Biomedical subjects

A Maruyama

Publications and source records attributed to A Maruyama.

At least 19 recordsLinked to original sources

PubMed2006

pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells.

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.

Animals↗

PubMed2006

Determinants for prediction of malignant potential by metalloproteinase:E-cadherin ratio in prostate core needle biopsy.

According to a good correlation between in situ hybridization-based metalloproteinase-2/9:E-cadherin ratio (MER) and the pathological stage of prostate cancer, we set the cutoff line of MER at 6.0 (MER>6) to distinguish between organ-confined (pT2) and advanced diseases (pT3a-b/N1). In this study, we looked at the factors affecting MER and leading to a misprediction of the pathological stage. We examined MER in 39 paired specimens of prostate core needle biopsy and prostatectomy from the same patient and compared these MERs. In 34 (87%) of 39 cases, the MER of biopsy was correlated with the final pathological stage (pT2 vs. pT3a-b/N1). MER ranges in pT3a-b/N1 cancer were significantly wider than those in pT2 cancer (p < 0.01). The number of MER>6 fields in Gleason score 8-9 cancer was larger than that in Gleason score 7 cancer (p < 0.0001). In 5 cases where there was a failure to distinguish pT2 from pT3a-b/N1, the misdiagnosis was significantly associated with a small number of biopsies (4 or 6 specimens; p = 0.0469), a small amount of tumor tissue in biopsy specimens (less than 5 mm; p = 0.0492), and a wide MER range (more than 5.0; high intratumoral heterogeneity; p = 0.0202). Considering these factors increases the usefulness of preoperative prediction of the final pathological stage by MER in prostate cancer.

Biomarkers, Tumor↗

PubMed2004

Brain-derived neurotrophic factor increases inhibitory synapses, revealed in solitary neurons cultured from rat visual cortex.

To elucidate chronic actions of brain-derived neurotrophic factor (BDNF) on GABAergic synapses, we examined effects of a long-term application of BDNF for 10-15 days on autapses (synapses) of solitary GABAergic neurons cultured from rat visual cortex. Solitary neuron preparations were used to exclude a possible contamination of BDNF actions on excitatory neurons in dissociated neuron culture or slice preparations. Neurons were confirmed to be GABAergic pharmacologically with bicuculline, a selective antagonist for GABAA receptors and immunocytochemically with antibody against glutamic acid decarboxylase 65, a GABA synthesizing enzyme. To evaluate GABAergic synaptic function, evoked and/or miniature inhibitory postsynaptic currents (IPSCs) were recorded in the whole-cell voltage-clamp mode. The treatment with BDNF at a concentration of 100 ng/ml enhanced the amplitude of evoked IPSCs and the frequency of miniature IPSCs. In contrast, BDNF did not have a detectable effect on the amplitude of miniature IPSCs and the paired pulse ratio of IPSCs evoked by two, successive activations. To evaluate morphological changes, neurons were immunocytochemically stained with antibodies against microtubule-associated protein 2, to visualize somatodendritic region and synapsin I, to visualize presynaptic sites. The quantitative analysis indicated that BDNF increased the area of soma, the numbers of primary dendrites and dendritic branching points, the total length of dendrites and the number of synaptic sites. Such an action of BDNF was seen in both subgroups of GABAergic neurons, parvalbumin-positive and -negative neurons. To visualize functionally active presynaptic sites, neurons were stained with a styryl dye, FM1-43. BDNF increased the number of stained sites that was correlated with the frequency of miniature IPSCs. These results suggest that the chronic treatment with BDNF promotes dendritic and synaptic development of GABAergic neurons in visual cortex.

Analysis of Variance↗

PubMed2003

Dynamics of microbial populations and strong selection for Cycloclasticus pugetii following the Nakhodka oil spill.

