Synthesis and phosphorylation of cytoskeleton components in foetal,regenerating and adult normal rat hepatocytes during culture

Summary Detergent insoluble material (DIM) was prepared by gentle treatment with detergent from foetal, regenerating and adult normal rat hepatocytes cultured for various times. It retained to some degree the morphology of the cells. After incubation of intact cells with ³⁵S-methionine, most of the labelled DIM proteins were found to be components of the cytoskeleton. They included several cytokeratins, vimentin and actin. The synthesis rate varied with the age of animals and culture conditions. The high synthetic rate of vimentin in foetal and regenerating hepatocytes could be associated with cell proliferation. No correlation was found between cytokeratin synthesis and hepatocyte growth. Most of the cytoskeleton proteins could be phosphorylated in intact cells and in DIM from cultured hepatocytes. However the degree of phosphorylation of these proteins was not related to their synthetic rate. The decreased phosphorylation level in cultured adult rat hepatocytes could be related to the rapid loss of specific functions.     

Use of tritium labeled amino acid conjugates of prostaglandins and thromboxanes as labeled ligands in prostanoid radioimmunoassay

Conjugates of prostaglandins and thromboxanes with tritium labeled amino acids were prepared and employed as labeled ligands in porstaglandin and thromboxane radioimmunoassays. Assays for PGF_2α, 15-keto-13, 14-dihydro-PGF_2α, TXB₂ and 15-keto-13, 14-dihydro-TXB₂ were evaluated in comparative studies using either these heterologous ligands or the corresponding homologus tritiated eicosanoid as tracers. Binding properties for the respective antibodies were found to be similar using either tracer.Three biological studies were also conducted, viz. study of the release of TXB₂ during collagen induced platelet aggregation, of 15-keto-13, 14-dihydro-TXB₂ during guinea pig pulmonary anaphylaxis, and of PGF_2α (measured as 15-keto-13, 14-dihydro-PGF_2α in peripheral plasma) during bovine luteolysis. The analyses gave comparable results using either the heterologous or the homologous assay.Thus, this type of labeled prostanoid conjugates may serve as a convenient alternative to homologous tracers in radioimmunoassay. Heterologous tracers may even in certain cases provide the only simple solution to the problem of preparing a labeled ligand of high specific activity.     

Microlocating lithium in the mouse embryo by use of a (n, α) nuclear reaction

Summary The quantitative imaging of lithium distribution, in histological sections of 15-days old mouse embryos (whose mother had been submitted to Li-treatment), was performed using⁶Li isotope as tracer,⁶Li(n,)³H nuclear reaction for detection, and dielectric track detectors. Despite the particular difficulties of cryosectioning the embryos without disturbing the lithium distribution, the Li regionalization appeared to be very clear-cut. The ectomesodermic tissues were significantly more loaded with lithium than the endodermic ones. This is probably related to the ectomesodermic tissues being also those most sensitive to the teratogenic effect of lithium. The Li-distribution in the embryo brain was almost homogeneous, instead of being heterogeneous as in adult brain. The mean Li-concentration in the embryo brain was not much below the Li concentration in the grey matter of the mother brain, but it was significantly larger than that in the white matter of the mother brain. Our results are discussed in the context of teratogenic effects observed in situ during mammalian development.     

DNA-polymorphic patterns linked to the β-globin genes in German families affected with hemoglobinopathies and thalassemias: a comparison to other ethnic groups

Summary DNA haplotype constellations of the β-globin gene cluster have been analyzed in German families with hemoglobinopathies (Hb Freiburg, Hb K?ln, Hb Presbyterian) and β-thalassemias. The polymorphis patterns obtained were compared to those found in families from Greece, Italy, and Turkey affected by β-thalassemia syndromes. With the combined analysis of seven restriction site polymorphisms a DNA-diagnostic prediction for additional offspring could be made with an overall frequency of 75% in the four ethnic groups.     

