Immunocytochemical evidence for α-melanocyte-stimulating hormone in the hypothalamus of the frog Rana esculenta

Summary The distribution of immunoreactive -melanocyte-stimulating hormone (-MSH) within the brain of the frog, Rana esculenta, has been studied on adjacent serial sections using an indirect immunofluorescence technique. Immunoreactive cell bodies are found in the anterior part of the preoptic nucleus and in some ventral subependymal cerebrospinal fluid-contacting elements, and in the nucleus infundibularis ventralis. Numerous -MSH-like immunoreactive fibers are present in the preoptic area, in the pars ventralis of the tuber cinereum, and in the outer layer of the median eminence. This staining pattern is completely eliminated after preabsorbing the antiserum with the corresponding antigen, but blocking tests with -MSH-related peptides do not lead to any change in the immunoreaction. From these results it may be inferred that an -MSH-like system is present in the hypothalamic neurosecretory area of R. esculenta, and is probably related to its hypophysiotropic functions.The results are compared to the distribution of -MSH within the hypothalamus of reptiles and mammals.This work was supported by a grant from the M.P.I. (60%)     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Production of secreted guar α-galactosidase by Lactococcus lactis

A plant -galactosidase gene was inserted in the expression vector pGKV259. The resulting plasmid pGAL2 consisted of the replication functions of the broad-host-range lactococcal plasmid pWV01, the lactococcal promoter P59, and the DNA sequences encoding the -amylase signal sequence from Bacillus amyloliquefaciens and the mature part of the -galactosidase from Cyamopsis tetragonoloba (guar). Lactococcus cells of strain MG1363 harbouring this vector produced the plant -galactosidase and secreted the enzyme efficiently as judged by Western blotting and activity assays. Expression levels of up to 4.3 mg extracellular -galactosidase g (dry weight) of biomass^–1 were achieved in standard laboratory batch cultures. The -galactosidase produced by Lactococcus was active on the chromogenic substrate 5-bromo-4-chloro-3-indolyl -d-galactopyranoside, the trisaccharide raffinose and on the galactomannan substrate, guar gum.     

Production of N-glycolylneuraminic acid derived from chemically modifiedE. coli Kl bacterial polysaccharide by membrane enclosed enzymatic catalysis

Summary N-Glycolylneuraminic acid (Neu5Gc) has been prepared by enzymatic hydrolysis of its -(28) linked homopolymer. The rate of hydrolysis of the natural poly -(28)-(Neu5Ac) and the semi-synthetic poly -(28)-(Neu5Gc) were compared with the neuraminidases fromClostridium perfringens andVibrio cholerae. The natural Neu5Ac polysaccharide was a better substrate for both enzymes. For comparison, acid hydrolysis of the two polysaccharides showed extensive degradation.     

Glycosylation patterns of membrane proteins of the jellyfish Cyanea capillata

Integral and membrane-associated proteins extracted from neuron-enriched perirhopalial tissue of the jellyfish Cyanea capillata were probed with a panel of lectins that recognize sugar epitopes of varying complexity. Of the 13 lectins tested, only concanavalin A, jacalin lectin and tomato lectin stained distinct bands on Western blots, indicating the presence of repeating -1,6-mannoses, terminal Gal--1,6-GalNAc and repeating -1,4-linked GlcNAc, respectively. In whole-mounted perirhopalial tissue, jacalin lectin stained several cell types, including neurons, muscle, cilia and mucus strands. Tomato lectin stained secretory cells intensely, and neurons in a punctate fashion. Concanavalin A stained cytoplasmic epitopes in both ecto-and endodermal cells, and ectodermal secretory cells and the mucus strands emanating from them. With the exception of tomato lectin's sugar epitope, the other sugar epitopes identified in this study are non-complex. This study suggests that while glycosylation of integral and membrane-associated proteins occurs in Cyanea, the sugars post-translationally linked to these proteins tend to be simple.     