Microbial population changes were monitored immediately after the Nakhodka oil spill accident in January 1997 at the heavily oil-contaminated Mikuni coast along the Sea of Japan. The total cell number was almost stable for one year at 2-5 x 10(5) cells mL(-1), while the relative occurrence of culturable heterotrophs and degraders of oil components such as C-heavy oil, kerosene, and n-tetradecane varied, showing a maximum (>50% of the total) immediately following the accident. Gene amplification and phylogenetic analysis of a dilution culture using C-heavy oil as the sole carbon and energy source revealed that one of the predominant oil degraders at the oil-contaminated coast in 2 weeks after the accident closely resembled the aromatic hydrocarbon decomposer Cycloclasticus pugetii. Microbial community composition in oil-contaminated seawater was estimated at the molecular level using newly developed oligonucleotide probes, probe wash-off curve estimation, and quantitative fluorescence dot-blot hybridization techniques. At two different oil-polluted sites, harbor and intertidal regions, the C. pugetii group was estimated to make up 23-25% of the total Bacteria population, followed by the aliphatic hydrocarbon decomposer Alcanivorax borkumensis, which formed 4-7% of the Bacteria. In incubation experiments using floated oil slick and indigenous microbes collected at the harbor, oil degradation activities were enhanced by the addition of both organic and inorganic nutrients. Significant decreases were found in aromatic and aliphatic hydrocarbon fractions: 54-60% and 22-24% in 2 weeks to 68-77% and 23-32% in 2 months, respectively.

Base Sequence↗

PubMed2003

Long-term follow-up of hepatitis C virus infection: HLA class II loci influences the natural history of the disease.

Hepatitis C virus (HCV) causes various grades of chronic liver disease, ranging from an asymptomatic state to cirrhosis. To assess genetic factors of disease severity, we selected two HCV patient groups according to the following stringent criteria: (i) asymptomatic carrier state (ASC) defined by HCV infection for more than 20 years, normal alanine aminotransferase levels for the past 5 years as well as normal liver histology and/or shape and (ii) liver cirrhosis (LC) as diagnosed by clinical symptoms, liver biopsy and/or ultrasonography. A total of 103 chronically infected Japanese HCV patients (43 ASC and 60 LC) were analyzed. HLA class I and II alleles were established using low resolution DNA typing. HLA-DRB1 and DQB1 genotypes were inferred upon polymerase chain reaction-restriction fragment length polymorphism analysis. Two hundred and one anti-HCV-negative ethnically matched controls were included. The frequencies of DRB1*12 (*1201 and *1202), DQB1*0301 and DRB3*03 alleles were higher in patients with ASC than in those with LC (odds ratio (OR) 11.23, OR 4.25, and OR 3.22, respectively). The frequency of DQB1*0503 were lower in ASC patients compared to LC patients (OR 0.05). No significant differences between groups were observed for age, sex, source of infection, HCV genotype or viral loads. Our findings establish that certain HLA class II alleles strongly influence disease progression following HCV infection.

Aged↗

PubMed2001

Design of polymer materials enhancing nucleotide hybridization for anti-gene technology.

Stabilization of nucleotide hybridization is considered important for improving gene therapy using oligonucleotides. We have designed comb-type copolymer consisting of polycation backbone (polylysine) and hydrophilic side chains as a stabilizer for double and triple helical DNAs. The copolymer considerably increased the thermal stability of triple helical structure but did not affect the reversible transition between triple helical and single-stranded DNA. An in vitro electrophoretic mobility shift assay revealed that the copolymer remarkably increased association constants of both Hoogsteen and reverse Hoogsteen-type triple helix formation. Moreover the triple helix-stabilizing efficiency of the copolymer was significantly higher than that of other oligocations like spermine and spermidine. Not only being good DNA triple helix stabilizer, it has also been shown to accelerate DNA strand exchange reactions between double helical DNA and its complementary oligonucleotides. From these, we conclude that this copolymer is capable of either 'stabilizing' or 'activating' DNA hybrids, and may useful for gene targeting employing oligonucleotides.

Animals↗

PubMed2001

Overexpression of sigma factor, sigma(B), urges Staphylococcus aureus to thicken the cell wall and to resist beta-lactams.

Whole genome sequence analysis revealed that Staphylococcus aureus is provided with only a few sigma factors, including the alternative sigma factor, sigma(B), which is thought to regulate some stress responses. Since the sigB knock-out mutant did not show remarkable phenotypic difference, we constructed the over expressed mutant to examine the role of the sigB. Electron microscopic observation revealed that the mutant showed a variety of cell sizes compared with the parent strain which showed almost homogeneous cell sizes. The mutant delivered a thicker cell wall, about 20% thicker than the parent strain. It became resistant to the lytic activity of lysostaphin and also raised MICs to the cell-wall-affecting antibiotics. The yield of carotenoids and transcripts of pbps were also increased in the mutant. The result suggests that sigB plays some important roles in cell wall synthesis and in resistance to antibiotics that perturb the cell wall synthesis.

