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Biomedical subjects

N Otsuka

Publications and source records attributed to N Otsuka.

At least 73 records · Page 4Linked to original sources
PubMed1994

[Detection of methicillin-resistant Staphylococcus aureus using PCR and non-radioactive DNA probes: III. Mutations of the fem A gene in clinical strains of Staphylococcus aureus].

Methicillin resistance in S. aureus is primarily due to the presence of the mec A gene. However, in addition to mec, the phenotypic expression of methicillin resistance requires the presence of an additional gene(s), fem A which is chromosomally encoded. Previous studies suggest an increase in the biochemical function of fem A gene products due to base substitutions in the region upstream of the fem A gene and in its coding frame. The partial nucleotide sequences of fem A regions in reference and clinical strains of S.aureus were therefore analyzed by PCR-direct solid-phase sequencing and suitable DNA probes. Amplified target DNAs of 251, 330 and 271 bp were resolved on ethidium bromide-stained gels and hybridized with DNA probes conjugated to alkaline phosphatase. In ATCC 12600 strain, a palindromic sequence was conserved in the region upstream of fem A. However, it was destroyed by the occurrence of mutations in other reference, and clinical strains tested regardless of whether they are methicillin-susceptible or resistant. Furthermore, in the coding frame of fem A, two missense mutations were present in MSSA and MRSA without any regularity. These findings suggest that mutations in the fem A region may not be a single factor essential for regulation of methicillin resistance, although fem A probably functions cooperatively with mec A.

Base Sequence↗
PubMed1994

[A case of membranoproliferative glomerulonephritis due to type II cryoglobulinemia probably associated with hepatitis C virus infection].

In recent years, several laboratories have suggested that chronic hepatitis C virus (HCV) infection is strongly associated with type II cryoglobulinemia (CG) and/or membranoproliferative glomerulonephritis (MPGN). We report here a case of MPGN due to type II CG probably associated with chronic HCV infection, and discuss the pathogenesis and treatment of such cases. A 60-year-old-female was referred to us from a local hospital because of progressive peripheral edema, purpura on the lower limbs, pleural effusion, ascites, hypertension, and renal failure. Laboratory findings indicated proteinuria, abnormal urinary sediments, normochromic normocytic anemia and azotemia. Other laboratory findings included positive rheumatoid factor, elevated serum IgM, hypocomplementemia and elevated circulating immune complexes. Cryoglobulin was detected and found to consist of a mixture of a monoclonal IgM kappa with polyclonal IgG. Renal biopsy showed MPGN. These observations suggested a close association between MPGN and type II CG. We did not find any causes of type II CG except for positive HCV antibody and HCV RNA. Therefore, we made the diagnosis of type II CG associated with chronic HCV infection. Symptoms related to CG was responsiveness to steroid, but development of liver dysfunction developed. Treatment with alfa-interferon (alpha IFN) was added and thereafter, the liver dysfunction improved. However, the serum Cryo level was not reproducibly lowered. While in this case it was unclear whether IFN therapy was beneficial, several reports in addition to the findings of this case suggest a close relation between HCV infection and type II CG and MPGN.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryoglobulinemia↗
PubMed1993

[Detection and identification of HBV DNA and DNA subtypes of HBs-antigen by the use of the polymerase chain reaction and non-radioactive probes].

A technique of nested polymerase chain reaction (PCR) and hybridization with non-radioactive probes was developed for the detection and identification of HBV DNA and HBs-subtypes in very small volumes of human sera. Four oligonucleotide primers (20 mer) complementary to DNA sequences in the S region of HBV and probes (18 or 20 mer) conjugated to alkaline phosphatase were used for the present PCR assay. The results of the PCR assay coincide with those of enzyme immunoassay (EIA) in 14 HBe-positive and 59 HBe-negative samples with 98.6% of specificity. The HBV subtypes adr and adw were identified using an 18-mer DNA probe in 30 samples with an accuracy of 100%. Further, the DNA subtypes were clearly demonstrated in 3 samples where HBs-antigen was undetectable. These results indicate that amplification of HBV DNAs by PCR and their detection with non-radioactive probes is a reliable tool for diagnosis of HBV infection in clinical laboratories.

