An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae

We have identified a recessive -mating-type-specific gene agl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agl showed sexual agglutination with cells but cells carrying agl showed sexual agglutination with neither cells nor a cells. cells carrying agl produced pheromone and responded to a pheromone just like wild cells. cells carrying agl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agl mutant is different from any -specific ste mutants found so far in many sexual activities. The agl gene is recessive, and unlinked to the mating type locus. Biological significance of the mating type agglutinability is discussed based on the results obtained with the mutant.     

Assignment of the gene locus for human α-l-fucosidase to chromosome 1 by analysis of somatic cell hybrids

The -l-fucosidases (EC 3.2.1.51) from human and mouse cells could be separated by isoelectric focusing of neuraminidase-treated cell extracts in acrylamide slab gels. Fourteen hybrid clones derived from the fusion of mouse and human cultured fibroblasts and 37 hybrid clones derived from the fusion of human long-term lymphoid lines with mouse RAG cells were tested for expression of human -l-fucosidase. A strong correlation between the expression of the human enzyme and the presence or absence of human chromosome 1 was found. The presence of human -l-fucosidase in clones scored as positive by isoelectric focusing was confirmed by Ouchterlony double immunodiffusion against IgG from rabbits immunized with purified human -l-fucosidase. It is concluded that the structural gene locus for human -l-fucosidase is located on chromosome 1.     

Binding of α-actinin to F-actin or to tropomyosin F-actin is a function of both α-actinin concentration and gel structure

Summary We have studied by electron microscopy as well as by measurements of low shear viscosity, rigidity and binding, the effect of-actinin on the gel formed at 37° C with F-actin and with tropomyosin-decorated F-actin. Contrary to previous reports in the literature,-actinin at nanomolar concentrations is an efficient actin gelling protein, even at 37° C, provided that the concentration of actin (or of tropomyosin-decorated F-actin) is low (1.2–2.4 m). The binding of-actinin to F-actin, as a function of actin concentration, is anomalous. The amount of bound-actinin increases when actin concentration increases from 0 to 1.2 m but does not change significantly when actin concentration is further increased up to 48 m. A similar result is obtained with tropomyosin-decorated F-actin.These observations can be explained by an hypothesis that binding is a function of the-actinin — F-actin association constant as well as of the rigidity of the gel. When the concentration of actin increases, the rigidity of the gel also increases and more work is required to bring two actin filaments to the reaction distance with-actinin and, consequently, a larger-actinin concentration is required to attain the same ratio of bound-actinin to actin monomers in the filaments.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Immunohistochemical localization of αl-antitrypsin and αl-antichymotrypsin in salivary pleomorphic adenomas

Summary Immunohistochemical identification of l-antitrypsin (l-AT) and l-antichymotrypsin (l-ACh) in pleomorphic adenomas of salivary glands is reported in order to compare their distribution profiles with those of lysozyme and lactoferrin, already described elsewhere.Normal salivary glands indicated positive l-AT staining in ductal segments and had no l-ACh in any glandular cell. Pleomorphic adenomas displayed moderate positivity to l-AT staining in duct-like, tubular and glandular epithelia which was particularly intense in luminal cells. The limited number of tumour cells which showed duct-like structures with a single cellular layer arrangement, displayed the highest staining to l-ACh. Strongly l-AT positive tumour cells located on the inner side of luminal cavities were also markedly positive to l-ACh. Spindle shaped tumour cells existed outside tubular and ductal structures and were negative to l-AT and l-ACh.Distribution of l-AT in salivary glands was similar to that of lysozyme as is usual in ductal segments or their transformed cells, and occurrence of l-ACh localization rather resembled that of lactoferrin, with occurrence in acinar compartments and changed epithelia within acini.The biological role of a specific immunohistochemical distribution of l-AT and l-ACh in pleomorphic adenomas may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases.     

Bioactive 5-Reduced 16α,17α-Cyclohexanoprogesterone Derivatives Weakly Interact with Proteins of the Soluble Uterine Fraction

We studied competitive activities of 16,17-cyclohexano-5- and 5-dihydroprogesterone in replacing ₃H-progesterone and ₃H-16,17-cyclohexano-6-methylprogesterone from protein complexes. Direct binding of ₃H-5-reduced derivatives with proteins of soluble fractions from rat and rabbit uteri was also assayed. Cd values for 5-reduced derivatives were in the micro- or submicromolar range. The data suggest that biological effects of these analogues are not mediated via soluble uterine receptors.     

