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Notes: Chromatographic methods (paper chromatography, thin layer chromatography, high performance liquid chromatography, gas chromatography and gas chromatography-mass spectrometry) for the determination of clinically important steroids in biological specimens are reviewed. The emphasis is on the use of gas chromatography, gas chromatography-mass spectrometry and high performance liquid chromatography as reference rather than routine techniques. Chromatographic methods are compared with colorimetric, fluorimetric and radioimmunoassay procedures in terms of simplicity of operation, cost and ability to analyse large numbers of specimens. The importance of correct specimen collection and storage are discussed. Sample preparation techniques for the various analytical methods are described. These include extraction of free and conjugated steroids from serum, plasma, urine and saliva by solvent partition, with polymer-based resins such as Amberlite XAD-2, DEAE-Sephadex and Sephadex resins bonded with various other function groups and, more recently, with chemically bonded reversed-phase silicas.

Notes: An optimization strategy for an isocratic reversed phase high performance liquid chromatographic system (RP-HPLC) is described. Factorial design and a computer program are used to predict the retention time and resolution of fourteen steroids. An optimized rapid (〈25 min) isocratic RP-HPLC system for the satisfactory separation of cortisone, cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17α-hydroxyprogesterone, progesterone, androstenedione, testosterone, estrone, estradiol, estriol, prednisone acetate and dexamethasone acetate has been developed using this strategy through eight experiments.

Notes: The isocratic reversed phase high performance liquid chromatographic method proposed for quinidine metabolic studies facilitates particularly the separation of 10(R) and (S) isomers of quinidine 10,11-dihydrodiols. The finding of each of these forms following a new synthetic pathway allows us to identify and quantify them in biological fluids. These two isomers have especially been observed in rat bile and hepatocyte secretions. The metabolic inducing effect of phenobarbital on the oxidative metabolism of quinidine is verified in rat isolated hepatocytes. Simultaneous secretion of the two dihydrodiols is also verified in human urine by a gas chromatography/mass spectrometry procedure.

Notes: The catalytic effect of bases (imidazole, pyridine, Tris and triethylamine) on the peroxyoxalate chemiluminescence (PO-CL) reaction for high performance liquid chromatography (HPLC) was investigated. Imidazole increased PO-CL intensity extraordinarily, whereas the other bases (pyridine, Tris and triethylamine) did not. The peak heights of dipyridamole (coronary vasodilator) obtained using the eluents containing buffers were largest at pH 7.0, a few times less at pH 6.0 and pH 5.0, 100 times less at pH 4.0 and a few hundred times less at pH 3.0. The eluents containing buffers at pH 3, 4, 5, 6 or 7 each with imidazole increased the peak heights by a few to ten times as compared with those without imidazole, and those peak heights were within one order of magnitude. On the other hand, the eluent containing buffer at pH 2 did not affect the peak heights with or without imidazole. Bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) alone and bis(2,4-dinitrophenyl)oxalate (DNPO) plus TDPO were recommended to be used against eluents containing buffers of pH 5 - 7 and pH 3 - 4, respectively. Dipyridamole and benzydamine hydrochloride (anti-inflamatory drug) were separated on the ODS column and detected by the present system. The detection limits of dipyridamole and benzydamine hydrochloride were 40 amol and 270 fmol, respectively.

Notes: A simple, reliable and highly sensitive procedure was devised for measuring the levels of Amicar in blood and urine. 100 μL of serum or urine sample was added to 10 μL of a 10% w/v zinc sulfate solution and 100 μL of methanol, as previously described (Lam et al., 1980) for the removal of proteins by precipitation. 50 μL of the supernatant was then mixed with 300 μL of 1 M borate buffer containing D-valine as the internal standard before derivatization with o-phthalaldehyde. The amino acids were then separated by a stereoselective reversed-phase system using a mobile phase containing 10% of acetonitrile in 2.5 mM Cu(II) complexes of L-proline. The chromatography is highly selective, resolving Amicar from L-valine which in turn is resolved from its unnatural D-antipode, the internal standard. The procedure including sample preparation and separation required a total of 15 min. As little as 50 ng/mL of Amicar in body fluids could be detected as the o-phthalaldehyde derivative by fluorescence.

Notes: A high performance liquid chromatographic (HPLC) method for analysis of 4 free and 8 conjugated bile acids in submicromolar quantities in serum is described using precolumn derivatization with 4-bromomethyl-7-methoxy-coumarin (BMC) and fluorescence detection. Bile acids were extracted from serum with 0.4 M sodium bicarbonate, adsorbed onto a Sep-Pak C18 cartridge and eluted with methanol. The extract was derivatized with BMC in acetonitrile using 18-crown-6 crown ether as catalyst and the BMC labelled glycine conjugates and free bile acids were analysed using acetonitrile + methanol + water gradient elution and detection at 320/385 nm.Using a novel and simple approach, taurine conjugates were isolated by extracting the dried, derivatized material with water, in contrast to previous methods which required column chromatography cleanup to isolate the taurine conjugates prior to derivatization. The isolated taurine conjugates were then hydrolysed enzymatically, extracted, derivatized and analysed as free-bile acids.Recoveries of individual bile acids varied from 83 - 96% for free and glycine conjugates and 72 - 83% for taurine conjugates. Coefficients of variation were in the range of 5.1 - 12.5%. In addition to the simpler and shorter procedure for taurine conjugates, this method has increased sensitivity over most other procedures and improved HPLC separation for the various bile acids and conjugates with equivalent recovery and reproducibility compared with other published methods.

Notes: High performance liquid chromatography (HPLC) with a TSK-4000SW gel filtration column was used to compare envelope polypeptides from four strains of herpes simplex virus type 1 (HSV-1). The chromatographic profiles demonstrated polypeptide variability among three clinical strains and the wild-type F strain. Radioimmunoprecipitation of the HPLC fractions with polyclonal anti-HSV-1 followed by SDS-polyacrylamide gel electrophoresis (PAGE) of the immunoprecipitates revealed molecular weight differences of various polypeptides in fractions from the area containing major peaks. This HPLC method could prove useful for the analysis of polypeptide polymorphism in clinical isolates of HSV-1, as well as in other viruses.

Notes: A fast and sensitive method is given for analysing urinary tryptophan and six of its metabolites on the nicotinic acid pathway. Kynurenine, tryptophan, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid and xanthurenic acid were isocratically eluted and completely resolved with a mobile phase of acetonitrile + sodium acetate buffer, pH 4.76 (4:96, v/v). The flow rate was 0.8 mL/min at the beginning and was then linearly increased to 1.2 after 6 min; after 14 min the flow was augmented from 1.2 to 2 mL/min. The effluent was monitored with a variable UV detector set at 254 nm for the first five peaks and at 280 and 325 nm for the penultimate peak and final peak. Analytical recoveries of the compounds after deproteinization varied between 64% and 98%. The reported method should enable one to examine easily, extensively, quantitatively and routinely urinary tryptophan and the most important metabolites of the nicotinic acid pathway.

Notes: Reversed phase HPLC methodology has been developed for separation of peptide leukotrienes and indomethacin in porcine bile. Reproducible recoveries were obtained using radioactive leukotrienes ([3H]LTC4, 57.1 ± 2.5%; [3H]LTE4, 62.7 ± 1.9%; [3H]LTD4, 54.3 ± 2.7%). Radioimmunoassay of column eluant demonstrated that as little as 300 pg of exogenous leukotrienes could be measured in bile fluids, with similar recoveries. Analysis of bile sampled 60-90 min after initiation of experimental endotoxic shock in indomethacin treated pigs revealed a leukotriene concentration of 5.24 ± 1.16 ng/mL(LTD4). This was significantly greater (p 〈 0.05, n = 3) than that observed in samples collected prior to endotoxin (0.42 ± 0.23 ng/mL), or from untreated animals (0.85 ± 0.51 ng/mL). This method is thus applicable to investigation of the role of 5-lipoxygenase products in porcine models of human disease, including shock conditions such as endotoxaemia, during cyclooxygenase inhibition by indomethacin.

Notes: The application of reductive coulometric electrochemical detection for analysis of the monoamine neurotransmitters norepinephrine, dopamine, and serotonin and their common metabolites in brain and cerebrospinal fluid following separation by isocratic high performance liquid chromatography is described. The high sensitivity and screening capabilities of coulometric electrodes permits the accurate quantitation of as little as 3-5 pg of these compounds in tissue following a simple single step purification procedure. Moreover, comparison of peak height ratios obtained from analysis of authentic reference standards and tissue samples at selected multiple electrode potentials provides a straightforward means for qualitative evaluation of peak identification and purity during analysis of biological samples. The method is comparatively inexpensive and precise within and between day coefficients of variation for most compounds range from 2-5%. Thirty samples can be run in duplicate in a 24 h period.

Notes: Some useful high performance liquid chromatographic methods for the determination of amines, amine metabolites and amine metabolizing enzymes are described. These include the separation of tyramine in wines and beers, determination of tryptamine in urine, assay of monoamine oxidase and catechol-O-methyltransferase and analysis of amine-aldehyde condensation products.

Notes: Catechol-O-methyltransferase (COMT) and aromatic L-amino acid decarboxylase (AADC) activities were determined in human gastrointestinal samples. L-dopa was used as the substrate and the reaction products 3-O-methyldopa (3OMD) and dopamine were separated by reversed phase HPLC and detected by electrochemical or UV detection. COMT activities varied between 40-350 pmol/mg/min and AADC activities between 100 - 3300 pmol/mg/min in different parts of the gastrointestinal tract. COMT inhibitors nitecapone (OR-462) and OR-611 effectively inhibited human gastrointestinal COMT activity in vitro, the IC50 values ranging from 10-20 nM and 5-75 nM, respectively. In vitro carbidopa inhibited AADC slightly more effectively than benserazide.

Notes: Analysis of small amounts of glycoproteins by high performance liquid chromatography (HPLC) and liquid secondary ion mass spectrometry (LSIMS) together with enzyme digestion has been investigated using fetuin as a model. Preliminary data indicates that 71% of the expected peptides were detected by LSIMS analysis of 200 pmol total digest. HPLC profiles of peptides and glycopeptides were obtained from 2 nmol of digest using a reversed phase (C18) column eluted in a solvent system containing TFA, water and acetonitrile. This has provided glycopeptides for subsequent oligosaccharide analysis. Strategies are reviewed for the chromatographic characterization of oligosaccharides following their release from glycopeptides by chemical and enzymatic procedures.

