Biomedical subjects

K Matsubara

Publications and source records attributed to K Matsubara.

At least 55 records · Page 3Linked to original sources

PubMed2001

Five mouse homologues of the human dendritic cell C-type lectin, DC-SIGN.

DC-SIGN, a human C-type lectin, is expressed on the surface of dendritic cells (DC), while a closely related human gene, DC-SIGNR or L-SIGN, is found on sinusoidal endothelial cells of liver and lymph node. Both DC-SIGN and DC-SIGNR/L-SIGN can bind ICAM-3 and HIV gp120, and transmit HIV to susceptible cells in trans. Here, we report the cloning of five mouse genes homologous to human DC-SIGN and DC-SIGNR/L-SIGN. Only one gene, named mouse DC-SIGN, is highly expressed in DC, and is not found in a panel of mouse macrophage and lymphocyte cell lines. The other four genes, named mouse SIGNR1 (SIGN-Related gene 1), SIGNR2, SIGNR3 and SIGNR4, are expressed at lower levels in various cells according to RT-PCR and Northern blot analyses on RNA. All the genes of mouse DC-SIGN and SIGNRs map to adjacent regions of chromosome 8 A1.2-1.3. However, like human DC-SIGN, only the mouse DC-SIGN gene is closely juxtaposed to the CD23 gene, while the other four SIGNR genes are located close to each other in a neighboring region. mRNAs of mouse DC-SIGN and three SIGNR genes encode type II transmembrane proteins (DC-SIGN, 238 amino acids; SIGNR1, 325 amino acids; SIGNR3, 237 amino acids; SIGNR4, 208 amino acids), but the SIGNR2 gene only encodes a carbohydrate recognition domain (CRD) without a cytosolic domain and a transmembrane domain (SIGNR2, 178 amino acids). Amino acid sequence similarities between the CRD of human DC-SIGN and the mouse homologues are 67% for DC-SIGN, 69% for SIGNR1, 65% for SIGNR2, 68% for SIGNR3 and 70% for SIGNR4 respectively. However, the membrane proximal neck domains in the mouse genes are much shorter than their counterparts in human DC-SIGN and DC-SIGNR/L-SIGN. This family of mouse C-type lectins is therefore complex, but only one of the new genes, DC-SIGN, is juxtaposed to CD23 and is expressed at high levels in DC.

Amino Acid Sequence↗

PubMed2001

Change of antimicrobial susceptibility of group B streptococci over 15 years in Japan.

We compared antimicrobial susceptibility patterns of 206 group B streptococcal (GBS) strains isolated from pregnant women and six from neonates/infants with invasive infection during the two periods 1985-1986 and 1999-2000. All strains in both periods were susceptible to the penicillins, cephalosporins and carbapenem tested. Seven (3%) isolates were resistant to erythromycin and three (1%) were resistant to clindamycin. There were no significant differences between the two study periods in the incidence of GBS resistant to the 14 antibiotics tested. These results showed that penicillins are still the first choice to prevent vertical transmission of GBS in Japan.

Anti-Bacterial Agents↗

PubMed2001

Molecular and functional analyses of the gene (eshA) encoding the 52-kilodalton protein of Streptomyces coelicolor A3(2) required for antibiotic production.

Analysis of proteins recovered in the S100 precipitate fraction of Streptomyces griseus after ultracentrifugation led to the identification of a 52-kDa protein which is produced during the late growth phase. The gene (eshA) which codes for this protein was cloned from S. griseus, and then its homologue was cloned from Streptomyces coelicolor A3(2). The protein was deduced to be 471 amino acids in length. The protein EshA is characterized by a central region that shows homology to the eukaryotic-type cyclic nucleotide-binding domains. Significant homology was also found to MMPI in Mycobacterium leprae, a major antigenic protein to humans. The eshA gene mapped near the chromosome end and was not essential for viability, as demonstrated by gene disruption experiments, but its disruption resulted in the abolishment of an antibiotic (actinorhodin but not undecylprodigiosin) production. Aerial mycelium was produced as abundantly as by the parent strain. Expression analysis of the EshA protein by Western blotting revealed that EshA is present only in late-growth-phase cells. The eshA gene was transcribed just preceding intracellular accumulation of the EshA protein, as determined by S1 nuclease protection, indicating that EshA expression is regulated at the transcription level. The expression of EshA was unaffected by introduction of the relA mutation, which blocks ppGpp synthesis.

