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Biomedical subjects

S Hayashi

Publications and source records attributed to S Hayashi.

At least 307 records · Page 17Linked to original sources
PubMed1999

Introduction of plasmid DNA and oligonucleotides into lung epithelial cells by the hemagglutinating virus of Japan (HVJ)-liposome method.

The hemagglutinating virus of Japan (HVJ) fused with liposomes provides a unique transfection vehicle with characteristics of both virus vector and liposome. Here we investigate the efficiency and safety of the HVJ-liposome technique in delivering foreign genes and oligonucleotides into the lung of the Wistar rat. A plasmid vector containing the Escherichia coli beta-galactosidase (beta-gal) gene and the chicken beta-actin promoter was transfected via the trachea using the HVJ-liposome method. Cytochemical staining showed expression of exogenous beta-gal activity in airway epithelial cells, alveolar macrophages, and alveolar type II cells. This activity persisted at least 28 days after administration of the genes. FITC-labeled oligonucleotides also were introduced into the same types of lung cells as those expressing beta-gal. After instillation of HVJ-liposome, anti-HVJ antibodies were detected in the sera of the rats, but even after repeated administration of HVJ-liposome, no marked histopathologic change was observed while exogenous beta-gal expression was detected in pulmonary cells.

Actins↗
PubMed1999

The expression of p73 is increased in lung cancer, independent of p53 gene alteration.

p73 gene, a new p53 homologue, has been identified: it supposedly acts as tumour suppressor gene in neuroblastoma. To clarify whether p73 might be involved in lung carcinogenesis, we examined p73 expression in resected lung cancer and paired normal lung in 60 cases using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We also examined p73 gene status in three representative cases using Southern blot, and p53 gene alteration in 49 cases using PCR-single-strand conformation polymorphism (PCR-SSCP) and direct sequence. In 87% of the cases (52/60) p73 expression in tumour was more than twice as high as that in paired normal lung tissues, and the difference between p73 expression in tumour and normal lung tissue was significant (P < 0.0001). However, Southern blot analysis revealed that none of the cases showed p73 gene amplification. Compared with clinicopathological characteristics, p73 expression correlates significantly with histological differences and age of patient, independently (P < 0.05). Concerning p53 gene status, 43% (21/49) showed p53 gene alteration, but there was no correlation between p73 overexpression and p53 gene alteration. Our results suggest that need for further functional analysis of the role of p73 in lung carcinogenesis.

Adult↗
PubMed1999

Multiple autoimmune haemopoietic disorders and insidious clonal proliferation of large granular lymphocytes.

We report a patient with clonal proliferation of CD3+8+TCRalphabeta+ large granular lymphocytes (LGL) presenting multiple episodes of autoimmune cytopenia, including autoimmune neutropenia, idiopathic thrombocytopenic purpura, autoimmune haemolytic anaemia, and pure red cell aplasia. Each disorder appeared separately or as a combination during an 11-year clinical course. The increase of blood CD3+8+TCRalphabeta+ LGL was detected 6 years after the initial diagnosis of cytopenia, but the absolute number of LGL cells was always < 1.0 x 109/l. LGL cells were of monoclonal origin and had a chromosomal abnormality. LGL cells transiently responded to cyclosporine A therapy, which was also effective on all of these autoimmune cytopenias. Accordingly, an undetectable level of proliferation of a clonal LGL population could cause various autoimmune haemopoietic disorders.

Adolescent↗
PubMed1999

Kinase-independent activity of Cdc2/cyclin A prevents the S phase in the Drosophila cell cycle.

BACKGROUND: The Cdc2-dependent inhibition of S phase is required in G2 for the correct ordering of the S and M phases in yeasts and Drosophila. This function of Cdc2 has been ascribed to its ability to phosphorylate replication factors to prevent the assembly of a preinitiation complex at the origin of replication. Whether this is the sole mechanism of S phase inhibition by Cdc2 in higher metazoans is not known because the pleiotropic functions of this essential cell cycle regulator make genetic analysis difficult. RESULTS: We show that Cdc2 co-expressed with Cyclin A inhibits the S phase in Drosophila salivary glands and diploid abdominal histoblasts. A kinase defective mutant of Cdc2 failed to promote mitosis, but was still able to inhibit the S phase with the same efficiency as the wild-type protein. In addition, Cdc2 and Cyclin A cooperatively inhibit transcriptional activation by the essential S phase regulator E2F. Cdc2 binds to E2F in vitro, and post-transcriptionally promotes its accumulation in vivo. Furthermore, the inhibitory effect of Cdc2 on S phase is overridden by E2F. CONCLUSION: The inhibition of S phase by Cdc2 is achieved in part by a kinase-independent mechanism, which is likely to be mediated by the inhibition of E2F.