Notes: A rapid gradient method has been established to measure simultaneously the tissue levels of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy). The method involves application of a 15%-25% linear gradient of methanol over a period of 10 min to an initial mobile phase of 15% methanol with 25 mM sodium phosphate, 10 mM 1-heptanesulfonic acid solution at pH 3.2 and a flow rate of 1 mL/min. AdoHcy elutes at 9.5 min and AdoMet at 12.6 min. The assay has a detection limit of 10 pmol and is linear in the concentration range 30-800 pmol. The method was used to assess changes in AdoMet and AdoHcy concentrations of pig tissues after seven days exposure to the anaesthetic gas nitrous oxide which irreversibly inactivates methionine synthase and induces an inability to recycle homocysteine, particularly in neural tissues. The treatment caused significant alterations in cellular AdoMet: AdoHcy ratios which were principally due to a dramatic rise in AdoHcy concentrations.

Notes: During postnatal development of the pigeon, a large portion of the skeleton becomes pneumatized, displacing the hemopoietic bone marrow. The consequences of pneumatization on distribution and quantity of bone marrow as well as the availability of other sites for hemopoiesis have been investigated. Hemopoietic marrow of differently aged pigeons divided into five groups from 1 week posthatching (p.h.) up to 6 months p.h. was labeled with Fe-59 and examined by serial whole-body sections. Autoradiography and morphometry as well as scintillation counts of single bones and organs were also carried out. No sign of a reactivation of embryonic sites of erythropoiesis was found. Bone marrow weight and its proportion of whole-body weight increased during the first 4 weeks p.h. from 0.54% to 2.44% and decreased in the following months to about 1.0%. The developing bone marrow showed a progressive distribution during the first months of life, eventually being distributed proportionally over the entire skeleton, except for the skull. At the age of 6 months p.h. bone marrow had been displaced, its volume decreasing in correlation to increasing pneumaticity and conversion to fatty marrow. This generates the characteristic pattern of bone marrow distribution in adult pigeons, which shows hemopoietic bone marrow in ulna, radius, femur, tibiotarsus, scapula, furcula, and the caudal vertebrae.

Notes: Artificially incubated fertile eggs from wild alligators have a significantly better hatch rate than those of captive, pen-reared alligators, possibly due to differences in the morphology of the eggshells. We compared the morphology of eggshells of wild alligators to those of captive alligators living in semi-natural environmental pens. Lengths and widths of eggs were measured and volume was determined, assuming an ellipsoid shape. Eggs were also evaluated for the quality of the eggshell (the presence or absence of rough deposits). Pieces of shell were cut from unincubated eggs and from eggs incubated for 55 days (just before hatching) and examined by scanning electron microscopy. Open pores on the outer surface of the shells were counted and thickness of the pieces was measured from micrographs. Results indicated that the number of pores on eggshells was lowest in eggs of captive alligators with early embryonic death. The number of pores was intermediate in eggs with early embryonic death from wild alligators, and the number of pores was highest in eggs with full-term embryos from wild or captive alligators. It is suggested that decreased porosity of eggshells may be associated with early embryonic death, is more prevalent in captive animals, and may, therefore, be related to poor hatch rate among penreared alligators.

Notes: Adult hornets (Vespa orientalis; Hymenoptera, Vespinae) build the brood combs out of organic or mineral matter. The cement that serves to glue the building material together is secreted in their saliva, the latter hardening within seconds to form fibers or plates. This saliva-derived spittle overlies and unites the building particles laminally and vertically. The hornet larvae spin a cocoon within the brood cells, which is largely fastened to and supported by the cell walls and is composed of a network of silk fibers and interlinking flat surfaces. On the outside of the cocoon fibers are spherical button-like structures that are very rich in phosphorus. The chemical composition of the adult salivary cement and the larval cocoon fibers is similar: both contain the elements P, Mg, S, Cl, K, and Ca. The possible biological significance of these findings is discussed.Among social insects belonging to the Hymenoptera, there are three main groups that build a multitude of cells, namely, the paper wasps (Polistinae), the other social wasps or hornets (Vespinae), and the social bees (Apinae). The constructed cells serve mainly for rearing the brood, but in many instances (particularly among various Apinae) they also serve for storing honey and pollen. For building material, Apinae rely primarily on beeswax (a product secreted by the bee itself to which various amounts of plant resins are added). On the other hand, wasps (Polistinae and Vespinae) build mainly from matter collected in the nearby environment, be it organic matter such as tree bark, mineral matter, or a combination of the two.Much information has accumulated on comb and cell building among these insect groups (e.g., Wheeler, '23; Van der Vecht, '57, '65; Lindauer, '61; Michener, '61; Kemper and Döhring, '67; Wilson, '71; Guiglia, '72; Spradbery, '73; Edwards, '80; Brian, '83; Schremmer et al., '85). Species of Polistinae and Vespinae are prevalent in forest areas in Southeast Asia and in central and South America or in the temperate regions in both the northern and southern hemispheres, and they mainly use vegetable matter to build their combs. In contrast, species prevalent in the Mediterranean region, which is dry and relatively unvegetated during the wasp and hornet active season, rely more on nearby mineral than on plant matter. Comb building in the Oriental hornet is well known (Darchen, '64; Ishay et al., '67; Schaudinischky and Ishay, '68; Ishay, '73, '75a, b, '76; Ishay and Sadeh, '75, '76; Ishay and Perna, '79; Ishay et al., '82). Recently Ganor et al. ('86) described the cell wall in the V. orientalis comb, showing it to be composed of mineral particles collected near the subterranean nest, in contrast to the comb of two European species (V. crabro and Vespula) (Paravespula) germanica, which is built primarily of organic matter. Regardless of whether the building material is mineral or organic, the hornets utilize particles of sand or other minerals or pieces of wood wrapped and melded together by saliva. However, nothing is known about the cement or mortar used to glue together these “bricks.” The present investigation was initiated to increase our knowledge of this cementing substance.Combs of V. orientalis were collected from fields in the Tel-Aviv district in 1987 during the active season, which extends over most of the summer months. Because the combs are easily damaged, care was taken to remove them intact from the natural nest. Once removed, the combs were cleared of the existing brood (eggs, larvae, or pupae) and then stored carefully in dry glass vessels until examined. For the present study, combs were collected only from nests in Khamra soil, which is common in the Tel-Aviv district and along the coastal area of Israel. To investigate the morphology of the cement material in the comb, strips of comb wall as well as segments of the pupal silk dome were removed from each comb and prepared for examination. The comb wall strips were cut to a size of 3 × 6 mm and fastened to the stub of a scanning electron microscope (SEM). The silk pieces were processed in two ways: (1)strips of 3 × 4 mm were fastened onto a stub with the convex (outer surface) facing up or (2) with the concave (interior) side upward.Silk strips were boiled for 2 hours in distilled water to remove all impurities, such as foreign matter adhering to the silk secreted by the larvae. Examination of these silk fibres was carried out in the three ways: (1)SEM micrography of the exterior (white portion) of the silk dome; (2) micrography of the interior; (3) micrography of both exterior and interior aspects after boiling for 2 hours in distilled water to remove water-suspended foreign materials that may have clung to the silk dome in the course of ordinary nest activity.The chemical composition of selected specimens was investigated by x-ray analysis. They were done on JEOL 840 Scanning Electron Microscope (SEM) equipped with Link 10,000 Energy-Dispersive System (EDS). (With the EDS System, the spectrometer separates the elements according to energy rather than wavelength). Quantitative analysis was by ZAF4 program. Five strips of comb cell wall were examined from a randomly selected comb, each comb from a different nest. Micrographs were taken of isolated silk samples from each of the examined combs.

Notes: Dissections were performed to document buccal anatomy in three species of the pulmonate genus Helisoma Swainson, 1840. The 28 muscles which are responsible for radular feeding in these animals are organized in three concentric and integrated envelopes. The deepest of these includes muscles which manipulate the radula about the odontophoral cartilage. Elements of the middle envelope direct movements of the cartilage within the buccal cavity, and muscles of the outer envelope control movements of the buccal mass within the cephalic haemocoel. Motion analysis by videomicrography showed that muscles of the middle and outer envelopes contribute to the action of radular feeding by acting as antagonists to other muscles and to hydrostatic elements of the buccal apparatus. Observations of radular dentition showed that although each of the three species examined has a unique radula, especially with regard to the specific details of tooth shape, all resemble a radula characteristic of the Planorbidae with regard to other, more general, aspects of ribbon architecture.

Notes: Crocodilians and birds are the extant representatives of a monophyletic taxon known as archosaurs. Their limbs are highly derived in terms of reduction in number of skeletal elements in both the carpus and the tarsus. It is necessary to have a detailed description of crocodilian limb ontogeny to address the evolutionary issue dealing with the origin and organization of the avian limb. In this paper, we present an analysis of the early development of the crocodilian limb skeleton. Contrasting with earlier observations, we redefine the number and composition of carpal, tarsal, and phalangeal elements. This ontogenetic information is then used to introduce a revision of the homologies of the skeletal elements in the crocodilian limb. Some invariances are pointed out in the developmental organization of tetrapod limbs and this evidence serves to readdress several issues concerning the evolution of the avian limb. We present further embryological data in support of the hypothesis that digits 2-3-4 are the components of the wing skeleton in birds. In general, our comparative survey indicates that the elements that appear late in ontogeny are the ones lost in phylogeny. By comparing turtle (primitive) limb development with crocodilian and bird development, we propose a hypothesis in which the derived skeletal patterns found in crocodilians and birds have originated by a heterochronic process of paedomorphosis.

Notes: A unique pathway that utilizes endoplasmic reticulum (ER) networks is proposed for screening pigment granule formation in the retina of adult Eumesosoma roeweri. Smooth endoplasmic reticulum (SER) functions to transport pigment particles to the formative site. Each site is composed of concentric, interconnected rings of SER that are filled with dense-cored, spherical pigment particles. Formation of the screening pigment granule begins by the release of particles from the innermost rings of carrier SER. Continued release followed by fusion and condensation of the pigment particles results in the formation of a mature pigment granule.

Notes: Light and scanning electron microscopy of vascular replicas from the facultative air-breathing fish Heteropneustes fossilis show modifications in the macrocirculation of the respiratory organs and systemic circulation, whereas, gill microcirculation is similar to that found in typical water-breathing fish. Three and sometimes four ventral aortae arise directly from the bulbus. The most ventral vessel supplies the first pair of arches. Dorsal to this another aorta supplies the second gill arches, and a third, dorsal to, and larger than the other two, supplies the third and fourth arches and the air sacs. Occasionally a small vessel that may be the remnant of a primitive aortic arch arises from the first ventral aorta and proceeds directly to the mandibular region without perfusing gill tissue. The air sac is perfused by a large-diameter extension of the afferent branchial artery of the fourth gill arch and its circulation is in parallel with the gill arches. Blood drains from the air sac into the fourth arch epibranchial artery. A number of arteries also provide direct communication between the efferent air sac artery and the dorsal aorta. All four gill arches are well developed and contain respiratory (lamellar) and nonrespiratory (interlamellar and nutrient) networks common to gills of water-breathing fish. Air sac lamellae are reduced in size. The outer 30% of the air sac lamellar sinusoids are organized into thoroughfare channels; the remaining vasculature, normally embedded in the air sac parenchyma, is discontinuous. A gill-type interlamellar vasculature is lacking in the air sac circulation. Despite the elaborate development of the ventral aortae, there is little other anatomical evidence to suggest that gill and air sac outflow are separated and that dorsal aortic oxygen tensions are maintained when the gills are in a hypoxic environment. Physiological adjustments to hypoxic water conditions probably include temporal regulation of gill and air sac perfusion to be effective, if indeed they are so.

