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Biomedical subjects

M Azuma

Publications and source records attributed to M Azuma.

At least 91 records · Page 5Linked to original sources
PubMed2000

Treatment with anti-CD86 costimulatory molecule prevents the autoimmune lesions in murine Sjögren's syndrome (SS) through up-regulated Th2 response.

Intraperitoneal administration with anti-CD86 (B7.2) MoAb into the murine model for primary SS in NFS/sld mutant mice resulted in dramatically inhibitory effects on the development of autoimmune lesions, while no significant effects were observed when the mice were administered with anti-CD80 (B7.1) MoAb. We found that spleen cells in the murine SS model treated with anti-CD86 MoAb showed a significant impairment of autoantigen-specific T cell proliferation. T cell activation markers (CD44high, CD45RBlow, Mel-14low) were significantly down-regulated in the spleen cells gated on CD4 in anti-CD86-treated mice. We detected a higher level of cytokine production of IL-4 from splenic T cells in anti-CD86-treated mice, but not of IL-2, and interferon-gamma (IFN-gamma), compared with those in the anti-CD80- and PBS-treated SS model. Moreover, serum autoantibody production against alpha-fodrin autoantigen was almost entirely suppressed in anti-CD86-treated mice. These data provide strong evidence that in autoimmune exocrinopathy resembling SS in NFS/sld mutant mice, the CD86 costimulatory molecule plays a crucial role in the initiation and subsequent progression of Th1-mediated autoimmunity in the salivary and lacrimal glands.

Animals↗
PubMed2000

Tumour rejection by gene transfer of 4-1BB ligand into a CD80(+) murine squamous cell carcinoma and the requirements of co-stimulatory molecules on tumour and host cells.

NRS1 is a murine squamous cell carcinoma that constitutively expresses the co-stimulatory molecule CD80 at a high level yet grows as a tumour in syngeneic C3H mice. We examined the effect of gene transfer of the 4-1BB ligand (4-1BBL) into NRS1 cells. Introduction of the 4-1BBL gene efficiently elicited anti-tumour immune responses in syngeneic mice which acquired specific immunity against wild-type tumour. T-cell depletion studies showed that CD8(+), but not CD4(+) T cells were essential for tumour eradication. Our results suggest that the transduced 4-1BBL is more effective than the spontaneously expressed CD80 for generation of primary anti-tumour CD8(+) T-cell responses. In addition to CD80 and CD86, the host-derived 4-1BBL is also involved in the secondary anti-tumour responses. This study indicates the complicated contribution of 4-1BBL, CD80 and CD86 on tumour and host cells in anti-tumour immune responses and a possible therapeutic application of 4-1BBL for human tumour vaccination and gene therapy.

4-1BB Ligand↗
PubMed2000

Impaired heart function and noradrenaline release after ischaemia in stroke-prone spontaneously hypertensive rats.

1. Stroke-prone spontaneously hypertensive rats (SHRSP) are a strain of rat that exhibit severely high blood pressure and stroke attacks at an early age, but their heart function in vitro has seldom been studied in detail. Although the activity of the sympathetic nervous system is known to increase after myocardial ischaemia, there is little information about the cardiac release of noradrenaline (NA) associated with heart function after ischaemia in SHRSP. The aim of the present study was to examine heart function and cardiac NA release after ischaemia in SHRSP. 2. Isolated hearts of 4- and 8-month-old SHRSP and age-matched Wistar-Kyoto (WKY) rats were perfused in a working heart preparation and were subjected to 30 min ischaemia followed by 30 min reperfusion. Heart function and coronary flow were monitored throughout the experiment. Coronary effluent was collected for determination of NA using high-performance liquid chromatography coupled with electrochemical detection. 3. Under baseline conditions, cardiac output of 4-month-old SHRSP was slightly but significantly decreased compared with that of WKY rats (P < 0.05), although coronary flow was maintained normally at this age. Eight-month-old SHRSP showed a further impairment of systolic heart function, with lower coronary flow and higher coronary vascular resistance under baseline conditions. Elevated left ventricular end-diastolic pressure was evident in SHRSP at both ages before ischaemia. Heart function was severely damaged after 30 min global ischaemia in SHRSP from both age groups. Stroke-prone spontaneously hypertensive rats also showed lower coronary flow and higher coronary vascular resistance during reperfusion. 4. Coronary NA was not detectable in WKY rats or SHRSP at 4 months of age under baseline conditions. In 8-month-old SHRSP, pre-ischaemic NA release was significantly higher than that in age-matched WKY rat controls. The concentration of NA in the coronary effluent of SHRSP during reperfusion was also significantly higher than that of WKY rats at both ages. 5. These data demonstrate that SHRSP have early impairment of both systolic and diastolic heart function compared with WKY rats. Severe damage of heart function and coronary flow after ischaemia in SHRSP was accompanied with an increased release of NA, which may play a harmful role in heart function impairment in SHRSP after ischaemia.

