Molecular cloning and characterization of the alkB gene of Escherichia coli

Summary Using methods of in vitro recombination we constructed hybrid plasmids that can suppress the increased methylmethane sulfonate sensitivity caused by alkB mutation. Since the cloned DNA fragment was mapped at 47 min on the Escherichia coli K12 genetic map, an area where the alkB gene is located, we concluded that the cloned DNA fragment contains the alkB gene itself but not other genes that suppress alkB mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkB codes for a polypeptide with a molecular weight of about 27,000. Introduction of a small deletion into the alkB region of the bacterial chromosome resulted in inactivation of both the alkB and ada genes, thereby suggesting that the two genes are adjacent on the E. coli chromosome.Abbreviations Ap ampicillin - Cm chloramphenicol - HPLC high performance liquid chromatography - kb kilobases - kd kilodaltons - MMS methylmethane sulfonate - MNU methylnitrosourea - MNNG N-methyl-N-nitro-N-nitrosoguanidine - Tc tetracycline - SDS sodium dodecyl sulfate     

Conditional VHL Gene Deletion Causes Hypoglycemic Death Associated with Disproportionately Increased Glucose Uptake by Hepatocytes through an Upregulated IGF-I Receptor

Our conditional VHL knockout (VHL-KO) mice, having VHL gene deletion induced by tamoxifen, developed severe hypoglycemia associated with disproportionately increased storage of PAS-positive substances in the liver and resulted in the death of these mice. This hypoglycemic state was neither due to impaired insulin secretion nor insulin receptor hypersensitivity. By focusing on insulin-like growth factor I (IGF-I), which has a similar effect on glucose metabolism as the insulin receptor, we demonstrated that IGF-I receptor (IGF-IR) protein expression in the liver was upregulated in VHL-KO mice compared to that in the mice without VHL deletion, as was the expression of glucose transporter (GLUT) 1. The interaction of the receptor for activated C kinase (RACK) 1, which predominantly binds to VHL, was enhanced in VHL-KO livers with IGF-IR, because VHL deletion increased free RACK1 and facilitated the IGF-IR-RACKI interaction. An IGF-IR antagonist retarded hypoglycemic progression and sustained an euglycemic state. These IGF-IR antagonist effects on restoring blood glucose levels also attenuated PAS-positive substance storage in the liver. Because the effect of IGF-I on HIF-1α protein synthesis is mediated by IGF-IR, our results indicated that VHL inactivation accelerated hepatic glucose storage through the upregulation of IGF-IR and GLUT1 and that IGF-IR was a key regulator in VHL-deficient hepatocytes.     

Symbiotic Bacteria Associated with Stomach Discs of Human Lice

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation “Candidatus Riesia pediculicola” for the louse symbionts.     

Infection of Actinomycin-Permeable Mutants of Escherichia coli with Urea-Disrupted Bacteriophage

Intact cells of actinomycin-permeable mutants of Escherichia coli could be infected with urea-disrupted phage T4 (designated as T4pi). The parental strains and the revertants, which are impermeable to actinomycin, were not susceptible to T4pi unless they had been treated with agents which altered their permeability. The permeable mutants developed competence for pi infection during the growth cycle; cells in the early stationary phase produced 10- to 100-fold more plaques on plating with T4pi than did exponentially growing cells. Colistin (polymyxin E) was effective in converting noncompetent cells of either permeable or nonpermeable strains to the competent state. Treatment with lysozyme resulted in a considerable increase in susceptibility to T4pi of permeable mutants but not of nonpermeable cells. It appears that development of competence for pi infection is mainly due to alterations in the permeability barriers of the cell.     

