Localization of CRF immunoreactivity in the central nervous system of three vertebrate and one insect species

Summary By use of a specific antiserum against synthetic ovine corticotropin-releasing factor (CRF) in the peroxidase-antiperoxidase (PAP) immunocytochemical procedure (Vandesande and Dierickx 1976), CRF-like antigenic determinants were demonstrated in the central nervous system of a human fetus, the Wistar rat, the frog Rana ridibunda, and the American cockroach Periplaneta americana. The immunoreactive CRF-producing cells occur mainly in the nucleus paraventricularis of the rat, while in Rana ridibunda these cells occur in the nucleus praeopticus. Immunoreactive CRF-containing fibres were also visualized. Very clear CRF-immunoreactive products were observed in the brain as well as the corpora cardiaca (CC) and corpora allata (CA) of the cockroach Periplaneta americana. ACTH-immunoreactivity was also demonstrated in the brain-CC-CA complex of this insect. Double immunohistochemical staining (Vandesande 1983) also revealed that both the CRFand ACTH-like substances occur in different neurosecretory neurons and nerve fibres. These results suggest that the antigenic determinants of CRF are very similar in vertebrates and insects bespeaking their very long evolutionary history.     

Electron microscopic study on the larval and adult corpus allatum of Oncopeltus fasciatus dallas (insecta,heteroptera)

Summary 1. The ultrastructure of the corpora allata of last larval instars and adults of Oncopeltus was studied. The unpaired gland undergoes submicroscopic alterations and shows signs of degradation in old animals. The organ is partly covered and penetrated by corpus cardiacum tissue. Axons with different types of neurosecretory granules form synaptoid contacts with the corpus allatum cells.2. Dark and light gland cells can be differentiated on account of the degree of electron density. The former predominate during the last larval stage and in the young imago, the latter in mature males and females. It is highly probable that the light cells are the active (i.e. hormone producing) ones and the dark cells the inactive ones.3. The active cells are characterized by rough endoplasmatic reticulum (often in whorls), small amounts of smooth endoplasmatic reticulum and many multivesicular bodies. Abundant free ribosomes, a not particularly well developed Golgi apparatus, dense bodies, and cytolysomes are present in active and inactive cells.4. The nuclei contain one to four prominent and variously shaped nucleoli, which show differences between adult males and females with respect to their location in the nucleus.5. The corpus allatum cells of Oncopeltus are obviously engaged in extensive protein synthesis. Tangible structural indications for the manufacture of juvenile hormone were not observed. Possible sites of hormone release are discussed.This study was made possible by a fellowship and grants from the Deutsche Forschungsgemeinschaft and was supported by research grants, administered by Prof. Scharrer, from the U.S.P.H. Service (NB-05219; NB-00840 and NS-07512). Present address of author: Institut für Allgemeine Zoologie der Johannes Gutenberg-Universität, D-6500 Mainz, Saarstraße 21, Bundesrepublik Deutschland.I am indebted to Prof. Scharrer for guidance and criticism. I also wish to express my appreciation to Mrs. S. Wurzelmann and Mr. S. Brown for their excellent technical assistance.     

The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3 beta-hydroxy-delta 5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

Summary A technique which allows for the continuous microdensitometric monitoring (at 3.3 s intervals) of an enzyme reaction has been applied to a study of 3-hydroxy-⁵-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5µm thick) of the pro-oestrous rat ovary. Initial reaction rates for NAD⁺ reduction by the activity of the enzyme were maintained for less than 3 min at all the concentrations (0-500µmol/l) of co-factor employed. The relationship between NAD⁺ concentration and enzyme activity under initial velocity rate conditions was hyperbolic and maximum enzyme activity was seen when the NAD⁺ concentration was greater than 250µmol/l. The apparent Michaelis—Menten constant (K _m) was 29µmol/l and V_max 0.63µmol NAD⁺ reduced/min/cm³ corpora lutea.     

