Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Ether phospholipids in control and 20∶4-depleted rat PMN: Additional evidence for a 1-0-alkyl-2-20∶4-sn-glycerol-3-phosphocholine specific phospholipase A2

Earlier studies from our laboratory have shown that PAF and LTB₄ biosynthesis are inhibited in rat PMN depleted of its 203 (JBC. '86,261, 7592). To test whether these cells contain sufficient 1-O-alkyl-2-acyl-GPC to support PAF synthesis, phosphotidyl choline was isolated from these 204-depleted cells, fractionated into different subclasses and their fatty acid composition determined. These results were compared with those obtained with control PMN. Both control and 204-depleted PMN contained significantly large amounts of alkylacyl-GPC and diacyl-GPC. Small amounts (4%) of alkenylacyl-GPC were also present. The amount of 203 in 204-depleted cells was more or less equal to the amount of 204 in control cells. About 62% of PC-bond 204 in control PMN and about 56% of PC-bound 203 in the 204-depleted PMN was found associated with the alkylacyl species. These results show that both control and 204 depleted PMN have ample precursor substrates to support PAF biosynthesis and these substrates are enriched with 204 in control cells and with 203 in 204-depleted cells. These findings are consistant with the existance of a highly specific phospholipase A₂ capable of distinguising 204 from 203 containing phospholipids.     

The influence of Poly I∶C on the course of infection in mice inoculated with West Nile Virus

Summary In mice infected intraperitoneally with a hundred per cent lethal dose of West Nile virus a significant reduction in mortality was found if treatment with the complex of synthetic polyriboinosinic and polycytidylic acids (Poly IC) was given four hours prior to or twenty hours after virus challenge.Treatment induced large amounts of circulating interferon a few hours after inoculation. Only a slight difference in maximum viraemia in the various groups was found, but viraemia developed later in the mice given Poly IC a few hours before virus injection. Infection of the brain developed later in the groups treated with Poly IC.Using various doses of West Nile virus almost the same mortality was found in the group given a lethal virus dose but treated with Poly IC and the group receiving sublethal virus dose and no Poly IC treatment. Maximum of viraemia was high in the former group, while in the latter group it was significantly lower. Therefore it is supposed that Poly IC in these experiments did not protect through an interferon mediated suppression of the viraemia but rather through an effect of the interferon exerted directly upon the target organ. A reduction of circulating HI antibodies was found in the group on which Poly IC had the most pronounced effect.     

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.     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

Biochemical characteristics,phage patterns,and O1 factor analysis ofEscherichia coli O1∶K1∶H7∶F11 and O1∶K1∶H−∶F9 strains isolated from patients with urinary tract infections

Biochemical characteristics, O1 antigen factors and phage patterns were examined in 35 urinary O1K1 isolates ofEscherichia coli different in H and F antigen. Fermentation of dulcitol, decarboxylation of ornithine, requirement for nicotinamide, and determination of O1 factor d allowed maximum differentiation. On the basis of these tests the strains could be divided into two major groups which are obviously of different clonal origin. Members of clone 1 represented by serotypes O1K1H7F11 (12 strains) and O1K1H^–F11 (5 strains) were characterized by positive biochemical reactions and absence of O1 antigen factor d. Negative biochemical tests and presence of O1 antigen factor d were shown by strains of clone 2 which were of serotypes O1K1H^–F9 (14 strains) and O1K1H^–F^– (3 strains). Phage patterns are less well correlated with clonal assignment. However, strains of clone 2 were not susceptible to K1-specific phage D and were non-typable with another set of 13 phages.     

Recognition by peptide mapping of three different structural groups of outer membrane protein YOP-1 of Yersinia enterocolitica and Yersinia pseudotuberculosis

The structural relation of YOP-1 of european and american Yersinia enterocolitica serotypes O3, O9, O5, 27, and O8 and O20, respectively, and Y. pseudotuberculosis serotypes I, II, and III was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping using Staphylococcus aureus protease V8. Apparent molecular weights of YOP-1 ranged from 206,000 (O3) to approx. 180,000 (O8). According to their respective peptide maps YOP-1 of the european and american Y. enterocolitica serotypes and Y. pseudotuberculosis serotypes could be assigned to three different groups. Evaluation of several isolates of Y. enterocolitica serotypes O3, O9, and O8 by peptide mapping indicated that YOP-1 is conserved within a serotype. However, one serotype O8 isolate differed from the consensus peptide pattern of the other serotype O8 and O20 isolates. The similarity of the peptide patterns of Yersinia serotypes which predominate in certain geographical locations, i. e., european and american Y. enterocolitica serotypes, suggest common evolution of YOP-1 of these serotypes independent of the evolution of the other serotypes.     