Base Sequence↗
PubMed1993

[Detection of methicillin-resistant Staphylococcus aureus using PCR and non-radioactive DNA probes (II)].

Methicillin resistance in staphylococci is primarily due to the presence of a mec A gene which encodes the novel penicillin-binding protein 2'. Some chromosomal factors, fem A and fem B, also participate in the expression of methicillin resistance in S. aureus. This study was designed to detect mec A, fem A and fem B genes for identification of staphylococcal species and for discrimination of methicillin-resistant cells. Three different pairs of DNA primers (PBP2'AF-PBP2'AR, fem AF-fem AR and fem BF-fem BR) complementary to unique regions of mec A, fem A and fem B genes were synthesized for use in polymerase chain reaction with DNAs of methicillin-sensitive S. aureus (MSSA), S. epidermidis, methicillin-resistant S. aureus (MRSA) and S. epidermidis. Amplified target DNAs of 630, 509, and 651 bp were resolved on ethidium bromide-stained gel, and hybridized to DNA probes conjugated to alkaline phosphatase. When applied to pure cultures on the MRSA screen agar, all three DNA probes tested detected MRSA in 47 of 61 culture-positive specimens (77.1%); the detection ratio of MRSA with mec A and either fem A or fem B probes was increased to 95.9%. By contrast, the fem A and fem B probes did not detect S. epidermidis. The result of detecting these species streaked on mannitol-salt agar were similar, while the detection of MSSA with the fem A and fem B probes was incomplete irrespective of the presence or absence of mec A. These findings suggest a good correlation between cultivation and DNA probe assay with respect to MRSA detection.

DNA Probes↗
PubMed1992

Paired helical filaments in astrocytes: electron microscopy and immunohistochemistry in a case of atypical Alzheimer's disease.

A 54-year-old man who had cerebellar ataxia and pseudobulbar palsy at the age of 29 years, and soon developed dementia, myoclonus and convulsions, died after about 20 years in a vegetative state. Histological examination of the extensively atrophic and devastated brain (680 g) revealed the almost total loss of cerebral cortical neurons associated with numerous beta-protein amyloid plaques, many extracellular tangles and a large number of hypertrophic astrocytes, and prominent amyloid angiopathy. The astrocytes were frequently immunopositive for anti-human tau antibody (anti-htau) and anti-ubiquitin antibody (anti-ubi). Double immunostaining with anti-htau and anti-glial fibrillary acidic protein (GFAP) antibody clearly demonstrated htau-positive domains within the GFAP-positive perikarya/and processes of several astrocytes. Electron microscopy of the hippocampal CA1, which was completely devoid of pyramidal neurons, revealed, in astrocytes, abnormal filaments indistinguishable from the paired helical filaments (PHFs) seen in neurons. On immunoelectron microscopy, the filaments were observed to be labeled with anti-htau and anti-ubi, exhibiting the same immunohistochemical features as neuronal PHFs. This is the first demonstration of clearly constricted and both tau- and ubiquitin-positive PHFs in astrocytes, indicating that, in some special conditions like in our case, processes similar to those that attack neurons also affect astrocytes and ultimately make the latter form PHFs.

Adult↗
PubMed1992

Changes of phorbol ester binding sites in rat brain following intracerebroventricular administration of thyrotropin-releasing hormone (TRH): an in vitro macroautoradiographic investigation.