Bacterial Proteins↗

PubMed2001

Loss of motility-related protein 1 (MRP1/CD9) and integrin alpha3 expression in endometrial cancers.

BACKGROUND: MRP1/CD9 and integrin alpha3 have played crucial roles in cell adhesion, motility, and signaling events. The loss of MRP1/CD9 and integrin alpha3 has been involved in tumor growth and metastasis of cancer cells. The aim of the current study was to clarify the clinical significance of MRP1/CD9 and integrin alpha3 in endometrial cancer. METHODS: The expression of MRP1/CD9 and integrin alpha3 from the same tissue sample were examined immunohistochemically in 15 patients with normal endometrium and in 56 patients with uterine endometrioid adenocarcinoma. Disease-free survival curves were estimated using the Kaplan-Meier method and analyzed by the log-rank test between the positive and reduced expression statuses of both MRP1/CD9 and integrin alpha3. These expressions and clinicopathologic variables were analyzed univariately and multivariately. RESULTS: In normal endometrium, MRP/CD9 was expressed at the cell membrane of cell contact sites, and the expression of integrin alpha3 was detected also at the cell membrane of cell contact sites and at borders of stromal tissues. In patients with endometrioid adenocarcinoma, 17 cases showed reduced expression of MRP1/CD9, and 20 cases had reduced expression of integrin alpha3. Fourteen cases indicated a reduced expression of both MRP1/CD9 and integrin alpha3. Each reduced expression of MRP1/CD9 or integrin alpha3 was significantly correlated with histologic grade and metastasis. Multivariate analysis using the Cox regression model disclosed that age at surgery, metastasis, and expression status of MRP1/CD9 were significant prognostic factors for disease-free survival. CONCLUSIONS: These findings suggested that the analysis for the expression statuses of MRP1/CD9 and integrin alpha3 may provide important information on the clinical behavior of endometrial cancer.

Antigens, CD↗

PubMed2001

Clinicopathologic and familial characteristics of endometrial carcinoma with multiple primary carcinomas in relation to the loss of protein expression of MSH2 and MLH1.

BACKGROUND: The frequency of synchronous or metachronous multiple primary carcinomas in patients with endometrial carcinoma has been reported to be between 10% and 23% and is highest among all gynecologic carcinomas. However, clinical characteristics and underlying genetic abnormalities in endometrial carcinoma with multiple primary carcinomas has not been well clarified. Endometrial carcinoma is the most commonly associated extracolonic malignancy in hereditary nonpolyposis colorectal carcinoma in which germ line mutations in DNA mismatch repair genes, particularly in MSH2 and MLH1, are known to cause this syndrome. The purpose of the current study was to investigate clinicopathologic and familial characteristics including MSH2, MLH1, and p53 expression in endometrial carcinoma with multiple primary carcinomas, by comparing them to endometrial carcinoma without other primary malignancies. METHODS: Patients were divided into two groups: 30 patients with synchronous or metachronous multiple primary carcinomas other than endometrial carcinoma and 116 patients with endometrial carcinoma without other primary malignancies. Clinicopathologic characteristics, family history of cancer, and immunohistochemical protein expression of MSH2, MLH1, and p53 expression were investigated in both groups, and 15 endometria from benign disease were used for normal controls in immunohistochemistry. RESULTS: The frequency of high risk clinicopathologic factors of endometrial carcinoma and 5-year survival rates and the frequency of p53 overexpression were not statistically different between the two groups. However, the loss of MSH2 and/or MLH1 expression was significant in endometrial carcinoma with multiple primary carcinomas, when compared with endometrial carcinoma alone (22 of 30 vs. 31 of 116). In cases with multiple primary carcinomas, particularly those diagnosed before the patient was 55 years of age or those in which the patient had a family history of cancer, the frequency of this loss was especially high (11 of 13 and 10 of 11, respectively). CONCLUSIONS: The clinical or biologic nature of endometrial carcinoma with multiple primary carcinomas seems to be similar to endometrial carcinoma alone. A high incidence of defective MSH2 and MLH1 protein in endometrial carcinoma with multiple primary carcinomas, however, suggests that abnormalities in the function of MSH2 and MLH1 may play an important role in tumorigenesis for patients with endometrial carcinoma with multiple primary carcinomas and their families.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Whole genome sequencing of meticillin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS: Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS: The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION: The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Animals↗

PubMed2001

Comb-type cationic copolymer expedites DNA strand exchange while stabilizing DNA duplex.