Abdomen↗
PubMed1999

Swelling-induced, CFTR-independent ATP release from a human epithelial cell line: lack of correlation with volume-sensitive cl(-) channels.

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(-) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(-) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56-80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 microM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 microM. Glibenclamide (500 microM) and arachidonic acid (100 microM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(-) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(-) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach approximately 13 microM by a biosensor technique using P2X(2) receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl(-) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 microM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(-) channel; and third, the ATP release is not a prerequisite to activation of the Cl(-) channel.

Adenosine Triphosphate↗
PubMed1999

Elevation of urinary enzyme levels in rat bladder carcinogenesis.

Urinary enzyme levels were investigated in rats administered different promoters in their diet for 32 weeks after being initiated by treatment with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in their drinking water for 4 weeks. All groups were composed of 10 rats each. Group 1: females treated with 3% uracil (100% carcinoma incidence). Group 2: control females kept on basal diet only (0% carcinoma incidence). Group 3: males treated with 5% sodium L-ascorbate (100% carcinoma incidence). Group 4: control males (0% carcinoma incidence). Urine was collected at the end of weeks 12, 24 and 36 and tested for lactate dehydrogenase (LDH), alkaline phosphatase, N-acetyl-beta-D-glucosaminase and aspartate aminotransferase activity. To facilitate comparison, data were related to the corresponding excreted creatinine levels. All measurements were made using a centrifugal automatic analyzer. The urine of rats with cancer lesions (groups 1 and 3) showed significant elevation in all enzyme activities at weeks 24 and/or 36 except for LDH in females (group 1). The M/H ratio of the LDH isozymes was reversed (1.10 +/- 0.10) in the tested rats with carcinomas at week 36. This study thus provides evidence of a correlation between high urinary enzyme levels and cancer development in the rat bladder. Measurement of the tested enzymes might thus provide a method to detect malignant changes in bladder epithelium by direct urine analysis.

Acetylglucosaminidase↗
PubMed1999

A synthetic peptide inhibitor for alpha-chemokines inhibits the tumour growth and pulmonary metastasis of human melanoma cells in nude mice.

Growth-related oncogene-alpha (GROalpha) was first described as an autocrine mitogen and growth factor for melanoma cells. More recent studies show that GROalpha, interleukin-8 (IL-8) and other members of the alpha-chemokine superfamily are also angiogenic. Therefore, we sought to determine if inhibitors of the alpha-chemokine receptor would be effective in inhibiting the tumour growth and pulmonary metastasis of human melanoma cells. We determined that melanocytes and 12 human melanoma cell lines produce both GROalpha and IL-8. The proliferation of A375SM, a highly metastatic cell line, and C8161-C were significantly increased by human recombinant GROalpha and inhibited by anti-human GROalpha monoclonal antibody. Antileukinate, a potent inhibitor of alpha-chemokine receptor binding, inhibited the binding of GROalpha to its receptors in melanocytes and all 12 melanoma cell lines tested. Antileukinate also suppressed proliferation of A375SM and C8161-C cells in a dose-dependent manner, and the suppression was not due to cytotoxic effects. Furthermore, continuous administration of antileukinate inhibited the tumour growth and pulmonary metastasis of A375SM cells in athymic BALB/c nude mice. These findings suggest that antileukinate inhibits the growth of melanoma cells by preventing GROalpha from binding to its receptors. This suggests a possible use of alpha-chemokine receptor inhibitors such as antileukinate in the treatment of malignant melanoma.

Animals↗
PubMed1999

Self-similar renormalization approach to barrier crossing processes.

An algebraic self-similar renormalization method developed recently for summation of divergent field-theoretical series is applied to the thermally activated escape of a Brownian particle over an arbitrarily shaped barrier. Based on the Mel'nikov-Meshkov result for the underdamped Brownian motion and the inverse friction expansion of the underlying Fokker-Planck equation for strong friction, an overall rate formula is constructed. This formula agrees in the weak friction regime with the rate obtained from a diffusion equation in energy variables and, in the limiting case of strong friction with the rate following from a Smoluchowski equation. Its validity is tested for Brownian motion in bistable potentials with parabolic, cusped, and quartic barriers of different heights. The proposed formula is found to give a reasonable description of activated rate processes even though the barrier is quite low. Our comparison also includes results from various different crossover theories. In most of the cases considered the present formula is in considerably better agreement with exact numerical rates than the other interpolation formulas.

Journal Article↗
PubMed1999

The characterization of Shiga toxin-non-producing Escherichia coli serotype O157:H7 isolated from carcasses of cattle at a slaughter house.