Amino Acid Sequence↗

PubMed2001

Comparative FISH mapping of human cDNA clones to chromosomes of the musk shrew (Suncus murinus, Insectivora).

Forty-one cDNA clones of human functional genes were newly mapped to chromosomes of the musk shrew (Suncus murinus, Insectivora) by fluorescence in situ hybridization, and a comparative cytogenetic map of 51 genes, including 10 genes reported in our previous study, was constructed between human (HSA) and musk shrew (SMU) chromosomes. In this comparative map, the 51 genes localized to human autosomes, except HSA 8, 16, and 20, were mapped to 15 shrew autosomes, except SMU 4, 16, 17 and 18. Twelve conserved segments were identified between human and shrew chromosomes, and six segments among the musk shrew, human, and mouse. Our results defined the presence of at least one inversion and several interchromosomal rearrangements that occurred during evolution after the two species diverged from a common ancestor. Localization of three major histocompatibility complex (MHC) genes to shrew chromosome 3 suggested that the MHC genes of the musk shrew are located in a cluster on chromosome 3. The cytogenetic map constructed in this study is the first cytogenetic map with many functional genes in insectivore species. This approach provides clues for clarifying the chromosomal evolution in this order.

Animals↗

PubMed2001

NeuroD/beta2 gene G-->A polymorphism may affect onset pattern of type 1 diabetes in Japanese.

OBJECTIVE: The majority of type 1 diabetes is considered to be autoimmune with, for the most part, abrupt development. However, type 1 diabetes with slow onset, or the so-called slowly progressive type 1 diabetes or latent autoimmune diabetes in adults, has been recently recognized and is considered to be autoimmune-related. Although some investigators tried to explain the difference in onset pattern by the genetic background, including HLA type, it has not been established thus far. We hypothesized that the difference in onset pattern may relate to regeneration or differentiation of pancreatic beta-cells, and we therefore focused on the NeuroD/BETA2 gene, which encodes a transcription factor for the insulin gene and beta-cell differentiation. RESEARCH DESIGN AND METHODS: We examined the NeuroD/BETA2 gene polymorphism in 105 Japanese type 1 diabetic patients and in 122 nondiabetic Japanese subjects in a case-control study, and we stratified the patients according to their onset pattern and islet-associated autoantibody positivity. RESULTS: Regardless of the existence of islet-associated autoantibody, we found a significant difference in A allele frequency between type 1 diabetic patients with acute-onset type and control subjects. However, no difference was found between type 1 slow-onset diabetic patients and control subjects. CONCLUSIONS: These results support our hypothesis that NeuroD/BETA2 may affect the ability of regeneration of beta-cells, leading to a difference in the onset pattern and clinical course of type 1 diabetes.

Adolescent↗

PubMed2001

Pyridoxal 5'-phosphate and pyridoxal inhibit angiogenesis in serum-free rat aortic ring assay.

