Polarization of Lambda hyperons produced by the quasifree ( pi +,K+) reaction on 12C.
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Journal Article↗
Biomedical subjects
T Shibata
Publications and source records attributed to T Shibata.
Epitope mapping of anti-recA protein IgGs by region specified polymerase chain reaction mutagenesis.
Monoclonal IgGs were shown to be useful for the specific inhibition of a set of activities of the recA protein, a key protein in homologous genetic recombination. The mapping of the epitopes for these IgGs and site-directed mutagenesis based on the mapping will facilitate location of the functionally active sites on the tertiary structure of the protein, which is being solved by means of physicochemical techniques. We developed a novel technique for region-specified mutagenesis and applied the technique to epitope mapping. Using the polymerase chain reaction in the presence of deoxyinosine triphosphate, we introduced random base substitutions specifically into a region of the recA gene defined by a pair of primers. RecA mutants exhibiting altered antigenicity were selected, in plaque-immunoblotting experiments, from libraries of mutagenized recA genes constructed on the lambda gt11 expression vector. Mutant recA genes were obtained at the frequency of about 10(-2) among the plaques expressing fused recA genes and then each one was expressed as a whole protein, which was characterized by enzyme-linked immunosorbent assay. Analyzing the DNA sequences of the mutant recA genes, we located at the amino acid sequence level the epitopes for two anti-recA IgGs which could not be located in previous studies. One of the antibodies was shown to prevent self-assembly of the recA protein and the other was suggested to inhibit the binding of double-stranded DNA. Thus, the active sites involved in these functions would be located in the space around or near the relevant epitope.
Amino Acid Sequence↗
A multicomponent protein of a fission yeast that promotes joint molecule formation from homologous DNAs.
We developed a quantitative assay ("homologous pairing gel assay") adequate for the purification of the activity promoting the formation of joint molecules, an intermediate of homologous recombination ("homologous pairing"). With this assay, one can measure the extent of homologous pairing between a single-stranded DNA and a strand of 3H-labeled double-stranded DNA by crude enzyme preparations. Since the total activity did not significantly change during the sporulation process, we tried to purify the activity from a whole cell extract of mitotic cells of a fission yeast, (Schizosaccharomyces pombe). Through quantitative assaying of a single fraction or of mixed fractions, we obtained three fractions, all of which were required for the maximum level of the ATP-independent homologous pairing: Fractions 65, 100, and 30. In Fractions 100 and 30, polypeptides of approximately 100 and approximately 30 kDa (the 100- and 30-kDa polypeptides), respectively, were the sole detectable components. Fraction 65 contained a polypeptide of approximately 65 kDa (the 65-kDa polypeptide) as the major component and also small amounts of the 30- and 100-kDa polypeptides. Fraction 65 by itself promoted homologous pairing, but the reaction was saturated at a level of approximately 20% of the maximum level achieved with the recA protein. Even when added in excess, Fraction 30 or 100 alone did not promote detectable homologous pairing. A mixture of Fractions 65 and 100 at a rather strict optimum ratio only promoted homologous pairing, the level being 50-70% that with the recA protein, suggesting a stoichiometric complex of these polypeptides as the active form. Fraction 30 alone did not enhance the reaction with Fraction 65, but stimulated homologous pairing promoted by the optimum mixture of Fractions 65 and 100 to the maximum level achieved with the recA protein. Therefore, the homologous pairing-promoting protein from the fission yeast is likely to be a multicomponent protein.
Chromatography, Affinity↗
Acute megakaryoblastic leukemia with translocation t(1;22)(p13;q13) in a 10-week-old infant.
A 10-week-old girl without Down syndrome developed an acute megakaryoblastic leukemia (AMKL). Bone marrow aspirates and biopsy showed megakaryoblastic infiltration with myelofibrosis. The diagnosis was made based on the findings that the positive reactions of leukemic cells to platelet peroxidase and to monoclonal antibodies which recognize platelet-specific surface glycoprotein (GP) IIb/IIIa and GP78. The blasts also showed myeloid and monocytoid differentiation antigens. The leukemic cells had a karyotype of 46,XX,t(1;22)(p13;q13). Our case and two other infantile cases reported by other investigators establish the novel association of the t(1;22) with AMKL.