Animals↗
PubMed2000

Involvement of cysteine proteases in bFGF-induced angiogenesis in guinea pig and rat cornea.

Overexpression and activation of matrix metalloprotease (MMP) have been implicated in angiogenesis. However, the involvement of cysteine proteases, such as calpains (EC 34.22.17), is obscure. Thus, the purpose of this experiment was to study the involvement of cysteine proteases in angiogenesis induced by basic fibroblast growth factor (bFGF) in guinea pig and rat corneas using cysteine protease inhibitors. Sustained-release polymers containing bFGF were implanted into guinea pig and rat corneas to induce angiogenesis. For treatment of corneal angiogenesis, polymers containing cysteine protease inhibitors, leupeptin or SJA6017, were also implanted into corneas. Using the slit lamp, the corneas were observed for nine days after polymer implantation. Soluble proteins and albumin levels were used as markers of corneal injury by angiogenesis. bFGF induced angiogenesis in guinea pig and rat corneas. In guinea pig cornea, wet weight, the amount of soluble protein and albumin was highest at four days after bFGF-containing pellet implantation. In rat cornea, the amount of soluble protein and albumin was highest at six days, and wet weight increased within four days. One hundred nmole of leupeptin showed a tendency to reduce bFGF-induced angiogenesis in guinea pig cornea, and 10 nmole of SJA6017 was effective in reducing bFGF-induced angiogenesis in rat cornea, although SJA6017 showed a stronger effect than leupeptin. Ten nmole of SJA6017 significantly reduced the number of new blood vessels. These data suggested involvement of cysteine proteases in angiogenesis in guinea pig and rat cornea.

Albumins↗
PubMed2000

Long-term regulation of catecholamine formation by ouabain in cultured bovine adrenal chromaffin cells.

The long-term effects of ouabain, an inhibitor of Na+/K+ -ATPase, on catecholamine formation in cultured bovine adrenal chromaffin cells were examined. The increase in [14C]catecholamine formation from [14C]tyrosine induced by ouabain was dependent on incubation time, and its maximal effect was observed after incubation for 8 h. The stimulatory effect of ouabain was concentration dependent (10-300 nM), causing maximal stimulation at 300 nM. The formation of [14C]catecholamines induced by ouabain was not increased by incubation with [14C]DOPA instead of [14C]tyrosine. Ouabain-induced [14C]catecholamine formation was influenced by decreases in extracellular Ca2+ concentration, but not by the presence of cycloheximide or actinomycin D. These results suggested that ouabain stimulates continuous activation of hydroxylation of tyrosine through a Ca2+ -dependent mechanism in cultured bovine adrenal chromaffin cells.

Animals↗
PubMed2000

The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection.

The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRDelta41 strain) and its revertant (the VRDelta41R strain). In the mouse encephalitis model, the replication of strain VRDelta41 was inhibited after 2 days post-infection, resulting in low virulence, by gamma-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRDelta41-inoculated brains, activate and induce the migration of gamma-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (IL)-1beta, IL-8 and macrophage inflammatory protein-1alpha by VRDelta41 infection were observed. Moreover, the VRDelta41 strain showed 20- and 5-fold higher sensitivity to interferon-alpha and -beta compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-alpha and -beta.

