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Biomedical subjects

T Kitagawa

Publications and source records attributed to T Kitagawa.

At least 271 records · Page 15Linked to original sources
PubMed1993

A new enzyme-linked immunosorbent assay (ELISA) for studying immunocytochemical procedures using an antiserum produced against spermidine as a model.

Antiserum was produced in rabbits against the polyamine spermidine (Spd) conjugated to bovine serum albumin (BSA). The reactivity of the serum to Spd and a variety of structurally related compounds was quantified by a new immunocytochemical model system incorporating an enzyme-linked immunosorbent assay (ELISA) binding test. This is based on the principle of coupling these compounds to the wells of microtiter plate activated with poly-L-lysine and glutaraldehyde and incubating the wells by the indirect immunoperoxidase method. The antiserum showed a 25% cross reaction with spermine (Spm), putrescine (Put), and cadaverine (Cad), and a 1% cross reaction with 1,3-diaminopropane (Dap), but no cross reaction with monoacetyl polyamines and amino acids. The antibody binding was inhibited most effectively by absorption of the antiserum with N1-acetylspermidine and Spd in the ELISA inhibition test. Also, immunoblot analysis of the antiserum with nitrocellulose paper gave completely identical results to the ELISA binding tests. Spd-like immunoreactivities in human melanoma BD and neuroblastoma IMR 32 cell lines are presented as examples of the staining pattern obtained with the antiserum. Absorption of the serum with N1-acetylspermidine and Spd was demonstrated to abolish the immunostaining reaction. The immunohistochemical model is simple: amines and amino acids are bound in the same way as in aldehyde-fixed tissues and, in comparison to immunoblot analysis, the immunoreactivity can be more easily and accurately quantified by assay with the antibody. The model should prove useful in assessing the specificity of other antisera.

Enzyme-Linked Immunosorbent Assay↗
PubMed1993

Changes in plasma dopamine-beta-hydroxylase activity during the perioperative period of cardiac surgery: an index of sympathetic nerve activity.

To evaluate the usefulness of plasma dopamine-beta-hydroxylase (DBH) activity as an index of sympathetic nerve activity during cardiac operations, we examined the serial changes in plasma DBH activity, in relation to the plasma noradrenaline (NA) level and hemodynamic parameters, in patients who underwent cardiac surgery. The plasma DBH activity decreased significantly after cardiopulmonary bypass, and remained low during dopamine (DA) infusion until 72 h after the operation. However, recovery of the hemodynamic parameters, being the mean arterial pressure, heart rate and cardiac index, was seen as early as 1-3 h postoperatively. It was therefore assumed that the plasma DBH activity takes a long time to recover after an operation. The time-course changes in the plasma NA level were quite different from the changes in DBH activity, with an apparent negative correlation being observed between them. Thus, there is a possibility that exogenously administered DA, as well as increased plasma NA, might inhibit DBH activity during cardiac surgery. Moreover, since catecholamines are often administered upon completion of cardiac surgery, measurement of the plasma catecholamine level would be inappropriate for evaluating real sympathetic nerve activity. From the results of this study, it is surmised that measurement of the plasma DBH activity could be useful for estimating the intrinsic sympathetic nerve activity of patients who have undergone cardiac surgery.

Adult↗
PubMed1993

Management problems in continuous care of male patients with mild hypertension with special reference to alcohol-intake.

Forty two male patients with borderline and mild hypertension were followed up for 3.9 years without medication. During this period, all subjects had nonpharmacological interventions including salt restriction, weight reduction, anti-smoking or anti-alcohol instruction, and physical exercise after the established protocol. Comparisons of biomedical data between pre- and post-observation revealed no significant improvements. Although the smoking habit decreased during this period, the drinking habit rarely changed and even significant increase of the total amount of alcohol ingestion was observed. These data indicate great limitation of nonpharmacological intervention in the care of these asymptomatic hypertensive patients for a lifelong base.

