Biomedical subjects

N Hanada

Publications and source records attributed to N Hanada.

At least 73 records · Page 4Linked to original sources

PubMed1992

Mechanism of water-insoluble glucan synthesis in Streptococcus sobrinus.

Synthesis of water-insoluble glucan (IG) by 1,3-alpha-D-glucan synthase from Streptococcus sobrinus was examined using methylation analysis. The purified enzyme was incubated with sucrose and dextran T2000 (DT2000) for a given time and only IG was harvested by centrifugation. The remaining supernatant was incubated again, and IG was obtained. By repeating the above method using the residual supernatant, we obtained 5 varieties of IG precipitated in different periods. These IGs were methylated and examined using gas chromatograph mass spectrometry. The DT2000 water-insolubilized in the early reaction stage tended to have a highly ramified structure, with 1,3-alpha-D-glucan on a 1,6-alpha-D-glucan chain as the main chain. On the contrary, the DT2000 water-insolubilized in the late stage tended to have sparse side chains of 1,3-alpha-D-glucan which elongated with incubation. Specifically, the greater the number of side chains, the sooner the DT2000 was insolubilized. These results suggest that water-insolubilization of the water-soluble glucan not only depends on the increase of the ratio of 1,3-alpha-glucoside linkages to 1,6-alpha-glucoside linkages but also on the degree of branching of the 1,3,6-alpha-branched glucoside linkages.

Glucans↗

PubMed1992

Transfer of specific IgG and IgG subclasses to herpes simplex virus across the blood-brain barrier and placenta in preterm and term newborns.

The kinetics of virus-specific IgG subclasses (IgG 1-4) among newborns and their mothers has not yet been determined. In this report, we examined anti-herpes simplex virus IgG activities (HSV-IgG) and its subclasses in CSF and serum of premature or term newborns without HSV infection and in the serum of their mothers using ELISA. We found that CSF/serum ratios of HSV-IgG and IgG subclasses (IgG 1-4) in newborns with a gestational age less than 38 weeks were higher than those of term newborns. These findings indicate that the blood-brain barrier against HSV-IgG and IgG subclasses is insufficient in newborns. Furthermore, we found that HSV-IgG subclasses, which were transferred across the placenta and later transferred across the blood-brain barrier had a tendency to be proportional to each of the maternal HSV-IgG subclasses.

Antibodies, Viral↗

PubMed1992

Prophylactic oral acyclovir in outbreaks of primary herpes simplex virus type 1 infection in a closed community.

Oral acyclovir was given prophylactically to 37 children in the early stages of three outbreaks of herpes simplex virus type 1 (HSV-1) infection and the results were compared with those in untreated control subjects in two other outbreaks. The rates of seroconversion to HSV were significantly reduced in children treated with acyclovir compared with control subjects (91% vs 27%, P less than .001). The incidence of symptomatic disease was also significantly reduced (82% vs 0%, P less than .001). In some children receiving prophylactic acyclovir, anti-HSV antibody titers did not rise despite the presence of replicative HSV on throat swabs just before the start of treatment. Restriction endonuclease analysis of isolated HSV-DNA confirmed that one strain was responsible for the five outbreaks. No resistance to acyclovir was detected during the study, and no adverse effects of treatment were noted. In conclusion, short-term prophylactic acyclovir may limit the spread and reduce clinical manifestations of HSV infections in closed communities, although this use should be restricted to communities where severe symptoms are observed.

Acyclovir↗

PubMed1992

Relapse of herpes simplex encephalitis in children.

