Engagement of T-cell receptor (TcR)/CD3 complexes on T-cells rapidly provokes tyrosine phosphorylation of cellular proteins, which is thought to be an essential step to the following events of T-cell activation. p56lck, a member of src-related, non-receptor type protein tyrosine kinases, is expressed predominantly in lymphocytes. Accumulating data suggest that p56lck is one of the kinases responsible for TcR-mediated protein tyrosine phosphorylation. To investigate the role of p56lck in TcR-signaling in detail, we injected anti-Lck monoclonal antibody (mAb), MOL171 or MOL294, both specifically suppress Lck kinase activity in vitro, into Jurkat T-cells by the erythrocyte-ghost procedure in order to block the activity of p56lck. In Jurkat cells injected with anti-Lck mAb, intracellular Ca2+ mobilization induced by TcR-stimulation was markedly reduced in comparison with control mouse IgG-injected samples. This block of Ca2+ influx seems to be specific for TcR-signaling because anti-Lck mAb-injection did not cause significant suppression of phytohaemagglutinin-induced Ca2+ increase. Furthermore, injection of anti-Lck mAb inhibited TcR-mediated protein tyrosine phosphorylation of 100 kDa protein and phospholipase C gamma 1. These results confirm that p56lck is an indispensable element of TcR-signaling and p100 and phospholipase C gamma 1 are strongly presumed to be candidates for substrates for p56lck.

Antibodies, Monoclonal↗

PubMed1994

The difference in gp160 and gp120 of HIV type 1 in the induction of CD4 downregulation preceding single-cell killing.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is synthesized as a precursor glycoprotein, gp160, and is then processed into gp120 and gp41. In the present study, CD4+ cell clones expressing either gp160 or gp120 of HIV-1 under the transcriptional control of an inducible promoter were made in order to examine the effect of these env products on the downregulation of surface CD4 and cell injury. A complete disappearance of surface CD4 preceding single-cell death occurred in the cell clones expressing gp160, in which a complex between CD4 and gp160 was formed and then accumulated intracellularly. In contrast to this, the cell clones expressing gp120 neither exhibited any such depletion of surface CD4 nor showed any apparent cytopathic effect. Therefore, it is thought that gp160 but not gp120 plays a crucial role in both the downregulation of surface CD4 and the resultant cell death in the cells infected with HIV-1.

Base Sequence↗

PubMed1994

Molecular and biological characterization of fusion regulatory proteins (FRPs): anti-FRP mAbs induced HIV-mediated cell fusion via an integrin system.

Anti-FRP mAbs induced polykaryocyte formation of U2ME-7 cells (CD4+U937 cells transfected with the HIV gp160 gene). Anti-FRP-1 mAb immunoprecipitated gp80-85, gp120 and homodimers of these peptides, and anti-FRP-2 mAb reacted with gp135 identically to the alpha 3 subunit of integrin. Both anti-FRP-1 and anti-FRP-2 mAb-induced cell fusion was blocked by anti-beta 1 integrin antibody, fibronectin or inhibiting anti-FRP-1 antibody. Therefore, anti-FRP mAbs were thought to induce the fusion via an integrin system(s). FRP-mediated fusion was temperature, cytoskeleton, energy and Ca2+ dependent. These experiments showed a possible regulatory function of cell fusion by an integrin system(s).

Amino Acid Sequence↗

PubMed1994

Possible gene dose effect of a mutant cardiac beta-myosin heavy chain gene on the clinical expression of familial hypertrophic cardiomyopathy.

We have examined for a mutation in the cardiac beta myosin heavy chain gene from Japanese patients with familial hypertrophic cardiomyopathy. A missense mutation due to a G to A transition in codon 935, leading to a replacement of Glu with Lys, was found in one patient. Family members of this patient were then examined. It was revealed that both the proband and his elder brother, who was also a symptomatic patient, were homozygous for the mutation. The proband eventually died of intractable heart failure, and his brother died suddenly in their thirties. On the other hand, his parents, who were first cousins and heterozygous for the mutation, had cardiac hypertrophy without clinical symptoms. His elder sister was also heterozygous for the mutation, however, she did not manifest with cardiac hypertrophy. These observations suggest a gene-dose-like effect of the mutant cardiac beta myosin heavy chain gene on the clinical manifestation of familial hypertrophic cardiomyopathy.