Adult↗
PubMed1993

Malignant transformation of a mouse liver epithelial cell line by transfection of an activated c-H-ras gene with a point mutation at codon 12.

In order to scrutinize the reason why in mouse liver system only activated H-ras gene with a point mutation at codon 61 but not codon 12 is frequently seen although the latter mutation is highly frequent in methylnitrosourea-induced rat mammary tumors, transforming activity of these two types of mutated H-ras gene was investigated utilizing an immortalized but not fully transformed mouse liver epithelial cell line MLE-10, established in our laboratory. MLE-10 cells were transfected with activated human c-H-ras gene having a point mutation at either codon 12 (PT24) or 61 (PSK2), together with PSV2neo, or with PSV2neo only. G418 resistant colonies, propagated separately, gave rise to 6, 3 and 6 lines respectively. All the PT24 and PSK2-transfected cell lines were growth capable in both soft agar and nude mouse subcutis, with similar growth rate and morphological features whereas none of the cell lines transfected with the PSV2neo only revealed such growth capability. The results thus revealed that c-H-ras with a mutation at codon 12 has oncogenic activity to the mouse hepatocyte, although after immortalization, at the degree similar to the same gene with a mutation at codon 61.

Animals↗
PubMed1993

The molecular features and catalytic activity of CuA-containing aco3-type cytochrome c oxidase from a facultative alkalophilic Bacillus.

Cytochrome aco3 of Bacillus YN-2000 was purified by an improved procedure and its molecular features and catalytic activity were extensively studied. The enzyme molecule was composed of three subunits with M(r)s of 50,000, 41,000, and 22,000, and contains 1 molecule each of cytochrome a, cytochrome c, and cytochrome o3 in the minimal structural unit (M(r), 113,000). The 41,000 subunit (subunit II) contains heme c. The EPR, optical, and resonance Raman spectra of the oxidized enzyme demonstrated the presence of CuA whose coordination environment bore close resemblance to that of the aa3-type cytochrome c oxidase. Resonance Raman studies demonstrated that the cytochrome a moiety was similar to that of an aa3-type oxidase and also that the cytochrome o3 contained a five-coordinated high-spin heme with histidine as an axial ligand. The Fe-CO stretching mode of the cytochrome o3.CO complex was observed at 520 cm-1, which is the same frequency as that of cytochrome aa3.CO. The oxygen consumption activity of cytochrome aco3 was measured using several kinds of cytochromes c as the electron mediators. The reaction between cytochrome aco3 and eucaryotic cytochromes c was completely inhibited by poly-L-lysine. In contrast, poly-L-lysine was indispensable for sufficient reaction between the oxidase and Bacillus YN-2000 cytochrome c-553, the physiological electron donor. The combined results on the structure and enzymatic properties suggest that the cytochrome aco3 is very similar to cytochrome caa3 except that the cytochrome aco3 has cytochrome o3 in place of cytochrome a3 and the cytochrome c component has a very low redox midpoint potential (95 mV).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1993

A new enzyme-linked immunosorbent assay (ELISA) for spermidine using glutaraldehyde coupling of the hapten to carrier-coated microtiter plates.

An enzyme-linked immunosorbent assay (ELISA) for small molecular haptens needs conjugates of the hapten with larger protein molecules for coating the wells of microtiter plates. The formation of such conjugates is not always reproducible. This makes it difficult to evaluate hapten-protein stoichiometries and to understand the precise orientation of the hapten on the protein. In this paper we describe an assay in which the polyamine spermidine (Spd) is coupled with glutaraldehyde (GA) to carrier poly-L-lysine (PL) or human serum albumin (HSA) coated on polystyrene microtiter wells. Each step of the assay was tested for maximum efficiency. This ELISA detected Spd with excellent reproducibility (coefficient of variation = 5.9%), an EC50 of 4.3 x 10(-6) M and a detection limit of 0.1 x 10(-6) M. The present ELISA was about 100 times more sensitive in detecting Spd than a conventional ELISA for Spd using a Spd-HSA conjugate for coating microtiter wells. The Spd antiserum showed 215% cross-reaction with N1-acetyl-Spd, 30.7% with N8-acetyl-Spd, 10% with spermine, 3.3% with cadaverine, 1.7% with putrescine, and less than 0.43% with 1,3-diaminopropane in the new ELISA system. A much higher degree of hapten binding to the plate occurred with the present method than with the previously reported method using microtiter wells activated directly with GA. The hapten conjugation is simple, reproducible and should provide a general method for developing ELISAs, not only for polyamines but also for amino acid and small peptides.

