction was detected directly by an amperometric detector using a platinum working electrode. The detection limit was 0.03 ppm (1.5 × 10-7 M, 3 pmol/injection). The linear dynamic range was three orders of magnitude at least. The system was stable and reproducible both in short-and long-term operation. The proposed method is suitable for analysis of complicated matrices of biological samples because of its good selectivity and sensitivity.

Notes: Plasma B-6 vitamer and plasma and urinary 4-pyridoxic acid concentrations of 21 young white women, 21-27 years, having radiomonitored pyridoxal 5′-phosphate and coenzyme stimulation of erythrocyte alanine amino-transferase activities indicative of adequate vitamin B-6 status were determined in an effort to establish normal ranges for plasma B-6 vitamers. B-6 vitamers and 4-pyridoxic acid were quantitated using reversed phase high performance liquid chromatography with fluorometric and ultraviolet detection. Pyridoxal phosphate values obtained by radioenzymatic and chromatographic, fluorometric and ultraviolet, assays were highly correlated as were pyridoxine phosphate values determined using both detectors. The B-6 vitamer and 4-pyridoxic acid values of these subjects should be of use in the establishment of normal ranges of these congeners in women.

Notes: We have compared anion exchange chromatography on APS-Hypersil (4.6 × 100 mm) eluted with a phosphate gradient with reversed phase chromatography on ODS-Hypersil (4.6 × 100 mm) in the presence of either tetrabutylammonium (TBA) or triethylammonium (TEA) ions with a methanol gradient. The systems have been compared both for ease of operation and for their resolving power with standard mixtures and acid extracts of both normal red cells (RBC) and ischaemic tissues. The two chromatographic modes exhibited similar separating efficiencies for standard mixtures of nucleotides but retention times were most stable using reversed phase liquid chromatography (RPLC) with TEA. Anion exchange columns slowly lost ion exchange capacity but selectivity was unchanged. RPLC in the presence of TBA gave reproducibile capacity factors only when operated isocratically due to irreversible changes to the silica surface. For RBCs the RPLC with TEA and anion exchange sysems resolved 17 and 15 peaks, respectively, and for the ischaemic samples 22 and 14 peaks, respectively. However, nucleosides and bases were also resolved by the ODS column causing chromatographic crowding and uncertain peak identification.

Notes: The effect of peroxidizing herbicides on the liver porphyrin content of experimental animals was examined. Mice treated with the herbicide oxadiazon accumulated uroporphyrin and protoporphyrin in the liver. Fomesafen-treated mice accumulated uroporphyrin and heptacarboxylic porphyrin.

Notes: The measurement of the nucleoside deaminases - cytidine deaminase, guanosine deaminase and adenosine deaminase - by reversed phase high performance liquid chromatography is reviewed. The clinical value of assaying the enzyme activity is discussed for each of these enzymes. Both cytidine deaminase and adenosine deaminase measurements have proven clinical value, although the use of the assay of cytidine deaminase in the diagnosis of pre-eclampsia is probably not helpful.

Notes: The cupula of the supraorbital neuromast in the lateral line canal of the clown knifefish contains vertical columns. In the central region of the cupula overlying the macula, these columns are densely packed, are relatively constant in size, and run from the base of the cupula to the surface of the cupula which is exposed to canal fluid. There are two types of columns, dark and light, which form elliptical compartments in planes of section that cut across the columns; the cupula therefore has the appearance of mosaic tile in such sections. The dark compartments contain tubules that extend from the base of the cupula at the junction with the macula to the top of the cupula. Each tubule is associated with the kinocilium of a single hair cell. The lateral parts of the cupula, not overlying the macula, also contain compartments, but these compartments differ in size and structure from those in the central region. In addition to the compartments, the central region of the cupula also contains spherical aggregates of droplets. These small aggregates, termed mora, are found principally in a layer within the central region of the cupula, but are also found outside this layer. Because of their light-reflecting properties, the mora can be used for noninvasive optical measurements in vivo of the motion of the cupula.

Notes: Light and electron microscopic examination demonstrated two types of non-endocrine agranular cells, cavity boundary cells and stellate cells, in the adenohypophysis of the South American lungfish, Lepidosiren paradoxa. The cavity boundary cells line the hypophyseal cleft and diverticulum and display few microvilli, occasional cilia, prominent junctional complexes, and many cytoplasmic microfilaments. The stellate cells are scattered in the glandular parenchyma and are devoid of microvilli and cilia. When adjacent, they are connected to one another by desmosomes. Pinocytotic vesicles or caveolae are frequently seen along the plasma membrane of the agranular cells adjoining the endocrine cells or abutting on the basement membrane. Possible roles of the agranular cells, physically and metabolically supportive functions, are discussed on the basis of their ultrastractural features.

Notes: The paired organ of Bellonci protrudes from the optic lobe of the giant Antarctic isopod, Glyptonotus antarcticus. It is linked to the cortex by a broad peduncle. No connection to the cuticle or “sensory pore organ” was found. A cluster of sensory-like cells forms two outer ciliary segments branching into numerous microvilli with microtubules. The putative sensory somata are irregular in shape and contain a very high density of glycogen granules. The two outer segments sprout from two pits of the soma in different directions, forming a right angle. Glial cells wrap around the sensory cells and also delimit lacunae into which bundles of microvilli project. These lacunae contain electron-dense granules of small size and with species-specific patterns. Lacunae and dense granules show features typical of a degeneration process in the sensory cells.This general morphology corresponds to the unilobular type of organ of Bellonci, known in other isopods; it differs from the plurilobular type with onion bodies found in other Crustacea.

Notes: Neuromast structure in Rana cancrivora larvae was observed by scanning and transmission electron microscopy. Neuromast units, each being composed of two or three neuromasts, are arranged in several well-defined lines in the head, body, and tail regions. The structure of neuromasts in these three regions is basically identical. The neuromast is composed of sensory, sustentacular, and mantle cells. The top of each neuromast has a hillocklike appearance, and is surrounded by four to six epidermal cells with tight intercellular junctions. Long kinocilia and many stereocilia occur in the apex of the neuromasts and are surrounded by numerous microvilli. Numerous granules are present on the apical portions of the mantle and the sustentacular cells. Four or five trapeziform mantle cells are connected closely with each other to form the shell of the neuromast. Large intercellular spaces occur between the mantle cells and the cells of the inner epidermal layers, and between the cells of the inner epidermal layer. Thus, at the apical parts of the neuromast intercellular junctions are tight and the intercellular spaces are more dilated in more basal areas. Morphologically the neuromasts of R. cancrivora larvae resemble those of generalized pond anurans, based on the grouping of Lannoo (Journal of Morphology 191:115-129, 1987a), although larvae of this species inhabit brackish water.

Notes: Orbital gland structure of the Atlantic bottlenose dolphin, Tursiops truncatus, was examined at the macroscopic, light microscopic, and electron microscopic levels. The gland completely encircles the ocular globe in a belt-like fashion near the conjunctival fornix but is considerably more developed medially. Duct openings are scattered throughout the fornix and over the surface of the palpebral conjunctiva. Microscopically, the gland has a tubuloalveolar arrangement; alveolar cells contain numerous secretory vesicles which can be interpreted as two structural types by light and electron microscopy. Histochemical staining demonstrates that both types contain glycosaminoglycans. Lipid analysis of the glandular secretion (dolphin tears) shows them to be non-oily and to contain only negligible amounts of cholesterol, triglycerides, phospholipids, and free fatty acids. The secretion is clear, slippery, and viscoelastic and well-adapted to protecting the eye and to reducing frictional forces between the eye surface and surrounding seawater.

Notes: The muscle fibers of the feline biceps femoris have tapered ends, across which tension is transmitted to the endomysium. The angle of taper of 11 ends, measured on scanning electron micrographs, varied between 0.16° and 1.18°. The muscle fibers are highly variable in cross-sectional shape. The shape of the fibers has been quantified as the ratio (form factor [FF]) of the measured perimeter to the calculated circumference of a circle having an area equal to that contained by the fiber perimeter. The FF for 173 terminal portions of fibers varied between 1.06 and 1.85 and was found to have a highly significant negative correlation with sarcomere length. The slope of the regression line suggests that the fibers maintain both volume and surface area as they change length. These studies suggest that isovolumic muscle fibers maintain a constant surface area by changing shape as they change length.

Notes: Salt glands of the domestic duck Anas platyrhynchos differ from those of the herring gull Larus argentatus and other birds. In ducks, each salt gland consists of distinct medial and lateral segments. Centrally located drainage ducts that extend along the entire length of these medial and lateral segments collect hypertonic fluid secreted by an array of lobules. Each lobule is formed by a single mass of branched tubules in which the direction of capillary blood flow is opposite to that of the secreted fluid. This fluid drains from the medial segment through an external duct that opens into the nasal cavity at the base of the vestibular fold. A duct from the lateral segment loops and opens onto the surface of the nasal septum. The structure and function of the secretory cells is reviewed briefly within the context of our study of the configuration of duck nasal salt glands.

Notes: A contrast radiographic study of pelvic fin drainage in rays reveals considerable differences in patterns of drainage among the species studied. The “typical” shark pattern of drainage, to the lateral abdominal vein, is also found in rays with shark-like morphology. However, variation in the connections of pelvic fin veins to muscular and cutaneous vessels of the pectoral fin occurs in the more “derived” batoid groups, with marked differences between rays of similar external morphology and mode of locomotion. There is a positive association between the pattern of fin drainage and the number of radial cartilages in the posterior (metapterygial) lobe of the pectoral fin. Variation in shark pelvic fin drainage may also be related to differences in pectoral fin morphology.

Notes: Dissections of Sudan black B stained specimens reveal that, of a complex of medial, intermediate, and lateral muscles of skates, presumed homologous to the cucullaris of sharks, only the lateral muscle is innervated by a branch or branches of the vagus and is inserted, in part, to the fused pharyngobranchials of the caudal visceral arches. The medial and intermediate muscles are supplied by separate branches of rostral spinal nerves and do not attach to the branchial skeleton. The lateral muscle therefore is the most likely homologue of the cucullaris (trapezius) of sharks and perhaps other fishes and tetrapods. The medial and intermediate muscles appear to be part of the axial musculature.