TRP channels: a TR(I)P through a world of multifunctional cation channels

The transient receptor potential (TRP) family of ion channels comprises more than 50 cation-permeable channels expressed from yeast to man. On the basis of structural homology, the TRP family can be subdivided in to seven main subfamilies: the TRPC (Canonical) group, the TRPV (Vanilloid) group, the TRPM (Melastatin) group, the TRPP (Polycystin), the TRPML (Mucolipin), the TRPA (Ankyrin) and the TRPN (NOMP) family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of cell types, tissues and species. This paper briefly reviews the TRP superfamily and the basic properties of its many members as a readers guide in this Special Issue. Hopefully, a better understanding of TRP channel physiology will provide important insight into the relationship between TRP channel dysfunction and human diseases.     

High predictive value ofToxoplasma gondii IgG antibody levels in HIV-infected patients for diagnosis of cerebral toxoplasmosis

The results of serological tests forToxoplasma gondii IgG in 31 HIV-infected patients with toxoplasmic encephalitis (TE) and 49 HIV-infected patients seropositive forToxoplasma gondii but without TE were compared. All patients had a CD4+ lymphocyte count < 1 50/l. Of the TE patients, 22 (71%) were designated as having relatively high IgG levels on the basis of the followingToxoplasma IgG titre combination: Sabin-Feldman test 1256, indirect hemagglutination test 11024, direct agglutination test 114,580. Only 3 patients without TE had relatively high IgG titres. Relatively high IgG titres indicated TE with a positive predictive value of 88% in HIV-infected patients with CD4+ cell counts < 150/l, and could be observed in most patients several months prior to the first clinical and radiological signs of TE.     

SpecialEscherichia coli serotypes among enterotoxigenic strains from diarrhoea in adults and children

106 enterotoxigenicEscherichia coli strains from children and adults from many parts of the world were serotyped for O and H antigens. Some OH types,i.e. O6H16, O8H9, O15H11, O25H42, O78H11 and O78H12, were found repeatedly from different geographical locations. Some of these OH serotypes were only found rarely among more than 20000E. coli strains collected over many years from different locations and sources. It is suggested that these special OH serotypes represent clones which have been selected to the special conditions in the small intestine and selected to carry the plasmids necessary to provoke diarrhoea.     

Mating-type suppression of the DNA-repair defect of the yeast rad6Δ mutation requires the activity of genes in the RAD52 epistasis group

The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MAT a rad6 strain can be suppressed by the MAT2 gene carried on a multicopy plasmid. The a1-2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-2 suppression of the rad6 mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 mutant into the RAD52 recombinational repair pathway. The a1-2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.     

Die Nebennierenrindenfunktion unter Applikation von 9α-Fluorcortisol

Zusammenfassung Untersuchungen über den Einfluß von 9-Fluorcortisol auf die Nebennierenrindenfunktion ergaben — in Verbindung mit in der Literatur mitgeteilten Werten — eine dosisabhängige Einschränkung der Ausscheidung von Nebennierenrindenhormonen. Die Ansprechbarkeit der Nebennierenrinde auf exogenes ACTH bleibt erhalten. Es ist daher eine Hemmung der hypophysären ACTH-Sekretion anzunehmen, die durch die Struktur des synthetischen Steroids erklärbar ist. — In geringer Dosierung, wie sie als Erhaltungsdosis bei Langzeittherapie verabfolgt wird, verursacht 9-Fluorcortisol keine wesentliche Einschränkung der Hormonausscheidung.

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Effect of 9-fluorocortisol on adrenocortical function

Summary Investigation of adrenal cortical function during administration of 9-fluorcortisol revealed—in connection with results obtained from the literature—a dose-related inhibition of the secretion of adrenocortical hormones. Adrenal cortical response to exogenous ACTH remains unaffected. An inhibition of hypophyseal ACTH-secretion is therefore assumed, caused by the structure of the synthetic steroid. At low dosage, as applied in long-term treatment, no significant alteration of steroid excretion patterns was observed.

