An enantioselective NADP+-dependent alcohol dehydrogenase responsible for cooxidative production of (3S)-5-hydroxy-3-methyl-pentanoic acid

A soil bacterium, Mycobacterium sp. B-009, is able to grow on racemic 1,2-propanediol (PD). The strain was revealed to oxidize 3-methyl-1,5-pentanediol (MPD) to 5-hydroxy-3-methyl-pentanoic acid (HMPA) during growth on PD. MPD was converted into an almost equimolar amount of the S-form of HMPA (S-HMPA) at 72%ee, suggesting the presence of an enantioselective MPD dehydrogenase (MPD-DH). As expected, an NADP⁺-dependent alcohol dehydrogenase, which catalyzes the initial step of MPD oxidation, was detected and purified from the cell-free extract. This enzyme was suggested to be a homodimeric medium-chain alcohol dehydrogenase/reductase (MDR). The catalytic and kinetic parameters indicated that MPD is the most suitable substrate for the enzyme. The enzyme was encoded by a 1047-bp gene (mpd1) and several mycobacterial strains were found to have putative MDR genes similar to mpd1. In a phylogenetic tree, MPD-DH formed an independent clade together with the putative MDR of Mycobacterium neoaurum, which produces opportunistic infections.     

Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.     

Neural network development in late adolescents during observation of risk-taking action

Emotional maturity and social awareness are important for adolescents, particularly college students beginning to face the challenges and risks of the adult world. However, there has been relatively little research into personality maturation and psychological development during late adolescence and the neural changes underlying this development. We investigated the correlation between psychological properties (neuroticism, extraversion, anxiety, and depression) and age among late adolescents (n?=?25, from 18 years and 1 month to 22 years and 8 months). The results revealed that late adolescents became less neurotic, less anxious, less depressive and more extraverted as they aged. Participants then observed video clips depicting hand movements with and without a risk of harm (risk-taking or safe actions) during functional magnetic resonance imaging (fMRI). The results revealed that risk-taking actions elicited significantly stronger activation in the bilateral inferior parietal lobule, temporal visual regions (superior/middle temporal areas), and parieto-occipital visual areas (cuneus, middle occipital gyri, precuneus). We found positive correlations of age and extraversion with neural activation in the insula, middle temporal gyrus, lingual gyrus, and precuneus. We also found a negative correlation of age and anxiety with activation in the angular gyrus, precentral gyrus, and red nucleus/substantia nigra. Moreover, we found that insula activation mediated the relationship between age and extraversion. Overall, our results indicate that late adolescents become less anxious and more extraverted with age, a process involving functional neural changes in brain networks related to social cognition and emotional processing. The possible neural mechanisms of psychological and social maturation during late adolescence are discussed.     

Comparison of Nitrogen mineralization from 15N-labeled organic amendments under flooded and upland conditions

Knowledge of N availability from organic amendments is a key to improve N use efficiency and reduce environmental pressure from agriculture. Nitrogen mineralization from ¹⁵N-labeled cattle dung compost and rapeseed cake was investigated under flooded and upland (60% of water holding capacity) conditions in an incubation experiment for 63 d at 25 °C. The relative abundance of N in the cattle dung compost by the simple step-wise acid hydrolysis method was in the following order: labile N (37% of total N, refluxing with 1 M HCl for 3 h, H1-N) > non-hydrolyzable N (32%) > recalcitrant N (18%, 3 M HCl for 3 h, H2-N). There was no significant difference in the ¹⁵N abundance between total N and N in each fraction of the cattle dung compost. For the rapeseed cake, the H1-N accounted for 81% of total N and the ¹⁵N abundance of total N and H1-N was higher than the ¹⁵N abundance of H2-N and non-hydrolyzable N. In the cattle dung treatment, inorganic ¹⁵N was the highest at 21 d of incubation and then decreased thereafter under flooded conditions, whereas it remained constant from 21 to 63 d under upland conditions. In the rapeseed cake treatment, inorganic ¹⁵N was the highest at 42 d under flooded conditions and inorganic ¹⁵N increased until 42 d and remained stable thereafter under upland conditions. The N mineralization rate from the cattle dung compost was slow both under flooded and upland conditions. More than half of N in the rapeseed cake was mineralized during the incubation period both under flooded and upland conditions. There was no significant difference in ¹⁵N recovery in the soil between flooded and upland conditions at 63 d in the cattle dung treatment, while the ¹⁵N recovery in the soil at 63 d was higher under upland than under flooded conditions in the rapeseed cake treatment. Although N mineralization from the rapeseed cake was greater under flooded conditions than upland conditions, there was no significant difference in N mineralization from the cattle dung compost between both conditions. Therefore, N mineralization from organic amendments is not always more rapid under flooded than upland conditions depending on the amendment type.     

