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Biomedical subjects

K Hamaguchi

Publications and source records attributed to K Hamaguchi.

At least 19 recordsLinked to original sources
PubMed1992

Stabilization of a protein by removal of unfavorable abnormal pKa: substitution of undissociable residue for glutamic acid-35 in chicken lysozyme.

Glu35 in chicken lysozyme has an abnormally high pKa (6.1) partly due to the hydrophobic environment provided by Trp108. The relationship between protein stability and abnormal pKa was investigated in detail by using mutant lysozymes in which Glu35 was replaced by undissociable residues and an oppositely ionizable residue. It was found that lysozyme was stabilized at alkaline pH range by the replacement of Glu35 with an undissociable residue, Gln (E35Q lysozyme) or Al (E35A lysozyme). On the other hand, when Glu35 was replaced by His (E35H lysozyme), which could have an opposite charge to Glu by ionization, the introduced His35 was found to have an abnormally low pKa (3.6), leading to the destabilization of lysozyme at acidic pH. These observations are completely consistent with the situation that the environment around Glu35 is highly hydrophobic and therefore the placement of either a positive or negative charge in such an environment leads to destabilization of lysozyme. These observations also indicate that the replacement of an acidic residue having abnormally high pKa or a basic residue having abnormally low pKa by an undissociable residue is a very efficient and general method for stabilization of a protein.

Animals↗
PubMed1992

Purification of serine hydroxymethyltransferase from Bacillus stearothermophilus with ion-exchange high-performance liquid chromatography.

The gene of serine hydroxymethyltransferase (SHMT) of a thermophilic bacterium Bacillus stearothermophilus was expressed in Escherichia coli, and SHMT was successfully purified from the crude extract of E. coli in two steps while maintaining the enzymatic activity. The purification steps involved ammonium sulphate precipitation followed by high-performance liquid chromatographic separation using the anion-exchange column Fractogel EMD DEAE-650(S). In addition to the DEAE column, three other types of anion- and cation-exchange columns were also studied for their ability to separate SHMT, and the performance of the four columns were compared.

Ammonium Sulfate↗
PubMed1992

Significance of the serum CA125 level in recurrent ovarian cancer.

Thirty-four recurrent cases of 89 patients with ovarian cancer treated in our department between 1985 and 1989 were examined for changes in serum CA125 level. 1) Upon confirmation of recurrence, 17 patients were positive for CA125 and 15 were negative. 2) According to the histopathological type, the rate of CA125 positivity in patients with recurrence was high for serous adenocarcinoma, suggesting that determination of CA125 is useful for detection of recurrence. In contrast, all patients with mucinous adenocarcinoma or endometrioid adenocarcinoma were negative for CA125. 3) The CA125 positivity rate upon confirmation of recurrence was 9% in patients whose CA125 was less than 1,000 U/ml on initial examination, suggesting that close management of such patients is necessary. 4) Elevation of CA125 by 3 steps or more within the normal range (less than 35 U/ml) was useful for predicting recurrence. 5) The cut-off level of CA125 during follow-up should be set at 16 U/ml. 6) It was difficult to evaluate remission with only the serum CA125 level. It is impossible to avoid second look operation at present. These results indicate that pretreatment values and changes of the parameter within the normal range (less than 35 U/ml) have to be considered when using CA125 as a marker of tumor recurrence.

Adenocarcinoma↗
PubMed1992

Beta cell expression of endogenous xenotropic retrovirus distinguishes diabetes-susceptible NOD/Lt from resistant NON/Lt mice.

Endogeneous retroviral expression in beta cells is a feature of prediabetes in nonobese diabetic (NOD) mice. The purpose of this study was to characterize the class-specific pattern of retroviral gene expression in NOD/Lt beta cells versus a related, but diabetes-resistant strain, NON/Lt. Electron microscopic comparison of beta cells from both strains indicated low constitutive expression of the intracisternal type A (IAP) retroviral class. However, NOD beta cells, in contrast to NON beta cells, expressed an additional intracisternal retroviral form resembling a type C particle. Antibodies against both IAP and type C were detected in NOD, with the humoral response to type C, but not IAP, preceding decline in beta cell function. RNA was extracted from freshly isolated islets from NOD and NON males. Comparative Northern blot analysis of total type C retroviral gene expression using a gag-pol DNA probe corroborated expression of endogenous type C proviruses in both NOD and NON islet cells and thymus. Use of class-specific retroviral probes identified the class of expressed endogenous retrovirus distinguishing the two inbred strains. The single ecotropic provirus present in both the NOD and NON genome (Emv-30) was not expressed in islets or thymus of either strain. Comparison of endogenous xenotropic provirus content by Southern blot analysis revealed two unique xenotropic loci (Xmv-65, -66) in NOD; 8.4 and 3.0 kb xenotropic envelope (env) RNA transcripts were detected in NOD, but not NON islets and thymus. NON contained three xenotropic loci common to other inbred strains (Xmv-21, -25, and -28). Both strains were partially characterized for content of recombinant (polytropic and modified polytropic) proviruses. IAP RNA expression was common to both NOD and NON islets and hence could not be specifically associated with the unique intracisternal type C particle found in NOD, but not NON beta cells. In conclusion, this study shows that expression of xenotropic type C but not IAP distinguishes retroviral activity in NOD/Lt versus NON/Lt beta cells. The potential pathogenic role of retroviral gene expression in NOD beta cells is discussed.

