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Biomedical subjects

K Hara

Publications and source records attributed to K Hara.

At least 307 records · Page 17Linked to original sources
PubMed1997

Intracellular degradation of secretion defect-type mutants of antithrombin is inhibited by proteasomal inhibitors.

To examine the cellular basis for secretion defect-type antithrombin deficiency, we expressed two mutants, P --> stop (Pro429 to stop codon) and deltaGlu (deletion of Glu313). Pulse-chase experiments using stably transfected BHK cells showed that little (< 5%) of P --> stop mutant as well as deltaGlu mutant was secreted and the total amount of radioactivity was significantly reduced, suggesting an intracellular degradation. The degradation was not inhibited by brefeldin A, indicating it occurring in a preGolgi apparatus. However, the degradation was strongly inhibited by proteasomal inhibitors, such as carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL), carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV) and lactacystin. By endoglycosidase H digestion and immunofluorescence staining, these mutants were shown to localize in the endoplasmic reticulum (ER). These results suggest that the secretion defect-type mutants of antithrombin are degraded by proteasome through the ER-associated quality control mechanism in the cells.

Acetylcysteine↗
PubMed1997

Urinary 1-hydroxypyrene levels of garbage collectors with low-level exposure to polycyclic aromatic hydrocarbons.

Because garbage collectors work in the street, they are exposed to polycyclic aromatic hydrocarbons (PAHs) in motor vehicle exhaust gas as they work. Urinary 1-hydroxypyrene (1-OH-pyrene) began to be used as a biological monitoring index for human exposure to high concentrations of PAHs. The objective of this study was to examine the applicability of urinary 1-OH-pyrene as a biological monitoring index for human low-level PAH exposure, such as the PAH exposure experienced while working in the street. The subjects were fifteen male garbage collectors. We measured individual exposure to PAHs, urinary 1-OH-pyrene concentrations and urinary cotinine concentrations. Individual air samplers were attached to the collar of the clothing of five workers to capture PAHs. Urine samples were collected before work, around noon and after finishing the day's work. In all, five PAH samples and 45 urine samples were collected. As control data, we analyzed the urinary 1-OH-pyrene and urinary cotinine levels of six smoking and four non-smoking control subjects who were not occupationally exposed to PAHs. The benzo[a]pyrene level in the air sampled for 5-6 h was 2.5-10.5 ng/m3, and the pyrene level as 10.3-70.3 ng/m3. These levels were similar to those in the vicinity of streets in Japan. A positive correlation between total PAH levels and the pyrene levels was observed. The average urinary 1-OH-pyrene level of the smokers was 0.21 +/- 0.13 mumol/mol creatinine, vs. 0.15 +/- 0.11 mumol/mol creatinine in the non-smokers. The urinary 1-OH-pyrene level obtained in this study was slightly higher than in the control group. No correlation was found between pyrene exposure and the urinary 1-OH-pyrene level of the five workers who wore the personal samplers. A significant positive correlation was observed between the urinary 1-OH-pyrene level and urinary cotinine level of the smokers. A significant positive correlation was also observed between the urinary 1-OH-pyrene and urinary cotinine levels of the control group smokers. In conclusion, urinary 1-OH-pyrene is not applicable for biological monitoring of extremely low levels of exposure to PAHs, as in the case of working in the street. Caution is required to exclude the effects of smoking when evaluating PAH exposure.

Adult↗
PubMed1997

Stimulation of human keratinocyte growth by alginate oligosaccharides, a possible co-factor for epidermal growth factor in cell culture.

Oligosaccharides, involved in regulation of plant developmental and defensive processes, were tested to determine their ability to enhance proliferation of human keratinocytes. A mixture of alginate oligosaccharides remarkably stimulated keratinocyte growth and [3H]thymidine uptake in the presence of epidermal growth factor (EGF). The activity was comparable to bovine pituitary extract, a common complement in keratinocyte culture, and additive on BPE-induced stimulation. The most effective oligosaccharide in the mixture was identified and its chemical structure was determined. These findings demonstrate a novel activity of alginate oligosaccharide(s) in keratinocyte growth and suggest a possible co-factor for EGF-dependent stimulation in medium for keratinocytes.