A bacteriological investigation of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 was performed on 298 carcasses of cattle at slaughter houses between July 1996 and January 1997 in Gifu Prefecture, Japan. As a result, four Stx-non-producing Escherichia coli O157:H7 strains were isolated from two slaughtered carcasses of cattle. The purpose of this study was to examine the characterization of isolates. Isolates possessed the E. coli attaching and effacing gene (eaeA), and hemolysin gene (hlyA), and harbored 3.0-MDa and 60-MDa plasmids. The Xba I pulsed-field gel electrophoresis (PFGE) pattern showed three similar patterns. Consequently, a closely related genotype of Stx-non-producing E. coli O157:H7 may widely exist in cattle.

Abattoirs↗
PubMed1999

Overexpression of MDM2 in MCF-7 promotes both growth advantage and p53 accumulation in response to estradiol.

The overexpression of the oncogene product MDM2 is often observed in human breast cancer cells, especially in estrogen receptor (ER)-positive ones. To study the role of MDM2 protein in ER-positive breast cancer, we have established cell lines derived from MCF-7 which stably express increased and decreased levels of MDM2 by transfection of a mammalian expression vector containing human mdm2 cDNA in sense and antisense orientations, respectively. Interestingly, MDM2 overexpression in MCF-7 cells afforded a remarkable growth advantage under estradiol (E2)-supplemented condition. Then, we analyzed the expression of p53, which is an important regulator of growth and the cell cycle. Unexpectedly, the p53 accumulation induced by E2 was remarkably higher in MCF-7 cells stably overexpressing MDM2 than in the parent MCF-7 cells. On the other hand, reduction of MDM2 suppressed the E2-induced increase in p53 protein. Moreover, mdm2 antisense oligonucleotides prevented E2-induced accumulation of p53. In the steady state, the cellular levels of p53 were also correlated with those of MDM2. These interactions are not consistent with the well-known role of MDM2, which acts as a negative regulator for p53 by inhibiting its function and promoting its rapid degradation. These results suggest that MDM2 may regulate the expression of p53 in the steady state and in response to E2 in breast cancer cells, and imply a novel and important role of MDM2 during breast carcinogenesis.

Breast Neoplasms↗
PubMed1999

Post-stroke treatment with imidapril reduces learning deficits with less formation of brain oedema in a stroke-prone substrain of spontaneously hypertensive rats.

The present study was undertaken to examine the effects of the ACE (angiotensin converting enzyme) inhibitor imidapril, on the brain, when administered after the onset of stroke in a stroke-prone substrain of spontaneously hypertensive rats (SHRSP). Learning deficits and induced lesions in the brain as well as in the kidneys and heart were investigated in detail. SHRSP were divided into two groups with or without salt loading at the age of 4 weeks. The salt loading was performed for 7-9 weeks to increase the incidence of stroke. Within 24 h after the first observation of stroke, animals were subsequently treated with 5 mg/kg imidapril orally once a day or the vehicle for up to the age of 27 weeks. Imidapril attenuated progression of neurological abnormalities such as irritability, hyperkinesia and motor dysfunction, and increased survival rate. In three-panel runway testing, learning deficits did not develop significantly in the imidapril-treated group, and was comparable to that in the non-salt-loaded/non-stroke group. Imidapril reduced oedema formation in the cortex, hippocampus and striatum, and also suppressed lesion formation in the kidneys and heart. Imidapril thus suppressed progression of neurological deficits with loss of learning ability following onset of stroke, and also suppressed formation of oedema in the brain and decreased the number of lesions in other organs. Imidapril-induced reduction of cerebrovascular damage, which presumably occurs in the brain after stroke, may account for the inhibitory effects of imidapril on lesion formation and learning impairment.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Thoracic duct: visualization at nonenhanced MR lymphography--initial experience.

In six healthy volunteers, the thoracic duct was examined at magnetic resonance (MR) lymphography without contrast agent. With a 1.5-T clinical imager, a short echo spacing, three-dimensional, half-Fourier fast spin-echo sequence with electrocardiographic gating was employed to depict the thoracic duct along the thoracic aorta. The images clearly depicted the main duct in the region of the thoracic aorta in all six volunteers, drainage into the left subclavian region in four, and the cisterna chyli in one.

Adult↗
PubMed1999

Endotoxin-specific NF-kappaB activation in pulmonary epithelial cells harboring adenovirus E1A.