Antibodies, Monoclonal↗
An endonuclease with multiple cutting sites, Endo.SceI, initiates genetic recombination at its cutting site in yeast mitochondria.
Endo.SceI is a mitochondrial sequence-specific endonuclease which has multiple cutting sites. In order to examine the possible role of Endo.SceI in homologous recombination, we analyzed the mode of recombination upon mating using antibiotic resistance markers on the mitochondrial genome. The segregation of a marker located very close to one of the Endo.SceI cutting sites showed a disparity (polarized segregation, i.e. gene conversion). This gene conversion depended on the presence of the functional Endo.SceI gene. In vivo cutting of mitochondrial DNA upon mating was detected at the cutting site in the antibiotic marker region, which also depended on the Endo.SceI activity. These results suggest that mitochondrial recombination is induced by cleavage of mitochondrial DNA by this sequence-specific endonuclease. This is the first demonstration that a sequence-specific endonuclease with multiple cutting sites induces genetic recombination.
Base Sequence↗
Enzyme immunoassay and immunochemical characterization of pancreatic stone protein in human serum.
Monoclonal antibodies were raised against human pancreatic stone protein (PSP) and used for one-step enzyme immunoassay (EIA). PSP-S2-5 was employed as the standard in the assay. The assay's measurable range was 25-1,500 ng/ml and within run coefficient of variation was 3.7-6.4%. Analytical recovery of the assay was 101.5 +/- 5.65% (mean +/- SD). The results of experiments in which serum was fractionated by Mono S (cation exchange chromatography) suggested that most of immunoreactive material in human serum is PSP-S2-5. The EIA offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis.
Antibodies, Monoclonal↗
Enzyme immunoassay for specific analysis of pancreatic stone proteins in human pancreatic juice.
In order to study the concentration of pancreatic stone protein (PSP) in human pancreatic juice, we investigated the influence of the insoluble form of PSP-S1 converted from PSP-S2-5 on PSP determination and the assay method for PSP-S1 precipitate after solubilizing PSP-S1. When bovine trypsin was added to pancreatic juice, PSP-S1 was converted from PSP-S2-5 and precipitated about 45-85% after 1 h. The precipitated PSP-S1 was dissolved in 0.1 M sodium acetate buffer, pH 4.0, and the concentration was measured by the enzyme immunoassay, with similar reactivity to PSP-S1 and PSP-S2-5. The proposed method can offer accurate and specific analysis of the PSP level in pancreatic juice. The results of the fractionation of pancreatic juice and duodenal juice on Mono S cation-exchange chromatography suggested that the major component of PSP was PSP-S2-5 in pancreatic juice and PSP-S1 in duodenal juice.
Calcium-Binding Proteins↗
Cellular immunosuppression in children with acute lymphoblastic leukemia: effect of consolidation chemotherapy.
