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Biomedical subjects

T Igarashi

Publications and source records attributed to T Igarashi.

At least 19 recordsLinked to original sources
PubMed1999

Sequential analysis of apoptosis induction in peripheral blood mononuclear cells and lymph nodes in the early phase of pathogenic and nonpathogenic SIVmac infection.

To investigate the role of apoptosis in the early phase of HIV infection, we used macaques infected with simian immunodeficiency virus strain mac (SIVmac) as a primate model and examined sequentially the characteristics of apoptosis of lymphocytes in peripheral blood mononuclear cells (PBMCs) and lymph nodes in the early phase of SIVmac infection. Five macaques infected with a pathogenic strain of SIV, SIVmac239, were analyzed during the first 4 weeks after infection. Peripheral CD4+ and CD8+ cells transiently decreased at 1 week postinfection. The percentage of apoptotic cells in cultured PBMCs increased from about 2 weeks postinfection. The number of apoptotic cells in lymph node sections was higher on days 13 and 28 postinfection than before infection and on day 5 postinfection. Fas antigen expression on peripheral lymphocytes was upregulated from day 8 postinfection. These results indicate that apoptosis is induced about 2 weeks after SIVmac239 infection, following the upregulation of Fas antigen expression on lymphocytes. Since apoptosis was induced about 1 week after the decrease in peripheral CD4+ and CD8+ cell counts, it appears that the apoptosis induction does not play an important role in the transient lymphopenia in the early phase of SIVmac infection. In macaques infected with a nonpathogenic derivative of SIVmac239, SIVmac delta nef, apoptosis of lymphocytes was induced as it was in SIVmac239-infected macaques, but to a lesser degree, suggesting a correlation between the extent of apoptosis induction in lymphocytes in the early phase of SIVmac infection and the pathogenicity of SIVmac.

Animals↗
PubMed1999

Selection of HIV-specific immunogenic epitopes by screening random peptide libraries with HIV-1-positive sera.

Efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing broad neutralizing Ab responses. In fact, the high mutation rate occurring in HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism. To overcome these obstacles, we screened random peptide libraries using sera from HIV-infected subjects to identify antigenic and immunogenic mimics of HIV-1 epitopes. After extensive counterscreening with HIV-negative sera, we isolated peptides specifically recognized by Abs from HIV-1-infected individuals. These peptides behaved as antigenic mimics of linear or conformational HIV-1 epitopes generated in vivo in infected subjects. Consistent with these findings, sera of simian HIV-infected monkeys also recognized the HIV-specific epitopes. The selected peptides were immunogenic in mice, where they elicited HIV-specific Abs that effectively neutralized HIV-1 isolates. These results demonstrate that pools of HIV-1 mimotopes can be selected from combinatorial peptide libraries by taking advantage of the HIV-specific Ab repertoire induced by the natural infection.

Animals↗
PubMed1999

c-fos induction by heat, arsenite, and cadmium is mediated by a heat shock element in its promoter.

c-fos mRNA is induced by many types of noxious stimuli that are known to enhance transcription mediated by the heat shock element (HSE). Nonetheless, it has been accepted that the c-fos promoter does not contain an HSE. In this report, we revealed that the human and rodent c-fos promoters do contain HSEs which are highly responsive to heat, arsenite, and cadmium. These HSEs may play important roles in regulating c-fos expression in various pathological and physiological situations.

Animals↗
PubMed1999

Metastasis suppressor gene(s) for rat prostate cancer on the long arm of human chromosome 7.

Allelotype analyses of human prostate cancer indicate that allelic losses on human chromosome arms 7q, 8p, 10q, 13q, 16q, 17q, and 18q are observed frequently. For the study of the possible biological significance of the frequently observed deletions on chromosome arm 7q in human prostate cancer, human chromosome 7 was introduced into highly metastatic rat prostate cancer cells by use of a microcell-mediated chromosome transfer technique. The introduction of human chromosome 7 resulted in the suppression of metastatic ability of the microcell hybrids, whereas no suppression of tumorigenicity was observed. To identify the portion of chromosome 7 containing the metastasis-suppressive function gene, the derivative chromosome 7 that was generated with the initial transfer was retransferred into rat prostate cancer cells. Human chromosome 7-containing rat prostate cancer cells could be used as the donor cells, because rodent cells produced a sufficient number of microcells with colchicine treatment. Cytogenetic and molecular analyses of these clones demonstrated that loss of segments on 7q was related to the reexpression of the metastatic phenotype. These results show that human 7q contains a metastasis suppressor gene or genes for rat prostate cancer. The findings also suggest that this gene may play an important role in the progression of human prostate cancer.

