Noncompetitive immunoassays for small molecules with high sensitivity and specificity.
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Amino Acid Sequence↗
Biomedical subjects
N Kobayashi
Publications and source records attributed to N Kobayashi.
Inhibition of nuclear export of ribonucleoprotein complexes of influenza virus by leptomycin B.
We have studied nuclear export of influenza virus components using an in vitro transport system with digitonin-treated infected cells. We first monitored the efficiency of export of the viral ribonucleoprotein (vRNP) complex by analyzing viral components with western blotting. We used leptomycin B (LMB), an inhibitor of nuclear export signal (NES)-and its receptor, CRM1/Exportin1-mediated protein export. LMB efficiently inhibited vRNP export, while it did not affect the subcellular localization and export of matrix protein (M) 1 and nonstructural protein (NS) 2. Second, indirect immunofluorescence assays also revealed that vRNP export is sensitive to LMB. NS2 in NS2-transfected cells was not accumulated in nuclei in the presence of LMB, while NS2 in infected cells was found slightly accumulated in nuclei in the presence of LMB. Finally, we performed in vitro RNA synthesis assays using digitonin-treated infected cells and exported fractions. The exported vRNP was RNA synthesis-competent. Analyses using glycerol density gradients showed that a major fraction of M1 and NS2 was not complexed with the exported vRNP. These results suggest that nuclear export of RNA synthesis-competent vRNP is dependent on a LMB-sensitive pathway and that there would be two types of NS2, i.e. LMB-sensitive and -insensitive NS2. The involvement of viral late proteins in vRNP export during late stages of infection is discussed.
Active Transport, Cell Nucleus↗
Virtual MR microscopy for unruptured aneurysm.
PURPOSE: This study was performed to evaluate the usefulness of virtual magnetic resonance microscopy (VMRM) for the diagnosis of cerebral aneurysms. MATERIALS AND METHODS: We reviewed 11 patients with unruptured aneurysms confirmed by angiography or surgical therapy. We evaluated the ability of VMRM to represent aneurysms and the findings of the form and neck of the aneurysms. RESULTS: VMRM revealed 17 aneurysms, one of which was not detected by MR angiography (MRA). One suspected aneurysm by MRA was denied by VMRM. Although VMRM did not clearly demonstrate either one giant aneurysm or distal middle cerebral artery, two aneurysms in the cavernous sinus were clearly visualized by VMRM. VMRM seems to be almost equivalent to computed tomography angiography when detecting aneurysms without additional radiation exposure. CONCLUSION: VMRM is found valuable not only to plan the microscopic surgical therapy, but to visualize aneurysms.
Aged↗
Analysis on distribution of insertion sequence IS431 in clinical isolates of staphylococci.
Distribution of insertion sequence IS431 in clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus was investigated. Except for methicillin susceptible-S. aureus (MSSA), IS431 was detected in all isolates of the three staphylococcal species. In MSSA, only 20% of isolates with distinct coagulase types and genetic types possessed IS431. In a few MSSA isolates, an IS431 variant with internal deletion was found.
Base Sequence↗
Effects of quinapril on expression of eNOS, ACE, and AT1 receptor in deoxycorticosterone acetate-salt hypertensive rats.
Angiotensin II and nitric oxide (NO) may play a role in hypertensive cardiovascular remodeling. We evaluated the effects of long-term treatment with quinapril, an angiotensin converting enzyme (ACE) inhibitor, on expression of endothelial NO synthase (eNOS), ACE, and angiotensin II type 1 (AT1) receptor in the left ventricle and evaluated these relations to myocardial remodeling in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Deoxycorticosterone acetate-salt rats were induced with weekly injections of DOCA (30 mg/kg) and 1% saline in drinking water after right nephrectomy. Quinapril (DOCA-QUI, 10 mg/kg/day, subdepressor dose) or AT1 receptor antagonist TCV-116 (DOCA-TCV, 5 mg/kg/day, subdepressor dose) or vehicle (DOCA-V) were given after induction of DOCA-salt hypertension for 5 weeks, and age-matched sham-operated rats (ShC) served as a control group. The eNOS expression in the left ventricle were significantly decreased in DOCA-V compared with ShC, and were significantly increased in DOCA-QUI and DOCA-TCV compared with ShC and DOCA-V. The gene expression of ACE, AT1 receptor, and type I collagen mRNA were significantly increased in DOCA-V compared with ShC, and significantly suppressed in DOCA-QUI compared with DOCA-V. The DOCA-V rats demonstrated a significant increase of the wall-to-lumen ratio, perivascular fibrosis, and myocardial fibrosis, with all these parameters being significantly improved by quinapril. Myocardial remodeling in DOCA-salt hypertensive rats was significantly ameliorated by a subdepressor dose of quinapril, which may be due to an increase in eNOS mRNA and protein expression and a decrease in ACE and AT1 receptor mRNA expression in the left ventricle.