Purification of α-glucosidae and invertase from bakers' yeast on modified polymeric supports

In the present work Amberlite® XAD-16 and Indion® NPA-1, Polystyrene Divinylbenzene macroreticular spherical resins, have been evaluated quantitatively as supports for the adsorption and isolation of the yeast proteins and the enzymes, invertase and -glucosidase. Modification of these supports has been carried out by surface grafting using acrylate polymers to reduce the hydrophobicity and nonspecific adsorption of proteins. Good grafting efficiency, in excess of 90%, has been obtained using ultrasonic irradiation for the surface activation of polystyrene resins. XAD-16 has higher adsorption capacities for the total yeast proteins as well as for both the enzymes, -glucosidase and invertase, than NPA-1 in its respective native and grafted form. Adsorption capacities of XAD-16 and NPA-1 in their respectivenative and grafted forms for -glucosidase are higher than the capacities for invertase. Nonspecific adsorption of total proteins has been reduced considerably after the grafting of acrylate polymers on hydrophobic supports. At the same time selectivity for the adsorption of both the enzymes has been enhanced on grafted supports. The overall solid-liquid adsorption mass transfer coefficient values (K _l a) estimated for adsorption of invertase on XAD are lower than those for -glucosidase. Native and grafted resins could be regenerated and reused for adsorption of -glucosidase for two regeneration cycles studied. Storage stability of invertase and -glucosidase is the same on native and grafted form of XAD-16 and is more than the enzymes in the free form.     

Denaturation behavior of α-chymotrypsinogen A in urea and alkylurea solutions: Fluorescence studies

The solvent denaturation of-chymotrypsinogen (-ctg A) in aqueous solution of urea, methyl-,N,N-dimethyl-, ethyl-, propyl- and butylurea was studied by fluorescence measurements. Data were analyzed on the assumption of a two-state approximation to obtain the apparent equilibrium constant,K and the apparent Gibbs free energy of transition G ⁰ . It has been observed that alkylsubstitution of urea significantly lowers the denaturant concentration needed to denature-ctg A at 25°C. Denaturation was accompanied by the red shift of emission maxima, the increase of the half-width of the fluorescence spectra, the increase of the fluorescence intensity, and the decrease of the fluorescence polarization. The differences of these fluorescence parameters observed for-ctg A in alkylureas and urea can be ascribed to different unfolded states of the protein in different denaturant solutions. Minor differences in the extent of unfolding were confirmed by size-exclusion chromatography.     

The fine structure of the hypothalamic secretory neurons of the White-crowned Sparrow,Zonotrichia leucophrys gambelii (Passeriformes: Fringillidae)

Summary The fine structures of the neurons and neuropils of the magnocellular supraoptic nucleus and the parvocellular nuclei of the rostral hypothalamus, including the suprachiasmatic and medial, lateral and periventricular preoptic nuclei, and the neuronal apparatus of the organum vasculosum laminae terminalis, have been examined in the male White-crowned Sparrow, Zonotrichia leucophrys gambelii, by correlated light and electron microscopy.The magnocellular supraoptic nucleus is characterized by large neurosecretory perikarya which contain a well developed Golgi complex and densecored granules 1,500–2,200 Å in diameter. The neuropil displays axons, dendrites and glial fibers. Some axonal profiles contain dense-cored vesicles 800–1,000 Å in diameter and clear vesicles 500 Å in diameter. Axo-somatic and axo-dendritic synapses are conspicuous in this nuclear region.The suprachiasmatic nucleus is characterized by an accumulation of small neurons with moderately developed cellular organelles and some dense-cored granules, approximately 1,000 Å in diameter. The profiles of axons within the neuropil contain dense-cored granules 800–1,000 Å in diameter and clear vesicles 500 Å in diameter.The neurons of the medial preoptic nucleus are relatively large and exhibit well developed cellular organelles and dense-cored granules 1,300 to 1,500 Å in diameter. Granular materials are formed within the Golgi complex. The medial preoptic nucleus is rich in secretory perikarya.Occasionally, neurons with granules 1,500–2,200 Å in diameter are encountered in the lateral preoptic and periventricular preoptic nuclei. They may be considered as scattered elements of the magnocellular (supraoptic and paraventricular) system.The organum vasculosum laminae terminalis consists of three layers, i.e., ependymal, internal and external zones, and exhibits a vascular arrangement similar to that of the median eminence. The perikarya of the parvocellular neurons and their axons in the internal zone contain numerous secretory granules ranging from 1,300 to 1,500 Å in diameter.This investigation was supported by Grant No. 5R040 Japan-U.S. Cooperative Science Program of the Japan Society for the Promotion of Science to Professor H. Kobayashi and Professor S.-I. Mikami, by a Scientific Research Grant No. 56019 from the Ministry of Education of Japan to S.-I. Mikami, by support from the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm Biologie der Zeitmessung) to Prof. A. Oksche and by Grant No. GF 33334, U.S.-Japan Cooperative Science Program of the National Science Foundation to Prof. D.S. Farner.Herrn Professor Dr. Dres h.c. Wolfgang Bargmann zu seinem 70. Geburtstag am 27. Januar 1976 gewidmet.     