Notes: The antennae of adult Damalinia ovis, the sheep louse, were studied using light and scanning electron microscopy. Sensory structures are located on all three antennal segments with the predominant sensilla type being tactile. Nine different types of sensilla are described on the basis of external appearance. One of the sensilla, designated a “pit organ” because of its unusual shape, has not been described previously. A pair of these sensilla are present on each antenna, and their function is unknown. A group of 11 sensilla on the tip of each antenna contains olfactory and chemosensory pegs, and a possible thermohygroreceptor. The antennae are sexually dimorphic, the male having more tactile sensilla, two well-developed terminal hooks, and a different cuticular architecture on the posterior surface of antennal segment 1.

Notes: The formation of the unpaired structure ventral to the basibranchial region, the so-called urohyal, differs within osteichthyans. A cartilaginous preformed, unpaired “urohyal” is present in sarcopterygians. A three-tendon ossification is present in Polypterus. An “urohyal” or urohyal is absent in both Amia and Lepisosteus. The urohyal formed as an unpaired ossification of the tendon of the sternohyoideus muscle is a feature of teleosts. A new structure, the parurohyal, arises as a double ossification of the tendon of the sternohyoideus muscle in siluroids; during ontogeny an anterodorsal crest or cup-like structure derives from the anterior basibranchial region and the tendon bone; therefore, the parurohyal is compound in origin. Judging from their formation and their distribution within osteichthyans the cartilaginous preformed “urohyal” and the teleostean urohyal are nonhomologous, whereas the urohyal and parurohyal are homologous. The urohyal is connected by ligaments with the ventral hypohyals in most teleosts, whereas it articulates with the ventral hypohyals in teleosts such as Anguilla and Chanos. The parurohyal is a synapomorphy of siluroids. The parurohyal in siluroids is articulated with both ventral and dorsal hypohyals, and with the basibranchial region in catfishes such as diplomystids and ictalurids, whereas it articulates only with the ventral hypohyals in other catfishes such as trichomycterines. The passage of the hypobranchial artery through the hypobranchial foramen of the parurohyal is a unique feature of siluroids, like the absence of the basihyal bone.An ossified dorsal hypohyal appears late in ontogeny in Amia, as do tooth plates related to the medial side of the hyoid arch and branchiostegal rays in Amia, and tooth plates on the hyoid arch and branchiostegal rays in Elops (unique features within extant teleosts). Two ossified hypohyals, a synapomorphy of teleosts, are present early in ontogeny.There is intraspecific variation in the onset of ossification of the bones of the head, but the sequence of ossification between bones in a defined structural system is conserved (e.g., branchiostegal rays ossify first, then bones of the hyoid arch).

Notes: The vas deferens of Ambystoma macrodactylum is composed of a peritoneal epithelium, connective tissue layer with fibroblasts, circular smooth muscle, capillaries, cells containing lipid, and a luminal epithelium composed of a single layer of cuboidal cells covered by a net of interconnected ciliated squamous cells. The cuboidal cells have abundant rough endoplasmic reticulum, mitochondria, and PAS + secretory vesicles. Squamous cells of breeding males consistently have tufts of ∼100 cilia located at one end of the long axis of each cell. These cilia may help distribute secretory products. The squamous cells, absent in post-breeding males, are apparently sloughed into the lumen. Lipid vesicles are present throughout the cytoplasm of the cuboidal and squamous epithelial cells and are also in some cells of the connective tissue layer. These vesicles increase dramatically in number during the first 4 weeks after breeding and may serve as an energy pool for the next breeding season. Enzyme-histochemical tests for testosterone synthesis were negative. In addition to the accumulation of lipid and the loss of squamous cells in the vas deferens, after breeding PAS + vesicle production is terminated. These alterations appear to represent energy conservation strategies employed by the sperm-depleted vas deferens.

Notes: The concept of parallel muscle combinations, in which spindle density is significantly higher in small muscles compared to their larger counterparts in large-small muscle combinations acting across a joint, is supported by the results of this study regardless of the joint. Analysis of the canine data as well as previously published guinea pig forelimb and human pelvic limb data revealed no significant difference in spindle density between antigravity and non-antigravity muscles. Furthermore, a gradual increase in spindle density from proximal to distal on the limb was not found, although spindle density was significantly higher in the intrinsic manus or pes muscles compared to more proximal limb muscles in all three species. The significant differences in spindle densities in parallel muscle combinations and in manus/pes versus proximal muscles are discussed relative to their possible role in the control of locomotion.

Notes: The choanocyte chambers of eight different haplosclerid sponge species have been investigated with regard to their histological structure, their relation to the mesohyl, and their arrangement between the canal systems. Two fundamentally different organizational types occur in the investigated sponge species. In Haliclona rosea, H. oculata, H. elegans, H. simulans, H. rava, H. fistulosa, and Acervochalina limbata the choanocytes are separated from the mesohylar tissue, being more or less covered over the outer surface by pinacocytes belonging to the incurrent canal walls. In Haliclona indistincta, on the other hand, the choanocytes are, as in most other Demospongiae, in contact with the mesohyl at their outer surfaces. This indicates that the present order Haplosclerida is polyphyletic and contains sponges of a presumably poecilosclerid origin.

Notes: The three-dimensional structures of the carotid labyrinth in five species of anurans representing four families (Rana nigromaculata, Rana catesbeiana, Bufo japonicus, Hyla arborea, and Xenopus laevis), and three species of caudates representing three families (Cynops pyrrhogaster, Hynobius nebulosus, Ambystoma mexicanum) were compared using vascular corrosion castings and scanning electron microscopy (SEM). Anuran carotid labyrinths are spherical in shape and are classified into two groups according to the origin of the external and internal carotid arteries. One group, which included Rana, Hyla, and Bufo, is characterized by the presence of a vascular ring at the proximal end and some vascular routes at the distal end of the labyrinth. The external and internal carotid arteries originate from these structures. The other group, which includes only Xenopus, is characterized by the external carotid artery opening directly from the central chamber or the common carotid artery, and by the internal carotid artery originating from within the vascular maze. The vascular maze is most complex in Xenopus, less so in Rana and Bufo, and simplest in Hyla. The carotid labyrinths in Cynops and Hynobius are oblong in shape. The fundamental organization in salamanders is similar to that in anurans. The vascular maze, however, is much simpler than in Hyla. There is no specialized swelling in Ambystoma mexicanum. The present findings suggest that most amphibian carotid labyrinths have the appropriate architecture for controlling vascular tone.

Notes: The vomeronasal sensory epithelium of a crotaline snake, Trimeresurus flavoviridis, was shown to consist of a superficial supporting cell layer and an underlying sensory cell layer composed of columns of sensory cells. The supporting cell layer consists of both supporting cells and dendrites of the underlying sensory neurons. The apical regions of sensory cell dendrites contain numerous microtubules, many elongated mitochondria, centrioles, and electron-dense bodies. The dendrites terminate as dendritic knobs from which microvilli project into the vomeronasal lumen. Smooth vesicles are abundant in the dendritic terminals and their vicinity. Supporting cells also bear microvilli, and these cells contain large electron-opaque granules and dense vesicles near their free surfaces. Cytoplasmic extensions of the supporting cells form a meshwork which separates dendrxites from each other in the vicinity of the luminal surface. The meshwork becomes obliterated in the infranuclear region of each supporting cell. Bipolar-shaped sensory cells with lightly stained round nuclei contain the characteristic cell organelles of neurons and are thought to be sensory neurons. These cells are especially characterized by well-developed lamellae of rough endoplasmic reticulum and extensive arrays of smooth endoplasmic reticulum. The perikarya of cells located in the apical region of the cell columns tend to contain larger amounts of smooth endoplasmic reticulum and lipofuscin granules than the perikarya of cells located in lower regions. Undifferentiated cells are found in the basal region of the columns. Satellite cells form the framework of the columns and are also found among neuronal elements.

Notes: The protandric hermaphrodite Ophryotrocha puerilis possesses one pair of eyes. They are located in the peristomium. Each light-sensitive organ consists of one sensory cell and one to two supporting cell(s) embedded in a cup-shaped reflector. The sensory-supporting cell complex is enveloped by a basal lamina. This lamina is supposed to be identical with the neural lamella. Thus the eyes proper have to be regarded as protrusions from the brain, while epidermal cells seem to differentiate to crystalline cells (reflector) and are deposited onto the sensory complex.The reflector is built up by several cup-shaped cells (juveniles, 4-5; adults, 10-12). Each of these cells comprises a multilayer of parallel-oriented, membrane-bound crystalline platelets which are thought to be guanine. The sensory cell is of the inverted rhabdomere type. Submicrovillar cisternae, typical for most polychaete eyes, are lacking. The first and always present supporting cell entirely envelops the sensory cell, thus forming the extracellular space around the rhabdomere. It does not contain any pigment granules. Often but not always a second supporting cell has been observed surrounding the sensory cell and first supporting cell. It is interpreted as a glial cell.In the sensory cell beneath the rhabdomere, pino- and phagocytosis can be observed and secondary lysosomes are found in high densities. Preliminary results seem to demonstrate that there is no distinct diurnal cycle of receptoral membrane recycling. In comparison with the ocelli of Dinophilidae, which have been interpreted as a dorvilleid-related family, morphological differences and their application to phylogenetic considerations are discussed.

Notes: The appearance pattern of pharyngeal tooth germs was investigated in the larval Japanese dace, Tribolodon hakonensis, which has a bilaterally asymmetrical dentition. Teeth develop in a series of replacement waves beginning with the initial central tooth (Ce) and continuing with teeth of anterior (An) and posterior (Po) positions relative to the initial one. Identified by wave number (n) and tooth position (r), according to the formula n-1[r], tooth germs appeared in the order of tooth 0[Ce0], 1[Po1], 1[Anl], 2[Ce0], 2[An2], 3[Po1], 3[An1], 4[Ce0], 4[An2], 5[Po1], 5[An1], 5[An3], 6[Ce0], 6[An2] during the larval period. Dentition on the right side, however, lacks the first tooth at position An2 (tooth 2[An2]) and teeth at position An3. Tooth germs on the first, second, and third replacement waves appeared simultaneously on the arches of both sides. During following waves, tooth germs on the left side appeared later than those on the right. Delay of tooth germ appearance On the left side is interpreted as an inhibitory influence of existing tooth germs in accordance with Osborn's (Proc. R. Soc. Lond. Ser. B 179:261--289, '71) theory. The delay of tooth germ appearance on the left arch is most pronounced on the seventh replacement wave. Teeth of the right major row in adults of this species are replaced more frequently than those of the left major row, apparently in correlation with the absence of the first larval tooth at position An2 and teeth at position An3. It is hypothesized that cyprinids evolved the minor rows and specialized teeth of their adult dentition as apomorphic characteristics by the process of neoteny.

