Analysis of T cell receptor Vβ gene usage during the course of disease in patients with chronic hepatitis B

The T cell receptor (TCR) is a heterodimeric molecule expressed on the surface of T cells and recognizes foreign peptides presented by the major histocompatibility complex on the surface of antigen-presenting cells or virusinfected cells. Analysis of TCR usage by T cells which recognize hepatitis B virus (HBV) provides further insight into the participation of T cell populations during the course of disease. In this study, we examined the T-cell-proliferative response and the TCR V gene usage of peripheral blood mononuclear cells in 3 patients with clinical evidence typical of chronic hepatitis B. All 3 patients had significant T-cell proliferative responses against HBV core antigen (HBcAg) during the remission stage, while no responses were detected during the acute exacerbation stage. In addition, the TCR V₇ gene was utilized more frequently in T cells recognizing HBcAg during remission, while TCR V₁ and V₂ were utilized at a higher percentage during acute exacerbation. On the contrary, the T cell proliferative response against HBV surface antigen was undetectable and no specific V gene was utilized more frequently by all 3 patients, regardless of disease state. Our longitudinal studies, although based on a small sample of patients, demonstrate that the population of HBcAg-activated T cells alters during the course of disease in chronic hepatitis B patients.     

Presence of adrenocorticotropin and β-Endorphin immunoreactivities in the magnocellular neurosecretory system of the rat hypothalamus

Summary Antisera raised against ACTH (1–39), -endorphin and the 16K proopiocortin were used, in association with the immunoperoxidase reaction, to localize positively-staining cell bodies and nerve fibres in the hypothalamus of the rat. Antigens, cross-reactive against anti-ACTH (1–39) serum were detected in a fibre system in the rostro-dorsal hypothalamus situated between the optic chiasm and the third ventricle while immunoreactive 16K-like material was present in fibres localized in the caudal hypothalamus, dorso-lateral to the arcuate nucleus. This latter system was also associated with the appearance of ACTH (1–39) and ACTH (17–39) immunoreactivity.Cells of the arcuate nucleus stained positively for ACTH (1–39), 16K antigen and -endorphin, and on examining adjacent thin sections it was observed that cells that contained 16K antigen-like material, also gave a positive immunoreaction with ACTH (1–39) and -endorphin antisera. In the magnocellular system, cells of the supraoptic (SON) and paraventricular (PVN) nuclei also gave a positive immunoreaction with anti-ACTH (1–39), 16K antigen and -endorphin serum. As in the case of the arcuate nucleus, common cells stained for these three antigens.On the basis of the precursor theory for the synthesis of ACTH, 16K antigen and -endorphin, it was not unexpected to find these three fragments of pro-opiocortin localized together in cells of the arcuate nucleus. That ACTH (1–39), 16K antigen and -endorphin-like materials are present in the magnocellular neurosecretory system would suggest that cells of the SON and PVN are not only involved in the synthesis of neurophysin and the neurohypophysial hormones, but also of some products of the pro-opiocortin molecule. Whether the biochemical nature of the ACTH and -endorphin in cells of the SON and PVN is identical to that of anterior pituitary origin remains to be established, as does the biosynthetic relationship between neurophysin and oxytocin/ vasopressin and these fragments of pro-opiocortin.Drs. M.M. Wilkes, S.S.C. Yen, G. Pelletier, B.A. Eipper and R. Walter are thanked for supplying some of the antisera and antigens used in this study. Thanks also go to Ciba-Geigy Ltd. and Organon Inc. for supplies of ACTH (17–39) and ACTH (1–24) respectively. This work was financed by The Medical Research Council of New Zealand     

Cystatins from bovine brain: Purification,some properties,and action on substance P degrading activity

Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80°C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. K_i values for the complexes of papain and the inhibitors were estimated to be 2.8×10^–10 M for HMM-cystatin and 1.3×10^–9 M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.Abbreviations HMM-cystatin high molecular mass inhibitor - LMM-cystatin low molecular mass inhibitor - SP substance P - SPM synaptosomal plasma membranes - p-CMB 4-chloromercuribenzoic acid - BK bradykinin - Bz-Arg-Nap N-benzoyl-dl-arginine--naphthylamide - Arg-Nap dl-arginine--naphthylamide - P-Pxy-Hb hemoglobin initially coupled with pyridoxal-5-phosphate     

Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Glutaraldehyde-fixation of yeast cells: Kinetics of a model system for enzyme cytochemistry

