Beiträge zur funktionellen Morphologie der Neurohypophyse

Zusammenfassung Beidseitige Adrenalektomie und Hypophysektomie führen bei der Ratte zu gleichartigen histologischen Veränderungen in der Zona externa infundibuli. In beiden Fällen treten in der normalerweise weitgehend goniorinegativen Zona externa infundibuli große Mengen gomoripositiver Granula auf. Sie scheinen Fasern anzugehören, die senkrecht zur Längsachse des Infundibulum verlaufen.Die Befunde werden als weiterer Hinweis dafür betrachtet, daß die gomoripositiven Substanzen der Zona externa infundibuli eine Bedeutung für die Steuerung der Nebennierenrindenfunktion haben.

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Studies on the functional morphology of the neurohypophysisII. Comparison of histological changes in the median eminence of the rat after bilateral adrenalectomy and hypophysectomy

Summary Bilateral adrenalectomy and hypophysectomy in the rat produce similar histological changes in the outer layer of the median eminence. In both cases, abundant gomoripositive granules are observed in the outer layer, which normally reacts gomorinegative. The gomoripositive granules seem to belong to fibres running vertically through the infundibulum.These findings are regarded as a further indication, that the gomoripositive substances of the outer layer of the median eminence play a significant role in controlling adrenocortical function.

     

Comparative cellular localization of corticotropin and melanotropin in lerot adenohypophysis (Eliomys quercinus)

Summary Corticotropin and melanotropin producing cells were localized in the adenohypophysis of normal Lerots by using antibodies against synthetic corticotropins (anti 1–24 ACTH, anti 17–39 ACTH, anti 25–39 ACTH), and melanotropins (anti MSH, anti MSH). All the anticorticotropin sera stained the same cells both in the anterior lobe and in the intermediate lobe. The anti MSH serum only stained a few cells, exclusively located in the intermediate lobe. These MSH cells were not stained with anticorticotropin antibodies. The anti MSH serum revealed all the cells stained with anticorticotropin and anti MSH sera. Absorption tests showed that the 4–10 heptapeptide common to ACTH and MSH, is not responsible for the immunohistochemical staining. The staining of only some corticotrophs with the anti 4–10 ACTH serum might indicate the presence in these cells of a peptide with an accessible 4–10 site. These results are discussedWe thank A. Pillez for technical assistance (C.N.R.S.). This work was supported by a grant from U.E.R. III Lille 1976Attaché de Recherche INSERM     

Prevalence and molecular heterogeneity of alfa+thalassemia in two tribal populations from Andhra Pradesh,India

Summary We describe here the screening of a small group of apparently healthy individuals belonging to the tribal communities of Koya Dora and Konda Reddi. A remarkably high incidence of deletion and nondeletion ⁺ thalassemia mutants has been found with allele frequencies and distributions characteristic to each tribe. We have confirmed the strict relationship between Hb S levels and the number of globin genes in double heterozygotes for the S gene and thalassemia. In this population sample we did not find either heterozygous carriers of ⁰ thalassemia (deletion of both alpha genes in cis) or individuals showing hemolytic anemia due to inactivation of three -globin genes (Hb H disease). Selection by malaria is most probably responsible for the prevalence of the various ⁺ thalassemia haplotypes among the two tribal populations of Andhra Pradesh.     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

Post-translational modifications of α-tubulin in<Emphasis Type="Italic"> Zea mays</Emphasis> L. are highly tissue specific

To further understand post-translational modifications (PTMs) of plant -tubulin, post-translationally modified -tubulin isoforms from selected tissues of Zea mays L. were examined using two-dimensional electrophoresis and immunoblotting. Except for polyglycylated tubulin, tyrosinated, detyrosinated, acetylated and polyglutamylated -tubulin isoforms were all present in maize tissues. Tyrosinated -tubulin was the predominant variant in all cases, with isoforms 1–4 (5) being the most common components. Leaves exhibited a striking difference in PTM patterns of -tubulin isoforms compared to other tissues examined. In leaves, several major specific isoforms were highly modified by detyrosination, acetylation and polyglutamylation. In pollen and anthers, only the most abundant isoform 3 was acetylated to an appreciable extent, and no acetylated isoform was found in roots. Similarly, in pollen, anthers and roots, only 3 was appreciably polyglutamylated. Additionally, a detyrosinated isoform 6 was present in anthers and in leaves, while the tyrosinated isoform 6 seemed to be pollen specific. These results indicate that certain types of PTM of plant -tubulin preferentially occur in a tissue-specific way.Abbreviations 1-, 2-D one-, two-dimensional - MT microtubule - PTM post-translational modification     