Antibodies, Monoclonal↗
PubMed1993

Rapid accumulation of deleted mitochondrial deoxyribonucleic acid in postmenopausal ovaries.

To study the aging process in human ovary, we analyzed the accumulation of mitochondrial DNA (mtDNA) mutation in ovary in women of various ages. Thirty-nine tissues from ovaries and abdominal muscles, ranging from those of a fetus to those of a 77-yr-old woman, were collected at artificial abortion or gynecological surgery. Total DNA was extracted from these tissues, and parts of mtDNA were amplified via polymerase chain reaction. Through these experiments, we found a 4977-bp deletion of mtDNA between the ATPase8 and ND5 genes in ovarian samples obtained from menopausal and postmenopausal women and in all muscle samples. Quantitative analysis of the deleted mtDNA also showed that the accumulation of deleted mtDNA in the ovary occurs rapidly around the menopausal period while the accumulation found in abdominal muscle occurs gradually with aging. These results indicated that the mtDNA deletion occurs in muscular tissues even at very young ages but that accumulation of the deletion in ovarian tissue starts at the menopausal period and may have a relationship to dysfunction of the ovary in aging.

Adult↗
PubMed1993

Alpha-particle dose to the liver and spleen tissues of Japanese Thorotrast patients.

We set out to establish an appropriate and convenient method for calculating alpha-particle absorbed doses to the liver and spleen of Thorotrast patients and to estimate a representative dose rate to the liver for the whole population of surviving and deceased Thorotrast patients in Japan. First, we determined steady-state activity ratios of 232Th progeny from 13 autopsy cases and found them to be identical to those reported in German subjects. Second, we estimated the alpha dose rates in 206 subjects at autopsy from radioactivity measurements and terminal weights of the organs. Combining these results with measurements of exhaled 220Rn in survivors, we considered the representative dose rates at injection to be 0.22 Gy y-1. Additionally, our data suggest that deposition in the spleen has previously been overestimated. This is an error with some bearing on the evaluation of leukemia risks from alpha-particle irradiation.

Adult↗
PubMed1993

Ductectatic-type mucinous cystadenoma and cystadenocarcinoma of the human pancreas: a novel clinicopathological entity.

A new type of mucinous cystic pancreatic neoplasm is described. The lesions, which have been recognized only recently with the progress of diagnostic techniques and have not previously been described in the literature, are characterized by multilocular cysts with papillary proliferation of the lining epithelium. They occur exclusively in the head and body, predominantly in males, and coexistence of well-differentiated adenocarcinoma as well as adenoma components is frequently encountered. These lesions are quite different from hitherto described mucinous cystic neoplasms of pancreas, not only in terms of gross features but also with regard to intrapancreatic location, and sex and age distributions. We propose to classify these lesions as a new pancreatic tumor entity: the ductectatic-type mucinous cystadenoma/cystadenocarcinoma.

Aged↗
PubMed1993

Atypical mitosis in gastric intestinal metaplasia in Japanese patients.