Notes: Development of the internal structures of the caudal region (muscles, bones, cartilages, nerves, and blood vessels) of the Da/Da mutant medaka and of wild-type +/+ fish was compared. Muscles and nerves were immunochemically stained by using an antibody to troponin T and antibodies to neurofilament proteins, respectively. Bones and cartilages were stained with alizarin red and alcian blue. In stages 31-32 of the Da/Da embryo, the rudiments of epurals 1, 2, and 3 differentiated dorsally opposite the sites of the ventral rudiments of the parhypural, the lower hypural plate, and the upper hypural plate, respectively. In the +/+ embryo, the rudiments of epurals 1 and 2 differentiated in the later developmental stages (after stage 34), but the rudiment of epural 3 did not differentiate. In the Da/Da embryo, the deep dorsal flexor passed dorsalwards and ended at the rudiment of epural 2, whereas the muscle passed ventralwards and ended at the rudiment of the upper hypural plate in the +/+ embryo. In the Da/Da fry, the epichordal region of the caudal fin fold grew and the notochord extended straight into the tail without bending, while in the + /+ fry, the epichordal region did not grow and the notochord turned dorsalwards within the tail. In the Da/Da fry, extra skeletal rudiments and musculature differentiated in the epichordal region of the caudal fin, whereas these supernumerary structures did not develop in the +/+ fry. The pattern of the epichordal skeleton and musculature in the caudal region of the adult Da/Da fish was an almost mirror-image duplication of the hypochordal structures. These observations suggest that the characteristic expansion of the dorsal fin and epichordal lobe in the caudal region of the adult Da/Da fish is due to a process of ventralization, whereby the dorsal skeleton, musculature, and fin fold are stimulated to differentiate in a pattern like that which is restricted to the anal fin and hypochordal lobe of the wild type.

Notes: Tongue musculature in 24 genera of snakes was examined histologically. In all snakes, the tongue is composed of a few main groups of muscles. The M. hyoglossus is a paired bundle in the center of the tongue. The posterior regions of the tongue possess musculature that surrounds these bundles and is responsible for protrusion. Anterior tongue regions contain hyoglossal bundles, dorsal longitudinal muscle bundles and vertical and transverse bundles, which are perpendicular to the long axis of the tongue. The interaction of the longitudinal with the vertical and horizontal muscles is responsible for bending during tongue flicking. Despite general similarities, distinct patterns of intrinsic tongue musculature characterize each infraorder of snakes. The Henophidia are primitive; the Scolecophidia and Caenophidia are each distinguished by derived characters. These derived characters support hypotheses that these latter taxa are each monophyletic. Cylindrophis (Anilioidea) is in some characters intermediate between Booidea and Colubroidea. The condition in the Booidea resembles the lizard condition; however, no synapomorphies of tongue musculature confirm a relationship with any specific lizard family. Although the pattern of colubroids appears to be the most biomechanically specialized, as yet no behavioral or performance feature has been identified to distinguish them from other snakes.

Notes: Wall and internal organelles of the encysting salt marsh heterotrich ciliate Fabrea salina were examined by bright field and Nomarski interference contrast microscopy and by transmission and scanning electron microscopy. A mucoid sheath believed to be derived from bacteria covers the ectocyst. The possible bacterial origin of this sheath has been demonstrated experimentally by its removal and reappearence after 24 hours. Control ectocyst maintained in sterile seawater did not replace the sheath. The ectocyst has a crinkled appearance. The endocyst is composed of two layers. The inner endocyst material is continuous with that of the plug located at one end of the cyst. The two membranes are separated from one another by an interwall space filled with fibrous material. The cytoplasm, covered by an outer double membrane pellicle, contains mucocysts, pigment granules, microtubules, non-ciliated kinetosomes, Golgi complexes, ribosome-studded endoplasmic reticulum, and mitochondria. The endoplasm contains numerous autophagosomes, mitochondria, and food reserve materials. The macronucleus is centrally located in the cytoplasm of the encysting organism.

Notes: Analysis of lateral and dorsoventral radiographic films shows that ingestion, transport, and mastication in Pedetes capensis (Rodentia) are cyclic and their movement patterns are essentially similar for the three food types offered. During the ingestion cycle, closing of the mouth is accompanied by a backward translation of the condyles, so that movement is predominantly orthal. During the opening stage, the extent of the anterior condylar translation is smaller. As a result the mandibular incisors move ventrally and posteriorly. During the ingestion cycles, food is transported to the back of the tongue, with the transverse rugae and the folds of the upper lip playing important roles.Springhares show a bilateral masticatory pattern; food is chewed on both sides simultaneously. During chewing, the condyles lie in their most forward position at maximum opening of the mouth. The mouth is closed by rotation of the lower jaw around the temporomandibular joint coupled with posterior condylar translation. At the beginning of the slow-closing stage, the upward rotation of the mandible slows and the jaw slowly shifts forward. During the grinding stage, the mandible is shifted forward with both toothrows in occlusion. During the opening stage, the jaw returns to its starting position.Comparison of kinematic and anatomical data on rodent mastication suggests that some dental characteristics form the most important factors regulating the masticatory pattern and consequently allow reasonably reliable prediction of rodent masticatory patterns.

Notes: Avian embryos can be completely paralyzed by injection of neuromuscular-blocking agents. We used a single injection of decamethonium iodide to paralyze embryos at 7, 8, or 10 days of incubation and analyzed the growth of individual bones (clavicle, mandible, ulna, femur, tibia, humerus) and of individual muscles that act upon some of those bones (clavicular and sternal heads of m. pectoralis, and mm. biceps brachii, depressor mandibulae, pseudotemporalis, and adductor externus). Growth of the bones is not equally affected by paralysis. Only 27% of clavicular growth (by mass) but 77% of mandibular growth occurred in paralyzed embryos, whereas the four long bones exhibited 52-63% of their normal growth. Analysis of muscle weight, fiber length and physiological cross-sectional area (weight/fiber length) indicate that there was greater reduction of the muscles acting on the limbs than of those acting on the mandible, i.e., diminished growth of the skeleton is correlated with reduced muscular activity. Specific retardation of clavicular growth is due to fusion of sternal rudiments and collapse of the thorax, as well as virtual absence of the musculature that normally attaches to the clavicle. We discuss these results in the light of intrinsic and extrinsic factors governing growth of tne embryonic skeleton. Paralysis reduces skeletal growth by reducing both the movements taking place in ovo, and the loads imposed on the bones by muscle contraction, changes that represent alterations in the mechanical environment of the skeleton.

Notes: This study using light and electron microscopy indicates that the saccus vasculosus is distinguishable in 9-mm embryos and grows continuously throughout embryonic development to the adult stage. In the saccus vasculosus, epithelial mitoses are observed in all stages studied. Phases of centriologenesis, ciliogenesis, and globule formation have been characterized in developing coronet cells. During the phase of centriologenesis, new centrioles appear in association with pre-existing centrioles and not on deuterosomes. After ciliogenesis, each cilium differentiates to a globule almost at the same time as the other cilia of the coronet cell. The inner membrane system of the globules seems to derive from the ciliary plasma membrane. This membrane system often produces membrane whorls during the development. The different phases of coronet cell development have been found in the same individual and in all the stages studied except the 9-mm embryo. Cerebrospinal fluid-contacting neurons are observed in the saccus epithelium from the 12-mm embryos on and are distinguishable from coronet cells in their early formative stages. The three cell types of the saccus vasculosus increase continuously in number during development. Nerve processes are found in the saccus vasculosus of embryos, whereas differentiated synapses appear later in the fry. The significance of continued coronet cell formation is discussed in relation to a putative coronet cell and/or a globule renewal cycle in the adult.

Notes: Changes of architecture of adult rat gastrocnemius medialis muscle (GM) due to growth were studied in relation to length-force characteristics. Myofilament lengths were unchanged, indicating constant sarcomere length-force characteristics. Number of sarcomeres within fibers was unchanged as a consequence of growth, allowing persistence of differences between proximal and distal fibers in all age groups. Distal fiber length at muscle optimum length was shorter for the 14- than for the 10- and 16-week age groups despite a lack of difference of number of sarcomeres. This is indicative of a shift of optimum fiber length with respect to muscle length to lengths higher than optimum length. Some evidence for the occurrence of distribution of fiber optimum lengths with respect to muscle optimum length was found in other age groups as well, albeit of a smaller magnitude. Muscle and aponeurosis length increased substantially with growth. Functional effects of increased aponeurosis lengths were increased contributions to muscle length changes by the aponeurosis, allowing smaller fiber contributions in older animals. Fiber angle increased approximately 5° with growth. Despite the differences of architecture indicated above, muscle length range between optimum length and active slack length was constant. This was probably caused by widening of this length range in the youngest age group by variations of architecture within the muscle. It is concluded that adaptation of aspects of muscle architecture is an important mechanism for adult muscle growth in rat GM. Of these aspects regulation of muscle length seems a dominant factor.

Notes: A capillary gas chromatographic method is described for the determination of polyamines (putrescine, spermidine and spermine) in the urine of normal human and cancer patients. Morning urine after acid hydrolysis is cleaned up on a silica gel column and derivatized with trifluoroacetic-anhydride. Creatinine in human urine is used as internal standard. Recoveries of polyamines are 96.7% putrescine, 102.6% spermidine (Spd), and 98.7% spermine. SD of the method for Spd is 1.949 ± 0.041 (μg/mg creatinine, mean ±SD, n = 5). The results show that the mean level of polyamines in cancer patients urine is much higher than that in normal human urine. The mean of total polyamines in the normal human and the cancer patients is 2.01 and 44.74, respectively (g/mg creatinine).

Notes: An alumina stationary phase has been assessed in the study of the retention behaviour of the anticancer drug cisplatin and its major hydrolysis products. Parameters such as buffer concentration in the mobile phase, pH, organic modifier and competing ion have been investigated in order to optimize chromatographic separation with ultraviolet detection. The separation scheme developed has been used to monitor the hydrolysis of cisplatin in aqueous and saline media, and to monitor the interaction of hydrolysed solutions of cisplatin with the amino acid cysteine. A new peak was observed in the chromatograms of such mixtures when they had been allowed to stand for periods of greater than 16 h and, from analysis of the data obtained, it was concluded that this new peak was due to a complex formed between the mono-aquo hydrolysis product of cisplatin and the amino acid.

