Biomedical subjects
Publications and source records attributed to T Kubota.
A cutaneous hemangioma of the penis extending from the proximal portion of the glans to the prepuce of an 18-year-old man was successfully treated by sclerotherapy with polidocanol and interrupted ligation of the hemangioma.
OBJECTIVE: To evaluate the diagnostic value of insulin-like growth factor binding protein-1 (IGFBP-1) as an indicator of ruptured fetal membranes. METHODS: IGFBP-1 concentrations were measured on 42 paired cervicovaginal samples before and after membrane rupture, using an immunoenzymatic assay. A PROM test [dipstick detecting IGFBP-1 with a cutoff value of 25 micrograms/l (A)], a BTB test (B), a ROM-Check (C), and a fern test (D) were compared in diagnosing ruptures of the membranes in 48 patients. RESULTS: The optimal cutoff concentration was between 20.1 micrograms/l and 148.4 micrograms/l by receiver operating characteristic curve analysis. The PROM test had the highest sensitivity (94.7%) and highest specificity (93.3%) (sensitivity of A-D, B-D, C-D and specificity of A-B: p < 0.05 by chi 2-test). Unlike the other tests, the PROM test is unaffected by contamination, cervical dilatation, or uterine contraction. CONCLUSIONS: The measurement of IGFBP-1 in vaginal fluids is useful for the diagnosis of ruptured fetal membranes.
The prognosis and evolutional changes of 77 patients with West syndrome (WS) were studied after patients were classified into four groups on the basis of their magnetic resonance imaging (MRI) findings: anomaly, perinatal injury, normal, and the other groups. The average age at onset of spasms was earliest in the patients with anomalies and latest in patients with normal MRI findings. Patients with normal MRI findings had the shortest duration of spasms, and patients with anomalies had the longest duration of spasms. Antecedent seizures were observed in 6 patients (3 patients with anomalies, 1 patient with normal MRI findings, and 2 patients with other abnormalities). Thirty-five patients had subsequent seizures. Patients with anomalies often had partial seizures and patients with perinatal injuries often had generalized seizures. Seizures were infrequent in patients with normal MRI findings. Developmental outcome was best in the patients with normal MRI findings and worst in patients with perinatal injuries. Various types of epileptic syndromes occurred subsequent to WS in patients with anomalies, although nonspecific symptomatic generalized epilepsy was common in patients with perinatal injuries. These results suggest that seizure prognosis, evolutional changes in seizures, and developmental outcome are different among the types of brain lesions.
The reversed elephant trunk operation has been applied in patients with extensive aortic involvement as a scheduled staged operation. We report application of the same technique in two patients with Marfan's syndrome. The two patients underwent total replacement of the thoracoabdominal aorta for a DeBakey IIIb aortic dissection. The proximal end of the prosthetic graft was invaginated to facilitate future proximal operation. No complication related to the trunk had been observed during the follow-up period.
Although several proteoglycans (PGs) have been reported in bovine periodontal ligament (PDL), the composition of PGs in PDL has been poorly characterized. In the present study, we isolated and characterized keratan sulfate-substituted PG (fibromodulin) in bovine PDL. Fibromodulin was purified from 4 M guanidine hydrochloride (GdmCl) extracts of bovine PDL tissues using DEAE Sephacel ion-exchange chromatography and preparative electrophoresis. Fibromodulin appeared as a single polydisperse band with an apparent molecular weight (MW) of 80,000 (80 kDa) on SDS-PAGE. Digestion of fibromodulin with keratanase or neuraminidase reduced the apparent molecular size, and N-glycanase treatment produced core protein bands of around 40 kDa. Fibromodulin reacted with keratan sulfate monoclonal antibody (5D4) and fibromodulin polyclonal antibodies (alpha-FM). The keratanase-digested fibromodulin reacted with alpha-FM, but not with 5D4. These data suggest that fibromodulin is one of the small PGs in the PDL-matrix and may fulfill construction and maintenance functions in this tissue.