The characteristics of the mitotic figures occurring in intestinal metaplasia of the gastric mucosa were investigated in 50 consecutive gastrectomy specimens. The specimens contained, in addition, early gastric cancer of intestinal type (n = 38) or of diffuse type (n = 12). One hundred or more consecutive mitoses/specimen were studied by high-power microscopy (x 1000). Of the 5,140 mitoses, 1,030 (i.e. 20.0%, range 8-34%) were considered as atypical according to a previous classification. The presence of atypical mitosis was not influenced by the gender or age of the patient, or the histological type of the tumor present in the specimen. Studies of gastrectomy specimens from Swedish patients with early gastric cancer showed a much lower frequency of mitotic figures/specimen and occasional atypical mitosis. The high frequency of atypical mitosis in intestinal metaplasia in the gastric mucosa of Japanese nationals may mirror a profound genetic instability in that mucosa, and suggests that intestinal metaplasia with atypical mitosis may be a precancerous lesion in the Japanese.

Female↗
PubMed1993

bcl-2 gene rearrangement analysis of Japanese follicular lymphomas by polymerase chain reaction in formalin-fixed, paraffin-embedded tissue specimens.

The prevalence of chromosomal translocation t(14;18) was examined in 41 cases of follicular lymphoma covering all histopathological subtypes, using the polymerase chain reaction and hybridization method with non-radioactive oligonucleotide probes. DNAs were extracted from unfixed fresh-frozen and/or formalin-fixed paraffin-embedded biopsy specimens. The t(14;18) chromosomal translocation, resulting in bcl-2-JH (immunoglobulin heavy chain gene joining segment) fusion gene, was detected in 7 (36.8%) of 19 follicular small cleaved cell lymphomas and 6 (54.5%) of 11 follicular mixed, small cleaved cell and large cell lymphomas. Meanwhile, 11 cases of follicular large cell lymphoma revealed no bcl-2-JH fusion gene. There was a statistically significant difference in the frequency of bcl-2-JH rearrangement between follicular, small cleaved cell and large cell lymphoma (two-sided Fisher exact test, P = 0.049). The difference of bcl-2 rearrangement frequency between follicular, mixed and large cell lymphoma was also statistically significant (P = 0.012).

Base Sequence↗
PubMed1993

Recombinant human granulocyte colony-stimulating factor therapy for cyclic neutropenia associated with common variable immunodeficiency.

A 14 year old boy with common variable immunodeficiency (CVID) had regularly recurring episodes of severe infections independently of the serum gamma-globulin level. Serial blood counts revealed that this patient also had cyclic neutropenia. Recently, recombinant human granulocyte colony-stimulating factor (rhG-CSF) was reported to be an effective treatment for this disease. We tried rhG-CSF therapy for this patient and a prompt increase in the neutrophil count was noted. However, the cyclic alterations and duration of the nadir of the neutrophil count were not altered, which suggested that rhG-CSF has a variable efficacy in at least some patients with cyclic neutropenia.

Adolescent↗
PubMed1993

Human ovarian aging and mitochondrial DNA deletion.

The functions of human ovary change dynamically around the menopausal period. A decrease of the number of primordial follicles and an increase of fibrous tissues are observed histologically in the aged ovary. As endocrinological aspects at menopause, the synthesis and secretion of ovarian steroid hormones such as estrogens and progesterone decrease, followed with the resultant increases of pituitary gonadotropins. However, the mechanism of menopause and ovarian aging is not well understood. Thus, to study the regulatory mechanism of ovarian dysfunction by aging, we analyzed the accumulation of mitochondrial DNA (mtDNA) mutation in the human ovary in women of various ages. The amplification of a 5.5-kb region in mtDNA with polymerase chain reaction revealed a 0.5-kb band in ovarian samples obtained from menopausal and postmenopausal women, which means that the 5.0-kb deletion of mtDNA in ovarian tissue starts at the menopausal period. The close relationship between the occurrence of ovarian mtDNA deletion and the menstrual irregularity was also observed. These observations suggest that the accumulation of the deleted mtDNA may be a regulating factor of dysfunction of the ovary by aging.

Adult↗
PubMed1993

Enzyme-linked immunosorbent assay (ELISA) for luteinizing hormone releasing hormone (LH-RH) using a heterobifunctional cross-linking agent, N-[beta-(4-diazophenyl)ethyl]maleimide.