Alginates↗
PubMed1997

Stent placement in surgically reimplanted left main coronary artery in patient with anomalous origin of left main coronary artery from pulmonary artery.

Surgical repair of a 29-year-old woman's left coronary artery with an anomalous origin from the pulmonary artery was performed by reimplantation of the left main artery into the aorta, together with coronary artery bypass grafting. Subsequent stenosis of the reimplanted left main artery was treated with successful Palmaz-Schatz stent placement.

Adult↗
PubMed1997

Processing of cathepsins L, B and D in psoriatic epidermis.

Proteinase activity is increased in psoriatic epidermis. To elucidate the involvement of enzymes in psoriatic epidermis, the expression of cathepsins, L, B and D was investigated by Western blotting and immunohistological studies. Normal epidermis contained abundant inactive precursors (39 kDa) of cathepsins L and B and an inactive intermediate form (45 kDa) of cathepsin D. Cathepsin L in psoriasis was processed to a variable extent from the precursor to a single-chain form (30 kDa) and a mixture of single- and heavy-chain (25 kDa) forms of the active mature enzyme, corresponding to the immunohistological staining patterns 'diffuse dense', 'small granular', and unevenly distributed 'condensed granular'. Cathepsin B showed a mixture of precursor form (39 kDa) and single-chain (30 kDa) forms and was expressed as a 'diffuse dense' staining pattern in the mid-spinous layer and as a 'condensed' pattern in the upper spinous and granular layers. Cathepsin D was processed to the heavy-chain (31 kDa) form of activated mature enzyme with small granular staining and a mixture of heavy-chain and degraded protein (28 kDa) with larger and more condensed granular staining. The distribution patterns of 'small granular' cathepsin L, and of cathepsins B and D expression in suprabasal keratinocytes were very similar to that of involucrin. After complete clinical resolution of psoriasis by 8-methoxypsoralen plus UVA treatment, the expression of the three cathepsins was normalized. These results suggest that cathepsins L, B and D in forms activated to a variable extent may be involved in the pathology of psoriasis.

Adolescent↗
PubMed1997

Characterization of complement receptor type 1 and type 3 mRNA expression on polymorphonuclear leucocytes (neutrophils) in gingival crevicular fluid from periodontitis-affected patients.

A previous study has demonstrated that complement receptors on the surface of polymorphonuclear leucocytes (neutrophils) in gingival crevicular fluid significantly increased compared with those in autologous peripheral blood obtained from periodontitis-affected subjects. The present study attempted to determine the mRNA levels of complement receptor types 1 and 3 (CR1, CR3) on neutrophils in gingival crevicular fluid using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Gingival crevicular fluid samples were obtained from 11 adult periodontitis patients by gingival crevicular washing, and venipunctured peripheral blood was used as a control. RT-PCR analysis was performed using the primer sets for CR1, CR3 and beta-actin. Digoxigenin-labelled RNA probes were synthesized from RT-PCR products for in situ hybridization. Both CR1 and CR3 mRNA levels relative to beta-actin were significantly lower in crevicular fluid neutrophils than in peripheral blood neutrophils (crevicular fluid-CR1, 32.75 +/- 22.93%, peripheral blood-CR1; 65.30 +/- 43.25%, p < 0.005; crevicular fluid-CR3; 9.09 +/- 5.34%, peripheral blood-CR3; 30.14 +/- 18.80%, p < 0.005). In in situ hybridization, a greater majority of neutrophils showed positive CR1 and CR3 mRNA expression, while only a few neutrophils showed positive signals in gingival crevicular fluid. Data in the present study suggest that increased expression of complement receptors on the neutrophil cell surface appears to be unrelated to de novo synthesis.

Female↗
PubMed1997

Linear focal elastosis: a review of three cases in young Japanese men.

Linear focal elastosis in three young Japanese men is described. The lesions are asymptomatic palpable yellow strialike bands extending horizontally across the middle and lower parts of the back. They are histologically composed of many fine wavy bundles of elastic fibers separating the dermal collagen bundles. Electron microscopy demonstrates numerous elongated and fragmented elastic fibers.