The polymerase chain reaction method was used to diagnose herpes simplex encephalitis in children. Initial samples of cerebrospinal fluid from 15 patients with herpes simplex encephalitis were all positive for the herpes simplex virus DNA by polymerase chain reaction assay. In terms of early diagnosis, polymerase chain reaction assay became positive significantly earlier than the detection of intrathecally produced anti-herpes simplex virus antibody using the enzyme-linked immunosorbent assay (4.4 vs 8.9 days after onset; P less than .01). Serial examinations showed that the presence of virus DNA in cerebrospinal fluid continued for 3 to 18 days after the neurologic onset (mean 10.1 days). Four of the 15 patients had a relapse of encephalitis after completing acyclovir therapy. The mean duration of initial acyclovir therapy in the recurrent group was significantly shorter than that in the nonrecurrent group. In recurring cases, herpes simplex virus DNA reappeared temporarily in the cerebrospinal fluid of two patients. These results show that polymerase chain reaction assay is a useful diagnostic tool for the early and noninvasive diagnosis of herpes simplex encephalitis in children. Results also suggest that a comparatively short duration of acyclovir therapy may be related to a relapse of herpes simplex encephalitis in some children.

Acyclovir↗

PubMed1991

The extension of alpha-D-1,3-branch linkages by 1,3-alpha-D-glucan synthase from Streptococcus sobrinus.

In the presence of an acceptor, 1,3-alpha-D-glucan synthase of Streptococcus sobrinus synthesizes water-insoluble glucans from sucrose. Under such conditions, 1,3-alpha-D-glucoside linkages were extended without any change in the glucose-residue number between the 1,3,6-branch points on the acceptor. From these results, the mechanisms of water-insoluble-glucan formation were proposed as follows: (i) the attachment of an acceptor to the glucan binding sites of 1,3-alpha-D-glucan synthase occurs during the initiation of the reaction, and concurrently determines the positions of the branched portions of 1,3,6 on the acceptor, and (ii) the 1,3-alpha-D-glucoside linkage extends from these positions.

Carbohydrate Conformation↗

PubMed1991

Cloning of a Streptococcus sobrinus gtf gene that encodes a glucosyltransferase which produces a high-molecular-weight water-soluble glucan.

The gtf gene coding for glucosyltransferase (GTF), which produces a water-soluble glucan, was cloned from Streptococcus sobrinus OMZ176 (serotype d) into plasmid vector pBR322. This gene was expressed in Escherichia coli, and the product was purified to near homogeneity. The antigenicity of recombinant GTF (rGTF) was examined with the antisera raised against purified GTF P1, P2, P3, and P4 obtained from S. sobrinus AHT (serotype g). The rGTF reacted only with anti-GTF P1 serum in a Western blot (immunoblot) analysis. The rGTF closely resembled GTF P1 in its molecular mass, Km value for sucrose, optimal pH, primer dependency, and immunological properties. The high-molecular-weight, water-soluble glucan produced by the rGTF also resembled that of GTF P1, which is the most efficient primer donor for primer-dependent, water-insoluble glucan synthesis. Properties of the rGTF were also compared with those of rGTFS, which was purified from E. coli carrying the gtfS gene isolated from Streptococcus downei (previously S. sobrinus serotype h) MFe28. Both rGTF and rGTFS synthesized water-soluble glucan from sucrose without primer dextran, but their characteristics in Km values for sucrose, optimal pHs, and polymer sizes of the glucan were different. Furthermore, the gtf gene did not hybridize with the gtfS gene in a Southern blot analysis. These results showed that rGTF is similar to S. sobrinus AHT GTF P1 but distinct from rGTFS that has been previously purified from E. coli carrying the gtfS gene.

Amino Acid Sequence↗

PubMed1991

Rapidly progressive renal failure associated with angiofollicular lymph node hyperplasia.

The authors report a case of rapidly progressive renal failure associated with Castleman's disease. Renal biopsy revealed crescentic glomerulonephritis associated with marked tubulointerstitial nephritis. This kind of renal lesion has never been reported in association with angiofollicular lymph node hyperplasia (Castleman's disease) or other immunoblastic disorders. The patient initially required hemodialysis therapy. However, steroid therapy was effective in treating the systemic manifestations of Castleman's disease and some renal function was recovered. The patient was finally withdrawn from hemodialysis therapy.

Acute Kidney Injury↗

PubMed1991

Substrate specificity of hydrolase activity of the primer-dependent glucosyltransferases from Streptococcus sobrinus.