Mapping of cysteine genes on the chromosome of Pseudomonas aeruginosa PAO

Summary Three loci coding for different steps in the pathway of cysteine biosynthesis have been mapped by R68.45-mediated coconjugation analysis. The cysteine auxotrophic mutants could be subdivided into sulfite and sulfide-requiring mutants. Sulfide-requiring mutants (cysIV group) were localized at a single position between pyrF and pur-67, while sulfite-requiring mutants (cysI and cysII) mapped at two different regions. The cysI group was also localized between pyrF and pur-67, although more distal to pyrF than the cysIV group. This group included the cys-54 marker, which has been mapped previously. The second group of sulfite-requiring mutants, designated as cysII, was cotransducible with hisI and localized at the end of the PAO chromosomal map. This location was also confirmed for the marker cys-59.The marker cys-59 (which was cotransducible with his1) was cotransferred by R68.45-mediated conjugations with both the late marker pur-67 and the early marker ilv-226. As the late marker hisI was positioned at about 60–65 min (Herrmann and Günther, in press) the length of the PAO chromosome was estimated to be about 70 min.     

pH dependence of the in situ formation of an organicN-chloramine water disinfectant

Summary Staphylococcus aureus was used to assess the bactericidal efficacy of aqueous solutions of the organicN-chloramine compound 3-chloro-4,4-dimethyl-2-oxazolidinone (agent I) formed in situ. The rate of in situ formation, accomplished by reacting free chlorine with the amine precursor, was a function of pH. When the reagents were combined under acidic conditions (pH5.5) and allowed to react for 22 h, sufficient residual free chlorine was present to inactivate the bacteria in less than 5 min. When combined under less acidic conditions (pH6.0), comparable bacterial inactivation required 30–60 min due to the extensive reaction of the free chlorine to form agent I. The kill rates present under less acidic and neutral conditions are equivalent to those for pre-formed agent I. In water disinfection applications for pH6.0, in situ formation of agent I would provide a combination of rapid initial and slower long-term disinfection.     

New derivatization and separation procedures for reducing oligosaccharides

4-Trifluoroacetamidoaniline was reacted with reducing oligosaccharides in the presence of sodium cyanoborohydride to give aminoalditol derivatives, useful for linkage to proteins or solid matrices. A mixture of reducing oligosaccharides, difficult to separate by HPLC, was treated in the same way. The resulting derivatives were easily separated by HPLC.Abbreviations TFAN 4-trifluoroacetamidoaniline - LcOse₄ lacto-N-tetraose - IV²Fuc-LcOse₄ lacto-N-fucopentaose l - III⁴Fuc-LcOse₄ lacto-N-fucopentaose II - III³Fuc-nLcOse₄ lacto-N-fucopentaose III - IV²Fuc, III⁴Fuc-LcOse₄ lacto-N-difucohexaose I - II⁶Galß1-4GlcNAc-LcOse₄ lacto-N-hexaose - II³NeuAc-Lac 3-sialyllactose - GlcNAcß1-4GlcNAcß1-4GlcNAc chitotriose - GalNac1-3|Fuc1-2|Galß1-4Glc A-tetrasaccharide     

Primary astroglial cultures

Conclusion Primary cultures from neonatal rat brain consist mainly of astroglial cells, immunohistochemically identified by GFAp and S-100. As other cells than astrocytes may survice in the culture, specific markers for the expected cells were used. Cells with phagocytic properties, endothelial-like cells, oligoblasts, ependymal cells and mesenchymal cells were identified. No neurons have so far been detected.The astroglial cells have a high-affinity uptake for glutamate, aspartate GABA, taurine and hypotaurine, while there is probably a non-saturable uptake of norepinephrine, dopamine and 5-HT. The enzymes MAO, COMT, GABA-T and GS have been demonstrated. It thus seems that astrocytes take part in the inactivation of neurotransmitters, although amino acids and monoamines are taken up with different mechanisms.The presence of receptors for different neurotransmitters and neuromodulators has been demonstrated on astrocytes.Astroglial-enriched cultures from various brain regions have shown that the cells express specialized functional properties concerning neurotransmitter uptake, metabolizing enzymes and receptor density.Astroglial cell differentiation in culture is shortly reviewed and one possibility to affect this maturation by co-cultivation with neuronal containing cultures is point out.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by ¹H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.     

Proton-translocating ATPase and lysosomal cystine transport

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.