Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase

5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.     

Characterization and sequence of tomato 2S seed albumin: a storage protein with sequence similarities to the fruit lectin

We found a 2S storage albumin from the seed of tomato ( Lycopersicon esculentum L. cv. Cherry) that cross-reacted with antiserum to the fruit lectin, and named it Lec2SA. According to its size and basicity, Lec2SA was classified into four isoforms. These isoforms have an M(r) of approximately 12,000, and are composed of a small subunit (M(r) 4,000) and a large subunit (M(r) 8,000) linked by disulfide bonds. The complete amino acid sequence of Lec2SA was determined. The small subunit was composed of 32 amino acids, whereas the large subunit contained 70 amino acids with a pyroglutamine as the N-terminal residue. The sequence of Lec2SA was similar to that of 2S albumins from different plants, such as Brazil nut and castor beans. Furthermore, a sequence similarity was found between the large subunit of Lec2SA and the peptide sequence from tomato lectin. Although these similarities were found, Lec2SA did not show hemagglutinating activity or sugar-chain-binding activity, indicating that Lec2SA lacks the carbohydrate-binding domain. These results suggest that tomato lectin is a chimeric lectin sharing the seed storage protein-like domain that is incorporated into the gene encoding tomato lectin through gene fusion.     

Inhibition of beta-fructofuranosidases and alpha-glucosidases by synthetic thio-fructofuranoside

A synthetic beta-thio-fructofuranoside of mercaptoethanol inhibited not only beta-fructofuranosidases but also alpha-glucosidases. The compound was hardly hydrolyzed by the glycosidases. The thio-fructoside competitively inhibited beta-fructofuranosidases from Aspergillus niger, Candida sp., and Saccharomyces cerevisiae, but not Arthrobacter beta-fructofuranosidase at all. Sucrase activity of rat intestinal sucrase/isomaltase complex was also suppressed in the presence of the thio-fructoside. The thio-fructoside showed noncompetitive inhibition toward maltase activity of the rat intestinal enzyme complex and Saccharomyces sp. alpha-glucosidase. Inhibition against the Bacillus stearothermophilus alpha-glucosidase, Rhizopus glucoamylase, and porcine kidney trehalase were more slight than that against these two alpha-glucosidases.     

Novel evidence suggesting Clostridium difficile is present in human gut microbiota more frequently than previously suspected

Prevalence rate of Clostridium difficile in healthy human adults is believed to be very low. Our RT-PCR system using glass powder, which can eliminate PCR inhibitors, detected C. difficile toxin B mRNA in 16 of 30 fecal samples (53.3%) from healthy human adults. In contrast, we failed to detect toxin B in the same fecal samples by PCR using DNA templates extracted with phenol-chloroform. Our results suggest that PCR inhibitors in feces carried through phenol-chloroform extraction procedure might suppress the sensitivity of PCR and that C. difficile is actually present in human gut microbiota more frequently than previously suspected.     

Salinity decreases nitrite reductase gene diversity in denitrifying bacteria of wastewater treatment systems

Investigation of the diversity of nirK and nirS in denitrifying bacteria revealed that salinity decreased the diversity in a nitrate-containing saline wastewater treatment system. The predominant nirS clone was related to nirS derived from marine bacteria, and the predominant nirK clone was related to nirK of the genus ALCALIGENES:     

Spermidine synthase genes are essential for survival of Arabidopsis

The cellular polyamines putrescine, spermidine, and spermine are ubiquitous in nature and have been implicated in a wide range of growth and developmental processes. There is little information, however, on mutant plants or animals defective in the synthesis of polyamines. The Arabidopsis genome has two genes encoding spermidine synthase, SPDS1 and SPDS2. In this paper, we describe T-DNA insertion mutants of both of these genes. While each mutant allele shows normal growth, spds1-1 spds2-1 double-mutant seeds are abnormally shrunken and they have embryos that are arrested morphologically at the heart-torpedo transition stage. These seeds contain significantly reduced levels of spermidine and high levels of its precursor, putrescine. The embryo lethal phenotype of spds1-1 spds2-1 is complemented by the wild-type SPDS1 gene. In addition, we observed a nearly identical seed phenotype among an F2 seed population from the cross between the spds2-1 allele and SPDS1 RNA interference transgenic lines. These data provide the first genetic evidence indicating a critical role of the spermidine synthase in plant embryo development.     

Isolation and functional analysis of the melanoma specific promoter region of human GD3 synthase gene

Human GD3 synthase gene consisted of five exons and span about 135 kilobases. The 5'-flanking region lacked canonical TATA and CAAT boxes, but contained SP1 binding site(s) as in rat and mouse. The promoter activity in the 5'-flanking region (-2262 approximately +1) became definite when SV40 enhancer was added to the reporter plasmid. Luciferase assay with deletion mutants suggested the existence of a silencer region between -2262 and -978 nt similarly with those in mouse and rat. They also commonly contained a GT/CG repeat sequence at upstream of -1200 approximately -1300 nt, suggesting that they form Z-type DNA, and are involved in the gene regulation.     

Studies on pathogenic Vibrio parahaemolyticus during a warm weather season in the Seto Inland Sea,Japan

Vibrio parahaemolyticus is a potentially pathogenic bacterium, occurring naturally in estuarine and marine environments throughout the world. The incidence of this organism in an aquatic environment depends upon many ecofactors. Sea water and organic material were collected during the warm weather season from a coast of the Seto Inland Sea, Japan, and analysed to determine V. parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 99% of samples were positive for V. parahaemolyticus with densities of 3 to >1400 cells per 100 ml of water or 10 g of organic samples by the most-probable-number (MPN)-PCR technique, but only 76.6% were positive by the conventional MPN culture technique, with densities ranging from 3 to >1400 cells per 100 ml of water or 10 g of organics. Furthermore, the tdh and trh genes were positive in 41.5% and 8.5% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN culture procedure. The difference in detection between the MPN culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.     

Comparison of glycoprotein hormone alpha-subunits of laboratory animals

The common alpha-subunit of glycoprotein hormones (CGalpha) is a core protein shared by follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH). In order to obtain a molecular basis for an efficient superovulation technique applicable to a wide range of animal species and to discuss the phylogenetic aspect based on molecules related to the reproductive system, we determined cDNA sequences of CGalpha in seven laboratory animals: the guinea pig, Mongolian gerbil, golden hamster, mastomys, Japanese field vole, the JF1 strain of Mus musculus molossinus, and rabbit. Comparison of the inferred CGalpha amino acid sequences of these animals and other mammals (human, mouse, rat, cow, pig, and sheep) showed that the signal peptides and the first ten residues at the N-terminus of the apoprotein were variable, while the rest of the apoproteins were highly conserved. In particular, all rodents had a leucine residue at the apoprotein N-terminus, except the guinea pig, which had a phenylalanine residue, as in the cow, pig, sheep, and rabbit. Phylogenetic trees constructed from amino acid sequences suggest a closer relationship between the guinea pig and artiodactyls than to rodents, confirming the taxonomic peculiarity of the guinea pig.