Angiotensin Receptor Antagonists↗
Suppression of allergic reactions by royal jelly in association with the restoration of macrophage function and the improvement of Th1/Th2 cell responses.
We studied the immunomodulatory effects of royal jelly (RJ), the principal food source of the queen honeybee. In this study, suppression of allergic reactions by RJ was investigated in DNP-KLH immunized mice (DNP-KLH mice). Oral administration of RJ (1 g/kg) to DNP-KLH mice significantly decreased the serum levels of antigen-specific Ig E and significantly inhibited DNP-KLH mediated-histamine release from mast cells, resulting in the suppression of immediate hypersensitivity reactions of ear skin. In DNP-KLH mice, IFN-gamma (Th1 cytokine) production from CD4+ T cells was suppressed and IL-4 (Th2 cytokine) production from CD4+ T cells was increased as compared to normal mice. On the other hand, RJ improved the balance of Th1/Th2 cell responses from Th2-dominant to Th1-dominant. RJ significantly increased GSH levels in macrophages from DNP-KLH mice. In addition, the administration of RJ to DNP-KLH mice increased IL-12 p40 mRNA expression and NO production, and decreased PG E2 production from macrophages as compared to untreated DNP-KLH mice. These results suggested that RJ suppressed antigen-specific Ig E production and histamine release from mast cells in association with the restoration of macrophage function and improvement of Th1/Th2 cell responses in DNP-KLH mice.
Adjuvants, Immunologic↗
Distribution of aminoglycoside resistance genes in recent clinical isolates of Enterococcus faecalis, Enterococcus faecium and Enterococcus avium.
Aminoglycoside modifying enzymes (AMEs) are major factors which confer aminoglycoside resistance on bacteria. Distribution of genes encoding seven AMEs was investigated by multiplex PCR for 279 recent clinical isolates of enterococci derived from a university hospital in Japan. The aac(6')-aph(2"), which is related to high level gentamicin resistance, was detected at higher frequency in Enterococcus faecalis (42.5%) than in Enterococcus faecium (4.3%). Almost half of E. faecalis and E. faecium isolates possessed ant(6)-Ia and aph(3')-IIIa. The profile of AME gene(s) detected most frequently in individual strains of E. faecalis was aac(6')aph(2") + ant(6)-Ia + aph(3')-IIIa, and isolates with this profile showed high level resistance to both gentamicin and streptomycin. In contrast, AME gene profiles of aac(6')-Ii+ ant(6)-Ia+aph(3')-IIIa, followed by aac(6')-Ii alone, were predominant in E. faecium. Only one AME gene profile of ant(6)-Ia+aph(3')-IIIa was found in Enterococcus avium. The ant(4')-Ia and ant(9)-Ia, which have been known to be distributed mostly among Staphylococcus aureus strains, were detected in a few enterococcal strains. An AME gene aph(2")-Ic was not detected in any isolates of the three enterococcal species. These findings indicated a variety of distribution profiles of AME genes among enterococci in our study site.
Aminoglycosides↗
Herpes simplex virus in the vestibular ganglion and the geniculate ganglion-role of loose myelin.
This study presents the first direct evidence for herpes simplex virus type 1 (HSV-1) infection in the neurons of the vestibular ganglion. Although many investigators have reported electron microscopic evidence of HSV-1 infection in sensory ganglia, HSV-1 infection in the vestibular ganglion has not been described. Vestibular ganglion neurons have a unique structure, with a loose myelin sheath instead of the satellite cell sheath that is seen in other ganglia. This loose myelin is slightly different from compact myelin which is known as too tight for HSV-1 to penetrate. The role of loose myelin in terms of HSV-1 infection is completely unknown. Therefore, in an attempt to evaluate the role of loose myelin in HSV-1 infection, we looked for HSV-1 particles, or any effects mediated by HSV-1, in the vestibular ganglion as compared with the geniculate ganglion. At the light microscopic level, some neurons with vacuolar changes were observed, mainly in the distal portion of the vestibular ganglion where the communicating branch from the geniculate ganglion enters. At the electron microscopic level, vacuoles, dilated rough endoplasmic reticulum and Golgi vesicles occupied by virus were observed in both ganglia neurons. In contrast, viral infections in Schwann and satellite cells were observed only in the geniculate ganglion, but not in the vestibular ganglion. These results suggest that loose myelin is an important barrier to HSV-1 infection, and it must play an important role in the prevention of viral spread from infected neurons to other cells.
