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Biomedical subjects

M Sugai

Publications and source records attributed to M Sugai.

At least 37 records · Page 2Linked to original sources
PubMed2001

Extraction of bismuth(III) with 2-bromoalkanoic acid in nondonating solvent from highly acidic aqueous solution.

The extraction behaviors of bismuth(III) with carboxylic acid (HL), which have not yet been clearly elucidated, because of the precipitation of hydroxide, were studied using the 2-bromoalkanoic acid in benzene and in hexane systems under aqueous conditions of high acidity at I = 1.0 M ((H,Li)NO3). The extraction equilibria were analyzed based on the-initial concentration of nitric acid and the concentration of bismuth(III) extracted in the organic phase. The extracted species and the logarithmic values of the extraction constant (log Kex) were found to be a single species of BiL3(HL)3 for the systems of 2-bromooctanoic acid/benzene (log Kex = -1.66) and 2-bromohexadecanoic acid/benzene (-1.58), and to be two species of BiL3(HL)4 (-1.01) and Bi3L9HL (-1.62) for the system of 2-bromooctanoic acid/hexane, where the monomer was dominant at a higher reagent concentration.

Journal Article↗
PubMed2000

Identification of a fmtA-like gene that has similarity to other PBPs and beta-lactamases in Staphylococcus aureus.

We identified a gene from Staphylococcus aureus, flp (fmtA-like protein), encoding a protein of 489 amino acid residues with a molecular mass of 56.4 kDa. The deduced amino acid sequence shows similarity to previously characterized penicillin binding proteins (PBPs) and FmtA of S. aureus (one of the factors which affect methicillin resistance). FLP protein has three motifs, which are conserved in PBPs and beta-lactamases, suggesting that it might be associated with cell wall synthesis. Recombinant FLP protein, however, lacks penicillin binding activity, and the inactivation of flp in two methicillin-resistant S. aureus strains did not cause a reduction in the methicillin resistance.

Amino Acid Motifs↗
PubMed2000

Triton X-100-induced lipoteichoic acid release is correlated with the methicillin resistance in Staphylococcus aureus.

We previously reported that Triton X-100 (TRX) reduced methicillin resistance in Staphylococcus aureus, although the degree of reduction varied among strains. One of the biological effects of TRX on S. aureus cells was enhancement of lipoteichoic acid (LTA) release. We investigated the correlation between the amount of LTA released and the degree of reduction in methicillin resistance induced by TRX. The strains showing the greatest reduction of methicillin resistance released the largest amount of LTA, compared to those showing slight or moderate reduction. A mutant whose resistance was not affected by TRX did not increase its release of LTA. These findings suggest that LTA release is associated with a reduction in methicillin resistance in the presence of TRX.

Humans↗
PubMed2000

Phage conversion of exfoliative toxin A production in Staphylococcus aureus.

The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (phiETA) that encodes ETA and determined the complete nucleotide sequence of the phiETA genome. phiETA has a head with a hexagonal outline and a non-contractile and flexible tail. The genome of phiETA is a circularly permuted linear double-stranded DNA, and the genome size is 43 081 bp. Sixty-six open reading frames (ORFs) were identified on the phiETA genome, including eta, which was found to be located very close to a putative attachment site (attP). phiETA converted ETA non-producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that phiETA or related phages are responsible for the acquisition of eta genes in S. aureus.

Animals↗
PubMed2000

Tn551-mediated insertional inactivation of the fmtB gene encoding a cell wall-associated protein abolishes methicillin resistance in Staphylococcus aureus.

A Tn551 insert in a gene termed fmtB was shown to reduce oxacillin as well as Triton X-100 resistance in highly methicillin-resistant Staphylococcus aureus (MRSA) COL. Backcrosses of fmtB::Tn551 into S. aureus COL and into two genetically distinct MRSA strains, KSA8 and NCTC10443, confirmed the linkage of fmtB::Tn551 with loss of oxacillin resistance. The fmtB gene codes for a protein of a deduced molecular mass of 263 kDa that contains 17 tandem repeats of 75 amino acids and a C-terminal LPXTG cell wall-sorting motif. Immunoblots with anti-FmtB antibodies confirmed its localization in the cell wall fraction. The fmtB gene was mapped downstream of the phosphoglucosamine mutase operon glmM which catalyses formation of glucosamine-1-phosphate. Oxacillin resistance was not restored in fmtB mutants by trans-complementation with fmtB. However, although GlmM production was not affected by fmtB inactivation, oxacillin resistance was increased in fmtB mutants by introducing a plasmid-borne glmM gene, presumably by GlmM overexpression. Interestingly, a similar phenotypic complementation was obtained in fmtB mutants by including substrate level concentrations of N-acetylglucosamine or glucosamine in the growth medium. Inactivation of the fmtB gene seems therefore to have an indirect effect on methicillin resistance which can be relieved by increasing the production of the cell wall precursor glucosamine-1-phosphate.

