Hb Niigata [beta 1 (NA1) Val-->Leu]: the fifth variant with retention of the initiator methionine and partial acetylation.
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Biomedical subjects
Publications and source records attributed to Y Ohba.
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One year's experience on a polymerase chain reaction (PCR)-based test for the detection of mycobacteria (AMPLICOR Mycobacterium, Roche Japan) was reviewed in the light of the results of a prior questionnaire study regarding to the expected merits and demerits of the test. Rapidity in reporting, the top-ranked merit, was realized in 22 patients with positive results (8.7% of all patients tested). The PCR-based test saved 30.7 days on average in reporting a positive result in these patients. False negative results (PCR negatives in culture positives) the top-ranked demerit, was encountered in 5 samples from 5 patients (0.7% of all samples, or 4.5% of culture positive). The presence of some PCR inhibitors was suspected in these samples, totaling to 45 samples (6.5% of all samples) from 35 patients as well. The frequency of these cases was higher in outpatients than in hospitalized patients, indicating the necessity of some improvement in public health. Furthermore, the character of sputum is a particularly important factor for the precise test by PCR as well as culture.
To provide clinical testing that is useful for medical treatment, it is essential to provide correct, reliable test results to doctors and patients, i.e. high-quality test data are always required for appropriate medical treatment. To fulfill this requirement, it is necessary to establish and smoothly manage a test quality control system designed to maintain a fixed level of data accuracy and precision. The history of quality control in clinical testing spans nearly 30 years and new developments and innovations are required in this field. In this memorial lecture, I will review the development of clinical laboratory testing and explore the potential for improved test result control in the near future.
The effects of allogeneic lymphocytes on the rat thymus following sublethal irradiation were investigated using immunofluorescence. The recovery of thymus weight following irradiation was delayed in rats 6 days after receiving lymphocytes compared to controls. Allogeneic cells forming colonies were detected by immunofluorescence in both the cortex and medulla of the host thymus, most frequently on day 15 when an appropriate number (3 x 10(6)) was injected. The allogeneic cells detected in the host thymus, presumably T lymphocytes, appeared to disturb thymic reconstitution following irradiation. However, double-immunofluorescence staining revealed that allogeneic cells did not affect the thymic stromal microenvironment. Allogeneic cells may have subsequently affected thymic tissue via cytokines. It is important to investigate not only the character of allogeneic cells in the host thymus but also the interactions of donor allogeneic cells, host immature lymphocytes and thymic epithelial cells because of the possibility that these allogeneic cells in the host thymus could prevent the rejection of allogeneic transplants.
An abnormal hemoglobin (Hb) found in a 63-year-old Japanese woman presenting with asymptomatic chronic microcytic anemia was extensively characterized by the conventional protein chemistry, restriction mapping of the genomic DNA with Bgl II digestion, and direct sequencing of a relevant segment of the mutant non-alpha-globin gene. The results identified a hybrid globin with delta- and beta-like sequences, from position 1 to 87 and from 105 to 146 (C-terminal), respectively. The delta beta fusion gene was produced by deletion of 7.4 kb involving parts of the two closely linked genes. The break point was somewhere within the homologous 58 bps, from codon 88 to IVS-II-7. The same deletion had repeatedly been reported in Hb Lepore Washington-Boston. The expression of a mild (delta beta)(+)-thalassemia trait in the present case completely agreed with previous reports on representative Hb Lepore heterozygotes. The basis for the activity of Hb Lepore gene which is several times higher than the delta-globin gene was discussed.
The first reported case of congenital Heinz body hemolytic anemia was subsequently shown to be caused by an unstable hemoglobin, Hb Bristol [beta 67(E11) Val-Asp]. This has become one of the classic models of an unstable hemoglobin, the hydrophilic aspartate disrupting the hydrophobic heme pocket. We have restudied this original case, who remains clinically well after nearly 50 years of severe hemolysis with a hemoglobin level of about 7 g/dL and two unrelated Japanese cases. Surprisingly, all three cases show the same DNA changes, predicting a valine to methionine change at beta 67, rather than the expected aspartate. Further analysis with electrospray ionization mass spectrometry and globin chain biosynthesis strongly suggests that this anomaly is because of a novel posttranslational mechanism, with slow conversion of the translated methionine into an aspartate residue. The proximity of the heme and oxygen may be important in facilitating the reaction. These findings show the importance of complete characterization of variant hemoglobins using protein, DNA, and biosynthetic analyses.
The effects of the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (baf.A1) and the carbonic anhydrase II inhibitor acetazolamide (AZ) on bone resorption and procathepsin L secretion of rat osteoclasts were investigated using the bone slice assay method, pit formation test. Baf.A1 completely suppressed osteoclastic bone resorption stimulated by parathyroid hormone (PTH), but did not affect procathepsin L secretion, while AZ suppressed both bone resorption and procathepsin L secretion. These findings suggest that bone resorption by procathepsin L secretion and its processing are regulated by proton production and proton secretion.