Animals↗
PubMed1999

Thymidine phosphorylase expression is predominantly observed in stroma of well-differentiated adenocarcinoma of endometrium and correlates with a frequency of vascular involvement.

Thymidine phosphorylase (dThdPase) expression was studied in 20 cases of well-differentiated endometrial carcinomas to examine the clinicopathological significance. Immunohistochemical study showed predominant dThdPase expression in tumor stroma. No expression was detected in coexisting normal endometrial stroma or stroma of endometriosis. Seven cases (35%) showed strong stain and 13 (65%) showed weak stain. Based on the strength of immunoreactivity, patients were grouped into two groups: group S (strong) and group W (weak). In group S, 5 (71%) showed vascular involvement whereas in group W, only 3 (23%) cases were positive, giving a significant difference in the frequency of vascular involvement between the two groups (P < 0.03, chi2 analysis). No correlation was found between dThdPase expression and myometrial invasion. Patients with vascular involvement resulted in poorer outcome (P < 0.003) whereas between group S and W, there showed no difference in survival (group S vs group W; P < 0.15). A multivariate analysis including stage, vascular involvement, and dThdPase stain as variables showed none as independent risk factors. However, univariate analysis showed that the presence of a vascular involvement was a risk factor for a shorter survival (relative risk = 6.277, 95% range = 1.2-32.6, P < 0. 03). It is concluded that expression of dThdPase in endometrial carcinoma is a marker of a desmoreaction and a risk factor for vascular invasion. Since vascular invasion is a risk factor for poor outcome, the significance of dThdPase expression in endometrial carcinoma requires further clinical evaluations.

Adenocarcinoma↗
PubMed1999

Interferon-alpha and 5-fluorouracil therapy in patients with metastatic renal cell cancer: an open multicenter trial. The Japanese Study Group Against Renal Cancer.

OBJECTIVES: Recent clinical trials have implied the cytotoxic and antiproliferative effects of combining 5-fluorouracil and interferon-alpha in the treatment of metastatic renal cell cancer. We therefore conducted an open multicenter trial to test the efficacy of such a combination on this cancer. METHODS: Human lymphoblastoid interferon (3 MIU per patient) was administered subcutaneously three times weekly for 12 weeks, while 5-fluorouracil was administered (600 mg/m2/day) as a continuous infusion for the first 5 days, followed by an intravenous bolus infusion of 600 mg/m2 once a week from the 3rd week until the 12th week. RESULTS: Of the 63 patients entered into the trial, 55 were eligible and evaluable for systemic toxicities, and 53 were evaluable for their response. All patients had undergone a prior nephrectomy, and their European Cooperative Oncology Group (ECOG) performance status ranged from 0 to 3 (median 0). Three complete and eight partial responses were induced, with an overall response rate of 20.0%. The median time to progression and the median survival time were 11 and 33 months, respectively. World Health Organization grade 3 toxicities were observed in 8 patients; however, no grade 4 toxicities or toxicity-related deaths were noted. CONCLUSIONS: Combination therapy of interferon-alpha plus 5-fluorouracil at the above-described dosage and schedule produced no better responses than interferon monotherapies. Prolongation of survival could be attributable to the fair performance status of the patients. This regimen has limited value for the treatment of patients with advanced renal cell cancer.

Adult↗
PubMed1999

Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys.