Bacterial Proteins↗
PubMed2000

Photoregulation of asymmetric cell division followed by rhizoid development in the fern Ceratopteris prothalli.

Strap-shaped prothalli of CERATOPTERIS: richardii grown in the dark have an apical meristem, a subapical elongation zone and a basal growth cessation zone [Murata et al. (1997) Plant Cell Physiol. 38: 201]. When the dark-grown prothalli were irradiated with continuous white light, marginal cells of the elongation zone divided asymmetrically, and the resulting smaller cells developed into rhizoids. The asymmetric division was also induced by brief irradiation of red light. The effect of red light was cancelled by subsequent irradiation of far-red light, indicating that the asymmetric division was regulated by phytochrome. Since the response to red light was not observed at 10(1) J m(-2) and saturated at 10(2) J m(-2) and the response is photoreversible by far-red light, the photoresponse was classified as a low-fluence response of phytochrome. Although the asymmetric division was induced by brief irradiation of red light, continuous irradiation of white, blue or red light was necessary to induce rhizoid growth. These results indicate that asymmetric division and subsequent cell growth are independently regulated by light in CERATOPTERIS: prothalli.

Cell Division↗
PubMed2000

Modification of autolysis by synthetic peptides derived from the presumptive binding domain of Staphylococcus aureus autolysin.

The autolytic cell wall hydrolase of Staphylococcus aureus, Atl, contains three highly cationic repeats in the central region of the amino acid sequence, and the repeats are presumed to have the role of binding the enzyme to some components on the cell surface. To explain the possible function of the repeats, we synthesized a number of 10- to 30-mer oligopeptides based on the Atl amino acid sequence (Thr432-Lys610) containing repeat 1, and examined their effects on the autolysis of S. aureus cells. When the peptides were added to a cell suspension of S. aureus under low ionic strength conditions, five peptides, A10, A11, A14, A16 and B9, showed immediate increases in optical density (OD) of the cell suspension accompanied by decreases in viable cell counts. After the immediate increases, the ODs for A10 and A14 changed little in the first 2 hr. In contrast, the ODs for A11 and A16 decreased rapidly. When peptide A10 was added to suspensions of heat-killed whole cells, crude cell walls and a crude peptidoglycan preparation, their ODs were increased approximately 2-fold. In contrast, the OD was not increased when the peptide was added to a suspension of pure peptidoglycan from which anionic polymers had been removed. Light microscopic and transmission electron microscopic study showed that A10 and A14 inhibited autolysis and that A11 and A16 induced autolysis earlier than the control. These results suggest strongly that the peptides adsorb to and precipitate on the anionic cell surface polymers such as teichoic acid and lipoteichoic acid via ionic interaction. The effects of peptides on the autolysis may be the results of the modification of S. aureus autolysin activities. These peptides, especially the 10-mer peptide B9 (PGTKLYTVPW) that represents the C-terminal half of A10 and N-terminal half of A11, may be important segments for Atl to bind to the cell surface.

Amino Acid Sequence↗
PubMed1999

Selective diapedesis of Th1 cells induced by endothelial cell RANTES.

Differentiated CD4 T cells can be divided into Th1 and Th2 types based on the cytokines they produce. Differential expression of chemokine receptors on either the Th1-type or the Th2-type cell suggests that Th1-type and Th2-type cells differ not only in cytokine production but also in their migratory capacity. Stimulation of endothelial cells with IFN-gamma selectively enhanced transmigration of Th1-type cells, but not Th2-type cells, in a transendothelial migration assay. Enhanced transmigration of Th1-type cells was dependent on the chemokine RANTES produced by endothelial cells, as indicated by the findings that Ab neutralizing RANTES, or Ab to its receptor CCR5, inhibited transmigration. Neutralizing Ab to chemokines macrophage-inflammatory protein-1alpha or monocyte chemotactic protein-1 did not inhibit Th1 selective migration. Whereas anti-CD18 and anti-CD54 blocked basal levels of Th1-type cell adherence to endothelial cells and also inhibited transmigration, anti-RANTES blocked only transmigration, indicating that RANTES appeared to induce transmigration of adherent T cells. RANTES seemed to promote diapedesis of adherent Th1-type cells by augmenting pseudopod formation in conjunction with actin rearrangement by a pathway that was sensitive to the phosphoinositol 3-kinase inhibitor wortmannin and to the Rho GTP-binding protein inhibitor, epidermal cell differentiation inhibitor. Thus, enhancement of Th1-type selective migration appeared to be responsible for the diapedesis induced by interaction between CCR5 on Th1-type cells and RANTES produced by endothelial cells. Further evidence that CCR5 and RANTES play a modulatory role in Th1-type selective migration derives from the abrogation of this migration by anti-RANTES and anti-CCR5 Abs.

