Detection of point mutations by SSCP of PCR-amplified DNA after endonuclease digestion.
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Base Sequence↗
Biomedical subjects
K Yoshimoto
Publications and source records attributed to K Yoshimoto.
Molecular and genetic analysis of a compound heterozygote for dysprothrombinemia of prothrombin Tokushima and hypoprothrombinemia.
The molecular and genetic basis of a compound heterozygote for dys- and hypoprothrombinemia was analyzed. Abnormal nucleotide sequences of the human prothrombin gene were screened by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion and mutated primer-mediated PCR-RFLP. A single nucleotide substitution responsible for dysprothrombinemia of prothrombin Tokushima was detected, as were three polymorphisms. The mutation for hypoprothrombinemia was detected by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion in exon 6, near MboII-RFLP and NcoI-RFLP. Sequencing of PCR-amplified genomic DNA revealed a single base insertion of thymine (T) at position 4177. The resulting frameshift mutation caused both an altered amino acid sequence from codon 114 and a premature termination codon (i.e., TGA) at codon 174 in exon 7. Because exon 7 encodes the kringle 2 domain preceding the thrombin sequence, this frameshift leads to the null prothrombin phenotype. The inheritance of the hypoprothrombinemia gene from the father to the proband was proved by PCR-SSCP with endonuclease digestion and mutated primer-mediated PCR-RFLP.
Amino Acid Sequence↗
Detection of a single base substitution of the gene for prothrombin Tokushima. The application of PCR-SSCP for the genetic and molecular analysis of dysprothrombinemia.
The genetic and molecular basis of a mutant prothrombin of 'prothrombin Tokushima' was studied by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses. The abnormal gene was detected by altered migration by PCR-SSCP and by the loss of an MspI site by PCR-RFLP. The gene for prothrombin Tokushima was shown to be inherited from the mother of the proband. Sequencing analysis using PCR-amplified genomic DNA clarified a substitution of thymine (T) for cytosine (C) at position 9,490, changing arginine (Arg) to tryptophan (Trp) at position 418 of the polypeptide chain. This point mutation is assumed to be the molecular basis of prothrombin Tokushima, firstly, because of the absence of distinct changes in Southern blot analysis of the proband's DNA (using a full-length human prothrombin cDNA as a probe), secondly, because it has the same molecular weight as the abnormal gene product, and, thirdly, because of the absence of other amino acid abnormalities in the proteolytic peptide-fragments. It is concluded that PCR-SSCP and PCR-RFLP were useful for detecting the abnormal gene and for directly diagnosing the carrier status of dysprothrombinemia. This is the first report of gene analysis of dysprothrombinemia.
Amino Acid Sequence↗
[Norfloxacin-induced infectious mononucleosis (IM)-like syndrome with Stevens-Johnson syndrome].
A 77-year-old male who had suffered from an upper respiratory infection and had been given Norfloxacin (NFLX) on May 2, 1990, developed generalized erythema which did not subside with prednisolone. He was hospitalized on May 8, and Stevens-Johnson syndrome was diagnosed. The WBC was 115,400/microliter (Ly 61.0%, Aty Ly 39.5%). Sternal tap revealed hypercellular marrow with increased lymphocytes (48.5%; Aty Ly 24.5%) and eosinophils (7.0%). Clinical chemistry revealed slightly abnormal liver and renal function with LDH (1,745 IU/l) and IgE (803 IU/ml) elevation. No pathognomonic result was obtained with several viral antibodies. CD4+, CD8+ lymphocytes and the 4/8 ratio were 39%, 43% and 0.9, respectively. Clinical and laboratory abnormalities were normalized within 3 weeks after the discontinuance of all drugs. Positive lymphocyte stimulation test results were obtained by NFLX. While drug allergy is known to be a cause of IM-like syndrome, there are few reports regarding the subset characterization of the increased T lymphocytes. In this case, T lymphocytosis was remarkable, but the 4/8 ratio declined only slightly, indicating that CD4+ as well as C8+ cells were activated and increased, unlike IM. The record of this case helps to clarify the mechanisms of the lymphocyte activation shared and not shared by EBV-induced IM and IM-like syndrome.
Aged↗
Occurrence in vivo of 5-hydroxytryptophol in the brain of rats treated with ethanol.
The effect of ethanol (EtOH) on the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) and the efflux of their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindol-3-ylacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOL) from the striatum of the freely moving rat were studied in vivo using brain microdialysis. Striatal DA and 5-HT release was maximally enhanced at first fraction after the administration of EtOH (2 g/kg, i.p.). The level of the DA-oxidized metabolite, DOPAC, decreased significantly. In the 5-HT metabolic pathway, the oxidized metabolite, 5-HIAA, did not show significant changes, whereas levels of the biogenic alcohol 5-HTOL were increased to 180% at 90 min following EtOH administration. It is suggested that EtOH, most probably via acetaldehyde, could shift 5-HT metabolism from the oxidative to the reductive pathway in the rat brain.