Responsiveness of the adult cricket (Gryllus bimaculatus andAcheta domesticus) retrocerebral complex to allatostatin-1 from a cockroach,Diploptera punctata

Brain-retrocerebral complexes of female crickets,Gryllus bimaculatus andAcheta domesticus, treated with antibody to allatostatin-1 from a cockroach,Diploptera punctata, show extensive immunoreactivity. The results suggest that allatostatins or allatostatin-like molecules are produced in neurosecretory cells of the brain and are delivered to the corpora allata through nervous connections and/or via haemolymph. Radiochemical measurements of juvenile hormone III biosynthesis by isolated corpora cardiaca-corpora allata complexes from adultG. bimaculatus have been used to demonstrate an in vitro sensitivity of these glands to allatostatin-1 fromD. punctata. Allatostatin-1 is a relatively potent inhibitor of juvenile hormone III biosynthesis in corpora allata of both young adult females and males. In glands taken from 3-day virgin females, 50% inhibition of hormone biosynthesis is reached at ca. 3 nmol·l^-1 allatostatin-1. The inhibitory action of allatostatin-1 is rapid, dose-dependent and reversible. Addition of 200 mol·l^-1 farnesol to the incubation medium prevents inhibition of juvenile hormone III biosynthesis by allatostatin-1. Juvenile hormone III biosynthesis by isolated corpora allata of 3-day female house crickets,A. domesticus, is also susceptible to inhibition by 1 mol·l^-1 allatostatin-1.Abbreviations ASB2 Diploptera punctata allatostatin-5 - CA corpora allata - CC corpora cardiaca - Dip A-1 Diploptera punctata allatostatin-1 - HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid - JH juvenile hormone(s) - Mas-AS Manduca sexta allatostatin - MF methyl farnesoate - NCA nervus corporis allati - NCC nervus corporis cardiaci - SEM standard error of mean - TRIS Tris(hydroxymethyl)aminomethane     

Immunoreactive material resembling vertebrate neuropeptides in the corpus cardiacum and corpus allatum of the insect Leucophaea moderae

Summary The presence and differential distribution of substances antigenically related to known vertebrate neuropeptides demonstrated within the corpus cardiacum of the insect Leucophaea are as follows: Of ten mammalian antisera tested, six yielded substantial immunoreactive deposits resembling oxytocin, somatostatin, Substance P, met-enkephalin, bombesin, and neurotensin, respectively. In the remaining four, the reaction was moderate (vasopressin, -endorphin) or marginal (LH-RF, calcitonin). With regard to their regional distribution, these biochemically distinct reaction products seem to fall into two groups: (1) Materials resembling oxytocin, vasopressin, met-enkephalin, -endorphin (and presumably also neurotensin and LH-RF) predominate in the central release area of the organ and are considered to be of extrinsic (cerebral) origin. (2) Substances localized primarily in areas rich in intrinsic glandular cells of the corpus cardiacum, and revealed by antisera raised against somatostatin, Substance P, and bombesin, are judged to be synthesized and stored within this organ. In peptidergic fibers entering the adjacent corpora allata, thus far Substance P-, -endorphin-, and LH-RF-like immunoreactivities have been demonstrated. Some of these new neuropeptides may be contained in classical neurosecretory neurons, formerly identified by less specific methods, others must be assigned to additional peptidergic neurons heretofore unknown.Supported by NSF grant BMS 74-12456 (B.S.). The excellent technical assistance of Mrs. Sarah Wurzelmann is gratefully acknowledged     

The ultrastructure of the corpus luteum of the guinea-pig

Summary An electron-microscopic investigation, based on the suggestion that differences seen in progesterone levels under differing hormonal conditions might be reflected in the ultrastructural organisation of the lutein cells of the guinea-pig was undertaken. Comparisons were made between corpora lutea taken from animals during the normal oestrous cycle, pregnancy and lactation, and after hysterectomy or hypophysectomy.The lutein cells from the oestrous cycle corpus luteum appeared to be of two types, light and dark. The former were more numerous. The main difference between them lay in the arrangement of the endoplasmic reticulum. Lutein cells from corpora lutea (with the exception of the old degenerating corpora lutea) all contained well-developed agranular endoplasmic reticulum, little granular endoplasmic reticulum, several electron-dense lipid granules, lysosomal bodies which ranged from small spherical bodies to large autophagic vesicles and mitochondria. The mitochondria were numerous, and in the corpus luteum of pregnancy, they were closely associated with the parallel arrays of granular endoplasmic reticulum.With minor exceptions, the lutein cells of the guinea-pig present a strikingly uniform picture despite their hormonal condition.The manner in which this uniformity of ultrastructure may be related to observed differences in progesterone levels in the corpus luteum of the guinea-pig is discussed.Meat and Livestock Commission (MLC) Scholar.The authors wish to thank Dr. J. S. Perry for doing the surgery involved in this work and for the specimens of corpora lutea of hysterectomy. They are also grateful to him for his helpful discussions and interest throughout.     