Notes: Midazolam concentrations in patients' plasma was determined after extraction with high performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). GC was selected for routine plasma assays in terms of selectivity, simplicity, precision, accuracy and sensitivity (0.02 μg/ml); HPLC analysis was less sensitive (0.1 μg/mL) than GC; GC/MS was used for analysis validation. Plasma protein binding of midazolam was determined by GC in patients' plasma after in vitro incubation with midazolam, ultrafiltration and extraction; 5% of the drug was unbound to plasma proteins. Midazolam distribution in lipoprotein fractions separated by ultracentrifugation of plasma obtained from patients on prolonged midazolam treatment was also assayed by GC.

Notes: The metabolités of lappaconitine in the urine of humans having been previously administered intramuscularly with lappaconitine hydrobromide were studied using high performance liquid chromatography with electrochemical and ultraviolet detection. The urine was extracted by means of liquid- and solid-phase extractions. Each of the metabolites of lappaconitine was purified by high performance liquid chromatography on a reversed phase column and identified on the basis of the chromatographic behaviour and the detector response. It was proved that lappaconitine, N-deacetyl-16-O-demethyllappaconitine and N-deacetyllappaconitine were excreted in urine from humans receiving lappaconitine.

Notes: A reversed phase high performance liquid chromatographic procedure was developed to quantify 6-thioguanine, 6-mercaptopurine, methylthioguanine, and methylmercaptopurine in red blood cells. The free base of each thiopurine was liberated from the respective nucleoside and nucleotide moiety by acid hydrolysis, which allowed for a determination of the total thiopurine present. 6-Thioguanine and 6-mercaptopurine were analyzed on an octadecyl- silane column using methanol + 20 mM sodium phosphate (15:85), pH 7.5, containing 0.07% tetrabutylammonium chloride. Detection was by potassium permanganate oxidation and fluorescence detection at 290 nm excitation and 400 nm emission. Methylmercaptopurine and methylthioguanine were analyzed on a cyanopropylsilane column using methanol + 40 mM sodium phosphate (18:82), pH 2.7, and then ultraviolet absorption at 314 nm and 290 nm, respectively. The method was used to quantify the four primary thiopurines present in red blood cells of an acute lymphoblastic leukemia patient. The procedure may be a therapeutic monitoring technique that quantifies the cytotoxic drug burden in patients receiving azathioprine or 6-mercaptopurine therapy.

Notes: A sensitive and selective high performance liquid chromatographic method for the simultaneous determination of nitrated polycyclic aromatic hydrocarbons and their reduced compounds has been developed. As the model compounds for the proposed method, pyrene, aminopyrene, nitrosopyrene, and several nitrated pyrenes were used. After separation on a reversed phase column, both nitropyrenes and nitrosopyrene which were not fluorescent, were electrochemically reduced to strongly fluorescent aminopyrenes, and detected chemilumigenically by using bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide as post column reagents. Their detection limits were all at the fmol levels, which were one or two orders lower than those by fluorescence detection.By the proposed method, nitropyrene, 1,3-, and 1,8-dinitropyrenes were detected in sooty emissions of cars after simple purifications.

Notes: Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1μ L of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.

Notes: A high performance liquid chromatographic system with chemiluminescence detection (HPLC-CL) was used for determining phospholipid hydroperoxides in human plasma low density lipoprotein (LDL). This system involved separation of phospholipids from LDL-total lipids with normal phase silica gel HPLC and post-column detection of hydroperoxide-dependent chemiluminescence produced by luminol oxidation during the reaction of hydroperoxide with cytochrome c-haeme. By using HPLC-CL, we could detect phosphatidylcholine hydroperoxide (PCOOH) in human plasma LDL, and the LDL-PCOOH concentration was significantly higher in patients with atherosclerosis and hyperlipidemia than that of healthy volunteers. The LDL-PCOOH level was proportional to the plasma total cholesterol concentration.

Notes: A sensitive and accurate reversed phase liquid chromatographic assay was developed for the determination of 6-mercaptopurine (6MP) (the active metabolite of azathioprine) in human plasma. The assay involved extraction into acetonitrile and dichloromethane from plasma pretreated with 0.038 M of dithiothreitol solution. The residue was analyzed by isocratic chromatography on a C18 analytical column with UV detection at 326 nm. The average extraction recovery of 6MP was 85%.The method has been applied successfully to the determination of 6MP and its metabolites in pharmacokinetic studies.

Notes: A method for the determination of doxorubicin and daunorubicin in plasma is described. The plasma is injected directly into a loop column and then washed with water. After switching the injection valve, the sample is separated on a phenyl column using detection at 254 nm. The detection limit is 10 ng/mL, the coefficient of variation is 7% for 100 ng/mL of doxorubicin and 4% for 200 ng/mL of daunorubicin.

Notes: A method for the determination of lorazepam in rat brain is described using liquid/liquid and solid-phase extraction, followed by high performance liquid chromatography. After addition of chlordiazepoxide as the internal standard, 100 mg brain tissue was homogenized and incubated with alkaline protease. Lorazepam and chlordiazepoxide were extracted three times with toluene. After treatment through a C 18-Bond Elut column, lorazepam and chlordiazepoxide were analyzed isocratically on a reversed-phase column with a mobile phase consisting of methanol + 0.025 M sodium phosphate buffer (66:34, v/v). The eluted drugs were monitored by their absorption at 240 nm. The sensitivity limit of this method was 10 ng of lorazepam per 100 mg of brain tissue sample. The standard curve was linear over the range of 20 to 200 ng lorazepam. The coefficient of variation for day-to-day precision established by 21 replicate analyses was 4.5 to 13.6%.

Notes: A simple high performance liquid chromatographic method has been devised for the measurement of bisoprolol in plasma or serum. The sample (200 μL) is vortex mixed for 30 s with 2 M Tris solution (50 μL), aqueous internal standard (benzimidazole, 2.0 mg/L, 50 μL) and methyl t-butyl ether (200 μL). After centrifugation (9950 × g, 2 min), a portion of the resulting extract is analysed on a microparticulate (5 μm) silica column using 1 mM camphorsulphonic acid in methanol as the mobile phase. Detection is by fluorescence at an excitatioh wavelength of 215 nM. The lower limit of accurate measurement for the assay is 10 μ/L (CV% = 8.9, n = 9) with a lower limit of detection of 5 μg/L. There is minimal interference from either commonly prescribed drugs or endogenous compounds.

Notes: A procedure is described for the removal of sodium dodecyl sulphate (SDS) from proteins isolated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins separated by SDS-PAGE were stained with Coomassie Blue and extracted with Tris-HCI buffer containing SDS. The obtained extracts were subjected to gel permeation chromatography in an acidic aqueous acetonitrile solution. The procedure allows purification of the isolated proteins not only from SDS, but also from Coomassie Blue, buffer salts and other small molecular weight contaminants.

Notes: In experimentally produced alcoholic fatty liver microsomal fatty acid composition was measured using gas chromatography. The results showed an increase in linoleic acid (18:2, n-6) and hexadecaenoic acid (22:6, n-3) and a decrease in arachidonic acid (20:4, n-6) in alcohol-fed rats. Using high performance liquid chromatographic separation of radiolabelled substrate and products, Δ9, Δ6 and Δ5 desaturase enzymes were assayed. The activity of Δ9 and Δ5 desaturase was decreased in alcohol-fed rats and Δ6 desaturase activity was similar in control and alcohol-fed groups. These results indicated there was no causal relationship between desaturase activity and membrane fatty acid changes. Increased amounts of eicosatrienoic acid (20:3, n-9) in rats fed 〈5% fat were observed in both control and alcohol-fed rats. The results indicated that essential fatty acid deficiency was not due to alcohol consumption.

Notes: The activities of coproporphyrinogen oxidase, protoporphyrinogen oxidase and ferrochelatase in liver homogenates of iron-overloaded and acutely or chronically ethanol-treated rats were assayed by high performance liquid chromatographic methods. Decreased coproporphyrinogen oxidase activity was found in chronically carbonyliron-fed rats while the activities of protoporphyrinogen oxidase and ferrochelatase remained unchanged. Coproporphyrinogen oxidase and protoporphyrinogen oxidase activities in both chronically and actuely ethanol-treated animals were not significantly different from controls. In chronically ethanol-fed rats normal ferrochelatase activity was found.

Notes: A light and electron microscopic study of the skin of domestic chickens, seagulls, and antarctic penguins revealed abundant extracellular dermal lipid and intracellular epidermal lipid. Dermal lipid appeared ultrastructurally as extracellular droplets varying from less than 1 μm to more than 25 μm in diameter. The droplets were often irregularly contoured, sometimes round, and of relatively low electron density. Processes of fibrocytes were often seen in contact with extracellular lipid droplets. Sometimes a portion of such a droplet was missing, and this missing part appeared to have been “digested away” by the cell process. In places where cells or cell processes are in contact with fat droplets, there are sometimes extracellular membranous whorls or fragments which have been associated with the presence of fatty acids. Occasionally (in the comb) free fat particles were seen in intimate contact with extravasated erythrocytes. Fat droplets were seen in the lumen of small dermal blood and lymph vessels. We suggest that the dermal extracellular lipid originates in the adipocyte layer and following hydrolysis the free fatty acids diffuse into the epidermis. Here they become the raw material for forming the abundant neutral lipid contained in many of the epidermal cells of both birds and dolphins. The heretofore unreported presence and apparently normal utilization of abundant extracellular lipid in birds, as well as the presence of relatively large droplets of neutral lipid in dermal vessels, pose questions which require a thorough reappraisal of present concepts of the ways in which fat is distributed and utilized in the body.

Notes: Development of neurons in the area triangularis of Gallotia galloti was investigated in Golgi-impregnated brain tissue. Four major neuronal types present in adults were found to originate from two migratory neuroblast types, which were followed from embryonic stage S.32. One type has a thick main medial process, whereas the second type has a long main lateral process. As they migrate toward the periphery of the nucleus, morphological characteristics of maturation appear, including growth cones, filopodia, and outgrowth of axons. Neuroblasts with a main lateral process differentiate into two immature neuronal types, bipolars and pyramidals, observed at S.33 and thereafter. The neuroblasts with a main medial process undergo some somatic translocation through a transitory tangential shaft. Then they develop into monopolar immature forms with a long varicose medial, process, appearing from S.36. onward. Immature bipolar neurons do not experience great changes in their dendritic arborization during development to the adult stage, but pyramidals and monopolars undergo a rapid development of the dendritic tree after S.36. By S.38 archetypes of adult neuronal forms are established. Hairlike appendages first appear on neurons at S.36 They decrease suddenly in S.38 and then proliferate in S.39 when spines first appear. Around the time of hatching, the hairlike appendages begin to disappear and spines become established. Reduction of spines occurs after hatching and continues to the adult stage. Possible influences of several external factors on neuronal maturation are discussed.