Supraphysiological doses of vitamin B6 has been reported to suppress tumor growth and metastasis in rodents. To examine if its anticancer effect is due to suppression in angiogenesis, this study was conducted to investigate the antiangiogenic effect of pyridoxal 5'-phosphate (PLP), pyridoxine, pyridoxal and pyridoxamine in an ex vivo serum-free matrix culture model using rat aortic ring. Rat aortic rings were incubated with PLP or pyridoxine (25 micromol/l to 5 mmol/l). Higher concentrations of PLP (2.5 and 5 mmol/l) and pyridoxine (5 mmol/l) caused complete inhibition of microvessel outgrowth. However, the addition of pyridoxine at 2.5 mmol/l did not show complete inhibition of angiogenesis. PLP inhibited microvessel outgrowth almost completely at a concentration of 500 micromol/l and showed antiangiogenic effect in a dose-dependent manner within the range of 25-500 micromol/l. At 250 micromol/l, pyridoxal was as effective as PLP, but pyridoxamine was inactive, implying that the aldehyde group relates to the antiangiogenic effect. These results indicated the antiangiogenic effect of PLP and pyridoxal, and suggested that the antitumor effect of high levels of vitamin B6 might be mediated through suppression of angiogenesis.

Angiogenesis Inhibitors↗

PubMed2000

Gene expression profiling of mouse postnatal cerebellar development.

Expression patterns of 1,869 genes were determined using adapter-tagged competitive PCR (ATAC-PCR) at 6 time points during mouse postnatal cerebellar development. The expression patterns were classified into 12 clusters that were further assembled into 3 groups by hierarchical cluster analysis. Among the 1,869 genes, 1,053 known genes were assigned to 90 functional categories. Statistically significant correlation between the clusters or groups of gene expression and the functional categories was ascertained. Genes involved in oncogenesis or protein synthesis were highly expressed during the earlier stages of development. Those responsible for brain functions such as neurotransmitter receptor and synapse components were more active during the later stages of development. Many other genes also showed expression patterns in accordance with literature information. The gene expression patterns and the inferred functions were in good agreement with anatomical as well as physiological observations made during the developmental process.

Animals↗

PubMed2000

Microarray hybridization with fractionated cDNA: enhanced identification of differentially expressed genes.

Molecular indexing is a technique to select a subpopulation of cDNA by ligation of adapters to cDNA fragments digested by a class IIS restriction enzyme(s). By this technique, 3' end cDNA fragments are divided into 16 fractions by selective ligation of adapters and subsequent PCR amplification. Each fraction is used as a hybridization target for microarray hybridization. This fractionated target has a relatively lower nucleic acid complexity, including more fractions for rare transcripts, and is useful for their detection. In an experiment with mouse cerebellum and cerebrum RNA, analysis of a subset of candidate genes indicated that 10 times more differentially expressed genes were detected by this method than by the conventional method using the unfractionated target.

Animals↗

PubMed2000

A portion of the nucleotide sequence corresponding to the N-terminal coding region of livJ is essential for its transcriptional regulation.

We investigated the regulation of the livJ and livKHMGF operons, which are involved in branched-chain amino acid high-affinity transport in Salmonella typhimurium. When livJ was fused to lacZ at the second codon of livJ to make a livJ-lacZ protein fusion, expression from the livJ promoter was not repressed even under repressing growth conditions; however, expression of an analogous construct of livK-lacZ was repressed. When livJ was fused to lacZ at the twelfth codon of livJ, the expression level under unrepressing growth conditions was elevated, resulting in apparent repressibility of the livJ-lacZ protein fusion. Expression from the livJ-lacZ operon fusion, in which livJ was fused to lacZ 159 bp downstream from the A of the start codon of livJ, was relatively normal under unrepressing growth conditions. Deletion analysis and site-directed base-substitution analysis strongly suggested that cis-acting element for regulation of livJ transcription, 5'-GGCAGGATGTATCG-3', starting at +21 and ending at +34 downstream from the A of the start codon of livJ, was present in the N-terminal coding region of livJ.

Bacterial Proteins↗

PubMed2000

Enantio-selective occurrence of (S)-tetrahydropapaveroline in human brain.

