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M Shirai

Publications and source records attributed to M Shirai.

At least 163 records · Page 9Linked to original sources
PubMed1995

In vitro selection analysis of trans-acting HDV ribozyme.

In order to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. After 10 generations, a randomized pool of trans-acting ribozymes accumulated in which the secondary structure of each ribozyme confirmed to the pseudoknot model and important bases in single-stranded regions were all conserved. We were surprised that mutated ribozymes derived from genomic sequence were changed to anti-genomic-like sequences. Further investigations of the most active variant confirmed that each mutated base was the most appropriate nucleotide at every position of HDV ribozyme.

Base Sequence↗
PubMed1994

A multifunctional expression vector for an anti-HIV-1 ribozyme that produces a 5'- and 3'-trimmed trans-acting ribozyme, targeted against HIV-1 RNA, and cis-acting ribozymes that are designed to bind to and thereby sequester trans-activator proteins such as Tat and Rev.

We previously constructed a multiribozyme expression vector by combining cis- and trans-acting ribozymes and we showed that several ribozymes, each directed against a different target in the HIV genome and acting independently in a 'shotgun' manner, markedly increased the efficiency of cleavage of HIV RNA in vitro [Ohkawa et al., Proc. Natl Acad. Sci. USA 90, 11302 (1993)]. However, the cis-acting ribozymes that had trimmed the 5' and 3' ends of each trans-acting ribozyme were designed merely to await for degradation by RNases when they were used in vivo. Since several trans-activator proteins are essential for viral replication of HIV-1, we wondered whether a decoy function could be coupled with the cleavage activity of ribozymes. We therefore introduced the TAR or the RRE sequence into the stem II region of each cis-acting ribozyme. When the activity of each resulting cis-acting ribozyme that had been endowed with the decoy function was examined in vitro, it was found to retain almost full trimming activity. Moreover, cis-acting ribozymes with either the TAR or the RRE sequence were shown to be able to trap Tat or Rev protein successfully. It is, therefore, possible to endow the stem II region with a specific protein-binding function without the loss of ribozyme function. Thus, cis-acting ribozymes, endowed with the decoy function, can first trim the 5' and 3' ends of each trans-acting ribozyme and are then still available for trapping trans-activator proteins possibly prior to their degradation by RNases when they are to be used in vivo. Furthermore, it is also expected that the reduction in production of HIV RNA that is achieved by sequestering the trans-activator proteins might provide the trans-acting ribozymes, targeted to HIV RNA, with a better chance of eliminating the remaining HIV RNA.

Base Sequence↗
PubMed1994

Helper-cytotoxic T lymphocyte (CTL) determinant linkage required for priming of anti-HIV CD8+ CTL in vivo with peptide vaccine constructs.

CTL are a critical component of protective immunity against viral infections, but requirements for in vivo priming of CTL are not completely understood. Covalent linkage of a helper determinant to a CTL determinant, analogous to that required for cognate help for antibody production, does not appear to be necessary in vitro, but its necessity has not been extensively explored in vivo, especially at a molecular level. We previously defined peptides encompassing multideterminant regions of HIV-1 gp160 (cluster peptides) recognized by Th from mice and humans of multiple MHC types. To investigate the requirement for Th in the development of CTL in vivo, in the context of developing a synthetic peptide vaccine for HIV active in multiple strains of mice, we immunized with compound peptides representing an immunodominant CTL epitope, P18, of gp160, co-linearly synthesized at the C-terminus of three cluster peptides. Spleen cells from compound-peptide-immunized mice of three MHC haplotypes sharing the Dd class I MHC molecule but with different class II molecules exhibited enhanced gp160-specific CD8+ CTL activity and CD4+ Th. In contrast, immunization with P18 alone or a mixture of cluster peptide and P18 elicited only marginal CTL activity. These results imply a requirement for determinant linkage in CTL induction in vivo similar to that already well recognized for cognate help for antibody induction. The results also define promising peptide HIV vaccine candidates for induction of CTL, as well as neutralizing antibodies, in diverse MHC types.