Animals↗
The DSL domain in mutant JAG1 ligand is essential for the severity of the liver defect in Alagille syndrome.
Alagille syndrome (AGS) is a congenital multi-system anomaly mainly characterized by paucity of intrahepatic bile ducts caused by haploinsufficiency of the Jagged 1 gene (JAG1). To explore the relationship between genotype and phenotype, we analyzed the JAG1 gene in 25 Japanese AGS families at the genomic DNA level and identified 15 point mutations and one large deletion. Analysis of the genotype and phenotype strongly indicated that the Delta/Serrate/Lag-2 (DSL) domain in JAG1 protein played an essential role in determining the severity of the liver disorder. In four sporadic cases, missing an entire DSL domain in mutant JAG1 resulted in progressive liver failure and all 4 patients needed a liver transplant at a very young age. This correlation was further confirmed by statistical analysis (chi2=9.143, p<0.001). Our finding demonstrated that the DSL domain in JAG1 appears to be essential for normal liver development and function.
Alagille Syndrome↗
Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology.
The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scanning confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.
Animals↗
Paediatric absorbed doses from rotational panoramic radiography.
OBJECTIVES: To determine the paediatric doses in rotational panoramic radiography with film/screen and photostimulable phosphor receptors. STUDY DESIGN: A paediatric anthropomorphic head and neck phantom was used. Absorbed doses were measured for two panoramic systems, the Orthophos (Sirona Dental Systems, Bensheim, Germany) and the PM 2002 CC (Planmeca Oy, Helsinki, Finland), with and without programmable child settings, using both screen/film and photostimulable phosphor receptors. RESULTS: Absorbed doses to the eye ranged from 5 to 24 micro Gy. Doses to the dental arches with the Orthophos unit ranged from 50 to 555 micro Gy with the adult and from 27 to 436 micro Gy with the child program; using the PM 2002 CC unit, doses ranged from 56 to 1040 micro Gy using the adult settings, and from 60 to 890 micro Gy with the paediatric settings. The paediatric exposure settings reduced doses at most locations for both panoramic systems. The highest doses were measured near the rotational axes of the X-ray beam. Paediatric settings with the Orthophos P10 resulted in the dose reduction more than 50% to the thyroid but not with the PM 2002 CC. When lower kVcp or mA settings were used, absorbed doses were effectively reduced for all combinations of machines, programs and detectors. CONCLUSIONS: Specific program settings for children reduced the absorbed doses from panoramic radiography irrespective of the machine or receptor used.
Absorption↗
In situ visualization of ultraviolet-light-induced DNA damage repair in locally irradiated human fibroblasts.
We have developed a novel method that uses a microfilter mask to produce ultraviolet-induced DNA lesions in localized areas of the cell nucleus. This technique allows us to visualize localized DNA repair in situ using immunologic probes. Two major types of DNA photoproducts [cyclobutane pyrimidine dimers and (6-4) photoproducts] were indeed detected in several foci per nucleus in normal human fibroblasts. They were repaired at those localized sites at different speeds, indicating that DNA photoproducts remain in relatively fixed nuclear positions during repair. A nucleotide excision repair protein, proliferating cell nuclear antigen, was recruited to the sites of DNA damage within 30 min after ultraviolet exposure. The level of proliferating cell nuclear antigen varied with DNA repair activity and diminished within 24 h. In contrast, almost no proliferating cell nuclear antigen fluorescence was observed within 3 h in xeroderma pigmentosum fibroblasts, which could not repair either type of photolesion. These results demonstrate that this technique is useful for visualizing the normal nucleotide excision repair process in vivo. Interestingly, however, in xeroderma pigmentosum cells, proliferating cell nuclear antigen appeared at ultraviolet damage sites after a delay and persisted as late as 72 h after ultraviolet exposure. This result suggests that this technique is also valuable for examining an incomplete or stalled nucleotide excision repair process caused by the lack of a single functional nucleotide excision repair protein. Thus, the technique provides a powerful approach to understanding the temporal and spatial interactions between DNA damage and damage-binding proteins in vivo.