We examined the influence of the intracerebroventricular (icv) administration of thyrotropin-releasing hormone (TRH) on protein kinase C (PKC) activities in various rat forebrain regions in order to cast light on the mechanism of extra-pituitary non-endocrine physiological actions of TRH in the central nervous system. An in vitro macroautoradiographic method, with [3H]phorbol 12, 13-dibutyrate (PDBu) as the radioactive ligand, was used to investigate quantitative alterations of PKC activities. The optical densities for PDBu binding sites in the striatum and hippocampal formation were significantly increased after the icv administration of TRH, while those in the frontal cortex and septum were unchanged. These findings suggest that TRH may exert some of its non-endocrine functions through striatal and hippocampal neurons which used PKC in their second messenger systems.

Animals↗
PubMed1992

Accumulation of 99mTc-HM-PAO in photon deficient areas in bone scan of bone metastasis from hepatocellular carcinoma.

To evaluate bone metastasis from hepatocellular carcinoma (HCC), both bone and 99mTc-HM-PAO scintigraphies were performed in six patients with clinically and pathologically confirmed HCC. Two patients had a bone scintigram which revealed abnormal accumulation in the skull base, pelvic bone and thoracic spine. The 99mTc-HM-PAO scans of both these patients also showed abnormal accumulation in the same sites. The bone scintigrams in one patient revealed not only abnormal accumulation in the ribs but also photon deficient areas in the sternum, thoracic spine and femur, while 99mTc-HM-PAO scans showed abnormal accumulation in all these sites. In three patients, bone scintigraphy revealed photon deficient areas in the ribs, pelvic bone and femur, and their 99mTc-HM-PAO scintigrams showed abnormal accumulation in the same sites. Thus, it was shown that, in the detection of bone metastasis from HCC by means of bone scintigraphy, it was necessary to pay attention to hot and cold lesions, and that a combination study with 99mTc-phosphorous compounds and 99mTc-HM-PAO was useful in evaluating these lesions.

Aged↗
PubMed1992

[Genetic diagnosis of phenylketonuria: identification of the mutations of phenylalanine hydroxylase gene by PCR direct sequencing].

To investigate the mutations of the phenylalanine hydroxylase (PAH) gene in Orientals, direct sequencing was conducted on DNA fragments amplified by the polymerase chain reaction, using solid phase technology involving the biotin-streptavidin system. Four mutations possibly associated with phenylketonuria (PKU) were identified in a Chinese and four Japanese patients. A novel Arg158 (CGG)-to-Trp158 (TGG) mutation was identified in exon 5 of the PAH gene in a Chinese PKU patient. The second change was due to a G-to-A transversion at the last base of intron 4. The third change was a compound heterozygote; one mutation was a G-to-A transversion at the last base of intron 4. The other was a G-to-C transversion at the second base of codon 413, which resulted in a substitution of Arg(CGC) by Pro(CCC) in exon 12. The last change was due to a Tyr204(TAT) -to-cys204(TGT) mutation in exon 6 of the PAH gene in two Japanese. This preliminary study revealed a novel PKU mutation and considerable genetic heterogeneity in the PAH gene among Orientals.

Asian People↗
PubMed1992

[Detection of Campylobacter species by using polymerase chain reaction and nonradioactive DNA probes. II. PCR direct sequencing of the Campylobacter DNA].

We have developed a sensitive DNA hybridization assay for the detection and identification of Campylobacter species which are recognized as important pathogens of acute diarrheal disease in humans. This technique utilizes DNA probes complementary to nucleotide sequences present in 16S ribosomal RNA (rRNA) of C. jejuni, C. coli, C. laridis, C. fetus, C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis, and polymerase chain reaction. The partial sequence of DNAs encoding the Campylobacter rRNA was first analyzed by direct solid phase sequencing in order to select suitable DNA probes. Amplified target DNA of 240 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with DNA probes conjugated to alkaline phosphatase. In identification experiments, one of the 10 probes tested here gave a positive hybridization reaction with C. jejuni, C. coli and C. hyointestinalis but not with other Campylobacter species. The other was specifically reactive with C. fetus, C. fetus subsp. fetus and C. fetus subsp. venerealis. When applied to stool specimens, a good correlation was found between the results obtained by the present assay and by biochemical tests. These findings suggest that the nonradioactive probe assay can be used as the practical criterion for differentiating Campylobacter species.