3,4-Dihydroxyphenylacetic Acid↗
Novel determination of glycated proteins in biological samples.
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Chromatography, Thin Layer↗
NcoI RFLP in the human prothrombin (F2) gene.
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Asian People↗
[Clinical characteristics in multiple endocrine neoplasia type 1 in Japan: a review of 106 patients].
A review of 106 patients with multiple endocrine neoplasia (MEN) type 1 reported between 1966-1989 in Japan was conducted in order to clarify the natural history of this disease. Sporadic MEN 1 was found in 61 patients, and familial MEN 1 was found in 45 patients from 15 families. The mean ages at diagnosis of the two groups were 46.2 and 41.3 years, respectively, and the male to female ratio was 3:4. With regards to the involvement of the pituitary, parathyroid and endocrine pancreas, the combination of three endocrine glands was 31%, and that of two was 48%, in which the pituitary, parathyroid and endocrine pancreas had tumorous lesions in 60%, 88% and 63%, respectively. The first clinical manifestations of MEN 1 were the symptoms of hyperparathyroidism (32%), pituitary tumors (26%), peptic ulcer (28%) and hypoglycemia (13%). These symptoms appeared between 6 and 57 years of age (mean 34 years). The main clinical symptoms of the pituitary tumors were acromegaly and gigantism (37%), galactorrhea-amenorrhea syndrome (20%), Cushing's disease (10%) and visual disturbance due to compression of the tumor (20%). The clinical manifestations of hyperparathyroidism were mainly asymptomatic hypercalcemia (41%), nephrolithiasis (42%) and osteitis fibrosa (5%). In the patients with pancreatic tumor, Zollinger-Ellison syndrome (52%) and hypoglycemic symptoms (42%) were found. Tumors in the adrenal cortex, thyroid, carcinoid and lipoma were detected in association with MEN 1 in the frequencies of 28%, 21%, 9% and 5%, respectively. Death was reported in 37 patients between 9 and 86 years of age (mean 50.0 years). The cause of death was gastrointestinal bleeding and perforation (45%), surgery (24%) and disseminated carcinomatosis (18%). Recently, the gene predisposing to this syndrome has been assigned to chromosome 11 (11q13) in non-Japanese cases but not yet in Japanese MEN 1 patients.
Adolescent↗
Morphine-induced thymic hypoplasia is glucocorticoid-dependent.
Mice administered morphine as a s.c. pellet implant exhibit a marked and sustained thymic hypoplasia as well as suppression of T lymphocyte functions. In the present study, the effects of morphine on thymocyte differentiation were characterized. Morphine produced a significant decrease in both the number and proportion of CD4+/CD8+ double positive (DP) cells. The percentage of the CD4+/CD8-, CD4-/CD8+, and CD4-/CD8- double negative subsets in these mice was proportionally increased. Morphine also increased the proportion of cells expressing either the epsilon-chain of the CD3 complex or the IL-2R. The initial reduction in the proportion of DP thymocytes appeared fully recovered by 10 days post-implantation, although the number of DP thymocytes gradually returned to normal over a 3-wk period. Morphine administration resulted in a marked increase in serum corticosterone levels, and a single injection of dexamethasone mimicked the effects of morphine on thymus differentiation. Furthermore, adrenalectomy abolished the morphine-induced decrease in CD4+/CD8+ thymocytes relative to a sham-operated group. The present findings are consistent with the hypothesis that morphine-induced thymic hypoplasia may be mediated by an increase in the circulating levels of corticosterone.
Adrenalectomy↗
Inhibition of calcium mobilization is an early event in opiate-induced immunosuppression.
Morphine administered as a subcutaneous implant inhibits the initial increase in cytoplasmic free-calcium [Ca2+]i induced by mitogens in mouse splenocytes. This effect was not reproduced by incubation of splenocytes with morphine (10(-8)-10(-4) M). Analysis of splenocyte subpopulations demonstrates that this effect was manifest in both B and T cells. However, within T cell subpopulations, CD4+ but not CD8+ cells were affected. Adrenalectomy abolished this effect of morphine in CD4+ T but not CD4-, CD8- spleen cells (most likely Thy 1.2- B cells). Moreover, simultaneous administration of the opiate antagonist naltrexone blocked the effect of morphine in CD4-, CD8- spleen cells, but not in CD4+ T cells. These data indicate that the effects of morphine on mitogen-stimulated increase in [Ca2+]i may be mediated through distinct glucocorticoid-dependent and -independent mechanisms. The morphine-induced inhibition of an increase in [Ca2+]i in immune cells reported here may be an early event mediating opiate-induced immunosuppression.