Animals↗
PubMed2000

Rapid phenotypic characterization method for herpes simplex virus and Varicella-Zoster virus thymidine kinases to screen for acyclovir-resistant viral infection.

A rapid phenotypic screening method for herpes simplex virus (HSV) and varicella-zoster virus (VZV) thymidine kinase (TK) genes was developed for monitoring acyclovir-resistant viruses. This method determines the biochemical phenotype of the TK polypeptide, which is synthesized in vitro from viral DNA using a procedure as follows. The TK gene of each sample virus strain is amplified and isolated under the control of a T7 promoter by PCR. The PCR products are transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Using this method, enzymatic characteristics and the size of the TK polypeptides encoding HSV and VZV DNA were defined in less than 2 days without virus isolation. The assay should be a powerful tool in monitoring drug-resistant viruses, especially in cases in which virus isolation is difficult.

Acyclovir↗
PubMed2000

Comparison of polymorphism of thymidine kinase gene and restriction fragment length polymorphism of genomic DNA in herpes simplex virus type 1.

The polymorphism of the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) was analyzed and was compared with the restriction fragment length polymorphism (RFLP) of the whole genome to evaluate the relative efficiency of the TK gene as a potential probe for identification and discrimination of HSV-1. The effectiveness of using the polymorphism of the TK gene in classifying HSV-1 strains was comparable to that of RFLP analysis of 66 sites, suggesting that TK gene sequencing may have important applications in epidemiological studies of HSV-1.

DNA, Viral↗
PubMed2000

Effects of Ethanolamine as a nitrogen source on hydrogen production by Rhodobacter capsulatus.

Ethanolamine was examined as a nitrogen source in the production of hydrogen by Rhodobacter capsulatus ST-410, a hydrogenase-deficient mutant of the strain B-100. It was found that ethanolamine supports cell growth as the sole nitrogen source and permits a large amount of hydrogen evolution, detected at 138 micromol/ml-culture from 3.5 mM ethanolamine and 30 mM DL-malate. The amount corresponded to a stoichiometric yield of 77% and was close to that obtained from 7.0 mM L-glutamate and 30 mM DL-malate. The hydrogen evolution rate per unit biomass (cells) was higher than that with L-glutamate, and the cells grown with ethanolamine had higher nitrogenase activity than the cells grown with L-glutamate. In terms of bioconversion of cellulosic and hemicellulosic biomass to hydrogen, D-glucose, D-xylose, and D-cellobiose were tested as substrates. The results indicated that those sugars permit a large evolution of hydrogen through cultivation with ethanolamine as a nitrogen source. For instance, the cells grown with 3.5 mM ethanolamine evolved hydrogen of 289 micromol/ml-culture (80% yield) from 30 mM D-glucose under a controlled pH of 6.4 to 6.9.

Ethanolamine↗
PubMed2000

Lp85 calpain is an enzymatically active rodent-specific isozyme of lens Lp82.

PURPOSES: To clone and sequence the cDNA for Lp85 calpain from young rat lens, and to test for Lp85 protein expression and proteolytic activity. METHODS: RT-PCR and molecular cloning were performed on total RNA from 12 day-old rats. Lp85 protein expression was visualized by immunoblotting using a specific antibody developed to the unique peptide sequence in Lp85. Proteolytic activity was assessed by casein zymography. Transient expression of Lp85 and previously characterized lens-specific calpain Lp82 were separately performed in mammalian COS-7 cells. RESULTS: The 2410-bp cDNA for rat lens Lp85 encoded a protein of 737 amino acid residues with a calculated molecular weight of 85.0 kDa and a predicted pI of 5.67. The amino acid sequence of Lp85 was identical to Lp82 except for an insert region of 28 amino acids in domain IV of the calcium-binding region. mRNA and protein for Lp85 were present only in rat and mouse lenses and not in other tissues or species. Lp85 protein concentrations were highest in the nuclear region, most concentrated in the insoluble fraction, disappeared with lens maturation, and Lp85 exhibited migration similar to Lp82 on native PAGE gels. Lp85 was enzymatically active when expressed in COS-7 cells. CONCLUSIONS: Lp85 is a newly classified, lens- and rodent-specific, enzymatically active, member of the AX1 (alternative exon 1) subclass of calpains. In conjunction with Lp82 and m-calpain in lens, Lp85 may be responsible for proteolysis during normal lens development and maturation or during cataract formation in young rodents.