AIDS Vaccines↗
PubMed1994

Diameter and flow velocity changes of feline small pulmonary vessels in response to sympathetic nerve stimulation.

Using an X-ray television system, we measured directly changes in the internal diameter (ID), flow velocity, and volume flow of the small pulmonary vessels (100-500 microns ID) in response to electrical sympathetic nerve stimulation (SNS) in anaesthetized cats before and after adrenergic receptor blockade. Flow velocity was obtained by measuring the distance that the leading edge of the contrast medium moved per 0.1 s in the small arteries. Volume flow was obtained from the product of flow velocity and cross-sectional area calculated from the ID of the small arteries. SNS was accomplished with 10- to 15-V square-wave pulses of 2-ms duration at 20-30 Hz for 20-s periods. In response to SNS, arterial ID decreased significantly by 8-13% in the 200- to 500-microns vessels but not in the 100- to 200-microns vessels. In the veins, on the other hand, there was no significant ID decrease in any of the 100- to 500-microns vessels. After alpha-receptor blockade (phentolamine, 2 mg/kg i.v.), there were significant ID increases (4-9%) in the 100- to 500-microns arteries in response to SNS, the maximum increases being in the 100- to 200-microns arteries. After beta-blockade (propranolol, 2 mg/kg i.v.), the ID decrease due to SNS in the 200- to 500-microns arteries was enhanced (24-27%) and, in addition, the 100- to 200-microns arteries exhibited a significant ID decrease (18%). Combined alpha- and beta-blockade completely abolished the ID decrease due to SNS. In the veins, on the other hand, no ID change occurred even after alpha- or beta-blockade.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Cross-reactivity and neutralizing ability of monoclonal antibodies against microcystins.

Monoclonal antibodies (MAbs) against the microcystin-leucine-arginine variant (MCYST-LR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. The specificity of the MAbs and their ability to neutralize the toxin were investigated by an indirect enzyme-linked immunosorbent assay (ELISA) and by a neutralizing test in mice, respectively. All MAbs reacted with MCYST-LR and also with the microcystin-arginine-arginine variant (MCYST-RR), 3,7-didesmethylmicrocystin (MCYST-3, 7-dDMLR) and 7-desmethylmicrocystin (MCYST-7-DMLR). Furthermore, the antibodies reacted with cell-extracts of toxic and non-toxic M. aeruginosa strains. The MAbs can apparently recognize the common configuration, but not the variant-specific structure, in the microcystin molecules. The non-toxic strains apparently contain some substance(s) related antigenically to microcystin. The in vivo toxin-neutralizing ability of MAbs was minimal.

Animals↗
PubMed1994

An epitope in hepatitis C virus core region recognized by cytotoxic T cells in mice and humans.

Several cytotoxic T-lymphocyte (CTL) epitopes have been defined in hepatitis C virus (HCV) proteins. CTL may play an important role in the control of infection by HCV. Here, we identify a highly conserved antigenic site in the HCV core recognized by both murine and human CTL. Spleen cells from mice immunized with a recombinant vaccinia virus expressing the HCV core gene were restimulated in vitro with 11 peptides from the core protein. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 129-144). This conserved epitope was presented by a murine class I major histocompatibility molecule (H-2Dd) to conventional CD4- CD8+ CTL mapped by using transfectants expressing Dd, Ld, or Kd, but was not seen by CTL restricted by H-2b. The murine epitope was mapped to the decapeptide LMGYIPLVGA. The same 16-residue peptide was recognized by CTL from two HCV-seropositive patients but not by CTL from any seronegative donors. CTL from two HLA-A2-positive patients with acute and chronic hepatitides C recognized a 9-residue fragment (DLMGYIPLV) of the peptide presented by HLA-A2 and containing an HLA-A2-binding motif, extending only 1 residue beyond the murine epitope. Therefore, this conserved peptide, seen with murine CTL and human CTL with a very prevalent HLA class I molecule, may be a valuable component of an HCV vaccine against a broad range of HCV isolates. This study demonstrates that the screening for CTL epitopes in mice prior to human study may be useful.