Tetrahydropapaveroline is an endogenous complex alkaloid derived from dopamine through the oxidation by monoamine oxidase. This alkaloid is considered to be involved in the pathogenesis of alcoholism and to act as a false neurotransmitter. Recently the (S) enantiomer was proposed to be a precursor of morphine biosynthesis in the opium poppy. In this paper stereo-chemical characteristic of tetrahydropapaveroline in human brains was examined. In all four control human brains examined, only the (S)-tetrahydropapaveroline was detected. The concentrations were 0.12-0.22 pmol/g wet weight of brain tissue, and the presence of alcohol in blood did not affect the concentration. The results suggest that (S)-tetrahydropapaveroline may be enantio-selectively synthesized in human brain and it may be an intermediate of the de novo synthesis of morphine analogues.

Aged↗

PubMed2000

Primary structures of two hemagglutinins from the marine red alga, Hypnea japonica.

As the first examples among marine algal hemagglutinins, the primary structures of two hemagglutinins, named hypnin A-1 and A-2, from the red alga Hypnea japonica, were determined by Edman degradation. Both hemagglutinins were single-chain polypeptides composed of 90 amino acid residues including four half-cystines, all of which were involved in two intrachain disulfide bonds, Cys(5)-Cys(62) and Cys(12)-Cys(89). Hypnin A-1 and A-2 had calculated molecular masses of 9146.7 and 9109.7 Da which coincided with determined values, 9148 and 9109 Da, by electrospray ionization-mass spectrometry, respectively. Both hemagglutinins only differed from each other at three positions; Pro(19), Arg(31) and Phe(52) of hypnin A-1 as compared with Leu(19), Ser(31), and Tyr(52) of hypnin A-2. Approximately 43% of total residual numbers consisted of three kinds of amino acids: serine, glycine and proline. The hemagglutination activities were lost by reduction and alkylation of the disulfide bonds. The nature of the small-sized polypeptides, including disulfide bonds, may contribute to the extreme thermostability of the hemagglutinins. Sequence having overall similarity to hypnin A-1 or A-2 was not detected in databases. Unexpectedly, however, hypnins contained a motif similar to the alignment of the C-terminal conserved amino acids within carbohydrate-recognition domains of C-type animal lectins. Furthermore, interestingly, the hemagglutination activities were inhibited by a protein, phospholipase A-2 besides some glycoproteins, suggesting that hypnins may possess both a protein-recognition site(s) and a carbohydrate-recognition site(s).

Amino Acid Sequence↗

PubMed2000

Gene expression in mouse cerebellum during its development.

Genes expressed during the cerebellar development of the mouse were identified in 3'-directed cDNA libraries prepared from the postnatal day 4, day 12, and week 6 cerebellar tissues. Among about 5500 clones selected randomly from each library, there were approximately 3500 distinct species. A total of 7728 species were identified in the three libraries, 1346 of which were known genes in the GenBank, 3041 EST-matching genes, and 3341 new genes. Relative expression levels at the three postnatal stages were quantitated by adapter-tagged competitive PCR for 130 known genes that appeared six times or more in one of the libraries. Genes for ribosomal proteins and some cytoskeletal and nuclear proteins were abundantly expressed at the early stage, coincidently with extensive proliferation of granule cells as the major cerebellar component. Genes related to brain functions, including those for mitochondrial activities and some ion channel systems, were more active at a later stage when the majority of granule cells were engaged in axon extension and synapse formation or the cerebellum had reached maturity. Compared to these stage-specifically expressed genes, genes for transcriptional regulation, signal transduction, protein modification, and basic cellular functions, in general, were not abundantly expressed at any stage of development.

Animals↗

PubMed2000

Fundamental study of hot spot detectability in 3-dimensional positron emission tomography.