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Astonin-H, Hersteller: Fa. E. Merck A.G., Darmstadt.

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In der Arbeit werden folgende Abkürzungen verwendet: 17-KS=17-Ketosteroide; 17-OH-CS=17-Hydroxycorticosteroide; F=Cortisol=Pregn-4-en-11,17,21-triol-3,20-dion; E=Cortison=Pregn-4-en-17,21-diol-3,11,20-trion; THF=Tetrahydrocortisol=5-Pregnan-3,11,17,21-tetrol-20-on; allo-THF=allo-Tetrahydrocortisol=5-Pregnan-3,11,17,21-tetrol-20-on; THE=Tetrahydrocortison=5-Pregnan-3,17,21-triol-11,20-dion; Andro=Androsteron=5-Androstan-3-ol-17-on; Ätio=Ätiocholanolon=5-Androstan-3-ol-17-on; DHA=Dehydroepiandrosteron=Androst-5-en-3-ol-17-on.

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Herrn Prof. Dr. med. H. Franke zum 60. Geburtstag gewidmet.     

The contractile behaviour of EGTA- and detergent-treated heart muscle

Summary Tension responses of rat ventricular trabeculae subjected to successive treatment with EGTA and Triton X-100 are described in order to investigate the effects of chemical skinning techniques. In some preparations the alkaloid saponin was also used before Triton. Ultrastructural evidence is cited that the EGTA-treatment fails to render cells hyperpermeable, i.e. freely permeable to small ions, whereas both saponin and Triton do so. In this paper we show that contractile responses like those described previously for the EGTA-treated tissue can be obtained. However, more detailed examination shows that such behaviour is quantitatively distinct from that of conventionally skinned fibres in a way that is incompatible with the notion of hyperpermeability. The Ca-sensitivity after treatment with either EGTA, saponin or Triton is identical in our hands. However, this is not explained by free access of Ca (and EGTA) to the intracellular space in the EGTA-treated preparation: contractures develop with very different time courses, being fastest after Triton and only marginally slower when first exposed to saponin but a factor of five times slower after EGTA-treatment alone. This applies to contractures evoked direct from Ca²⁺ concentration 10^–9 m to the test Ca²⁺ concentration at constant total buffer concentration.EGTA-treated fibres develop tension when ATP or creatine phosphate (CrP) are removed from the bath. However, responses to ADP and to CrP changes persist with millimolar levels of ATP present, quite unlike the Triton-skinned muscle. Exposure to each of a variety of solutions for 24h produce preparations showing similar behaviour: whatever the explanation for the EGTA-skinning phenomenon it is not dependent upon low bathing Ca²⁺ concentration. On the basis of the functional characteristics described here, and the structural results cited, we conclude that the cell membrane continues to function as a selective permeability barrier after EGTA-treatment: this treatment does not produce a model of a selectively skinned cardiac cell.     

Adrenaline inhibits muscarinic transmission in bullfrog sympathetic ganglia

The effect of adrenaline (Ad) on muscarinic transmission was examined in B neurones of bullfrog sympathetic ganglia by using intracellular and voltage-clamp recording methods. Bath-application of Ad (5–500 M) caused a depression of the slow excitatory postsynaptic potential (EPSP) elicited by repetitive stimulations of preganglionic nerve fibres in the presence of curare (30 M). Ad also depressed the muscarinic ACh potential induced by ionophoretic application of ACh directly to curarized sympathetic neurones in a concentration-dependent manner. Isoprenaline mimicked the effect of Ad in producing the inhibition of the muscarinic ACh potential. Propranolol antagonized the inhibitory action of Ad. Dibutyryl adenosine 3,5-monophosphate had no significant effect on the muscarinic ACh potential. Under voltage-clamp conditions, Ad caused an inward current associated with inhibition of the M-current (Brown and Adams 1980). Ad depressed the amplitude of slow postsynaptic currents produced by applications of ACh and muscarine. At a concentration of 100 M, Ad produced a 68±8% (n=12) depression of the amplitude of the muscarinic ACh current. The inhibition of muscarinic transmission induced by Ad is due to a direct suppression of the muscarinic current at the postsynaptic membrane in bullfrog sympathetic ganglia.     