Four kinds of glucosyltransferases, P1, P2, P3 and P4, were separately purified from the culture supernatant of Streptococcus sobrinus. Their dependencies on primer were analysed. There were two primer-dependent glucosyltransferases (P3 and P4). In the absence of primer 1,6-alpha-D-glucan, P3 was not able to produce glucan from sucrose. However, P3 showed sucrose hydrolase activity, whereas P4 was still able to produce glucan without primer 1,6-alpha-D-glucan. Consequently, glucosyltransferase activity of P4 was incompletely primer-dependent. Both P3 and P4 showed high substrate specificity for sucrose, failing to use melezitose, raffinose, or stachyose as the substrates.

Chromatography, High Pressure Liquid↗

PubMed1991

Clinical manifestations of primary herpes simplex virus type 1 infection in a closed community.

The clinical features and the molecular epidemiology of primary herpes simplex virus type 1 (HSV-1) infection among children younger than 3 years of age were investigated in day-care nursery. Serial sera were assayed for anti-HSV-1 glycoprotein B antibody by enzyme-linked immunosorbent assay. Serologic examinations revealed 55 cases of primary HSV infection during the observation period. Fifty-one of them (93%) had typical herpetic gingivostomatitis, showing a high rate of clinically overt infection. Four outbreaks of herpetic gingivostomatitis were observed during the observation period. Forty-one children were infected with HSV-1 in the outbreaks. The rates of infection in the susceptible children were 81%, 73%, 78%, and 100%, respectively, in the four outbreaks. Restriction endonuclease analysis of DNA of isolated HSV revealed that only one strain of HSV-1 had been transmitted among children for a long period.

Age Factors↗

PubMed1990

Human cytomegalovirus infection during childhood: detection of viral DNA in peripheral blood by means of polymerase chain reaction.

Polymerase chain reaction (PCR) technique was applied to detect cytomegalovirus (CMV) DNA. Two pairs of synthetic oligonucleotide primers were used to amplify DNA from the immediate early 1 and the late antigen genes of CMV, respectively. Either primer sets could detect as few as 0.01 plaque-forming unit of CMV strain AD 169 by Southern blot hybridization. Sixteen CMV clinical isolates were examined and all were found to be positive by the both primer sets. The PCR was used to detect CMV DNA in peripheral blood from six children with elevated anti-CMV antibody titers, who showed abnormal liver-function tests. In three immunocompromised patients, all blood samples were positive for CMV DNA. In three immunocompetent young infants with primary CMV infection, CMV DNA was detected from peripheral blood of one patient during acute phase. Presence of CMV DNA in peripheral blood seemed to be related with the extent of CMV infection, and possibly diagnostic for CMV hepatitis.

Child, Preschool↗

PubMed1990

Characterization of the product of the gtfS gene of Streptococcus downei, a primer-independent enzyme synthesizing oligo-isomaltosaccharides.

The gtfS gene, coding for a glucosyltransferase which synthesizes water-soluble glucan and previously cloned from Streptococcus downei strain MFe28 (mutans serotype h) into a bacteriophage vector, was subcloned into a plasmid vector. The gtfS gene products expressed in Escherichia coli were compared to the primer-independent, oligo-isomaltosaccharide synthase in Streptococcus sobrinus strain AHT (mutans serotype g) and shown to resemble it closely in molecular mass, isoelectric point, immunological properties, optimum pH and Km values. The glucans produced from sucrose by the gtfS gene products are alpha-1,6-linked linear oligo-isomaltosaccharides without any branching sites. A similar gtfS gene was also detected on chromosomal DNA from S. sobrinus strain AHT.

Blotting, Western↗

PubMed1990

Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381.