The accelerating effect of cationic substances on the DNA strand exchange reaction between a 20 bp DNA duplex and its complementary single strand was studied. A polycationic comb-type copolymer, that consists of a poly(L-lysine) backbone and a dextran graft chain (PLL-g-Dex) and known to stabilize triplex DNA, expedites the strand exchange reaction under physiological relevant conditions. Electrostatically a small excess of the copolymer let to a 300-1500-fold increase in the DNA strand exchange while large excess of spermine or cetyltrimethylammonium bromide, a cationic detergent known to promote markedly hybridization of complementary DNA strands, shows only a slight effect. The efficacy of the copolymer was not affected by a 10 mM Mg2+ concentration. Notably the copolymer promotes the strand exchange reaction while it stabilizes double-stranded DNA. The stabilization of strand exchange intermediates consisting of the parent duplex and the single strand by the copolymer is believed to be responsible for the observed acceleration behavior.

Cations↗

PubMed2001

Clinical value of lung uptake of iodine-123 metaiodobenzylguanidine (MIBG), a myocardial sympathetic nerve imaging agent, in patients with chronic heart failure.

This study investigated the clinical value of I-123 MIBG pulmonary accumulation and washout in patients with chronic heart failure (CHF). Nineteen patients with CHF and 15 normal volunteers (NL) were included. The uptake ratio of heart to mediastinum (H/M), that of lung fields to mediastinum (L/M), and washout rate (WR) of the heart and lung fields were calculated in anterior planar images and compared with results of echocardiography and cardiac catheterization. In the CHF group, the lung uptake in delayed images increased and lung WR was decreased, suggesting pulmonary endothelial lesions. Furthermore, there was a negative correlation between right and left lung WR and pulmonary arterial diastolic pressure (PA(D)) and pulmonary arterial systolic pressure (PA(s)) in the CHF group. Since the WR of MIBG reflected PA, it may be used as an index of severity of cardiac dysfunction.

3-Iodobenzylguanidine↗

PubMed2001

Genetic transformation system for a psychrotrophic deep-sea bacterium: isolation and characterization of a psychrotrophic plasmid.

A versatile system that permits genetic manipulation of a psychrotrophic deep-sea bacterium, Pseudoalteromonas sp. PS1M3, has been developed. A cryptic indigenous plasmid, pPS1M3, of 3.1 kb from the above strain was isolated and characterized. The nucleotide sequence analysis of plasmid pPS1M3 revealed the presence of one open reading frame, and its deduced amino acid sequence was identified as the essential protein for plasmid maintenance. Transformation with the pPS1M3 harboring antibiotic resistance genes by electroporation was fully successful using the pPS1M3-cured strain as a host. This plasmid was quite stable under nonselective culture conditions for about 100 generations at 4 degrees C. The copy number of this plasmid in the cell was about 5 copies per chromosome.

Journal Article↗

PubMed2001

Receptor-mediated cell modulator delivery to hepatocyte using nanoparticles coated with carbohydrate-carrying polymers.

Cell modulators such as colchicine (CO), cytochalasin B (CY) and taxol (TX) loaded nanoparticles coated with carbohydrate-carrying polymers, as hepatocyte-specific targeting material using galactose ligands as recognition signals to asialoglycoprotein receptors were prepared by the diafiltration method. Effects of cell modulators from their loaded nanoparticles on morphology of hepatocytes were studied. Receptor-mediated endocytosis of the nanoparticles were examined by fluorescence and confocal laser microscopy. It was found that the shapes of most hepatocytes were changed for the CY-loaded, TX-loaded, or CO-loaded nanoparticles whereas their shapes were not changed in comparison with control when CY, TX, or CO were mixed with the nanoparticles. From the fluorescence and confocal laser microscopic studies, it is suggested that the nanoparticles coated with sugar-carrying polymers were internalized by the hepatocytes through the receptor-mediated mechanism.

Animals↗

PubMed2001

Beta-cyanoalanine synthase and cysteine synthase from potato: molecular cloning, biochemical characterization, and spatial and hormonal regulation.