Production,isolation and characterization of extracellular protease of an alkaliphilic strain of Arthrobacter ramosus,MCM B-351 isolated from the alkaline lake of Lonar,India

An extracellular protease was produced by Arthrobacter ramosus isolated from the alkaline lake of Lonar, Buldhana District of Maharashtra, India when grown on a synthetic medium of pH 10 containing casein. The optimum conditions for production were 3.0% initial casein concentration, 2% inoculum of 1 × 10⁸ cells/ml, pH 9.0, temperature 30 °C and shaken culture conditions. The protease was purified by ammonium sulphate precipitation followed by Sephadex G-100 chromatography. Two proteases viz. Arthro I and Arthro II, having molecular weights 21 and 11.4 kDa respectively were isolated. The Arthro II fraction had K _m 395 g/ml and V _max 10.55 g/min for azocasein. The maximum activity of enzyme was at 55 °C and pH 8. It was thermostable (up to 80 °C), alkali stable (pH 12) and stable in commercial detergent. The enzyme may contain a thiol group at the active site.     

Isolation, physiological characterization, release and sequence elucidation of a hypertrehalosaemic neuropeptide from the corpus cardiacum of the stick insect, Sipyloidea sipylus

ABSTRACT. The corpora cardiaca of the stick insect, Sipyloidea sipylus Westwood, contain peptidic material which elevates blood lipids in migratory locusts, blood carbohydrates in American cockroaches, and activates glycogen phosphorylase in the fat body of the cockroach in a time- and dose-dependent manner. The active principle is found in appreciable amounts only in the corpora cardiaca; slight hyperlipaemia is caused by extracts made from corpora allata and abdominal ganglia, whereas brain, suboesophageal and thoracic ganglia are not active. The adipokinetic activity is already present in corpora cardiaca from second instar (first day) nymphs. The factor retains its adipokinetic activity after boiling for up to 1 h. Conspecific injections of extracts from corpora cardiaca of S.sipylus cause hypertrehalosaemia in ligated stick insects and activate glycogen phosphorylase in non-ligated S.sipylus. After incubation of corpora cardiaca in vitro in saline with high concentrations of potassium and calcium, one fraction with adipokinetic (in locusts) and hypertrehalosaemic (in stick insects) activity can be isolated from the medium by RP-HPLC. Fractionation of a methanolic extract of corpora cardiaca from S.sipylus by RP-HPLC shows that active compounds are confined to apparently three absorbance peaks. The material of the highest absorbance peak was purified to homogeneity by RP-HPLC, and its amino acid composition determined after acid hydrolysis with HCl and with methanesulfonic acid revealed the residues Asx, Thr₍₃₎, Glx, Pro, Gly, Leu, Phe and Trp. The primary structure of this hypertrehalosaemic factor is assigned as a blocked decapeptide, pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH₂, from its FAB spectrum and metastable scans of its FAB spectrum. The structure is confirmed by synthesis; the synthetic and natural peptide co-chromatograph, and the synthetic peptide elevates blood carbohydrates in ligated stick insects and activates fat body phosphorylase in non-ligated S.sipylus.     

Macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) resins—Versatile immobilization supports for biocatalysts

Crosslinked macroporous hydrophilic poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)s [abbreviated poly(GMA-co-EGDMA)] with identical chemical structure (60% of glycidyl methacrylate) but with varied average pore sizes (from 30 to 560 nm), specific surface areas (from 13.2 to 106.0 m²/g), specific volumes (from 0.755 to 1.191 cm³/g) and particle sizes (less than 100–650 μm) were synthesized via suspension polymerization. The influence of the resin properties on the loading of Candida antarctica lipase B (Cal-B) during immobilization and on the hydrolytic (hydrolysis of para-nitrophenyl acetate) and synthetic (ring-opening polymerization of -caprolactone) activity of the immobilized Cal-B were studied. Immobilization of Cal-B was performed at different temperatures and pH values. Cal-B immobilized at 30 °C and pH 6.8 was leading to increased activities. By decreasing the resin diameter: (i) the amount of Cal-B adsorbed onto the resin decreases, (ii) the conversion of para-nitrophenyl acetate increases (hydrolytic activity) and (iii) the conversion of -caprolactone and the molecular weight of the synthesized poly--caprolactone increases (synthetic activity). Varying the porosity parameters results in different hydrolytic and synthetic activities. Pore sizes of all synthesized resins (from 30 to 560 nm) are big enough to overcome diffusion limitations. Therefore increasing the pore size of the resins resulted in a large increase in the hydrolytic and synthetic activity. Increasing the specific surface area resulted in an increase of activities, as the result of alleviated substrate approach to the immobilized enzyme zones. The obtained results were compared to results from dried Cal-B powder and Novozyme 435. Resin with particle size less than 100 μm and pore size 48 nm had much higher hydrolytic activity than both dried Cal-B powder and Novozyme 435. Nearly similar trends were observed for the synthetic activity.Via the DMSO leaching technique we could show that about 80% of Cal-B was covalently attached to the macroporous resin.     

Origin,destination and mapping of tritocerebral neurons of locust

Summary The connectivities of the tritocerebrum of locust (Locusta migratoria L., Schistocerca gregaria (Forsk.)) were studied histologically and by means of cobalt chloride infusion. Its neuropil consists partly of fibers which traverse the tritocerebrum and areas consisting of neuropilar agglomerizations (glomeruli). The following direct connections between the tritocerebrum and other regions were observed: connections to 1) dorsal and lateral brain regions (mushroom body, optic lobe), 2) the ventral nerve cord, 3) the stomatogastric nervous system (here the protocerebrum and the subesophageal ganglion are also involved in these connections), 4) the retro-cerebral glands (corpora cardiaca, corpora allata), and 5) muscles of the foregut. Acknowledgements. This study was supported by grants from the Deutsche Forschungsgemeinschaft to N.K. The authors wish to thank Dr. H.-W. Honegger (Fachbereich Biologie, Universität Konstanz) and Dr. N.J. Strausfeld (E.M.B.L., Heidelberg) for helpful comments and for assisting with the English text     

Ultrastructure of the reactivated corpus luteum after embryonic diapause in the marsupial,Macropus rufogriseus banksianus

Summary During embryonic diapause in the red-necked wallaby, M. r. banksianus, both the corpus luteum and uterine blastocyst remain dormant, and are reactivated following removal of the suckling pouch young (RPY). The morphology of dormant and reactivated corpora lutea has been studied throughout the 26.5 days of delayed gestation. Corpora lutea at 0, 2 1/2, 4, 9, 14, 21 and 25 days after RPY were fixed by perfusion. From day 4 to day 14 after RPY there was a progressive increase in the amount of smooth endoplasmic reticulum and the numbers of mitochondria. However there was a decrease in mitochondrial size from 1–2 m in diameter (0 days after RPY) to 0.5–1 m (14 days after RPY). Densely-staining granules (approximately 0.2 m in diameter) were first observed in the luteal cells at 4 days after RPY. The maximum density of granules was observed at 21 days after RPY. Shortly before birth (25 days after RPY) the number of secretory granules had significantly decreased and the features of cellular regression were evident. As with the eutherian mammals, the wallaby luteal cells have all the structural organelles associated with steroid hormone production. The numbers of densely-staining granules are greatest at 21 days after RPY and may reflect the luteal progesterone content since similar granules in the sheep and cow have been shown to be associated with elevated levels of progesterone.     