The purpose of this study was to determine the usefulness of the hybrid capture HPV DNA assay, a new nonradioactive solution hybridization assay, as a diagnostic tool for human papillomavirus infection. In a total of 234 women, samples for the hybrid capture assay and polymerase chain reaction (PCR) assay were obtained by wiping a swab across the cervix and external os (either a Dacron swab or a cotton swab was used). The papanicolaou smear test (Pap smear) was carried out on all 234 women. Tissue samples for biopsy were obtained by colposcopy from 118 of the women. Fisher exact test was used for statistical analyses. Using the hybrid capture assay, HPV DNA of high- and intermediate-oncogenic-risk type was detected in 23 (13.9%) of 166 samples from women with Pap smear Class I or II, and 48 (70.6%) of 68 with Pap smear Class III, IV or V (p < 0.0001). The HPV DNA type was detected in 18 (29.0%) of 62 samples from those with no evidence of cervical intraepithelial neoplasia and 44 (78.6%) of 56 with cervical intraepithelial neoplasia or squamous cell carcinoma (p < 0.0001). Correlation of the test results between the hybrid capture test and PCR was determined by using the 217 samples in which both test results were available (PCR test results were not obtainable in 17 samples. When PCR is set as a gold standard, the hybrid capture test has high sensitivity (74.6%) and specificity (92.7%). These findings suggest that the hybrid capture HPV DNA assay is a useful method for diagnosing HPV infection in the clinic.
The drug-resistance patterns and plasmid profiles of 147 isolates (patient origin 142 and food origin 5 isolates) from the outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection in Obihiro-city Hokkaido in late October, 1996, were examined. Thirty-six isolates were resistant to tetracycline (TC) (24.5%), 15 of which were resistant to both streptomycin and TC. The minimal growth inhibitory concentration (MIC) of fosfomycine (FOM) was examined, confirming that MIC changed by the cultivation conditions, that is 12.5 micrograms/ml at the aerobic condition, 1.6 micrograms/ml at the anaerobic condition and 3.2 micrograms/ml on blood agar plates. Furthermore, though E. coli O157 could not be detected once by the FOM medication, FOM sensitivity of the patient origin O157 isolates who became O157-positive again was examined. Any changes in FOM sensitivity were not observed. Plasmid profiles of all isolates were divided by 4 patterns from A to D. The most dominant pattern was type A, and plasmid profiles of food origin O157 belonged to pattern A. In 9 examples of the person-to-person infection in the family, plasmid patterns of O157 isolates were the same to each other, even though drug-resistant patterns were different. In 13 patients developing the duration of excretion of EHEC, the changes of the drug-resistance patterns were correlated with the changes of plasmid profiles. By comparing plasmid profiles and TC resistance, it was suggested that TC resistance was controlled on a plasmid. Since food origin O157 isolates were sensitive to all drugs and presenting the same plasmid profiles, demonstrating that TC resistance and plasmid are newly added to the bacterial cells while food origin O157 isolates passe inside the human body.
Eradication of Helicobacter pylori infection cures gastritis and prevents recurrence of peptic ulcers. Endoscopy is usually used to evaluate the effectiveness of eradication therapy. We designed a new noninvasive assay system for the early evaluation of eradication of H. pylori infection in which a crude H. pylori outer membrane protein preparation (HPOmp) is used as an antigen, and we determined the sensitivity and specificity of the serological assay system. Immunoblot analysis showed that anti-HPOmp antibodies reacted to a protein with a molecular mass of approximately 29 kDa. In those patients who responded to therapy, the anti-HPOmp immunoglobulin G (IgG) titers measured by enzyme-linked immunosorbent assay (ELISA) at 1 month after the end of therapy were significantly lower than those before treatment (34.8% reduction; P < 0.001), and the posttreatment reduction in the antibody titer was significantly greater than that of the titer measured with a commercially available anti-H. pylori IgG ELISA (34.8% versus 16.1%; P < 0.001). When a 25% reduction of anti-HPOmp IgG titer at 1 month after the end of treatment was taken as the cutoff value for H. pylori eradication, the sensitivity and specificity of our new assay were 75% (51 of 68 treatment responders) and 96% (22 of 23 nonresponders), respectively. Our results indicate that the novel serological test with HPOmp might be a clinically useful tool for assessment of eradication of H. pylori.