The alpha-subunit of glycoprotein hormones exists in the prolactin secretory granules of the bullforg (Rana catesbeiana) pituitary gland

Summary Our recent finding that the number of immunoreactive -subunit cells was invariably greater than the total number of immunoreactive gonadotropin (GTH) and thyrotropin (TSH) cells in the bullfrog (Rana catesbeiana) pituitary gland raises the possibility that the -subunit also exists in pituitary cells other than GTH and TSH cells. The present study demonstrates that there are a considerable number of immunoreactive prolactin (PRL) cells that are also stained with antibody against the -subunit when adjacent sections are immunocytochemically examined. Neither immunoreactive growth hormone nor adrenocorticotropin cells are stained with the antibody against the -subunit. The specificity of the antibody against the -subunit and of that against PRL was demonstrated by preabsorption test, non-competitive binding test, and immunoblot analysis. Double-immunolabeling with gold particles of different sizes for the -subunit and PRL revealed that most of the immunolabeled PRL-secretory granules are also labeled with the -subunit antibody. The gold particles indicating the presence of the -subunit were mostly found in the peripheral zone of the secretory granules.     

IR and turbidity studies of vitamin E-cholesterol-phospholipid membrane interactions

Binary and tertiary mixture of -tocophenol, cholesterol and dimyristoylphosphatidylcholine in the form of multilamellar liposomes were investigated by Fourier Transform Infrared and visible spectroscopy. Results of the FTIR and turbidity experiments indicate that T decreases or diminishes the effect of cholesterol on the frequency and the bandwidth of the C-H stretching, CH₂ scissoring and C=O stretching bands in FTIR spectra and the turbidity measurements (recorded as absorbance values at 440 nm) in phospholipid model membranes.     

Cytochemical evidence for the presence of actin in the nucleus of the voodoo lily appendix

Immunoflorescence microscopy of sections of the voodoo lily Sauromatum guttatum appendix stained with monoclonal antibodies against -smooth muscle actin and cytoplasmic actin revealed different staining intensity of different parts of the cell. The anti-cytoplasmic-actin recognized antigens present mainly in the cytoplasm, and the anti--smooth muscle-actin recognized more intensively antigens present in the nuclei. A positive staining of the nucleus was also obtained with FITC–phalloidin confirming the presence of actin in its filamenous form in the nucleus. The presence of a nuclear -smooth muscle-actin-like protein was further confirmed by confocal laser microscopy. On Western blots, the two anti-actins labelled a protein band that comigrated with standard actin at the approximate molecular weight of 43kDa. Several other proteins interacted with the two antibodies to a different degree. The monoclonal antibodies against -tubulin subunit stained only the periphery of the cytoplasm and anti-pan cytoplasmic myosin stained the cytoplasm weakly. On a Western blot, anti--tubulin subunit primarily recognized a protein band at the appropriate molecular weight of 50kDa. This is the first cytochemical evidence for the presence of -smooth muscle-actin-like protein in the plant nucleus.     