Virus-specific antibodies protect individuals against a wide variety of viral infections. To assess whether human immunodeficiency virus type 1 (HIV-1) envelope-specific antibodies confer resistance against primate lentivirus infections, we purified immunoglobulin (IgG) from chimpanzees infected with several different HIV-1 isolates, and used this for passive immunization of pig-tailed macaques. These monkeys were subsequently challenged intravenously with a chimeric simian-human immunodeficiency virus (SHIV) bearing an envelope glycoprotein derived form HIV-1DH12, a dual-tropic primary virus isolate. Here we show that anti-SHIV neutralizing activity, determined in vitro using an assay measuring loss of infectivity, is the absolute requirement for antibody-mediated protection in vivo. Using an assay that measures 100% neutralization, the titer in plasma for complete protection of the SHIV-challenged macaques was in the range of 1:5-1:8. The HIV-1-specific neutralizing antibodies studied are able to bind to native gp120 present on infectious virus particles. Administration of non-neutralizing anti-HIV IgG neither inhibited nor enhanced a subsequent SHIV infection.

Animals↗
PubMed1999

Human immunodeficiency virus type 1 neutralizing antibodies accelerate clearance of cell-free virions from blood plasma.

The concentration of human immunodeficiency virus type 1 (HIV-1) particles in blood plasma is very predictive of the subsequent disease course in an infected individual; its measurement has become one of the most important parameters for monitoring clinical status. Steady-state virus levels in plasma reflect a balance between the rates of virions entering and leaving the peripheral blood. We analyzed the rate of virus clearance in the general circulation in rhesus macaques receiving a continuous infusion of cell-free particles in the presence and absence of virus-specific antibodies. Here we show, by measuring virion RNA, particle-associated p24 Gag protein and virus infectivity, that the clearance of physical and infectious particles from a primary, dual-tropic virus isolate, HIV-1DH12, is very rapid in naive animals, with half-lives ranging from 13 to 26 minutes. In the presence of high-titer HIV-1DH12-specific neutralizing antibodies, the half-life of virion RNA was considerably reduced (to 3.9-7.2 minutes), and infectious virus in the blood became undetectable. Although physical virus particles were eliminated extravascularly, the loss of virus infectivity in the blood reflected the combined effects of extravascular clearance and intravascular inactivation of HIV-1 infectivity due to antibody binding.

Animals↗
PubMed1999

Improvement of exercise capacity of rats with chronic heart failure by long-term treatment with trandolapril.

1. The effects of long-term treatment with trandolapril, an angiotensin I-converting enzyme inhibitor, on exercise capacity of rats with chronic heart failure (CHF) following coronary artery ligation were examined. CHF was developed by 8 weeks after the coronary artery ligation. 2. The running time of rats with CHF in the treadmill test was shortened to approximately 65% of that of sham-operated rats (16.3+/-1.2 vs. 25.1+/-1.6 min, n = 7; P<0.05). ATP, creatine phosphate (CP), and lactate contents of the gracilis muscle of rats with CHF were similar to those of sham-operated rats before running. After running, ATP and CP were decreased and lactate was increased in both rats with CHF and sham-operated rats. There were no significant differences in the levels of energy metabolites between rats with CHF and sham-operated rats. The rates of decrease in ATP and CP and rate of increase in lactate in the gracilis muscle of rats with CHF during exercise were greater than those of sham operated rats (2.5, 2.0 and 1.5 fold high, respectively), suggesting wastage of energy during exercise in the animals with CHF. 3. Myofibrillar Ca2+ -stimulated ATPase (Ca-ATPase) activity of skeletal muscle of rats with CHF was increased over that of the sham-operated control (62.03+/-1.88 vs. 52.34+/-1.19 micromol Pi mg(-1) protein h(-1) n = 7; P<0.05). The compositions of myosin heavy chain (MHC) isoforms of gracilis muscle were altered by CHF; decreases in MHC types I and IIb and an increase in MHC type IIa were found (P<0.05). 4. Rats with CHF were treated with 1 mg kg(-1) day(-1) trandolapril from the 2nd to 8th week after surgery. Treatment with trandolapril prolonged the running time, reversed the rates of decrease in ATP and CP and the rate of increase in lactate, and restored the Ca-ATPase activity (51.11+/-0.56 micromol Pi mg(-1) protein h(-1), n = 7; P<0.05) and composition ratio of MHC isoforms in the gracilis muscle. 5. The results suggest that long-term trandolapril treatment of rats with CHF may restore their ability to utilize energy without wastage and thus improve exercise capacity.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Gene-mutated HIV-1/SIV chimeric viruses as AIDS live attenuated vaccines for potential human use.