The purpose of this study was to investigate the detectability of small hot lesions with the 3-dimensional transmission/emission (3D T/E) acquisition mode in FDG-PET scan. The correlation of target detectability, target size, target to non-target uptake ratio (T/N ratio) and standardized uptake value (SUV) were studied. Small hot lesions ranged from 4.4 mm to 36.9 mm in diameter were located in cylindrical phantom. The images of phantoms with a T/N ratio of 2.0, 4.0, 6.0, 8.0, 9.6, 13.2, 17.5, 23.8 and 30.3 were obtained with 2-dimensional transmission/emission (2D T/E) scan and 3D T/E scans. Targets in diameter more than 10.6 mm in diameter with an actual T/N ratio ranged from 6.0 to 30.3 could be identified on the images obtained with all the 2D T/E and 3D T/E acquisition modes. The detectability efficiency of small hot target in 2D T/E and 3D T/E scans was as same (77.8%). The T/N ratio of targets from 2D T/E images was 30% to 48.4% different to that from 3D T/E image, and the SUV of the target from the 2D T/E images was almost the same as that from 3D T/E images. This study revealed that 3D T/E scanning had similar hot spot detectability to 2D T/E scanning; 3D T/E and 2D T/E scanning had the same faculty for semiquantitative analysis using SUV. These findings may be helpful for the diagnosis and understanding of 3D T/E FDG-PET in hot lesion detection.

Fluorine Radioisotopes↗

PubMed2000

N-methylation ability for azaheterocyclic amines is higher in Parkinson's disease: nicotinamide loading test.

The discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) leads to the hypothesis that Parkinson's disease (PD) is may be initiated or precipitated by endogenous toxins by the mechanism similar to that of MPTP in genetically-predisposed individuals. The higher cerebrospinal fluid levels of N-methylated azaheterocyclic amines, such as beta-carboline and tetrahydroisoquinoline, have been found in parkinsonian patients compared with age-matched controls. To estimate the N-methylation ability for azaheterocyclic amines in parkinsonian patient, nicotinamide was dosed with 100 mg to 26 parkinsonians and 20 controls consisted of 16 other neurogenic disease patients and 4 healthy volunteers. The urine was collected for 4 h, and then analyzed urinary its metabolites by an improved HPLC method. Nicotinamide has a pyridine ring in its structure and may be metabolized through the pathways similar to those for the endogenous neurotoxins. The urinary excretions of nicotinamide metabolites were significantly affected by aging. The excretion of N1-methylnicotinamide decreased along with aging both in PD patients and controls. In younger (65 years old or younger) PD patients, the excretion amount of N1-methylnicotinamide was significantly higher than that in younger controls. The decline rate of N1-methylnicotinamide excretion in parkinsonians was significantly greater than that in controls; the rate is more than 2-fold higher in parkinsonian patients. The age-associated decrease in 1-methyl-2-pyridone-5-carboxyamide excretion was observed only in parkinsonian patients, but not in controls. The total excreted amount of N-methylated metabolites (N1-methylnicotinamide plus 1-methyl-2-pyridone-5-carboxyamide) was also observed the age-related decline in both groups. The urinary excretions of nicotinamide and nicotinamide-N-oxide were not influenced by aging. These results would indicate that the excess N-methylation ability for azaheterocyclic amines before the onset had been implicated in PD. On the other hand, the present results suggested that the contribution of aberrant cytochrome P450 or aldehyde oxidase activity acting on the pyridine ring, that could act as detoxification routes of endogenous neurotoxins, would be small in the etiology of PD.

Aged↗

PubMed2000

The major immediate-early genes of human cytomegalovirus induce two novel proteins with molecular weights of 91 and 102 kilodaltons.

Mouse monoclonal antibody MAB810 is known to recognize the major immediate-early (IE) proteins, 68 kDa IE1 (IE1p68) and 80 kDa IE2 (IE2p80), of human cytomegalovirus (HCMV). Using this antibody we found that two additional proteins with higher molecular weights of approximately 91 (p91) and 102 kDa (p102) are also synthesized in HCMV-infected cells. p91 and p102 were produced in cells stably transfected with plasmid expressing IE1p68 and IE2p80, respectively, and shown to be related with IE1p68 and IE2p80, respectively, in primary amino acid sequence. Taken together, these results indicate that p91 and p102 are expressed from the IE1 and IE2 genes, respectively.