The present study was designed to evaluate the chemotherapy-induced cellular immunosuppression in 20 children with acute lymphoblastic leukemia (ALL) in remission and receiving maintenance chemotherapy. Peripheral blood was serially obtained from leukemic children during vincristine/cyclophosphamide/6-mercaptopurine/prednisone combined consolidation chemotherapy. The mean absolute number of peripheral blood lymphocytes as well as the mean absolute numbers of lymphocyte subsets (T cells, T cell subsets, B cells, and natural killer cells) from leukemic children before consolidation chemotherapy were all significantly lower than in control subjects; however, the percentages of lymphocyte subsets were similar in both groups. After consolidation chemotherapy, the percentages of CD4+ T lymphocytes and natural killer (NK) cells were significantly decreased and the percentages of monocytes and CD8+ T lymphocytes were significantly increased. Phytohemagglutinin- and 12-O-tetradecanoylphorbol-13-acetate-induced production of interleukin-2 (IL-2) and NK-cell-mediated cytotoxic activity by peripheral blood mononuclear cells (PBMC) were also substantially decreased in the post-therapy groups. NK activity correlated with the percentage of NK cells in PBMC. In contrast, OK432-induced production of tumor necrosis factor alpha (TNF alpha) and killer activity against NK-resistant target cells were significantly increased after therapy as compared with the pre-therapy and control groups. TNF alpha production correlated with the percentage of monocytes in PBMC. These results demonstrate that substantial quantitative and qualitative chemotherapy-induced abnormalities of the cellular immune system are present in the majority of patients treated with ALL. It is also suggested that the increased TNF alpha production by monocytes and the appearance of potent killing activity against NK-resistant targets might compensate for the defects of IL-2 production and NK activity during intensive consolidation chemotherapy.
Animals↗
Acute and subacute inhalation toxicity of silane 1000 ppm in mice.
Male ICR mice were exposed to silane 1000 ppm, a concentration 200 or 2000 times higher than the recommended occupational exposure limits by many countries and academic associations, for 1, 2, 4, and 8 h (phase I study) and for 6 h/day, 5 days/week, over 2 and 4 weeks (phase II study). Hematological and biochemical studies were performed, and the animals were examined for histopathological lesions of the cornea, nasal cavity, respiratory tract, lung, liver, kidney, spleen, pancreas, thymus, thyroid, bone marrow, salivary glands, esophagus, and testis. All mice in both studies survived until they were sacrificed. In the phase I study, no exposure-related changes were found as a result of the hematological, biochemical, or histopathological examinations. In the phase II study, hematological and biochemical examinations failed to reveal any exposure-related changes, but mild irritation, manifested in the form of a small amount of exudate (eight out of ten animals), and inflammatory cells and/or necrotic cells on the nasal mucosa (six out of ten animals) was observed in the mice exposed to silane for 4 weeks. These findings suggest that silane toxicity and irritation are not severe.
Administration, Oral↗
Peritoneal absorption of pancreatic enzymes in dogs.
To elucidate peritoneal absorption of pancreatic enzymes, plasma levels of amylase, lipase, and trypsinogen were measured after the intraperitoneal injection of 10 ml human pancreatic juice in dogs. Plasma pancreatic amylase, lipase, and trypsinogen were determined using the immunoassay specific to the corresponding human pancreatic enzyme to exclude cross-reaction with the endogenous enzyme activities of the canine plasma. Plasma immunoreactivity of human amylase persistently rose during 24 h after the injection, whereas elevation of plasma amylase enzyme activity become significant only at 24 h. Increase of plasma lipase was not remarkable. Significant increase of the immunoreactivity was observed only at 24 h but there was no significant increase of the enzyme activity during this period. Plasma trypsinogen immunoreactivity peaked at 2 h remained significantly elevated during 24 h. The transperitoneal absorption of pancreatic enzymes in dogs was confirmed using intraperitoneal injection of human pancreatic juice combined with immunoassay specific to human pancreatic enzymes.
Amylases↗
Molecular form of pancreatic elastase 1 in human plasma.
In order to study the molecular forms of immunoreactive pancreatic elastase 1 (IRE) in human plasma, we investigated the characteristics of proelastase 1, elastase 1, and their alpha 1-protease inhibitor (alpha 1-PI) complexes on anion-exchange (Mono Q) chromatography, gel (Superose 12) chromatography, and enzyme immunoassay systems with monoclonal antielastase 1 antibodies of different affinities for alpha 1-PI-elastase 1 and alpha 1-PI-proelastase 1 complexes. The rate of complex formation between proelastase 1 and alpha 1-PI was dependent on temperature of incubation and concentration of alpha 1-PI. At 37 degrees C, 90% of proelastase 1 was bound to alpha 1-PI during 2 h of incubation of purified human proelastase 1 with human alpha 1-PI. Similar results were also obtained by 2 h of incubation of proelastase 1 with human plasma at 37 degrees C. Therefore, under physiological conditions, neither free proelastase 1 nor elastase 1 can be detected in the human blood stream, and most IRE exists as the complex of proelastase 1 with alpha 1-PI in human plasma.
