A high-molecular-weight,alkaline, and thermostable β-1,4-xylanase of a subseafloor <Emphasis Type="Italic">Microcella alkaliphila</Emphasis>

An endo β-1,4-xylanase (XynE15) from a culture broth of a deep subseafloor microorganism, Microcella alkaliphila JAM-AC0309, was purified to homogeneity. The molecular mass of XynE15 was approximately 150 kDa as judged by SDS-PAGE. The optimal pH and temperature for hydrolysis of xylan were pH 8 and 65 °C. The enzyme was stable to incubation for 30 min at up to 75 °C, and the half-life at 50 °C was 48 h. XynE15 hydrolyzed arabinoxylan, oat spelt xylan, and birchwood xylan well, but not avicel, carboxymethylcellulose, or arabinan. Xylooligosaccharides were hydrolyzed to mainly xylobiose from higher than xylotetraose. The genome sequencing analysis of strain JAM-AC03039 revealed that XynE15 was composed of 1,319 amino acids with one catalytic domain and three carbohydrate-binding domains belonging to glycoside hydrolase (GH) family 10 and carbohydrate-binding module (CBM) family 4, respectively.     

Cyclin E2 is required for embryogenesis in Xenopus laevis

In mammalian cells, E-type cyclins (E1 and E2) are generally believed to be required for entry into S phase. However, in mice, cyclin E is largely dispensable for normal embryogenesis. Moreover, Drosophila cyclin E plays a critical role in cell fate determination in neural lineages independently of proliferation. Thus, the functions of cyclin E, particularly during early development, remain elusive. Here, we investigated the requirement for E-type cyclins during Xenopus embryogenesis. Although cyclin E1 has been reported as a maternal cyclin, inhibition of its translation in the embryo caused no serious defects. We isolated a Xenopus homologue of human cyclin E2, which was zygotically expressed. Sufficient inhibition of its expression led to death at late gastrula, while partial inhibition allowed survival. These observations indicate distinct roles for Xenopus cyclins E1 and E2, and an absolute requirement of cyclin E2 for Xenopus embryogenesis.     

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.     

Flow cytometry: an improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry.

In previous work, we clarified the relationship between the productivity and stability of gene-amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed near the telomeric region, was named the "telomere type." The other type of cell group, in which the amplified genes were observed in other chromosomal regions, was named the "other type." The phenotypes of these two types of cells are very different. In this experiment, using a fluorescein isothiocyanate-labeled methotrexate (F-MTX) reagent with flow cytometry, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene-amplified cell pools more easily than by the method of limiting-dilution assay. The limiting-dilution method requires several months to obtain highly productive gene-amplified cells, while our flow-cytometry-based method of selection requires only a few weeks.     

In vivo gene transfer of an extracellular domain of platelet-derived growth factor beta receptor by the HVJ-liposome method ameliorates bleomycin-induced pulmonary fibrosis

A number of investigators have reported augmented expression of PDGF in lungs with idiopathic pulmonary fibrosis (IPF) or with other types of pulmonary fibrosis. To accomplish such a regulation of PDGF activity, we constructed an expression plasmid of the extracellular domain of PDGF receptor beta chain (XR), which lacks intracellular tyrosine kinase domain and transmembrane portions, and estimated the therapeutic effects of XR gene transfer through the trachea on bleomycin-induced lung fibrosis of C57BL/6 mice using the hemagglutinating virus of Japan(HVJ)-liposome method. The XR gene transfer ameliorated the increases in the wet weight and hydroxyproline content and the histopathologic changes of the lung induced by bleomycin. These findings suggest that PDGF plays a crucial role in the pathogenesis of pulmonary fibrosis, and that XR gene transfer using the HVJ-liposome method may limit the progression of pulmonary fibrosis.     

Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer

Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45(+) hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.