Luteinizing hormone-releasing hormone (LH-RH) was first labeled with an enzyme, beta-D-galactosidase (beta-Gal; EC 3.2.1.23), using N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional cross-linking agent. An antigen was similarly prepared by coupling LH-RH to mercaptosuccinylated bovine serum albumin with DPEM and was used for the immunization of rabbits for antibodies against LH-RH. A new, simple enzyme-linked immunosorbent assay (ELISA) for LH-RH was developed by using the principle of direct competition between LH-RH and beta-Gal-labeled LH-RH for anti-LH-RH IgG which had been adsorbed to the plastic surface of microtiter plates. LH-RH concentrations lower than 50 pg/assay well were measurable reproducibly by the ELISA, the sensitivity of which was found to be about 6250 times greater than the corresponding high performance liquid chromatography (HPLC) procedure. The specificity of this ELISA seems to be primarily toward the C-terminal region of LH-RH, showing a cross-reaction with the LH-RH6-10 fragment to the same extent as with LH-RH, but no cross-reaction with the LH-RH1-3 and LH-RH4-6 fragments. Using this assay, LH-RH levels were easily measured in the blood and urine of rats following the administration of LH-RH in a single dose of 0.5 mg/kg i.v. The present, newly developed ELISA is a nonradioactive, inexpensive and rapid method, and might be useful for elucidating experimental hypothalamic-pituitary-gonad interactions.

Amino Acid Sequence↗
PubMed1993

[Control of membrane permeabilities in mammalian cells and its application to pharmaco-biology].

The plasma membrane in mammalian cells possesses unique permeability properties serving as a selective permeability barrier as well as transporters for nutrients and ions in maintaining cellular homeostasis. External ATP modulates the permeability barrier in transformed cells. The characteristics and possible mechanism for this permeability change are summarized. Application of this membrane change for cancer chemotherapy was also examined in both in vitro and in vivo. The uptake of D-glucose by mammalian cells was carried out by a facilitated diffusion through a specific transporter protein in the membrane. The control mechanism for glucose transport by growth factors based on the changes in the glucose transporter levels is summarized. Modulation of glycosylation in the transporter protein and its possible role are discussed.

Adenosine Triphosphate↗
PubMed1993

Abnormal signal averaged ECG after surgical repair of tetralogy of Fallot--a combined analysis in the time and frequency domain.

PURPOSE: The clinical significance of abnormal signal averaged ECG (SA-ECG) determined by time and frequency domain analyses was assessed in tetralogy of Fallot patients after surgical repair, and the methods of analysis were compared. MATERIALS AND METHODS: SA-ECG was performed in 42 patients (mean age, 9.4 years) after radical surgical repair of tetralogy of Fallot, and in 11 preoperative patients (mean age, 2.6 years). Abnormal SA-ECGs were defined by time domain analysis (vector magnitude method) and frequency domain analysis (fast Fourier transformation). RESULTS: Abnormal SA-ECGs were recognized in 10 postoperative, patients (3 by time domain and 9 by frequency domain, analysis), but in none of the preoperative patients. Three patients with abnormal SA-ECGs had nonsustained ventricular tachycardia, 5 others had premature ventricular contractions, and the remaining 2 had no ventricular tachyarrhythmias documented by 24 h Holter monitoring. Patients with abnormal SA-ECGs more commonly had ST-T segment depression on standard ECG during exercise (8/10 versus 8/32, p < 0.001), a history of resection of a hypertrophic septoparietal muscle band (8/10 versus 2/32, p < 0.001) and histologically documented myocardial fibrosis at radical surgical repair (9/10 versus 5/19, p < 0.002). CONCLUSION: A Combination of time and frequency domain analyses was necessary to detect abnormal SA-ECGs in postoperative patients because of ventricular conduction disturbance. This technique might increase our ability to identify patients at risk of ventricular tachyarrhythmia, or those with underlying myocardial abnormalities.

Adolescent↗