Animals↗
PubMed1992

[An examination of Metzger's theory on geometrical illusions by using the illusion of major and minor axes in ellipse].

An experiment with ellipses was conducted to evaluate the validity of Metzger's theory on geometrical illusions (the law of good Gestalt) by examining whether its major axis is underestimated and its minor axis overestimated. If the horizontal or vertical dimension of a circle with a fixed diameter is reduced, " smaller" ellipses with major axes equal to the circle's diameter will be constructed. On the other hand, if the dimension is enlarged, "larger" ellipses with minor axes equal to the circle's diameter will be produced. Ten university students estimated the apparent lengths of such axes. It was found that the major axes were underestimated, while the minor axes were overestimated, irrespective of the relative sizes of the ellipses. The same result was obtained when the orientation of the axis was rotated 90 degrees. These results may be interpreted in line with Metzger's theory to the illusion of major and minor axes in ellipse.

Adult↗
PubMed1992

[A case of giant pigmented nevus with multiple meningiomas].

A 36-year-old female had a giant congenital pigmented nevus on her anterior trunk. She complained of muscle weakness of the right arm and leg in August 1988, and she was admitted to our hospital to take medical examinations in September 1988. Neurological examination revealed right hemiparesis and exaggerated deep tendon reflexes. Three small falx meningiomas were shown on CT scans and MR images, but they did not appear to be causes of her symptoms. CT-myelography and MR images revealed an intradural extramedullary tumor spreading from the foramen magnum to C2. Oncotomy and C1 laminectomy were performed. Histological finding was compatible with transitional meningioma. Neurological symptoms markedly improved after operation. This patient is considered as a case of neurocutaneous syndrome with central nervous system tumors. Considering that both giant pigmented nevus and meningioma originate from neural crest, they appear to be closely related each other. Such a combination of giant pigmented nevus and meningioma has not yet been reported.

Adult↗
PubMed1992

[Studies on the erythrocyte membrane skeleton in a patient with chorea-acanthocytosis--theoretical speculation on the mechanism of neurological involvement].

Patients with chorea-acanthocytosis exhibit symptoms of self-biting, choreic movement, and acanthocytosis, but not dementia. The mechanism of choreic movements is still unknown. In order to clarify the etiologic mechanism underlying these movements, we evaluated the erythrocyte membrane in one patient with chorea-acanthocytosis. A 35-year-old female was admitted to Saitama Medical School Hospital because of involuntary movements. She was alert, well-oriented, and had no gross memory defects. She had slurred speech, choreic movements and lip biting. Laboratory examination showed acanthocytes in her peripheral red blood cells, normal serum lipid values, and caudate atrophy on her brain CT scan. In analyzing the acanthocytes, we initially evaluated the size of the acanthocyte population by incubating her red blood cells with plasma. The cell population approximately doubled after 2 hours incubation. Next we examined the protein composition of erythrocyte ghost by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). There was no significant difference between the patient's erythrocyte ghosts and those of a control. Then we investigated morphological changes in the patient's erythrocyte by scanning and transmission electron microscopy (SEM and TEM). SEM showed the typical acanthocyte shape. The quick-freeze, freeze-substitution method confirmed that the routine TEM section was not artifactual, and was in fact in accurate reflection of the actual features of acanthocytes. TEM of the sections prepared from erythrocyte ghosts demonstrated that spectrin tended to be accumulated in the thorn region. Furthermore, TEM of quick-freeze, deep-etched replica of the ghost revealed more clearly a spectrin network densely packed on the inner hydrophilic surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Acanthocytes↗
PubMed1992

Growth regulation of ovarian cancer.