Notes: An immunocytological study of four different parts of the gut of Helix aspersa clearly demonstrates the presence of many cells and fibers immunoreactive toward antibodies directed to vertebrate (α, β-endorphin, α, β-MSH, ACTH 1-24 and ACTH 17-39, met-enkephalin, somatostatin, insulin, glucagon, P.P., serotonin) or invertebrate (FMRF-amide) peptides.These results are evidence of the presence of different substances related to known peptides or amines in the epithelial and connective tissue cells and nerve fibers of the snail gut. Immunocytochemistry may help to elucidate the morpho-functional characteristics of the enteroendocrine cells of H. aspersa.

Notes: A histochemical investigation of kidney and lower intestine of the European starling (Sturnus vulgaris) shows no carbonic anhydrase activity in proximal convoluted tubules, although activity is seen in similarly prepared sections of rat proximal tubules. Early distal tubule cells in the starling are stained throughout the cytoplasm and at the apical and highly infolded basolateral membranes. Late distal tubules lose apical activity and have reduced basolateral infolding, resulting in less intense staining. Darkly stained intercalated cells appear in the connecting tubules and cortical collecting ducts. Both of these segments also show intense basolateral staining. Medullary cones of the starling are highly organized, with central zones containing unstained thin descending limbs of loops of Henle, surrounded by both medullary collecting ducts with only scattered cells staining for enzyme, and by thick ascending limb segments. The latter contain many uniformly stained cells intermingled with occasional unstained cells. Scattered cells of the starling colonic villi demonstrate intense apical brush border membrane staining as well as cytoplasmic staining. Cells lining the cloaca stain less intensely. A biochemical assay for carbonic anhydrase was used to quantify enzyme activity in these tissues. Starling kidney contained 1.96 ± 0.33 (mean ± SEM) enzyme units/mg protein, less than half the activity seen in rat kidney. Stripped colonic epithelium contained 0.66 ± 0.15 enzyme units/mg protein. These quantitative results correlate well with the interpretations derived from the histochemical observations. The lack of proximal tubule carbonic anhydrase activity suggests that the avian kidney relies more on distal nephron segments to achieve net acidification of the urine.

Notes: The orientation of the fibers in the dermis of the tiger salamander, Ambystoma tigrinum, undergoes a dramatic repatterning at metamorphosis. The pre-metamorphic, larval dermis is a tight layer composed of crossed fibers that wind helically around the trunk. This condition is retained by neotenic adults which do not undergo metamorphosis. In contrast, the metamorphosed adult dermis consists of a superficial, loose network of fibers invested with large multicellular glands - -the stratum spongiosum - and a deeper tight layer of fibers - the stratum densum. However, unlike the crossed fibers of the pre-metamorphic dermis, there is no preferred orientation to the fibers in either layer of the post-metamorphic dermis.In order to evaluate whether these two distinctly different fiber patterns are constructed from biochemically similar fibers, the collagen types present in the pre- and post-metamorphic dermis were determined using SDS-polyacrylamide gel electrophoresis. Type I collagen is the predominant collagen of the dermis and the same major collagen types are present for all individuals, whether preor post-metamorphic. Thus, the major types of collagen that compose the dermal fibers do not change during metamorphic repatterning of the dermis.

Notes: In the first half of this century, several workers observed small, seemingly glandular structures attached to the ampullate glands of spiders. Hence, they were termed accessory ampullate glands. In juvenile Araneus cavaticus, two pairs of these structures are present (starting at least with third instars), one pair attached to the major ampullate (MaA) glands and the other pair attached to the minor ampullate (MiA) glands. In adults, two pairs of accessory MaA glands and two pairs of accessory MiA glands are present. The two latter-formed pairs of accessory ampullate glands are clearly the remnants of those ampullate glands which atrophy shortly after adulthood is reached. Morphological similarities between these accessory ampullate glands and those present in juveniles provide an indication that the latter also have their origin in functional ampullate glands.A reduction in the number of ampullate glands following the last molt occurs in many spiders. The reason(s) for these reductions is unknown. In penultimate spiders close to ecdysis, we have observed that while the larger pairs of MaA and MiA glands (those that are retained in the adult) are undergoing molt-related changes which apparently render them nonfunctional, their smaller counterparts are seemingly unaffected and functional. This raises the possibility that the principal role of the smaller ampullate glands may be to assume functions during the pre-ecdysial period which are normally in the domain of the larger ampullate glands. If true, then their degeneration after the last molt would make economic sense.The presence of cylindrical spigots in juvenile females starting with fourth instars is documented.

Notes: Three basic types of cells are distinguished in the rat vomeronasal epithelium at birth: bipolar neurons, supporting cells, and basal cells. Neurons at this time include both immature and differentiated cells. By the end of the first postnatal week, all neurons show morphological signs of maturity in their cytoplasm, including abundant granular and smooth endoplasmic reticulum, neurotubules, dense lamellar bodies, apical centrioles, and tufts of microvilli. During the third week microvilli are more frequently encountered and appear to be longer and more branched. Supporting cells appear well-developed by the second day after birth. During the first ten days of life, supporting cells lose their centrioles and all of the complex associated with ciliary generation in the apical zone. Basal cells appear to be more numerous in newborns than in older animals. Protrusions projecting into the lumen are frequently observed in the epithelium of newborn animals, both on the dendrites of neurons and on supporting cells. After the third week, such protrusions are only observed in the transitional zone between the sensory and the non-sensory epithelia of the vomeronasal tubes. In this transitional zone, a fourth cell type showing apical protrusions with microvilli differentiates. Cytoplasm in this type resembles that of neighboring ciliated cells but has no cilia or centrioles. These transitional cells are considered to be cells in an intermediate state of differentiation, between that of the differentiated neurons and supporting cells of the sensory epithelium and that of the predominate ciliated cells of the non-sensory epithelium. The results suggest that by the end of the third week the vomeronasal epithelium is morphologically mature.

Notes: The wall of the stomach of the tigerfish is described and compared with that of other vertebrates. Light microscopic and ultrastructural characteristics of the stomach wall correspond to a large extent to those of other vertebrates, although some differences are found. The mucosa contains (1) surface epithelium characterized by narrow columnar cells with abundant mucous granules; (2) gastric glands consisting of pepsinogenic cells of variable height, containing tubulovesicles and bearing microvilli; (3) five granulated cell types located basally in the epithelium (types 1-5); and (4) lamina propria and muscularis mucosae. Connective tissue separating smooth muscle fibers of the muscularis mucosae constitutes a stratum compactum. The submucosa contains a loose connective tissue, a tunica muscularis of inner circular and outer longitudinal layers, and a serosa of mesothelium and subjacent connective tissue. Immunocytochemical tests with antisera to five polypeptides show gastrin/cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP) immunoreactivities in some cells of the gastric glands, and somatostatin in cells lying among epithelial cells lining the gastric luminal surface or gastric pits.

Notes: Previous studies by Stephens and McNulty and Strecker and Stephens have demonstrated that foil barriers placed between the mesonephros and lateral plate at stages 12 to 15 inhibited limb development, but foil barriers placed between the neural tube and somites at stages 11 to 12 resulted in limbs with normal skeletal patterns. It was concluded that some influence present in the paraxial region of the embryo at stages 11 to 15 is necessary for normal limb development. The present study was undertaken to localize that influence more precisely. Foil barriers were placed in the lateral edge of the somites or segmental plate of stage 10 to 15 chick embryos. Barriers placed into stage 13 to 15 embryos resulted in chicks with normal limbs, but barriers placed into stage 10 to 11 embryos resulted in chicks with defective limbs. Barriers inserted just lateral to Hensen's node at stages 6 to 8 resulted in embryos with defective or absent wings. We also grafted stage 4 to 9 presumptive limb territories with and without Hensen's node. Explants without Hensen's node formed limb-like structures in 1% of the cases. Explants with Hensen's node formed limb-like structures in 27% of the cases. When barriers were implanted and a node was placed on the lateral side of the barrier, limbs formed in 40% of the cases. These data suggest a medial to lateral progression of some as yet unknown morphogenetic influence necessary for normal limb development and we hypothesized that the influence may initially emanate from Hensen's node.

Notes: The limb bone skeleton of the moa, a family of extinct ratite birds endemic to New Zealand, is described with particular reference to the anatomical and functional significance of osteological landmark form variation. The results generally support the existing classification of moa. Four genera, Megalapteryx, Anomalopteryx, Dinornis, and Pachyornis, were found to be evolved within moa, with Emeus and Euryapteryx possibly being more primitive. Megalapteryx was found to be less mobile than other moa genera. The Dinornis species were found to be more cursorial and more mobile relative to other moa. They may also have had a different center of gravity. A marked development of the lower leg in Anomalopteryx suggested a digging habit associated with food procurement.

Notes: A detailed description of muscular and skeletal features of the pelvi-cloacal region of the amphisbaenian Blanus cinereus reveals that the species has real hind limbs articulating with a real pelvic girdle. Arguments support this homology: 1) a link with the vertebral column; 2) a movable articulation, showing all the features of a diarthrosis, between the femur and the acetabulum; 3) all the long bone characteristics for the femur which distally bears a horny element. The morphological peculiarities of the amphisbaenian pelvic girdle are generally close to those of lizards, but the pubis seems to be more reduced. For the same number of precloacal vertebrae, the amphisbaenians have more appendicular elements than lizards have, and this composition recalls that of the Leptotyphlopidae, Aniliidae, and Boidae. The account provides more information concerning the aponeuro-tendinous system associated to the skeleton of the girdle and the hindlimb, the musculature, and the interrelations between the different structures of the pelvi-cloacal region.

Notes: Programmed cell death is an integral and ubiquitous phenomenon of development that is responsible for the reduction of wing size in female moths of Orgyia leucostigma (Lymantriidae). Throughout larval and pupal life, cells of the wing epithelium proliferate and interact to form normal imaginal discs and pupal wings in both sexes. But at the onset of adult development, most cells in female O. leucostigma wings degenerate over a brief, 2-day period. Lysosomes and autophagic vacuoles appear in cells of the wing epithelium shortly after it retracts from the pupal cuticle. Hemocytes actively participate in removing the resulting cellular debris. By contrast, epithelial cells in wings of developing adult males of O. leucostigma do not undergo massive cell death. Wing epithelium of female pupae transferred to male pupal hosts behaves autonomously in this foreign environment. By pupation, cells of the female wing apparently are committed to self-destruct even in a male pupal environment. Normal interactions among epithelial cells within the plane of a wing monolayer as well as between the upper and lower monolayers of the wing are disrupted in female O. leucostigma by massive cell degeneration. Despite this disruption, the remaining cells of the wing contribute to the formation of a diminutive, but reasonably proportioned, adult wing with scales and veins.

