Showing papers in "Histochemistry and Cell Biology in 1982"

A Comparative Microphotometric Study of Succinate Dehydrogenase Activity Levels in Type I, IIA and IIB Fibres of Mammalian and Human Muscles

Abstract: Activity levels of succinate dehydrogenase (SDH) were determined kinetically by means of comparative microphotometric measurements in situ. Activities were correlated with fibre types classified histochemically according to Brooke and Kaiser (1970). Analyses of tibialis anterior muscles in the mouse, rat, guinea pig, rabbit, cat and the human showed pronounced variations in the activity profiles of type I, type IIA and IIB fibres of these muscles. Large scattering of enzyme activity existed in the three fibre populations. Overlaps of varying extent were found for the SDH profiles between the different muscles. Type I fibres reveal species diffeences in aerobic oxidative capacity. Whereas the majority of the IIB fibres in rabbit muscle tended to be low in SDH activity, the main fraction of this fibre population was characterized by high activities in mouse muscle. Similarly, the IIA fibre populations revealed opposite properties in mouse and rabbit muscles. These extremes as well as intermediate activity patterns indicate that no general scheme exists according to which the histochemically assessable myosin ATPase is correlated with the aerobic oxidative capacity of muscle fibres in various mammalian muscles.

The influence of tissue section thickness on the study of collagen by the Picrosirius-polarization method.

Abstract: The influence of tissue section thickness on the color and intensity of birefringence displayed by collagen in tissue sections studied by means of the Picrosirius-polarization method, is reported in this paper. When dermal collagen sections of different thicknesses (ranging from 0.25 to 11 micrometers) were studied by this method, it became evident that not only did the intensity of birefringence increase proportionally to tissue section thickness, as was to be expected, but also a gradual shift in color from green through a yellow to red could be observed as tissue section thickness increased. The limitations of the Picrosirius-polarization method for the localization of collagen types I, II, and III in routinely used histological slides is discussed, showing that this method is useful for the study of the distribution of the different types of interstitial collagen in normal adult vertebrate organs.

Exogenous selenium in the brain. A histochemical technique for light and electron microscopical localization of catalytic selenium bonds.

Abstract: Transcardial perfusion or intraperitoneal injections with sodium selenite result in the creation of selenium bonds that can be visualized by physical development. The present paper describes how these catalytic bonds are made visible in the tissues by surrounding them with shells of metallic silver. Based on experiments with chelating agents, the possibility that selenium-metal bonds are the catalysts is discussed. In the brain, the selenium pattern is delicate and highly laminated, the grains of silver being orderly arranged corresponding with the neuropil morphology. The precipitate is most densely packed in cortical regions. The difference in staining intensity seen in different regions of the CNS reflects the density of selenium reactive terminals. The visualized selenium bonds are predominantly located within boutons, and examination in the electron microscope reveals accumulation in the presynaptic regions. In a few places precipitates can also be found in axons, but have not been observed in perikarya or dendrites. The only non-neuronal locations of selenium were sparsely scattered, astrocyte-like neuroglia, predominantly found in the cerebellum and the hypothalamus; infrequently a few blood vessels were also stained. Sections from kidney and liver are presented as examples of localizations outside the CNS of exogenous selenium.

No classical type IIB fibres in dog skeletal muscle.

Abstract: To analyse the fibre type composition of adult dog skeletal muscle, enzyme histochemistry, immunohistochemistry for type I, IIA and IIB myosins, and peptide mapping of myosin heavy chains isolated from typed single fibres were combined. Subdivision of type II fibres into two main classes according to the activity of the m-ATPase after acidic and alkaline preincubation proved to be rather difficult and was only consistently achieved after a very careful adjustment of the systems used. One of these sub-classes of type II fibres stained more strongly for m-ATPase activity after acidic and alkaline preincubation, was oxidative-glycolytic and showed a strong reaction with an anti-type IIA myosin. The other one, however, although unreactive with anti-IIA myosin, was also oxidative-glycolytic, and only showed a faint reaction with an anti-type IIB myosin. Peptide mapping of the myosin heavy chains of typed single fibres revealed two populations of heavy chains among the type II fibre group. Thus, in dog muscle, we are confronted with the presence of two main classes of type II fibres, both oxidative-glycolytic, but differing in the structure of their myosin heavy chains. In contrast to some reports in the literature, no classical type IIB fibres could be detected.

