Molecular cloning of a cDNA encoding mouse DNA helicase B, which has homology to Escherichia coli RecD protein, and identification of a mutation in the DNA helicase B from tsFT848 temperature-sensitive DNA replication mutant cells

DNA helicase B is a major DNA helicase in mouse FM3A cells. A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B. In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein. By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members. A database search revealed similarity between DNA helicase B and the α subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast. The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.     

Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage

This study has used in situ hybridization, Northern blot analysis, and immunohistochemistry at the light and electron microscope levels to localize mRNAs and core proteins of biglycan in developing tibial epiphyseal cartilage of 10-day old Wistar rats. The expression of mRNAs and core proteins of biglycan appeared prominent in hypertrophic and degenerative chondrocytes associated with the epiphyseal ossification centre and the growth plate cartilage, but was not seen in the rest of epiphyseal cartilage. Northern blot analysis confirmed biglycan mRNA expression in the epiphyseal cartilage. Ultrastructural immunogold cytochemistry of the growth plate revealed that prominent immunolabelling was confined to the Golgi apparatus and cisternae of rough-surfaced endoplasmic reticulum of the hypertrophic and the degenerating chondrocytes, the early mineralized cartilage matrices of the longitudinal septum of the lower hypertrophic and the calcifying zones, and fully mineralized cartilage matrices, which were present in the metaphyseal bone trabeculae. Furthermore, Western blot analysis of biglycan in extracts of fresh epiphyseal cartilage revealed that an EDTA extract, after chondroitinase ABC digestion, contains core proteins of biglycan, indicating the presence of biglycan in mineralized cartilage matrices. These results indicate that the distribution of biglycan is associated with cartilage matrix mineralization.     

Cloning of a rat glia maturation factor-gamma (rGMFG) cDNA and expression of its mRNA and protein in rat organs

We isolated a rat glia maturation factor-gamma(rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-beta (rGMFB). Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen. GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs.     

Presence of oxidized protein hydrolase in human cell lines, rat tissues, and human/rat plasma

Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [(3)H]DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [(3)H]DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [(3)H]DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.     

Cleavage of CD14 on human gingival fibroblasts cocultured with activated neutrophils is mediated by human leukocyte elastase resulting in down-regulation of lipopolysaccharide-induced IL-8 production

Activated polymorphonuclear leukocytes (PMNs) release various types of proteases and express them on the cell surface. The proteases play important roles in PMN-mediated events. In the present study, flow cytometric analysis revealed that CD14 expression on human gingival fibroblasts (HGF) was markedly reduced by PMA-activated PMNs in a coculture system. We found that this reduction was caused by both secreted and cell surface proteases produced by activated PMNs. A protease responsible for the reduction was found to be human leukocyte elastase (HLE) secreted from the activated PMNs by use of various protease inhibitors, although HLE was only partially involved in CD14 reduction caused by cell-bound molecule(s) on fixed PMNs. Analysis with purified HLE revealed a time- and dose-dependent reduction of CD14 on HGF, and complete reduction was observed by 20 microg/ml HLE treatment for 30-60 min, but the other molecules such as CD26, CD59, CD157, and MHC class I on HGF were only slightly reduced. This reduction of CD14 resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD14 on fixed HGF and also on purified cell membranes. As a result of CD14 proteolysis, IL-8 production by HGF was suppressed when triggered by 10 ng/ml LPS, but not by IL-1alpha, indicating that HLE inhibited a CD14-dependent cell activation. These findings suggested that activated PMNs have a potential negative feedback mechanism for HGF function at the inflammatory site, particularly in periodontal tissues.     

Strongyloides ratti: chemotactic responses of third-stage larvae to selected serum proteins and albumins

Experiments were carried out in vitro to investigate whether the sera of several animals as well as albumins and peptides might act as attractants for larvae of Strongyloides ratti. Samples of sera from several mammal species were dialysed and the aliquots were further centrifuged using ultrafiltration cartridges to remove any remaining small molecules. Additional test substances included commercially obtained ovalbumin, rat and bovine serum albumins, polypeptides such as peptone, tryptone and tryptose, amino nitrogens, monosaccharides, and reduced glutathione (triaminopeptide). Larvae were strongly attracted to the dialysed mammalian sera, which mainly consisted of serum albumin and globulins. Ov- and serum albumins, and polypeptides also acted as attractants. On the other hand, reduced glutathione, 16 kinds of amino acids and four kinds of monosaccharides did not attract this nematode.     

Use of oligotrophic bacteria for the biological monitoring of heavy metals

Oligotrophic bacteria exhibited active growth even in nutritionally deficient medium made with nutrient broth that had been diluted with distilled water, 1 : 10 000. The oligotrophic bacteria, Sphingomonas paucimobilis KPS01 and Burkholderia cepacia KPC01 and KPC02 were found to be highly susceptible to heavy metals and to be potentially useful as sensors for the assessment of toxicity. The susceptibility of the bacteria to metals was measured by incubating the bacteria with metals of varying concentrations in the nutritionally deficient medium at 30 degrees C for 24 h. Bacteria were considered susceptible when the growth inhibition rate (EC50 was more than 50% of the control. The EC50 value of Ag+, Pb2+ and Cd2+ was 10(5)mmol l(-1) and Zn2+, Cr3+, Cr6+, Cu2+ and Hg2+ was 10(-4) mmol l(-1) in S. paucimobilis KPS01. Other strains also showed similar results. No difference in the EC50 was found using either the chloride or sulphate forms of these metals. The optimum incubation time was 24 h and a longer incubation time did not necessarily lead to more inhibition. The EC50 value rose in proportion to the concentration of nutrition in media. Environmental samples were tested and 14 out of 88 samples inhibited the growth of S. paucimobilis KPS01.     

An evaluation of hard palate mucosa graft as a lining material in alar reconstruction: a 7-year experience applied to the full-thickness alar defect

The authors present their experience with 25 hard palate mucosa grafts used as lining material in the reconstruction of full-thickness alar defects. Good "take" was obtained in 22 grafts; the other three grafts incurred necrosis of the overriding skin flaps and postoperative infection. Degree of shrinkage was 11 to 15 percent of grafted size in patients with the type of defect that did not include the alar margin; shrinkage was 26 to 35 percent in patients with the type that included more than 50 percent of the alar margin. In all patients who had a good graft take, the nasal cavities were maintained and there was no nasal obstruction or collapsing during strong breathing. The healing time of the palate donor site varied from 7 days to 5 weeks, depending on the size of the defect. No patients experienced any symptoms at the donor site after healing. The authors concluded that hard palate mucosa can be considered a useful material in alar reconstruction because of the ease in graft harvesting and its support features. When the defect is large enough to involve the total unilateral ala nasi, even though the degree of postoperative shrinkage is comparatively high, hard palate mucosa may be the most suitable material to ensure good take of the graft and less possibility of donor-site morbidity.