Notes: We describe the localization of lipids in the wall and superficial ooplasm of the largest avian ovarian follicles by the use of different fixatives and light and electron microscopy. We demonstrate that each yolk globule is always accompanied by one or more highly osmiophilic and sudanophilic alcohol insoluble yolk masses, which we have called satellite yolk. Together with the protein containing yolk globule it forms an integral morphological part of a compartmentalized, bipartite yolk system. Cytochemical, histoautoradiographic, biochemical, and light and electron microscopical aspects of satellite yolk were studied. At the start of satellite yolk formation in the 3-4 mm diameter follicle (when the oocyte begins to yellow) the distribution of the microcirculation of the follicle wall becomes printed on the underlying superficial ooplasm of the oocyte. The oocyte then presents so-called yolk mountains (containing satellite yolk), only localized below the thecal capillary sinus and not below the efferent and radially perforating thecal veins (black hole regions). We also describe the structural continuity between the thecal intercellular spaces and the microvilli-associated extracellular spaces of the granulosa cells via the basement membrane. The thecal cells present centripetal extensions into the basement membrane and the basement membrane material extends centripetally into the granulosa microvillar channels. Therefore, at least two cellular barriers are crossed when fat or fat precursors are transported from the thecal capillary sinus to the ooplasm.

Notes: Embryos of the poeciliid Heterandria formosa develop to term in the ovarian follicle in which they establish a placental association with the follicle wall (follicular placenta) and undergo a 3,900% increase in embryonic dry weight. This study does not confirm the belief that the embryonic component of the follicular placenta is formed only by the surfaces of the pericardial and yolk sacs; early in development the entire embryonic surface functions in absorption. The pericardial sac expands to form a hood-like structure that covers the head of the embryo and together with the yolk sac is extensively vascularized by a portal plexus derived from the vitelline circulation. The hood-like pericardial sac is considered to be a pericardial amnion-serosa. Scanning and transmission electron microscopy reveal that during the early and middle phases of development (Tavolga's stages 10-18 for Xiphophorus maculatus) the entire embryo is covered by a bilaminar epithelium whose apical surface is characterized by numerous, elongate microvilli and coated pits and vesicles. Electron-lucent vesicles in the apical cytoplasm appear to be endosomes while a heterogeneous group of dense-staining vesicles display many features characteristic of lysosomes. As in the larvae of other teleosts, cells resembling chloride cells are also present in the surface epithelium. Endothelial cells of the portal plexus lie directly beneath the surface epithelium of the pericardial and yolk sacs and possess numerous transcytotic vesicles. The microvillous surface epithelium becomes restricted to the pericardial and yolk sacs late in development when elsewhere on the embryo the non-absorptive epidermis differentiates. We postulate that before the definitive epidermis differentiates, the entire embryonic surface constitutes the embryonic component of the follicular placenta. The absorptive surface epithelium appears to be the principle embryonic adaptation for maternal-embryonic nutrient uptake in H. formosa, suggesting that a change in the normal differentiation of the surface epithelium was of primary importance to the acquisition of matrotrophy in this species. In other species of viviparous poeciliid fishes in which there is little or no transfer of maternal nutrients, the embryonic surface epithelium is of the non-absorptive type.

Notes: The flagellum of the thysanopteran spermatozoon has been examined by electron microscopy and computer-aided image analysis. The flagellum consists of 27 microtubular elements that probably are formed as outgrowths from three separate basal bodies. Nine of the elements are normal microtubular doublets that carry dynein arms and nine are doublets without dynein arms. The remaining nine elements are microtubular singlets that apparently bear dynein arms and have the same appearance as A-subtubules of microtubular doublets. The 27 elements are arranged in a fixed pattern that consists of nine groups, each of which begins with a microtubular singlet and ends with an arm-less microtubular doublet. Computer-aided image analysis has shown that the A-subtubules of the doublets and the microtubular singlets have lumens with very similar patterns. The sperm tail is known to have some motility; it generates fast waves running along its length. The amalgamated axonemes hence act as a functional flagellum. The thysanopteran sperm tail is the only type of flagellum known to us that consists of microtubules in a highly asymmetric array.

Notes: Many arachnids lack extensor muscles at the femoropatellar (knee) joint of their legs and extend this joint with hydraulic pressure during locomotion. Pressure is generated through compression of the prosoma, but there is disagreement about which muscles are involved in this process. Many arachhnologists consider contraction of the musculi laterales, a group of modified extrinsic leg muscles, as the cause of high prosomal pressure and regard hydraulic extension as a derived feature. However, integration of results from phylogenetic and comparative anatomical studies supports the view that hydraulic extension is primitive in Arachnida and that fluid pressure is generated by contraction of endosternal suspensor muscles.The functional predictions of the musculi laterales and endosternite hypotheses were tested by measuring muscle activity and prosomal pressure during unrestrained locomotion in a primitively “extensorless” arachnid, the giant whipscorpion. The results corroborate the endosternite model and refute the musculi laterales model. Changes in the prosomal pressure baseline were correlated with changes in endosternal muscle activity, while the musculi laterales fired in a step-coupled pattern of discrete bursts that appeared to be incapable of generating the pressure observed during locomotion. Step-coupled fluctuations in prosomal pressure were observed but were apparently caused by rapid flexing of the femoropatellar joints of the fourth leg pair rather than contraction of the musculi laterales.

Notes: In the fungiform papilla of Rana esculenta (Anura Ranidae), the Ca++-ATPase is mainly distributed on the basolateral membrane of the sensory area cells (i.e., neuroepithelial, supporting, and mucous cells). Apical membranes of all cells facing the surface present a slight enzymatic activity. Lateral wall cells have a strong Ca++-ATPase activity on basolateral and apical membranes. Strong Na+, K+-ATPase activity occurs on the apical surface of neuroepithelial cells. Ca++-ATPase activity is absent on the surface of endothelial cells of the capillaries located under the sensory area. These observations lead us to conclude that the sensory area of fungiform papilla is the selective way for calcium influx. Furthermore the absence of ATPase activity on the surface of the endothelial cells indicates that there is no functional barrier to calcium influx into capillary, and that calcium can be removed by vessels from the sensory area.

Notes: Spermatogenesis and sperm ultrastructure were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) in the longidorid Xiphinema theresiae. All germ cell stages, except spermatogonia, are present in the testes of young adult males. The nonflagellated, slightly elongated sperm displays little intraspecific variation and, although never polarized into a head and tail region, has a remarkably precise form, with a high degree of internal organization. Incipient fingerlike pseudopodia appear in the young spermatid and increase to such an extent that the adult sperm has a conspicuous “woolly” appearance. Microfilament bundles encircle the perinuclear mitochondria in the spermatid, and seem to be closely associated with the evaginated plasma membrane, especially in the spermatozoon. A large nucleus with nuclear envelope is prominent in the spermatocyte, but the envelope is absent in the young spermatid. Mitochondria are present in all germ cell stages and undergo certain morphological changes (e.g., in size and number, presence or absence of cristae), as well as changes in intracellular movements during spermatogenesis. Membranous organelles are prominent in the spermatocyte, but disappear in the older spermatid. Annulate lamellae and a residual body (i.e., cytophore) are conspicuous in the spermatocyte and spermatid, respectively; the spermatozoon clearly lacks a refringent body (i.e., acrosome).

Notes: The morphology and distribution of muscle spindles of jaw and tongue muscles in the mallard were examined in serial transverse sections of single muscles and in horizontal sections of a whole head. Our observations on spindle morphology are in agreement with previous descriptions of spindles in birds. Some spindles differ in their innervation and the pattern of intrafusal muscle fibers. The spindles of individual adductor and pterygoid muscles are distributed unevenly. Some adductor muscles lack spindles, whereas those of other muscles are confined to limited areas. Jaw opening muscles and extrinsic tongue muscles lack spindles. The stretch of extrafusal muscle fibers could be estimated from the difference in sarcomere length for birds with the beak open and closed. Not all muscle fiber groups are stretched evenly over the whole range of jaw opening. Only those fiber groups that are continuously stretched during jaw opening contain spindles.

Notes: Living embryos of three species of South American annual fishes, Cynolebias constanciae, C. nigripinnis, and C. whitei, were observed from fertilization through the 10-somite stage. A description of normal stages of development applicable to all three species of Cynolebias is presented. Cleavage (stages 1-10) is meroblastic and produces a typical teleost blastoderm. Following cleavage (stages 11-13) blastomeres segregate into two populations, viz., (1) a population of deep blastomeres that will disperse as single motile cells, and (2) a hemispherical shell of outer blastomeres that flattens to form an enveloping cell layer (EVL). When epiboly of the EVL and the yolk syncytial layer (YSL) commences (stage 14), deep blastomeres clump together as a consolidation mass and then migrate outward as single cells on the YSL. When epiboly is concluded (stage 19), deep blastomeres have completely dispersed. If diapause does not intervene, the dispersed phase lasts only a few days. Subsequently, the dispersed cells come together to form a definitive aggregate (stage 27). Embryogenesis within the reaggregated mass of previously dispersed cells produces a typical teleost embryo.Early development in Cynolebias resembles that of other South American annual fishes, such as Austrofundulus, in that a phase of deep blastomere dispersion and reaggregation spatially and temporally separates epiboly from embryogenesis. Several features of development markedly differ from Austrofundulus. There are far fewer (250 vs. 2,500) deep blastomeres. Deep cells of Cynolebias are flattened rhomboids with filipodial extensions in contrast to the amoeboid cells of Austrofundulus. Blastomeres of dispersion and reaggregation stages in Cynolebias send out numerous cell surface extensions onto the YSL and in contact with one another, and often line up in rows as do some African annual fishes, e.g., Nothobranchius. During Dispersion II (stage 21), Reaggregation I (stage 22), and Reaggregation II (stage 23), deep cells move in an oriented pattern with respective mean velocities of 3.48 ± 0.91, 1.28 ± 0.46, and 1.31 ± 0.31 μm/minute. Cells move toward a granular mass of unknown composition, located at the YSL-yolk interface in the lower hemisphere of the egg. This mass appears to coincide with the site of cell reaggregation.