Adolescent↗
PubMed1997

Purification and characterization of myofibril-bound serine proteinase from carp Cyprinus carpio ordinary muscle.

1. A novel myofibril-bound serine proteinase (MBP) has been purified from ordinary muscle of the carp Cyprinus carpio. 2. It was solubilized from the myofibril fraction with acid treatment (under the conditions of 0.6 M KCl, pH 4.0), then purified by column chromatographic steps on Ultrogel AcA 54, and Arginine-Sepharose 4B. 3. The purified enzyme revealed a single protein band on SDS-PAGE, and its molecular mass was estimated to be 30 kDa by SDS-PAGE and gel filtration. 4. The optimum pH and temperature of the enzyme were 8.0 and 55 degrees C, respectively, when Boc-Phe-Ser-Arg-MCA and casein were used as substrates. 5. The enzyme hydrolyzed Boc-Gln-Arg-Arg-MCA most rapidly, and also hydrolyzed the substrates for trypsin-type proteinase, but not for chymotrypsin. The enzyme was inhibited by serine proteinase inhibitors such as DFP, STI and leupeptin. These results suggested that the enzyme was a trypsin-type serine proteinase. 6. Boc-Phe-Ser-Arg-MCA hydrolyzing activity of the purified enzyme was reduced by addition of NaCl, but the caseinolytic activity and Boc-Phe-Ser-Arg-MCA hydrolyzing activity of the partially purified enzyme were activated by NaCl.

Amino Acid Sequence↗
PubMed1997

Substrate specificity of carp Cyprinus carpio cathepsin H with methylcoumarylamide substrates.

Cathepsin H was purified from the crude extract of carp (Cyprinus carpio) hepatopancreas by a reformed method involving six stages, and the specific activity increased about 11,500-fold with a 23% recovery. Of varying fluorescent synthetic substrates tested, carp cathepsin H possessed an ability to hydrolyze four N-terminal unblocked substrates those are composed of a single amino acid bound to 4-methylcoumaryl-7-amide (MCA), namely Leu-MCA, Arg-MCA, Lys-MCA and Ala-MCA. In contrast the enzyme was only marginally able to hydrolyze an unblocked substrate such as Pro-Phe-Arg-MCA and totally unable to degrade all blocked derivative employed. From the kinetic constants with four unblocked substrates, car cathepsin H had the highest affinity toward Leu-MCA with a Km value of 35.4 microM. Besides both the hydrolytic rates and molecular activities of the enzyme decreased from Lys-MCA > Arg-MCA > Ala-MCA > Leu MCA as judged by their Vmax and Kcat values, respectively. Otherwise, optimal pHs for hydrolysis of cathepsin H were different for four substrates. The enzyme exhibited maximum level of the activity at pH 6.5 for Arg-MCA and Lys-MCA and at pH 7.0 for Leu-MCA and Ala-MCA.

Animals↗
PubMed1997

Purification and characterization of cathepsin L from hepatopancreas of carp Cyprinus carpio.

Cathepsin L was purified from carp hepatopancreas by a method involving ammonium sulfate precipitation and a series of column chromatographies, in which the enzyme had an affinity toward Concanavalin A and Cibacron Blue F3GA. Its homogeneity was established by Native-PAGE, but two protein bands corresponding to molecular masses of 30,000 (single chain) and 24,000 (heavy chain) migrated on SDS-PAGE. The enzyme exhibited a maximum activity for carbobenzoxy-L-phenylalanyl-L-arginyl-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA) at pH 5.5-6.0 and 50 degrees C and the remarkable stability at pH 5.0-6.5 and below 40 degrees C. All tested cysteine protease inhibitors and TLCK and chymostatin markedly inhibited its activity, whereas the other serine protease inhibitors and a metalloprotease inhibitor negligibly affected it. In addition, several metal compounds reduced either its activity or stability to differing extents. Although EDTA alone caused an only marginal activation of the enzyme, its maximum activation required both 2 mM cysteine and 1 mM EDTA. The enzyme had an ability to hydrolyze three peptidyl-MCA substrates including Z-Phe-Arg-MCA, but all kinetic constants indicate that Z-Phe-Arg-MCA is the optical substrate to the enzyme.