Cell Line↗
Expression of cell cycle regulatory proteins in the multistep process of oesophageal carcinogenesis: stepwise over-expression of cyclin E and p53, reduction of p21(WAF1/CIP1) and dysregulation of cyclin D1 and p27(KIP1).
AIMS: Cell cycle regulatory proteins were analysed by immunohistochemistry in order to clarify how their expression changes with the degree of atypia as oesophageal surface squamous epithelium progresses from normal mucosa, through reactive change, low-grade dysplasia, and high-grade dysplasia to mucosal invasive carcinoma. METHODS AND RESULTS: Immunostaining for cyclin D1, cyclin E, p21, p27, p53 and Ki67 proteins was performed using 22 normal mucosa, 17 reactive change, 22 low-grade dysplasia, 15 high-grade dysplasia and 22 mucosal invasive carcinoma specimens. Normal mucosa, low-grade dysplasia and high-grade dysplasia samples were taken from patients without any oesophageal invasive carcinoma by endoscopic biopsy or endoscopic mucosal resection, and reactive change and mucosal invasive carcinoma were obtained from oesophagectomy material. Stepwise over-expression of cyclin E (P < 0.0001) and p53 (P < 0.0001), reduction of p21 (P=0.0189) and dysregulation of cyclin D1 and p27 were observed in the multistep process of oesophageal carcinogenesis. Significant differences in expression of p27 (P < 0.0001), p53 (P=0.0299) and Ki67 (P=0.0101) were observed between reactive change and low-grade dysplasia. Furthermore, expression of cyclin D1, cyclin E, p27 and p53 in mucosal invasive carcinoma were significantly different from those in high-grade dysplasia (P=0.0079, P=0.0237, P=0.0042 and P= 0.0299, respectively). CONCLUSIONS: Cell cycle regulatory proteins, cyclin E, p53 and p21 show stepwise over-expression or reduction with progression of oesophageal carcinogenesis, correlating with the increased cell proliferation observed with Ki67 labelling. We conclude that immunohistochemical analysis for p27, p53 and Ki67 is practically useful for the discrimination between low-grade dysplasia and reactive change. Cyclin D1, cyclin E, p27 and p53 help to distinguish high-grade dysplasia from mucosal invasive carcinoma.
Cell Cycle Proteins↗
Plasma brain natriuretic peptide levels in chronic hemodialysis patients: influence of coronary artery disease.
A noninvasive biochemical testing method for early detection and monitoring the condition of cardiac complications in hemodialysis (HD) patients would be useful and might lead to improved survival. The aim of this study is to clarify the pathophysiological significance of plasma brain natriuretic peptide (BNP) levels in HD patients with and without coronary artery disease (CAD). We measured plasma atrial natriuretic peptide (ANP) and BNP levels on Monday, Wednesday, and Friday before and after HD in 28 consecutive patients who underwent HD three times weekly. In addition, we measured plasma ANP and BNP levels in 21 HD patients with CAD and 27 HD patients without CAD and studied the relationships between BNP levels and cardiac function and clinical variables. Plasma ANP levels significantly decreased after HD on Monday, Wednesday, and Friday, and predialysis plasma ANP levels on Monday were significantly greater than those on other days. Plasma BNP levels did not change after HD on Monday; however, they significantly decreased after HD on Wednesday and FRIDAY: Predialysis plasma BNP levels on Monday were greater than those on other days, and postdialysis plasma BNP levels on Monday were greater than predialysis plasma BNP levels on WEDNESDAY: Plasma BNP levels in HD patients with CAD were significantly greater than those in HD patients without CAD and significantly correlated with left ventricular (LV) ejection fraction (r = -0.69), end-diastolic volume index (r = 0.59), and end-systolic volume index (r = 0.84) determined by left ventriculography. Conversely, plasma BNP levels in HD patients without CAD significantly correlated with LV mass index (r = 0.54) determined by echocardiography and mean systolic blood pressure (r = 0.72) determined by 48-hour ambulatory blood pressure monitoring. These results suggest the following: (1) plasma BNP levels before and after HD in chronic HD patients directly correlate with the degree of body fluid retention, and the day of the week on which the sample is obtained should be considered for its evaluation; (2) plasma BNP levels reflect LV function in HD patients with CAD; and (3) plasma BNP levels reflect LV mass and blood pressure in HD patients without CAD.
Adult↗
Transfusion transmitted virus infection in patients on maintenance haemodialysis and in hospital workers.
