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Biomedical subjects

H Sakai

Publications and source records attributed to H Sakai.

At least 343 records · Page 19Linked to original sources
PubMed1998

Generation of protein carbonyls by glycoxidation and lipoxidation reactions with autoxidation products of ascorbic acid and polyunsaturated fatty acids.

Accumulation of carbonyl derivatives of proteins (protein carbonyl) is taken as a biomarker of oxidative protein damage in aging and in various diseases. We detected protein carbonyls in situ in human diabetic arteriosclerotic tissues and characterized the formation of protein carbonyls. Protein carbonyls were identified in the thickened intima of arterial walls and co-localized with protein adducts formed by carbonyl amine chemistry between protein and carbonyl compounds derived from autoxidation of carbohydrates, lipids, and ascorbate, i.e. advanced glycation end products or glycoxidation products, such as carboxymethyllysine (CML) and pentosidine, and lipoxidation products, such as malondialdehyde (MDA) and 4-hydroxy-nonenal (HNE). In vitro incubation of proteins with ascorbic acid accelerated the production of protein carbonyls as well as CML and pentosidine, and incubation with arachidonate accelerated the production of protein carbonyls as well as CML, MDA, and HNE. By contrast, incubation of proteins with glucose resulted in the production of CML and pentosidine, but not protein carbonyls. Schiff base inhibitors, (+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylace tanilide and aminoguanidine, inhibited the production of protein carbonyls after incubation with ascorbate and arachidonate. The present study suggests that ascorbate and polyunsaturated fatty acids, but not glucose, represent potential sources of protein carbonyls, and that both the glycoxidation and lipoxidation reactions contribute to protein carbonyl formation in aging and various diseases.

Arachidonic Acid↗
PubMed1998

Different response to inflammation of the multiple mRNAs of rat N-acetylglucosaminyltransferase I with variable 5'-untranslated sequences.

We found that there are at least five subclasses of N-acetylglucosaminyltransferase I (GnT-I; EC 2.4.1.101) mRNA with different 5'-untranslated regions in rat brain. These five subclasses were also expressed in many tissues with distinct tissue-specific patterns. Moreover, they were regulated differently in response to acute-phase inflammation. The expression of the most abundant subclass of GnT-I mRNA in rat liver decreased 2.5-fold in response to inflammation, concomitantly with a significant decrease in the total amount of GnT-I mRNA. In contrast, one of the minor subclasses of GnT-I mRNA was induced 10-fold by inflammation.

Animals↗
PubMed1998

Molecular cloning and cell-cycle-dependent expression of a novel NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk) in Tetrahymena cells.

With the intention of investigating the signal-transduction pathway that mediates the cold-stress response in Tetrahymena, we isolated a gene that encodes a novel protein kinase of 561 amino acids, termed Tetrahymena pyriformis NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk), by differential display from Tetrahymena cells exposed to temperature shift-down. TpNrk possesses an N-terminal protein kinase domain that is highly homologous with other NIMA-related protein kinases (Neks) involved in the control of the cell cycle. The TpNrk protein is 42% identical in its catalytic domain with human Nek2, 41% identical with mouse Nek1 and 37% with A. nidulans NIMA. In addition, TpNrk and these NIMA-related kinases have long, basic C-terminal extensions and are therefore similar in overall structure. In order to further explore the function of the TpNrk gene and the association of the cold stress with the cell cycle of Tetrahymena, changes of TpNrk mRNA were determined during the course of the synchronous cell division induced by the intermittent heat treatment. The level of TpNrk transcription increased immediately after the end of the heat treatment, with a peak at 30 min, and declined thereafter reaching the minimum level when nearly 80% of the cells synchronously entered cell division (75 min after the end of heat treatment). The accumulation of TpNrk mRNA starting from 0 min to 30 min after the end of the heat treatment was assumed to be a prerequisite for the start of synchronous cell division. These results suggest that TpNrk may have a role in the cell cycle of Tetrahymena, and that mRNA expression, at least, is under tight cell-cycle control.