The disease referred to as ovarian cancer is composed of those tumors derived from the ovarian surface epithelium. Ovarian cancer incidence peaks in postmenopausal women, when the hormonal milieu is in a state of flux. The ovarian carcinomas have histologic characteristics of classical endocrine responsive tissues. For example, the serous tumors are similar in appearance to the epithelium of the fallopian tube, the mucinous tumors to that of the endocervix, and the endometrioid tumors to that of the endometrium. These observations alone suggest a role for hormones in disease etiology and progression. In addition, much experimental evidence has accumulated that shows direct hormonal and growth factor effects on the normal and neoplastic ovarian surface epithelium and the potential for responsiveness based on the presence of specific receptors. The concept that growth stimulatory substances, by their impact on mitosis and cell number, may influence the rate of mutations that could confer malignant transformation serves as an additional mechanism by which growth stimulatory substances can influence ovarian cancer initiation. With the emergence of new molecular biology techniques, future studies should unravel the mechanisms by which steroid hormones, peptide hormones, and peptide growth factors influence the development of ovarian cancer.

Female↗
PubMed1992

[A case of complete mediastinal thyroid cancer: aberrant goiter].

A 65-year-old man was admitted with the chief complaints of hoarseness and an abnormal mass shown by chest x-ray films. CT scan, MRI, thyroid scintigram, and angiogram showed a right upper mediastinal tumor. This was histologically diagnosed as malignant mediastinal goiter by percutaneous needle biopsy. Operation was performed using a collar incision and median sternotomy. The tumor was located in right upper mediastinum and had no relation to the cervical thyroid gland. It was 7 x 5 x 3.5 cm in size. Histological examination revealed papillary adenocarcinoma of the thyroid gland. To our knowledge, 17 cases of complete mediastinal malignant goiter have been reported in Japan, including our case. These 17 patients are reviewed with regard to their clinical features in this article.

Adenocarcinoma, Papillary↗
PubMed1991

Structure of the active 27-residue fragment of human calpastatin.

A synthetic 27-residue peptide corresponding to exon 1B of the endogenous inhibitor calpastatin contains a well-conserved region and has an ability to inhibit the cysteine endopeptidase calpain specifically. We examined the solution structure of this peptide in DMSO-d6 by two-dimensional 1H NMR spectroscopy. Although regular secondary structures such as alpha-helix and beta-sheet were not found, the region from Ile18 to Arg23 formed a well-defined structure with a type I beta-turn. This region coincided well with the highly conserved region of calpastatin. The result strongly suggests that this turn structure is essential for the inhibitory activity of calpastatin.

Amino Acid Sequence↗
PubMed1991

Conformation and disulfide bond formation of the constant fragment of an immunoglobulin light chain: effect of truncation at the C-terminal region.

Constant fragments with different carboxyl terminals, CL(109-211), CL(109-207), and CL-(109-200), were prepared by limited carboxypeptidase P or Y proteolysis of the constant fragment, CL-(109-214), of a type lambda immunoglobulin light chain, and their conformations and stabilities, and formation of the disulfide bond from the reduced fragments, were studied. No change in conformation or stability was observed on removal of three residues from the C-terminal end. Removal of seven or more residues from the C-terminal end destabilized the CL fragment. The rate of disulfide bond formation from reduced CL(109-207) was about 7 times faster than that for CL(109-214). These findings suggest that elongation of the polypeptide chain at least beyond the 207th residue is necessary for folding of the CL fragment into a definite conformation.

Amino Acid Sequence↗
PubMed1991

Use of fluorescence energy transfer to characterize the compactness of the constant fragment of an immunoglobulin light chain in the early stage of folding.

The CL fragment of a type-kappa immunoglobulin light chain in which the C-terminal cysteine residue was modified with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (CL-AEDANS fragment) was prepared. This fragment has only one tryptophan residue at position 148. The compactness of the fragment whose intrachain disulfide bond was reduced in order for the tryptophan residue to fluoresce (reduced CL-AEDANS fragment) was studied in the early stages of refolding from 4 M guanidine hydrochloride by fluorescence energy transfer from Trp 148 to the AEDANS group. The AEDANS group attached to the SH group of a cysteine scarcely fluoresced when excited at 295 nm. For the reduced CL-AEDANS fragment, the fluorescence emission band of the Trp residue overlapped with the absorption band of the AEDANS group, and the fluorescence energy transfer was observed between Trp 148 and the AEDANS group in the absence of guanidine hydrochloride. In 4 M guanidine hydrochloride, the distance between the donor and the acceptor was larger, and the efficiency of the energy transfer became lower. The distance between Trp 148 and the AEDANS group for the intact protein estimated by using the energy-transfer data was in good agreement with that obtained by X-ray crystallographic analysis. By the use of fluorescence energy transfer, tryptophyl fluorescence, and circular dichroism at 218 nm, the kinetics of unfolding and refolding of the reduced fragment were studied. These three methods gave the same unfolding kinetic pattern. However, the refolding kinetics measured by fluorescence energy transfer were different from those measured by tryptophyl fluorescence and circular dichroism, the latter two giving the same kinetic pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1991

