A comprehensive survey for polymorphisms in the bovine IFN-gamma gene reveals a highly polymorphic intronic DNA sequence allowing improved genotyping of Bovinae.

This study aimed to identify interferon-gamma (IFN-gamma) gene variants in cattle for diagnostic purposes. Therefore, the entire bovine IFN-gamma gene (BoIFNG) and 2605 bp of its promoter DNA were sequenced. The BoIFNG DNA sequence conforms to the published part of Bo-IFN-gamma cDNA. Primer extension experiments show the presence of a 5' extension of exon 1 by 42 nucleotides (nt). One SINE element (Bov-A2) is located in the 5'-region, and two SINE elements (Bov-tA, Bov-B) are contained in the 3'-region of BoIFNG. The variants were detected by comparative sequence analysis of PCR amplicons from different bovine species. Four polymorphic mononucleotide repeats are situated in the promoter and in intron 1. Four distinct series of single nucleotide polymorphisms (SNP) were found in functionally important regions of BoIFNG. The region between the two intron 1 microsatellites contains the highest density of SNPs in Bos taurus breeds. One G-T transversion in the coding region of exon 1 causes a Gly(14) to Val(14) exchange in the BoIFNG signal peptide of different bovine species. A G-A transition in exon 2 encodes a Ser(19) to Asn(19) change in the mature protein of the Tibetan yak. Genotyping of randomly sampled Holstein Friesian cows at selected SNPs and of both intron 1 microsatellites revealed two dominant BoIFNG microhaplotypes. The detected SNPs improve the recently reported genotyping system of cattle.     

Increased drug cytotoxicity at reduced pH counteracts cyclophosphamide resistance in cultured rat mammary carcinoma cells

The sensitivity of a cyclophosphamide (CP)-resistant MIR rat mammary carcinoma cell variant (MIRCPr) in monolayer culture towards the cytotoxic effect of mafosfamide (an analogue of "activated" CP) was measured as a function of extracellular pH (pHe). An inverse correlation was found between cell survival and the H+ ion concentration in the culture medium. At pHe 7.4, the fraction of clonogenic MIRCPr cells exposed to mafosfamide (7.5 micrograms/ml) for 24 hr was 1 X 10(-1) in relation to untreated control cells. At pHe 6.2, however, this value was reduced to 3 X 10(-4), i.e., a value equal to that for the CP-sensitive parental MIR cells exposed to the same concentration of mafosfamide at pHe 7.4. Our data indicate complete compensation of CP resistance in MIRCPr cells at pHe 6.2. MIRCPr cells were not resistant to the cytotoxic effect of nornitrogen mustard. This suggests that resistance to CP in MIRCPr cells is due to enzymatic inactivation of the primary intermediates in CP bioactivation. The alkylating activity of nornitrogen mustard (and less so that of phosphoramide mustard) is strongly enhanced at low pH. In MIRCPr cells shifted to an acidic environment, therefore, a (putative) decrease in the intracellular concentration of alkylating CP metabolites may be counteracted by an enhancement of their alkylating activity (on a molar basis). By parenteral administration of glucose, the pH in malignant tumors of both animal and human origin can be lowered to values between 5.6-6.6. Our data suggest that an upshift of H+ ion concentration in malignant tissues may at least partially counteract CP resistance in cancer cells in vivo.     

Enhancement of immunogenicity of Jeg3 cells by ectopic expression of HLA-A*0201 and CD80

PROBLEM: The choriocarcinoma cell line Jeg3 suppresses immunity in vitro by secretion of soluble factors like leukemia inhibitory factor suppressing leukocyte activation. The cells lack expression of classical human leukocyte antigen (HLA)-A and -B alleles but express some HLA-C, and non-classical HLA-G and -E. Upon binding to killing inhibitory receptor on natural killer (NK) cells, HLA-G prevents activation of cytolytic activity. We investigated whether Jeg3 cells are capable of immune stimulation after complementation with classical HLA and T cell costimulatory signal CD80. METHOD OF STUDY: Jeg3 cells were transduced to express HLA-A*0201 and/or CD80. Parental Jeg3 or transfectants Jeg3-A2, Jeg3-CD80 or Jeg3-CD80-A2 were used to stimulate allogeneic resting and activated peripheral blood lymphocytes (PBL). The different cell lines were loaded with a HLA-A2-restricted Epstein-Barr virus (EBV) recall antigen peptide epitope and antigen presenting ability was examined. T cell lines specific for Jeg3 and transfectants were generated from HLA-A2 matched and nonmatched donors and compared for expansion, phenotypes and cytolytic activity. RESULTS: While all Jeg3 cell lines induced only marginal proliferation of resting T cells, phytohemagglutinin (PHA)-activated T cells were stimulated by CD80 or CD80-A2 expressing Jeg3. Only the transfectant Jeg3-CD80-A2 was capable of specific T cell stimulation by EBV recall antigen presentation. T cell lines of HLA-A2 non-matched donors stimulated with the Jeg3 transfectants showed significant expansion only when HLA-A2 and the costimulus CD80 were present. T cells from HLA-A2 positive donors did not expand significantly or differentially. No NK cells grew under any condition. In Jeg3-CD80-A2 stimulated T cells lines CD8+ cells expanded preferentially. These T cells exerted cytolytic activity toward all Jeg3 cell lines. CONCLUSION: Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by Jeg3 cells when classical HLA- and/or costimulatory signals are present on the cells.     