Zinc uptake by proximal cells isolated from rabbit kidney: Effects of cysteine and histidine

The aim of this study was to characterize the mechanisms of zinc transport in proximal cells isolated from rabbit kidney cortex. Uptakes of ⁶⁵Zn were assessed under initial rate conditions, after 0.5 min of incubation. The kinetic parameters obtained at 20°C were a K _m of 15.0±1.5 M, a J _max of 208.0±8.4 pmol min^–1 (mg protein)^–1, and an unsaturable constant of 0.259±0.104 (n=8). Cadmium competitively inhibited the zinc uptake, with a K _i value of 13.0±2.8 M, while zinc competitively inhibited ¹⁰⁹Cd uptake by isolated cells. Cysteine and histidine stimulated zinc transport at an amino acidzinc molar ratio ranging from 11 to 81. This stimulation was not observed in the absence of a sodium gradient. At a molar ratio greater than 161 (i.e., 400 M cysteine or histidine and 25 M Zn), there was evidence of inhibition. These data suggest that zinc enters renal proximal cells (a) as a free ion via a saturable carrier-mediated process or an unsaturable pathway and (b) complexed with cysteine or histidine, by means of a sodium/amino acid cotransport mechanism.     

Isolation of the Bα and Bβ mating-type loci of Schizophyllum commune

The B mating type of the basidiomycete fungus, Schizophyllum commune is determined by two, tightly linked, multi-specificity (also called multi-allelic) loci: B and B. A plasmid library was used in DNA-mediated transformation to obtain transformants that displayed B-directed development. Plasmids that conferred B1 and B1 mating-type specificities were rescued from the transformants. Fragments of DNA from each plasmid hybridized to genomic DNA from the strain used to make the plasmid library; however, they did not hybridize, or hybridized only weakly, to genomic DNA from strains with mating-type specificities different from B1 or B1. The cloned fragments are presumed to correspond to active regions of each B mating-type locus.     

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.     

Increasing incidence of meningococcal disease in Spain associated with a new variant of serogroup C

Serogroup B has been the main cause of meningococcal disease in Spain since at least 1979, but in recent years an increase in the prevalence of infection due to serogroup C meningococci has been detected. In 1996, for the first time, most cases of meningococcal disease were caused by serogroup C strains. The sero/subtype of all serogroup C meningococci received from 1993 to June 1996 was determined, and the results showed that C2bP1.2,5, the most common phenotype in 1995 and 1996 (63% and 65%, respectively), represented only 4.8% of strains in 1993. The C2bP1.2,5 epidemic strains appear to be responsible for the high prevalence of serogroup C in Spain. One hundred fifty-one randomly selected serogroup C strains were analyzed by multilocus enzyme electrophoresis, ribotyping, and pulsedfield gel electrophoresis. Pulsed-field gel electrophoresis provided the most accurate information: more than 80% of the C2bP1.2,5 and C2bP1.2 isolates exhibited one of two very closely related profiles, while most of the C:2b:NST and C2bP1.5 strains had a pattern located at a genetic distance of 0.24 from those two profiles. The results show that C2bP1.2,5 strains represent a subclone or a genetic variant of the previously identified Spanish epidemic clone C2bnon-subtypable strains.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Intrakranielle und intraspinale Blutungen unter Behandlung mit Cumarinderivaten

Summary From 1978–1986, 63 patients (48–79 years) under coumarin derivatives had to be hospitalized neurosurgically because of intrcranial or intraspinal bleedings. This corresponds to a twelvefold increased risk compared to the untreated people. The male/female ratio was 1.5. At the time of the bleeding there was no true indication for anticoagulation in at least 60% of the patients. 80% with coma on admission died. Only for 2/7 with an intraspinal hemorrhage the outcome was better than paraplegic. Women proved to have a better chance of survival. — There is a need for more concise indications for chronic anticoagulation.

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Abkürzungen CT Röntgen-Computertomographie - Cumarine für Cumarinderivate; hier zu 97% Phenprocoumon - ZNS-Blutung nur der Kürze wegen fürbegrifflich korrekt: intrakranielle und intraspinale Blutung Meinem Lehrer, Herrn Dr. med. Czeslaw Andrzejewski, zum 70. Geburtstag gewidmet!