The kinetics of glutaraldehyde inactivation of a protoplasmic (-fructofuranosidase) and an extracytoplasmic (acid phosphatase) enzyme inSaccharomyces rouxii cells were studied at pH 5.5 and 30°C. The effects of glutaraldehyde concentration (0.5–3%), pH value, and temperature were surveyed by varying the fixation conditions. Cells from 1- to 10-day cultures retained 50–75% of their acid phosphatase activity and 15–24% of their -fructofuranosidase activity after 1-h exposures to 0.5% glutaraldehyde. The surviving -fructofuranosidase activity remained physically cryptic and was revealed only after further membrane perturbation with ethyl acetate. This crypticity barrier disappeared after overnight incubation of the treated cells at 4°C, with or without added glutaraldehyde, during which time the enzyme was resistant to further inactivation. The velocity ratio for raffinose versus sucrose, as substrate, decreased in treated cells, and changes inV _max andK _m were indicative of frank destruction of some enzyme molecules as well as modification of survivors. A comparable set of changes was also generated by treating cell-free extract with glutaraldehyde. Glutaraldehyde (0.5%) killed all yeast cells at 30°C within 5 min; at 4°C survival rates were quite high—81% after 15 min and 65% after 1 h. The bearing of these examples of enzyme inactivation, permeability barrier abolition, and structural stabilization on the general problems of yeast cytochemistry is discussed.     

2-Phenylethylamine-induced changes in catecholamine receptor density: Implications for antidepressant drug action

It is now established that (1) concentrations of 2-phenylethylamine (PEA) are greatly increased in brain following administration of monoamine oxidase inhibitor (MAOI) antidepressants; (2) PEA is a metabolite of the MAOI antidepressant phenelzine; and (3) PEA may be a neuromodulator of catecholamine activity. On the basis of these observations, the effects of long term increases in brain PEA on catecholamine receptors have been assessed. Both PEA and antidepressants induced a reduction in the behavioural response to the ₂ adrenoceptor agonist salbutamol. Radioligand binding measurements revealed that 28 day administration of PEA in combination with the type B MAOI (–)-deprenyl results in a decrease in the density of ₁ adrenoceptors but not ₂ adrenoceptors in rat cerebral cortex and cerebellum. (–)-Deprenyl alone also induced a significant decrease in ₁-adrenoceptors but when PEA was added to this treatment there was a further decrease in ₂-adrenoceptor density. Only changes in ₁ adrenoceptor density were evident following 28 day administration of MAOI antidepressants. PEA also induced a decrease in the density of D1-like dopamine (DA) receptors in the rat striatum. MAOI antidepressants induced a decrease in the density of both D1-like and D2-like DA receptors. These data are discussed in terms of a possible role of PEA-catecholamine interactions in antidepressant drug action.     

Zur Cytochemie der Lysosomen in der Rattenniere unter normalen und experimentellen Bedingungen

