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Biomedical subjects

S Matsuzawa

Publications and source records attributed to S Matsuzawa.

At least 19 recordsLinked to original sources
PubMed1998

Conditioned fear stress induces ethanol-associated place preference in rats.

The purpose of the present study was to establish ethanol-induced place preference under conditioned fear stress (exposure to an environment paired previously with electric foot shock) in rats by using the conditioned place preference paradigm. The administration of ethanol (300 mg/kg, i.p.) with conditioned fear stress, but not without conditioned fear stress, induced a marked and significant place preference. Furthermore, additional exposure to conditioned fear stress immediately before the post-conditioning test further enhanced the development of ethanol-induced place preference. These results suggest that psychological stress may play an important role in the development of ethanol-induced place preference, and the present method may be useful for studying the mechanism of the rewarding effect of ethanol.

Animals↗
PubMed1997

BAG-1 modulates the chaperone activity of Hsp70/Hsc70.

The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.

Adenosine Triphosphatases↗
PubMed1997

The interconversion of protein phosphatase 2A between PP2A1 and PP2A0 during retinoic acid-induced granulocytic differentiation and a modification on the catalytic subunit in S phase of HL-60 cells.

Alterations in protein phosphatase 2A (PP2A) during retinoic acid-induced differentiation of HL-60 cells have been investigated. PP2A activity of HL-60 cells for phosphorylated myelin basic protein showed a sharp and transient increase after 18-h treatment with 1 microM retinoic acid, which corresponded to G1/S boundary of the cell cycle. This PP2A of the 18-h treated cells was eluted from a DEAE-Sepharose column with 0.13 M NaCl, while PP2A from control cells was eluted with 0.23 M NaCl. The phosphorylase phosphatase activity of PP2A in the 0.13 M eluate was greatly enhanced in the presence of protamine compared with that of the later eluting PP2A. Immunoblot analyses with antisera against B' and B alpha subunits showed that the PP2A in the 0.13 M NaCl eluate from 18-h retinoic acid-treated cells was PP2A0 (AC-B'), whereas the PP2A eluted with 0.23 M NaCl from 24-h retinoic acid-treated cells and 0-, 18-, and 24-h control cells was PP2A1 (AC-B alpha). These results strongly suggest that PP2A undergoes a transient and reversible interconversion of holoenzyme forms during the initial stage of retinoic acid-induced granulocytic differentiation. PP2A activity assayed after dissociation of the catalytic subunit, for phosphorylase as substrate, showed a sharp and transient decrease in S phase of HL-60 cells irrespective of the presence or absence of retinoic acid. Immunoblot analyses with antisera against C-terminus and N-terminus of the catalytic subunit of PP2A suggested that a modification at the C-terminus is responsible for the decrease in PP2A activity. Immunoreactivity to the C-terminal antibody was restored after treatments of the S-phase extract with alkali or ethanol, the conditions which remove the methyl group from the C-terminus. These results suggest that the C-terminus of PP2A catalytic subunit is transiently methylated in S phase of HL-60 cells.

Blotting, Western↗
PubMed1997

Alterations in type-1 serine/threonine protein phosphatase PP1alpha in response to B-cell receptor stimulation.

In response to stimulation of B-cells through cell surface IgM, the activity of the serine/threonine protein phosphatase PP1, but not PP2A, was transiently decreased and reached a minimum 10-20 min after the stimulation. The decrease was more profound in the immature B-cell line WEHI-231, than in the mature B-cell line BAL-17. Under these conditions, PP1alpha, an isoform of PP1, showed unique alterations in the patterns of several spots with distinct isoelectic points in the Western blot after two-dimensional electrophoresis, whereas another isoform, PP1delta, did not show any alteration. PP1gamma1 and PP1gamma2 were not detected in B-cells. Similar alterations in these spots were observed in B-cells stimulated by PMA. When partially purified PP1 consisting of PP1alpha and PP1delta was incubated with [gamma-32P]ATP and PKC, radioactive spots of PP1alpha could be detected, but no spot of PP1delta was detected. Because differences in sequence among PP1 isoforms are mostly restricted to their C-terminals, phosphorylation rates of the C-terminal peptides containing the PKC-phosphorylation motif were compared. The C-terminal peptide of PP1alpha is a better substrate for PKC than those of PP1gamma1 and PP1gamma2, and is phosphorylated at the serine residue corresponding to Ser-325 of PP1alpha. The corresponding C-terminal region of PP1delta does not contain the phosphorylation site. On the other hand, there was a large difference in subcellular distribution of PP1delta, but not PP1alpha, between immature and mature B-cells. From these results, it was strongly suggested that PP1alpha is involved, via phosphorylation by PKC, in the regulation of signal transduction in response to the stimulation of B-cells through cell surface IgM.