The accumulation and intracellular compartmentation of cadmium, lead, zinc and calcium in two earthworm species (Dendrobaena rubida and Lumbricus rubellus) living in highly contaminated soil.

Abstract: The earthworm Lumbricus rubellus contained more Ca and Zn, and less Pb and Cd, than Dendrobaena rubida living in the same contaminated disused-mine soil. Differences in the kinetics of Ca turnover may account for some of the inter-specific differences in heavy metal burdens, although the calciferous glands do not seem to be directly involved in heavy metal excretion. A comparison of the present findings with published data indicated that the concentration of soil Ca and the bioavailability of heavy metals, both factors being allied to soil pH, are important exogenous determinants of heavy metal accumulation by different earthworm populations. Electron microprobe X-ray analysis of air-dried smears of chloragogenous tissue showed that the metals were fairly specifically compartmentalized into two distinct organelles in both worms: Ca, Pb and Zn were found (associated with P) in the chloragosomes; Cd was found (with S and probably in stoichiometric association) in a more electron-lucent vesicular component, designated the ‘cadmosome’, but which may be identical with the debris vesicles which are characteristic inclusions in conventionally-fixed chloragocytes. The in vivo incorporation of Pb by the chloragosomes of D. rubida was accompanied by the loss of Ca, Zn and P.

Demonstration of laminin, a basement membrane glycoprotein, in routinely processed formalin-fixed human tissues

Abstract: Laminin was demonstrated by immunoperoxidase and immunofluorescence staining in sections of normal human tissues fixed in formalin and routinely processed in paraffin. Exposure of the sections to a solution of pepsin (Burns et al. (1980) Histochemistry 67∶73–78) revealed the antigenicity of this basement membrane glycoprotein. Sections from paraffin blocks stored for years at room temperature could be stained with this procedure. Normal human tissues, developing fetal tissues and tumors could be stained with this method. The staining patterns were similar to those seen in unfixed frozen sections. It thus appears that basement membrane components can be detected by immunohistological means from routinely processed histological samples, once the sections are pretreated with proteases. Staining for laminin could be used in embryonic studies and in histopathology to study the relation of cells to basement membranes and for the visualization of normal and abnormal vascularization.

Mapping, Quantitative Distribution and Origin of Substance P- and VIP-Containing Nerves in the Uvea of Guinea Pig Eye

Abstract: VIP- and substance P-like immunoreactivities were found in considerable concentrations (VIP: 17.3±4.8 pmol/g, mean ± SEM; substance P:11.1±1.8 pmol/g) in the uveal portion of the guinea pig eye.d Immunocytochemistry localised these two regulatory peptides to nerve fibres found principally in a plexus in the iris (substance P) and in an extensive network surrounding the blood vessels of the choroid (VIP). A remarkable anatomical demarcation of the two types of peptide-containing nerves was established by the staining of substance P-containing nerves, which stops at the level of the ciliary body. This uveal area is known to be involved in the ocular responses to nociceptive stimuli. At the ultrastructural level, immunoreactivity for both peptides was localised to distinct subpopulations of p-type nerves, distinguishable by the size of their large dense-cored vesicles. Those immunoreactive for VIP were significantly larger (p<0.0005) than those immunoreactive for substance P (95±7 nm and 82±9 nm respectively; mean ± SD). Interruption of the trigeminal pathway produced a remarkable decrease of substance P immunoreactivity in the anterior portion of the uvea (9.1±1.5 pmol/g, mean ± SEM, control; 5.3±1.3 pmol/g, denervated), but not of VIP immunoreactivity in the choroid. Following colchicine treatment, VIP-immunoreactive neuronal cell bodies were localised in the choroid. The separate anatomical localisations and distributions of the two uveal peptides appear to be related to their different origins and functional roles in the response of the eye to noxious stimuli.

FMRFamide-like immunoreactivity in the nervous system of hydra

Abstract: FMRFamide-like immunoreactivity has been localized in different parts of the hydra nervous system. Immunoreactivity occurs in nerve perikarya and processes in the ectoderm of the lower peduncle region near the basal disk, in the ectoderm of the hypostome and in the ectoderm of the tentacles. The immunoreactive nerve perikarya in the lower peduncle region form ganglion-like structures. Radioimmunoassays of extracts of hydra gave displacement curves parallel to standard FMRFamide and values of at least 8 pmol/gram wet weight of FMRFamide-like immunoreactivity. The immunoreactive material eluted from Sephadex G-50 in several components emerging shortly before or after position of authentic FMRFamide. The presence of FMRFamide-like material in coelenterates shows that this family of peptides is of great antiquity.