Amino Acid Sequence↗
PubMed1994

beta-Adrenergic mechanisms attenuated hypoxic pulmonary vasoconstriction during systemic hypoxia in cats.

In this study, we examined how locally mediated hypoxic pulmonary vasoconstriction is modulated by autonomic nervous system activation during global alveolar hypoxia (GAH) accompanied by systemic hypoxemia. Using an X-ray television system on the in vivo cat lung, we measured changes in the internal diameter (ID) during GAH and regional alveolar hypoxia (RAH) without systemic hypoxemia in identical small pulmonary arteries and veins (100-600 microns ID). We also analyzed the effects of the autonomic nervous system blockade on the hypoxic ID changes. During GAH the ID of the arteries reduced by 5 +/- 1 and 3 +/- 1% with 10 and 5% O2 inhalations, respectively, whereas during RAH the arterial ID reduced by 12 +/- 1 and 18 +/- 1% with 10 and 5% O2 inhalations, respectively. The magnitude of the ID reduction was significantly smaller during GAH than during RAH. After pretreatment with propranolol, however, GAH induced large ID reductions (16 +/- 1 and 23 +/- 1% with 10 and 5% O2 inhalations) with patterns very similar to those seen during RAH. Phentolamine and atropine had no effect on the response during GAH. The ID reductions during RAH, on the other hand, were unaffected by all the blockers. The results indicate that, in the cat, alveolar hypoxia per se acts locally to constrict the small pulmonary vessels and that the hypoxic vasoconstriction is attenuated by a beta-receptor-mediated vasodilator effect during GAH with systemic hypoxemia. In addition, we found that, after adrenalectomy plus ganglion blockade with hexamethonium bromide, the GAH-induced ID reduction with 5% O2 inhalation was enhanced from 3 to 19%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1994

Effect of maternal paraquat administration on the fetal ductus arteriosus in the rat.

The ductus arteriosus (DA) in the fetal rat was calibrated using a whole-body freezing method, 3 hr after maternal treatment with paraquat at a dose of 25 mg/kg of body weight on days 19-21 of gestation. On days 20 and 21, the DA was significantly constricted. The DA was also significantly constricted on day 19 2/3 but not on day 19 1/2. It is concluded that paraquat has a constrictive effect on the DA and that the onset of this effect occurs in the first half of day 19 of gestation.

Animals↗
PubMed1994

In vitro study of teratogenic effects of caffeine on cultured rat embryos and embryonic cells.

The teratogenic potential of caffeine was examined in vitro by a whole embryo culture system (WECS) and an embryonic cell culture system (micromas teratogen assay: MTA) in the rat. In the WECS, hyperemia of the tail, and a reduction of the placental size was induced by caffeine at concentrations higher than 50 micrograms/ml; hypoplasia of the forelimb bud was induced at concentrations higher than 100 micrograms/ml; hematoma in the yolk sac and dysmorphogenesis of the fore- and hind-limb buds, prosencephalon and tail were induced by 200 micrograms/ml caffeine. In the MTA, even with 200 micrograms/ml caffeine, the toxicological parameters obtained by proliferation and differentiation assays of the midbrain and limb bud cells were almost the same as in the control. In conclusion, caffeine induced various morphological anomalies, but did not affect proliferation or differentiation of cells in these experimental systems.

Abnormalities, Drug-Induced↗
PubMed1994

Constrictive effects of paraquat on the ductus arteriosus in fetal rats.