Zusammenfassung Es wird das Vorkommen der sauren Phosphatase (sP) und der -Glucu-ronidase (-Glu) in den Lysosomen von Rattennieren vom 16. Embryonaltag (ET) bis 33. Lebenstag (LT) sowie von erwachsenen unbehandelten, kastrierten und nach der Kastration mit gleich- bzw. gegengeschlechtlichen Hormonen behandelten Tieren untersucht. Bis zur Geburt entwickelt sich die lysosomale sP und -Glu in den einzelnen Nephronabschnitten und Sammelrohren annähernd parallel. Am 1. LT nimmt in den S₁-Segmenten der iuxtamedullären Nephrone Zahl, Durchmesser und Aktivität der sP positiven Lysosomen sprunghaft zu, um zwischen 5. und 9. LT wieder schnell abzunehmen. Die Aktivität der -Glu steigt dagegen kontinuierlich an. Zwischen 24. und 25. LT nimmt die Aktivität der -Glu in den Lysosomen der S₁- und S₃-Segmente ab, die der sP deutlich zu. Geschlechtsunterschiede treten bei der sP zum ersten Mal am 18. LT in den S₃-Segmenten, bei der -Glu am 25. LT in den S₁-Segmenten auf. Das ausdifferenzierte Enzymmuster für sP und -Glu kann erst bei erwachsenen Tieren nachgewiesen werden. Dann fallen bei beiden Geschlechtern unter den Lysosomen der S₁-Segmente zwei Größenklassen besonders auf: a. große Lysosomen (beim Weibchen 7 m, beim Männchen 5 m) und b. kleinere Lysosomen (bei beiden Geschlechtern 2–3 m). Die großen Lysosomen sind bei Weibchen zahlreicher und sP aktiver als bei Männchen. In den S₂-Segmenten (Durchmesser der Lysosomen 1,5–2,5 m) ist die -Glu in den Lysosomen der Männchen aktiver als bei den Weibchen; jedoch sind die Geschlechtsunterschiede in S₂ geringer als in S₁ In den S₃-Segmenten sind sP und -Glu in den Lysosomen weiblicher Nieren aktiver als in denen männlicher Tiere. — In den übrigen Abschnitten des Nephrons und in den Sammelrohren bestehen keine Geschlechtsunterschiede. — Die Lysosomen der Sammelrohre haben eine höhere Aktivität für -Glu als für sP.Durch Kastration werden die Geschlechtsunterschiede geringer, bleiben aber grundsätzlich erhalten; in den S₁-Segmenten können jedoch Lysosomen über 2,5 m nicht mehr nachgewiesen werden. Nach Testosteronbehandlung männlicher Kastrate nimmt die Aktivität der sP und -Glu gegenüber unbehandelten Kastraten ab. Bei kastrierten weiblichen Tieren treten nach Testosteronbehandlung wieder große Lysosomen auf, das Enzymmuster unbehandelter Tiere wird jedoch nicht erreicht. Östradiolbehandlung kastrierter Tiere führt bei beiden Geschlechtern in etwa zu einer Restitution der normalen Enzymverteilung, insbesondere bei den Männchen. Bei den Weibchen sind die Lysosomen größer und reagieren intensiver als bei Normaltieren, die Anzahl der Lysosomen scheint aber geringer zu sein.Insgesamt ergibt sich, daß die Lysosomen und die lysosomalen Enzyme unter dem Einfluß der Geschlechtshormone stehen.

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On the cytochemistry of lysosomes in the rat kidney under normal and experimental conditions

Summary Acid phosphatase (ap) and -glucuronidase (-glu) have been investigated in the lysosomes of the rat kidney between the 16th embryonic and 33th postnatal day. Furthermore these enzymes were demonstrated in the kidney of adult normal as well as of orchiectomized or ovarectomized rats following treatment with homosexual and heterosexual hormones. — Up till birth, the development of ap and -glu runs nearly parallel to one another in the nephron and in the collecting tubules. At the first day of life an irregular increase with respect to number, diameter, and activity of lysosomes containing ap can be observed in the S₁ segments of the iuxtamedullary nephrons; a decrease occurs between the 5th and 9th postnatal day. On the contrary the -glu activity increases continously. Between the 24th and 25th day of life its activity decreases in the lysosomes of the S₁ and S₂ segments; the ap activity, however, increases. For the first time sex-specific differences concerning the distribution pattern of ap can be revealed around the 18th day of life in the S₃ segments; in the case of -glu ca one week later in the S₁ segments. The final ap and -glu pattern only exists in the kidney of adult animals. Here, in the male and female kidney two classes of lysosomes exhibit striking peculiarities in the S₁ segments: a. big lysosomes (in male 5 m, in female 7 m in diameter) b. small lysosomes (in both sexes 2–3 m in diameter). The big lysosomes are more numerous. Moreover their ap activity is higher in female rats in comparison with the male kidney. In the S₂ segments (lysosomal diameter 1.5–2.5 m) the -glu activity of male rats surpasses that of females. However sex differences in the S₂ are as obvious as in the S₁ segments. In S₃ the strongest ap and -glu reaction appear in female kidneys. — In the other parts of the nephron and in the collecting tubules sexspecific differences have never been observed. — In the lysosomes of the collecting tubules more -glu than ap activity can be detected.Castration induces only a decrease of sex differences, but they do not disappear completely; in the S₁ segments lysosomes being bigger than 2.5 m are absent. In male castrates treated with testosterone the activity of ap and -glu is lowered in comparison with unsubstituted animals. Following application of testosterone the big lysosomes reappear in overectomized rats; but the enzyme pattern of untreated animals will never be obtained. In both sexes treatment of castrated animals with estradiol is accompanied by restitution of the normal ap and -glu pattern especially in males. In the female kidney lysosomes are bigger and more active in comparison with the controls; the number of lysosomes seems to be reduced. Summarizing strong evidence occurs that in the rat kidney lysosomes and lysosomal enzymes are controlled by sex hormones.