Intracellular lectin-binding sites in symbiont-bearingCrithidia species

Crithidia oncopelti, C. deanei, andC. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectinsUlex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, andCanavalia ensiformis, which recognize -l-fucose, - and -N-acetylglucosamine, -galactose and -N-acetylgalactosamine, -galactose, -galactose, -N-acetylgalactosamine, sialic acid and -d-mannose, and -d-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Rat Macrophage Inflammatory Protein-1α, a CC Chemokine, Acts as a Neutrophil Chemoattractant in Vitro and in Vivo

Recombinant rat macrophage inflammatory protein-1 (rMIP-1) at a concentration of 3 × 10^–8 M had strong neutrophil chemotactic activity, though the potency of rMIP-1 was less than that of cytokine-induced neutrophil chemoattractant (CINC)-1 at lower concentrations. In addition, rMIP-1 induced neutrophil chemotaxis in vivo when rMIP-1 was injected into the preformed air-pouch on the back of rats. The adhesion of rMIP-1-treated neutrophils to fibrinogen significantly increased, reaching a maximum adhesion at 10^–8 M. Stimulation of neutrophils with rMIP-1 induced a transient increase in intracellular free [Ca²⁺] dose-dependently. rMIP-1 still induced an increase in the intracellular [Ca²⁺] of rat neutrophils stimulated first with CINC-1, CINC-3 or C5a, suggesting that rat neutrophils have a specific receptor for rMIP-1. Supporting these findings, an additive increase in chemotactic potency was found when both rMIP-1 and CINC-1 were added to the lower wells of Boyden chamber in vitro. In addition, high levels of rMIP-1 were detected in the inflammatory site of air-pouch/carrageenan-induced inflammation in rats. Our results suggest that rMIP-1 acts as a neutrophil chemoattractant and, together with CINCs, plays an important role in infiltration of neutrophils into inflammatory sites in rats.     

Glomerulonephritis as late manifestation of severe αl-antitrypsin deficiency

An association of ₁-antitrypsin deficiency with glomerulonephritis is rare and has so far been observed only in children or young adults. We report a 63-year-old man with severe ₁-antitrypsin deficiency associated with pulmonary emphysema, cirrhosis of the liver, and mesangioproliferative glomerulonephritis with nephrotic syndrome. Following initial presentation with nephropathy, further work-up revealed ₁-antitrypsin deficiency of proteinase inhibitor Z. In the absence of glomerular ₁-antitrypsin deposits the relationship between renal disease and ₁-antitrypsin deficiency remains unclear. ₁-Antitrypsin deficiency should be considered in adults with abnormal renal function and chronic liver disease.Abbreviations A1AT ₁-antitrypsin - Pi proteinase inhibitor     

Functionally active α2-macroglobulin and kinin release in synovial fluids of rheumatoid arthritis

The function of ₂M (₂-macroglobulin) as a proteinase inhibitor in synovial fluid of rheumatoid arthritis was investigated. Low esterase activity was found in the 19S sieve fraction of Sephadex G-200 gel filtered synovial fluid samples, comparable with that obtained with normal human plasma. The molar binding ratio of ₂M isolated from the synovial fluid and trypsin was estimated according to earlier established methods. The formation of an equimolar complex indicated that the synovial ₂M was functionally intact.Esterase activities were measured on N-tosyl-l-arginine [³H]methyl ester. Synovial fluid samples were all found to contain proteinase enzyme which did not bind to the synovial ₂M nor to the added functionally intact, immunologically pure ₂M prepared from normal human plasma.Albumin, ₁-acid glycoprotein, ₂HS-glycoprotein, ₂M and kininogen in synovial fluids were determined by single radial immunodiffusion. Only the ₂M contents were clearly lower than in normal human serum per ml.The higher than 1 ratio between the immunoreactive kininogen determined with monospecific anti-human kininogen serum and the pharmacologically active kininogen indicated that kinin release had occurred possibly in the synovial membrane. Proteinase enzymes present in the synovial fluids and unbound to ₂M caused further depletion of the active kinin segment in synovial kininogen. A model of the regulation by ₂M of the synovial kininogen-kinin system is given.     

Antiandrogentherapie der pathologisch gesteigerten und abartigen Sexualität des Mannes

Zusammenfassung Mit 100 mg Cyproteronacetat (1.2-Methylen-6-chlor-^4,6-pregnadien-17-ol-3.20-dion-17-acetat) pro Tag per os konnten innerhalb von 10–14 Tagen Libido und Potenz hypersexuell bzw. abartig sexuell erregter Männer unabhängig vom Lebensalter in bisher 17 behandelten Fällen ohne Therapieversager und ohne wesentliche Nebenwirkungen gehemmt werden.