Rats on day 21 of pregnancy received a subcutaneous injection of paraquat (2, 7 or 25 mg/kg). The inner diameter of the ductus arteriosus (DA) of their fetuses was measured using the whole-body freezing method 1, 3, 6 and 24 hours after injection. The 7 mg/kg dose group showed a significant constriction of the DA 3 hours after paraquat treatment. The 25 mg/kg dose group showed a significant constriction 3 and 6 hours after treatment. The constrictive effect was dose-dependent, that is, the magnitude of constriction observed 3 hours after paraquat injection was greater in the 25 mg/kg dose group than in the 7 mg/kg dose group. The 2 mg/kg dose group showed no significant change in the inner diameter of the DA at any point of measurement (1, 3, 6 and 24 hours after injection), compared to the saline treated controls. These results indicate that paraquat induces constriction of the fetal rat DA in a dose-dependent manner when administered in late stages of gestation, and that this effect of paraquat is not seen at a dose level of 2 mg/kg.

Animals↗
PubMed1994

Weekly low-dose methotrexate therapy for sarcoidosis.

Low-dose methotrexate therapy has been used to treat a variety of chronic inflammatory diseases; a few studies have discussed the efficacy of this therapy for sarcoidosis. We present a patient with symptomatic sarcoidosis who was successfully treated with low-dose methotrexate after being refractory to steroids. This patient showed significant clinical improvement without development of any severe side effects. These findings suggest that low-dose methotrexate therapy may be a useful alternative treatment for sarcoidosis.

Adult↗
PubMed1994

[The significance of biochemical data of patients with sarcoidosis].

The clinical laboratory has a significant role in sarcoidosis. We summarized the biochemical data of laboratory tests in serum of patients with sarcoidosis. To clarify their importance, we put emphasis on the following aspects, including: 1. The data reflecting pathophysiology of sarcoidosis, such as angiotensin converting enzyme, lysozyme, adenosine deaminase, beta 2-microglobulin and intercellular adhesion molecule-1, 2. The data resulting from organ involvement, such as amylase, LDH, and Ca, 3. The data serving as an indicator of disease activity, 4. The data related to prognostic outcome, Keeping these differences in mind helps us make the best use of the clinical data of sarcoidosis.

Adenosine Deaminase↗
PubMed1994

Safer colonoscopic polypectomy with local submucosal injection of hypertonic saline-epinephrine solution.

BACKGROUND: In view of the popular acceptance of the adenoma-carcinoma sequence, endoscopic polypectomy is indicated for the removal of colorectal adenomas. Larger or sessile lesions should be removed by an experienced endoscopist, but complications such as hemorrhage and perforation still may occur. METHODS: To render the removal of sessile lesions feasible by endoscopic polypectomy and to reduce the risk of complications, we attempted local submucosal injection of hypertonic saline-epinephrine solution [(HSE) a mixture of 4.7% sodium chloride and 0.005% epinephrine] before electrosurgical snare excision; 0.5-2 ml HSE were injected into the base and immediate vicinity of polyps to produce mucosal bulging. RESULTS: Between August 1990 and April 1992, 645 polyps in 403 patients were removed by this method; sessile lesions could be more readily removed. Of these, 17 (2.6%) revealed invasive carcinoma and 19 (3.0%) carcinoma in situ. The largest sessile polyp removed was 40 mm in diameter and 7 mm in height and contained a carcinoma in situ. Even a 6-mm depressed adenoma and two rectal carcinoid tumors could be excised completely. No serious complications were encountered with this method. CONCLUSION: We conclude that local injection of HSE makes the endoscopic removal of colorectal polyps much easier and safer.

Adenocarcinoma↗
PubMed1993

Multiple pathways are involved in the extracellular processing of MHC class I-restricted peptides.

T cell stimulation by certain class I-restricted antigenic peptides, such as the HIV 1 gp160-derived peptide, P18, requires peptide processing by angiotensin-1 converting enzyme (ACE) in FCS. We observed that longer versions of P18 and the murine cytomegalovirus pp89-derived core peptide, pMCMV, which could stimulate T cell hybridomas in FCS, were not as sensitive to the ACE inhibitor captopril as P18. Using cell-free soluble murine class I MHC molecules and protease inhibitors, we found that there are pathways of differing efficiency that use enzymes other than ACE for the proteolytic processing of peptides in serum. The kinetics of the generation of T cell stimulatory activity among P18 variant peptides in serum differed with peptide length, and with the nature of amino and COOH-terminal extensions. Such processing occurs in human plasma as well as in FCS. The understanding of this processing, its kinetics, and its inhibitors can lead to better design of peptide-based therapies, including vaccines.