     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

Liquid water and the origin of life

Liquid water has many peculiarities which confer special properties. The most important among them probably are the ability to establish hydrogen bonds, a high polarity and a high dielectric constant. In the presence of liquid water, large organic molecules have to manage the conflict between hydrophobic groups and hydrophilic groups. This duality can generate interesting prebiotic situations such as the stereoselective aggregation of short peptide sequences of alternating hydrophobic — hydrophilic residues into thermostable -sheets endowed with chemical acrivity. Liquid water is also a powerful hydrolytic agent. As such, it allows pathways which would have few chances to occur in an organic solvent.Presented at the Session Water in the Solar System and Its Role in Exobiology during the 26th General Assembly of the European Geophysical Society, 22–26 April 1991 in Wiesbaden, Germany.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Influence of thyroid hormones on the human ATP synthase β-subunit gene promoter

The action of thyroid hormones on the expression of the mitochondrial ATP synthase -subunit gene (ATPsyn) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsyn gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5 upstream region of ATPsyn gene were unresponsive to T₃ when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T₃ in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsyn expression occur through indirect mechanisms.     

Neuronal apoptosis versus necrosis induced by glutamate or free radicals

The type of cell death encountered in neuronal cell cultures exposed to excitatory amino acids — such as glutamate, the major excitatory neurotransmitter in the central nervous system, or free radicals, such as nitric oxide (NO^.) and superoxide anoin (O2^{. –}), which react to form peroxynitrite (ONOO^–) — appears to depend on the intensity of the exposure and may involve two temporarily distinct phases. Following relatively fulminant insults, an initial phase of necrosis — associated with extreme energy depletion — may simply reflect the failure of neurons to carry out the default apoptotic death program used to efficiently dispose of aged or otherwise unwanted cells. Neurons recovering mitochondrial energy potential after an initial fulminant insult or following a more subtle inciting injury may subsequently undergo apoptosis, possibly associated with a factor released from mitochondria that triggers this death program. The maintenance of balanced energy production may be a decisive factor in detemining the degree, type, and progression of neuronal injury caused by excitotoxins and free radicals. Similar events could possibly occur in vivo after ischemia or other insults.     

Role of Different Types of Adrenoreceptors in Regulation of Functional Activity of Vasopressinergic Neurosecretory Cells of Rat Hypothalamus

The goal of the present study was to find out by which adrenoreceptors noradrenaline affects synthesis and release of vasopressin from perikarya of neurosecretory cells in paraventricular and supraoptic nuclei of rat hypothalamus and what the character of this effect. In experiments in vitro with incubation of surviving hypothalamic slices in the medium containing noradrenaline or one of antagonists of adrenoreceptors (cirazoline—of ₁-, UK14304—of ₂-, cimaterol—of -adrenoreceptors) it was shown that application of different adrenoreceptor agonists resulted in different changes of the functional state of vasopressinergic cells in the supraoptic and paraventricular nuclei. Comparison of the content of vasopressin mRNA (method of hybridization in situ) and of vasopressin-immunoreactive substance (immunohistochemical method) has allowed us to conclude about different ratios of intensities of vasopressin synthesis and release in these cells under effects of noradrenaline and adrenoreceptor agonists studied. The results obtained indicate that noradrenaline has no effect on intensity of synthesis of vasopressin, but can inhibit its release from perikarya. Meanwhile, noradrenaline via ₁-adrenoreceptors is able to inhibit synthesis of vasopressin and, possibly, its release; via ₂-adrenoreceptors it also inhibits release of vasopressin, but does not affect its synthesis, whereas activation of -adrenoreceptors stimulates vasopressin synthesis by neurons of supraoptic and paraventricular nuclei of rat hypothalamus. The diversity of responses of vasopressinergic system to action of noradrenaline, which has been reported by many authors, seems to be due to predominant involvement of particular types of adrenoreceptors.     

Reappraisal of osmium tetroxide and OTAN histochemical reactions

Summary Osmium tetroxide and OTAN histochemical reactions have been reappraised following recent publications in this journal by Ellbder and Lojda. Provided that the standard OTAN reaction is used on frozen sections of conventional thickness, unsaturated hydrophilic polar lipids are stained in an orange or red shade while unsaturated hydrophobic non-polar lipids are stained brown-black or black. We have confirmed the anomaly — pointed out by Elleder and Lojda — that the polar lipid reaction of the atherosclerotic plaque is essentially extinguised by acetone. Solubility and blockading methods provide circumstantial evidence that Elleder and Lojda's assumed OsO₄-protein reactions are due to the lipid moiety of a lipoprotein complex.     