The use of β-galactosidase as a tracer in immunocytochemistry

Abstract: A new immunocytochemical method using β-galactosidase as a tracer is described. The positive staining appears blue on an unstained background. The present method has the high sensitivity and specificity of the immunoperoxidase method and appears to be a practical alternative. The substrate has no carcinogenic activity. Staining is permanent and the sections can be dehydrated and mounted in synthetic media. Enzyme and substrate solutions are stable for several months.

Morphological studies on the epiphyseal growth plate combined with biochemical and X-ray microprobe analyses

Abstract: The epiphyseal growth plate of the domestic pig was investigated topologically combining biochemical methods with electron microprobe microanalyses both correlated to histological controls. A lateral resolution of about 50 μm was reached. Highest nuclease activity was found in the lower columnar cell zone, while alkaline phosphatase showed maximal activity in the hypertrophic area, connected with maximal values for extractable, organically bound phosphorus, and extractable Ca and Mg. Acid phosphatase activity reached maximal values in the zone of the lower primary spongiosa, while the extractable Pi had maximal values at the end of the zone of bone remodelling. Microprobe analyses have shown that the extracellular Ca content (per dry mass) remained relatively constant at 0.7% (about 58 mM/kg wet weight for 66% tissue fluid) in all zones of the plate increasing to 1% in the vicinity of the first foci of mineralization. The intracellular P content (per dry mass) was about 4.5%, the extracellular 0.1–0.2% (about 10–20 mM/kg wet weight) increasing also to about 1% in the vicinity of the first foci of mineralization. Thus the Ca x P product was much higher than the ion-product of 2 mM2 which is necessary for an in vitro mineralization of connective tissue. The extracellular S content (per dry mass) as a probable indicator of sulfated proteoglycans was relatively constant at about 3.5% in the different zones but decreased to about 0.3% in the fully mineralized regions. This indicates a loss of sulfur containing substances with mineralization which is not so high since the concentrations per dry mass must be normalized to a unit volume of equal density of mass.

Catecholamine- and acetylcholinesterase-containing nerves in human lower respiratory tract.

Abstract: The innervation of human lower respiratory tract was studied with special emphasis on airways with sodium-potassium glyoxylic acid (SPG) and acetylcholinesterase (AChE) methods to demonstrate catecholamine-containing and acetylcholinesterase-containing nerve fibers. AChE-method revealed a rich network of cholinesterase positive nerves both inside the bronchial glands where they run around and between the acini, and the airway smooth muscle from secondary bronchi to terminal bronchioli. No AChE-positive fibers were found in connection with the blood vessels or within the epithelium of bronchi or bonchioli. The AChE-positive nerve fibers in bronchial smooth muscle greatly outnumbered those containing catecholamine. The SPG-method revealed the presence of adrenergic nerves from the level of secondary bronchi to that of terminal bronchioli. These nerve fibers were most abundant in bronchial glands, where their amount was equal and distribution similar to those of AChE-containing nerve fibers. Outside the glands adrenergic fibers were constantly seen in connection with the bronchial blood vessels in connective tissues surrounding bronchi. A few nerve fibers were also present in airway smooth muscle from the secondary bronchi to terminal bronchioli.

Somatostatin and VIP neurons in the retina of different species.

Abstract: Neurons displaying somatostatin or vasoactive intestinal polypeptide (VIP) immunoreactivity were detected among the amacrine cells in the retina of baboon, cynomolgus monkey, squirrel monkey, cow, pig, cat, rabbit, guinea-pig, rat, mouse, frog and goldfish. Generally, immunoreactive cell bodies were located in the inner nuclear layer with processes ramifying in three more or less well-defined sublayers in the inner plexiform layer. The density of the sublayers and their location varied with the peptide and species investigated. In most cases there was a sublayer in the outermost part (Ramon y Cajal's sublamina 1) of the inner plexiform layer and this sublayer was usually the best developed. In some species a few somatostatin fibres were also detected in the outer plexiform layer, suggesting that some interplexiform cells contain somatostatin. In the baboon VIP was found exclusively in interstitial amacrine cells which have their cell bodies and processes entirely within the inner plexiform layer.