Beta-cyanoalanine synthase (CAS, L-3-cyanoalanine synthase; EC 4.4.1.9) is the most important enzyme in cyanide metabolism. In addition to CAS, cysteine synthase (CS, EC 4.2.99.8) possesses CAS activity. To explore the physiological significance of cyanide metabolism, we isolated the cDNA clones corresponding to purified CAS (designated PCAS-1 and PCAS-2) and CS (designated PCS-1 and PCS-2) from potato using the information of these amino acid sequences. The recombinant proteins of PCS-1, PCS-2 and PCAS-1 catalyzed both CAS and CS reactions, although the ratios between CAS and CS activity were remarkably different. PCAS-1 preferred the substrates for the CAS reaction to the substrates for the CS reaction. From the kinetic characters and homology of amino acid sequences with known CS-like proteins, PCS-1, PCS-2 and PCAS-1 were identified as cytosolic CS, plastidic CS and mitochondrial CAS, respectively. The highest level of CAS activity, CAS protein and its mRNA were detected in potato buds. Stimulation of CAS activity and protein accumulation by ethylene without the concomitant increase of its mRNA suggested that ethylene induces CAS protein accumulation at the post-transcriptional level.

Amino Acid Sequence↗

PubMed2001

Polycation graft copolymers accelerating DNA strand exchange: involvement of ionic interaction.

In the previous study (Chem. Eur. J., 7, 176 (2001)) we demonstrated that the comb-type polycationic copolymer (PLL-g-Dex) which is composed of poly (L-lysine) backbone and dextran graft chains expedited the DNA strand exchange reaction. In this study, fluorescence resonance energy transfer (FRET) was employed to explore the copolymer-mediated DNA strand exchange with higher time-resolution. To initiate strand exchange reaction the duplex prepared from 3'-fluorescein isothiocyanate (FITC)--and 5'-carboxytetramethylrhodamine(TAMRA)-labeled complementary DNAs was added to its non-labeled complementary single strand. DNA strand exchange was monitored by observing the recovery of the FITC quenching. More than 20,000 times increase in strand exchange rate at 37 degrees C by the copolymer was estimated. To investigate the accelerating mechanisms of the copolymer, the same reactions but at various ionic strenghts were studied. With increasing ionic strengths the strand exchange rate in the absence of the copolymer increased, suggesting that ionic repulsion among DNAs is unfavorable for the strand exchange to occur. Hence, alleviation of the electrostatic repulsion through interpolyelectrolyte complex formation is probably a role of the copolymer for accelerating the strand exchange reaction.

DNA↗

PubMed2001

Synergistic stabilization of triplex by combination of comb-type cationic copolymer and oligo-N3'-->P5' phosphoramidates.

In the present study, we describe rapid formation of stable pyrimidine motif triplex at physiological pH. The triplex formation was achieved by the synergistic effect of poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO). Either the PLL-g-Dex copolymer or the PN modification alone increased the binding constant by nearly two orders of magnitude for the triplex formation at neutral pH. The combination of both stabilizing factors that was the triplex formation with the PN TFO in the presence of the copolymer increased the binding constant by nearly four orders of magnitude. The kinetic study indicated that the copolymer increased the association rate constant, whereas the PN modification decreased the dissociation rate constant. No negative interference between these stabilizing effects was observed. The kinetically orchestrated effects in which the copolymer and the PN TFO contribute to distinct ingredients in triplex equilibrium achieved the rapid formation of the stable triplex.

Amides↗

PubMed2001

Protein/oligonucleotide conjugates as a cell specific PNA carrier.

We have focused on proteineus ligand conjugate with oligonucleotides (ODNs) as a cell-specific delivery vector for peptide nucleic acids (PNAs). Asialofetuin (AF), a hepatocyte-specific proteineus ligand, was conjugated with ODNs that served as binding sites for PNAs. Succinimidyl-transe-4(N-maleimidylmethyl)-cyclohexane-1-carboxylate (SMCC) modified AF was coupled with 5'-thiolated oligodeoxynucleotide (HS-ODN). The resulting conjugate held PNAs with sequence-specific manner. The PNA/DNA conjugate complex has resistance against nucleases in serum. The efficient release of PNA from the complex was observed when the complex was made in contact with a target nucleotide. PNA uptake to hepatocytes was greatly enhanced when hepatocytes was incubated with PNA/conjugate complex. Free AF thoroughly inhibited PNA uptake with the conjugate, evidencing asialoglycoprotein receptor (ASGP-R) mediated endocytosis to be a major-route for the cellular uptake.

Animals↗