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Summary With 100 mg Cyproteroneacetate (1.2-methylene-6-chloro-^4,6-pregnadiene-17-ole-3.20-dione-17-acetate) orally applied per day libido and potency of men suffering from hyper- or abnormal sexuality were inhibited within 10 to 14 days independent from the age of the patients in 17 cases up to this time without any failure in therapy and without fundamental side effects.

     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the α subunit in agonist sensitivity and desensitization

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes 4/n1 and 3/n1 were readily distinguishable from one another by ACh sensitivity and desensitization. 3/n1 receptors showed lower ACh sensitivity and stronger desensitization than 4/n1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n1 subunit was unchanged, the subunits must be responsible for these functional differences. Symmetric hybrid cDNAs, 43 and 34, were constructed and functional receptors were obtained by co-injection with n1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the subunit. In contrast, no part of the subunit was found fully to determine desensitization.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Does TNF-α directly increase endothelial cell monolayer permeability?

The effects of dexamethasone (DEX) and methyl ester (l-NAME) on the tumour necrosis factor- (TNF-)-induced increase in permeability of human umbilical vein endothelial cell (HUVEC) monolayer to [¹²⁵I] labelled bovine serum albumin (BSA) were examined. Preincubation of HUVEC monolayers with DEX (1M, 2 h) completely abolished the effect of TNF- (5 ng/ml, 18 h). Administration of DEX 2 h after TNF- also reduced the effect of TNF-, whilel-NAME (5 ng/ml, 1 mM, 18 h) had no significant effect.The observed inhibition of the TNF--induced permeability increase on preincubation with DEX would suggest a role for nitric oxide (NO) in mediating the permeability response. However, this is not confirmed by the experiments withl-NAME. The inhibition caused by DEX administered after TNF- would suggest alternative mechanisms by which DEX may be acting in addition to inhibition of NO synthase induction.     

Temps de renouvellement de la noradrénaline tissulaire: influence du traitement par l'α-méthylparatyrosine sur la vitesse de disparition de la noradrénaline endogène et la fixation de la noradrénaline3H dans différents organes du rat

Summary The norepinephrine (NE) turnover times in various peripheral sympathetically innervated organs of the rat were determined by the decay in endogenous NE after inhibition of tyrosine hydroxylase by -methylparatyrosine (-MPT). The values for turnover times were: heart (36,0 h), sub-maxillary gland (13,7 h), femoral muscle (11,6 h), spleen (10,1 h), small intestine (4,3 h). In vas deferens, no significant depletion of the NE was observed 8 h after -MPT administration.The influence of -MPT treatment on the tissular uptake of a tracer dose ofL ³H-NE was investigated. In rats pretreated with -MPT, the uptake of³H-NE was significantly reduced in salivary glands; it was weakly but not significantly (0.1<p<0.2) reduced in spleen, muscle and small intestine and was not impaired in heart. A significant augmentation of the hepatic radioactivity was seen in treated animals which could be interpreted as the consequence of the decrease of the neuronal³H-NE uptake.The validity of -MPT utilization for measuring NE turnover is discussed and the values for turnover time yielded by -MPT have been compared with that obtained from an isotopic method (decline in radioactivity after labeling with³H-NE): the results of the two methods showed obvious discrepancies. In heart and submaxillary gland, the turnover times determined by isotopic method were shorter than those obtained by means of -MPT: in spleen, muscle and small intestine, inverse variations were observed, the -MPT method leading to shorter turnover times. This suggests that the sources of error inherent to each method are of variable importance according to the nature of the organ.

     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

Detection and quantitation of cell-surface sugar receptor(s) ofLeishmania donovani by application of neoglycoenzymes

Promastigote culture forms of the log growth phase ofLeishamania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme. Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety, was the only variable feature of the applied tools. Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of -galactosidase fromEscherichia coli: -d-lactose, -d-thiogalactose, -d-mannose, -l-rhamnose, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylglucosamine, the - and -glucosides maltose and cellobiose, -d-xylose, -d-mannose-6-phosphate, the -galactoside melibiose, -l-fucose, and -d-glucuronic acid as well as sialic acid. Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group. This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigoteLeishmania. Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, andN-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealedK _D values of around 100mm and around 10⁴ binding sites for the polyvalent ligands.     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.