Organochlorine and organophosphorus pesticide concentrations in water,sediment, and selected organisms in Lake Naivasha (Kenya)

Water, sediment, red swamp crayfish (Procambarus clarkii) and black bass (Micropterus salmoides) from Lake Naivasha were analyzed for selected organochlorine and organophosphorus pesticide residues. The mean p,p'-DDT, o,p'-DDT and p,p'-DDE residue levels recorded in black bass (28.3 (± 30.0), 34.2 (±54.0) and 16.1 (±16.1) g kg^–1, respectively) and crayfish (4.6 (±5.1), 3.2 (±2.8), and 1.4 (±1.1) g kg^–1, respectively), were higher than previously recorded. This indicated recent usage of technical DDT in the lake's catchment. Levels of p,p'-DDT, higher than those of p,p'-DDE further emphasized this. Mean lindane, dieldrin, -endosulfan and aldrin concentrations in black bass were 100.5, 34.6, 21.6 and 16.7 g kg^–1, respectively. The same residues were detected at lower concentrations in crayfish at 2.0, 2.0, 2.0 and 1.9 g kg^–1, respectively. The higher fat content (3.7 ± 2.7% SD) in black bass (compared to 0.6 ± 0.3% in crayfish) accounted for the significantly higher residue concentrations in black bass. Organophosphate pesticides were the most commonly used pesticides in the lake's catchment, but none was detected in any of the samples. The results indicate that there is need for further work to identify sources and fate of pesticide contaminants, as well as to improve monitoring of pesticide use throughout the catchment.     

Conséquences du traitement préalable par le précocène 2 sur les réponses du criquet migrateur,Locusta migratoria, à un apport en hormone juvénile exogène

Un apport unique de 10 g d'hormone juvénile 1, réalisé chez des criquets du dernier stade larvaire à corpora allata intacts ou préalablement soumis à une application de precocène 2, provoque, à la mue suivante, l'apparition d'individus dont les caractéristiques sont intermédiaires entre celles du dernier stade larvaire et celles de l'imago. Cependant, les criquets traités par le précocène 2 présentent des variations phénotypiques plus prononcées, se traduisant par un maintien accru des caractéristiques larvaires. Les études ultrastructurales montrent que les corpora allata dégénèrent rapidement sous l'action des 400 g de précocène 2.

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Summary In fifth instar locusts, with intact corpora allata or previously subjected to precocene 2 action, a single injection of 10 g juvenile hormone 1 leads, at the next molt, to the appearance of individuals displaying characters ranging from the typical larval to the typical imaginal ones. But, unexpectedly, the phenotypic variations are more marked in the precocene 2 treated locusts, and are typified by an increased retention of larval characters. Electron microscopic studies indicate that the use of 400 g of precocene 2 rapidly leads to the degeneration of the corpora allata.

     

Hormonally-regulated differential expression of the two duplicated insecticyanin genes,ins-a and ins-b,during development of the tobacco hornworm,Manduca sexta

The effects of juvenile hormone (JH) and 20-hydroxyecdysone (20E) on the developmental expression of the two insecticyanin genes, ins-a and ins-b, were investigated with two gene-specific probes. Removal of the corpora allata (-CA, source of JH) clearly delayed and down-regulated the epidermal expression of these genes but enhanced their expression in the fat body during the early development of the fifth instar. Application of JH I to the -CA larvae at the time of head capsule slippage completely restored the normal epidermal expression pattern of the two genes in the early fifth instar, then INS-a mRNA declined prematurely whereas INS-b mRNA remained similar to that in the intact larvae. By contrast, in the fat body of -CA larvae, the exogenous JH had little effect on the levels of INS-a mRNA, but enhanced expression of INS-b mRNA relative to intact larvae. Culture of epidermis from day 1 fifth instar larvae with 40 ng/ml 20E for up to 24 h accelerated the loss of INS-a mRNA without affecting the levels of INS-b mRNA. Both mRNAs declined in isolated larval abdomens over a 24 h period, and this decline was slowed by 1 g methoprene (a JH analog). Together these results indicate that JH controls the levels of the two mRNAs in both the epidermis and fat body, with additional factors involved in regulating these genes in the fat body during the molt and in the epidermis during the growth phase.     