Notes: The female reproductive system of Sphaerodema rusticum consists of a pair of ovaries, two lateral oviducts, a median common oviduct, and a median spermatheca. Accessory glands are absent. Each ovary has five free ovarioles branching from the oviduct. Each ovariole consists of a terminal filament, germarium, vitellarium, brown mass, and an exceptionally long pedicel. The terminal filament consists of a central core, interstitial cells, and an outer sheath. In the germarium, which consists of trophic and prefollicular regions, the trophic region or nurse cell chamber is divided into four histologically differentiated zones, distinguished as zones I-IV. Nutritive cords, originating from the posterior end of the trophic core in zone IV extend centrally and join the developing oocytes in the prefollicular chamber and the vitellarium. The compact prefollicular tissue at the base of the trophic core gives rise to prefollicular cells which, after encircling the young oocytes, become modified into follicular epithelial cells, the interfollicular plug, and epithelial plug. The young oocytes descend into the vitellarium and gradually develop into mature oocytes. A compound corpus luteum is observed simultaneously in all the ovarioles of both ovaries after ovulation. Below the epithelial plug there is an accumulation of material, the “brown mass,” which develops cyclically in correlation with the ovulation cycle. Each pedicel stores five mature chorionated eggs ready for oviposition. The epithelium of the anterior region of the pedicel secretes a PAS-positive material. General morphology and histology of the subdivisions of the ovarioles are described.

Notes: The feeding mechanisms of two labrid fishes (Cheilinus chlorurus and C. diagrammus: Labridae: Perciformes) are modeled using four-bar linkage theory from mechanical engineering. The actions of the feeding mechanisms are simulated by a computer program that uses morphometric data to calculate the geometry of mechanism structure. The predictions of three different four-bar linkages regarding the kinematics of feeding are compared to the movements observed through hign speed (200 fps) cinematography. A previously unidentified four-bar chain was found to be an accurate model of the mechanism by which upper jaw protrusion, maxillary rotation, and gape increase occur in Cheilinus. This mechanism involves the anterior jaws including the mandible, maxilla, premaxilla, palatine, and suspensorium. The accuracy of two previously described four-bar linkages was also tested by comparison of model predictions and film results. The opercular linkage proposed by Anker ('74) as a mechanism of jaw depression via opercular levation was found to be a poor predictor of feeding movements. This four-bar chain involves the opercle, suspensorium, interopercle, and mandible. Muller ('87) proposed a mechanism of hyoid depression involving cranial elevation due to epaxial muscle contraction as input motion The links in this mechanism include the neurocranium and hyomandibula, hyoid, sternohyoideus muscle, and pectoral girdle. This model was an accurate predictor of hyoid depression in Cheilinus when simultaneous cranial elevation and sternohyoideus contraction were simulated. Quantitative kinematic models involve simplifying assumptions when applied to complex musculoskeletal systems, but such models have a wide range of applications to vertebrate functional morphology.

Notes: The morphology of both the main nasal cavity and the vomeronasal organ differs among species representing six families of caecilians. The main nasal cavity is either divided or undivided. The vomeronasal organ differs in position (mediolateral, lateral), size (large vomeronasal organ in the aquatic species), and shape (mediolateral extension, vomeronasal organ with a lateral rostral projection). The great amount of respiratory epithelium of the main nasal cavity, the large vomeronasal organ, and its extensive innervation in typhlonectids may reflect both phylogeny and habitat adaptation, for these taxa are secondarily aquatic or semiaquatic and have several concomitant morphological and physiological modifications. The vomeronasal organ is associated with the caecilian tentacle as the tentacular ducts open into it. This association is further evidence for the involvement of the caecilian tentacle in vomeronasal chemoperception and may represent the mechanism by which these animals smell though the main nasal cavity is closed during burrowing or swimming. Labelings of primary olfactory and vomeronasal projections by means of horseradish peroxidase reaction reveal that the pattern of vomeronasal projections is similar in Ichthyophis kohtaoensis, Dermophis mexicanus, and Typhlonectes natans, even though T. natans possess stronger vomeronasal projections relative to olfactory projections than I. kohtaoensis and D. mexicanus. However, there are differences with respect to the patterns of olfactory projections. The olfactory projection of I. kohtaoensis is characterized by many displaced glomeruli. T. natans has the smallest olfactory projection. The nervus terminalis is associated with the olfactory system as shown by selective labelings of olfactory projections.Six characters potentially useful for phylogenetic analysis emerge from this study of comparative morphology. The characters were subjected to analysis using PAUP to see (1) if any resolution occurred and (2) if any groups were distinguished, whether they corresponded to phylogenetic arrangements based on other morphological characters. The characters are too few to produce nested dichotomous sets for all cases, but they do support the two typhlonectid genera examined and Dermophis and Gymnopis as sister taxa discrete from other groups, and they show that species within genera cluster together.

Notes: A kidney from the budgerigar (budgie, parakeet; Melopsittacus undulatus) is composed of cortical reptilian-type nephrons (without loops of Henle) and mammalian-type nephrons (with loops) grouped together in medullary cones. The loop of the mammalian-type nephrons has a descending segment composed of thin and highly interdigitated cells. These thin limb cells have few mitochondria (15% of cell volume), undetectable Na+, K+-ATPase activity, and virtually no basolateral surface amplification. Prior to the hairpin turn, the descending limb thickens, but the cells continue to lack basolateral amplification. Cells just prior to and within the hairpin turn resemble cells of the entire ascending limb. These cells are thick (there is no thin ascending segment in the avian loop), with extensive infoldings of the basolateral membrane surrounding numerous mitochondria (45% of cell volume). The area of basolateral membrane is 25 times that of the apical membrane. The basolateral membrane (but not the apical membrane) is enriched in Na+, K+-ATPase activity. The structure of the avian mammalian-type nephron (as epitomized by the budgie nephron) and the fact that NaCl accounts for over 90% of the osmotic activity of avian urine leads to the conclusion that the countercurrent multiplier of the avian kidney functions by active NaCl transport from the entire ascending limb. No explanation is offered for the transport specializations found in the thick descending segment of the loop, just prior to the hairpin turn.

Notes: The cells-of-origin and the mode and site of termination of the interhemispheric connections passing through the anterior and posterior pallial commissures in the telencephalon of two lizards (Podarcis hispanica and Gallotia stehlinii) were investigated by studying the anterograde and retrograde transport of unilaterally injected horseradish peroxidase. The commissural projections arise mainly from pyramidal cells in the medial, dorsomedial, and dorsal cortices (medial subfield). Additionally some non-pyramidal neurons in the medial and dorsal cortices contribute to the commissural system. Medial cortex neurons project to the contralateral anterior septum through the anterior pallial commissure. The dorsomedial cortex projects contralaterally via the anterior pallial commissure to the dorsolateral septum and to the medial, dorsomedial, and dorsal cortices. The projection to the medial cortex terminates in two bands at the inner and outer border, respectively, of the cell layer; the projection to the dorsomedial and dorsal cortex ends in a zone in layer 1 which previously has been described to be Timm-negative, and in a diffuse band in the inner half of layer 3. The medial subfield of the dorsal cortex projects through the anterior pallial commissure to the dorsomedial and dorsal cortices with a similar pattern of termination to that found for the dorsomedial cortex. The posterior pallial commissure contains only the projections from the ventral cortex to its contralateral counterpart and to the ventral part of the caudal medial cortex. The similarities found between this commissural system and the mammalian hippocampal interhemispheric connections are discussed.

Notes: The distribution of structural and secretory glycoconjugates in the gastric region of metamorphosing Xenopus laevis was studied by the avidin-biotinperoxidase (ABC) histochemical staining method using seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin I, UEA-I; and wheat germ agglutinin, WGA). Throughout the larval period to stage 60, the epithelium consisting of surface cells and gland cells was stained in various patterns with all lectins examined, whereas the thin layer of connective tissue was positive only for RCA.-I. At the beginning of metamorphic climax, the connective tissue became stained with Con A, SBA, and WGA, and its staining pattern varied with different lectins. The region just beneath the surface cells was strongly stained only with RCA-I. With the progression of development, both the epithelium and the connective tissue gradually changed their staining patterns. The surface cells, the gland cells, and the connective tissue conspicuously changed their staining patterns, respectively, for Con A and WGA; for Con A, PNA, RCA-I, SBA, and WGA; and for Con A, RCA -I, and WGA. At the completion of metamorphosis (stage 66), mucous neck cells became clearly idpntifiable in the epithelium, and their cytoplasm was strongly stained with DBA, PNA, RCA-I, and SBA. These results indicate that lectin histochemistry can provide good criteria for distinguishing among three epithelial cell types, namely, surface cells, gland cells, and mucous neck cells, and between adult and larval cells of each type.

Notes: Ultrastructural features of the ovotestes, spermatogenesis, and the mature sperm are described for three galeommatid bivalves, Divariscintilla yoyo, Divariscintilla troglodytes, and Scintilla sp., from stomatopod burrows in eastern Florida. All three species yielded similar results except with respect to mature sperm dimensions. The ovotestis contains three types of somatic cells within the testicular portion: flattened myoepithelial cells defining the outer acinal wall; underlying pleomorphic follicle cells containing abundant glycogen deposits; and scattered, amoeboid cells containing lysosomal-like inclusions which are closely associated with developing sperm. Early spermatogenesis is typical of that reported from other bivalves. In contrast, the late stages of spermiogenesis involve the migration and gradual rotation of the acrosomal vesicle, resulting in a mature acrosome tilted about 70° from the long axis of the cell. The mature sperm possesses an elongated, slightly curved nucleus; a subterminal, concave acrosome with a nipple-like central projection; five spherical mitochondria and two centnoles in the middlepiece; and a long flagellum. The rotational asymmetry and the presence of perimitochondrial glycogen deposits in these sperm are unusual in the Bivalvia and may be associated with fertilization specializations and larval brooding common among galeommatoideans.

Notes: Polychaetes normally possess one pair of nuchal organs at the posterior edge of the prostomium or peristomium. They have been regarded as chemosensory organs. The nuchal organs of four marine polychaete species with different habits were investigated by electron microscopy.Although the shapes of nuchal organs can vary greatly from simple ciliary bands (Scolelepis squamata, Spionidae) to retractile tongue-like, piston- or finger-shaped forms (Eteone longa, Anaitides mucosa, Phyllodocidae; Heteromastus filiformis, Capitellidae), the structural components, including the ciliated supporting cells, sensory cells, and nuchal epidermal cells, are essentially similar. The differences basically concern 1) the position of the sensory cells with relation to the ciliated supporting cells, 2) the location and structure of the nuchal nerve, and 3) the structure of the nuchal cuticle.The diverging nature of this modified cuticle is described and discussed in detail. Comparisons are made with the fine structure of nuchal organs of other polychaete species. Similarities of cellular components of nuchal organs are found not only in the four species studied here but also in all nuchal organs investigated so far. This is hypothesized to be due to the fact that the polychaete stem species already possessed nuchal organs with the respective cell types. Differences in the number and distribution of cellular components and in the overall shape of nuchal organs are thought to have evolved in correlation with the equipment of other cephalic appendages and with different habits and modes of nutrition.