Actins↗
PubMed1999

Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.

We have identified a novel gene referred to as activation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.

APOBEC-1 Deaminase↗
PubMed1999

Hepatolobectomy-induced depression of hepatic circulation and metabolism in the dog is counteracted by isoflurane, but not by halothane.

BACKGROUND: The effects of isoflurane and halothane anesthesia on hepatic circulation and oxygen metabolism during hepatolobectomy were investigated in the dog, in an attempt to assess which of the anesthetics was the better one for hepatic resection. METHODS: Mongrel dogs (n=24) were divided into two groups and accordingly anesthetized with isoflurane (n=12) or halothane (n = 12). Each test anesthetic was administered in air. Electromagnetic flowmeters were used to measure hepatic arterial and portal venous blood flows 1) before the inhalation of each anesthetic (baseline); 2) 1 h inhalation of 1.5 MAC (minimum alveolar concentration) of each anesthetic; and 3) 1 h after hepatolobectomy with each anesthesia. Measurements of systemic hemodynamics, blood gas tensions, and the arterial ketone body ratio were made at the same time. RESULTS: Isoflurane maintained portal venous, hepatic arterial and total hepatic blood flows better than halothane anesthesia before and after hepatolobectomy. With halothane anesthesia, hepatolobectomy decreased prominently hepatic arterial blood flow. Hepatic arterial and mesenteric vascular resistance increased in the halothane group, but remained constant in the isoflurane group after hepatolobectomy. Hepatic oxygen delivery was significantly suppressed in the halothane group, but did not change in the isoflurane group. No significant difference was found in hepatic oxygen consumption between the two groups, but the arterial ketone body ratio decreased significantly only in the halothane group before and after hepatolobectomy. CONCLUSION: The present data indicate that isoflurane has less adverse effect than halothane anesthesia on hepatic circulation, oxygen delivery and energy charge in hepatolobectomy cases.

Analysis of Variance↗
PubMed1999

Cloning and characterization of a gene, pbpF, encoding a new penicillin-binding protein, PBP2B, in Staphylococcus aureus.

A previously unrecognized penicillin binding protein (PBP) gene, pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and beta-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed in Escherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region of pbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.

Amino Acid Sequence↗
PubMed1999

Characterization of fmtA, a gene that modulates the expression of methicillin resistance in Staphylococcus aureus.

FmtA is a factor which affects the methicillin resistance level in methicillin-resistant Staphylococcus aureus. Since FmtA has two of three conserved motifs which are typically found in penicillin-binding proteins (PBPs) and beta-lactamases, we investigated the penicillin-binding activity of recombinant FmtA and found no such activity. Immunoblotting analysis revealed that FmtA localizes in the membrane fraction. To investigate the function of FmtA, high-pressure liquid chromatography analysis of cell wall muropeptides was performed with an fmtA-inactivated mutant and its parent. The mutant showed a reduced cross-linking and partially reduced amidation of glutamate residues in the peptidoglycan of the mutant. The transcription of fmtA was dose dependently increased by the addition of beta-lactam antibiotics, fosfomycin, and bacitracin, while its transcription was not changed by the addition of vancomycin or tetracycline. These results reveal that Fmt is a membrane-located, non-penicillin-binding protein and that mutation of fmtA affects the cell wall structure, although its precise function is still unknown.

Anti-Bacterial Agents↗
PubMed1999

Cytotoxic necrotizing factor type 2 produced by pathogenic Escherichia coli deamidates a gln residue in the conserved G-3 domain of the rho family and preferentially inhibits the GTPase activity of RhoA and rac1.