Amino Acid Sequence↗
PubMed2000

Evaluation of portosystemic collaterals by SPECT imaging after endoscopic variceal sclerotherapy: usefulness for predicting recurrence.

UNLABELLED: Bleeding from esophageal varices is a major cause of morbidity and mortality in cirrhotic patients. Identification of patients at high risk for bleeding is particularly important. The aim of this study was to determine whether detection of portosystemic collaterals by SPECT could predict the outcome of endoscopic injection sclerotherapy of esophageal varices and be useful for selecting appropriate therapy. METHODS: Sixty-two patients with liver cirrhosis who were considered at high risk of bleeding were treated with endoscopic injection sclerotherapy. Endoscopy was performed every 3 mo after therapy or until bleeding occurred. Before and within 2 wk after therapy, tomographic images of intra-abdominal blood pool were constructed by SPECT. RESULTS: Before therapy, the following portosystemic collateral routes were observed: coronary veins in 53 (85.5%) of 62 patients, short gastric veins in 8 patients (12.9%), splenorenal shunts in 10 patients (16.1%), and paraumbilical veins in 6 patients (9.7%). Patients positive for imaging of coronary veins were divided into 3 groups on the basis of changes in images after therapy: complete responders (n = 17), whose coronary vein images disappeared completely; partial responders (n = 18), whose images became smaller; and nonresponders (n = 18), whose images did not change significantly before or after therapy. The rates of recurrence after endoscopic injection sclerotherapy until 6 mo in complete responders (4/17, 23.5%) and partial responders (7/18, 38.9%) were significantly less (P < 0.05) than that in nonresponders (11/13, 84.6%). The rate of recurrence of esophageal varices until 6 mo in nonresponders treated with additional submucosal injection sclerotherapy (1/5, 20.0%) was significantly less (P < 0.05) than that in nonresponders without additional submucosal injection sclerotherapy (11/13, 84.6%). CONCLUSION: Abdominal blood-pool SPECT, a noninvasive method, is useful for evaluating the therapeutic effectiveness of endoscopic sclerotherapy, for predicting the recurrence of varices, and for selecting appropriate management after sclerotherapy.

Collateral Circulation↗
PubMed2000

Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice.

PURPOSE: The purpose of the present experiments was to provide a biochemical mechanism for the involvement of lens-specific calpain Lp82 in experimental cataractogenesis in mice. METHODS: Nuclear cataracts were produced by culturing lenses from 4-week-old mice and rats in calcium ionophore A23187 or by injection of buthionine sulfoximine (BSO) into 7-day-old mice. Casein zymography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, calcium determinations, in vitro precipitation, and cleavage site analysis by mass spectrometry were performed on lens samples. RESULTS: Amino acid sequences for Lp82 were found to be highly conserved in lenses from mouse to cow, and expressed Lp82 proteolytic activity was high in the mouse and rat. Lenses from mice were more susceptible to A23187-induced cataract and BSO cataracts than rats. Both types of cataracts showed rapid elevation of calcium, activation of Lp82 and m-calpain, and proteolysis of crystallins. Lp82 caused in vitro precipitation of crystallins; and in contrast to m-calpain, Lp82 truncated only the first five amino acids from the C-terminus of alphaA-crystallin. CONCLUSIONS: Under pathologic conditions of massive elevation of lens calcium found in young rodent lenses, overactivation of Lp82 and m-calpain leads to rapid truncation of crystallins at both common and unique cleavage sites, precipitation of truncated crystallins, and cataract.