Adenine↗

PubMed1994

Asymmetrical topology of diether- and tetraether-type polar lipids in membranes of Methanobacterium thermoautotrophicum cells.

We investigated the distribution of diether polar lipids between the inner and outer leaflets of the membrane of Methanobacterium thermoautotrophicum comparing the orientation of tetraether polar lipids, which constitute a monolayer in the same membrane. Three kinds of reactions were employed for intact cells or protoplasts and unsealed membrane fragments prepared from the organism: glycosidase digestion for glycolipids, NaIO4 oxidation for glycolipids and inositol lipids, and trinitrophenylation for aminophospholipids. The results indicated that (a) most gentiobiose residues of both diether and tetraether polar lipids were exposed on the outside of the cells; (b) serine and inositol residues of both diether and tetraether polar lipids were mainly oriented to the cytoplasmic surface of the membrane; and (c) approximately 80% of archaetidylethanolamine (diether type) was distributed in the outer leaflet of the membrane bilayer, while only 25% of the ethanolamine residue of gentiobiosyl caldarchaetidylethanolamine (tetraether type) was oriented to the outer surface of the membrane. These results, except for ethanolamine lipids, are consistent with the hypothesis that the tetraether polar lipids are synthesized from the corresponding diether polar lipid precursors that have been already substituted by polar groups in the membrane by head-to-head condensation without rearrangement of lipids.

Carbohydrate Conformation↗

PubMed1994

Role of Coulomb energy in promoting collisionally activated dissociation of multiply charged peptides formed by electrospray ionization.

Electrospray ionization tandem quadrupole mass spectrometry has been applied to a series of lysine-substituted octaalanines and some naturally occurring peptides containing more than two basic amino acid residues. Unusually high fragmentation efficiency along with site-specific cleavages at the outer side of and remote from basic residues were observed for collisionally activated dissociation spectra of multiply charged peptides (MCP) with basic residues in close proximity. It was suggested that Coulomb energy (CE) rather than Coulomb repulsion (CR) was responsible for promoting fragmentation of the MCP studied. A possible fragmentation mechanism of MCP is proposed, in which the conversion of CE into internal vibrational energy was brought about by intramolecular proton migration in the presence of CR between charged sites.

Amino Acid Sequence↗

PubMed1994

Therapeutic management of dilated cardiomyopathy.

The therapeutic approach to dilated cardiomyopathy (DCM) still remains nonspecific and symptomatic, since no specific etiology is identified. Nevertheless, the recent introduction of angiotensin converting enzyme (ACE) inhibitors and beta-blockers greatly improved the treatment of DCM. The poor prognosis of patients with DCM encourages maximal aggressive attempts to prevent progression of ventricular dysfunction rather than to wait for treatable symptoms. To achieve this goal, vasodilators, particularly ACE inhibitors, now appear to be essential for the treatment of DCM. Digitalis is added unless contraindicated by adverse effects. Diuretics should be used only to relieve congestive symptoms. In the presence of sinus tachycardia or ventricular arrhythmias, beta-blockers are the next choice in our practice. When congestive symptoms or low output state are not controlled with vasodilators, diuretics, and digitalis, inotropic agents are indicated, with or without mechanical assist devices. For severely ill patients unresponsive to maximal medical management, heart transplantation is needed.

Adrenergic beta-Antagonists↗

PubMed1994

Neutrophil activation after percutaneous transluminal coronary angioplasty.