Base Sequence↗
PubMed1992

[A fundamental study of 99mTc-ethyl cysteinate dimer (99mTc-ECD)].

In vitro fundamental studies of 99mTc-ethyl cysteinate dimer (99mTc-ECD), a newly developed radiopharmaceutical for cerebral perfusion scintigraphy, were performed, and were compared with those of 99mTc-HM-PAO, which has been widely applied to clinical use. 99mTc-HM-PAO should not only be injected soon after reconstitution, but also be avoided mixture with saline or drugs such as Diamox, because of its radiochemical purity decreases with the passage of time and the addition of saline and Diamox. On the other hand, the radiochemical purity of 99mTc-ECD reached a constant level of 94-96% at 30 to 180 min after reconstitution, and it remained stable against the addition of saline and Diamox. Therefore, it was suggested that 99mTc-ECD could be injected from an already saline-infused venous-line or mixed with Diamox. However, early injection after reconstitution should be avoided, because 99mTc-ECD did not become stable enough to maintain sufficient radiochemical purity until 30 min after reconstitution. The present studies are suggested that the red blood cell component is largely related to the metabolism of 99mTc-ECD in blood. Furthermore, from study of the metabolic rate using rabbit blood, it was suggested that a species difference existed in esterase activity in blood.

Animals↗
PubMed1992

[Usefulness of 99mTc-ECD SPECT in diseases of the central nerve system: special reference to a comparison with 123I-IMP and 99mTc-HM-PAO SPECT].

The usefulness of a new cerebral perfusion imaging radiopharmaceutical, 99mTc-ethyl cysteinate dimer (ECD), was clinically evaluated. The subjects of this study were 14 patients with neurological disorders including 10 patients with cerebral infarction and 4 patients with other diseases. A total of 15 examinations was performed. 99mTc-HM-PAO or 123I-IMP SPECTs were performed simultaneously, and the findings from those examinations were compared with 99mTc-ECD. As to the count ratio of lesions to normal area (L/N), the L/N ratio in severe ischemic patients was lower in 99mTc-ECD than in 99mTc-HM-PAO or 123I-IMP. In mild ischemic patients, on the other hand, the L/N ratio was the lowest in 123I-IMP. When the relationship between rCBFs obtained from 123I-IMP and the values of L/N in 99mTc-ECD or 99mTc-HM-PAO was compared, the values of L/N in 99mTc-ECD or 99mTc-HM-PAO were found to have decreased linearity with increasing rCBF. In a patient showing luxury perfusion, the accumulation pattern of 99mTc-ECD was different from that of the other two radiopharmaceuticals, and focal defect was revealed in 99mTc-ECD SPECT. On the dynamic SPECT of 99mTc-ECD in a patient with meningioma, the tumor showed a change from high to low perfusion with the passage of time. This finding indicated that care should be taken in the evaluation of accumulation of 99mTc-ECD. Therefore, 99mTc-ECD was found to be useful as a cerebral perfusion agent. In addition, as accumulation of 99mTc-ECD might somehow reflect metabolism in some cases, further careful investigation of many cases should be carried out.

Adult↗
PubMed1992

[Detection of methicillin-resistant Staphylococcus aureus using PCR and non-radioactive DNA probe].