Amino Acid Sequence↗
PubMed1998

A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy.

Mesangial cells play an important role in maintaining a structure and function of the glomerulus and in the pathogenesis of glomerular diseases. To identify a specific gene expressed in human mesangial cells, we used a rapid large-scale DNA sequencing and computerized data processing to compare the transcripts in cultured human mesangial cells with various different cells and organs. Using this novel approach, we discovered a new mesangium-predominant gene termed "megsin." We obtained a full-length cDNA clone of megsin, which coded for a novel 380-amino acid protein. Amino acid homology search revealed that megsin belonged to the serpin (serine protease inhibitor) superfamily. The amino acid sequences in the reactive loop site of megsin showed characteristic features of functional serpins. Northern blot and reverse-transcribed PCR analyses of various tissues and cells demonstrated that megsin was predominantly expressed in human mesangial cells. In situ hybridization studies showed the megsin expression in the mesangium of normal glomeruli, while it increased in the expanded mesangium of glomeruli from patients with IgA nephropathy with the degree of mesangial proliferation. Here we report a new human mesangium-predominant gene that may function as an inhibitory serpin in normal and abnormal biological processes of glomerulus.

Amino Acid Sequence↗
PubMed1998

Seroprevalence of Taenia solium cysticercosis in pigs in a rural community of Honduras.

Several retrospective studies have shown that human Taenia solium cysticercosis is endemic in Honduras, but very few reports of porcine cysticercosis in rural communities have been published. To determine the local prevalence of this disease in pigs, a serological survey has been undertaken in a rural community, Salamá, in the Department of Olancho in central Honduras. Eighty-five families raising pigs in the community were randomly selected and sera were obtained from pigs older than one month of age. The sera were examined by the enzyme-linked immunoelectrotransfer blot assay (EITB). Of 192 porcine sera, 27.1% (52) were positive by the EITB. Seropositivity did not correlate with age and sex by statistical analysis. With respect to the number and the frequency of recognition of the seven diagnostic glycoprotein bands in the EITB, 67.3% of the positive serum specimens recognized only one band and 80.8% of them recognized GP42-39. Since recognition of GP42-39 has been reported as a characteristic of late infection, these results suggest that most of the seropositive pigs were in the late stage of infection (more than 5-8 weeks postinfection). It seems that pigs in this community may be infected with this parasite soon after the birth and be in a hyperendemic steady state. In view of a high prevalence of antibodies to T. solium in pigs or characteristics of the antigen detected by the EITB, the infection pressure of T. solium appears to be very high in this community. This is probably the case in most of the communities in Honduras.

Animals↗
PubMed1998

Insulin-like growth factor I stimulates proliferation and Fas-mediated apoptosis of human osteoblasts.

In vitro studies have shown that insulin-like growth factor I (IGF-I) is a potent growth factor for osteoblasts, although both bone formation and resorption are upregulated by IGF-I in vivo. To understand the difference in the action of IGF-I observed in vitro and in vivo experiments, we examined the effect of IGF-I on the proliferation and Fas-mediated apoptosis of human osteoblasts in vitro. Human osteoblastic cell line MG63 and human primary osteoblast-like cells obtained from biopsy specimens were used as human osteoblasts. Cells were cultured with or without various concentrations of IGF-I followed by determination of the proliferative response and Fas-mediated apoptosis. IGF-I dose dependently stimulated the proliferation of cultured human osteoblasts. Both Fas expression and the degree of anti-Fas IgM-induced apoptosis of human osteoblasts was also augmented by IGF-I. Furthermore, the cytotoxicity of Fas ligand (FasL) cDNA transformants against human osteoblasts was increased when IGF-I-stimulated osteoblasts were used as target cells, indicating that stimulation of IGF-I increased functional Fas expression on human osteoblasts as well as their proliferation. The addition of DEVD-CHO, a specific inhibitor of CPP32, to the culture resulted in a significant inhibition of Fas-mediated apoptosis of both unstimulated and IGF-I-stimulated osteoblasts, although it did not affect the proliferative response or Fas expression. Our data suggest that activation of CPP32 is necessary for Fas-mediated apoptosis of human osteoblasts, and treatment of IGF-I increased this signaling pathway. In contrast, regulation of proliferation and Fas expression of the cells were probably not affected by CPP32 activation. Our results suggest that IGF-I acts on cultured human osteoblasts by increasing their proliferation and induction of Fas-mediated apoptosis by neighbouring FasL+ cells such as osteoclasts, thus probably functioning as a local coupling factor in the bone in vivo, stimulating both bone formation and resorption.