The suppressive effect of N-(benzyloxycarbonyl)-L-phenylalanyl-L-tyrosinal on bone resorption was examined in vitro and in vivo. This synthetic peptidyl aldehyde was found to be a potent and selective cathepsin L inhibitor in our screening for cysteine protease inhibitors. In the pit formation assay with unfractionated rat bone cells, 1.5 nM of this compound markedly inhibited parathyroid hormone-stimulated osteoclastic bone resorption. In addition, intraperitoneal administration of this peptidyl aldehyde (2.5-10 mg/kg) for 4 weeks suppressed bone weight loss dose dependently in the ovariectomized mouse, experimental model of osteoporosis. Hydroxyproline measurement of the decalcified femurs from these ovariectomized mice suggested that this compound acts as a bone resorption suppressor through the inhibition of collagen degradation.
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Lentinan (LNT), a (1-->3)-beta-D-glucan with (1--6)-beta-D-glucopyranoside branches, has marked antitumor effects in syngeneic and autochtonous hosts. Clinically, LNT has proved effective with chemotherapeutic agents in patients with recurrent gastric and colorectal cancer. However, the mechanism that triggers subsequent immunologic reactions remains obscure. We hypothesized that LNT must first bind to the host cells. Accordingly, we analyzed LNT binding to host cells in healthy volunteers after incubating their cells under a variety of conditions as well as intravenously injecting LNT then subjecting them to flow cytometry and immunofluorescent staining using monoclonal antibody (anti-LNT mAb). LNT bound to monocytes and neutrophils, but not to lymphocytes in vitro. The most avid LNT binding was to monocytes. The percentage of LNT-bound monocytes after 60 min incubation at 4 degrees C was greater than that at 37 degrees C. The binding of LNT to monocytes was inhibited slightly by anti-iC3b receptor (anti-CR3) mAb, strongly by anti-C3b receptor (anti-CR1) mAb, and completely by anti-CR1 and anti-CR3 mAb together. The percentages of LNT-binding monocytes in the peripheral blood increased significantly 3 and 4 after 2 mg LNT-injection and returned to low levels 5 h later. However, no increase in LNT-binding neutrophils and lymphocytes was observed. We concluded that binding of LNT to human monocytes may initiate the influence of this compound on the immune system and differ between individuals. Its binding site may be similar to the C3b receptor.
A set of acute-phase proteins was found to have been induced in rat after triiodothyronine (T3) treatment. To study the mechanism of gene activation by T3, the rat gene of alpha 1-acid glycoprotein (AGP), one of the major acute-phase proteins, was cloned and analyzed. CAT assay identified a thyroid hormone responsive element (TRE) in the first intron of the AGP gene and showed that mutations introduced into the TRE region destroyed the TRE activity. Gel shift assay and methylation interference assay showed that the thyroid hormone receptor bound to the TRE. The TRE identified here had a palindromic structure and was highly homologous to other known TREs.
We report a rare case of thoracic vertebral hemangioma which developed into angiosarcoma during the course of repetitive operations and irradiation. A 44 year old female was operated on for hemangioma of the first thoracic vertebra. The diagnosis of hemangioma was confirmed histopathologically with the specimen from the first operation. The tumor developed multiple lesions later in the clinical course after the first operation, these lesions were removed in four consecutive operations and each histological diagnosis was that of hemangioma. Throughout the period of these operations, the patient was treated with steroid, and with radiotherapy simultaneously. The patient underwent the fifth operation for the recurrence of the tumor on 26 March 1990, and the histopathological diagnosis was not hemangioma but hemangiosarcoma which was considered a malignant transformation. The tumor cells immunohistochemically revealed positive staining with UEA-I, Factor-VIII, as the tumor immunohistochemically showed a vascular endothelioid nature.
Effects of concanamycin B, a specific inhibitor of the vacuolar type H(+)-ATPase (V-ATPase), on the stimulation of bone resorption induced by parathyroid hormone (PTH) were examined in vitro. Concanamycin B was found to inhibit PTH-stimulated osteoclastic pit formation and to suppress the acidification of vacuolar organelles by V-ATPase in the osteoclasts. PTH-stimulated 45Ca release from prelabelled chick embryonic calvariae was also inhibited by concanamycin B in a dose-dependent manner. These results suggest that osteoclastic acidification of lacunae by V-ATPase plays an essential role in mineral dissolution and degradation of the organic matrix during bone resorption.
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We established an improved production of an antitumor polypeptide anti biotic, phenomycin (PHM), by using a genetically engineered Escherichia coli. Phenomycin consists of 89 natural amino acids without intramolecular disulfide bridge. PHM gene was synthesized as a fusion gene in which PHM at the C-terminus and the residues 1 approximately 20 of Hirudin variant 1 (HV1) at the N-terminus connected by a factor Xa recognition sequence (Ile-Glu-Gly-Arg). E coli JM 109 transformed with a plasmid containing the synthesized gene expressed a fusion protein and the trypsinization of the fusion protein purified by ultrafiltration and ion-exchange chromatography gave efficiently recombinant PHM at a final yield of 50 mg/liter of culture. This PHM yield was six times higher than that obtained by a natural PHM producing strain of Streptoverticillium baldacci. Recombinant PHM was not distinguishable from natural PHM in all aspects observed.