Amino Acid Sequence↗
PubMed1997

Gene expressions and activities of protein phosphatases 1 alpha, 2A and 2C in hepatocarcinogenesis and regeneration after partial hepatectomy.

The mRNA levels of protein phosphatases (PP) 1 alpha, 2A, and 2C were determined both in hepatocarcinogenesis and in liver regeneration. In the precancerous stage and during regeneration, the mRNA levels of PP1 alpha, PP2A, and PP2C were markedly increased compared with those in normal livers. In primary hepatomas, all three of these mRNA levels were decreased to the control levels. In poorly differentiated hepatomas, however, only PP1 alpha mRNA was specifically increased, in contrast to PP2A and PP2C, which were at the control levels or below. While PP1 activity in the non-nuclear fraction of partially hepatectomized livers was nearly constant, the activity in nuclei was increased about 2.5-fold over control levels at 12 h after partial hepatectomy, the time that corresponds to the G1 to S transition in the cell cycle of hepatocytes. On the other hand, PP2A activity in both fractions was nearly constant throughout. These results appear to suggest some involvement of protein phosphatases in regulation of hepatocyte proliferation.

3T3 Cells↗
PubMed1996

Ultrasensitive heterogeneous immunoassay using photothermal deflection spectroscopy. 2. Quantitation of ultratrace carcinoembryonic antigen in human sera.

We previously proposed a photothermal immunoassay based on highly sensitive detection by a photothermal beam deflection method using colloidal gold as a labeling material. The assay system was significantly improved in terms of background reduction of ultrasensitive laser spectrometry through the newly designed reaction and separation procedure using microspheres for a solid phase. Highly sensitive laser spectrometric analysis is often susceptible to interference by even a small amount of admixtures. In this subsequent study, we improved our photothermal immunoassay so that it possesses further selectivity, matching that of the highly sensitive photothermal detection method. It was proved that our method could be applied to determine ultratrace quantities of a carcinoembryonic antigen (CEA) in cancer patients' and normal sera. The lower detection limit of CEA was 0.078 ng/mL (9.8 amol, absolute quantity). This is 10 times superior to that of any other immunoassay method and has enough detectability to measure a low level of CEA in healthy individuals. Intraassay coefficient of variation is about 8% at eight different concentrations (n = 6). We assayed 61 serum samples, and there was a good correlation between this method and previously established ELISA. We obtained clearer demarcation of healthy persons and colon cancer patients in terms of the CEA value than was obtained by ELISA. We believe this is the first report in which the laser-based ultrasensitive immunoassay is proved to be applicable to human sera, although the matrix admixtures causing interference in the assay were quite different in each sample serum.

Carcinoembryonic Antigen↗
PubMed1996

Alterations in activity of protein tyrosine phosphatase SH-PTP1 in autoimmune MRL/mpj-lpr/lpr mice.

Activities of protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) in autoimmune MRL/MpJ-lpr/lpr mice (lpr mice) were measured and compared with the activities in the tissues from MRL/MpJ-+/+ mice (+/+ mice) as the control. In the spleen and liver, PTK activities in cytosol and membrane fractions were about 1.7- and 1.3-fold, respectively, higher in lpr mice than +/+ mice. PTP activities in cytosol and membrane fractions from lpr mice were 1.7- and 1.3-fold, respectively, higher in spleen, and 2.5- and 1.3-fold, respectively, higher in liver compared with those of the controls. These results demonstrate that the mutation of lpr gene resulted in elevation of PTK and PTP activities. Then, we measured the amounts and activities of SH-PTP1, a cytosolic PTP playing a crucial role in intracellular signaling from Fas antigen. The amounts of SH-PTP1 were about 4-fold larger in thymus, spleen, and lymphnodes than in liver, but there was no marked difference in the amounts between lpr and +/+ mice. On the other hand, activity of SH-PTP1 was definitely lower in lpr spleen and lymphnodes than +/+ spleen, but several times higher in lpr liver than +/+ liver. Tyrosine phosphorylation levels of SH-PTP1 in spleen of lpr and +/+ mice were similar. However, in liver, it was less phosphorylated in lpr than in +/+ mice. This hypophosphorylation might cause the activation of SH-PTP1 activity in lpr liver.

Animals↗
PubMed1995

[Simple determination methods for blood grouping from biological stains. 1. Immunologic gold colloid aspiration test and dot ELISA test by membrane aspiration method for determining ABO blood groups from saliva specimens].