Evidence for distinct polygenic regulation of antibody responses to some unrelated antigens in lines of mice selected for high or low antibody responses to somatic antigen of Salmonella

The effect of the selective breeding of mice for high or low antibody production to complex immunogens is largely nonspecific, since it modifies the responsiveness of high (H) and low (L) lines to many antigens unrelated to the selection antigen. However, the nonspecific effect of the polygenic control operating in these lines is not a general feature. For example, the group of genes in selection IV, carried out for responsiveness to somatic antigen of Salmonella, does not modify the responses to sheep erythrocytes (SE). Despite equivalent responses in H and L mice of selection IV, a large variability was found in individual responses of F₂ interline hybrids, which demonstrates the presence of alleles with high or low effect on responses to SE. A selective breeding (Selection IV-A) was therefore initiated from this F₂ population for responsiveness to SE. A progressive interline divergence occurred during the first seven generations of selection; the interline separation was due to polygenic regulation (about four independent loci from a preliminary estimate).Equivalent responses to the s antigen of Salmonella are observed in the two lines. This constitutes additional evidence for distinct polygenic regulation of responses to SE and to somatic antigen. Moreover, the pattern of responses to several unrelated antigens (nonspecific effect) also differs between Selections IV and IV-A.Abbreviations H high responder lines - L low responder lines - s somatic antigen of Salmonella - f flagellar antigen of Salmonella - R response to selection - S selection differential - F₀ foundation population - h² heritability (realized) - RGG rabbit gamma globulin - CE chicken erythrocyte - HE human erythrocyte - PE pigeon erythrocyte - SE sheep erythrocyte     

Cross-linking of the endogenous inhibitor protein (IF1) with rotor (gamma,epsilon) and stator (alpha) subunits of the mitochondrial ATP synthase

The location of the endogenous inhibitor protein ( IF₁) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F₁I and intact F₁F₀I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of –IF₁, IF₁–IF₁, –IF₁, and –IF₁ cross-linkages in soluble F₁I and in native F₁F₀I complexes. Cross-linking blocked the release of IF₁ from its inhibitory site and therefore the activation of F₁I and F₁F₀I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF₁ is at a distance 12 Å,to and subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF₁ inhibits conformational changes of the catalytic subunits, the inhibitory mechanism of IF₁ may involve the interference with rotation of the central stalk.     

Several phenotypic changes in the cell envelope of Agrobacterium tumefaciens chvB mutants are prevented by calcium limitation

The chvB gene of Agrobacterium tumefaciens encodes a 235 kDa proteinaceous intermediate involved in the synthesis of -1,2-glucan. chvB mutants show a pleiotropic phenotype. Besides not to produce cyclic -1,2-glucan, chvB mutants have been reported to be avirulent, attachment-deficient, and nonmotile. In this study we report additional differences from the parent strain, probably all linked to changes in the cell envelope. This pleiotropic phenotype — except for attachment and virulence — could largely be prevented by growing chvB cells with low levels of calcium. Although a role for -1,2-glucan in osmoadaptation has been proposed, the mode of action of -1,2-glucan is not known. We speculate that in A. tumefaciens -1,2-glucan stabilizes membranes, which would be important especially in hypotonic media containing calcium.Abbreviations Cb carbenicillin - Km kanamycin - TCA trichloroacetic acid - K_av fraction of the stationary gel volume available for diffusion - LPS lipopolysaccharide - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Beta-2 microglobulin types in mice of wild origin

To determine the distribution of beta-2 microglobulin (B2m) alleles in wild mice we have typed mice derived from natural populations in Europe, North Africa, South America, and East Asia. Mus musculus domesticus mice from Germany, France, Italy, and Peru were all B2m ^a as were most from the United Kingdom. M.m. musculus mice from Denmark and Czechoslovakia, several stocks of M.m. molossinus from Japan, and M.m. castaneus from China, Thailand, and the Philippines were of B2m ^b type. This is consistent with the notion that C57BL/6 may have obtained some of its genes, including B2m, from Eastern mice. A BgII restriction site characteristic of B2m ^b was also found in mice from Czechoslovakia and Japan, confirming that B2m ^b is a naturally occurring allele of B2m. A new type of ₂m ( ₂m^w1) was found in four stocks of M. spretus from Portugal, Spain, and Morocco. This molecule differs in apparent size and charge from the a and b types. ₂m^w2 was found together with ₂ ma in one stock of M.m. domesticus (brevirostris) from Morocco. ₂m^w3 and ₂m^w4 were found in a few M. m. bactrianus from Pakistan. In all cases tested, these new ₂m molecules associate with class I histocompatibility antigens.Abbreviations used in this paper ₂m beta-2 microglobulin - B2m gene for beta-2 microglobulin - IEF isoelectric focusing - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - MHC major histocompatibility complex - T. E. Tris-EDTA buffer     