Cytotoxic necrotizing factor types 1 and 2 (CNF1 and -2) produced by pathogenic Escherichia coli strains have 90% conserved residues over 1,014-amino-acid sequences. Both CNFs are able to provoke a remarkable increase in F-actin structures in cultured cells and covalently modify the RhoA small GTPases. In this study, we demonstrated that CNF2 reduced RhoA GTPase activity in the presence and absence of P122(RhoGAP). Subsequently, peptide mapping and amino acid sequencing of CNF2-modified FLAG-RhoA produced in E. coli revealed that CNF2 deamidates Q63 of RhoA-like CNF1. In vitro incubation of the C-terminal domain of CNF2 with FLAG-RhoA resulted also in deamidation of the FLAG-RhoA, suggesting that this region contains the enzymatic domain of CNF2. An oligopeptide antibody (anti-E63) which specifically recognized the altered G-3 domain of the Rho family reacted with glutathione S-transferase (GST)-RhoA and GST-Rac1 but not with GST-Cdc42 when coexpressed with CNF2. In addition, CNF2 selectively induced accumulation of GTP form of FLAG-RhoA and FLAG-Rac1 but not of FLAG-Cdc42 in Cos-7 cells. Taken together, these results indicate that CNF2 preferentially deamidates RhoA Q63 and Rac1 Q61 and constitutively activates these small GTPases in cultured cells. In contrast, anti-E63 reacted with GST-RhoA and GST-Cdc42 but not with GST-Rac1 when coexpressed with CNF1. These results indicate that CNF2 and CNF1 share the same catalytic activity but have distinct substrate specificities, which may reflect their differences in toxic activity in vivo.

Amino Acid Sequence↗
PubMed1999

Cloning of the gene encoding the Actinobacillus actinomycetemcomitans serotype b OmpA-like outer membrane protein.

The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of A. actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in E. coli perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A. actinomycetemcomitans serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34.

Aggregatibacter actinomycetemcomitans↗
PubMed1999

Expression of osteoprotegerin (osteoclastogenesis inhibitory factor) in cultures of human dental mesenchymal cells and epithelial cells.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) inhibits osteoclast differentiation, activity, and survival; therefore OPG/OCIF may regulate the resorption of dental hard tissues, such as alveolar bone, cementum, and dentin. To investigate this issue, reverse transcriptase-polymerase chain reaction using specific primers for OPG/OCIF was performed with total RNAs isolated from human gingival keratinocytes (HGKs), human gingival fibroblasts (HGFs), human periodontal ligament cells (HPDLs), and human pulp cells (HPCs) in culture. PCR products were found in HGFs, HPDLs, and HPCs, but not in HGKs, and the DNA sequence of these products was 100% identical to the reported sequence of the OPG gene. Northern blot analyses also showed that HGFs, HPDLs, and HPCs, but not HGKs, expressed OPG/OCIF transcripts of approximately 2.5 kb. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) increased OPG/OCIF mRNA levels in a dose-and time-dependent manner in HPDL. After 12 h of treatment, IL-1beta at 3 ng/ml and TNF-alpha at 3 ng/ml increased OPG/OCIF mRNA expression by 190% and 110%, respectively, with a maximal effect. The stimulatory effects of IL-1beta and TNF-alpha were also seen in HPC. However, IL-6 and transforming growth factor-beta had little effect on OPG/OCIF mRNA levels in HPDL. These findings suggest that OPG/OCIF synthesized by dental mesenchymal cells locally regulates the resorption of dental hard tissues through cytokines.

Cells, Cultured↗
PubMed1998

Identification and molecular characterization of a gene homologous to epr (endopeptidase resistance gene) in Staphylococcus aureus.

Certain Staphylococci possess a gene called epr or lif that renders the cells resistant to lysis by glycylglycine endopeptidase. The resistance is conferred by modifying the amino acid composition of interpeptide chains in cell-wall peptidoglycan by increasing serine content and decreasing glycine content. A gene homologous to epr/lif was cloned from S. aureus RN450 genomic libraries and designated eprh. eprh was found to localize 27bp downstream of a novel cell-wall hydrolase gene lytN, which is in the same orientation with eprh. By analogy with epr/lif, eprh is suggested to be involved in the transfer of certain amino acids, possibly serine or amino acids other than glycine, to interpeptide chains of cell-wall peptidoglycan. Unlike epr/lif, overexpression of eprh in S. aureus did not result in an increased resistance to lysostaphin. Insertional inactivation of eprh or lytN by Campbell-type integration did not affect the susceptibility of the cells to lysostaphin, either. These results suggest that eprh and lytN are not essential genes for S. aureus growth. The physiological function of eprh remains unknown.

Amino Acid Sequence↗