Apoptosis↗
PubMed1998

Polymorphism in the Ialpha1 germ-line transcript regulatory region and IgA productivity in patients with IgA nephropathy.

Enhanced in vivo and in vitro production of IgA has been reported in patients with IgA nephropathy (IgAN) and their family members. It is generally considered that IgA1 is a prominent subclass of IgA in IgAN. Although genetic mechanisms of IgA class switch recombination in IgAN have been studied enthusiastically, the critical factors that induce IgA1-specific class switching in IgAN have yet to be elucidated. A large body of data indicates that the germ-line transcript of Ig constant region (C(H)) genes that precedes actual class switching has regulatory effects on class switch recombination. To analyze structural abnormalities in the Ialpha1 germ-line transcript regulatory gene, a region about 1000 bp long located upstream of Ialpha1 exons was surveyed by the PCR-single strand conformation polymorphism method, and the polymorphism detected was confirmed by subsequent DNA sequencing. Three hot spots for point mutation were detected upstream of the promoter region of the Ialpha1 germ-line transcript, and the mutations were observed more frequently in patients than in controls. Patients with the mutations showed higher levels of serum IgA and higher in vitro IgA synthesis. In the luciferase assay, the regulatory gene with the mutations showed a potent effect for induction of the Ialpha1 germ-line transcript. The polymorphism in the Ialpha1 regulatory region possibly causes enhanced IgA production in some patients with IgAN.

Base Sequence↗
PubMed1998

ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis.

The plant hormone ethylene regulates a variety of processes of growth and development. To identify components in the ethylene signal transduction pathway, we screened for ethylene-insensitive mutants in Arabidopsis thaliana and isolated a dominant etr2-1 mutant. The etr2-1 mutation confers ethylene insensitivity in several processes, including etiolated seedling elongation, leaf expansion, and leaf senescence. Double mutant analysis indicates that ETR2 acts upstream of CTR1, which codes for a Raf-related protein kinase. We cloned the ETR2 gene on the basis of its map position, and we found that it exhibits sequence homology to the ethylene receptor gene ETR1 and the ETR1-like ERS gene. ETR2 may thus encode a third ethylene receptor in Arabidopsis, transducing the hormonal signal through its "two-component" structure. Expression studies show that ETR2 is ubiquitously expressed and has a higher expression in some tissues, including inflorescence and floral meristems, petals, and ovules.

Amino Acid Sequence↗
PubMed1998

Implication of altered redox regulation by antioxidant enzymes in the increased plasma pentosidine, an advanced glycation end product, in uremia.

