Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

Differential effects of prostaglandin F2 alpha on oxytocinergic and non-oxytocinergic neurones in the paraventricular nucleus of the lactating rat

The effects of the prostaglandin F2 alpha (PGF2 alpha) given into the third cerebral ventricle on the unit activity of neurosecretory neurones in the paraventricular nucleus (PVN) were studied in urethane-anesthetized rats. The firing activity of PVN neurones was recorded extracellularly and 50 neurones were antidromically identified as neurosecretory neurones. Thirty of them were classified oxytocinergic neurones because they gave a burst of action potential 12-15 sec before reflex milk ejection and the remaining twenty PVN neurones which showed no response prior to reflex milk ejections were regarded as non-oxytocinergic ones. Twenty-five (83%) of the30 oxytocinergic neurones increased in the firing rate following the intraventricular (IVT) injection of PGF2 alpha (500ng in 1 microliter of isotonic saline) and the responses lasted for about 20-30 min. The remaining 5 (17%) oxytocinergic neurones showed no response in the firing rate to IVT PGF2 alpha. Fifteen (75%) of the 20 nonoxytocinergic neurones decreased in the firing activity in response to IVT PGF2 alpha, and the remaining 5 (25%) of them showed no response. IVT injection of isotonic saline (1 microliter) did not affect the firing activity of both the oxytocinergic and nonoxytocinergic cells. The intramammary pressure was slightly increased by the IVT administration of PGF2 alpha. These findings indicate that IVT PGF2 alpha has a differential action on oxytocinergic and non-oxytocinergic neurones in rats.     

Ecdysteroids during early embryonic development in silkworm Bombyx mori: metabolism and functions

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various molecular species of ecdysteroids in free and conjugated forms. In B. mori eggs, 20-hydroxyecdysone (20E) is a physiologically active molecule. In nondiapause eggs, 20E is produced by the conversion of maternal conjugated ecdysteroids (ecdysteroid-phosphates) and by de novo biosynthesis. In contrast, in diapause eggs, neither of these metabolic processes occurs. In de novo biosynthesis of 20E in B. mori eggs, hydroxylation at the C-20 position of ecdysone, which is catalyzed by ecdysone 20-hydroxylase, is a rate-limiting step. Furthermore, we found that a novel enzyme, called ecdysteroid-phosphate phosphatase (EPPase), specifically catalyzes the conversion of ecdysteroid-phosphates to free ecdysteroids. The developmental changes in the expression pattern of EPPase mRNA correspond closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs. EPPase is localized in the cytosol of yolk cells, and the bulk of maternal ecdysteroid-phosphates is bound to vitellin and stored in yolk granules. The vitellin-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase. Therefore, to examine how ecdysteroid-phosphates are hydrolyzed by EPPase during embryonic development further investigations were focused on yolk granules. Recent data indicate that acidification in yolk granules, induced by vacuolar H(+)-ATPase, triggers the dissociation of ecdysteroid-phosphates from the vitellin-ecdysteroid-phosphates complex and the dissociated ecdysteroid-phosphates are released from yolk granules to the cytosol. To explain the process of the increase in the level of 20E during embryonic development in B. mori eggs, a possible model is proposed.     

Establishment and characterization of a new human glioblastoma cells line, NYGM

A cell line designated NYGM was established from a human cerebral glioblastoma multiforme (GBM) obtained from a 75-year-old Japanese woman. The cell line has grown slowly without interruption and has been propagated continuously by serial passages (more than 80 passage) during the past 3 years. The cultured cells were fusiform or polyhedral in shape. The population doubling time was 24 hours. The chromosomal number varied between 77 and 88, with modal chromosomal number of 84. NYGM cells concomitantly expressed MET receptor tyrosine kinase (a product of c-met protooncogene) and its ligand HGF/SF (hepatocyte growth factor/scatter factor), as well as HGF activator and HGF activator inhibitors. The cells might be useful for the study of pericellular regulation of HGF/SF-MET signaling and HGF activation of GBM cells.     

Disruption of the CAR1 gene encoding arginase enhances freeze tolerance of the commercial baker's yeast Saccharomyces cerevisiae

The effect of intracellular charged amino acids on freeze tolerance in dough was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.     

Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.     

Inhibition of active HIV-1 replication by NF-κB inhibitor DHMEQ

Previous reports indicate that nuclear factor (NF)-κB regulates induction of human immunodeficiency virus type 1 (HIV-1) gene expression in latently infected cells. However, the role of NF-κB in cells with active HIV-1 replication is not well understood. In this study, we examined the effect of a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on HIV-1 replication in a human T cell line and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMCs). We further explored the mechanism of DHMEQ-mediated inhibition of HIV-1 replication. DHMEQ inhibited HIV-1 replication in HIV-1-infected Molt-4 and PHA-PBMCs. DHMEQ inhibited constitutive NF-κB activity in HIV-1-infected PHA-PBMCs and HIV long terminal repeat promoter activity driven by tumor necrosis factor (TNF)-α and the trans-activator Tat. The single-round assay using vesicular stomatitis virus-pseudotyped virus in the human T cell line M8166 indicated that DHMEQ treatment resulted in decreased integration of HIV-1 provirus into the host genome and decreased HIV-1 expression. These results indicate that NF-κB regulates early events as well as the initial and accelerated expression of HIV-1 in its life cycle. Therefore, we conclude that NF-κB is a molecular target for controlling active HIV-1 replication.