Ultrastructural visualization of galactosyl residues in various alimentary epithelial cells with the peanut lectin-horseradish peroxidase procedure

Abstract: A conjugate of peanut lectin with horseradish peroxidase (PL-HRP) has been employed for ultrastructural localization of glycoprotein with presumed terminal galactose residues in mouse alimentary epithelial cells. The PL-HRP conjugate imparted electron opacity in sites that stain at the light microscopic level, as for example, Golgi cisternae in surface epithelial cells of the stomach and in superficial and deep crypt cells and goblet cells of the large intestine. Ultrastructural staining revealed that Golgi cisternae intermediate between the trans and cis faces stained selectively in these sites. Secretion stored in secretory granules or Golgi vesicles in the cells lacked affinity for PL-HRP conjugate, however. Selective staining of intermediate Golgi cisternae in cells with unreactive secretory product is interpreted as indicating the site of galactosyl transferase activity and a location where galactose occurs transitorily as the terminal sugar in the glycoprotein side chains. The luminal aspect of the surface epithelial cells in the stomach and columnar cells in the colon also stained, but with some variability. Staining of these surfaces was considered possibly attributable to PL affinity of some of the secretory glycoprotein which, after absorbing to the cell surface, lost terminal sialic acid through action of luminal enzyme. PL-HRP conjugate stained granules in pancreatic zymogen cells near the block surface but not in other cells, presumably because of limited penetration of reagent. Secretion on the surface of pancreatic acinar cells or in the lumen also exhibited affinity for PL-HRP complex as did the luminal surface of gastric chief cells. Staining of secretion in the pancreatic zymogen cells and gastric chief cells for galactose appeared inconsistent with lack of evidence for presence of glycoprotein in these sites which failed to stain with the periodic acid-Schiff or periodic acid-thiocarbohydrazide-silver proteinate methods for demonstrating glycoprotein at the light and electron microscopic levels. This discrepancy points to possible selective binding of PL-HRP conjugate to a moiety other than terminal galactose of glycoprotein in a few histologic sites. These results demonstrate the applicability of the PL-HRP technique at the ultrastructural level and provide information concerning the chemical structure of epithelial cell glycoproteins and their biosynthesis.

Localization of bombesin and GRP (gastrin releasing peptide) sequences in gut nerves or endocrine cells

Abstract: Five anti-gastrin releasing peptide (GRP) sera have been characterized against GRP, bombesin and related polypeptides spotted on cellulose acetate discs. Antibodies reacting with the C-terminal G-14 sequence of bombesin and the 19–27 sequence of GRP, were detected in all sera. Antibodies directed exclsively against the bombesin unrelated 1–17 sequence of GRP were found only in one serum (R-6902). With parallel immunohistochemical tests only the C-terminal immunoreactivity was detected in endocrine-paracrine cells of the chicken proventriculus, while both immunoreactivities were present in nerve fibres and a few nerve cell bodies of the mammalian gut. The distribution of GRP- and bombesin-like immunoreactive nerves in the gastric mucosa of both pyloric and oxyntic type the submucosal and myenteric plexus along the whole gastrointestinal wall and at sphincter regions is detailed.

A comparison of two ATPase based schemes for histochemical muscle fibre typing in various mammals.

Abstract: A comparative study was performed on the fibre populations in tibialis anterior muscles of mouse, rat, guinea pig, rabbit, cat and dog using the two different methods of histochemical staining for myofibrillar ATPase after acid (Brooke and Kaiser 1970) or alkaline preincubations (Guth and Samaha 1970). For all species a complete correspondence existed between type I (Brooke and Kaiser 1970) and beta fibres (Samaha et al. 1970). Gross correspondence (greater than 85%) existed between IIA and IIB (Brooke and Kaiser 1970) and alpha beta and alpha fibres (Samaha et al. 1970) respectively in mouse, guinea pig, rabbit, cat and dog. In the case of mouse and dog, this high degree of correspondence was based on the assumption that mouse tibialis anterior contains no type I and the dog no type IIB fibres. For the rat, a pronounced overlap existed between IIA fibres on the one hand and alpha beta and alpha fibres on the other hand as well as between IIB fibres and alpha beta and alpha fibres. These observations lead to the conclusion that the two classification schemes are not interchangeable for all species and that the two terminologies should be used only in relation with the methods from which they were derived.