To develop an AIDS vaccine for human use as well as a suitable animal model for AIDS research, we constructed a series of HIV-1/SIVmac chimeric viruses (SHIVs). We successfully generated a SHIV (designated as NM-3rN) having the HIV-1 env gene, which enabled the evaluation of the efficacy of HIV-1 Env-targeted vaccines in macaque monkeys instead of chimpanzees. Two NM-3rN derivatives (NM-3 and NM-3n) induced long-term anti-virus immunities without manifesting the disease. The monkeys vaccinated with NM-3 or NM-3n became resistant to a challenge inoculation with NM-3rN. Serum from a monkey vaccinated with NM-3 neutralized not only the parental HIV-1 (NL432), but also an antigenically different HIV-1 (MN). In vivo experiments confirmed the heterologous protection against an SHIV having the HIV-1 (MN) env. In addition to specific immunity including neutralizing antibodies and cytotoxic T lymphocyte activity, nonspecific immunity such as natural killer activity is associated with this protection. These data suggest that the live vaccine has the ability to protect individuals against various types of HIVs. These SHIVs should contribute to the development of future anti-HIV-1 live vaccines in humans.

AIDS Vaccines↗
PubMed1999

Characterization of the leukotriene B4 receptor in porcine leukocytes. Separation and reconstitution with heterotrimeric GTP-binding proteins.

Leukotriene B4 (LTB4) is a potent chemoattractant derived from arachidonic acid. When cDNAs for LTB4 receptor (BLT) were cloned it was found that they belong to a guanine nucleotide-binding regulatory protein (G-protein)-coupled receptor superfamily. However, purification of BLT from inflammatory cells and reconstitution with various types of G-proteins have not been successful. In the present study, BLT from porcine leukocytes was solubilized, separated from associated G-proteins by Ricinus communis agglutinin (RCA) 120 chromatography, and reconstituted with several endogenous and exogenous G-proteins, in combination with the fraction which contained endogenous phospholipids and Gbeta gamma. Kinetic studies of LTB4 were performed to determine the association with G-proteins. A partially purified BLT fraction (retained on an RCA120 column) free of G-proteins showed a lower affinity for LTB4 (Kd = 500 nm), but reconstitution of the BLT fraction with a G-protein-rich fraction (flow-through of an RCA column) increased the affinity for LTB4 10-fold (Kd = 50 nm). The partially purified BLT fraction was also reconstituted with exogenous G-proteins such as a heterotrimeric Gi2 purified from bovine brain or recombinant alpha subunits of Gi1, Gi2, Gi3, and Go expressed in Spodoptera frugiperda-9 cells. These increases in LTB4 bindings demonstrate that the BLT of porcine leukocytes can interact with pertussis toxin-sensitive G-proteins in vitro. The method is useful for the purification and reconstitution of other, as yet unisolated, G-protein-coupled receptors.

Animals↗
PubMed1999

Synthesis and degradation of 1-aminocyclopropane-1-carboxylic acid by Penicillium citrinum.

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M(r) 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the Km for S-adenosyl-L-methionine of 1.74 mM and kcat of 0.56 s-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M(r) 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a Km for ACC of 4.8 mM and kcat of 3.52 s-1. The presence of 7 mM Cu2+ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.

Amino Acids↗
PubMed1999

Apoptosis of human endometrium mediated by perforin and granzyme B of NK cells and cytotoxic T lymphocytes.

Endometrial stromal granulocytes (EGs) were found to be a major component of human endometrial stroma in the late secretory phase. However, the role of EGs in the mechanism of human endometrial menstruation has not been clarified. Immunohistochemistry of CD56, perforin, granzyme B, and vimentin, in situ detection of apoptosis by TUNEL (TdT-mediated dUTP-biotin nick and labeling) and electron microscopy were performed in endometrial tissue samples with normal menstrual cycles. We analyzed the number of immunostained cells in the functional layer of stroma and the number of apoptotic cells detected by TUNEL in the endometrial glandular cells. Double-staining revealed that CD56-positive endometrial stromal cells were simultaneously positive for both perforin and granzyme B, and negative for vimentin, which recognized stromal tissue. Vimentin was positive for the predecidual cells and negative for EGs. CD56-positive EGs involving perforin and granzyme B were progressively recruited during the secretory phases before menstruation. Apoptosis in endometrial glandular cells increased from the late secretory phase, which maximized at the menstrual period. This finding suggests that the cytotoxic granules released from EGs, which are derived from cytotoxic T lymphocytes and natural killer cells, are initiators of the apoptotic pathway that induces endometrial menstruation.