Two immunologic tests using nitrocellulose membrane and an aspiration device were applied to determine the ABO blood groups and secretor status from saliva specimens. First, immunologic gold colloid membrane aspiration test (IMAT) was performed with orange-red colloidal gold particles (GP) sensitized with anti-A or anti-B antibody or anti-H lectin (A chicken antiserum to O secretor saliva was also used as an anti-H reagent in some experiments). The boiled saliva specimens were serially diluted and dotted to the nitrocellulose membrane, which was equipped to a disposable aspiration kit, and the antibody- or lectin-coated GP was aspirated through the membrane. The stained saliva changed red by the immunologic binding of the GP. Test results could be obtained within some minutes. Detection limits of IMAT shown by saliva dilution were 1:100 to 1:10,000 for secretor salivas, whereas those for non-secretor salivas were 1:10 or lower. Second, a revised dot ELISA was performed by using the membrane aspiration device. The requisite time for this test (Dot ELISA-A) was 40 to 45 minutes, whereas that for conventional dot ELISA was 3 to 4 hours. The detection limits of the dot ELISA-A shown by the saliva dilutions were 1:1,000 to 1:100,000 for secretor salivas, whereas those for non-secretor salivas were 1:1,000 or below.

ABO Blood-Group System↗
PubMed1994

Thyrsiferyl 23-acetate is a novel specific inhibitor of protein phosphatase PP2A.

Thyrsiferyl 23-acetate (TF23A), a cytotoxic compound from marine red alga, has been shown to potently and specifically inhibit serine/threonine protein phosphatase 2A (PP2A) with IC50 values of 4-16 microM, depending on the enzyme concentration. TF23A did not affect activity of protein phosphatase 1 (PP1), 2B (PP2B), 2C (PP2C), or protein tyrosine phosphatases (PTP) up to 1 mM. It inhibited PP2A activity in a crude extract of a human T cell line, Jurkat cell, as well as the purified catalytic subunit. Thus, TF23A proved to be a novel useful probe for clearly distinguishing the activity of PP2A from those of the other protein phosphatases in crude cell extracts and identification of cellular processes that are regulated by PP2A.

Cell Line↗
PubMed1994

Rapid detection of human seminal plasma proteins by membrane aspiration test (MAT).

A new dot-immunobinding method, called the membrane aspiration test (MAT), was developed for the detection of human seminal plasma (HSP) antigens. Biological materials, such as macerated vaginal swabs or extracts from seminal stains, were dotted on a nitrocellulose membrane in a disposable aspiration kit. After the drying and aspiration of a quenching solution, a suspension of gold particles (GP) coated with an immune serum to HSP was aspirated through the nitrocellulose membrane. The dotted portion on the membrane turned red after several repeated aspirations, if HSP antigen was present. Results can be obtained within a few minutes. This test has broad applicability because of its rapidity and simplicity.

Animals↗
PubMed1993

IL-2 induces transient and specific decrease in cytosolic protein phosphatase PP1 activity in murine T cell lines.

In the IL-2-dependent murine cytotoxic T cell line CTLL-2, IL-2 induced a rapid and transient decrease in Co(2+)-trypsin-treated activity of protein phosphatase PP1. The PP1 activity declined to a minimum level, being 70% of control value, in 20 min after the addition of IL-2 but recovered to the control level within 45 min. The decrease of PP1 activity was dependent on IL-2 concentration and occurred specifically in cytosolic fraction. Similar alteration was observed in IL-2 sensitive murine T-lymphoblasts. Neither activity of protein phosphatase PP2A nor PP2C showed alteration during the IL-2 stimulation. These results suggest that PP1 plays an important role in early events of the intracellular growth signaling from the IL-2 receptor.

Animals↗
PubMed1993

Quantitation of IgE and carcinoembryonic antigen (CEA) by optical beam deflection (OBD) measurement of dot-immunobinding assay patterns visualized by an ELISA technique.

Dot-immunobinding assays of IgE and CEA were performed by a conventional dot-ELISA technique with diaminobenzidine staining, and the quantitative results were compared by densitometry and a new, spectroscopic, optical beam deflection (OBD) method using the same membrane. It was possible with the OBD method to detect quantities of these substances at least ten times smaller than with densitometry. Better intra-assay reproducibility for IgE and CEA measurements was obtained by the OBD method. The measurable ranges of the OBD method was broader than that of densitometry, because dark bands caused OBD in proportion to their color densities. When the dot-immunobinding assay with OBD measurement for CEA was also compared with a microtube ELISA using biotin-avidin conjugates, the sensitivities and reproducibilities of the two methods were found to be similar, with a correlation coefficient of 0.991.

Carcinoembryonic Antigen↗
PubMed1993

Hypercalcemia associated with all-trans-retinoic acid in the treatment of acute promyelocytic leukemia.

Recent reports have described clinical benefits of all-trans-retinoic acid (ATRA) therapy for acute promyelocytic leukemia (APL). This paper describes severe hypercalcemia (serum calcium: 18.7 mg/dl) in association with ATRA treatment in a 14 year old girl with APL. Serum parathyroid hormone (PTH) concentrations were normal (0.21 ng/ml), which precludes the possibility of primary hyperparathyroidism or ectopic PTH secretion as a cause of the hypercalcemia. As for the factors which can accelerate mineral resorption, there were no apparent increases in the levels of PTH-related protein (PTH-rP), prostaglandins and vitamin D metabolites. In our in vitro experiment, ATRA did not stimulate the leukemic cells to produce PTH-rP. We speculate that ATRA, like PTH, may increase osteoclastic activity and induce hypercalcemia.