Notes: The buccal glands of adults of the Southern Hemisphere lamprey Geotria australis consist of a pair of small, bean-shaped, hollow sacs, embedded within the basilaris muscle in the region below the eyes and to either side of the piston cartilage. Each gland, which is lined by a simple columnar epithelium and surrounded by an incomplete layer of skeletal muscle, discharges its contents into the oral cavity via a long, narrow duct. In downstream migrating young adults, the epithelial cells are low columnar, intermediate in electron density, and contain dark-staining inclusions and numerous lipid-like droplets. After saltwater acclimation, the epithelial cells become taller and the numbers of dark-staining inclusions increase whereas those of lipid-like droplets decline. By the end of the marine phase, the epithelium is more folded and now also contains dark and light cells. The ultrastructure of the epithelium shows the characteristics of both apocrine and merocrine secretion. Although intra-epithelial nerve endings were not observed, axons and occasional neurons are present in the lamina propria. Since the skeletal muscle capsule is also well innervated and contains neurons, a local feed-back mechanism may regulate the release of buccal gland fluid by monitoring the luminal pressure. Contractions of the skeletal muscle capsule and movements of the basilaris muscle during feeding would presumably assist the movement of secretion along the duct. The secretion possesses anticoagulating and haemolytic properties.

Notes: The early development of Pedetontus unimaculatus from maturation to germ rudiment formation has been described by light and electron microscopy. Newly laid eggs of P. unimaculatus are in the metaphase of the first maturation division, and two successive maturation divisions produce two polar bodies. Nuclear divisions up to the eighth or ninth are accompanied by cytoplasmic divisions, and are holoblastic. Each resulting blastomere contains a single nucleus. Most cleavages are radial, but a few are tangential resulting in the formation of primary yolk nuclei. After the eighth or ninth nuclear division, cytoplasmic divisions are restricted to the egg periphery, and these later cleavages are superficial. Boundaries of blastomeres gradually disappear. Nuclei, which settle in the peripheral cytoplasm, proliferate and differentiate into blastoderm cells, and they also give rise to secondary yolk nuclei. A posterior circular region of blastoderm thickens and concentrates to form a germ rudiment 50-100 μm in diameter. During the formation of a germ rudiment the serosal cuticle begins to form.A similar pattern of cleavage was observed in other species of the Machilidae belonging to four genera in two subfamilies (Machilinae, genus Haslundichilis; Petrobiinae, genera Pedetontus, Pedetontinus, Petrobiellus). The cleavage pattern of the machilids closely resembles that found in the myriapod groups, the Symphyla, Diplopoda, ana Pauropoda, as well as in the apterygote Collembola, but it differs from the purely superficial cleavage pattern characteristic of the apterygote Thysanura sensu stricto (Zygentoma) and the Pterygota.It is concluded 1) that the pattern of early total cleavage changing later to superficial cleavage is a plesiomorphic character for the Antennata, and 2) that the purely superficial pattern is an apomorphic character within the Hexapoda.

Notes: The digestive gland (midgut gland, hepatopancreas) of the American lobster, Homarus americanus (Decapoda: Nephropidae), is permeated with terminal arterioles of the hepatic artery. Fixed phagocytes associated with the terminal hepatic arterioles removed two types of non-biogenic, foreign particles (carbon particles and latex beads) that were injected into the blood. These cells play an important role in the cell-mediated immunity of lobsters.

Notes: The digestive gland (midgut gland, hepatopancreas) of the American lobster, Homarus americanus (Decapoda: Nephropidae), is supplied with blood by the hepatic artery. Numerous branches, ending in terminal hepatic arterioles, permeate the organ and discharge blood into the hemal sinuses. Histological and ultrastructural studies show the arterioles to be elongate tubes that lie in the hemal spaces, interspersed among the digestive tubules of the digestive gland. The terminal hepatic arterioles comprise three distinct populations of cells: endothelial cells form the wall of the blood vessel; hemocytes circulate through the lumen of the vessel; and fixed phagocytes are attached to the outer surface of the endothelium. In addition, extracellular membranes form an endothelial intima that lines the lumen and a perforated membrane that covers the outer surface of the arteriole.

Notes: mAb WE3 recognizes an antigen that is developmentally regulated in the wound epithelium of regenerating newt limbs. The antigen is precociously expressed when pieces of WE3-negative wound epithelium axe grafted subcutaneously (Tassava et al.: Recent Trends in Regeneration Research. New York: Plenum Publishing Co., pp. 37-49, 1989). In the present study, we investigated whether the WE3 antigen is expressed in epidermis of subcutaneous grafts of skin. Small pieces of limb skin were grafted into small tunnels in the lower jaw, limb, and tail, oriented either the same as (epidermis facing out) or opposite to (epidermis facing in) the orientation of the host skin. In most cases, the epithelium migrated from the graft along the wounded surface of the tunnel, closed onto itself, and formed a multilayered “emigrant” epithelium. Infrequently, the migrating epithelium combined with the wound epithelium of the insertion wound. In no case did the epithelium migrate over the cut edge of the grafted dermis. Reactivity to mAb WE3 was first seen at 4 days after grafting, when the migrating epithelium had almost closed over onto itself. By 6 days and thereafter, the entire emigrant epithelium was reactive to mAb WE3. While initially restricted to the emigrant epithelium, at 10 days after grafting and thereafter, reactivity was also seen in the epidermis that remained in contact with the dermis. Expression of the WE3 antigen was not influenced by the orientation of the graft nor by the graft site. The results show that, compared to amputated limbs, the epithelium originating from these grafts precociously expresses the WE3 antigen. Also, epidermis of grafted skin is capable of expressing the WE3 antigen.

Notes: Endocrine cells and brush cells at the bronchiolo-alveolar junctions of the lung of neonatal hamsters were studied by transmission electron microscopy. On both sides of the junctions (bronchiolar and alveolar), clusters of endocrine cells occur as neuroepithelial bodies (NEB). A few solitary endocrine cells are also present at the alveolar sides of the junctions. Some endocrine cells reach from the basement membrane to the air space but the area of apical cell membrane exposed to the airway is small as the cells are largely covered by Clara cells in the bronchioles and by thin attenuations of alveolar type 1 cells in the alveoli. Some Clara cells around NEB contain cytoplasmic lamellar bodies, similar to those characteristically associated with alveolar type 2 cells. A few brush cells are also seen at both sides of the junctions. Long wide microvilli with filamentous cores extend from the apices of the brush cells. Endoplasmic reticulum and Golgi apparatus are moderately developed. Well-developed bundles of intermediate filaments course throughout the cytoplasm of some of the brush cells. The functions of endocrine cells and brush cells are unknown. However, the presence of these cells at the bronchiolo-alveolar junctions of neonatal hamster lungs suggests a role in regulation of respiratory function.

Notes: Anolis carolinensis has two aggressive displays involving movements of the hyoid apparatus: erection of the throat and extension of the dewlap. Erection of the throat is an enlargement of the gular region and dewlap extension consists of a vertical erection of the gular flap. Cinefluoroscopy and high speed cinematography show that the dewlap is extended in three phases: 1) protraction of the entire hyoid apparatus; 2) forward pivoting movement of the ceratobranchials II; and 3) retraction of the ceratobranchials II and the entire hyoid apparatus. The cartilaginous elements of the hyoid apparatus are variably mineralized. The entoglossal process and the hypohyals are the most calcified elements. The mineralized portion of the hyoid body, to which the other elements articulate, presents a complex pattern. The calcification of entoglossal process and the hypohyals stop just where they are fused with the hyoid body. The hyoid body presents four mineralized masses, two central corresponding to the base of the ceratobranchials II and two lateral being the head of the ossified ceratobranchials I. The lateral masses articulate on the central masses by a synovial joint. Morphologically, the ceratobranchials II form the hyoid body and become separated at the mid length of the synovial articulation of the ceratobranchials I and the hyoid body. The calcified matrix of the ceratobranchials II gradually changes from a large calcified mass (within the hyoid body) to a semicircle, opened ventrally, which permits their bending during dewlap extension. The highly mineralized posterior tip of the entoglossal process and the hyoid body serve as a pivot to pivoting forward movement of the ceratobranchials II producing at the change of the pattern of mineralization. Forward movement of the ceratobranchials II is produced by electrical stimulation of the M. branchio hyoideus. The opposition of the throat skin to the movement of the ceratobranchials II produces the bending of those longest elements. Electrical stimulation of the hyoid muscles confirms the key role of M. branchiohyoideus during dewlap extension. Simultaneous contractions of all the hyoid and extrinsic tongue (retractor and protractor) muscles with the M. branchiohyoideus during dewlap extension may be a possible motor pattern for dewlap extension in Anolis lizards.

Notes: The ovotestis of Arion hortensis was investigated by electron microscopy, using serial ultrathin sections and freeze-fracture. Desmosome-like junctions between Sertoli-Sertoli and Sertoli-germ cells (spermatogonia, spermatocytes, spermatids, and spermatids just prior to spermiation) were studied. Desmosome-like junctions were circular when viewed en face and 90--180 nm in diameter. The intercellular space at the junctional site was 15-25 nm in width. An intermediate dense line was absent.Freeze-fracture replicas showed that these junctions formed groups, with one to ten junctions within each group. Groups of junctions were scattered over the Sertoli cell surface. The most prominent feature of this junction was an electron-dense plaque on the cytoplasmic face of the plasmalemma. We describe variation in the development of the electron-dense plaque on the germ cell side at different stages in the development of the germ cell. In the spermatogonium and spermatocyte stages, desmosome-like junctions had equally developed electron-dense plaques. The plaques are pronounced in junctions between Sertoli cells and spermatocytes/spermatids and less developed in relation to late spermatids where nuclear condensation is complete, and appear to be lost at spermiation. Finger-like processes arise from the Sertoli cell as desmosome-like junctions are lost at spermiation.Hypertonic fixative solutions containing dextrose were applied to the ovotestis resulting in cell shrinkage. The intercellular space between Sertoli cells and germ cells was increased except where the desmosome-like junctions were present. We suggest that desmosome-like junctions provide strong adhesive sites between Sertoli and germ cells.