Gnotobiotic mice inoculated with an enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain developed a flaccid paresis, usually culminating in death. The bacteria colonized feces at 10(9) to 10(10) CFU per g (inoculum size: 2.0 x 10(9) CFU/mouse), and Shiga-like toxins (SLTs) were detected in the feces. A microscopic examination of colons showed mild inflammatory cell infiltration, thinning of the intestinal wall, or necrotic foci. Necrosis of tubular cells was noted in these symptomatic mice. Microhemorrhage, thrombosis, and edematous changes of the brain were also seen. Inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), and IL-6, were detected in the kidney after EHEC infection, but not in the serum. In the brain, only TNF-alpha was detected. When 2.0 x 10(2) CFU of EHEC O157:H7 was fed to germ-free mice, the number of bacteria began to rise rapidly on day 1 and was maintained at 10(8)to 10(9) CFU/g of feces. SLTs were detected in the feces of the mice. However, the mice showed no histological changes and no cytokine responses, similar to what was found for controls. Treatment with TNF-alpha modified the clinical neural signs, histopathological changes, and cytokine responses; mice treated with TNF-alpha developed severe neurotoxic symptoms and had higher frequencies of systemic symptoms and glomerular pathology. Strong cytokine responses were seen in the kidney and brain. Serum cytokines were also detected in this group. In contrast, a TNF-alpha inhibitor (protease inhibitor) inhibited these responses, especially in the brain. However, local synthesis of the cytokines was observed in the kidney. Thus, TNF-alpha and the other proinflammatory cytokines could be important in modifying the disease caused by EHEC.
We have examined the antibody response to Helicobacter pylori lipopolysaccharides (LPS) in humans. We used sera from patients with gastroduodenal diseases and healthy adults infected or not infected with H. pylori. Data from the experiments for antibody binding to LPS suggested that the polysaccharide chains from many H. pylori strains showed high immunogenicity in humans. Sera from most (above 70%) H. pylori-infected individuals contained immunoglobulin G (IgG) antibodies against the polysaccharide region highly immunogenic H. pylori LPS. The IgG titers of individual serum samples that reacted strongly with highly immunogenic LPS were quite similar (r2 = 0.84 to 0.98). The results suggest wide distribution among H. pylori strains of a highly antigenic epitope in the polysaccharide moieties of their LPS. Also, the similarity in the titers of individual serum samples against highly immunogenic LPS points to the existence of epitopes sharing a common structural motif. However, some strains showed low antigenicity, even those with polysaccharide-carrying LPS. The dominant subclass of IgG that reacted with the highly immunogenic LPS was IgG2, which was preferentially raised against polysaccharide antigens. Recently, a structure that mimics that of the Lewis antigens was identified in the O-polysaccharide fraction of H. pylori LPS; however, no correlation between antigenicity of the polysaccharide chain in humans and the presence of Lewis antigens was found. The IgA and IgM titers against H. pylori LPS seemed to be mostly nonspecific and directed against lipid A. In a few cases, however, sera from individuals infected with H. pylori gave strong IgA and IgM titers against the highly immunogenic polysaccharide. In conclusion, the LPS of many H. pylori strains possess an antigenic epitope in their polysaccharide regions that is immunogenic in humans. However, our results show that the antigenic epitope is unlikely to be immunologically related to structures mimicking Lewis antigens.
Autoantibodies reactive with double-stranded (ds) DNA are a hallmark of systemic lupus erythematosus (SLE), but it is not fully elucidated how these antibodies contribute to the various pathological changes observed in lupus patients. In a previous study, we have found a significant enhancement effect of two murine monoclonal anti-dsDNA antibodies on DNA cleavage by the hydroxyl radical. We have extended this study and have found that purified polyclonal anti-dsDNA antibodies from sera of lupus patients and an MRL/lpr mouse, but not anti-ssDNA antibodies, also enhanced radical cleavage of DNA. The cleavage was inhibited by EDTA, DMSO and thiourea. The antibody preparations per se did not cleave DNA. These results suggest that such an enhancement effect on DNA cleavage is a feature of a significant part of anti-dsDNA antibody population and a possible clue in understanding the roles of anti-dsDNA antibodies in the pathophysiology of SLE under certain circumstances.