Chromatography, Ion Exchange↗
A case of rheumatoid arthritis associated with silent thyroiditis.
A 41-year-old female with rheumatoid arthritis had nontender enlarged thyroid gland. Thyroid function tests revealed increased concentrations of serum free T3 (FT3, 10.8 pmol/L) and free T4 (FT4, 31.1 pmol/L) with suppressed concentration of thyrotropin (TSH, lower than 0.1 mU/L) and low 24-hour thyroidal radioactive iodine uptake (1.6%). Serum thyrotropin receptor antibody (TRAb) was negative (0%) and she had positive anti-thyroglobulin and anti-microsomal antibodies. A diagnosis of silent thyroiditis was made based on laboratory findings. Serum concentrations of FT3 and FT4 normalized one month later without treatment. The causal relationship between the two diseases is discussed.
Adult↗
Pancreatic stone predominantly composed of fatty acid calcium.
A 41-year-old man with chronic pancreatitis and pancreatic stones predominantly composed of fatty acid calcium is reported. He had complained of occasional abdominal pain for 10 years and visited the hospital because of a severe attack of abdominal pain. Laboratory data supported a diagnosis of pancreatitis. Computed tomography (CT) showed a high-density area in the head of the pancreas, and the CT number of this high-density area was lower than usual for pancreatic stones. Ultrasonography and endoscopic retrograde pancreatography showed a cystic lesion with small pancreatic stones in the head of the pancreas and irregular dilatation of the main pancreatic duct. Pancreaticojejunostomy and resection of pancreatic cyst were carried out for repeated episodes of abdominal pain under the diagnosis of chronic pancreatitis. The pancreatic stones obtained at surgery were proved to be mainly composed of fatty acid calcium after analysis of chemical composition of the stones. Fatty acid calcium was sometimes found in the biliary stones but never in the pancreatic stones.
Adult↗
Imaging of subunit complexes of thermophilic bacterium H(+)-ATPase with scanning tunneling microscopy.
Using a scanning tunneling microscope (STM), we observed reconstructed subunit complexes of H(+)-ATPase of a thermophilic bacterium. The measurement was carried out in air without conductive coating on the samples deposited on a highly oriented pyrolytic graphite (HOPG). The F1 subunit complex of the H(+)-ATPase, and an H(+)-ATPase whose F0 portion was embedded into liposomes prepared from soybean lecithin were imaged. Overall structural images of the subunit complex F1 were obtained: the structural dimensions of the STM images are in agreement with those deduced from conventional methods such as an transmission electron microscopy (TEM) and small-angle X-ray scattering (SAX) experimentation. Regarding the STM imaging of these samples, we discuss the advantages and disadvantages of the STM over those of conventional methods such as a TEM and SAX.
Cloning, Molecular↗
Induction of "wire-loop" lesions by murine monoclonal IgG3 cryoglobulins.