Animals↗
Loss of heterozygosity on chromosome 1p in thyroid adenoma and medullary carcinoma, but not in papillary carcinoma.
We analyzed 53 loci on 21 chromosomes other than chromosome 4 to detect possible loss of heterozygosity in 31 thyroid tumors using polymorphic DNA markers that detect allelic deletions at specific chromosomal loci. Loss of heterozygosity on chromosomes 1, 7 and 12 was detected in one follicular thyroid adenoma, and on chromosome 1 in two medullary thyroid carcinomas. However, no loss of heterozygosity was detected at any of the loci examined in papillary thyroid carcinomas. These results suggest that chromosomal loss detected in thyroid adenoma is one of the signals for risk of premalignant transformation, and that inactivation of unknown genes on chromosome 1p contributes to tumorigenesis of medullary thyroid carcinoma. Some genetic changes other than chromosomal losses may participate in the tumorigenesis of papillary thyroid carcinoma.
Adenoma↗
Allele loss on chromosome 11 in a pituitary tumor from a patient with multiple endocrine neoplasia type 1.
We have examined the allele loss of chromosome 11 in a pituitary tumor from a patient with familial multiple endocrine neoplasia type 1 (MEN 1). The extensive loss of chromosome 11, including loci of D11S149, HRAS1 and F2, was detected by the loss of heterozygosity. All of the lost alleles of these loci were transmitted from the unaffected father and not from an affected mother. This is the first evidence of allele loss of chromosome 11 in a pituitary tumor of MEN 1 and supports the idea that similar allelic deletion of MEN1 locus on chromosome 11 is the common genetic basis for tumorigenesis in the pituitary, endocrine pancreas, and parathyroid gland in MEN 1.
Adult↗
A second neutralizing epitope of B19 parvovirus implicates the spike region in the immune response.
We used 18 monoclonal antibodies against B19 parvovirus to identify neutralizing epitopes on the viral capsid. Of the 18 antibodies, 9 had in vitro neutralizing activity in a bone marrow colony culture assay. The overlapping polypeptide fragments spanning the B19 structural proteins were produced in a pMAL-c Escherichia coli expression system and used to investigate the binding sites of the neutralizing antibodies. One of the nine neutralizing antibodies reacted with both VP1 and VP2 capsid proteins and a single polypeptide fragment on an immunoblot, identifying a linear neutralizing epitope between amino acids 57 and 77 of the VP2 capsid protein. Eight of nine neutralizing antibodies failed to react with either of the capsid proteins or any polypeptide fragments, despite reactivities with intact virions in a radioimmunoassay, suggesting that additional conformationally dependent neutralizing epitopes exist.
ATP-Binding Cassette Transporters↗
[The diagnosis of colorectal polyp by endoscopic ultrasonography].
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Colonic Polyps↗
[Possible mechanism of production of the musical second heart sound and its clinical significance].
To investigate the predisposing factors and the clinical significance of the musical aortic component of the second heart sound (musical S2), 18 patients with musical S2 (musical group) among the consecutive 2,000 patients with phonocardiographic examination were noninvasively studied by analyzing underlying diseases, phonocardiographic findings, organic changes of the aortic valve, severity of aortic regurgitation and left ventricular dysfunction. Organic changes of the aortic valve were assessed by two-dimensional echocardiography, and aortic regurgitation was assessed by color Doppler flow imaging. Twenty-two normal subjects (normal group) and 17 patients with essential hypertension (hypertensive group) served as controls. Mean ages were matched among the three groups. 1. Left ventricular dilatation (seven patients) and hypertension (six patients) were the dominant part of underlying disease in the musical group. 2. Musical S2 was classified in the following two types based on the phonocardiographic characteristics; musical vibrations followed immediately after the accentuated S2, and the S2 which was replaced by regular vibratory waves. 3. Frequency of the musical vibrations ranged from 120 to 200 Hz, and its duration ranged from 60 to 120 msec. Amplitude of the musical vibrations decreased by inhalation of amyl nitrite, but increased by infusion of methoxamine. In a case with mild rheumatic valve disease, methoxamine induced marked intensification of the amplitude and prolongation of the duration of the musical vibrations, finally giving a typical cooing murmur. 4. Echo intensity of the aortic valve tended to be higher in the musical group than in the other two groups. 5. Echocardiographically, aortic regurgitation appeared more frequently in the musical group (88%) than in the normal (36%) and hypertensive (41%) groups. Area of the aortic regurgitant signal was significantly larger in the musical group (4.1 +/- 1.4 cm2) than in the normal (1.2 +/- 0.8 cm2) and hypertensive (2.3 +/- 1.2 cm2) groups. 6. Left ventricular end-diastolic dimension was significantly larger in the musical group (5.8 +/- 0.6 cm) than in the normal (4.7 +/- 0.5 cm) and hypertensive (4.8 +/- 0.7 cm) groups. Fractional shortening of the left ventricle was significantly smaller in the musical group (26 +/- 10%) than in the normal (37 +/- 5%) and hypertensive (37 +/- 8%) groups. In a case of the musical group, musical vibrations following the S2, which was large in amplitude at the state of heart failure, decreased markedly after the recovery from heart failure.(ABSTRACT TRUNCATED AT 400 WORDS)
Adolescent↗
[Transesophageal echocardiographic study on systolic flow pattern of the pulmonary vein in patients with mitral stenosis and atrial fibrillation].