Notes: Several surfperches (Embiotocidae), including the black surfperch, Embiotoca jacksoni, exhibit a specialized prey handling behavior known as winnowing, in which ingested food and non-nutritive debris are separated within the oropharyngeal cavity. Prey items are swallowed, and unpalatable material is ejected from the mouth. Winnowing is believed to play an important role in the partitioning of food resources among sympatric embiotocids. We present a mechanistic model for this separative prey processing based on high-speed video analysis, cineradiography, electromyography, and buccal and opercular cavity pressure transducer recording.Winnowing by embiotocids is characterized by premaxillary protrusions repeated cyclically with reduced oral gape. Protrusion is accompanied by depression of the hyoid apparatus and adduction of the opercula. Alternating expansion and contraction of the buccal and opercular cavities generate regular pressure waveforms that indicate bidirectional water flow during processing. Separation of food from debris by Embiotoca jacksoni occurs in three phases. The prey-debris bolus is transported anteriorly and posteriorly within the oropharyngeal cavity and is then sheared by the pharyngeal jaws. Mechanical processing is complemented by the rinsing action of water currents during hydraulic prey transport.The feeding apparatus of Embiotoca jacksoni is functionally versatile, although not obviously specialized relative to that of nonwinnowing surfperches. Protrusion of the premaxillae and depression of the hyoid apparatus are critical to both prey capture and subsequent prey processing. The pharyngeal jaws exhibit kinematic patterns during separation of food from debris distinct from those observed during mastication of uncontaminated prey. This behavioral flexibility facilitates resource partitioning and the coexistence of E. jacksoni in sympatric embiotocid assemblages.

Notes: Quantifying shape is a broad problem in the morphological sciences. Most techniques for numerically describing shape abstract the shape into the most logical ideal Euclidean dimension. The fractional, or fractal, dimension is a simple computation that expresses shape in real, rather than ideal, space. The structured walk technique developed for the fractal analysis of rugged boundaries is applied here to the contour of the human sagittal suture in order to discriminate the separate morphological patterns of interfingering and interlocking. These attributes contribute differentially to the suture's “complexity,” a concept often used in biomechanical hypotheses. Previous techniques for estimating sutural complexity do not isolate small-scale from large-scale morphological patterns. Results indicate that despite the visual appearance of great variation, human sagittal sutures are remarkably consistent in the degree of complexity expressed separately by large-scale interfingering lateral excursions and small-scale interlocking ruggedness. There is no significant correlation between the absolute or bregma-lambda chord length of the human sagittal suture and its degree of complexity as determined by the structured walk technique.

Notes: Spermatophores in a squid, Todarodes pacificus, were observed by light and electron microscopy and were further analyzed by X-ray microanalysis (XMA) of frozen thin sections. Each spermatophore consists of a sperm mass, a cement body, an ejaculatory apparatus, and some fluid materials, all of which are covered by an outer tunic. The outer tunic consists of about 20 membranous layers, each containing straight, parallel microgrooves. Each layer's microgroove pattern is roughly in an orthogonal arrangement with respect to the next layer's pattern. The sperm mass, which is the only cellular component, consists of a sperm rope which is coiled more than 500 times. Most of the spermatozoa in the rope are arranged regularly and are enveloped in materials which are well-stained by Alcian blue. The cement body is located between the sperm mass and ejaculatory apparatus and has a hard outer shell with an arrowhead-like structure, presumably for penetration into the tissue of the female. Calcium and phosphorus are present in the shell of the cement body, which also has an affinity for alizarin red. The ejaculatory apparatus consists of two tubes, designated as the inner tunic and the inner membrane.After the spermatophoric reaction, a sperm reservoir is formed at the anterior end of the extruded and inverted ejaculatory apparatus. The sperm reservoir, which encases the sperm mass, is composed of the cement body at the anterior end and the inner tunic of the ejaculatory apparatus at the posterior end.

Notes: Spermiogenesis in the South American leptodactylid frog Odontophrynus cultripes was analyzed ultrastructurally. The spermatids undergo morphological modification while still enclosed in microtubule-rich processes of Sertoli cells. Electron-dense plates resembling junctional structures appear in regions at which the spermatids lie in close contact with the surface of Sertoli cell processes. Spermatid differentiation can be divided into five distinct stages based mainly on chromatin condensation. In the late stages, the densely compacted chromatin loses reactivity to ethanolic phosphotungstic acid (E-PTA). Helical arrangements of microtubules appear in the cytoplasm that surrounds the spermatid nucleus after the second stage. The acrosomal vesicle differentiates into a cone-shaped acrosome that caps the anterior region of the nucleus. The connecting piece, located in the flagellum implantation zone, has transverse striations, and is continuous with the axial rod. The tail is formed by a 9 + 2 axoneme, an undulating membrane, and an axial rod that is rich in basic proteins as demonstrated by E-PTA staining.

Notes: Morphology of the chromaffin cells of Triturus cristatus during a complete annual cycle has been investigated. General ultrastructural characteristics are similar for all chromaffin cells, including numerous small mitochondria, well-developed Golgi apparatus and rough endoplasmic reticulum with short cisternae. The primary difference among cells is the type of the chromaffin granules they posses. These are of two kinds: adrenalin (A) and noradrenalin granules (NA). Both types are simultaneously present in the chromaffin cells but with different ratios during the year. During December-January and May-August, NA granules largely prevail, while in September-November and February-April, A and NA granules are present in about equal quantities. The total quantity of catecholamine granules, however, is relatively constant throughout the year. These findings suggest that T. cristatus has a single type of chromaffin cell, the granule content of which varies according to different functional states. The catecholamines are apparently discharged by exocytosis.

Notes: The palatoquadrate and associated dermal bones have significant evolutionary transformations among teleostomes and provide numerous features that characterize teleostomian subgroups. The palatoquadrate forms the upper part of the mandibular arch and is present as a single cartilaginous element in the early ontogeny of teleostomes, except for some advanced teleosts such as siluroids where it is divided into pars autopalatina and pars pterygoquadrata. During ontogeny, the palatoquadrate may ossify as a unit, with a pars autopalatina (absent in Acanthodii), pars quadrata, and pars metapterygoidea in teleostomes (e.g., primitive acanthodians and actinopterygians, onychodonts, and rhipidistians). However, the palatoquadrate may remain cartilaginous (e.g., chondrosteans) or it may ossify as separate elements (e.g., autopalatine, metapterygoid, and quadrate) as occurs in advanced acanthodians, Polypterus and advanced actinopterygians, and advanced actinistians. From the single-unit pattern, separate autopalatine, metapterygoid, and quadrate evolve in parallel in the three teleostomian subgroups. Therefore, it is necessary to distinguish between actinopterygian and actinistian autopalatines and among acanthodian, actinopterygian, and actinistian metapterygoids and quadrates. A palatoquadrate fused with the neurocranium occurs in parallel in dipnoans.There are differences in the timing of ossification of the autopalatine, metapterygoid, and quadrate. The autopalatine ossifies late in ontogeny in Polypterus, Amia, and primitive teleosts (absent in lepisosteids and osteoglossmorphs), whereas both metapterygoid and quadrate ossify early in ontogeny. The early ossification of the autopalatine is characteristic of clupeocephalan teleosts. During ontogeny, tooth plates (not forming a separate dermometapterygoid) fuse with the metapterygoid in actinopterygians.Pars autopalatina, pars metapterygoidea, and pars quadrata are regions at the three corners of the single-unit palatoquadrate present in primitive teleostomes; there are no clear limits among these regions, but they may be identified by their processes, articular facets, and topographical relationships with surrounding bones and the orbit. Autopalatine, metapterygoid, and quadrate are chondral bones, perichondrally ossified. Dermal elements such as dermopalatine(s), entopterygoid, ectopterygoid, and tooth plates may cover the palatoquadrate medially. The predermopalatine that originates in front of pars autopalatina in Cladistia and the “dermopalatine” that lies medial to the ectopterygoid in Ginglymodi are specializations of these groups. A dermopalatine fused with the autopalatine is characteristic of clupeocephalan teleosts. Highly specialized tendon bone pterygoids are found in some teleosts (e.g., siluroids). The presence of both maxilla and lacrimal lateral to the pars autopalatina is synapomorphous of osteichthyans. The eye supported by the bony palatoquadrate is a teleostomian synapomorphy. Dermal elements support the eye in actinopterygians, the entopterygoid in advanced actinopterygians, but the ectopterygoid in lepisosteids.A quadratojugal is a synapomorphy of osteichthyans but exhibits a number of transformations in connection with the vertical pit-line and the preopercular canal; a quadratojugal bearing the vertical pit-line is the primitive condition for osteichthyans. Ontogenetic evidence does not support the homology of the membranous posterior process of the teleostean quadrate with the quadratojugal. The lack of a quadratojugal and the presence of the elongate posterior or posteroventral process of the quadrate is a synapomorphy of teleosts.

Notes: The optical purity of scopolamine derived from Datura sanguinea was determined using coupled column chromatography. A C18 column was used to separate scopolamine from the additional alkaloids and other biological material present in the vegetal extract. The C18 column was coupled through a six-port switching valve to two β-cyclodextrin columns in series which were used to resolve the scopolamine enantiomers. A single acetylated β-cyclodextrin column gives equivalent results to the native cyclodextrin columns because of slightly higher enantioselectivity for scopolamine. A multistep extraction procedure is used to isolate scopolamine from the vegetal material. 4-6% of the scopolamine in the final extract was found to be the d enantiomer. Sample extracts as well as commercial scopolamine hydrobromide were treated under various conditions commonly encountered during typical commercial extraction procedures and analyzed in order to determine if the d enantiomer was present in the original material or if it was produced during the extraction process and, if so, determine which step and conditions contribute to acemization. Both the salt and the extract were found to be susceptible to racemization under basic conditions (≥pH 9) although the extract appeared to be more susceptible than the salt. Tropic acid formed from the hydrolysis of scopolamine seemed to be completely racemized even though the remaining scopolamine was only partially racemized. Within experimental error, no d enantiomer was found in the original fresh plant material.