Establishment and characterization of a human cell line, HS-MM, derived from a case of clear cell sarcoma

The characteristics of a new human clear cell sarcoma (CCSa) cell line, HS-MM, established from the pleural effusion in a 39-year-old man with lung metastasis, have been morphologically studied in vitro and in vivo. HS-MM cells growing on a cover-slip were round or spindle in shape with round nuclei containing extremely prominent nucleoli. Heterotransplantation of the cells into nude mice was easily succeeded following tumor development. Light microscopically, HS-MM cells, both in vitro and in vivo, were positive for anti-S-100 protein and anti-melanoma specific antibodies with immunostain, but no melanin pigment was detected in them. Ultrastructurally, the cells had round euchromatin-rich nuclei with large nucleoli revealing conspicuous nucleolonema, and contained a few mitochondria, rough endoplasmic reticulum and lysosomal dense bodies, besides a large amount of glycogen, but no melanosome in their cytoplasm. HS-MM cells retained and fully expressed morphologically unique characteristics as a CCSa, compatible with amelanotic type of malignant melanoma also. This cell line, HS-MM, therefore, proves to be extremely useful for clinicopathological studies on a CCSa.     

Xylitol Production by a Corynebacterium Species

The washed cells of a gluconate-utilizing Corynebacterium strain grown in a gluconate- xylose medium produced xylitol from D-xylose in the presence of gluconate. The amount of xylitol was progressively increased with increasing gluconate concentration.

An extract of cells grown in the gluconate-xylose medium showed NADPH-dependent D-xylose reductase activity and NADP-dependent 6-phosphogluconate dehydrogenase activity.

These enzymes in the cell-free extract were purified by Sephadex G–100 gel filtration.

The reduction of D-xylose to xylitol was demonstrated by the coupling the D-xylose reductase activity to the 6-phosphogluconate dehydrogenase activity with NADP as a cofactor using the cell-free extract and the fractionated enzymes.     

Binding capacity of human YB-1 protein for RNA containing 8-oxoguanine

8-oxoguanine (8-oxo-7,8-dihydroguanine) is generated in the cellular nucleotide pool as well as in nucleic acids, by the action of oxygen radicals produced in cells. 8-oxoguanine has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms should have mechanisms for preventing the misincorporation of 8-oxoguanine-containing nucleotide into RNA and for removing 8-oxoguanine-containing RNA from processes of translation. We now report that mammalian Y box-binding protein 1 (YB-1 protein) possesses the activity to bind specifically to RNA containing 8-oxoguanine. On incubation with a purified preparation of YB-1 protein, 8-oxoguanine-containing RNA forms stable complexes with the protein while normal RNA scarcely forms such a complex. Using a series of deletion mutants which produce altered forms of YB-1 protein lacking some parts of the sequence, domains of the protein necessary for RNA binding were identified. Escherichia coli cells expressing normal or truncated forms of YB-1 protein with the binding capacity acquire resistance against paraquat, a drug that induces oxidative stress in cells, whereas cells with truncated proteins lacking such an activity do not. YB-1 protein may disturb the bacterial system in recognizing oxidatively damaged RNA, thus exerting a dominant negative effect on cell growth. We propose that YB-1 protein may discriminate the oxidized RNA molecule from normal ones, thus contributing to the high fidelity of translation in cells.     

Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease

Excessive production of airway mucus is a cardinal feature of bronchial asthma and chronic obstructive pulmonary disease (COPD) and contributes to morbidity and mortality in these diseases. IL-13, a Th2-type cytokine, is a central mediator in the pathogenesis of bronchial asthma, including mucus overproduction. Using a genome-wide search for genes induced in airway epithelial cells in response to IL-13, we identified pendrin encoded by the SLC26A4 (PDS) gene as a molecule responsible for airway mucus production. In both asthma and COPD mouse models, pendrin was up-regulated at the apical side of airway epithelial cells in association with mucus overproduction. Pendrin induced expression of MUC5AC, a major product of mucus in asthma and COPD, in airway epithelial cells. Finally, the enforced expression of pendrin in airway epithelial cells in vivo, using a Sendai virus vector, rapidly induced mucus overproduction in the lumens of the lungs together with neutrophilic infiltration in mice. These findings collectively suggest that pendrin can induce mucus production in airway epithelial cells and may be a therapeutic target candidate for bronchial asthma and COPD.