Fine structure and histochemistry of the tail fin ray in teleosts

Abstract: Ultrastructural and histochemical studies performed on the skeletal elements of the tail fins of six representative species of teleosts enabled the following observations to be made. The electron microscopic pattern of amorphous substance deposition, and the diameter of the collagen fibrils, in lepidotrichia closely resemble those which are typical of cartilage. In addition, lepidotrichia contain chondroitin sulfate AC as the only sulfated glycosaminoglycan, and this glycosaminoglycan shows high levels of interaction with collagen, both features being characteristic of cartilage. Furthermore, the histochemical data presented in this paper suggest that not all of the glycosaminoglycans present in lepidotrichia are bound to protein cores to form proteoglycans. Each actinotrichium consists of a single ultrastructural entity of remarkable width and, thus, is not composed of a bundle of discretely separated collagen fibrils but rather of hyperpolymerized collagen molecules. This aspect differs from the arrangement pattern of all the other interstitial collagens, suggesting that actinotrichia may contain a new type of collagen.

The cellular specificity of lectin binding in the kidney

Abstract: In order to estimate the usefulness of lectins in the study of the functional segmentation of the nephron, the sites of binding of five lectins were identified in the rat kidney. Lectin-peroxidase conjugates were applied to cryostat sections. The bound conjugates were stained with 3,3′-diaminobenzidine for light microscopical observation. Each lectin has a specific binding pattern along the nephron. Reversely, the different segments of the nephron defined by other histological methods can be identified on the basis of their affinity for lectins. The different parts of the thick ascending limb, namely the medullary segment, the cortical segment and the macula densa, can be distinguished even more readily with lectin histochemistry than with any other histochemical procedure. The binding of lectins to luminal membranes in some segments indicate the possibility to use lectins for the separation of particular cell types and for the modification of the transport properties of their membranes.

Capillary supply and cross-sectional area of slow and fast twitch muscle fibres in man

Abstract: The muscles triceps brachii, quadriceps femoris (part vastus lateralis) and soleus were analysed in 6 men and 6 women for fibre composition (% slow twitch, ST-fibres and % fast twitch, FT-fibres), fibre cross sectional areas, and capillarization. Also the fraction of fibres enclosed by their own fibre type was analysed together with the capillary supply of these fibres. Fibre composition was 39(19-60)% ST in m. triceps brachii, 60(29-78)% ST in m. vastus lateralis and 73(49-88)% ST in m. soleus. Fibre areas ranged from 2,320 to 16,667 microns2 being smallest in m. triceps brachii and largest in m. soleus (p less than 0.05) and with ST fibres being significantly smaller than FT fibres in some of the muscles. In all muscles the shape of the fibres was elliptical with the larger diameter being about twice the smaller diameter. Capillary density per cross sectional muscle area was not related to the fibre composition and was 379(302-500) cap/mm2 in m. triceps brachii, 404(284-529) cap/mm2 in m. vastus lateralis and 417(333-592) cap/mm2 in m. soleus. However, capillary supply expressed as fibre type area per capillary was up to 40% larger for FT-fibres than for ST-fibres within the same muscle (p less than 0.05). The capillary supply of enclosed fibres was not different from that of fibres surrounded also by the other fibre type. The results demonstrate that the difference in capillary supply to ST and FT-fibres is less distinct in humans than in other mammals, which is consistent with the metabolic potentials also being more alike.

Peptide-containing innervation of the human intestinal mucosa. An immunocytochemical study on whole-mount preparations.

Abstract: The three-dimensional distribution of the peptide-containing innervation in the human intestinal mucosa was studied by fluorescence immunohistochemistry on whole-mount mucosal preparations. An extensive VIP-immunoreactive nerve supply was demonstrated at all levels, but was markedly increased in density in the distal intestine, where it formed a particularly rich network in close contact with the luminal epithelium. In contrast, substance P-containing nerve fibres formed a looser and evenly distributed innervation at all levels. The muscularis mucosae was richly supplied by VIP- and substance P-containing fibres. Met-enkephalin immunoreactivity was confined to a few scattered nerve bundles running in the muscularis mucosae and around the bottom of epithelial crypts.

Vascular and tubular renin in the kidneys of mice.