Apoptosis and autoimmune diseases

Apoptosis is a process by which harmful or useless cells are eliminated. This process can be divided into two steps, death and engulfment. Molecules involved in apoptosis have been identified, and mouse lines deficient in these genes have been established. Among these deficiencies, those in the death receptor system or the engulfment of apoptotic cells cause systemic lupus erythematosus, whereas inefficient DNA degradation causes anemia by activating innate immunity, leading to death during embryogenesis. The apoptotic process and the diseases caused by defects in it are briefly reviewed.     

Opposite effects of rho family GTPases on engulfment of apoptotic cells by macrophages

The efficient engulfment of apoptotic cells by professional or nonprofessional phagocytes is critical to maintain mammalian homeostasis. To identify molecules involved in the engulfment of apoptotic cells, we established a retrovirus-based expression cloning system coupled with the engulfment assay. By screening a cDNA library of a mouse macrophage cell line, we identified two small GTPase family members (RhoG and Rab5) that enhanced the engulfment of apoptotic cells. By examining other small GTPase family members, we found that Rac1 enhanced the engulfment of apoptotic cells, whereas RhoA inhibited the process. Accordingly, the expression of a dominant-negative form of RhoG or Rac1 in primary macrophage cultures severely reduced the ability of the macrophages to engulf apoptotic cells, and a dominant-negative form of RhoA enhanced the process. These results indicated that the efficient engulfment of apoptotic cells requires the concerted action of small GTPase family members. We demonstrated previously that NIH3T3 cells expressing the alphav beta3 integrin efficiently engulf apoptotic cells in the presence of milk fat globule epidermal growth factor 8 via a phosphatidylserine-dependent mechanism. The dominant-negative form of RhoG or Rac1 inhibited this process, which suggested RhoG and Rac1 are also involved in the integrin-mediated engulfment.     

Vitamin E deficiency accelerates nitrate tolerance via a decrease in cardiac P450 expression and increased oxidative stress

Organic nitrates, such as nitroglycerin (NTG), have been used to relieve the symptoms of angina pectoris. However, their biochemical mechanisms of action, particularly in relation to the development of tolerance, are incompletely defined. It has been reported that supplemental antioxidants such as vitamin E attenuate the development of nitrate tolerance. Therefore, we examined the role of vitamin E in the regulation of nitrate tolerance. Continuous NTG infusion induced nitrate tolerance in rats after 48 h, and vitamin E concentrations decreased in a time-dependent manner in tissues and plasma. Vitamin E supplementation (0.5 g/kg diet) maintained higher concentrations of vitamin E during NTG infusion. The onset and extent of the tolerance, estimated by the decrease in blood pressure following NTG bolus injection during the infusion of NTG, were accentuated in the vitamin E-deficient group. Vitamin E supplementation inhibited nitrate tolerance 48 h after NTG infusion. Cardiac P450 expression (CYP1A2) assessed by immunoblotting, markedly decreased 48 h after NTG administration in control rats. The supplementation of vitamin E significantly attenuated the decrease in P450. Treatment of NTG enhanced vascular superoxide production (L-012 chemiluminescence, DHE fluorescence). The peak of lipid peroxidation and free radical generation in the heart was reached before tolerance developed. In contrast, vitamin E-deficient hearts had lower P450 expression and higher free radical generation than control hearts. To evaluate other vitamin E-inhibitable mechanisms of nitrate tolerance, we studied the NO-cGMP pathway. NTG markedly reduced the vasodilator-stimulated phosphoprotein (VASP) serine 239 phosphorylation (specific substrate of cGMP-activated protein kinase I; cGK-I) in tolerant hearts. Vitamin E inhibited the depletion of pVASP. In conclusion, because continuous NTG infusion causes vitamin E depletion as well as nitrate tolerance, vitamin E deficiency may further accelerate nitrate tolerance via an increase in oxidative stress, the reduced bioconversion because of decreased P450 expression, and impairment of the NO/cGMP pathway in tolerant heart tissues.     

Rheumatoid polyarthritis caused by a defect in DNA degradation

Macrophages phagocytose cells that die during programmed cell death and the nuclei that are expelled from erythroid precursor cells during erythropoiesis; subsequently, the ingested DNA is degraded in their lysosomes. A defect in this lysosomal DNA degradation activates the macrophages to produce cytokines such as IFNbeta and TNFalpha in a Toll-like receptor (TLR)-independent manner. IFNbeta thus produced in the mouse fetus induces the apoptosis of erythroid and lymphoid precursor cells, and kills the embryos. On the other hand, when the capacity for lysosomal DNA degradation is knocked out after birth, TNFalpha production increases in adulthood, causing chronic polyarthritis that resembles human rheumatoid arthritis. Here, I summarize recent findings on inflammatory diseases induced by such defects in DNA degradation.