Notes: The ultrastructural studies have shown three types of motor endings in the macaque intrafusal fibers: (1) unindented axon terminals with smooth or shallowly folded postsynaptic membrane; (2) indented terminals with few postsynaptic folds; and (3) indented terminals with heavily folded postsynaptic membrane. The terminals on bag 1 and chain fibers were generally more indented than those on the bag 2 fibers. Deeply indented terminals with highly folded postsynaptic membranes were noticed on the bag 1 and chain endings in spindles from lumbrical but not the biceps muscle. In the individual intrafusal fibers from the biceps and lumbrical spindles, the degree of indentation did not correlate with the extent of postsynaptic folding (P〉.01). Endings on bag 1 and chain fibers in the lumbrical spindles showed a positive correlation between indentation of terminals and their distance from the primary sensory endings (P〈.01), whereas the lumbrical bag 2 endings and the biceps intrafusal endings did not (P〉.01). The shape of the intrafusal motor endings thus is independent of their location but dependent on the type of intrafusal fibers.

Notes: Sockeye salmon were transferred rapidly from freshwater to seawater and the changes in gill morphology, in particular the distribution and sizes of chloride and mucous cells on the afferent filamental surface examined. Salmon that successfully adapted to seawater were compared with salmon that did not adapt to seawater and died as a consequence of osmoregulatory failure. The number of mucus cells (density), determined from scanning electron microscopy, increased significantly after seawater challenge. A greater increase in mucus cell density occurred in the salmon that failed to adapt to seawater. Light microscopy of transverse sections of gills detected no difference in mucus cell numbers after seawater challenge. It is proposed that mucus cells that lie just beneath the gill epithelium are activated in response to the seawater challenge, and migrate and open onto the epithelium. Freshwater-adapted salmon that had low densities of chloride cells prior to the seawater challenge failed to adapt, whereas salmon that had high densities of chloride cells adapted successfully to seawater. In the latter, the density of chloride cells on the afferent surface decreased after 30 days in seawater. The apical surface of the chloride cells of freshwater-adapted sockeye were either smooth or covered with microvilli. A greater proportion of microvilli-covered chloride cells occurred in the freshwater-adapted salmon that subsequently adapted to seawater.

Notes: The ultrastructure of scalariform junctions in the Malpighian tubules of the hemipteran Rhodnius prolixus and the dipteran Aedes taeniorhynchus is described. Both autocellular and intercellular scalariform junctions are illustrated. This is the first report of scalariform junctions in the Malpighian tubules of a dipteran. When combined with previous observations by other authors, the presence of scalariform junctions has now been reported in the Malpighian tubules of insects from five orders, including ametabolous, hemimetabolous, and holometabolous forms. The cell types in which scalariform junctions were found in R. prolixus and A. taeniorhynchus differ in the direction of ion and fluid transport. The cells share the capacity to transport KCl. These same cells also possess morphological features promoting close associations of mitochondria and plasma membranes in the apical region of the cell. The possible role of scalariform junctions is discussed in light of these observations.

Notes: A rapid sensitive and selective isocratic technique was developed for the analysis of plasma epirubicin and three of its known fluorescent metabolites epirubicinol, 4′-O-β-D-glucuronyl-4′-epidoxorubicin and 4′-O-β-D-glucuronyl 1,3-dihydro-4′-epidoxorubicin, with daunorubicin as an internal standard, by using high performance liquid chromatography (HPLC) with fluorescence detection and a ‘Hypersil ODS’ column. The drugs were easily and efficiently extracted with a Sep-Pak C18 cartridge and the mean recoveries were greater than 85%. Intraassay and Interassay coefficients of variation (plasma samples) were better than 8.25%. An example of pharmacokinetic study obtained in a cancer patient after intravenous injection of epirubicine is described.

Notes: An isocratic reversed phase high performance liquid chromatography procedure utilizing ultraviolet and fluorescence detectors linked in series is described for the analysis of cortisone (E), cortisol (F), corticosterone (B), 11-deoxycortisol (S), 11-deoxycorticosterone (DOC), androstenedione (A), testosterone (T), 17-hydroxyprogesterone (17-OHP), progesterone (P), estriol, estradiol, estrone, prednisone acetate and dexamethasone acetate in serum. Serum specimens were extracted with ethyl ether. The optimized mobile phase was methanol + tetrahydrofuran + water 26:18:56, v/v/v). A Shim-pack ODS column was used. The recoveries were 80 to 103%. lntra- and inter-day coefficient of variance were less than 8%. The detection limit is 0.5 pmol per injection volume for estriol, estradiol, E, F and B; 1 pmol for S, A, DOC and estrone; 2 pmol for T and 17-OHP; and 4 pmol for P. Serum from normal subjects and patients with congenital adrenal hyperplasia due to 21- or l7-hydroxylase deficiency were measured, as well as samples of maternal and umbilical cord serum.

Notes: A very sensitive, selective and simultaneous method for the analysis of salivary steroid hormones was examined by discharge-assisted thermospray liquid chromatography mass spectrometry (plasmaspray LC/MS). Plasmaspray LC/MS gave [M + M]+ or [MH  -  H2O]+ as a predominant ion in most of the steroids in this work and in some cases fragment ions were also observed. Eight salivary steroid hormones, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, aldosterone, and testosterone were identified within 10 minutes using the selected ion monitoring technique in conjunction with plasmaspray LC/MS. Progesterone, 17-hydroxyprogesterone, cortisol, aldosterone and testosterone were detected in 1 mL of saliva from a normal child and two patients with congenital adrenal hyperplasia.

Notes: A sensitive and selective high performance liquid chromatographic doxophylline assay with ultraviolet detection has been developed for plasma samples. The drug is isolated from biological samples with a reversed phase C18 disposable extraction column. Plasma standard curves are linear for concentrations of doxophylline from 0.03 to 10 mg/L. At the therapeutic range concentrations, the recoveries are better than 96.9%. The coefficients of variation for the procedure are 4.1% and 2.7% for the concentrations 0.03 mg/L and 10 mg/L, respectively. By this method, pharmacokinetic profiles are obtained for six adult volunteers.

Notes: A high performance liquid chromatographic method for the measurement of bemoradan levels in plasma/serum is described. This method uses Varian's AASP® (Varian Associates, Sunnyvale, CA, USA), a semi-automatic liquid/solid extraction sample preparation system. It requires only small volumes of plasma/serum samples (0.2-1 mL) and needs no organic solvent for sample preparation. The mean recovery of bemoradan at plasma or serum concentrations of 0.5-100 ng/mL is 82%. The assay has a detection limit of 0.5 ng/mL (when 1 mL of plasma/serum is used) and is linear in the concentration range 0.5-500 ng/mL.

Notes: It is of importance to differentiate heroin intake from the absorption of opiate-containing pharmaceuticals or opiates from other sources. A method for the routine determination of O6-monoacetylmorphine (6-MAM), the specific metabolite of heroin in human urine, by gas chromatography and classical detectors without having recourse to gas chromatography/mass spectrometry-selected ion mode (GC/MS-SIM) is described. With dual detection by nitrogen selective and flame ionization detectors, the limits of detection for 6-MAM were determined to be 2 ng/mL and 4 ng/mL urine for a 10 mL sample. When applied to urines preliminarily screened for opiates, the results appeared consistent in comparison with those obtained by GC/MS-SIM. The method was also developed for the simultaneous quantitative analysis of morphine and codeine. The linearity was tested up to 600 ng/mL for the three compounds of interest 6-MAM, morphine and codeine and their absolute recoveries were 76%, 78%, 75% respectively.

Notes: The mouse monoclonal antibody 2D4, which recognizes the terminal GalNAcβ1-4Gal-disaccharide of GgOse3Cer and GgOse5Cer, was used for the detection of ganglio-series gangliosides. The method involves separation of gangliosides on thin layer chromatography plates, followed by silica gel fixation, Arthrobacter ureafaciens neuraminidase treatment and final immunostaining of desialylated gangliosides with the monoclonal antibody 2D4. Both neuraminidase and the hybridoma 2D4 producing the specific monoclonal antibody are commercially available and therefore accessible to all researchers working in this field.Gangliosides from mouse T lymphocytes and the mouse T cell lymphoma YAC-1 have been used as examples. This technique may be used for fast screening of gangliosides with the GgOse5Cer core structure which have been described as T cell markers, antigens in human neuronal disease and receptors for certain pathogenic bacteria.

Notes: Purines and pyrimidines are now routinely separated by HPLC. By careful selection of chromatographic conditions which match the expected changes in hydrophobicity and/or ionic nature of the substrate and products most enzymes of the purine and pyrimidine salvage pathways can be routinely and accurately determined. Ion-paired reversed-phase systems are often the most advantageous. The relevance of such assays to biomedical analysis including their role in the diagnosis of inborn errors of metabolism is stressed.

Notes: The morphology, cell types, and innervation of the several small papillae (x̄ = 17) and two larger papillae, which together form a ring just outside the fimbriae surrounding the suctorial disc of adult Geotria australis, have been studied using various histological stains, including silver impregnation, and scanning and transmission electron microscopy. The epithelium of all papillae consists almost entirely of mucigenic cells. The multivillous and oligovillous cells, which are found elsewhere in the lamprey epidermis, were not observed, and Merkel and polyvillous cells are rare. Free nerve endings are common, however, in the basal layers of the epidermis. Unlike the small papillae, the two large papillae contain a core of skeletal muscle and a prominent layer of dermal collagen. In the submucosa of these large papillae, the nerves form a dense, compact layer that contains many large and probably sensory axons. It is suggested that the oral disc papillae of adult G. australis are encapsulated mechanosensory structures that play a role in enabling the animal to locate and attach to a suitable point on host fishes or other surfaces.

Notes: Although torsion (axial twisting) is rare in living structures, some starfish routinely use ray torsion in the righting process. This study examined the torsional properties of the ray and dorsal body wall of Echinaster spinulosus (Echinodermata: Asteroida: Spinulosidae). The body wall of the ray is composed of a reticular skeleton composed of CaCO3 ossicles sandwiched between collagenous layers in which the fibers are orthogonally oriented. G, the shear modulus of the dorsal body wall, determined from a torsion pendulum, was 95.3 MPa, and damping was extremely pronounced (Δ = 1.36). The torque-rotation curve was J-shaped, and G determined from this data was 176 MPa. Torsional stress relaxation was rapid, with the relaxation spectrum skewed to the left and τmax = 2.3 for anaesthetized tissue and 2.7 for unanaesthetized tissue.Analysis of ray geometry showed that the ambulacral groove reduced torsional stiffness by about two orders of magnitude compared to an ungrooved ray. Ray taper reduced the stiffness of the tip relative to the root. The shear stress distribution determined for the ray cross-section showed stress concentrations around the ambulacral arch, the outer body wall surface, and the inner body wall surface. The ossicles are situated to minimize torsional stiffness of the ray.