Physicochemical characterization of alpha-crystallins from bovine lenses: hydrodynamic and conformational properties

A detailed investigation of hydrodynamic and conformational behavior has been made of the HM-crystallin and -crystallins of bovine lens. Results from this study indicated that HM (high-molecular-weight -crystallin) and (low-molecular-weight -crystallin) possess considerable size and charge heterogeneities in their native structures and subunit polypeptides, respectively. Sedimentation velocity showed a heterogeneous polydisperse system of HM with an average sedimentation coefficient of about 50 S and a more homogeneous system of -crystallin of 20 S. Viscosity and circular dichroism studies pointed to a compact and globular shape of dominant -sheet conformation for -crystallin, yet a highly asymmetrical and aggregated form for HM. The conformational stability of -crystallin was investigated in the presence of various denaturants. The evidence presented shows that hydrogen bonding is the main force in maintaining the quaternary structure of compact native -crystallin. Conformational flexibility of -crystallin demonstrated in the equilibrium unfolding study indicated a multistep transition that made the extraction of thermodynamic data from the heat denaturation study difficult. Temperature perturbation on -crystallin suggested the possible involvement of hydrophobic interaction in the aggregation process, leading to the formation of HM from -crystallin. The comparison of conformational properties between HM and -crystallin strongly indicated that HM is a denatured form of -crystallin.     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

Frequency and molecular types of deletional α-thalassemia in Egypt

Summary The frequency of deletional -thalassemia in the Egyptian population was estimated at 0.08 by DNA analysis of a newborn random sample. No ⁰ determinants were found. The most frequent ⁺ determinant was the –^3.7 type I in association with the medium allele at inter-zeta HVR. The –^4.2 and ^{anti 3.7} arrangements were found at very low frequencies.     

The mode of secretion of α-amylase in barley aleurone layers

The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl₂ two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[³H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE N-carbobenzoxy-L-tyrosine p-nitrophenylester - Cyt oxidase Cytochrome oxidase - ER endoplasmic reticulum - EDTA ethylenediaminetetraacetic acid - GA₃ gibberellic acid - IDPase inosine diphosphatase - K⁺-ATPase pH 6.5 K⁺-stimulated adenosine triphosphatase - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - NADH: Cyt c reductase cyanide-insensitive NADH-linked cytochrome-c reductase - RER rough endoplasmic reticulum - Tris tris-(hydroxymethyl)-aminomethane     

Convergent synthesis of neoglycopeptides by coupling of 2-bromoethyl glycosides to cysteine and homocysteine residues in T cell stimulating peptides

The 2-bromoethyl -glycosides of the disaccharide galabiose [Gal(1-4)Gal] and the trisaccharides globotriose [Gal(1-4)Gal(1-4)Glc] and 3-sialyllactose [Neu5Ac(2-3)Gal(1-4)Glc] have been prepared by improved routes. The 2-bromoethyl glycosides were then used in cesium carbonate promoted alkylations of the sulfhydryl groups of cysteine and homocysteine residues in T cell stimulating peptides. This convergent and general approach was used to prepare 16 neoglycopeptides which were obtained in 52–95% yields after purification by HPLC. 1H NMR spectroscopy revealed that -elimination and epimerization of neoglycopeptide stereocentres did not occur during the synthesis.     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

Evidence of regional differences in the lectin histochemistry along the ductus epididymis of the lizard,Podarcis sicula Raf

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Ac(2,6)Gal(1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal(1,3)GalNAc, Gal (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Gal(1,4)GlcNAc and GalNAc, the luminal surface by GalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal (1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, and internal GlcNAc, expressed terminal Gal (1,4)GlcNAc and GalNAc in the caput, and terminal GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal GalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal GalNAc only in the corpus.     