Pentosidine is an advanced glycation end product (AGE) formed during Maillard or browning reaction by non-enzymatic glycation and oxidation (glycoxidation). Recent studies demonstrated the increased plasma pentosidine levels not only in diabetic patients with hyperglycemia but also in normoglycemic uremic patients. The mechanism of increased glycoxidation reaction in uremia, however, remains unknown. As superoxide dismutases (SODs) and glutathione peroxidase (GPx) are antioxidant enzymes involved in the metabolism of H2O2 which accelerates the glycoxidation reaction, we measured their activities by enzymatic assays in the plasma of normal and non-diabetic hemodialysis patients and examined a link between redox regulation by antioxidant enzymes and glycoxidation reaction. The activities of GPx were significantly lower in the plasma of hemodialysis patients than in normal subjects, whereas those of SODs were higher in the former than in the latter. As plasma SODs comprise three isozymes, i.e., Cu/Zn-SOD, Mn-SOD, and extracellular (EC)-SOD, we determined the levels of each SOD isozyme by ELISAs. The plasma concentrations of Cu/Zn-SOD and EC-SOD were significantly higher in hemodialysis patients than in normal subjects, whereas those of Mn-SOD did not differ between the two groups. It is of note that GPx activities correlated inversely with pentosidine in the plasma of hemodialysis patients (r2 = 0.262, P < 0.01). There was no significant correlation between total SOD activities and pentosidine levels in the plasma of hemodialysis patients, but, among the three SOD isozymes, the plasma EC-SOD levels correlated with the levels of pentosidine in hemodialysis patients (r2 = 0.286, P < 0.05). As decreased GPx and increased SOD activities result in the increased H2O2 generation, which accelerates the glycoxidation of protein, these data suggest a link of altered redox regulation by antioxidant enzymes to increased glycoxidation reaction in the uremic plasma. This paper provides the first time evidence for the possible involvement of enzymatic redox regulation in the non-enzymatic glycoxidation reaction in vivo.

Antioxidants↗
PubMed1998

Functional compensation of the low platelet count by increased individual platelet size in a patient with May-Hegglin anomaly presenting with acute myocardial infarction.

Platelets are known to play a crucial role in normal hemostasis as well as in thrombus formation at sites exposed to blood flow, as in coronary thrombosis. Thus, low platelet count is a strong negative risk factor for the occurrence of arterial thrombosis, such as occurs in acute myocardial infarction. We encountered a patient with May-Hegglin anomaly, presenting with acute myocardial infarction in his sixth decade, even though his platelet counts had always been less than 50 x 10(3)/microl. We investigated the characteristics of his platelets under the effect of shearing and found that shear-induced platelet aggregation and binding of soluble von Willebrand factor (vWF) to platelets could be induced, even when the patient's platelet count was less than 10 x 10(3)/microl, but that virtually no aggregation or vWF binding by normal platelets could be induced by shearing when platelet counts were less than 50 x 10(3)/microl. We conclude that the low platelet counts in a patient with May-Hegglin anomaly can be functionally compensated for by larger individual platelets, in view of the vWF-dependent platelet thrombus formation occurring under the effect of blood flow and that that is why most patients with May-Hegglin anomaly do not have a bleeding tendency, even though their platelet counts are very low.

Blood Platelets↗
PubMed1998

Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death. Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation.

PC12 cells undergo apoptosis as well as necrosis following exposure to hypoxia. Following a 6-h hypoxic treatment, a time-dependent increase in intracellular ceramide level was observed with a concurrent decrease in sphingomyelin. It was also shown that the hypoxia-induced ceramide accumulation resulted from activation of neutral magnesium-dependent sphingomyelinase. Comparative kinetic analyses of the neutral sphingomyelinase in the cells under normoxia and hypoxia showed that hypoxia increased Vmax but did not affect Km of the enzyme. In PC12 cells overexpressing Bcl-2 which show strong resistance to hypoxia, sphingomyelin hydrolysis was decreased and activation of neutral sphingomyelinase was reduced. Addition of exogenous C2-ceramide induced cell death and activated caspase-3 as markedly as the hypoxia treatment. On the other hand, in PC12 cells overexpressing Bcl-2, significant decreases in cell death and inhibition of caspase-3 activation were observed after exogenous addition of C2-ceramide. The inhibitors of caspase-3 prevented cell death by either hypoxia or C2-ceramide. These results suggest that ceramide generated by activation of neutral magnesium-dependent sphingomyelinase mediates hypoxic cell death and that Bcl-2 has inhibitory effects on ceramide formation and caspase activation.

