Differences in biochemical properties of two 5′-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions

Deep-sea Shewanella violacea 5′-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions.

Abbreviations: NTase: 5′-nucleotidase; SANTase: Shewanella amazonensis 5′-nucleotidase; SVNTase: Shewanella violacea 5′-nucleotidase; CD: circular dichroism     

Population genetic structure and demography of Magnolia kobus: variety borealis is not supported genetically

Species delimitations by morphological and by genetic markers are not always congruent. Magnolia kobus consists of two morphologically different varieties, kobus and borealis. The latter variety is characterized by larger leaves than the former. For the conservation of M. kobus genetic resources in natural forests, the relationships between morphological and genetic variation should be clarified. We investigated variations in nuclear microsatellites, chloroplast DNA (cpDNA) sequences and leaf morphological traits in 23 populations of M. kobus over the range of species. Two genetically divergent lineages, northern and southern were detected and their geographical boundary was estimated to be at 39°N. The northern lineage consisted of two genetic clusters and a single cpDNA haplotype, while the southern one had multiple genetic clusters and cpDNA haplotypes. The northern lineage showed significantly lower genetic diversity than the southern. Approximate Bayesian computation indicated that the northern and southern lineages had experienced, respectively, population expansion and long-term stable population size. The divergence time between the two lineages was estimated to be 565,000 years ago and no signature of migration between the two lineages after divergence was detected. Ecological niche modeling showed that the potential distribution area in northern Japan at the last glacial maximum was very small. It is thus considered that the two lineages have experienced different population histories over several glacial-inter-glacial cycles. Individuals of populations in the central to northern part of Honshu on the Sea of Japan side and in Hokkaido had large leaf width and area. These leaf characteristics corresponded with those of variety borealis. However, the delimitation of the northern and southern lineages detected by genetic markers (39°N) was not congruent with that detected by leaf morphologies (36°N). It is therefore suggested that variety borealis is not supported genetically and the northern and southern lineages should be considered separately when identifying conservation units based not on morphology but on genetic markers.

     

Severely reduced production of klotho in human chronic renal failure kidney

We recently identified a novel gene, termed klotho (kl) that is involved in the development of a syndrome in mice resembling human aging. A defect of the kl gene expression in mice leads to multiple disorders including arteriosclerosis, osteoporosis, ectopic calcification, and skin atrophy together with short life-span and infertility. Patients with chronic renal failure (CRF), develop multiple complications that are reminiscent of phenotypes observed in kl mutant mice. Furthermore, the kl gene is mainly expressed in kidney and brain. These evidences above suggest the possible involvement of Klotho function in the complications arising in CRF patients. To investigate the above possibility, we examined the kidneys of 10 clinically or histologically diagnosed CRF cases. The level of kl gene expression was measured by utilizing RNase protection assay. The expression of Klotho protein was assayed by utilizing Western blot analysis and by immunohistochemistry. The levels of kl mRNA expression were greatly reduced in all CRF kidneys. Moreover, the production of Klotho protein was also severely reduced in all CRF kidneys. These results suggest that the decrease in kl gene expression in CRF patients may underlie the deteriorating process of multiple complications in the CRF patients.     

Construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus

Because lentiviruses are able to infect nondividing cells, these viruses might be utilized in gene therapy applications where the target cell does not divide. However, it has been suggested that the introduction of primate lentivirus sequences, particularly those of human immunodeficiency virus, into human cells may pose a health risk for the patient. To avoid this concern, we have constructed gene transfer systems based on a nonprimate lentivirus, bovine immunodeficiency virus. A panel of vectors and packaging constructs was generated and analyzed in a transient expression system for virion production and maturation, vector expression and encapsidation, and envelope protein pseudotyping. Virion preparations were also analyzed for transduction efficiency in a panel of human and nonhuman primary cells and immortalized cell lines. The virion preparations transduced most of the target cell types, with efficiencies up to 90% and with titers of unconcentrated virus up to 5 x 10(5) infectious doses/ml. In addition, infection of nondividing human cells, including unstimulated hematopoietic stem cells and irradiated endothelial cells, was observed.     