Activated T lymphocyte subsets in experimental allergic neuritis.

Changes in activated T cell subsets in peripheral blood were examined during the course of experimental allergic neuritis (EAN), using two-color immunofluorescence flow cytometry. Both CD4+ and CD8+ activated T cells decreased transiently before the onset of clinical signs, and increased just around the time of onset of the disease. In contrast, during the recovery phase, the numbers of CD4+ activated T cells returned to the normal range, whereas CD8+ activated T cells continued to increase. These findings imply that activation of CD4+ helper/inducer cells contributes mainly to the evolution of EAN, and that of CD8+ suppressor cells are necessary for recovery.

Animals↗
PubMed1991

Study of lupus nephritis in males.

Clinical and pathological findings were studied in 23 male patients with lupus nephritis who were followed up for a period of 41 +/- 36 months after renal biopsy. Age at renal biopsy was 31 +/- 14 years and 19 patients (83 per cent) were between 15 and 50 years old. C3 and C4 levels were below normal in 23 (100 per cent) and 16 (70 per cent) respectively, CH50 was less than 25 u/ml in 67 per cent, and antinuclear and anti-DNA antibodies were found in 87 per cent and 82 per cent respectively. Serum albumin level increased from 2.9 +/- 0.8 g/dl to 3.7 +/- 0.8 g/dl during the follow up period (p less than 0.01), while urinary protein decreased from 2.0 +/- 2.3 g/day to 1.4 +/- 2.5 g/day. There was a significant improvement in the degree of haematuria (p less than 0.01), but serum creatinine levels showed no change (mean 1.5 mg/ml). Active proliferative lupus nephritis of moderate or severe degree was observed in 65 per cent of patients at the initial biopsy. A trend to regression in this activity was seen in most serial biopsies, but the chronicity index showed a slight increase. These data demonstrate that systemic lupus erythematosus in males, in comparison to our previous report of the disease in female patients, is accompanied by more active nephritis, but that it follows a benign course with therapy.

Adolescent↗
PubMed1991

NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse.

NOD/Lt mice harboring a hybrid rat insulin-promoter/SV40 large T-antigen gene spontaneously develop beta-cell adenomas. NIT-1 is a pancreatic beta-cell line established from one of these transgenic mice. Immunocytochemical staining of passage 18 cells showed most contained insulin, with less than 5% containing glucagon, and none containing pancreatic polypeptide or somatostatin. Glucagon content radioimmunoassayed in cell extracts was only 0.27% of the insulin content. Two-hour insulin secretion at 16.5 mM glucose was 638 ng/10(6) cells (41% of intracellular content) compared to only 1.3 ng glucagon (32% of intracellular content). Stimulated insulin secretion was consistently observed in response to 11 and 16.5 mM glucose between passages 11 and 19. At passage 19, both theophylline and tolbutamide stimulated insulin secretion at 5.5 mM glucose. Northern-blot analysis confirmed high levels of insulin mRNA but only trace glucagon mRNA and undetectable somatostatin mRNA. Interferon-gamma (IFN-gamma)-induced MHC class I RNA expression was correlated with markedly increased antigen expression at the cell surface. Similarly, a MHC-linked "occult" class I-like antigen detected by Cr release assay only after exposure of standard NOD/Lt islet cells to IFN-gamma was strongly induced by IFN-gamma in NIT-1 cells. Cell surface MHC class II antigen was not constitutively expressed on NIT-1 cells and could not be detected after IFN-gamma incubation, despite demonstration of IFN-gamma-induced Aa, Ab, and Li invariant-chain RNA transcripts. Similarly IFN-gamma induction of intercellular adhesion molecule 1 (Icam-1) transcripts was not accompanied by demonstrable cell surface expression of ICAM-1 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoma↗