Allatostatins and allatotropin in the corpus cardiacum/corpus allatum complex of larval and adult lepidopterans studied by confocal laser scanning microscopy: correlation to juvenile hormone biosynthesis

Peptidergic innervation of the corpus cardiacum/corpus allatum (CC/CA) retrocerebral complex, and neurosecretory areas of the brain of the lepidopterans Lacanobia oleracea, Heliothis virescens and Manduca sexta was studied by immunocytochemistry linked to confocal laser scanning microscopy. The patterns of immunostaining resulting from the simultaneous application of fluorochrome-conjugated antibodies against Manduca sexta allatostatin (Mas-AS), M. sexta allatotropin (Mas-AT), and a representative of the –Y/FXFGL-NH₂ superfamily of allatostatins was correlated with the physiological effects of these putative allatoregulatory peptides on juvenile hormone (JH) biosynthesis by the corpora allata. Whereas the two types of allatostatin immunoreactivity are present in both larval and adult CA of the three species, allatotropin immunoreactivity occurs only in the adult gland. The conclusion that withdrawal of the stimulatory effect of allatotropin is unlikely to be involved in the downregulation of CA activity prior to the onset of metamorphosis, but that an inhibitory influence of at least Mas-AS is important, is borne out in physiological experiments on JH biosynthesis in M. sexta larvae (Mas-AS inhibitory, Mas-AT without effect). Immunoreactivity to the Y/FXFGL-NH₂ allatostatins is present in both larval and adult CA and CC, frequently co-localised with Mas-AS. The function of this peptide family in the retrocerebral complex remains enigmatic since experiments on JH biosynthesis, either when the peptide is administered alone, or together with Mas-AS, show no effect on JH biosynthesis.Financial support was provided by The Wellcome Trust (063367/Z/00) (to A.T.) and by the Pesticide Safety Directorate of the Department for Environment, Food and Rural Affairs (to N.A. and R.J.W.)     

Binding and precipitating activities ofErythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins fromE. corallodendron,E. cristagalli,E. flabelliformis,andE. indica

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certainErythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 271034-41]. The present work describes a comparative study of the binding and precipitating activities of fourErythrina lectins,viz. E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, theErythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.Abbreviations EAL, ECorL, ECL, EFL, and EIL represent the lectins from the seeds ofErythrina arborescens, - E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica respectively - AFOS thetri-antennary complex type oligosaccharide from asialofetuin - AFGP the tri-antennary glycopeptide from asialofetuin - MeGal methyl -d-galactopyranoside Unless stated otherwise all sugars are in thed-configuration.     

Growth of the Thermophilic Bacterium Geobacillus uralicus as a Function of Temperature and pH: An SCM-Based Kinetic Analysis

The synthetic chemostat model (SCM), originally developed to describe nonstationary growth under widely varying concentrations of the limiting substrate, was modified to account for the effects of nontrophic factors such as temperature and pH. The bacterium Geobacillus uralicus, isolated from an ultradeep well (4680 m), was grown at temperatures ranging from 40 to 75°C and at pH varying from 5 to 9. The biomass kinetics was reasonably well described by the SCM, including the phase of growth deceleration observed in the first hours after a change in the cultivation temperature. At an early stage of batch growth in a neutral or alkalescent medium, bacterial cells showed reversible attachment to the glass surface of the fermentation vessel. The temperature dependence of the maximum specific growth rate (_m) was fitted using the equation _m = Aexp(T)/{1 + expB[1 – C/(T + 273)]}, where A, , B, and C are constants. The maximum specific growth rate of 2.7 h^–1 (generation time, 15.4 min) was attained on a complex nutrient medium (peptone and yeast extract) at 66.5°C and pH 7.5. On a synthetic mineral medium with glucose, the specific growth rate declined to 1.2 h^–1, and the optimal temperature for growth decreased to 62.3°C.     

Immuno-cytochemical demonstration of the inability of the homozygous Brattleboro rat to synthesize vasopressin and vasopressin-associated neurophysin

