Protease inhibitors reduce lysosomal acid phospholipase A1 activity in cultured rat hepatocytes

When rat hepatocytes were cultured in the presence of various specific protease inhibitors, lysosomal acid phospholipase A1 activity decreased progressively. Exposure of the cultured cells to 0.1 micrograms/ml of pepstatin, E 64, leupeptin or chymostatin also reduced the catalytic activities of several lysosomal marker enzymes. Irrespective of the protease inhibitor type employed, acid phospholipase A1 activity reacted most sensitively, followed by acid phosphatase, acid beta-N-acetyl-D-hexosaminidase and acid beta-glucuronidase. Of the protease inhibitors studied, pepstatin appeared to be most potent in reducing lysosomal enzyme activities in cultured hepatocytes. These findings suggest that proteolytic processes at as yet unknown, possibly extralysosomal sites play an important role in the turnover rates of lysosomal enzymes.     

Genetic variation underlying local adaptation of diapause induction along a cline in a butterfly

Diapause is a life history strategy allowing individuals to arrest development until favourable conditions return, and it is commonly induced by shortened day length that is latitude specific for local populations. Although understanding the evolutionary dynamics of a threshold trait like diapause induction provides insights into the adaptive process and adaptive potential of populations, the genetic mechanism of variation in photoperiodic induction of diapause is not well understood. Here, we investigate genetic variation underlying latitudinal variation in diapause induction and the selection dynamics acting upon it. Using a genomewide scan for divergent regions between two populations of the butterfly Pararge aegeria that differ strongly in their induction thresholds, we identified and investigated the patterns of variation in those regions. We then tested the association of these regions with diapause induction using between‐population crosses, finding significant SNP associations in four genes present in two chromosomal regions, one with the gene period, and the other with the genes kinesin, carnitine O‐acetyltransferase and timeless. Patterns of allele frequencies in these two regions in population samples along a latitudinal cline suggest strong selection against heterozygotes at two genes within these loci (period, timeless). Evidence for additional loci modifying the diapause decision was found in patterns of allelic change in relation to induction thresholds over the cline, as well as in backcross analyses. Taken together, population‐specific adaptations of diapause induction appear to be due to a combination of alleles of larger and smaller effect size, consistent with an exponential distribution of effect sizes involved in local adaption.     

Physiological characterization of a microbial sensor containing the yeast Arxula adeninivorans LS3

The yeast Arxula adeninivorans LS3 is a suitable organism for use as part of a microbial sensor. In combination with an amperometric oxygen electrode the sensor offered a possibility for the physiological characterization of this yeast. About 300-400 measurements could be carried out with a single Arxula sensor. The microbial sensor was remarkably stable for over 35 days, when kept at 37 °C during the operation time and at room temperature overnight. The physiological characteristics of Arxula adeninivorans LS3 obtained with the sensor technique were identical to the data obtained with the conventional techniques. However, the sensor technique makes it additionally possible to quantify the physiological data. So the substrates ribose, citric acid, glycerol, oil and benzoate produced signals lower than 10% in comparison to the glucose signal. Fructose, xylose, sucrose, maltose, gentianose, glucosamine, glutamic acid, tryptophan, butyric acid, lauryl acid and propionic acid reached 10-70%, galactose, alanine, glycine, lysine and methionine signals were similar to the glucose signal whereas acetic acid, ethyl alcohol, capron acid, capryl acid and caproic acid reached the highest signals up to 434%.     

Purification and Characterization of Class Alpha and Mu Glutathione S-Transferases From Porcine Liver

Six cytosolic GSTs from porcine liver were purified by a combination of glutathione affinity chromatography and ion-exchange HPLC. The isoenzymes were characterized by SDS-PAGE, gel filtration, isoelectric focusing, immunoblotting analysis and determination of substrate specificities and inhibition characteristics. The purified GSTs belong to the alpha and mu classes, respectively. No class pi isoenzyme was isolated or detected. The class alpha GST pA1-1* exists as a homodimer (M_r = 25.3 kDa), whereas GST pA2-3* consists of two subunits with different M_r values (27.0 and 25.3 kDa). The estimated pI values were 9.5 and 8.8, respectively. Furthermore, four class mu porcine GSTs, pM1-1*, pM1-2*, pM3-?* and pM4-?*, were isolated. The isoenzyme pM1-1* possesses a relative molecular mass of 27.2 kDa and a pI value of 6.2. Additional pM1 isoenzymes hybridize with the subunit pM2* (M_r = 25.2) to furnish a heterodimer, which shows a pI value of 5.8. The other class mu isoenzymes are heterodimers with pI values of 5.45 and 5.05. Substrate specificities and inhibition characteristics correlate very well with those of the corresponding human isoenzymes. The results are discussed with regard to the usefulness of porcine GSTs as an in vitro testing model.     

The myxochelin iron transport regulon of the myxobacterium Stigmatella aurantiaca Sg a15.