Adolescent↗
PubMed1993

Alterations in activities of protein phosphatases PP1 and PP2A in T and B lymphocytes of autoimmune MRL/MpJ-lpr/lpr mice.

Activities of protein phosphatases PP1 and PP2A were determined in T and B lymphocytes of autoimmune-prone MRL/MpJ-lpr/lpr mice (MRL/lpr mice) and two control strains, MRL/MpJ-(+)/+ mice (MRL/+/+ mice) and C3H/HeJ mice. Potential PP1 activity, which was measured after treatment of cell extract with Co(2+)-trypsin, was much higher in T lymphocytes than B lymphocytes. However, no difference in the activity was observed between MRL/lpr mice and the controls. Spontaneous PP2A activity showed similar levels in T and B lymphocytes from normal mice, but potential PP2A activity, which was measured after treatment with 2-mercaptoethanol, was significantly higher in T lymphocytes from MRL/lpr mice than those from controls. No differences were detected in PP1 or PP2A activities in B lymphocytes. From these results, our previous data [Matsuzawa, S. et al. (1992) J. Biochem. 111, 472-477] demonstrating increases in potential activities of PP1 and PP2A in lymphoid tissues from autoimmune MRL/lpr mice can be interpreted as follows. 1) The increase in potential PP1 activity of the lymphoid tissues from MRL/lpr mice is caused by replacement of B lymphocytes by abnormal T lymphocytes, which accumulate in enormous numbers. 2) The increase of potential PP2A activity in the lymphoid tissues from MRL/lpr mice is caused by the increase in this activity in their T lymphocytes.

Animals↗
PubMed1993

Increases in protein phosphatase 2B activity in lymphoid tissues and T-lymphocytes of autoimmune MRL/MpJ-lpr/lpr mice.

The assay conditions for protein phosphatase 2B (PP2B) in crude extracts from mouse lymphoid tissues and lymphocytes were extensively investigated. Under the conditions elucidated, the PP2B activity was measured in autoimmune-prone MRL/MpJ-lpr/lpr mice (MRL/lpr mice) and two control strains, MRL/MpJ- +/+ mice (MRL/+/+ mice) and C3H/HeJ mice. In the control mice, PP2B activity was distinctly higher in spleen and thymus than brain and liver. PP2B activity was further elevated in spleen of MRL/lpr mice than in the controls. Furthermore, we observed a specific increase in PP2B activity in T lymphocytes from MRL/lpr mice as compared with in those from control mice. On the other hand, no alteration was observed in PP2B activity in B lymphocytes. These results suggest the involvement of PP2B in the abnormal signal transduction and proliferation of T lymphocytes in MRL/lpr mice.

Animals↗
PubMed1993

Investigation of latex agglutination with different immunoglobulin classes of antibodies by a scanning electron microscopy.

The conformation of passive latex agglutinations was investigated by a scanning electron microscopy. The complexes of 2,4-dinitrophenyl group (DNP) and bovine serum albumin (BSA) coated microspheres were incubated with monoclonal antibodies to DNP belonging to different classes of immunoglobulins. Examination with a scanning electron microscope demonstrated that the connectors between microspheres, agglutinated with IgE antibody, tended to take long filamentary forms; whereas the connectors of microspheres, agglutinated with IgG or IgM antibodies, formed shorter filaments. These phenomena were confirmed by the agglutination reactions of tick suspension-coated latex and allergic patients' sera. This morphologic difference is probably attributable to the unusually high elasticity of IgE antibody and is an evidence of the high biologic activity of the IgE antibodies. We assumed the processes of latex agglutination whereby the filamentary substances would develop, expecting that the processes could be models of in vivo and in vitro agglutination of cells.

Animals↗
PubMed1993

A rapid dot-blot method for species identification of bloodstains.

A very simple and rapid test for species identification is reported. Extracts of bloodstains were applied to a synthetic porous membrane and dried. The membrane was then quenched with glycine buffered saline containing BSA and Tween 20. A suspension of colloidal gold particles (GP) coated with rabbit antiserum to human IgG was poured onto, gently whirled and aspirated through the membrane. Spots from the human and monkey bloodstains became red, whereas those from other species of animals remained unstained. This test was completed within 3 to 4 min, and the antibody-coated GP reagent was prepared within 20 min using a very small quantity of antiserum. Cellulose acetate membranes of 0.45 microns or more in pore size were appropriate to this test.

Animals↗