Probing the structural diversities of long alpha-neurotoxins by fluorescence quenching studies

Trp fluorescence of Ophiophagus hannah neurotoxins (Oh-4, Oh-6A, Oh-7, and Oh-8) and Bungarus multicinctus -bungarotoxin was quenched by acrylamide and iodide. Acrylamide quenching studies indicated that the degree of exposure of Trp residues in the neurotoxins followed the order Oh-8 > Oh-7 > Oh-6A > Oh-4 > -bungarotoxin, as did the accessibility for iodide. These results reveal that the exposed degree of Trp residues and the microenvironment surrounding Trp residues in the neurotoxins differ, even though their Trp residues and positively charged residues are located at the same or homologous positions. In contrast to unfolded Oh-4, Oh-6A, Oh-7, and -bungarotoxin, unfolding of Oh-8 by reduction and S-carboxymethylation caused a notable decrease in the susceptibility of their Trp residues for iodide. These observations support the view that the side chains of Trp residues and positively charged residues in their native structure do not point toward the same spatial positions. Computer models of the neurotoxins are in good agreement with this proposition. These results elucidate why the conserved Trp residues and cationic groups do not always play the same roles in the biological activities of the neurotoxins.     

Testosterone and progesterone metabolism in the central nervous system: Cellular localization and mechanism of control of the enzymes involved

Summary This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons and in the glia.1. The activities of 5-reductase (the enzyme that converts testosterone into dihydrotestosterone; DHT) and of 3-hydroxy steroid dehydrogenase (the enzyme that converts DHT into 5-androstane-3,17-diol; 3-diol) were first evaluated in primary cultures of neurons, oligodendrocytes, and type-1 and type-2 astrocytes, obtained from the fetal or neonatal rat brain. The formation of DHT and 3-diol was evaluated incubating the different cultures with labeled testosterone or labeled DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, also type-2 astrocytes and oligodendrocytes possess considerable 5-reductase activity. A completely different localization was observed for 3-hydroxysteroid dehydrogenase; the formation of 3-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3-diol is formed in very low yields by neurons, type-2 astrocytes, and oligodendrocytes. Moreover, the results indicate that, in type 1 astrocytes, both 5-reductase and 3-HSD are stimulated by coculture with neurons and by the addition of neuron-conditioned medium, suggesting that secretory products released by neurons might intervene in the control of glial cell function.2. Subsequently it was shown that, similarly to what happens when testosterone is used as the substrate, 5-reductase, which metabolizes progesterone into 5-pregnane-3,20-dione, (DHP), shows a significantly higher activity in neurons than in glial cells; however, also type-1 and type-2 astrocytes as well as oligodendrocytes possess some ability to 5-reduce progesterone. On the contrary, 3-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5-pregnane-3-ol-20-one (THP), appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3-hydroxysteroid dehydrogenase activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoenzymatic forms of the enzymes involved in androgen and progesterone metabolism is discussed.     

Immunohistochemical localization of opioid peptides in the brain of the leech Theromyzon tessulatum

Summary By use of antisera directed against met-enkephalin, leu-enkephalin, dynorphin or -neoendorphin, immunoreactive structures were visualized in the central nervous system and proboscis of the leech Theromyzon tessulatum. Their distribution in the various compartments of the supra- and subesophageal ganglia was mapped. No correspondence could be established between the neurons containing met- or leu-enkephalin-like substances and the different types of neurosecretory cells classically described in Hirudinea. Successive localization of leu- and met-enkephalin on the same section revealed that these two peptides occur in different neurons. Only one cell located in compartment 6 of the supraesophageal ganglion was both dynorphin- and leu-enkephalin-positive. The other dynorphinimmunoreactive cells were not stained with the anti-leuenkephalin serum. The -neoendorphin-immunopositive cells were leu-enkephalin immunonegative and vice versa.     