The taxonomic significance of the trunk limbs of the chydoridae (Cladocera)

Summary The differences in the structure of the trunk limbs allow to outline three sections of Chydoridae (see table I and fig. 1), coinciding with the sections distinguished according to the structure of the head pores.

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Chydoridae (Cladocera)

Chydoridae (. ), , .

     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins

Summary The functional R6K origin is composed of two DNA elements, one of 580 bp carrying the origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previosly identified as also required for and origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for origin activity. The function of the origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the origin depends on the R6K replication initiation protein .Within the 580 bp of the origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the replicon. Deletion of the 96 bp or 98 bp results in inactivation of the and the origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the and the origins of R6K. DNA homology analysis performed on , and origin sequences, also reveals 10–23 bp sequences in the and the origins that are related to the family of 22 bp direct repeats in the origin which were shown previously to be binding sites for the protein.     

Ascorbic acid spares α-tocopherol and prevents lipid peroxidation in cultured H4IIE liver cells

Ascorbic acid, or vitamin C, can recycle -tocopherol in lipid bilayers, but even sparing of -tocopherol has not been a consistent finding in intact cells. Therefore, we tested the ability of ascorbate loading to spare -tocopherol and to prevent lipid peroxidation of cultured H4IIE rat liver cells. Although -tocopherol was undetectable in H4IIE cells, its cell content was increased by overnight incubation with -tocopherol in culture. Cells incubated with ascorbate 2-phosphate accumulated ascorbate to concentrations as high as 0.6 mM after overnight loading, but also released ascorbate into the medium. Ascorbate loading of -tocopherol-treated cells spared -tocopherol in a concentration-dependent manner during overnight incubation. Lipid peroxidative damage, measured as a decrease in fluorescence of cell-bound cis-parinaric acid, was decreased in cells loaded with either -tocopherol or ascorbate 2-phosphate, and showed an additive effect. These results suggest that ascorbate loading of H4IIE cells spares cellular -tocopherol and either directly or through recycling of -tocopherol prevents lipid peroxidative damage due to oxidant stress in culture.     

Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

Glycerol-, inositol-, and reducing end hexose-containing oligosaccharides in human urine

Ten previously unreported oligosaccharides have been purified from the urines of human subjects using a combination of gel filtration, ion exchange, and thin-layer chromatographies. Their structures were determined by direct probe mass spectrometry, methylation analysis, and proton NMR spectroscopy of the permethylated oligosaccharide alditols.On the basis of composition, the oligosaccharides could be divided into three groups. Five oligosaccharides containing glycerol were characterized as glucosyl1-1glycerol; glucosyl1-1glycerol; galactosyl1-1glycerol; glucosyl-1-1(fucosyl-1-2)glycerol and/or fucosyl-1-1(glucosyl-1-2)glycerol; and glucosyl-1-1(galactosyl-1-2)glycerol or galactosyl-1-1(glucosyl-1-2)glycerol. Four inositol-containing oligosaccharides were characterized as galactosyl1 (fucosyl1)inositol,N-acetylgalactosaminyl1 (fucosyl1)inositol, fucosyl1-2galactosyl1 (N-acetylgalactosaminyl1)inositol and fucosyl1-2galactosyl1-4-N-acetylglucosaminyl1(N-acetylgalactosaminyl1)inositol. Finally, galactosyl1-3(fucosyl1-2)galactosyl1-6galactosyl1-4(fucosyl1-3)glucose, an oligosaccharide with glucose at its reducing end, was tentatively identified. The significance and possible origins of the carbohydrate structures are discussed.     

Regio- and stereo-selective synthesis of vinyl glucose ester catalyzed by an alkaline protease of Bacillus subtilis

The transesterification of -d-glucose with divinylsuccinate, divinyladipate and divinylsebacate in pyridine at 55 °C for 3 days was catalyzed by an alkaline protease from Bacillus subtilis to give corresponding 6-O-vinyl glucose esters at 30%, 53% and 35% yield, respectively. The stereo-selectivity of the alkaline protease toward the -anomer was affected by the acyl donor chain length. 6-O-Vinylsuccinyl-d-glucose was mixture of - and -anomers (/=44/56), the other two products were the pure -d-glucose derivatives.