Notes: Racemic phenylpropanolamine was resolved on a high performance liquid chromatographic (HPLC) chiral stationary phase (CSP) as the 3,5-dinitrophenyl ureide derivative. The CSP was prepared by a simple in situ procedure in which (R)-(1-naphthyl)ethyl isocyanate was bound to aminopropyl silanized silica through a urea linkage. The enantiomeric ureides were prepared by a room-temperature, 60-second procedure, accomplishing simultaneous extraction and derivatization and utilizing achiral 3,5-dinitrophenyl isocyanate as reagent. Baseline resolution was readily achieved under normal phase conditions, with a separation factor (α) of 1.16 and a resolution factor (Rs) of 2.2. Elution was complete within 10 min. A limit of detection, by UV at 235 nm, of 250 pg per isomer was established. Feasibility of the procedure for plasma determinations was demonstrated by assay of samples from a canine subject.

Notes: Peroxyoxalate chemiluminescence may be used for sensitive postcolumn detection of phenothiazine analytes separated by high performance liquid chromatography with appropriate optimization of measurement conditions such as solvent, pH and oxalate ester. Detectability of fluorescent analytes by chemical excitation varies greatly, but analytes with low oxidation potentials are generally more readily detected at low levels, as demonstrated for phenothiazines, an important class of fluorescent drugs. Some improvement in detection limits is observed for fluphenazine when chemiluminescence detection is compared to conventional fluorescence detection. Because of the specificity of chemical excitation, fewer interferences from fluorescent impurities in a urine matrix are observed.

Notes: Sensitive and specific methods for the simultaneous determination of gemfibrozil (Lopid®), a lipid-lowering agent, and its metabolites in plasma and urine are described. The methods are based on a fully automated high performance liquid chromatographic (HPLC) system with fluorescence detection. Urine samples, diluted with acetonitrile, were directly analysed by HPLC using a flow and eluent programming method. In the case of plasma, gemfibrozil and its main metabolites were extracted from acidified samples and the resulting extracts injected into the chromatographic system. The sensitivity was approximately 100 ng/mL for gemfibrozil and its four metabolites using 0.5 mL plasma or urine. An acyl glucuronide of gemfibrozil excreted in human urine after oral administration of the drug was isolated and its structure and stability examined.

Notes: A simple and sensitive high performance liquid chromatographic method for the analysis of tocopherols (α, β, γ and δ) in serum with UV detection is described. In normal phase mode excellent separation of positional isomers (β and γ) was achieved by adjusting the proportion of isopropanol in the mobile phase. Detection limits for both β and γ were 0.62 mg/L and 0.42 and 1.2 mg/L for α and δ isomers, respectively. Measurement of all the tocopoherol structural isomers (α, β, γ and δ) in a sample required only 400 μL of serum and each run was completed in less than 22 min.

Notes: A high performance liquid chromatographic (HPLC) technique for the determination of three metabolites of theophylline, 3-methylxanthine (3-MX), 1-methylxanthine (1-MX) and 1,3-dimethyluric acid (1,3-DMU) in human liver microsomes is described. The analytes were extracted from human liver microsomes with methylene chloride/isopropanol and stepwise gradient elution was employed for the resolution of peaks. The limits of quantitation were 15 ng/mL for 3-MX, 20 ng/mL for 1-MX and 20 ng/mL for 1,3-DMU. The calibration range was linear for the three metabolites and the calibration ranges were 15-250 ng/mL for 3-MX, 20-250 ng/mL for 1-MX and 250-4000 ng/mL for 1,3-DMU. The absolute recovery ranged from 63-84% for 3-MX, 65-79% for 1-MX and 77-89% for 1,3-DMU over the calibration curve range. Accuracy for all three metabolites was within ± 10% and adequate seletivity was demonstrated by the lack of interfering peaks in blank chromatograms. The within-run and interday precision were within 10% RSD for all three metabolites tested at two concentrations. The advantage of this method over previous methods is that the use of quaternary ammonium ion pair reagents in the mobile phase has been obviated. Also, unlike a previous radiometric HPLC method, the need for radiolabelled theophylline has also been eliminated. The method was used to characterize theophylline metasbolism in human liver microsomes for immunoinhibition studies and to investigate the interaction of theophylline with selected quinolone antibiotitics.

Notes: A new porphyrin, peroxyacetic acid uroporphyrin I, has been isolated from the urine of patients with congenital erythropoietic porphyria by reversed phase high performance liquid chromatography. The porphyrin was characterized by high resolution mass spectrometry and by typical chemical reactions of a peroxyacid.

Notes: A direct, isocratic, sensitive and precise liquid chromatographic method is presented for the enantiomeric separation of aminoglutethimide (AG) and its acetylated metabolite (AcAG) using cellulose tris-3,5-dimethyl phenyl carbamate (Chiralcel OD) and cellulose tris(4-methylphenyl benzoate) ester (Chiralcel OJ) columns in series. The enantiomeric elution order is determined by separate chromatography of the racemate AG and racemate AcAG and of their separate enantiomers under similar conditions. This method has been used to determine and identify the enantiomers of AG and AcAG in the urine sample collected from a metastatic breast cancer patient after administration of AG for 24 h. Large amounts of (+)-R-AG are excreted unchanged in the urine together with smaller quantities of (+)-R-AcAG, while most of the (-)-S-AG is metabolically converted into (-)-S-AcAG.

Notes: A high performance liquid chromatographic (HPLC) system is described for determination of the unsaturated disaccharide (ΔDi-HA) derived from hyaluronic acid (HA) in human urine by digestion with hyaluronidase SD. The effects of eluents on the separation of ΔDi-HA and ΔDi-OS, which is derived from the reaction of chondroitin with the enzyme, have been studied. The established chromatographic conditions were as follows-column: a stainless steel tube (4 mm i.d. × 250 mm) packed with TSKgel NH2-60; eluent: a mixture of acetonitrile and 0.1 M Tris-HCI buffer containing 0.1 M boric acid and 10 mM sodium sulphate, pH 7.0 (64:36, v/v). The strong fluorescence of unsaturated disaccharide after the reaction with 2-cyanoacetamide in alkaline medium was used for post-column detection. The calibration curve for ΔDi-HA was linear in the range 5 pmol-5 nmol with a practical detection limit of 2 pmol. The assay coefficients of variation (n = 5) at 200 pmol for ΔDi-HA and ΔDi-OS were 1.7 and 1.5%, respectively. This HPLC system has been applied to the determination of HA in human urine.

Notes: A rapid, reproducible and sensitive high performance liquid chromatography (HPLC) method for the determination and purification of metallothionein-I (MT-I) and metallothionein-II (MT-II) in mouse and rabbit livers has been developed. Methallothioneins (MTs) were separated by an HPLC anion exchange column, eluted through a linear gradient of Tris buffer and the peak containing MTs was determined by atomic absorption spectrophotometry. Furthermore, the content of MT-I or MT-II was calculated by protein peak area in a short time (about 20 min). The sample to be tested was homogenized, centrifuged and saturated by cadmium. MT-I and MT-II were eluted at 15.9 and 19.3 min, respectively. The following mouse liver cytosols were tested: controls, Cd-injected samples and 60Co-irradiated samples. A detection limit of 5 μg/g liver was established for this method. We have analysed more than 100 biological samples and obtained satisfactory results.

Notes: A high performance liquid chromatography (HPLC) method was developed for the determination of seven short-chain fatty acids in equine caecal liquor. Samples were cleaned up on a Sep-pak (C18) cartridge, and the analyte was eluted from the extraction cartridge and filtered through a 0.45 μm cellulose nitrate filter. The analyte was chromatographed by ion exchange HPLC. Detection was by UV at 210 nm. Recovery from phosphate buffer (0.05 M, pH 7.0) and equine caecal liquor was 76.95% (lactic), 76.76% (acetic), 84.40% (propionic), 89.35% (isobutyric), 88.73% (butyric), 80.33% (isovaleric) and 72.61% (valeric). The limit of detection of the short-chain fatty acids in phosphate buffer was 0.00006 M (lactic), 0.0001 M (acetic), 0.0002 M (propionic), 0.0001 M (isobutyric), 0.0002 M (butyric), 0.0002 M (isovaleric) and 0.0003 M (valeric). The specificity and sensitivity of this method was sufficiently high to allow the characterization of the pattern of these short-chain fatty acids in equine caecal liquor following intravenous administration of oxytetracycline at the recommended dose rate in a pony.

Notes: A gas - liquid chromatographic method for the determination of pentoxifylline and its secondary alcohol metabolite in serum has been developed. The method is based on the combination of solid phase extraction, capillary column separation and nitrogen - phosphorus detection of the analytes. Optimization of the solid phase extraction conditions permitted a low concentration determination, with limits of determination of 2 n/mL and 10 n/mL for pentoxifylline and its metabolite, respectively. The simplicity and rapidity of the extraction step was preserved.

Notes: REMEDi (Rapid EMErgency Drug identification; Bio-Rad) is an automated high performance liquid chromatographic (HPLC) system designed to detect, identify and measure a range of basic and neutral drugs in 0.5-1.0 mL of urine or plasm/serum. We have evaluated REMEDi in the analysis of the antiarrhythmic drug disopyramide in patient samples. The specimens were also analysed by a conventional HPLC method, based on solvent extraction and UV detection (254 nm), and by EMIT. There were good correlations between the results obtained with each method (r = 0.91 or greater). REMEDi gave a lower mean result than EMIT [means±SD (m/L): REMEDi 2.64±1.10, EMIT 3.14±1.51; t = 4.0, p〈0.01; n = 25], but there were no other significant differences in mean results. The principal disopyramide metabolite, mono-N-desalkyldisopyramide, did not interfere in any method. Clearly REMEDi can be used for therapeutic drug monitoring of disopyramide provided enough sample is available.

Notes: In this study we report the levels of 3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, homovallinic acid, tryptophan, 5-hydroxyindole-3-acetic acid and serotonin in lumbar cerebrospinal fluid (CSF) from patients with multiple sclerosis, cerebrovascular disease and muscular tension headache the latter, as healthy controls. The separation of these substances was performed on a reversed phase column by ion pair high performance liquid chromatography and detection was made by a glassy carbon electrode set at +900 mV vs Ag+/AgCI. The whole separation was achieved within 25 min. Concentrations of all substances (10-1000 pmole/L) were linearly proportional to areas obtained. The system is sensitive, stable and reproducible. The significance of CSF levels of these metabolites from patient groups compared with healthy controls are discussed.