Alkyl-phenyl cleavage of the lignin model compounds guaiacylglycol and glycerol-β-guaiacyl ether by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized guaiacylglycol--guaiacyl ether (I) in high nitrogen, shaking and stationary cultures. 2-(o-Methoxyphenoxy) ethanol (X), 2-(o-methoxyphenoxy) acetic acid (IX) and methoxy-phydroquinone (MHQ) were identified as products of the metabolism of (I). P. chrysosporium also metabolized guaiacylglycerol--guaiacyl ether (IV) in high nitrogen stationary cultures. 2-(o-Methoxyphenoxy)-1,3 propanediol (XII) and 3-hydroxy, 2-(o-methoxy-phenyxy) propionic acid (XIV) were identified as products of the metabolism of (IV). Finally, P. chrysosporium metabolized -deoxyguaiacylglycol--guaiacyl ether (VI) and -deoxyguaiacylglycerol--guaiacyl ether (VII) in limiting nitrogen cultures. 2-(o-Methoxyphenoxy) ethanol (X) and 2-(o-methoxyphenoxy)-1,3 propanediol (XII) were identified as products of the metabolism of VI and VII respectively indicating hydroxylation of those substrates with subsequent alkyl-phenyl bond cleavage. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography - MHQ methoxyhydroquinone     

The VD1/RPD2 neuronal system in the central nervous system of the pond snail Lymnaea stagnalis studied by in situ hybridization and immunocytochemistry

Summary VD₁ and RPD₂ are two giant neuropeptidergic neurons in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. We wished to determine whether other central neurons in the CNS of L. stagnalis express the VD₁/RPD₂ gene. To this end, in situ hybridization with the cDNA probe of the VD₁/RPD₂ gene and immunocytochemistry with antisera specific to VD₁ and RPD₂ (the 1-antiserum, Mab4H5 and ALMA 6) and to R₁₅ (the 1 and 16-mer antisera) were performed on alternate tissue sections. A VD₁/RPD₂ neuronal system comprising three classes of neurons (A1–A3) was found. All neurons of the system express the gene. Division into classes is based on immunocytochemical characteristics. Class A1 neurons (VD₁ and RPD₂) immunoreact with the 1-antiserum, Mab4H5 and ALMA 6. Class A2 neurons (1–5 small and 1–5 medium sized neurons in the visceral and right parietal ganglion, and two clusters of small neurons and 5 medium-sized neurons in the cerebral ganglia) immunoreact with the 1-antiserum and Mab4H5, but not with ALMA 6. Class A3 neurons (3–4 medium-sized neurons and a cluster of 4–5 small neurons located in the pedal ganglion) immunoreact with the 1-antiserum only. All neurons of the system are immunonegative to the R₁₅ antisera. The observations suggest that the neurons of the VD₁/RPD₂ system produce different sets of neuropeptides. A group of approximately 15 neurons (class B), scattered in the ganglia, immunostained with one or more of the antisera, but did not react with the cDNA probe in in situ hybridization.     

In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 1.25–5.0 M 6-benzyladenine. Addition of -naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 M combined, respectively, with 2.5, 1.25–5.0 or 5.0 M -naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 M indole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under ex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies.     

On the histochemical differences of aldehyd-fuchsin positive material in the fibres of the hypothalamo-hypophyseal tract of Rana temporaria

Summary Using a modified Alcian blue/Alcian yellow method, differences in stainability in the fibres of the preoptic-hypophyseal tract of Rana temporaria were observed. Near the preoptic nucleus most of the fibres stained yellow, in the tract the fibres stained lightgreen or showed green inclusions on a yellow background, whereas at the fibre-endings a colour switch to blue-green was found. This colour switch was the most pronounced in the neural lobe, where the neurosecretion stained blue-green, whereas in the fibre-endings in the outer zone of the median eminence a colour switch to green was noticed.The results can best be explained with the assumption that a modification or transformation of the neurosecretion occurs during its transport from the preoptic nucleus to the fibre-endings.Literature related to this hypothesis is discussed.