Autoregulation of contractility in the myocardial cell

Summary An investigation was carried out on isolated cat's papillary muscle in order to study displacement effects upon the intensity and the time course of the contractile activity. Displacements occurring before or very early during a contractile cycle produce effects which can be entirely explained on the basis of the cardiac active length-tension relation. Displacements occurring later exhibit additional effects in so far as either stretches or releases induce a drop of contractile activation such that the course of the subsequent tension development is markedly below that of the same displacement applied earlier. In order to separate these effects from those based on the active length-tension correlation experiments were performed in which very short release-stretch or stretch-release operations were applied so that the muscle length was virtually the same at the beginning and at the end of the operation. The results obtained under these conditions can be summarized as follows.The extend to which contractile tension drops after a stretch-release or a release-stretch cycle has been applied depends upon (1) the stimulus intervention interval (2) the length change performed (3) the velocity of displacement during the intervention. It is not dependent on the initial muscle length. Increasing the extracellular Ca-concentration considerably reduces the displacement effects. The results are tentatively explained by assuming an internal feedback loop between a variable of the contractile machinary and the preceding mechanism of activation.This investigation was supported by the Deutsche Forschungsgemeinschaft (grant Ka 287, 1+3).     

Complement-opsonized HIV: the free rider on its way to infection

The complement system (C) is one of the main humoral components of innate immunity. Three major tasks of C against invading pathogens are: (i) lysis of pathogens by the formation of the membrane attack complex (MAC); (ii) opsonization of pathogens with complement fragments to favor phagocytosis; and (iii) attraction of inflammatory cells by chemotaxis. Like other particles, HIV activates C and becomes opsonized. To escape complement-mediated lysis, HIV has adopted various properties, which include the acquisition of HIV-associated molecules (HAMs) belonging to the family of complement regulators, such as CD46, CD55, CD59, and the interaction with humoral regulatory factors like factor H (fH). Opsonized virus may bind to complement receptor positive cells to infect them more efficiently or to remain bound on the surface of such cells. In the latter case HIV can be transmitted to cells susceptible for infection. This review discusses several aspects of C-HIV interactions and provides a model for the dynamics of this process.     

Quartz glass pipette puller operating with a regulated oxy-hydrogen burner

Quartz glass electrodes are superior to conventional glass electrodes for low-noise recording. They have better electrical characteristics and hydrophobic surfaces which resist creeping of salt solutions. We used oxy-hydrogen heating with program-controlled gas pressure to melt quartz glass capillaries. Usually, the relative wall thickness (the quotient of the outer and inner diameters do/di) of capillaries is, at best, maintained up to the electrode tip. If tips with thicker walls can be produced, coating and other surface treatments can be avoided. We found that programmed heating periods without pull allowed an fivefold increase of do/di in the tip region. Since do/di is inversely proportional to input capacity, the recording noise was minimized and became insignificant relative to amplifier and holder noise. A sample patch-clamp recording is shown.     

Der Einfluß von N1-n-Butylbiguanid auf das Verhalten der Unveresterten Fettsäuren (UFS) bei Normalpersonen und bei Diabetikern und am epididymalen Fettgewebe der Ratte

Zusammenfassung Das Verhalten der Unveresterten Fettsäuren an Gesunden und Diabetikern und am epididymalen Fettgewebe der Ratte unter N₁-n-Butylbiguanid wurde untersucht. Normalpersonen mit und ohne Biguanid reagieren bei Blutzuckerabfall mit einem Anstieg der UFS, der unter Biguanid signifikant höher ist. Diabetiker zeigen einen geringfügigen Abfall der UFS nach Biguanid. Unter Biguanidkonzentrationen, die der therapeutischen Serumkonzentration entsprechen (5/ml), werden vom Fettgewebe in Gegenwart von Glucose vermehrt UFS utilisiert. Bei hohen Konzentrationen (100 bis 1000/ml) tritt ein Wirkungsumschlag ein, die Fettsäureutilisation wird gehemmt.Teile dieser Arbeit sind in den Dissertationen vonM. L'age undJ. Stehr enthalten.