Two commonly used diagnostic tests for Staphylococcus aureus (MRSA) are cultivation of bacteria from clinical samples on mannitol-salt agar plate or on MRSA-screen agar plate with oxacillin. However, the use of PCR and DNA probes is considered more rapid and sensitive, as staphylococcal cells have high resistance to beta-lactam due to the novel penicillin-binding protein, PBP2'. Therefore, three different pairs of DNA primers (P1F-P1R, P2F-P2R and P3F-P3R) complementary with unique regions of the MRSA PBP2' gene were synthesized for use in polymerase chain reactions with DNA of MRSA. Amplified target DNA of 466, 480 and 630 bp could be resolved on ethidium bromide-stained gels, and hybridized with DNA probes conjugated to alkaline phosphatase. When applied to pure cultures on the MRSA screen agar, the DNA probes detected MRSA in 180 of 196 (P1F-P1R), 72 of 83 (P2F-P2R) and 66 of 71 (P3F-P3R) culture-positive specimens (accuracy range, 86.7-93.0%), while 61.6-89.3% of MRSA were detectable in culture-positive specimens streaked on the mannitol-salt agar. When applied directly to clinical samples, this DNA probe assay detected MRSA in culture-positive specimens within an accuracy range of 50.0-79.3%. It was thus clear that comparison of cultivation and DNA hybridization results yields good correlation with respect to detection of MRSA. These results suggest that the DNA probe assay may be useful in complementing existing culture techniques.

DNA Probes↗
PubMed1992

[Detection of Campylobacter species by using polymerase chain reaction and nonradioactive labeled DNA probe].

We have detected Campylobacter species which are now recognized as major pathogens of acute diarrheal disease in humans using polymerase chain reaction (PCR) and a nonradioactive labeled DNA probe. Diagnosis of Campylobacter enteritis without doing culture from stool samples is of great benefit in the laboratory. Two oligonucleotide primers (20 mer) complementary to a unique sequence of the DNA encoding ribosomal RNA (rRNA) of Campylobacter jejuni for PCR were synthesized by solid-phase phosphoamidite method. Amplified target DNA of 275 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with an oligodeoxynucleotide probe (28 mer) conjugated to alkaline phosphatase. In identification experiments, it was shown that the nonradioactive probe was hybridized to clinical strains of C. jejuni (104), C. coli (5), C. laridis (5), C. hyointestinalis (1) and C. fetus subsp. fetus (1) with an accuracy of 99-100%, while it was not for Helicobacter pylori. Further, there was no evidence of amplification in strains of K. pneumoniae, S. marcescens and E. coli. Using direct detection to stool specimens, this method could be performed in C. jejuni in 39 of 43 culture-positive specimens (91%), and in 19 of 141 culture-negative specimens (13.5%), respectively. The results of this comparative study suggested that the DNA probe assay became a rapid and reliable technique to confirm culture of Campylobacter species.

Animals↗
PubMed1991

Rapid appearance of beta-amyloid precursor protein immunoreactivity in damaged axons and reactive glial cells in rat brain following needle stab injury.

Brains of rats that had received needle stab wounds were examined sequentially for 72 h by immunohistochemistry using antibodies to beta-amyloid precursor protein (APP). Immunoreactivity appeared 0.5 h after the injury in axons adjacent to the needle tract, becoming stronger over 15 h in neighboring swollen axons. APP-immunoreactive glial cells appeared 6 h after the injury around the needle tract and persisted throughout the experiment. Morphologically, these cells were thought to be mainly astrocytes.

Amyloid beta-Protein Precursor↗
PubMed1991

Presenile appearance of abundant Alzheimer's neurofibrillary tangles without senile plaques in the brain in myotonic dystrophy.

In 7 myotonic dystrophy (MyD) cases of 35- to 56-year old and 18 non-neurological age-matched controls paraffin-embedded temporal lobe sections were stained by the modified Bielschowsky method to count neurofibrillary tangles (NFTs) and senile plaques (SPs). In the parahippocampal gyrus, NFTs were observed in all the MyD cases; a few in the youngest, with an increase in number with age to the abundant appearance in the 4 cases in their 50s. The eldest also had many NFTs in the hippocampus. By contrast, the control subjects had, if any, only a few NFTs in the hippocampus and the parahippocampal gyrus. No SPs were observed in any MyD or control case. Thus, it was found that in MyD NFTs appear, unaccompanied by SPs, at an abnormally early age in the parahippocampal gyrus, with a rapid age-related increase in their number. This neuronal change may belong to the progeric features observed in this condition.

Adult↗