Notes: Samples of biomedical interest which have been analysed by field-flow fractionation techniques are surveyed. The list begins with whole cells and microorganisms, going through viruses, nucleic acids, cell fragments and organelles, down to proteins and their aggregates. The principles of separation in the normal and steric mode of retention are illustrated, and instrumentation and techniques are described. The review concentrates mainly on the two systems of choice for biomedical applications: sedimentation and flow field-flow fractionation.

Notes: Rickettsiae-like structures were found in the salivary gland cells of Drosophila auraria during different larval and prepupal developmental stages, from the early 3rd instar up to 14 hr after spiracle inversion. These microorganisms are surrounded by a membrane, are constantly intracellular, and occur singly or in groups. Their widespread occurrence in various tissues of other Drosophila species indicates that they can be considered as symbionts, but their actual functional significance (if any) is unknown.

Notes: Histology of each of the five segments of the oviduct of the female turtle Chrysemys picta was described for successive intervals throughout their annual cycle. Uterine and glandular segments showed marked seasonal variations in the extent and content of the submucosal and epithelial glands. Submucosal glands were most prominent in preovulatory and postovulatory animals (May to June), regressing in late summer (oviposited animals) and recrudescing the following spring. These changes correlated with variations in the muscularis layer, the number of uterine epithelial blebs, oviductal vascularity, and the presence of eosinophils in cervical segment cross-sections. These cyclic seasonal changes are discussed in relationship to reported seasonal changes in gonadal steroids in this species. Hormonal control was corroborated by oviductal response to estradiol-17β injected (1 mg/kg daily for 2 weeks) into mature, reproductively inactive (winter) animals. This treatment induced increases in glandular activity, vascularity, and distribution of eosinophils comparable to those of reproductively active (summer) animals.

Notes: Molluscan shells, including those of Gastropoda, are formed by accretionary growth at the mantle edge. The mantle is a thin membrane of skirt-like shape, which extends minutely beyond the aperture, and its edge adds a shell increment to the aperture margin so that each increment copies a configuration of the mantle edge at that time. Thus, regulation of shell morphogeny is almost equivalent to the factors which control the mantle form at the moment of shell growth. Form of the mantle skirt is considered to be kept in a state of balance between the force of its internal stress and forces acting on it such as fluid pressure or muscle contraction.The expansion behavior of the mantle skirt has been numerically analyzed by using an elastic model (DMS-tube), which represents the fundamental structure of the mantle tissue as a double membrane structure with internal springs (DMS). Four characteristic expansion patterns of the DMS-tube have been detected: (1) general outward expansion; (2) developing a ridge-like fold on an initial longitudinal protrusion of the tube edge; (3) drastic shift of the expanded state from a uniformly curved to an elliptical shape in outline, owing to the existence of a fixed boundary condition on the tube wall; and (4) constricted protrusion on the open region of the shell wall surrounding the DMS-tube. These results have the potential for answering the following questions relating to the morphogenesis of gastropod shells. How does the mantle skirt usually make contact with the inner surface of the shell wall so as to ensure continuous accretion of shell materials to the aperture margin? What is the cause of spiral ridges? Why do open coiling or minimally overlapping shells have generally circular apertures, while shells with apertures overlapped by whorls have non-uniformly curved apertural lips? What is the cause of long closed spines and why do they always appear on spiral ridges?

Notes: The larval antenna of Bombyx mori has 13 sensilla and about 52 sensory neurons in its distal portion. The axons form two nerve cords which unite in the cranial hemocoel to supply the brain as the olfactory nerve. The antennal imaginal disc, which is a thick pseudostratified epithelium continuous with the antennal epidermis, thickens markedly during the 5th instar by rapid cell proliferation. At the prepupal stage cell proliferation ceases and the disc everts to form a large pupal antenna. Simultaneously, an extensive cell rearrangement occurs in the antennal epidermis and the disc tissue becomes much thinner because of the abrupt expansion of antennal surface area. The two larval nerve cords thin down markedly by degeneration of axons, but they do not disintegrate totally even after the onset of pupation. The epidermis of the larval antenna forms the distal portion of the pupal antenna, while the imaginal disc forms the more basal portion. Development to the adult antenna occurs almost immediately after the onset of pupation; many adult neurons appear in the simple epidermis facing toward the thick outer side of the newly formed pupal cuticle. By 12 hours after the onset of pupation, these neurons align themselves in many transverse rows which are the first sign of the adult antennal configuration. Addition of these neuronal axons to the once-thinned nerve cords causes resumed thickening of the cords during the first 24 hours and thereafter. Differentiation of adult sensilla begins in the next 24 hours and is almost completed at the third day of pupation, which requires a total of 10 days.

Notes: Sensilla diversity and abundance were extremely high on the apex of the maxillary and labial palpi of two species of Gryllacrididae. The terminal segment of the maxillary palpi of these species had 9 and 15 sensilla types, respectively, and up to 2,834 sensilla. The labial palpi had 7 and 12 types, respectively, and up to 5,195 sensilla. Several types of multiporous smooth and ridged olfactory basiconic sensilla, and coeloconic, coelosphaeric, placoid, and multipapilliform sensilla occurred, as well as many trichoid sensilla and the more typical uniporous basiconic contact receptors. Two species of the closely related Stenopelmatidae were compared to the gryllacridids and found to have similar sensillar diversity and abundance, but three species of the more distantly related Tettigoniidae had only 4 or 5 sensilla types and a total number ranging from 320 to 960 on their maxillary palpi.

Notes: The results of volumetric measurements led Hanström ('28) to suggest that specialized neuropil in the brain, the so-called central body (CB), may be of particular importance in the nervous control of web building behavior in spiders. We compared the volumes of the various brain regions in four spider species clearly differing in lifestyle and web building behavior: an orb weaver (Nephila clavipes), a wandering spider (Cupiennius salei), a jumping spider (Phidippus regius), and a bird spider (Ephebopus sp.). Our results obtained in adult animals as well as our observations on the postembryonic development of the brain do not support Hanström's hypothesis. The relative share of the CB in the brain is very similar in all four species (ca. 3.1 to 5.1%). The differentiation of the CB into a clearly demarcated two-lobed structure does not coincide with the onset of web building behavior in Nephila. The CB of both Nephila and Cupiennius is already clearly recognizable in spiderlings which have not even left the egg sac.

Notes: The venom gland of Crotalus viridis oreganus is composed of two discrete secretory regions: a small anterior portion, the accessory gland, and a much larger main gland. These two glands are joined by a short primary duct consisting of simple columnar secretory cells and basal horizontal cells. The main gland has at least four morphologically distinct cell types: secretory cells, the dominant cell of the gland, mitochondria-rich cells, horizontal cells, and “dark” cells. Scanning electron microscopy shows that the mitochondria-rich cells are recessed into pits of varying depth; these cells do not secrete. Horizontal cells may serve as secretory stem cells, and “dark” cells may be myoepithelial cells. The accessory gland contains at least six distinct cell types: mucosecretory cells with large mucous granules, mitochondria-rich cells with apical vesicles, mitochondria-rich cells with electron-dense secretory granules, mitochondria-rich cells with numerous cilia, horizontal cells, and “dark” cells. Mitochondria-rich cells with apical vesicles or cilia cover much of the apical surface of mucosecretory cells and these three cell types are found in the anterior distal tubules of the accessory gland. The posterior regions of the accessory gland lack mucosecretory cells and do not appear to secrete. Ciliated cells have not been noted previously in snake venom glands.Release of secretory products (venom) into the lumen of the main gland is by exocytosis of granules and by release of intact membrane-bound vesicles. Following venom extraction, main gland secretory and mitochondria-rich cells increase in height, and protein synthesis (as suggested by rough endoplasmic reticulum proliferation) increases dramatically. No new cell types or alterations in morphology were noted among glands taken from either adult or juvenile snakes, even though the venom of each is quite distinct. In general, the glands of C. v. oreganus share structural similarities with those of crotalids and viperids previously described.

Notes: The organization of identified neurosecretory cell groups in the larval brain of the tobacco hornworm, Manduca sexta, was investigated immunocytologically. Computer-assisted three-dimensional reconstruction was used to examine the architecture of the neurosecretory cell groups. The group III lateral neurosecretory cells (L-NSC III) which produce the prothoracicotropic hormone are located dorsolaterally in the protocerebrum and extend axons medially that decussate to the contralateral lobe prior to exiting the brain through the nervi corporis cardiaci I + II. The group IIa2 medial neurosecretory cells (M-NSC IIa2) are located anteriorly in the medial dorsal protocerebrum. The axons of these cells also exit the brain via the contralateral nervi corporis cardiaci I + II. However, their axons traverse a different pathway through the brain from that of the L-NSC III axons. Each of the cell groups possesses elaborate dendrites with terminal varicosities. The dendrites can be classified into specific fields based upon their location and projection pattern within the brain. The dendrites for these two neurosecretory cell groups overlap in specific regions of the protocerebral neuropil. After the axons of these neurosecretory cells exit the brain through the retrocerebral nerve, they innervate the corpus allatum where they arborize to form neurohemal terminals in strikingly different patterns. The L-NSC III penetrate throughout the glandular structure and the M-NSC IIa2 terminals are restricted to the external sheath. A third group of cerebral neurosecretory cells, the ventromedial neurons (VM) which stain with the monoclonal antibody to prothoracicotropic hormone in Manduca, are located anteriorly in the medial region of the brain. The axons of these cells do not exit the brain to the retrocerebral complex, but rather pass through the circumesophageal connectives and ventral nerve cord. These neurons appear to be the same VM neurons that produce eclosion hormone. One dendritic field of the L-NSC III terminates in close apposition to the VM neurons. The distinct morphologies of these neurosecretory cell groups in relation to other cell groups and the distribution of neuropeptides within the neurons suggest that insect neurosecretory cells, like their vertebrate counterparts, may have multiple regulatory roles.