Animals↗
PubMed1997

Relevance of local Th2-type cytokine mRNA expression in immunocompetent infiltrates in inflamed gingival tissue to periodontal diseases.

It has been suggested that the types of inflammatory round cell infiltrates and the divergence in the cytokine production profile by macrophages and helper T cells regulate the course of infectious or inflammatory diseases, including periodontitis and gingivitis. We examined the expression of IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6 and tumour necrosis factor-alpha (TNF-alpha) mRNA in the inflamed gingiva by in situ hybridization. The results of single-cell analysis were used as data sets for statistical analyses. The density of cells expressing IL-1alpha, IL-4 and IL-5 mRNA was higher in periodontitis than in gingivitis. IL-2 mRNA-expressing cells were almost absent in gingivitis specimens. Principal component analysis disclosed three factors explaining 84.8% of the variance: one accounting for 40.5% of the variance and mainly regulated by IL-1alpha, IL-1beta, IL-6 and TNF-alpha, and two others, explaining 29.9% and 14.4% of the variance, describing the relationship between the types of cytokines derived from macrophages or Th2 type. These results suggest that the cytokines produced by inflammatory cells infiltrating in the gingival tissue are influential on the progression of gingivitis, an acute and reversible inflammatory condition, to chronic and destructive periodontitis. Thus, periodontal disease progression may be regulated by the local cytokine network, and the bias in this network towards a Th2-type cytokine dominance could be an exacerbating factor.

Adult↗
PubMed1997

Characterization of HIV-related periodontitis in AIDS patients: HIV-infected macrophage exudate in gingival crevicular fluid as a hallmark of distinctive etiology.

In an attempt to clarify the immunobiological events featuring periodontitis lesions of AIDS patients in the late stage of the disease, peripheral blood (PB) and gingival crevicular fluid (GCF) leucocytes from periodontitis lesions of 23 late-stage AIDS patients were analysed by three-colour flow cytometry for detection and identification of intracytoplasmic p24+ cell fractions. The cells were reacted with CD14 and CD68 for mononuclear phagocytes or with CD4 and CD14 for Th cells, then with anti-p24 MoAb. To detect HIV proviral sequences and intracellular p24 RNA sequences, genomic DNA and cellular RNA from leucocytes were extracted for semi-nested polymerase chain reaction (PCR) amplification. CD68+/p24+ and CD14+/CD68+/p24+ fractions were larger in GCF than in PB (P<0.0001; P < 0.003). CD14+/p24+ fraction was lower in GCF than in PB (P < 0.05). The fluorescence intensities (FI) for intracellular p24 in CD68+ and CD14+/CD68+ cells were higher in GCF than in PB (P < 0.003; P < 0.02), whereas those of CD14+ macrophages did not differ. The p24 FI of CD68+ macrophages in GCF correlated with CD4+ lymphocyte counts in PB (P < 0.005). p24 FI levels of CD14+ monocytes in GCF and PB significantly correlated (P < 0.02), whereas that of CD68+ macrophages did not. PCR and reverse transcriptase (RT)-PCR of cellular DNA and RNA yielded positive signals, demonstrating viral integration and production in GCF leucocytes. These results show that periodontitis lesions in AIDS patients can be characterized by a rapid macrophage turnover, and these HIV-infected macrophage exudates in GCF may be considered as a within-mouth source of virus.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Nodular granulomatous phlebitis of the skin: a fourth type of tuberculid.

We present five cases of granulomatous phlebitis of the skin and compare them with a case of miliary tuberculosis with granulomatous phlebitis. All five patients were hypersensitive to purified protein derivative, but without active tuberculosis. Although anti-tuberculous drugs were effective, no tubercle bacilli were isolated from the skin. Clinically, subcutaneous nodules were felt along the course of the leg vein. Histologically, epithelioid cell granulomas with Langhans' giant cells were observed within the walls of the cutaneous veins. In a later stage, granulomatous panniculitis was often associated. Using the polymerase chain reaction method. Mycobacterium tuberculosis DNA was detected in four of the five cases of granulomatous phlebitis of the skin. Granulomatous phlebitis of the skin seems to represent a relatively early phase of delayed-type hypersensitivity reactions to Mycobacterium tuberculosis and may represent a distinct entity different from other types of tuberculid-a new tuberculid. Nevertheless, before making the diagnosis, the possibility of true tuberculosis must always be excluded. Nodular granulomatous phlebitis of the skin would be an appropriate name for the newly described condition.

