Isolation and characterization of a sulfur-oxidizing chemolithotroph growing on crude oil under anaerobic conditions

Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the epsilon subclass of the class Proteobacteria constituted major populations in underground crude-oil storage cavities. In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan. 16S rRNA gene sequence analysis indicated that its closest relative was Thiomicrospira denitrificans (90% identity). Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor. Oxygen also supported its growth only under microaerobic conditions. Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction. Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources. A typical stoichiometry of its energy metabolism followed an equation: S(2-) + 4NO(3)(-) --> SO(4)(2-) + 4NO(2)(-) (Delta G(0) = -534 kJ mol(-1)). In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible. When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth. On the contrary, YK-1 could not utilize crude oil as a carbon source. These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds. Based on its physiology, ecological interactions with other members of the groundwater community are discussed.     

Biological evaluation of de-O-sulfonated analogs of salacinol, the role of sulfate anion in the side chain on the alpha-glucosidase inhibitory activity

De-O-sulfonated analogs (10a, Y(-)=CH(3)OSO(3) and 10b, Y(-)=Cl) of salacinol, a naturally occurring glycosidase inhibitor, and its diastereomer (12a, Y(-)=CH(3)OSO(3)) with L-thiosugar moiety (1,4-dideoxy-1,4-epithio-L-arabinitol) were prepared. Their inhibitory activities against intestinal maltase and sucrase were examined and compared with those of the parent alpha-glycosidase inhibitor, salacinol (1a). Compounds 10a and 10b showed a potent inhibitory activity equal to that of 1a against both enzymes, although 12a was a weak inhibitor against sucrase and maltase. These results indicated that the O-sulfonate anion moiety of 1a is not essential for the inhibitory activity.     

ROLE OF GLUCOCORTICOIDS IN TERMINAL DIFFERENTIATION AND TISSUE-SPECIFIC FUNCTION (A REVIEW)

Glucocorticoids (GCs) are required by many kinds of organs, tissues and cells for initiation or maintenance of their specific functions in vivo and in vitro. It is noticeable that most of these GC actions can be induced at much lower levels of dosage or concentration than the well-known actions of the steroids in gluconeogenesis, immune suppression and anti-inflammation. Such "differentiation-stimulating" actions of GC are regarded as main physiological roles of the steroid.     

Polysaccharide-polynucleotide complexes (15): thermal stability of schizophyllan (SPG)/poly(C) triple strands is controllable by alpha-amino acid modification

Schizophyllan (SPG), a beta-1,3-glucan polysaccharide which is known to form macromolecular complexes with certain polynucleotides, was modified by a reductive amination method with alpha-amino acids (Arg, Lys, and Ser). The thermal stability of the complexes as estimated by T(m) was enhanced in SPG-Arg and SPG-Lys conjugates which have pI values higher than the pH of the medium (8.0). The T(m) shift increased with the increase in the percentage of alpha-amino acid introduced and the highest T(m) values attained were 64 degrees C for SPG-Arg conjugate and 62 degrees C for SPG-Lys conjugate, which are higher by 13 and 11 degrees C, respectively, than those of the unmodified SPG+poly(C) complex. In the SPG-Ser conjugate with a pI lower than the medium pH (8.0), the T(m) values decreased with an increase in the percentage of Ser. Formation of the macromolecular complex was no longer detected above 13.2% Ser. The findings indicate that the T(m) values are easily controllable by the type and percentage of the introduced alpha-amino acids. We believe, therefore, that the present conjugates, consisting of naturally originated SPG and alpha-amino acids, provide an important lead for developing nontoxic artificial vectors and to control the affinity with polynucleotides in response to medium pH and temperature.     

Peptide probes containing a non-hydrolyzable phosphotyrosine-mimetic residue for enrichment of protein tyrosine phosphatases

We developed peptide probes containing a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F₂Pmp) for the enrichment of protein tyrosine phosphatases (PTPs). We found that different F₂Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F₂Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F₂Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies.     

Detecting amplicons of loop‐mediated isothermal amplification

Loop‐mediated isothermal amplification (LAMP) assays are used to detect diverse pathogens. Initially, LAMP amplicons were detected using electrophoresis; later, real‐time monitoring based on turbidity was developed to overcome the problem of contamination with environmental DNA. Recently, real‐time monitoring of fluorescence signals using a quenching primer and probe has improved the reliability of amplification signals. Here, methods of detecting LAMP amplicons are reviewed.     

Isolation of uracil auxotrophs of the fungus Acremonium cellulolyticus and the development of a transformation system with the pyrF gene

Acremonium cellulolyticus CF-2612 is a cellulase hyper-producing mutant that originated from A. cellulolyticus Y-94. In this study, we isolated a uracil auxotroph (strain CFP3) derived from CF-2612, and cloned a wild-type pyrF gene encoding orotate phosphoribosyl transferase (OPRTase) from Y-94. OPRTase activity was not detected in strain CFP3, which had one nucleotide substitution in its pyrF gene. The wild-type pyrF gene restored the defective growth of CFP3 on uracil-free medium, and PCR and Southern analyses revealed that wild-type pyrF was integrated into the genome. These results indicate that our transformation system for A. cellulolyticus with the pyrFgene as a selection marker was successful.     

Novel mastoparan and protonectin analogs isolated from a solitary wasp, Orancistrocerus drewseni drewseni

Two novel biologically active peptides, Eumenine mastoparan-OD and Orancis-Protonectin, were isolated from a solitary wasp, Orancistrocerus drewseni drewseni (Eumeninae, Vespidae). MALDI-TOF MS analysis of a small amount of the crude venom gave two intensive molecular-related ion peaks at m/z 1269.9 and 1552.9 that were expected to be novel based on a peptide database search. Purification of the crude venom by HPLC gave two peptide fractions, P-1 and P-2. The amino acid sequence of P-1 (GRILSFIKAGLAEHL-NH₂) and P-2 (ILGIITSLLKSL-NH₂) were determined by ESI-MS/MS, automated Edman degradation, and amino acid analysis. According to the high sequence homology with those of mastoparan and protonectin, P-1 and P-2 were labeled Eumenine mastoparan-OD and Orancis-Protonectin, respectively. Orancis-Protonectin is the first example of a protonectin analog isolated from the venom of a solitary wasp. The hemolytic activities of these new peptides were more potent than that of mastoparan.     

Porphyrin accumulation in mitochondria is mediated by 2-oxoglutarate carrier

Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (Ki = 15 microM). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.     

Effects of Age and Gender on Sleep Habits and Sleep Trouble for Aged People

The healthy 455 subjects above 60 years of age were questioned on their sleep habit inventory and the morningness-eveningness questionnaire. We analyzed the effects of age and sex on sleep habits and sleep-related trouble. Bedtimes on weekdays and weekends became earlier with aging, and women went to bed significantly later than men did. The length of sleep on weekdays slightly increased with aging, and it was longer for men than for women. The number of urinations and awakenings during nocturnal sleep and the amount of daytime napping increased with aging. The score on morningness-eveningness shifted toward the morning type with aging. In comparison with men, women had significantly longer sleep latency; and a higher percentage of subjects who reported that they sleep for only a short time, have sleep trouble, have received medical treatment for their sleep trouble, and take sleep medication. From these results, we deduced that the phase of sleep shifted forward in subjects above 60 years of age, and they showed frequent interruptions during nocturnal sleep and long daytime napping. We discussed the factor of gender difference in sleep in relation to social and cultural factors, particularly the household activities of women.