Notes: The micropylar apparatus (MA) in Rhagoletis cerasi (Diptera, Tephritidae) is located at the anterior pole of the egg and consists of two parts: an outer chorion and an inner vitelline membrane. Sperm entry takes place through the micropylar canal, 2.0-2.5 μm in diameter, which penetrates the micropylar endochorion and terminates in the thick vitelline membrane, thus forming the “pocket.” The pore of the micropylar canal, i.e., the micropyle, is covered by the exochorionic tuft.The formation of the MA is accomplished by 40 micropylar cells during oogenesis. These cells secrete the successive eggshell layers: the vitelline membrane, the wax layer, the innermost chorionic layer, the micropylar endochorion, and the exochorion. Two among 40 micropylar cells differentiate and form two tightly connected projections. The latter contain a bundle of parallel microtubules and participate in the formation of the micropylar canal and the pocket. At the tip of the projections there are two thin extensions full of microfilaments. In late developmental stages the two projections and the extensions degenerate and leave the canal and the pocket behind. We also discuss the structural features of the MA in relation to its physiology among Diptera.

Notes: It is uncommon to find acid phosphatase activity in mature secretory granules. This paper demonstrates by light and electron microscope cytochemistry an acid phosphatase in mature secretory granules in the cells of one region of the salivary gland of Bradysia hygida (Diptera, Sciaridae). These secretory granules increase in number during larval development up to the beginning of the pre-pupal period when they undergo massive exocytosis. Biochemical assays show that upon exocytosis of the majority of the granules the total acid phosphatase activity in the granular gland region drops to 10% of the maximum reached before exocytosis. During and after exocytosis, two other acid phosphatases, eletrophoretically different and much weaker in activity, become increasingly detectable in all gland regions. At the same time, in whole mount preparations, numerous tiny acid phosphatase-positive granules (probably secondary lysosomes) become evident in all major cell types of the salivary gland. These results indicate that the S2 region of the salivary gland has mature secretory granules containing an acid phosphatase destined for exocytosis which is different in molecular properties from other acid phosphatases (likely lysosomal) made by the gland.

Notes: The general histology and ultrastructure of the tongue and anterior process of the sublingual plica of four Taiwanese venomous snakes, the Chinese cobra (Naja naja atra), banded krait (Bungarus multicinctus), Taiwan habu (Trimeresurus mucrosquamatus), and bamboo snake (Trimeresurus stejnegeri stejnegeri) are described.The tongue fork exhibits a mid-dorsal invagination that broadens gradually toward its base. No mid-ventral invagination is observed. The epithelial cells on both dorsal and ventral aspects of the tongue fork have large and small microfacets, micropores and microvilli. The cell size, distribution pattern of the large microfacets, and the number of small microfacets present on both sides of the fork are essentially the same within a species, but vary among species. The function of these ultrastructures on the cell surface might be for the capture of chemical substances. The large microfacets are raised areas of the cell membrane, each with a pale granule contained within. The chemical nature of the pale granule is not yet known. The small pores surrounding the large microfacets are shallow hollows left after the release of the pale granules from the microfacets. The basic histological pattern of the tongue fork of these species is similar, being composed of a mucosal layer outside and dense musculature inside. No taste buds are discernible.The anterior processes are concave-like expansions of the anteriormost portions of the sublingual plicae. The oblique folds and micropapillae of this organ might be helpful for receiving the chemicals collected on the tongue, when the tongue makes contact with the elevated processes. The elevated processes may penetrate the ducts of Jacobson's organs to effect the final transfer.

Notes: The scaleless notothenioid Gymnodraco acuticeps is a bottom dweller beneath the sea ice of McMurdo Sound, Antarctica. Gymnodraco experience unusual environmental conditions, including highly oxygenated subzero water. Skin morphology is evaluated with reference to its potential as a barrier to ice propagation and as a surface for cutaneous respiration. Light and electron microscopy and histochemistry reveal skin structure that is generally similar to that of other teleosts. In the epidermis, epithelial cells are arranged in nine to fifteen layers, and two types of mucous cells are also present. Large mucous cells are most common on external epidermal surfaces, whereas small cells are more frequent on internal epithelial surfaces. Epithelial cell junctions have extensive areas of desmosomes as well as interdigitations of the cell membranes, especially in the basal and midepidermis. The dermis consists of an exceptionally dense stratum compactum. The skin is thicker than that of Bovichtus, a scaleless temperate notothenioid from New Zealand. Mean skin thicknesses at sites on the trunk are 371-711 μm. With the exception of fins that contact the substrate, epidermal thickness between rays of most fins is 70-118 μm. The epithelial surfaces of the oral and branchial cavities are 27-50 μm thick. An unusual type of connective tissue is present beneath the epidermis of the pelvic fin. It contains abundant ground substance and is similar to mucous connective tissue of the mammalian umbilical cord. Perfusions of a microvascular filling agent reveal a moderately developed cutaneous vasculature. These vessels have the dimensions of capillaries (mean external diameter 11 μm). They are confined to the dermis and are more prominent on the head than on the trunk. The skin is secondary to the gills as a respiratory surface in Gymnodraco.

Notes: To determine the separate spermatogenic actions of FSH and testosterone, adult male lizards Hemidactylus flaviviridis with recrudescent testes were administered the non-steroidal antiandrogen flutamide either alone or in combination with FSH or testosterone, and the histology and histochemistry of the testes and ductus epididymides were studied. Flutamide-treated animals displayed a marked hypertrophy of Leydig cells. A few spermatids were also seen in testis of more than half the animals treated with flutamide. Flutamide also produced a significant increase of primary spermatocytes; no spermatids were observed in controls. A significant inhibition of spermatogenesis was noted in lizards treated either with testosterone alone or in combination with flutamide. Ovine FSH treatment caused a significant stimulation of spermatogenesis, as indicated by the increase of primary and secondary spermatocytes and the transformation of secondary spermatocytes into spermatids or, in a few cases, into spermatozoa. A considerable depletion of sudanophilic lipid and moderate Δ5-3β-hydroxysteroid dehydrogenase activity was noted in the Leydig cells of FSH-treated animals indicating enhanced steroidogenesis. Similar results were obtained when lizards were treated with flutamide + FSH. The effects of simultaneous treatment of flutamide with FSH or testosterone on ductus epididymidis revealed that flutamide markedly inhibited the epithelial cell height and lumen diameter with a loss of luminal content when compared to FSH or testosterone-treated lizards.

Notes: An immunohistochemical study of the localization of cytotactin and cytotactin-binding (CTB) proteoglycan throughout embryonic development of the anuran Xenopus laevis reveals that both appear in a restricted pattern related to specific morphogenetic events.CTB proteoglycan expression is first detected during gastrulation at the blastopore lip. Later, it is seen in the archenteron roof around groups of cells forming the notochord, somites, and neural plate. Cytotactin first appears after neurulation, and is restricted to the intersomitic regions. Both molecules appear along the migratory pathways of neural crest cells in the trunk and tail. Later, cytotactin is present at sites where neural crest cells differentiate, around the aorta and in the smooth muscle coat of the gut; CTB proteoglycan is absent from these sites. In the head, cytotactin is initially restricted to the regions between cranial somites, while CTB proteoglycan is distributed throughout the cranial mesenchyme. The expression of both molecules is later associated with key events in chondrogenesis during the development of the skull. After chondrogenesis, CTB proteoglycan is distributed throughout the cartilage matrix, while cytotactin is restricted to a thin perichondrial deposit. Both molecules are expressed in developing brain.These findings are compared to studies of the chick embryo and although distinct anatomical differences exist between frog and chick, the expression of these molecules is associated with similar developmental processes in both species. These include mesoderm segmentation, neural crest cell migration and differentiation, cartilage development, and central nervous system histogenesis.

Notes: During metamorphosis of Xenopus laevis the extracellular matrix (ECM) proteins cytotactin and cytotactin-binding (CTB) proteoglycan, and the cell adhesion molecules N-CAM and Ng-CAM, appear in highly restricted patterns determined by immunofluorescence histology.During limb development, cytotactin appears from the earliest stages in a meshwork of ECM fibrils associated with migrating mesenchymal cells forming the limb bud. Cytotactin also appears in the ECM between the apical limb ectoderm and mesenchyme. Later, both cytotactin and CTB proteoglycan appear co-localized within the central (prechondrogenic) limb mesenchyme. During chondrogenesis in these areas, cytotactin becomes restricted to perichondrium, while CTB proteoglycan is expressed throughout the cartilage matrix. The premyogenic mesenchyme surrounding the chondrogenic areas expresses N-CAM. Later, N-CAM is concentrated at the myogenic foci where cytotactin appears at sites of nerve/muscle contact and in tendons.Expression of these molecules in the blastemas of regenerating limbs was also studied, and during development of the central nervous system, stomach, and small intestine.Analysis of the expression patterns of cytotactin and CTB proteoglycan throughout development and metamorphosis reveals several consistent themes. The expression of these molecules is highly dynamic, often transient, and associated with key morphogenetic events. Cytotactin appears at multiple sites where cells undergo a transition from an undifferentiated, migratory phenotype to a differentiated phenotype. One or both molecules appear at several sites of border formation between disparate cell collectives, and CTB proteoglycan expression is associated with chondrogenesis.

Notes: In artificial fluid, the spermatozoa move as linear cells or round up and rotate, propelled by spontaneous bending of their tails. Both linear and rounded cells can move forward and backward, but usually they move forward. The tails of all cells display, simultaneously, small primary bends and fewer, much larger secondary bends. Rounded cells form single secondary bends that remain unchanged as the cells rotate. They also form “node-like” primary bends that travel posteriorly or anteriorly as the cells rotate forward or backward, respectively. Linear cells move their anterior regions into and out of focus in a cyclic fashion. They form rather prominent primary bends, as well as two to four secondary bends that travel posteriorly as the cells move forward. Secondary bends change in shape continuously and are not sinusoidal. The cells follow approximately linear trajectories, but the distances traveled per cycle, speeds, and secondary bending patterns are variable. When methyl cellulose is added to artificial fluid, linear movement is improved, and forward speeds are approximately tripled. The movement of spermatozoa in natural fluid of the female reproductive tract is remarkably less stereotyped than that of cells in artificial fluid. The cells, usually resembling straight lines or arcs, are very flexible and active. They lack obvious cyclic activity and double bending patterns. They are capable of moving both forward and backward and of adjusting their bending activity and speed within rather wide limits. Their average forward speed is about nine times faster than that of cells in artificial fluid.