Abstract: Monospecific antisera and the immunocytochemical PAP-method have been used to localize renin in the kidneys of mice With this procedure, reaction product was not only observed in the epitheloid cells of kidney vessels but also in kidney tubules: in the apical portion of proximal tubule cells and in some cells of the connecting and the cortical collecting tubule To answer the question, whether the occurrence of renin in kidney tubule cells is the consequence of tubular synthesis or that of glomerular filtration of plasma renin followed by its uptake from the tubular lumen, tracer experiments with radioiodinated renin and with horseradish peroxidase were undertaken The results of these studies as well as other arguments suggest reabsorptive pinocytosis of the filtered hormone as the source of tubular renin

Induced Metachromasia in Bull Spermatozoa

Abstract: The availability of sequential DNA phosphates to bind toluidine blue molecules after acid hydrolysis was studied in normally shaped and misshaped spermatozoa from subfertile and highly fertile bulls. The aim was to associate induced spermatozoal metachromasia with infertility. Some few normally and abnormally shaped cells from highly fertile bulls exhibited an induced metachromasia after being treated with 4N HCl for 10–30 min at 25°C prior to staining. Subfertile bulls contained 12 times as many metachromatic spermatozoa as highly fertile animals. The induced toluidine blue metachromasia is suggested as a rapid and simple method for detecting bull spermatozoa bearing an anomalous DNA-protein complex. This nucleoprotein complex was found to be more frequent in subfertile bulls.

The localization of converting enzyme in kidney vessels of the rat

Abstract: An antibody against pure rabbit lung converting enzyme (CE) showing cross-reaction with CE from other species was used for immunocytochemical studies in the kidney of rats. Using the indirect labelling PAP-technique, specific immunostaining was found in the endothelial layer of all arteries and arterioles of kidney cortex and in some descending vasa recta. CE-positive reactions were also seen in most glomeruli, the reaction product being confined to only a few capillary loops in connection with the glomerular stalk. A few immunstained capillaries in the cortical labyrinth were suspected to belong to the first ramifications of the efferent arteriole. The bulk of all other of the glomerular and peritubula capillaries as well as all veins of the kidney showed no obvious immunostaining. The functional significance of this specific localization pattern of CE in the endothelium of kidney vessels is discussed with respect to the actions of the systemic and the local, intrarenal reninangiotensin-system on kidney functions.

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. I. Optimalization of the staining procedure.

Abstract: A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual human erythrocytes is described. This staining method can be used for the rapid routine discrimination of patients with a deficiency of the enzyme in its homozygote or heterozygote form, but also for quantitative localization of its activity in individual erythrocytes. The staining procedure in its optimal form consists of a treatment of the erythrocytes with sodium nitrite, then a "fixation" in 0.025% glutaraldehyde (under NADP+ protection of the active site of the enzyme), followed by incubation of the cells in suspension in the presence of tetranitro BT, 1-methoxyphenazine methosulphate and polyvinyl alcohol. Using this new technique, a sharp localization is obtained of the glucose-6-phosphate dehydrogenase activity, which enables discrimination between red cells with different levels of enzyme activity, as a consequence of enzyme deficiencies or age changes.

Quantitative succinic dehydrogenases histochemistry. A comparison of different tetrazolium salts.

Abstract: Activity of succinic dehydrogenase (SD, EC 1.3.99.1) was investigated in the livers of 17 male albino rats by means of the tetrazolium salts NBT, INT, MTT, BT and TT, incubations carried out at a determined pH optimum of 7.9 and activities calculated with regard to activity of “nothing dehydrogenase” (ND) measured in control sections incubated without substrate. The highest activity could be achieved by means of NBT, the ratios of activities measured by NBT, INT, MTT, BT and TT being 100:86:82:29:18 and the order of activities corresponding to the order of redox potentials of the tetrazoles. A determination of ND was necessary, because of its influence on the real activity of SD measured in the slices by means of NBT, INT and MTT. Exact measurements could be made if activity was related to the dry weight of the slices or to the content of nucleic acid — bound phosphorus of reference slices; if activity was related to the content of protein of reference slices, the standard deviation would be the 1.4 fold. Meldola Blue (MB) was not a suitable soluble redox dye, the use of it leading to an activity only 23% of that determined when phenazine methosulphate (PMS) applied. A special measuring of the half-formazan of NBT, the molar extinction coefficient determined being 21,840 l/Mol/cm, produced higher accuracy of measurement, but had no influence on the amount of activity of SD. A comparison of the measured activity with reference values obtained by biochemical methods and histochemically quantified with PMS applied, showed a good conformity.