Amino Acid Sequence↗
PubMed2000

Influence of specific regions in Lp82 calpain on protein stability, activity, and localization within lens.

PURPOSE: To determine the influence of specific regions within Lp82 calpain on protein stability, enzymatic activity, and localization within lens and to test the influence of an Lp82 knockout mouse on normal maturational proteolysis in lens. METHODS: DNA constructs for Lp82 and Lp82-related proteins were subcloned into the pcDNA 3.1 vector. The constructs contained a substitution of the novel sequence (NS) region from p94 for the AX1 N-terminal region of Lp82 and insertions of the p94 IS1 and IS2 regions into Lp82. Transient expression of these Lp82-related proteins was performed in COS-7 mammalian cells. Immunoblotting and casein zymography were used to measure protein stability and enzymatic activity of the expressed proteins. Homologous recombination was used to knock out p94 gene expression and p94 splice variants such as Lp82 and Lp85 in the lenses of 10-day-old mice. Confocal microscopy revealed the immunohistochemical localization Lp82 and Lp85 within lens. RESULTS: Insertion of IS1 into Lp82 resulted in a lack of stable protein and loss of enzymatic activity. In contrast, substitution of the NS region for AX1 and insertion of IS2 into Lp82 had no effect on the stability of the Lp82-related proteins. p94 knockout mice at 10 days of age exhibited a total absence of Lp82 activity in the lens but normal activity for the separate mu- and m-calpain gene products. Calcium-induced in vitro proteolysis was retarded in these Lp82/p94 knockout lenses. Lp82 and Lp85 immunostaining was intense throughout the cytoplasm of the cortical and nuclear fibers of newborn mouse lenses with little staining in the epithelium. In contrast, immunostaining for the ubiquitous m-calpain was highest in the epithelium and bow region, with much lower levels in the nucleus. The naturally occurring IS3 insert in Lp85 also promoted the association of Lp85 with the perinuclear region of the nucleated lens fibers. CONCLUSIONS: The lack of the IS1 region in Lp82 accounts for the stability and abundance of enzymatically active Lp82 protein in rodent lenses. Conversely, the presence of the IS1 region is responsible for the lability of p94 and Rt88 calpains in muscle and retina, respectively. The insert in Lp85 may promote membrane association. A consequence of the specific loss of Lp82 in the lens may be to retard normal maturational proteolysis.

Animals↗
PubMed2000

Identification and characterization of a retina-specific calpain (Rt88) from rat.

PURPOSE: To identify and characterize a newly discovered calpain termed Rt88 from rat retina. METHODS: Rt88 in retina under normal physiological conditions was characterized in Sprague-Dawley rats of various ages by competitive RT-PCR, Northern blot analysis, cDNA cloning and sequencing. Recombinant Rt88 was expressed in the baculovirus system and characterized by casein zymography and immunoblotting. RESULTS: Rt88 was sequenced and found to be similar to muscle calpain p94 except for three differences. A different exon 1 (as in lens Lp82 calpain) was present, and exons 15 and 16 in the unique IS2 region of muscle p94 were deleted. Of eleven tissues studied, mRNA for Rt88 was found only in retina where Rt88 increased with maturation and then remained constant. Casein zymography showed that rRt88 was proteolytically active after activation by calcium, but intact rRt88 was rapidly broken due to the presence of the IS1 region in domain II. CONCLUSIONS: Rt88 is a retina-specific, calcium activated protease from the calpain superfamily (EC 3.4.22.17) of cysteine proteases. Rt88 is a recently identified member of the AX1 subfamily of calpains showing alternative exon 1 usage. So far, all AX1 subfamily members are from eye. Rt88 may perform specific proteolytic functions during development, normal turnover, or pathological degeneration of retinal proteins.

Amino Acid Sequence↗