Notes: The neuromuscular organization of feline anterior sartorius was examined using three experimental approaches. First, the branching pattern of the nerve supplying anterior sartorius was inspected in muscles taken from a large number of feline cadavers. All muscles were found to be supplied by two major nerve branches, one directed proximally and the other directed distally, and most muscles (42/51) had a third distinct branch that entered the muscle centrally. Second, the motoneuronal populations supplying the three nerve branches were investigated by electrophysiological techniques. Motoneurons that supplied axons to the distally-directed branch did not appear to have collaterals in more proximally-located branches. In contrast, other motoneurons supplying the proximally-directed branch also appeared to supply axon collaterals to the centrally-directed branch. This result suggested that the motoneuronal population of the distally-directed branch was largely separate from that supplying the proximally- and centrally-directed branches. Third, the motor unit territories supplied by different nerve branches were mapped using glycogen-depletion methods. Muscle fibers supplied by the distallydirected nerve branch were mostly distributed to the medial portion of anterior sartorius, whereas the fibers supplied by the other two branches were generally found more anteriorly. Further, the muscle fibers supplied by an individual nerve branch were present in greater numbers at the end of the muscle closest to the entry point of that branch. Thus, the motor units supplied by discrete nerve branches were found to be distributed asymmetrically within anterior sartorius, but were arranged neither strictly in-parallel nor strictly in-series.

Notes: The larvae of Halicryptus spinulosus bear six rings of sensory/ locomotory appendages. Twelve pedicellate flosculi, 6 dorsal and 6 ventral, are associated with the neck-lorica junction. Newly described are two rows of 4-5 flosculi present on the dorsal and ventral plates, and 2 tubuli located on each dorsolateral and ventrolateral plate near their junction with the midlateral plate. The ultrastructure of all organ systems is described. The multilayered lorica differs significantly from that of other priapulids. The anterior fourth of the lorica is not underlain by epidermal cells. At the junction of the introvert and neck are a series of adhesive tubuli that have groups of secretory cells basally. A gland is described at the junction of the fore- and midgut.

Notes: High speed video recordings (200 fields per second) of prey capture and food processing in Agama agama permit the identification of strikes, chews and transport movements. Ten variables from strike movements and seven variables from chewing sequences are digitized; transport movements are inspected only. Univariate and multivariate statistical analyses disclose significant interindividual differences for three variables (maximum gape distance, maximum head angle, and maximum throat distance); but neither these nor principal components analysis show differences between strikes and chews for any of the gape change and hyoid depression variables. However, strikes and chews obviously differ in tongue protrusion and body movements. Chewing may be divided into four stages, comparable to those of transport cycles of other lizards and the generalized tetrapod model. Transport differs from chewing by having a shorter power stroke and relatively more cranial and less jaw movement. The kinematics of feeding in Agama agama are compared with those of other lizards studied previously.

Notes: The liquid chromatographic analysis of drugs in urine through direct injection without any sample pretreatment was extended to micellar chromatography with nonionic surfactants, the Pinkerton™ ISRP column and the shelded hydrophobic phase (Hisep™) column. The feasibility of using each was demonstrated through the determination of the diuretic, hydrochlorothiazide, in urine. Good separation, recovery, precision and linearity, and adequate limits of detection were obtained for this analysis with all three techniques. The advantages and limitations of the mobile phase approach of micellar chromatography and the two stationary phase approaches are discussed for the direct injection of urine as well as other biological fluids.

Notes: A method for determining Picumast, an antiallergic drug, in plasma by HPLC and column switching has been developed. The system consisted of two precolumns, an analytical column, three pumps, an autosampler and a fluorescence detector. The precolumns (17 × 4.6 mm i.d.) were packed with LiChroprep RPR (a moderately polar reversed phase) and the analytical column with Nucleosil ODS (RP 18, 5 μm). The columns were connected according to the alternating precolumn technique. The mobile phase consisted of 30% CH3CN/70% 0.05M KH2PO4, pH 2.5, with a flow gradient. Detection wavelengths were 333 nm for excitation and 383 nm for emission. The retention times of Picumast, M1 and M2 were 12, 3.6 and 4.0 min, respectively. Total run time was 15 min. The limit of detection was 3 ng/mL for M1 and 1 ng/mL for M2 and Picumast using an injection volume of 150 μL. The recoveries vary between 89% and 97% with standard deviations between 2.4 and 3.3%.

Notes: High performance hydrophobic interaction chromatography has been used to separate progestin receptors (PRs) from human uterus and from the T47D human breast cancer cell line. Reproducible separations of high resolution were achieved using a TSK Phenyl-5PW column and a reverse salt gradient of 400 mM to 0 mM sodium sulfate in phosphate buffer, pH 7.4. Peaks of radioactivity exhibiting hydrophobic behaviour were isolated, as well as a smaller proportion of specific bound receptors located in the void volume fraction. No differences in retention times were observed between uterine and breast cell line samples. When the technique was used in conjunction with rapid vertical tube sucrose density gradient centrifugation, the 8S sedimenting PR from fresh, low-salt cytosol always eluted with a retention time of 24 min. The natural 4S receptor chromatographed as a single peak at 29 min while the 4S receptor species from high-salt cytosol appeared as two distinct peaks of radioactivity with retention times of 29 and 33 min. While specific binding was shown to occur in the void volume of the column, the origin of these receptors were indeterminate. These results would suggest that under these conditions the 8S receptor occurs as a single hydrophobic class of protein, whereas the data provides evidence that transformed 4S receptor may be proportioned into two unequal entities as a function of exposure to salt.

Notes: A procedure for the preparative separation of proteins was developed by using consecutively sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed phase high performance liquid chromatography (HPLC). The proteins were separated by SDS-PAGE and afterwards extracted from the gel. The extracted proteins were separated from SDS and other small molecular weight contaminants on a Fractogel TSK HW-40 (F) column in acidic aqueous acetonitrile. The proteins eluted from the Fractogel column were fractionated by HPLC. The identity and purity of the recovered proteins was confirmed by SDS-PAGE analysis.

Notes: Reversed phase ion pair chromatography is a highly selective separation technique for the determination of free porphyrin carboxylic acids from human materials. Isocratic and gradient elution methods can be used to analyse porphyrin isomers and to establish porphyrin profiles for the biochemical diagnosis of porphyrias. Ion pair high performance liquid chromatography led to the discovery of the atypical isomers II and IV of uroporphyrin and coproporphyrin in human urine. Advantages and limitations of the ion pair technique are discussed.

Notes: Samples of pregnant urine were treated with three diatomaceous earth columns to fractionate organic solvent-soluble components. The first column was pre-injected with the urine sample. The second and the third columns were pre-injected with sodium bicarbonate and sodium hydroxide solutions, respectively; these played an important role in capturing acidic components in the urine extract from the first column. Neutral components could be eluted out from the columns with the organic solvent, while the basic components were retained in the first column. Once strongly and weakly acidic components were captured on the alkaline columns, they were separately treated with an acidic mobile phase and recovered. The digoxin-like activities of each fraction were examined for cross-reactivity to antidigoxin antibody and inhibitory effects on dog kidney Na+, K+-ATPase. Both types of reactivity were found in all three fractions and decreased in the following order: strongly acidic, neutral and weakly acidic. The neutral fraction of pregnant urine showed significantly greater cross-reactivity than that of the non-pregnant urine. Following hydrolysis of the pregnant urine with β-D-glucuronidase, the cross-reactivity of the neutral fraction increased significantly. High performance liquid chromatographic analysis showed that several components in the neutral fraction had apparently increased after the hydrolysis, showing the possible presence of unknown digoxin-like components in the pregnant urine.

Notes: Wide porous glass (WPG) chemically coated with a poly-N-(2-hydroxyethyl)acrylamide layer is proposed as a carrier of biospecific ligands in affinity chromatography. The method of WPG chemical modification includes synthesis of the γ-aminopropyl derivative followed by chemical adsorption of poly(p-nitrophenyl acrylate). Ester groups of the polyacrylate-coated WPG can be used for coupling the ligands bearing primary amino groups. Condensation of esters with ethanolamine yields a poly-N-(2-hydroxyethyl)acrylamide-coated support with non-specific adsorption properties resembling those of Sepharose 4B. Human IgG immobilized on the polyacrylate support was used for isolation of the first complement component from human serum and for its separation into subcomponents C1r, C1s and C1q by a one-step method. An unbound part of serum may be used as the R1 reagent for determining haemolytic C1 activity. The stepwise elution of C1r, C1s and C1q from the column reflects the course of C1 breakdown after its activation on immune complex formation.

Notes: Chromatographic techniques for the determination of trace amounts of neurotransmitters were reviewed. The two techniques found to be most useful were GC-MS and the reversed-phase mode of HPLC with an electrochemical or fluorescent detector. For structure determination or unequivocal peak identification, GC-MS is the method of choice. In addition the limits of detection of GC-MS were better than those obtained by HPLC. However for routine analyses, HPLC is now being used in studies of mental illness and other diseases. Good resolution, reproducibility and sensitivity can be obtained without the derivatisation steps required for GC-MS, and catecholamines, serotonin, and their acidic metabolites can be concomitantly determined in one analysis.

Notes: A rapid, sensitive and simple-to-operate high performance liquid chromatographic method for the simultaneous determination of oxcarbazepine, 10-hydroxycarbazepine and 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine in plasma is described. The drug and its metabolites were extracted from plasma using commercially available reversed phase octadecylsilane bonded-silica columns (Bond Elut C18, 1 mL capacity). Chromatographic separation of oxcarbazepine and its metabolites was achieved using a mobile phase consisting of acetonitrile/methanol/water (13:25:62 by volume) at a flow rate of 1.2 mL/min in conjunction with a Waters Associates Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a LiChrospher 5 μm C18 guard column. By measuring the UV absorbance at 214 nm, plasma levels in the region of 50-100 ng/mL for the drug and its metabolites can be detected with only 100 μL of plasma. The method has been applied to pharmacokinetic studies of oxcarbazepine and its metabolites in children with epilepsy; preliminary pharmacokinetic findings in two patients at steady-state are presented.