Uncoupling protein 2 (UCP2), a member of the family of mitochondrial carrier proteins, has been implicated in the control of whole-body energy balance. The coding region of the human UCP2 gene has now been shown to comprise six exons, and the sequences of the exon-intron boundaries were determined. With the use of this sequence information, 25 Japanese patients with obesity and noninsulin-dependent diabetes mellitus (NIDDM) and 25 subjects with simple obesity were screened for mutations in the entire coding region of UCP2 by PCR and single-strand conformation polymorphism analysis. Two nucleotide polymorphisms resulting in Ala55 --> Val and Ala232 --> Thr substitutions were detected. With the use of PCR and restriction fragment length polymorphism analysis, the allele frequencies for each of these polymorphisms were determined in 210 Japanese patients with NIDDM, 42 obese individuals, and 218 normal control subjects. The frequency of the Val55 allele did not differ significantly among the NIDDM group (46.0%), the obesity group (48.8%), and the normal control group (48.4%). The Thr232 allele was detected in only three subjects, who were heterozygotes and in the NIDDM group (allele frequency, 0.7%). However, expression in yeast of the human wild-type UCP2 protein and UCP2 containing Thr232 revealed no difference in functional activity. These results indicate that the Ala55 --> Val and Ala232 --> Thr variants of UCP2 do not play an important role in the pathogenesis of NIDDM or obesity in the Japanese population.
Heparin inhibited hemagglutination (HA) by equine arteritis virus (EAV) as well as did HA by Aujeszky's disease virus (ADV), but failed to inhibit HA by parainfluenza virus type 3 (PIV-3). The minimal concentration of heparin required to inhibit 8 HA U of EAV was 0.1 U/ml. In addition, most EAV hemagglutinin was retained by heparin acrylic beads, as was ADV hemagglutinin, but was not PIV-3 hemagglutinin. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. However, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by EAV. All these findings suggest that a heparin-like molecule on the surface of mouse erythrocytes serves as the virus-cell receptor.
The aim of this study was to investigate the influence of the nitric oxide/nitric oxide synthase (NO/NOS) system on the release of endothelin-1 (ET-1) in human endometrial cells. Human endometrial stromal cells in secretory phase were incubated for 72 h in serum-free RPMI 1640 medium in the absence or presence of different concentrations of interleukin-1beta (IL-1beta) and NG-monomethyl-L-arginine (LNMMA), a specific competitive inhibitor of NOS. ET-1 released from the cultured cells into the medium was determined by specific RIA. In all the experiments at various times, IL-1beta significantly increased the release of ET-1. LNMMA significantly attenuated the release of ET-1 when the cells were cultured with both IL-1beta and LNMMA, but LNMMA alone had no effect on ET-1 release. These results suggest that the NO/NOS system in human endometrium is involved in the regulation of ET-1 release via IL-1beta secretion. It can also be inferred that NO and ET-1 control the functions of endometrium in close association with IL-1beta.
We report a case of fungus ball due to Pseudallescheria boydii (pseudallescherioma) associated with systemic lupus erythematosus. Direct microscopical examination revealed a fungus with broad septate hyphae resembling Aspergillus and the fungus was identified as P. boydii on culture. Surgical resection was required to control episodes of hemoptysis. Cases of pulmonary pseudallescheriasis are rare, especially in Japan. However, some cases previously diagnosed as pulmonary aspergillosis may have been found to be caused by P. boydii, if adequate culture studies had been conducted. Unlike Aspergillus, P. boydii is resistant to amphotericin B. Therefore, we emphasize the importance of a correct diagnosis based on culture examination.