Animals↗
PubMed1998

Molecular cloning of a cDNA encoding a serine protease homologous to complement C1s precursor from rat C6 glial cells and its expression during glial differentiation.

A cDNA of rat C6 cells was cloned, which was considered to be involved in glial cell differentiation induced by dibutyryl cyclic AMP and theophylline. The cDNA fragment of the gene, termed r-gsp, was originally isolated by mRNA fingerprinting using arbitrarily primed polymerase chain reaction, and was homologous to complement C1s precursors of hamster and human. It encodes a protein of 694 amino acids containing a potential signal peptide, an epidermal growth factor-like domain surrounded by two complement C1r/C1s-related repeats, and a putative trypsin-type serine protease domain. Since the hamster and human C1s, and a protein encoded by r-gsp shared high similarity in primary structure, the r-gsp gene could encode a C1s counterpart of the rat. Messenger RNA expression of this gene was markedly increased during cyclic AMP-induced glial cell differentiation. Its expression profile was well correlated with those of glial fibrillary acidic protein (GFAP) and S100B, which are known as glial differentiation markers. It was, moreover, observed that the r-gsp expression in brain increased considerably after birth, like those of S100B and GFAP. The results presented here suggest that the rat C1s gene would be also implicated in glial differentiation besides the complement cascade.

Amino Acid Sequence↗
PubMed1998

Cellular expression of beta-microseminoprotein (beta-MSP) mRNA and its protein in untreated prostate cancer.

BACKGROUND: Previous studies have shown that beta-microseminoprotein (beta-MSP) may be used as a diagnostic marker for prostate cancer. However, the level of expression of beta-MSP in prostate cancer detected by immunohistochemistry (IHC) has varied from one study to another. METHODS: We analyzed the expression of both beta-MSP mRNA and its protein in a large sample of prostate tumors from 104 patients with untreated prostate cancer, using both nonradioactive in situ hybridization (ISH) and IHC. RESULTS: Our results showed that 72 and 96 of 104 specimens were negative for beta-MSP mRNA (69.2%) and beta-MSP (92.3%), respectively. Furthermore, a reduced expression of both beta-MSP mRNA and its protein was detected in all malignant epithelial tissues compared with benign epithelia. Not all malignant tissue samples negative for beta-MSP mRNA were negative for beta-MSP (6.7%), and vice versa (29.8%). Other tissue samples were either negative for both (62.5%) or positive for both (1.0%). CONCLUSIONS: Our results showed a lower level of expression of beta-MSP in prostate cancer tissue, compared with benign prostate tissue. This phenomenon may be mainly due to the presence of reduced levels of beta-MSP mRNA.

Aged↗
PubMed1998

Subcutaneous microvascular responses to hemodilution with a red cell substitute consisting of polyethyleneglycol-modified vesicles encapsulating hemoglobin.

Phospholipid vesicles encapsulating purified hemoglobin [Hb vesicles (HbV); diameter 259 +/- 82 mm; oxygen affinity 31 mm Hg; [Hb] 5 and 10 g/dL] were developed to provide oxygen-carrying capacity to plasma expanders. Their function as a blood replacement was tested in the subcutaneous microvasculature of awake hamsters during severe hemodilution in which 80% of the red blood cell mass was substituted with suspensions of the vesicles in 5% human serum albumin (HSA) solution. Vesicles were tested with membranes that were unmodified (HbV/HSA) or conjugated with polyethyleneglycol (PEG) on the vesicular surface (PEG-HbV/HSA). The viscosity of 10 g/dL HbV/HSA was 8 cP at 358 s-1 owing to the intervesicular aggregation, while that of 10 g/dL PEG-HbV/HSA was 3.5 cP, since PEG chains inhibit aggregation. Both materials yielded normal mean arterial pressure, heart rate, and blood gas parameters at all levels of exchange, which could not be achieved with HSA alone. Subcutaneous microvascular studies showed that PEG-HbV/HSA significantly improved microhemodynamic conditions (flow rate, functional capillary density, vessel diameter, and oxygen tension) relative to unmodified HbV/HSA. Even though the enhancement of PEG modification did not achieve the functional characteristics of the blood-perfused microcirculation, PEG reduced vesicular aggregation and viscosity, improving microvascular perfusion relative to the unmodified type. These results highlight the significance of microvascular analysis in the design of red cell substitutes and the necessity of surface modification of HbV to prevent aggregation.