Amino Acid Sequence↗
PubMed1993

Preferential V beta usage by cytotoxic T cells cross-reactive between two epitopes of HIV-1 gp160 and degenerate in class I MHC restriction.

The T cell response to HIV-1 gp160 is among the most thoroughly studied immune responses to HIV-1 products. In our previous work, the MHC class I molecule Dd as well as H-2u, p, and q, were found to present P18 and HP53, two determinants of HIV-1 gp160, to CD8+ CTL in mice. We have studied the TCR V beta chain expression in CTL lines, either cross-reactive for these two peptides or specific for P18 alone, in these four different MHC haplotypes. The usage of V beta in T cells showing cross-reaction between these two peptides was remarkably conserved (primarily V beta 8 family, with some use of V beta 14) despite the extensive TCR V beta diversity of the non-cross-reactive CTL, which did not use V beta 8 or 14. This correlation of V beta usage with fine specificity was consistent in H-2d, u, and p (p < 0.01), but not in H-2q. The correlation of V beta use with peptide fine specificity independent of MHC restriction was unexpected. The strong predominance of V beta 8 family TCR was all the more surprising in view of the finding that mice bearing a genomic deletion of V beta 8 can still produce T cells with the cross-reactive phenotype, implying that other V beta chains can produce this specificity. We therefore asked whether the complexes of P18 with H-2d, p, and u are recognized as identical, and observed the surprising result that H-2d, p, and u cells mutually cross-present the peptides P18 and HP53 to allogeneic CTL lines and individual clones of each of the other haplotypes, whereas none of these cross-present to H-2q CTL, nor do H-2q targets present to CTL of the other haplotypes. This degeneracy of MHC restriction is novel for class I molecules. Moreover, the observed restriction in V beta usage occurs only in the unique set of CTL that exhibit both peptide-cross-reactive fine specificity and MHC allogeneic cross-presentation. The observation that a strain of mice in which the V beta 8 family is genomically deleted can still make CTL of this phenotype using another V beta demonstrates the plasticity of the class I MHC-restricted repertoire when the dominating receptor is not available.

Amino Acid Sequence↗
PubMed1993

Helminth infection results in decreased virus-specific CD8+ cytotoxic T-cell and Th1 cytokine responses as well as delayed virus clearance.

During the time of egg deposition, schistosome-infected mice exhibit a downregulation in interleukin 2 and interferon gamma production toward parasite antigens, mitogens, and foreign nonparasite protein antigens. To determine whether this imbalance in cytokine response would impact on CD8+ cytotoxic T-lymphocyte (CTL) responses, as well as on immune clearance of viral infections, we challenged Schistosoma mansoni-infected BALB/c mice, when cytokine imbalance was prominent, with a recombinant vaccinia virus expressing human immunodeficiency virus type 1 gp160. In contrast to control vaccinia-infected animals, S. mansoni plus vaccinia-infected mice did not produce significant Th1 cytokine responses upon in vitro stimulation with recombinant gp120, consistent with previous results for nonparasite antigens. However, more striking was the downregulation of the virus-specific CTL response not previously studied. Spleen cells from vaccinia-infected control mice displayed strong CD8+ cytolytic activity against gp160-transfected fibroblasts and fibroblasts pulsed with a peptide (P18) representing a CTL epitope of gp160. In contrast, mice coinfected with S. mansoni and vaccinia manifested absent or markedly reduced in vitro CTL activity even in the presence of exogenous interleukin 2. To determine whether this immune dysregulation might impact on viral clearance, we measured virus titers in tissues as a function of time. Mice infected with vaccinia virus alone rapidly cleared the virus, whereas in animals coinfected with S. mansoni, viral clearance was delayed by as much as 3 weeks in the liver and by several days in the spleen and lungs. These observations suggest that helminth infection may influence immune responses to concurrent viral infections.

Animals↗