Adenovirus E1A DNA and proteins are detected in lung epithelial cells of patients with chronic obstructive pulmonary disease. In investigating E1A regulation of inflammatory mediator expression in human lung epithelial cells, we found increased intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 expression after lipopolysaccharide (LPS) stimulation of A549 cells stably transfected with adenovirus 5 E1A. We now show that E1A-dependent induction of interleukin-8 expression is specific to LPS, superinduced by cycloheximide, and not observed after tumor necrosis factor or phorbol 12-myristate 13-acetate stimulation. Electrophoretic mobility shift assays revealed that tumor necrosis factor or phorbol 12-myristate 13-acetate induced nuclear factor-kappaB binding complexes of Rel A and p50 in E1A and control transfectants, whereas LPS was effective only in E1A transfectants. Similarly, LPS-induced nuclear translocation of nuclear factor-kappaB was observed only in E1A transfectants. CCAAT-enhancer binding protein binding was undetected and activator protein-1 binding was unaffected by LPS in either cell type, whereas basal mRNA levels of c-jun were unchanged by E1A. We conclude that E1A enhances the expression of these inflammatory mediator genes by modulating events specific to LPS-triggered nuclear factor-kappaB induction in these cells.

Adenoviridae↗
PubMed1999

Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy.

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells and acts as a survival factor against endothelial cell death. Therefore we hypothesized that a decrease in local vascular HGF might be related to the pathogenesis of peripheral arterial disease. We initially evaluated vascular HGF concentration in the vessels of patients with arteriosclerosis obliterans. Consistent with in vitro findings that hypoxia downregulated vascular HGF production, vascular HGF concentration in the diseased segments of vessels from patients with arteriosclerosis obliterans was significantly decreased as compared with disease-free segments from the same patients (P<0.05), accompanied by a marked reduction in HGF mRNA. On the other hand, a novel therapeutic strategy for ischemic diseases that uses angiogenic growth factors to expedite and/or augment collateral artery development has recently been proposed. Thus in view of the decreased endogenous vascular HGF, rhHGF (500 micrograms/animal) was intra-arterially administered through the internal iliac artery of rabbits in which the femoral artery was excised to induce unilateral hind limb ischemia, to evaluate the angiogenic activity of HGF, which could potentially have a beneficial effect in hypoxia. Administration of rhHGF twice on days 10 and 12 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemic model as assessed by angiography (P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals. In addition, we examined the feasibility of intravenous administration of rhHGF in a moderate ischemia model. Importantly, intravenous administration of rhHGF also resulted in a significant increase in angiographic score as compared with vehicle (P<0.01). Overall, a decrease in vascular HGF might be related to the pathogenesis of peripheral arterial disease. In the presence of decreased endogenous HGF, administration of rhHGF induced therapeutic angiogenesis in the rabbit ischemic hind limb model, as potential cytokine supplement therapy for peripheral arterial disease.

Aged↗
PubMed1999

Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth.

Loss of activity of the p53 tumor suppressor gene product has been postulated in the pathogenesis of human restenosis. Although the antioncogenes p53 and retinoblastoma (Rb) susceptibility gene have been reported to play a pivotal role in cell cycle progression in various cells, the role of p53 and Rb in the growth of human vascular smooth muscle cells (VSMC) has not yet been clarified. We used antisense strategy against p53 and Rb genes by the viral envelope-liposomal method. Transfection of antisense p53 oligodeoxynucleotides (ODN) alone resulted in an increase in DNA synthesis compared with control (P<0.01). Similarly, transfection of antisense Rb ODN alone resulted in a higher DNA synthesis rate than control (P<0.01). Moreover, increase in VSMC number was only induced by transfection of antisense p53 ODN alone or cotransfection of p53/Rb ODN (P<0.01), whereas a single transfection of antisense Rb ODN had little effect on cell number. Therefore, we hypothesized that this discrepancy is due to the induction of apoptosis mediated by p53. Interestingly, apoptotic cells were markedly increased in VSMC transfected with antisense Rb ODN alone, accompanied by the induction of p53 protein. The number of apoptotic cells was attenuated by cotransfection of antisense p53 ODN (P<0.01). We finally examined the molecular mechanisms of apoptosis induced by the absence of Rb. In VSMC transfected with antisense Rb ODN, bax, a promoter of apoptosis, was significantly increased in VSMC transfected with antisense Rb ODN (P<0.01), whereas bcl-2 and Fas did not play a pivotal role in the induction of apoptosis. Overall, these data first demonstrated that the antioncogenes p53 and Rb negatively regulated the cell cycle in VSMC, suggesting that the modulation of their activity may mediate VSMC growth such as that in restenosis and atherosclerosis. The presence of p53 plays a pivotal role in the regulation of apoptosis in human VSMC growth, probably through the bax pathway. These results provide evidence that p53 is a functional link between cell growth and apoptosis in VSMC.

Analysis of Variance↗