Aged↗
PubMed1997

A multiparametric analysis of occlusal and periodontal jaw reflex characteristics in young adults with normal occlusion.

Periodontal jaw reflex, duration of percussion sounds, tooth mobility, and time-moment analysis of occlusal contacts by the T-scan system was recorded in nine periodontally healthy volunteers. The results showed that (i) reflex responses to the pressure applied to the uppercentageral incisors in the lingolabial direction varied, depending on the background jaw-clenching force (BCF) of the same-sided first molars. The BCF levels to elicit excitatory reflexes were 6-8 Kgf, and inhibitory reflexes were clearly elicited with a BCF of 10 Kgf and beyond. (ii) Duration of percussion sounds via an occlusal sound analyser (4.73-4.84 mS: upper first molars, 4.89-5.00 mS: uppercentageral incisors) and tooth mobility using a 'Periotest' (3.3-3.5: upper first molars, 5.5-5.6: uppercentageral incisors) showed a normal value. (iii) The time moments of occlusal contacts were symmetrical toward the midsagittal axis of the occlusal plane. The centre of the anteroposterior occlusal contacts was located in the first molar regions.

Adult↗
PubMed1997

Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)-signal transfer in chick limb development.

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1, in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.

Animals↗
PubMed1997

Reciprocal responses to dietary diacylglycerol of hepatic enzymes of fatty acid synthesis and oxidation in the rat.

The activities of hepatic enzymes of fatty acid synthesis and oxidation were compared in rats fed on diacylglycerol and triacylglycerol. In the first trial, rats were fed on diacylglycerol or triacylglycerol (rapeseed oil) for 14 d. The diacylglycerol preparation contained 65.2 g and 32.6 g fatty acids/100 g total fatty acids as 1,3-species and 1,2-species respectively. Fatty acid compositions of these dietary lipids were similar. Dietary acylglycerols were added to experimental diets to provide the same amounts of fatty acids (93.9 g/kg diet). Dietary diacylglycerol compared with triacylglycerol significantly reduced the concentrations of serum and liver triacylglycerol. The activities of enzymes of fatty acid synthesis (fatty acid synthetase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and malic enzyme (EC 1.1.1.40)) were significantly lower in rats fed on diacylglycerol than in those fed on triacylglycerol. In contrast, the rates of mitochondrial and peroxisomal oxidation of palmitoyl-CoA in liver homogenates were higher in rats fed on diacylglycerol than in those fed on triacylglycerol. In the second trial, varying amounts of dietary triacylglycerol were replaced by diacylglycerol while the dietary fatty acid content was maintained (93.9 g/kg diet). After 21 d of the feeding period the significant reductions in serum and liver triacylglycerol levels were confirmed in groups of rats fed on the diets in which diacylglycerol supplied more than 65.8 g fatty acids/kg diet (65.8 and 93.9 g/kg). Reductions in the activities of enzymes of fatty acid synthesis and increases in palmitoyl-CoA oxidation rates by both mitochondrial and peroxisomal pathways were also apparent when diacylglycerol replaced triacylglycerol in diets to supply more than 65.8 g fatty acid/kg. Increasing dietary levels of diacylglycerol also progressively increased the activities of enzymes involved in the beta-oxidation pathway (carnitine palmitoyltransferase (EC 2.3.1.21), acyl-CoA dehydrogenase (EC 1.3.99.3), acyl-CoA oxidase (EC 1.3.3.6), enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8)) in the liver. These results suggest that alteration of fatty acid metabolism in the liver is a factor responsible for the serum triacylglycerol-lowering effect of dietary diacylglycerol.

Animals↗