To determine the clinical significance and effect of cycle length on systolic backward (C) and forward (S) flow patterns of the pulmonary vein, we performed transesophageal and transthoracic echocardiography in patients with atrial fibrillation (Af). Study population consisted of 10 patients with mitral stenosis and sinus rhythm (MS-SR), 15 with MS and Af (MS-Af), 15 with mitral valve replacement and Af (MVR-Af), 10 with Af without organic heart disease (lone-Af) and 15 normal subjects. Various parameters, including peak velocities of C and S waves, closing amplitude of anterior mitral valve echogram during end-diastole, amplitude of the mitral annulus and interatrial septal motion during systole and left atrial pressure during the mitral closing period or end-diastole, were measured in each group. Results were as follows: 1. C wave was observed in all Af groups and six of 10 patients with MS-SR. Particularly, peak velocity of the C wave in MS-Af group was increased significantly compared with those of every other group. 2. Peak velocity of S wave in all Af groups, particularly in MS- and MVR-Af groups, decreased significantly compared with that of the normal group. 3. There were significant negative correlations between preceding R-R interval and peak velocity of the C wave or closing amplitude of anterior mitral valve echogram or left atrial pressure during end-diastole in MS-Af group. 4. There were significant positive correlations between preceding R-R interval and peak velocity of the S wave or amplitude of the mitral annulus or interatrial septal motion during systole in MS-Af group. 5. Peak velocities of the C and S waves had no correlations to preceding R-R interval in lone-Af group. We concluded that the C and S waves of pulmonary venous flow velocity pattern in MS-Af are affected by cycle length, and that the former is influenced by left atrial pressure and/or pliability of the mitral valve during the mitral closing period, and the latter by the grade of left atrial dilatation and/or preceding left atrial emptying.
Adult↗
[A case of Pneumocystis carinii pneumonia with bronchial asthma following long-term corticosteroid therapy].
A 50-year-old man, who developed bronchial asthma at the age of 43 and was treated continuously with corticosteroids from the beginning of the first treatment, was admitted with exacerbation of exertional dyspnea. In spite of various treatments for bronchial asthma, such as theophylline, beta-stimulants, anti-allergic drugs and steroid-inhalation, asthmatic symptoms did not improve and he continued to receive intravenous administration of steroids. Five months after admission, he suddenly developed severe respiratory failure and died after five days. Autopsy revealed Pneumocystis carinii pneumonia and marked atrophy of adrenal glands. No evidence of malignancy or hematologic disease was seen. Serum HIV antibody was negative. Secondary immunodeficiency induced by long-term corticosteroid administration of over 7 years was considered to be the cause of Pneumocystis carinii pneumonia. It seems that no case of Pneumocystis carinii pneumonia with bronchial asthma alone has ever been reported in medical literature.
Adrenal Glands↗
[Antimicrobial susceptibilities of clinical isolates of Morganella-proteus-providencia group of bacteria].
We examined in vitro susceptibilities of 3,109 isolates belonging to 5 species of Proteeae to 2 penicillins, 5 cephems and 2 aminoglycosides. The isolates were collected from 69 hospital laboratories throughout Japan between 1986 and 1988. Minimal inhibitory concentrations were determined using the agar dilution method with inoculation of 10(8) cells/ml of bacteria. Proteus mirabilis had marked susceptibilities to the penicillins and cephems tested. Proteus vulgaris and Morganella morganii were similar in their susceptibilities to ampicillin (ABPC), piperacillin (PIPC), cefazolin (CEZ), cefotiam (CTM), latamoxef, gentamicin (GM) and netilmicin (NTL), but M. morganii was slightly more resistant to cefmetazole and ceftizoxime (CZX) than P. vulgaris. Providencia rettgeri also had a susceptibility pattern similar to that of P. vulgaris, except that P. rettgeri showed higher resistances to CZX, GM and NTL. Providencia stuartii had a very similar susceptibility pattern to P. rettgeri, but P. stuartii was much more resistant to GM and NTL than the latter. Some major differences on susceptibilities were clearly evident among the 5 species of Proteeae tested. Notable species-specific differences included higher susceptibilities of P. mirabilis to ABPC, PIPC, CEZ and CTM than others and stronger resistances of P. rettgeri and P. stuartii to GM and NTL.
Ampicillin↗