We investigated whether percutaneous transluminal coronary angioplasty (PTCA) would induce neutrophil activation in patients with coronary artery disease. Blood samples were taken from the coronary sinus in 14 patients who underwent PTCA and in 9 control subjects who underwent coronary arteriography (CAG). Flow cytometry was used to measure membrane surface expression of beta 2 integrin (CD11b) and the generation of hydrogen peroxide in neutrophils after ex vivo phorbol myristate acetate stimulation by 2,'7'-dichlorofluorescein. Neutrophil elastase was measured by an immunoenzymatic method. Surface expression of CD11b increased significantly, approximately twofold, after PTCA but not after CAG. Mean fluorescence intensity of 2',7'-dichlorofluorescein in stimulated neutrophils decreased significantly after PTCA, suggesting a previous in vivo activation, but not after CAG. Neutrophil elastase increased significantly after PTCA but not after CAG. These data indicate that PTCA induces neutrophil activation and suggest that neutrophils may contribute to the ischemic injury.

Adult↗

PubMed1994

Protective effects of D-penicillamine and a thiazole derivative, SM-8849, on pristane-induced arthritis in mice.

To evaluate the antiarthritic properties of a novel thiazole derivative, the drugs SM-8849, D-penicillamine and indomethacin were administered to pristane-injected DBA/1 mice. The mice were treated daily with the agents for 32 weeks, starting from the day of the pristane injection. Treatment with SM-8849 (50 mg/kg) resulted in an amelioration of arthritic disease, as assessed by clinical, radiographic, and histologic examinations. Similar results were obtained in mice treated with 50 mg/kg D-penicillamine, although this disease modifying antirheumatic drug was slightly less effective than the same dose of SM-8849. In contrast, indomethacin at the maximum tolerated dose of 2 mg/kg did not alter the course of the disease. SM-8849 and D-penicillamine were also shown to reduce serum levels of rheumatoid factors and the acute-phase reactant, serum amyloid P component. Indomethacin failed to affect either parameter. Flow cytometric analysis revealed an elevation in the T-cell population that expressed CD44, a marker of murine memory T-cells, in spleens from pristane-injected mice. SM-8849, but not D-penicillamine, prevented the increase in this cell population. These results led us to conclude that pristine-induced arthritis was a useful model for the evaluation of antirheumatic agents, in that using this model, we were able to distinguish disease modifying antirheumatic drugs from nonsteroidal anti-inflammatory drugs. Our findings also indicate that SM-8849 shows antiarthritic activity, with a unique mechanism of action, differing from that of D-penicillamine.

Adjuvants, Immunologic↗

PubMed1994

Free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries.

OBJECTIVES: The purpose of this study was to test the hypothesis that free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured coronary arteries. BACKGROUND: Although free oxygen radicals, such as superoxide anion and hydrogen peroxide, have been shown to alter platelet function in vitro, the potential role of free oxygen radicals has not been fully described in an in vivo model of coronary artery thrombosis. METHODS: Cyclic flow variations were produced in dogs by an external constrictor placed at the site of the left anterior descending coronary artery with injured endothelium. Blood flow in this artery was monitored by a pulsed Doppler flow probe. If cyclic flow variations were observed during postoperative days, dogs intravenously received superoxide dismutase plus catalase. In anesthetized dogs that did not develop an episode of cyclic flow variations, the effect of intracoronary infusion of xanthine plus xanthine oxidase or hydrogen peroxide on arterial blood flow velocity was studied. In platelet studies, the effect of free oxygen radicals and radical scavengers on platelet aggregation was examined. RESULTS: In conscious dogs with cyclic flow variations, superoxide dismutase plus catalase significantly reduced cyclic flow variations (n = 7), whereas saline infusion had no effect (n = 7). The infusion of xanthine plus xanthine oxidase or hydrogen peroxide significantly induced cyclic flow variations in four of six dogs or in five of seven dogs, respectively. In vitro platelet studies showed that xanthine plus xanthine oxidase or hydrogen peroxide significantly enhanced platelet aggregation, and superoxide dismutase or catalase significantly inhibited such aggregation. CONCLUSIONS: Reduction of free radical formation decreases platelet aggregation and may eliminate cyclic flow variations, whereas promotion of free radical generation enhances platelet aggregation and may induce cyclic flow variations. Thus, free oxygen radicals are an important mediator in this model.

Animals↗

PubMed1994

Gestational age assessment in Japanese low birthweight infants.