A newly discovered DNA virus, transfusion transmitted virus (TTV), was isolated from a post-transfusional hepatitis patient in Japan. A high prevalence (32-46%) of TTV infections in patients receiving maintenance haemodialysis (HD) has been reported but the occupational risk of TTV on HD units has not yet been determined. We determined the prevalence of TTV in workers in the same HD unit and the risk factors for TTV infection in HD patients, using logistic regression analysis. The prevalence of TTV DNA was 59.6% in 198 HD patients, significantly higher than that in the HD unit (13 of 39, 33.3%;P= 0.002) and non-HD healthcare workers (20 of 75, 26.7%; P= 0.001). A logistic regression analysis showed that male gender and negative test results for hepatitis G virus RNA were risk factors for TTV infection, but prior blood transfusion and duration of HD were not. Stepwise selection of multiple regression analysis showed that the presence of hepatitis C virus RNA was the only significant predictor for high serum ALT activity, and that the presence of TTV DNA was not. These results indicate that TTV is one of the prevalent human viruses transmissible either parenterally or nonparenterally in HD patients, but the occupational risk of TTV infection in HD unit workers is as low as in other healthcare workers. The pathogenic effects of TTV on the liver appear to be limited.
Adult↗
Neuropathy of motor branch of median or ulnar nerve induced by midpalm ganglion.
Two cases of neuropathy of a motor branch caused by a midpalmal ganglion are presented. In the first case the ganglion originated from the midcarpal joint, protruded into the thenar muscle, and compressed the motor branch of the median nerve. In the second case the ganglion, distal to the fibrous arch of the hypothenar muscles, originated from the third carpometacarpal joint and compressed the motor branch of the ulnar nerve. In both cases muscle weakness and finger deformity recovered well after resection of the ganglion. This clinical condition is rare compared with carpal tunnel syndrome and Guyon's tunnel syndrome, which are caused by a ganglion in the wrist.
Adult↗
Endoscopic treatment of hydrocephalus in children: a controlled study using newly developed Yamadori-type ventriculoscopes.
Although cerebrospinal fluid (CSF) shunting is the most common neurosurgical treatment for hydrocephalus, the long-term results have still been unsatisfactory because of a wide variety of shunt complications. We have recently developed flexible ventriculoscopes (Yamadori-type) which have excellent image quality, maneuverability, and capabilities for endoscopic operation. Here we report the efficacy of the new treatment in 88 children with hydrocephalus who initially underwent either ventriculoscopic operation or shunting surgery. The primary outcome measures were the rate of shunt independency and/or shunt complications with a follow-up of 2 years in each group. We performed endoscopic third ventriculostomy in cases of aqueductal stenosis, cyst fenestration, and choroid plexus coagulation in limited cases of communicating hydrocephalus. Overall, thirty-three (75%) of the 44 children initially treated endoscopically did not require ventriculoperitoneal (VP) shunts. The endoscopic procedures were repeated in the remaining 11 children (25%) mostly less than 1-year-old who ultimately required endoscope-guided VP shunting. Even in such patients, there was virtually no need for shunt revisions and no major complications such as slit-like ventricle, meningitis, and intraventricular hemorrhage. These results were statistically highly significant (p < 0.0001) compared to a control group of 44 patients treated initially by VP shunting. Our data demonstrate that therapeutic ventriculoscopy is safe and clinically effective as the first-line treatment of hydrocephalus in children.
Brain↗
Intraventricular hemorrhage in neonates: endoscopic findings and treatment by the use of our newly developed Yamadori-type 8 ventriculoscope.
Profound intraventricular hemorrhage (IVH) has devastating effects on neonates, leading to significant neurodevelopmental morbidity. We have developed a flexible fiberoptic ventriculoscope (Yamadori-type 8), which has a conspicuously high resolving power and is equipped with a bipolar coagulator and a working channel for irrigation. Four neonates with IVH were examined and treated for the first time using the flexible ventriculoscope. The patients had grade III or IV IVH on computed tomography scans and received endoscopic operations three to four weeks after birth. In patients with IVH, fragments of old hematomas were observed as brown masses because of the presence of hemosiderin, a characteristic endoscopic finding. We have found, for the first time, IVH due to rupture of the varices of septal veins, which could be cauterized endoscopically. Hydrocephalus was relieved by irrigation, third ventriculostomy, coagulation of the choroid plexus, and adequate placement of ventriculoperitoneal shunting was performed endoscopically. The flexible ventriculofiberscope could be a useful modality for early treatment of IVH in neonates because of the minimal invasiveness. The pathogenesis of IVH should be reviewed to include septal vein varices as a possible cause of IVH.
Cerebral Hemorrhage↗