Fluorescent properties of monoamine neurons following glyoxylic acid treatment of intact leech ganglia

Abstract: The SPG modification (de la Torre and Surgeon 1976) of the glyoxylic acid method for amine condensation is a straightforward procedure which can be used upon intact ganglia from the leech. Intense fluorescence of the neurosomata of identified neurons which contain either indoleamine (serotonin, 5-HT) or catecholamine (CA) is obtained in less than 30 min. The fluorescence of the 5-HT containing neurons is yellow (518-526 nm) and decays more rapidly than the dominant blue emission (478-480 nm) of the CA neurons. Most importantly, the SPG technique greatly enhances the visibility of the axonal processes of neurons which contain both classes of amines over the fluorescence produced by formaldehyde condensation techniques. Both blue and yellow fluorescent varicosities are easily distinguished in the longitudinal connectives and lateral roots of the leech C.N.S. Because of its simplicity and high fluorescence yields, the SPG method for histochemical fluorescence should contribute to investigations of amine functions in invertebrate nervous systems.

A comparison of the ability of serum and monoclonal antibodies to gastric inhibitory polypeptide to detect immunoreactive cells in the gastroenteropancreatic system of mammals and reptiles

Abstract: A monoclonal antibody raised to gastric inhibitory polypeptide (GIP) has been compared with conventional rabbit and guinea-pig antisera to GIP. Four staining methods were tested and of these the peroxidase antiperoxidase (PAP) method proved to give the best results with both the mouse and rabbit antibodies. The monoclonal antibody, when used to stain pancreatic tissue, gave negative results whereas a distinct population of gut endocrine cells was readily demonstrable, suggesting that GIP is not a constituent of the mammalian pancreas. The monoclonal antibody was found to be the most sensitive for immunocytochemistry achieving the titre of 1:106 in rat gut. A C-terminal specific antibody, with a high affinity and avidity to GIP, it was clearly the preferred antibody for immunocytochemical studies.

Immunohistochemical demonstration of a dermorphin-like peptide in the rat brain.

Abstract: The new opioid heptapeptide dermorphin, first isolated from frog skin, has been localized immunohistochemically in nerve cell bodies and nerve fibers of the arcuate-periarcuate region, around the organum vasculosum of the lamina terminalis (OVLT) and in the subfornical organ of the rat brain. A role of dermorphin in modulating LHRH release is suggested.

The distribution of polypeptide YY (PYY)- and pancreatic polypeptide (PP)-immunoreactive cells in the domestic fowl

Abstract: The distribution of the polypeptide which has an N-terminal tyrosine and a C-terminal tyrosine (PYY)- and pancreatic polypeptide (PP)-immunoreactive cells were investigated in the gut of the domestic fowl. PYY-immunoreactive cells were observed in the duodenum and jejunum. PP-immunoreactive cells were seen in the duodenum, jejunum, ileum and colon. Both PYY- and PP-immunoreactive cells were extended from the basal lamina to the gut lumen i.e. of open type. PYY-immunoreactive cells occurred mainly in the basal and middle portion of the villi. On the other hand, PP-immunoreactive cells were located mostly in the crepts. The occurrence of PYY-immunoreactive cells in the upper part of the small intestine is rather similar to that of amphibians and reptiles, than to that of mammals, where PYY-immunoreactive cells are located in the distal part of the small intestine and in the large intestine.

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish, Poecilia formosa and its related, triploid unisexuals.

Abstract: Amounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wild-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3–3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6–1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5–3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0–5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4–2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of 3H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.

Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry.

Abstract: The micronucleus test is a cytogenetic method for the screening of mutagens and carcinogens which exhibit clastogenic mechanisms of action. After application of clastogenic agents chromosomal fragments or even whole chromsomes can remain as conspicuous structures (micronuclei) in a small fraction of anucleated polychromatic erythrocytes which can be visually scored using a microscope following staining with May-Grunwald-Giemsa solutions. These time-consuming, painstaking, and tedious manual evaluations are often sources of unreliability and uncertainty. Here, a fluorescence technique is presented which applies DNA and protein fluorochromes to discriminate normal anucleated erythrocytes from micronucleated erythrocytes using a fluorescence microscope. This method is particularly tailored to be applied to flow cytometric instrumentation. Data obtained manually and automatically in flow show a strong linear correlation with high significance (r=0.96) as far as the percentage of micronucleated erythrocytes as an indicator for the mutagenicity of a given drug is concerned. These results have been obtained by means of the established clastogens cyclophosphamide and mitomycin C.