Das unterschiedliche Verhalten der Exopeptidasen des Hepatopankreas und Magensaftes vom Flußkrebs Astacus astacus (L.) und Cambarus affinis (Say) bei der Gelfiltration auf Sephadex sowie gegenüber Effektoren

Zusammenfassung Das Hepatopankreas (HP) der Flußkrebse Astacus astacus (L.) und Cambarus affinis (Say) enthält eine hochmolekulare Carboxypeptidase A-Wirkung (Substrat: Carbobenzoxyglycyl-l-phenylalanin) (K _d-Wert auf Sephadex G-200=0,04) und eine Arylamidase-Wirkung [Substrat: l-Leucin--naphthyl-amid·HCl (LNA)], sowie Dipeptidase-Wirkung (Substrat: Glycyl-l-leucin) (K _d-Werte auf Sephadex G-200=0,46 bzw. 0,39). Carboxypeptidase B (Substrat: Hippuryl-l-arginin)-Aktivität wurde im HP nicht gefunden. Im Gegensatz zum HP ist die Carboxypeptidase A des Magensaftes (MS) niedriger molekular (K _d-Wert auf Sephadex G-150=0,62; Molekulargewicht: ca. 30000), die LNA-ase des MS höher molekular (K _d-Wert auf Sephadex G-150=0,26). Außerdem enthält der MS eine hochaktive Carboxypeptidase B-ähnliche Wirkung, die sich auf Sephadexgel wie die Carboxypeptidase A verhält. Chelatbildner (,-Dipyridyl, o-Phenanthrolin) hemmen die Hippurylarginin-Wirkung nicht. Die Carboxypeptidase A des HP wird durch EDTA und Hydrozimtsäure deutlich, durch p-Chlormercuribenzoat gering aktiviert und durch 2-Mercaptoäthanol (10^–3 m und höhere Konzentrationen) stark gehemmt.

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The differential behaviour of the exopeptidases from hepatopancreas and gastric juice of the crayfish Astacus astacus (L.) and Cambarus affinis (Say) during gelfiltration on sephadex and towards effectors

Summary Hepatopancreas (HP) of the crayfishes Astacus astacus (L.) and Cambarus affinis (Say) contains a high molecular carboxypeptidase A like action (substrate: carbobenzoxyglycyl-l-phenylalanine) (K _d-value on Sephadex G-200 =0,04), an arylamidase like action (substrate: l-leucine- -naphthylamide·HCl; LNA), and a dipeptidase (substrate: glycyl-l-leucine) (K _d-values on Sephadex G-200 0,46 and 0,39 respectively). Carboxypeptidase B (substrate: hippuryl-l-arginine) activity was absent in HP. Contrary to the exopeptidases of HP the carboxypeptidase A of the gastric juice is of lower molecular weight (K _d-value on Sephadex G-150=0,62; molecular weight approx. 30.000), and the arylamidase of the gastric juice is of higher molecular weight (K _d-value on Sephadex G-150=0,26). Moreover gastric juice contains a highly active carboxypeptidase like activity, with identical behaviour on Sephadexgel as carboxypeptidase A. ,-dipyridyl and o-phenanthroline are without effect on the hippurylarginine splitting activity. Carboxypeptidase A of HP is significantly activated by EDTA and hydrocinnamic acid, and slightly activated by p-chloromercuribenzoate. 2-mercaptoethanol (10^–3 molar and higher concentrations) inhibits strongly the carboxypeptidase A of HP.

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Herrn Prof. Dr. Hanson möchte ich für sein Interesse an dieser Arbeit sowie für die kritische Durchsicht des Manuskriptes vielmals danken. — Der medizinisch-technischen Assistentin Frau Johanna Scheel danke ich für ihre wertvolle Mit-arbeit bei den Versuchen.