The biosynthetic gene cluster of the myxochelin-type iron chelator was cloned from Stigmatella aurantiaca Sg a15 and characterized. This catecholate siderophore was only known from two other myxobacteria. The biosynthetic genes of 2,3-dihydroxybenzoic acid are located in the cluster (mxcC-mxcF). Two molecules of 2, 3-dihydroxybenzoic acid are activated and condensed with lysine in a unique way by a protein homologous to nonribosomal peptide synthetases (MxcG). Inactivation of mxcG, which encodes an adenylation domain for lysine, results in a myxochelin negative mutant unable to grow under iron-limiting conditions. Growth could be restored by adding Fe3+, myxochelin A or B to the medium. Inactivation of mxcD leads to the same phenotype. A new type of reductive release from nonribosomal peptide synthetases of the 2, 3-dihydroxybenzoic acid bis-amide of lysine from MxcG, catalyzed by a protein domain with homology to NAD(P) binding sites, is discussed. The product of a gene, encoding a protein similar to glutamate-1-semialdehyde 2,1-aminomutases (mxcL), is assumed to transaminate the aldehyde that is proposed as an intermediate. Further genes encoding proteins homologous to typical iron utilization and iron uptake polypeptides are reported.     

Inhibition of Na- and K-activated ATPase by the direct lytic factor of cobra venom (Naja naja)

The Mg²⁺-dependent, Na⁺-K⁺-activated ATPase of ox brain was inhibited by the direct lytic factor of cobra venom at concentrations of 10⁻⁷ g/ml or higher. Only weak inhibition was seen in ghosts of human red cells. Haemolysis of guinea-pig red cells by phospholipase A was not enhanced when the erythrocyte ATPase had been blocked by ouabain. It is concluded that direct lytic factor-induced haemolysis is not dependent on an ATPase inhibiting effect.     

Fluoreszenzzytophotometrische Bestimmung der Zellkern-DNS

Zusammenfassung Die Akriflavin-Feulgen-Färbung (Böhm und Sprenger, 1968) und die Ethidiumbromid-Fluorochromierung nach vorausgehender Pepsinbehandlung (Göhde et al., 1971) ergeben an Leberzellausstrich-Präparaten weitgehend gleichartige DNS-Häufigkeitsverteilungen. Bei der zeitlich aufwendigen (220 min) Akriflavin-Färbung ist eine optimale Darstellung der Zellmorphologie und eine jahrelange Archivierung der Ausstrichpräparate möglich.Die rasch durchführbare Ethidiumbromid-Färbung (70 min) bietet nur nach schwerer Alteration der Zellmorphologie durch die vorausgehende Pepsinbehandlung eine selektive Darstellung der DNS. Eine Archivierung von Ausstrichpräparaten ist nur für 2 Tage möglich. Die schwere Alteration der Zellmorphologie ist besonders bei durchflußphotometrischen Prescreeninguntersuchungen in der gynäkologischen Krebsvorsorge nachteilig. Zellpopulationen, die zu einer zytophotometrischen Verdachtsdiagnose geführt haben, sind durch Verlust des Zytoplasmas und der Kernstruktur einer konventionellen morphologischen Diagnostik nicht mehr zugängig.

---

Fluorescence cytophotometric determination of nuclear DNAComparative study of the acriflavine-feulgen and the ethidiumbromide staining reactions

Summary Acriflavine-Feulgen (Böhm und Sprenger, 1968) and ethidiumbromide fluorescence after previous pepsin digestion (Göhde et al., 1971) yield corresponding DNA distribution patterns when applied to liver smears. With the time-consuming acriflavine staining (220 min) the cellular morphology is best preserved and the stained specimen may be stored for long periods.The rapidly obtainable ethidiumbromide staining (70 min) results in a selective fluorescence of DNA only after heavy alteration of the cellular morphology by pepsin digestion. Storage of the material is only possible for two days. The heavily altered cellular morphology, however, is rather unfavorable for prescreening in automated cytology. Specimens that were found to be suspicious are no longer suitable for conventional cytological diagnosis, because they have lost their cytoplasm and nuclear chromatin structure.

---

---

Mit Unterstützung durch das Bundesministerium für Jugend, Familie und Gesundheit.     

PolyploideLupinus luteus

Zusammenfassung Die Gründe für die Suche nach polyloiden Pflanzen vonLupinus luteus werden dargelegt. Auf die Schwierigkeiten bei der künstlichen Auslösung von polyploiden Formen von großkörnigen Leguminosen wird an Hand der Literatur verwiesen.Für die beschriebenen polyploidenLupinus luteus wird die Abstammung aus derF ₁ einer Kreuzung morphologisch unterschiedlicher Formen nachgewiesen und der geringe Vermehrungsfaktor in 5 Jahren herausgestellt. Die im 4. Jahr nach der Auffindung erfolgte Rückregulierung des Chromosomensatzes einer Pflanze ergab sofort eine normale Fertilität.Die Organgrößenvergleiche fielen in allen Fällen zu Gunsten der polyploiden Formen aus. Es wurden dazu folgende Organe miteinander verglichen: Samenkorngewichte, Chromosomenzahlen, die Länge und Breite von 5-, 7-und 9fingerigen Laubblättern, die Größe der Spaltöffnungszellen, die. Größe und Anazahl der Blüten sowie die Größe der Pollenkörner.Mit 10 AbbildungenHerrn. Prof. Dr.F. Oberdorf zum 65. Geburtstag gewidmet     

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.

     

Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia

Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.