Cell-associated alkaline phosphatase (ALPase) of Bacteroides gingivalis 381 was found in the outer part of the periplasmic space by using an ultracytochemical procedure. Cell-associated ALPase was solubilized by extraction with 1% Triton X-100, and the solubilized enzyme was purified 904-fold with 5.6% recovery by using affinity column chromatography for mammalian intestinal-form ALPase. The purified enzyme gave a single protein band that corresponded to the enzyme activity band on polyacrylamide gel electrophoresis preparations. A single protein band at a molecular weight of 61,000 was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis preparations. The molecular weight of the native enzyme was estimated to be 130,000 by gel filtration with TSK-gel G3000SW. These findings indicate that B. gingivalis ALPase is a homodimer. The optimal pH of the enzyme was between 9.1 and 9.3 in the absence of divalent metal ions and was between 10.1 and 10.3 in the presence of manganese or zinc ions. The apparent km for p-nitrophenylphosphate was 0.037 +/- 0.003 mM (mean +/- standard deviation) at pH 9.2 in the absence of divalent metal ions and 0.22 +/- 0.02 mM at pH 10.2 in the presence of 1 mM manganese ions. Under both of the conditions described above, the purified enzyme was able to hydrolyze casein and O-phosphoserine, suggesting that B. gingivalis ALPase can act as a phosphoprotein phosphatase. ALPase that immunologically cross-reacted with the purified enzyme was found in the extracellular soluble fraction. This means that ALPase is released from the periplasmic space into the culture supernatant as a soluble form.

Alkaline Phosphatase↗

PubMed1989

Evidence for the presence of two distinct sites of sucrose hydrolysis and glucosyl transfer activities on 1,3-alpha-D-glucan synthase of Streptococcus mutans.

1,3-alpha-D-Glucan synthase of Streptococcus mutans catalyzes both the hydrolysis of sucrose to glucose and fructose, and the glucosyl transfer to glucosyl polymers to yield water-insoluble glucan. The enzyme catalyzes only sucrose hydrolysis, however, in the absence of 1,6-alpha-D-glucan as an acceptor. In the present study, we found that glucosyl transfer activity was completely inhibited by the antiserum against isolated 1,3-alpha-D-glucan synthase but that the sucrose hydrolysis activity was not. The antiserum did not impair the binding of the enzyme to the acceptor. These findings indicate that sucrose hydrolysis and glucosyl transfer occur at two distinct sites on the enzyme.

Binding Sites↗

PubMed1989

Combined effects of acyclovir and human interferon-alpha on herpes simplex virus replication in cultured neural cells.

The combined effects of Acyclovir [9-(2'-hydroxyethoxymethyl)guanine; ACV] and human interferon-alpha (IFN-alpha) on replication of the herpes simplex virus type I (HSV-1) were determined in human neural cell lines, neuroblastoma (IMR), glioblastoma (118MGC), and glioma (U251MG). HSV-1 grew well in all these cells, with final yields of more than 1 x 10(6) PFU/ml. In terms of virus-yield reduction, ACV was found to be highly effective in IMR, moderately effective in U251MG, but ineffective in 118MGC. By contrast, IFN-alpha reduced the virus yield significantly in 118MGC and in U251MG, but did not in IMR. Combined application of ACV and IFN-alpha strongly inhibited the virus replication in all three cell lines with various degrees of synergism or additive effect. These results were also confirmed by immunofluorescent examinations. The sensitivity of HSV-1 to ACV or IFN-alpha was found to be different among the three different cell types. By combining the two agents, the virus growth was strongly suppressed in all the cells. These results suggest the importance of combination therapy for severe type of herpes simplex encephalitis in clinical practice.

Acyclovir↗

PubMed1989

Periodontal disease prevalence in different age groups in Japan as assessed according to the CPITN.

A CPITN survey was conducted involving 12,832 Japanese subjects from 7 to 64 years of age. Subjects under 18 were schoolchildren, and 18-year-old and older subjects represented various social backgrounds, having been randomly selected from both urban and rural Japan. Fifty percent of the 7-yr-old children had signs of periodontal disease, and this percentage increased with increasing age. In those under 14, this increase in periodontal disease was mainly due to an increase in the proportion of children developing dental calculus. Subjects with 6 mm or deeper pockets were observed starting from the 20-29-yr-old age group. In subjects over this age, the prevalence of periodontal disease was higher in men than in women. The percentages of subjects having pathologic pockets had increased remarkably in the 30-44-yr-olds. In the 45-64-yr-old group, almost all subjects had some sign of periodontal disease, and the percentage of those with 4 or 5 mm pockets and 6 mm or deeper pockets were 37% and 21%, respectively.