Adult↗
PubMed1999

Mechanism of weight gain suppressing effect of ER-40133, an angiotensin I converting enzyme inhibitor, in growing rats.

Effects of ER-40133, an inhibitor of angiotensin converting enzyme (ACE), on weight gain and sodium and potassium balance were studied in growing SD male rats. Thirty-two animals (seven weeks of age) were divided into two groups; one received a standard diet containing 0.227% sodium and the other a low (0.065%) sodium diet. They were divided into four subgroups; one control group and three treated groups receiving 3, 10 or 30 mg/kg of ER-40133, by gavage, once a day for five consecutive days. Body weight gain (average of the standard and low sodium diet groups) was -32% in the 3 mg/kg group,-74% in 10 mg/kg group and -99% in 30 mg/kg group, when compared with the control group. There was a highly linear correlation between suppression of body weight gain and reduction in sodium and potassium retention for both groups of animals given the standard and low sodium diet. The reduced sodium retention, the primary effect of ACE inhibitors, accounted for about 30% of suppressed weight gain, and the reduced potassium retention, the secondary effect of sodium deficiency, could account for the rest about 70% of weight suppression by ER-40133.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1998

A novel strategy of cell targeting based on tissue-specific expression of the ecotropic retrovirus receptor gene.

Gene transfer into specific tissues or cell types is a key technique in the development of gene therapy. Modification of vector particles such that they selectively bind to the target cells has been attempted, but the limitation of this approach is the low transduction efficiency. Here, we show that a two-step gene transfer system can be used for efficient cell targeting. With this strategy, and using a high-titer adenoviral vector containing a tissue-specific promoter, we have engineered a system in which only target cells become susceptible to retrovirus-mediated transduction. In a model experiment, we constructed an adenoviral vector (Ad.AFPEcoRec) containing the ecotropic retrovirus receptor (EcoRec) gene under the control of the alpha-fetoprotein (AFP) promoter. A binding assay showed that after transduction with AD.AFPEcoRec, EcoRec molecules were efficiently expressed in AFP+HepG2 cells, but not in AFP-HeLa and AFP-HLE cells. The EcoRec-expressing HepG2 cells could be stably transduced with ecotropic retroviral vectors, whereas HeLa and HLE cells remained highly resistant to retrovirus-mediated gene transfer. The apparent titer on HepG2 cells was greater than 2 x 10(5) CFU/ml. Because various tissue-specific promoter/enhancer elements are available, the two-step system could be used as a general strategy for both ex vivo and in vivo targeted gene transfer.

3T3 Cells↗
PubMed1998

Molecular cloning and functional analysis of cynomolgus monkey CYP1A2.

Complementary DNA fragments encoding cynomolgus monkey CYP1A2 were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) method from the liver total RNA of a 3-methylcholanthrene (3-MC)-treated cynomolgus monkey. The nucleotide sequence determined was 1630 bp long and contained an open reading frame for a polypeptide of 516 residues. The nucleotide and the deduced amino acid sequences of cynomolgus monkey CYP1A2 showed 95.1 and 92.8% identities to those of human CYP1A2, respectively. The level of CYP1A2 mRNA in the liver of untreated cynomolgus monkey was very low. Treatment with 3-MC increased it. Still, it was one-fortieth that of CYP1A1. Cynomolgus monkey CYP1A2 expressed in recombinant yeasts activated 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8dimethylimidazo[4,5-flquinoxaline (MeIQx) at efficient rates in the umu mutagenicity test. This cytochrome P450 (CYP) also activated 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but less efficiently. These results indicate that cynomolgus monkeys have a functionally active CYPIA2 gene, but its expression level is very low in the liver of untreated cynomolgus monkeys.

Amino Acid Sequence↗