Magnesium insertion by magnesium chelatase in the biosynthesis of zinc bacteriochlorophyll a in an aerobic acidophilic bacterium Acidiphilium rubrum

To elucidate the mechanism for formation of zinc-containing bacteriochlorophyll a in the photosynthetic bacterium Acidiphilium rubrum, we isolated homologs of magnesium chelatase subunits (bchI, -D, and -H). A. rubrum bchI and -H were encoded by single genes located on the clusters bchP-orf168-bchI-bchD-orf320-crtI and bchF-N-B-H-L as in Rhodobacter capsulatus, respectively. The deduced sequences of A. rubrum bchI, -D, and -H had overall identities of 59. 8, 40.5, and 50.7% to those from Rba. capsulatus, respectively. When these genes were introduced into bchI, bchD, and bchH mutants of Rba. capsulatus for functional complementation, all mutants were complemented with concomitant synthesis of bacteriochlorophyll a. Analyses of bacteriochlorophyll intermediates showed that A. rubrum cells accumulate magnesium protoporphyrin IX monomethyl ester without detectable accumulation of zinc protoporphyrin IX or its monomethyl ester. These results indicate that a single set of magnesium chelatase homologs in A. rubrum catalyzes the insertion of only Mg(2+) into protoporphyrin IX to yield magnesium protoporphyrin IX monomethyl ester. Consequently, it is most likely that zinc-containing bacteriochlorophyll a is formed by a substitution of Zn(2+) for Mg(2+) at a step in the bacteriochlorophyll biosynthesis after formation of magnesium protoporphyrin IX monomethyl ester.     

Effect of endothelin-1(1-31) on intracellular free calcium in cultured human mesangial cells

We found that human chymase selectively produces 31-amino-acid length endothelins (1-31) (ETs(1-31)). We investigated the effect of synthetic ET-1(1-31) on intracellular free Ca2+ concentration ([Ca2+]i) in cultured human mesangial cells. ET-1(1-31) increased [Ca2+]i in a concentration-dependent manner to a similar extent as ET-1. The ET-1 (1-31)-induced [Ca2+]i increase was not influenced by removal of extracellular Ca2+ but was inhibited by thapsigargin. ET-1(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon. It was inhibited by BQ123, but not by BQ788. These results suggest that ET-1(1-31) by itself exhibits bioactive properties probably through endothelin ET(A) or ET(A)-like receptors. Since human chymase has been reported to exist in the kidney, ET-1(1-31) may be a candidate substance for mesangium-relevant diseases.     

Mice lacking Pin1 develop normally, but are defective in entering cell cycle from G(0) arrest

The peptidyl prolil cis/trans isomerase Ess1/Pin1 is essential for mitosis progression in yeast cells and is hypothesized to perform the same role in mammalian cells. To investigate the function of Pin1 in mammalian cells, we created mice lacking Pin1. These mice underwent normal development. Although the embryonic Pin1-/- fibroblasts grew normally, they proved significantly deficient in their ability to restart proliferation in response to serum stimulation after G(0) arrest. These results suggest that Pin1 is required for cell cycle progression from G(0) arrest as well as mitosis progression in normal mammalian cells.     

Matrix metalloproteinases and lysosomal cysteine proteases in osteoclasts contribute to bone resorption through distinct modes of action

The effects of inhibitors of matrix metalloproteinases (MMPs) and lysosomal cysteine proteases on osteoclastic pit formation in dentine slices were investigated. A nonspecific cysteine protease inhibitor, E-64, inhibited pit formation on naked slices in a concentration-dependent manner, and at 10 microM E-64 reduced the pit volume by 70%. However, up to 10 microM of the MMP inhibitor, BB-94, did not show any inhibition of pit formation. On the other hand, on slices coated with reconstituted basement membrane, both BB-94 and E-64 at 10 microM showed a marked decrease in pit volume by 73% and 68%, respectively. By a combination of treatment with both BB-94 and E-64, pit formation could be completely suppressed. These results suggest that MMPs are necessary for the migration of precursor and/or immature osteoclasts to bone surface through basement membranes, while cysteine proteases are essential for the osteoclastic degradation of bone collagen.     

Formation mechanism of 2,6-dimethyl-2,6-octadienes from thermal decomposition of linalyl beta-D-glucopyranoside

Thermally decomposed products of (+/-)-linalyl beta-D-glucoside were analyzed by GC and GC/MS. 2,6-dimethyl-2,6-octadienes produced by mild pyrolysis of linalyl beta-D-glucopyranoside under a vacuum were detected and characterized by MS and NMR spectroscopy. This suggests that 2,6-dimethyl-2,6-octadienes are produced during thermal decomposition of the glucoside via proton transfer from the anomeric position to C-6 in the aglycon moiety. A stable isotope labeling experiment directly indicated the new reaction mechanism.