The present study investigated basolateral K+ channels and their pH-sensitivity in isolated bullfrog proximal tubule cells by using the patch-clamp technique, and compared channel activity with the basolateral membrane potential (EM) and intracellular pH (pHi) monitored by using double-barreled H+-selective microelectrodes in perfused bullfrog proximal tubules. In the patch-clamp experiments, K+ channels with inward slope conductance of about 50 pS were observed in the basolateral membrane of isolated proximal tubule cells in cell-attached patches. Raising pH of the bath with HCO3(-)-free HEPES Ringer solutions from 7.7 (control) to 8.2 in the presence of an H+ ionophore, FCCP (2 micro M), enhanced channel activity to 126.4% of controls; lowering bath pH to 7.2 and to 6.7 reduced channel activity to 26.4 and to 1.7% of controls. Microelectrode experiments in bullfrog proximal tubules perfused with HCO3(-) -free HEPES Ringer solutions showed that EM and pHi in control conditions of peritubular pH 7.7 without FCCP were -52.6 mV and 7.45. Raising peritubular pH to 8.2 in the presence of FCCP (2 micro M) increased EM and pHi to -61.8 mV and 7.73; lowering it to 7. 2 and to 6.7 decreased EM and pHi to -28.6 mV and 7.25 and to -10.6 mV and 6.95. These results suggest that changes in EM in response to cellular alkalinization or acidification made by HCO3(-)-free HEPES solutions are produced primarily by changes in activity of the pH-sensitive K+ channels.
The inwardly rectifying K+ channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cell is activated by cyclic AMP-dependent protein kinase (PKA). In this study, we further examined the involvement of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent process in modulation of this K+ channel by using the patch-clamp technique. In cell-attached patches, channel activity was increased by the application of either N2, 2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (DBcGMP, 100 microM) or 8-bromoguanosine 3',5'-cyclic monophosphate (8BrcGMP, 100 microM), and it was inhibited by KT5823 (10 microM), a membrane-permeable specific inhibitor of cGMP-dependent protein kinase (PKG). The effect of DBcGMP on channel activity was abolished by the pretreatment of cells with KT5823 (10 microM), but it was observed in the presence of KT5720 (200 nM), a specific inhibitor of PKA. Furthermore, atrial natriuretic peptide (ANP, 10 nM) increased channel activity, which was also prevented by the application of KT5823 (10 microM). In inside-out patches, ATP (3 mM) was required to maintain channel activity, which was inhibited by KT5823 (10 microM), but it was not increased by cGMP (100 microM) alone. The channel activity was increased by the coapplication of PKG (500 U/ml) and cGMP (100 microM). These results suggest that cGMP activates the inwardly rectifying K+ channel in OKP cells through PKG-mediated phosphorylation processes independent of PKA-mediated processes, and that ANP is an agonist which stimulates PKG-mediated processes in the proximal tubule cell. Furthermore, it is suggested that the ATP-dependent channel activity in inside-out patches is maintained at least in part by PKG, which is the membrane-bound catalytic domain.
The differential display technique was applied to observe the molecular dynamism of messenger ribonucleic acid (mRNA) in this study. Using this technique, the changes in mRNAs in brain perturbation such as ischemia was observed to understand the molecular base of the reaction. The transient cerebral ischemia was induced by clipping both common carotid arteries for 5 minutes in Mongolian gerbils. The total RNA was extracted from the hippocampal tissue samples before ischemia, 6 hours and 2 days after ischemia. The mRNAs were reverse transcribed and subsequently amplified by polymerase chain reaction (PCR). PCR products were displayed by autoradiography as ladders on a denaturing polyacrylamide gel. According to the autoradiography, mRNAs were divided into three patterns: 1) mRNAs obtained from the control decreased at 6 hours after 5-minute ischemia and disappeared at 2 days completely; 2) decreased mRNAs at 6 hours after ischemia recovered at 2 days; and 3) new mRNAs appeared after cerebral ischemia. Located bands of interest on a gel were cut out and reamplification of complementary deoxyribonucleic acid was performed. The pGEM-T Vector System was used for subcloning of the amplified PCR products. The differential display technique is the powerful method for detecting genes that are unique to ischemic processes and reactions.