We evaluated the validity of the Ballard scoring system for assessing gestational age in Japanese low birthweight infants. Infants included in this study were 116 neonates who were admitted to seven hospitals in Hokkaido. Gestational ages of all infants were prenatally established by ultrasonographic measurement of the crown-rump length performed between 8 and 12 weeks of gestation (clinical age). Of these, 74 infants were appropriate for gestational age (AGA) and the remaining were small for gestational age (SGA). Mean birthweight was 1703 g and 34% (39/116) were < 1500 g. The correlation coefficient for the total population was 0.93 (P < 0.001). Although mean Ballard age tended to overestimate clinical age, mean differences between Ballard age and clinical age did not exceed 0.6 weeks. Agreement within 2 weeks among all infants was 86.2%. Percentages of agreement within 2 weeks did not differ to a statistically significant degree between AGA and SGA. These results indicate that the Ballard method is of value in assessing gestational age in Japanese low birthweight infants.

Evaluation Studies as Topic↗

PubMed1994

Apoptosis induced in CD4+ cells expressing gp160 of human immunodeficiency virus type 1.

In a previous study (Y. Koga, M. Sasaki, H. Yoshida, H. Wigzell, G. Kimura, and K. Nomoto, J. Immunol. 144:94-102, 1990), we demonstrated that the expression of gp160, a precursor form of envelope glycoprotein of human immunodeficiency virus type 1, in CD4+ cells causes the downregulation of surface CD4 and single-cell killing by forming intracellular gp160-CD4 complex. In the present study we investigated the events that lead to cell death in CD4+ cells expressing gp160. We found that apoptosis is induced in cells undergoing single-cell death. Moreover, even the cell clone, which expresses so little gp160 that it does not exhibit any apparent cytopathic effects, such as the inhibition of cell growth, was found to be highly susceptible to the apoptosis induction by the anti-Fas monoclonal antibody.

Antigens, Surface↗

PubMed1994

Priming of immature thymocytes to CD3-mediated apoptosis by infection with murine cytomegalovirus.

Cytomegalovirus (CMV) causes severe clinical manifestations in immunocompromised hosts; however, it remains unclear whether the virus itself is a cause of immunosuppression or whether it is involved as an opportunistic bystander pathogen. This study was performed to elucidate the effect of CMV infection on the host's immune system. The double-positive thymocytes of BALB/c mice inoculated with a sublethal dose of murine CMV (MCMV) were extensively depleted by a 10-micrograms amount of anti-CD3 monoclonal antibody, while such an amount was unable to induce any apparent elimination of thymocytes in noninfected mice. In immature thymocytes of infected hosts, a markedly high level of susceptibility to apoptosis induction was found on treatment with anti-CD3 monoclonal antibody. Analysis of the signal transduction pathway of such double-positive thymocytes demonstrated a profound elevation of the intracellular Ca2+ level after anti-CD3 stimulation, implying that this aberrant mobilization of Ca2+ plays a crucial role in the signaling pathway leading these cells to an extensive apoptosis. Examination of the thymus by PCR was able to detect a low copy number of MCMV DNAs in thymic stromal cells but none at all in thymocytes. Therefore, it is suggested that a mechanism which is not associated with virus replication within the cells exerts a critical effect on rendering the thymocytes highly apoptosis sensitive in hosts infected with MCMV.

Animals↗

PubMed1994

Murine cytomegalovirus-associated pneumonitis in the lungs free of the virus.

At 4 wk after intraperitoneal inoculation of murine cytomegalovirus (MCMV) in adult BALB/c mice, MCMV remained detectable only in the salivary glands. When T cells of these mice were activated by a single injection of anti-CD3 epsilon monoclonal antibody, mice died of interstitial pneumonitis at 24-48 h after injection, accompanied by elevation of serum levels of TNF-alpha and IFN-gamma. However, MCMV remained undetectable in the lungs during the period. Simultaneous injection of cyclosporin A reduced such effects of anti-CD3. In conclusion, although the presence of MCMV in the host may be required, MCMV-associated pneumonitis is not mediated by virus in the lung but probably by the cytokines released from T cells, of which responsiveness to stimulation via CD3 molecule has been presumably modified by MCMV infection.

Animals↗