Adolescent↗

PubMed1989

Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis.

Two glucosyltransferase genes from Streptococcus mutans GS-5, gtfB and gtfC, have been previously isolated and sequenced in this laboratory. In the present communication a third gtf gene, gtfD, was isolated and characterized. Isolation of the gene involved a novel procedure utilizing the integration plasmid pVA891. A peptide expressed by the 1.7-kilobase DNA fragment from strain NHS1 (containing deletions in both the gtfB and gtfC genes) was initially identified in a pUC18 clone bank with antiglucosyltransferase antibodies. This fragment was integrated into the GS-5 chromosome following ligation into pVA891 and transformation, yielding strain DP2. The vector together with one complete and one incomplete copy of the gtfD gene was removed from the chromosome of strain DP2 following EcoRI digestion, religation, and transformation of E. coli HB101. The resultant plasmid, pNH4, expressed glucosyltransferase S (GTF-S) activity. The enzyme was purified to near homogeneity and was shown to synthesize water-soluble glucan exclusively in a primer-dependent manner. The molecular mass (155 kilodaltons) and the kinetic parameters of the purified enzyme were similar to those observed for the GTF-S enzyme previously purified from culture fluids of strain GS-5. Insertional inactivation of the gtfD gene indicated that this gene is not required for in vitro sucrose-dependent adherence to smooth surfaces. Furthermore, inactivation of the gtfD gene in a gtfC gtfB mutant indicated that three distinct gtf genes involved in glucan formation are present on the S. mutans GS-5 chromosome. Southern blot analysis further suggested that the gtfD gene does not share demonstrable homology with the gtf genes from Streptococcus sanguis or Streptococcus sobrinus.

Cloning, Molecular↗

PubMed1989

Purification of a fourth glucosyltransferase from Streptococcus sobrinus.

Recently, we found a novel primer-independent, water-soluble glucan synthase as a fourth glucosyltransferase (GTF) in a culture supernatant of strain AHT-k of Streptococcus sobrinus (Y. Yamashita, N. Hanada, and T. Takehara, Biochem. Biophys. Res. Commun. 150:687-693, 1988). In the present study, four kinds of purified GTFs, including the novel GTF, were prepared. They were composed of two primer-dependent GTFs and two primer-independent GTFs. Of the primer-dependent GTFs, one was a water-insoluble glucan synthase and the other was a water-soluble glucan synthase; both of the primer-independent GTFs were water-soluble glucan synthases (GTF-Sis). Using antisera against four purified GTFs, we concluded that the immunological properties of each were completely different from those of the others. Additionally, it was shown that the novel GTF-Si, which was previously shown to have a molecular weight of 137,000, was proteolytically degraded and could be isolated at a molecular weight of 152,000 and that Streptococcus cricetus secreted an enzyme that immunologically cross-reacted with GTF-Si. While the product of the novel GTF-Si was not an effective primer for both of the primer-dependent enzymes (water-soluble and -insoluble glucan synthases), the product of the enzyme affected the molecular size of the products of the other GTF-Sis.

Chromatography, Affinity↗

PubMed1988

A novel glucosyltransferase from Streptococcus mutans produces oligo-isomaltosaccharides.

Streptococcus mutans secretes a sucrose-independent branalphang enzyme that utilizes isomaltosaccharides as donors for branalphang formation on dextran. Although the branching enzyme is necessary for the formation of extracellular polysaccharide complexes, the source of the donor for the enzyme is unknown. In this study, we purified a novel glucosyltransferase from S. mutans and characterized its properties. The glucosyltransferase was primer independent 1,6-alpha-D-glucan synthase, which produced oligo-isomaltosaccharides. The enzyme was thought to be a source of donor for the branching enzyme in S. mutans.

Disaccharides↗