Electron-transfer restoration by vitamin K3 in a complex III-deficient mutant of S. cerevisiae and sequence of the corresponding cytochrome b mutation

The yeast box-mutant W7 exhibits deficiencies in cytochrome b and in nuclear coded complex III subunits, a phenotype observed previously in a patient with mitochondrial myopathy. DNA sequence analysis of mutant W7 revealed a single base transition in the cytochrome b gene; the mutated residue Gly 131 is perfectly conserved in all known cytochromes b and belongs to the Qo domain. Mutant W7 provides a model system for evaluating the action of therapeutic agents, such as vitamin K3 which restored NADH-oxidase activity in the mutant as well as in the antimycin-inhibited wild type. However, with the mutant, a greater quantity of menadione was necessary due to a decrease in other complex activities, and a much lower electron-flow fraction passed through cytochrome oxidase.     

Tryptophan environments in glutathione transferase of human placenta from temperature-dependent phosphorescence studies

An investigation of the tryptophan emission properties of glutathione transferase from human placenta was conducted in order to characterize the environments of the two aromatic residues. The low-temperature phosphorescence spectra and temperature dependence of the phosphorescence quantum yield of the tryptophan residues revealed a difference in the chemical nature and dynamical structure of the surrounding protein matrix. Thus, one tryptophan residue seems to be deeply embedded within the polypeptide in a rigid weakly polar environment, characteristic of a beta-type secondary structure. The other is located in a more polar site, probably near the surface, in a rather flexible region of the macromolecule. At high temperature, the heterogeneity in the triplet lifetime of the internal residue attests to the presence of multiple conformers which are not in rapid equilibrium in the phosphorescence time scale. The anisotropy of the phosphorescence emission of glutathione transferase indicates that no energy transfer occurs between the two residues, and measurement of the rotational correlation time yields an hydrodynamic volume which is in good agreement with the molecular weight reported in the literature for the dimer.     

Influence of lactate on isoproterenol-induced lipolysis and beta-adrenoceptors distribution in human fat cells

The influence of lactate on human adipocytes lipolysis and the possible relationship between lactate-induced metabolic effects and beta-adrenoceptor binding sites were investigated. beta-sites were identified in membranes with (125I)-cyanopindolol and in intact cells with (125I)-cyanopindolol and (3H)-CGP 12177. Lactate reduced isoproterenol-induced lipolysis in a dose-response fashion and such inhibition became significant only at 16 mmol/l lactate. Exposure of human fat cells to 16 mmol/l lactate significantly reduced beta-adrenoceptors density on crude membranes. When the binding assay was performed on intact cells using (125I)-cyanopindolol at 37 degrees C, the radioligand identified the same number of receptors, regardless of the presence of lactate in the preincubation medium. When (3H)-CGP 12177 was used, it bound to about 35% less receptors in lactate pre-treated cells than in control. Seemingly, at 37 degrees C, because of its lipophilicity, (125I)-cyanopindolol can cross the plasma membrane and bind to intracellular sites whereas, (3H)-CGP 1277, due to its hydrophilicity, identifies surface receptors only. Thus, the present in vitro study provides evidence that high levels of lactate, similar to the concentrations usually achieved in overt lactic acidosis, are able per se to inhibit human lipolysis and to redistribute beta-adrenoceptors from cell surface to a domain not accessible to hydrophilic ligands.     

Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1

The small cryptic plasmid pMB1 (1.9 kb), previously isolated from Bifidobacterium longum, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.     

Transformation of NIH 3T3 cells by overexpression of the normal coding sequence of the rat neu gene.

While the normal human erbB-2 gene is potently transforming when overexpressed in NIH 3T3 cells, its rat homolog, the neu gene, seems to acquire transforming properties only upon alteration of its coding sequence. In this study, we compared the effects of different levels of expression of normal erbB-2 and neu in NIH 3T3 cells. Our results revealed that the normal rat neu gene acts as a potent oncogene when sufficiently overexpressed in NIH 3T3 cells.     

Synthesis of rhodanese in Hep 3B cells

Summary The biosynthesis of rhodanese was studied in human hepatoma cell lines by immunoblotting and pulselabeling experiments using polyclonal antibodies raised against the bovine liver enzyme. Rhodanese, partially purified from human liver, showed an apparent molecular weight of 33,000 daltons, coincident with that of rhodanese from Hep 3B cells. After pulse labeling of Hep 3B cells both at 37°C and 25°C, rhodanese in the cytosol fraction exhibited the same molecular weight as the enzyme isolated from the particulate fraction containing mitochondria. Moreover, newly synthesized rhodanese from total Hep 3B RNA translation products showed the same electrophoretic mobility as rhodanese from Hep 3B cells. These results suggest that rhodanese, unlike most mitochondrial proteins, is not synthesized as a higher molecular weight precursor.     

The myelodysplastic syndrome: prognostic factors in relation to the "in vitro" clonogenic assay.

The object of this study was to determine whether the "in vitro" parameters of medullary and blood granulopoiesis in patients with MDS, furnish information of either prognostic or diagnostic value. This study covered 94 patients with MDS. All patients were studied at the onset of disease. In order to identify the factors related to patients' survival, Cox Multiple Regression analysis was performed by the BMD P2L program. When analyzing by means of actuarial curves the survival probability of patients with benign development versus those of malignant development (those who developed ANLL), the significance between both groups was p = 0.0001. Different variables of patients included in this study were analyzed and all showed great significances. Fab: p = 0.0022, disease evolution: p = 0.0001 and presence of blastic aggregates: p = 0.0011. Cox's regression analysis revealed that the only predictable survival variable is the presence of blastic colonies and/or clusters. Accordingly, two groups were constructed: favourable and unfavourable. In the favourable group, 40% of the patients belonged to the RA group, while in the unfavourable group, 55% belonged to the RAEB group. This study shows the validity of the elaboration of prognostic groups in MDS according to the presence of blastic colonies and/or clusters in CFUGM medullary and/or peripheral cultures. The "in vitro" myeloid progenitors culture techniques may therefore be advantageously applied in these disorders for formulating a diagnosis and predicting the patient's short term evolution.     

大鼠隔—海马通路损伤对海马内递质含量及酶活力的影响

单侧切断大鼠海马缴和部分穹窿可使海马部分去神经。损伤后7d,海马内胆碱能系统中乙酰胆碱(ACh)含量下降72.5%,胆碱乙酰基转移酶(ChAT)活力下降45.7%,胆碱酯酶活力下降52.2%,在单胺能系统中,去甲肾上腺素(NA)含量下降16.3%,多巴胺(DA)含量下降31.3%,5-羟色胺含量下降30.3%。在损伤过程中,海马内氨基酸含量没有改变。实验结果表明,海马缴和穹窿是脑内胆碱能和单胺能传入神经到达海马靶区的部分共同通路。