We have recently demonstrated that an IgG3 rheumatoid factor (RF) monoclonal antibody (mAb), clone 6-19, derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice, is able to generate cryoglobulins via a non-immunological IgG3 Fc interaction, and to induce an acute glomerulonephritis associated with cryoglobulinemia. Using this experimental model, we have characterized the glomerular lesions induced by the 6-19 RF monoclonal cryoglobulin, in particular the ultrastructural localization of the cryoglobulin deposits. Although their initial localization was confined to the mesangium, the 6-19 cryoglobulins were progressively accumulated in the subendothelial spaces of glomerular capillary walls, leading to the formation of glomerular lesions resembling the "wire-loop" lesion characteristically described for lupus nephritis. In addition, we have found that identical glomerular "wire-loop" lesions were induced by the 6-19-J558 hybrid antibody, composed of the 6-19 gamma 3 heavy chain and J558 lambda 1 light chain, which loses the RF activity, but retains the cryoglobulin activity. These results strongly suggest that the direct deposition of IgG3 cryoglobulins by itself, without involvement of immune complex formation, results in the generation of the classical "wire-loop" lesion characteristic of lupus nephritis. In addition, we have found that similar "wire-loop" lesions were generated by one anti-DNA mAb derived from (NZB x NZW)F1 hybrid mice, and two of four IgG3 mAb of unknown specificities, derived from MRL/MpJ-lpr/lpr mice. The absence of significant glomerular lesions, in spite of large amounts of cryoglobulins, in mice receiving two IgG3 mAb suggests the importance of physicochemical property of cryoglobulins to provoke glomerular lesions.
Animals↗
Selective pathogenicity of murine rheumatoid factors of the cryoprecipitable IgG3 subclass.
To analyze the involvement of rheumatoid factors (RF) in the generation of cryoglobulins and the development of related tissue injuries, we have established a panel of anti-IgG2a RF mAbs derived from MRL/MpJ-lpr/lpr (MRL-lpr), C3H/HeJ-lpr/lpr, and 129/Sv mice. After injection of hybridoma cells to normal mice, all four IgG3 RF mAbs induced cryoglobulinemia, and various degrees of glomerulonephritis and skin leukocytoclastic vasculitis. In contrast, none of the RF mAbs of the other isotypes generated cryoglobulins or tissue lesions. Since the same observation was obtained with another panel of five clonally related anti-IgG2a RF mAbs of MRL-lpr origin with almost identical heavy and light chain variable (V) regions but five different isotypes, it seems unlikely that the absence of pathogenicity of non-IgG3 RF mAbs was due to differences in fine specificity or V framework regions. In addition, the analysis of serum RF in MRL-lpr mice has demonstrated that a majority of 4 month old MRL-lpr mice produced substantial amounts of IgG3 RF with cryoglobulin activity. Because the cryoglobulin activity is associated with the murine IgG3 heavy chain constant region, RF of this subclass may play a significant role in the development of autoimmune-related tissue injuries, especially in MRL-lpr mice.
Animals↗
Three-dimensional structure of soybean beta-amylase determined at 3.0 A resolution: preliminary chain tracing of the complex with alpha-cyclodextrin.
The three-dimensional structure of a complex of soybean beta-amylase [EC 3.2.1.2] with an inhibitor, alpha-cyclodextrin, has been determined at 3.0 A resolution by X-ray diffraction analysis. Preliminary chain tracing showed that the enzyme folded into large and small domains. The large domain has a (beta alpha)8 super-secondary structure, while the smaller one is formed from two long loops extending from the beta 3 and beta 4 strands of the (beta alpha)8 structure. The interface of the two domains together with shorter loops from the (beta alpha)8 structure form a deep cleft, in which alpha-cyclodextrin binds slightly away from the center. Two maltose molecules also bind in the cleft. One shares a binding site with alpha-cyclodextrin and the other is situated more deeply in the cleft.
Binding Sites↗
Bleomycin but not its derivatives inhibits the in vivo shedding of a rat tumor-associated antigen.
Rat fibrosarcoma (KMT-17) cells and their in vitro clone, 10% FCS A3 cells, shed a tumor-associated antigen (TAA), CE7, from the cell surface under growth-promoting conditions. We treated cells with the antitumor agent bleomycin (BLM) and its analogs peplomycin and liblomycin in vitro and in vivo in an attempt to increase expression of this antigen and induce an antitumor response. Although all three agents enhance antigen expression in vitro, proportionate to their direct antiproliferative effects, only BLM enhances antigen expression in vivo. The in vivo regulation of CE7 expression appears not to be related to the direct cytotoxic effects of the antitumor agents but rather to the immuno-augmenting effects of BLM.
Animals↗