Animals↗
PubMed1998

Follow-up study on urinary type IV collagen in patients with early stage diabetic nephropathy.

Type IV collagen is a major component released from the glomerular and tubular basement membranes. To investigate the alteration of renal type IV collagen turnover in early stage diabetic nephropathy, urinary type IV collagen was measured by a highly sensitive one-step sandwich enzyme immunoassay (EIA). Urinary samples were obtained from 94 diabetic patients without overt proteinuria. Among those patients, 61 were normoalbuminuric and 33 patients were in the microalbuminuric group. Levels of urinary type IV collagen were serially examined at the start of this study and again one year later. The levels of urinary type IV collagen in patients in the microalbuminuric group were significantly higher than those in the normoalbuminuric group (P < 0.01). There was a significant correlation between the concentration of urinary albumin and urinary type IV collagen in both groups (P < 0.05). Twenty-eight patients (45.3%) in the normoalbuminuric group who showed an abnormal elevation of urinary type IV collagen in comparison to the reference range of normal healthy adults (normal range; less than 3.5 microg/g x Cr). Seven (25%) out of these 28 normoalbuminuric patients with increased urinary type IV collagen progressed to the microalbuminuric group one year later. The levels of urinary type IV collagen in such patients were significantly increased. In the 21 patients who stayed within the normoalbuminuric group, the urinary type IV collagen levels were significantly decreased one year later. It appears that the levels of urinary type IV collagen might reflect ongoing alteration of the extracellular matrix (ECM) turnover and might define more specifically the early stage diabetic nephropathy than the detection of microalbuminuria. It is concluded that the serial measurement of urinary type IV collagen can be a useful marker for detecting renal injury in diabetes.

Adult↗
PubMed1998

Amplification of JC virus regulatory DNA sequences from cerebrospinal fluid: diagnostic value for progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease in the central nervous system caused by a ubiquitous human polyomavirus designated as JC virus (JCV). PML affects individuals with decreased immune competence and is now one of the common opportunistic infections in patients with AIDS. JCV DNAs in the brain of PML patients contain various PML-type regulatory regions that were generated from the archetypal regulatory region during persistence. Recently, many studies have suggested that detection of JCV DNA from the cerebrospinal fluid (CSF) may offer a tool for diagnosing PML. However, in all of these studies, coding sequences within the T antigen or capsid protein gene have been targeted for amplification. To amplify the JCV regulatory region, we established a nested PCR that could efficiently amplify the regulatory region from most JCV subtypes prevalent in the world. Using this PCR, we amplified JCV regulatory regions from the CSF samples from 4 patients strongly suspected of PML, whereas amplification was negative from 80 CSF samples from patients without PML. Sequencing of the amplified fragments revealed that they had unique deletions and/or duplications. Furthermore, in 3 PML patients, we analyzed the structures of regulatory regions derived from the brain as well as CSF. In each of these cases, the major regulatory sequence of both origins were identical. This finding indicates that JCV DNA in brain lesions is excreted in the CSF. Since the structures of PML-type JCV regulatory regions are unique to individual patients, the current PCR, if the amplified fragments are sequenced, can eliminate false positives that may arise from contaminations.

Brain↗