Summary Immuno-enzyme cytochemical investigations have shown that, (1) the hypothalamic supraoptic and paraventricular nuclei of the Brattleboro rat, as in the normal rat, contain separate neurons which produce oxytocin + neurophysin; (2) the hereditary inability of the Brattleboro rat to synthesize vasopressin and its associated neurophysin is due to a biochemical defect of separate neurophysin-vasopressin neurons in the supraoptic and the paraventricular nuclei. These observations strongly support the hypotheses that (1) vasopressin and its associated neurophysin are formed via a common precursor, and (2) the initial point of intracellular appearance of the hereditary defect in the Brattleboro rat lies in the synthesis of this precursor, which occurs on ribosomes.Moreover, observations have demonstrated that, in the Brattleboro rat, in addition to the hereditary inability of the hypothalamic magnocellular neurosecretory system to synthesize vasopressin, there also exists a similar hereditary defect in the hypothetical parvicellular suprachiasmatic-median eminence neurosecretory system.This paper is dedicated to Professor Dr. W. Bargmann, in honour of his 70th birthday.Presented in part at the meeting of the Belgian Society of Endocrinology May 17, 1975 (Vandesande et al., 1975d).     

The localization of oxytocin,vasopressin, somatostatin and luteinizing hormone releasing hormone in the rat neurohypophysis

Summary The hypothalamic hormones arginine-vasopressin (AVP), oxytocin (OXT), somatostatin (SOM), and luteinizing hormone-releasing hormone (LHRH) were localized in the rat neurohypophysis by the use of semithin serial sections and the unlabeled antibody enzyme method. Clusters of AVP fibres are present within the central region of the neural lobe, clusters of OXT fibres mainly in the peripheral part. The AVP fibres enter bilaterally into the neural lobe.The results call into question previous reports on the presence of AVP on receptors in the pars intermedia cells, since incubation with anti-AVP resulted in similar staining in the pars intermedia of the Wistar and homozygous Brattleboro rat, a mutant strain deficient in AVP. The same intermediate lobe cells are stained after incubation of serial sections with anti-AVP and anti--melanocyte-stimulating hormone (-MSH). This staining of anti-AVP could be removed by solid phase absorption to -MSH and is thus most probably due to cross reaction with -MSH. SOM fibres appear to be present in the peripheral parts of the proximal neurohypophysial stalk and mainly lateral in its more distal parts. In the neural lobe they rapidly decrease in number, although some fibres continue into the distal part of the neural lobe, running bilaterally and situated adjacent to the pars intermedia. The SOM staining within magnocellular elements, which has been reported in the literature, can most probably be explained by cross reaction of anti-SOM with neurophysins. LHRH fibres are very scarce in the neurohypophysial stalk and absent in the neural lobe.Supported by the Foundation for Medical Research FUNGOThe authors wish to thank Drs. J. De Mey (Beerse, Belgium), A. Arimura (New Orleans, U.S.A.), M.P. Dubois (Nouzilly, France), B.L. Baker (Ann Arbor, U.S.A.) and A.G.E. Pearse (London, U.K.) for their gifts of anti-somatostatin serum, Dr. B. Kerdelhué (Gif-sur-Yvette, France) for anti-LHRH serum, and Dr. F. Vandesande (Ghent, Belgium) for anti-neurophysin I and II serum and bovine neurophysin I and II. Dr. J.G. Streefkerk (Free University, Amsterdam) is acknowledged for critical comments and Mr. A.T. Potjer and Miss J. van der Velden for their skilled assistance     

Cytogenetic studies of the intergeneric hybrids between Secale cereale and Elymus caninus,E. brevipes,and E. tsukushiensis (Triticeae: Poaceae)

Summary Integeneric hybridizations were carried out between Secale cereale L. (2n = 14, RR) and three Elymus species, namely, E. caninus (L.) L. (2n = 28, SSHH), E. brevipes (Keng) Löve (2n = 28, SSYY) and E. tsukushiensis Honda (2n = 42, SSHHYY). Chromosome pairing was studied at metaphase I in the parental species and the hybrids. Meiotic configurations of the hybrids were 20.74 1+0.14 II for E. caninus x S. cereale (SHR), 16.35 I+2.17 II+0.09 III for E. brevipes x S. cereale (SYR) and 25.84 I+1.10 II+0.02 III for E. tsukushiensis x S. cereale (SHYR), in addition to some secondary associations in the different hybrids. It is concluded from the study that (1) a certain, different homoeologous relationship exists among S, H and Y genomes in the investigated Elymus species; (2) low homoeology is present between genomes of Elymus (S or H or Y) and rye (R); (3) the Secale genome affects homoeologous chromosome pairing between different genomes in E. brevipes and E. tsukushiensis.