Animals↗

PubMed2000

Intratumoral genomic heterogeneity in human hepatocellular carcinoma detected by restriction landmark genomic scanning.

BACKGROUND/AIMS: One of the unique features of advanced hepatocellular carcinoma (HCC) is the morphological heterogeneity in a single tumor nodule. In order to investigate the intratumoral genomic heterogeneity of HCC, we performed Restriction Landmark Genomic Scanning (RLGS), which allows genomic DNAs to be surveyed at approximately 2000 landmark sites in a single 2-dimensional gel electrophoresis. METHODS: RLGS profiles of two regions from a single HCC nodule in six patients were compared with non-tumorous liver tissue. Four HCCs consisting of moderately-differentiated cells were separated into several small parts by thin fibrous septa, but not encapsulated. DNA samples were obtained from both parts of these so-called "nodule-in-nodule" HCC. Two HCCs consisting of well-differentiated cells which did not have a definite partition appeared pathologically homogeneous, and two independent regions of the HCC were used for the analysis. RESULTS: All six HCCs demonstrated different RLGS profiles (in total about 160 different spots) from the corresponding non-tumorous liver, and the number of different spots was greater in the 4 moderately-differentiated nodule-in-nodule HCCs (39-68 spots) than in the 2 well-differentiated homogeneous HCCs (6 and 3 spots). RLGS profiles of the two parts were different to each other in all 4 nodule-in-nodule HCCs. On the other hand, two other homogeneous HCCs showed the same RLGS profiles in the two regions. CONCLUSION: Thus, intratumoral genomic heterogeneity was demonstrated in the advanced HCC samples, and the genomic alterations may relate to the progression of HCC.

Aged↗

PubMed2000

Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum.

A fibrinolytic enzyme was isolated from a marine green alga, Codium divaricatum, and designated C. divaricatum protease (CDP). This protease effectively hydrolyzed fibrinogen A alpha chain, while it had very low hydrolyzing efficiency for B beta and gamma chains. This property was similar to that of alpha-fibrinogenase isolated from snake venom. Protease activity peaked at pH 9, and was completely inhibited by diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), identifying it as a serine protease. Its molecular form was single polypeptide structure and molecular weight was estimated as 31,000 by SDS-PAGE. Fibrinogen clotting enzyme was also identified in a fraction by ion-exchange chromatography. Analysis of clots formed by the enzyme and by thrombin by SDS-PAGE showed that the fibrinogen clotting enzyme would act like thrombin and have high substrate specificity.

Blood Coagulation↗

PubMed2000

Expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human gallbladder lesions.

The aim of this study was to investigate the expression of platelet-derived endothelial growth factor (PD-ECGF) in human gallbladder carcinomas to elucidate its role in angiogenesis and tumour progression. To this end, 56 archival surgical specimens of gallbladder lesions were examined for PD-ECGF/thymidine phosphorylase (TP) expression by immunohistochemistry and the PD-ECGF/TP protein level was assessed in five fresh specimens of gallbladder carcinoma by enzyme-linked immunosorbent assay (ELISA). Hyperplastic epithelial cells and adenoma cells showed no or faint staining with PD-ECGF/TP. Out of 43 gallbladder carcinomas, 27 (63%) showed moderate to strong immunoreactivity in the cytoplasm and nuclei of the tumour cells. PD-ECGF/TP immunoreactivity in stromal infiltrating cells was detected in 43% (3/7) hyperplasias, 17% (1/6) adenomas and 86% (37/43) carcinomas. PD-ECGF/TP protein levels in carcinoma tissues were higher than those in corresponding normal mucosa. PD-ECGF/TP expression did not correlate with angiogenesis, but significantly correlated with depth of invasion, lymph node metastasis, and tumour stage. These results overall suggest that PD-ECGF/TP produced by both cancer cells and infiltrating cells is associated with tumour progression in human gallbladder carcinoma.

Adenocarcinoma↗