Biomedical subjects

T Harada

Publications and source records attributed to T Harada.

At least 307 records · Page 17Linked to original sources

PubMed1997

A novel peptide motif for platelet fibrinogen receptor recognition.

To develop a specific antagonist of platelet alphaIIbbeta3 using small linear peptides, we synthesized a series of hexapeptides that did not have an Arg-Gly-Asp (RGD) sequence and examined their anti-platelet activity and their specificity for alphaIIbbeta3. We found a novel motif sequence, Pro-X1-X2-X3-Asp-X4, where X1 to X4 were all L-form alpha-amino acids, which specifically inhibited aggregation of human platelets at submicromolar concentrations. The Pro residue at the N terminus was essential to the anti-platelet activity, and the acetylation of the imino group of this residue also resulted in the complete loss of the activity. The results of the binding assay using purified human platelet alphaIIbbeta3 and placental alphavbeta3 and those of the cell adhesion assay suggest that this motif peptide is highly specific for platelet alphaIIbbeta3 among other integrins. Flow cytometric studies using an fluorescein isothiocyanate-labeled RGD peptide showed that this motif peptide inhibited the binding of an RGD peptide to activated platelets, suggesting that it has the same inhibitory mode as RGD peptides. Conformational analysis of this motif peptide and an RGD-containing peptide suggests that the imino group of the Pro residue may substitute for the role of the guanidino group of the Arg residue of the RGD sequence.

Animals↗

PubMed1997

Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein.

We have characterized viral-cell interactions of hepatitis C virus (HCV) and liver cells to study the pathogenesis of HCV infection. HepG2 cells constitutively expressing HCV core protein showed apoptotic changes in response to stimulation with anti-Fas monoclonal antibody. Cells treated with the antibody showed extensive cell rounding, shrinkage, and cytoplasmic blebbing and finally detached from plates. Fragmentation of the chromatin was observed in the nucleus and DNA ladders were detected. In contrast, cells expressing HCV envelope, nonstructural proteins or normal HepG2 cells did not exhibit such Fas-mediated apoptosis. However, expression of Fas receptor was not upregulated on the surface of the cells expressing HCV core protein. Apoptotic cell death was prevented by pretreatment with a specific inhibitor of the cysteine protease CPP32, while the specific inhibitor of interleukin-1 beta-converting enzyme did not show the preventive effect. The results suggest (i) that intracellular expression of HCV core protein makes cells prone to apoptotic death without upregulation of surface Fas expression and (ii) that the CPP32 protease plays a part in the apoptosis effector pathway of HCV core-expressing cells. HCV core protein may have a role in immune-mediated liver cell injury.

Apoptosis↗

PubMed1997

Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets.

There is now abundant evidence to substantiate an important role of hepatitis C virus (HCV) core protein in cellular gene expression as well as in the viral cycle. Thus the subcellular localization of this protein has important implications. However, several studies have shown controversial results: the HCV core has been, indeed, described as cytoplasmic or nuclear depending on the size of the protein or on the genotype analyzed. We have studied the localization of the HCV core protein in two different cell lines, one nonhepatic (CHO) and the other hepatic (HepG2). Double immunofluorescence staining using a nuclear membrane marker and confocal analysis showed the core protein pattern to be cytoplasmic and globular. This pattern is not cell cycle-regulated. Electron microscopy analysis revealed the nature of the globular staining observed in immunofluorescence. The HCV core protein accumulated at the surface of lipid droplets that were also the unique morphological feature of nonhepatic core transfected cells. The lipid droplets were isolated by sequential ultracentrifugation on the basis of their density; biochemical analysis revealed a prevalence of triglycerides. In addition the core protein colocalized with apolipoprotein AII at the surface of the lipid droplets as revealed by confocal microscopy. Moreover analysis of liver biopsies from chronically HCV-infected chimpanzees revealed that HCV core is cytoplasmic and localized on the endoplasmic reticulum and on lipid droplets. These results clearly define the subcellular localization of the HCV core protein and suggest a relationship between the expression of the HCV core protein and cellular lipid metabolism.

Animals↗

PubMed1997

Decreased expression and rare somatic mutation of the CIP1/WAF1 gene in human hepatocellular carcinoma.

CIP1/WAF1, a critical downstream effector of tumor suppressor p53, encodes a cyclin-dependent kinase inhibitor. By Northern blot analysis, the CIP1/WAF1 mRNA level in the tumor was significantly lower than that in the corresponding normal liver from 19 Japanese patients with hepatocellular carcinoma (P < 0.05). In the tumor from only one out of 19 patients (5%), somatic mutations of the CIP1/WAF1 as well as that of p53 gene were identified by RT-PCR/SSCP analysis. These results suggest that the decreased CIP1/WAF1 expression is involved in the carcinogenesis or the progression of hepatocellular carcinoma.

Adult↗

PubMed1997

Maintenance of hypomethylation status and preferential expression of exogenous human MDR1/PGY1 gene in mouse L cells by YAC mediated transfer.

Selection of cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance (MDR) genes, which encode the cell surface P-glycoproteins, as a result of gene amplification, transcriptional activation, or mRNA stabilization. The LMD1 and LMD4 cell lines were established after the transfer into mouse L cells of two independent yeast artificial chromosome clones containing 300 and 850 kb, respectively, of the human MDR locus. The human MDR1/PGY1 gene, but not the endogenous mouse mdr1a and mdr1b genes, was overexpressed as a result of gene amplification and transcriptional activation in various sublines of LMD1 and LMD4 cells selected for resistance to vincristine. Then we asked why human MDR1/PGY1 gene, but not mouse relevant gene, was expressed. Determination of the methylation status of cytosine residues at Msp I/Hap II cleavage sites (CCGG) in the promoter regions of human MDR1/PGY1 and mouse mdr1a revealed hypomethylation and hypermethylation of the human and mouse genes, respectively in LMD1, LMD4, and their vincristine-resistant derivatives. Various vincristine-resistant sublines were also established after exposure of LMD1 cells for 48 h to 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase. These sublines exhibited overexpression of mouse mdr1a and mdr1b, but not of human MDR1/PGY1, as well as hypomethylation of the mouse mdr1a promoter region. Thus, the selective expression of human or mouse MDR genes in this cell system appears to be related to the methylation status of the respective gene promoter regions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Expression of transforming growth factor alpha (TGF-alpha) gene in mouse embryonic development.

PURPOSE: The expression of genes for TGF-alpha, epidermal growth factor (EGF), and the EGF receptor (EGFR) in mouse blastocysts was evaluated by the reverse transcription-polymerase chain reaction (RT-PCR). We evaluated the effects of TGF-alpha and EGF on the development of mouse embryo prior to implantation. RESULTS: The results revealed the presence of transcripts of TGF-alpha and EGFR. However, EGF mRNA was not observed in repeated experiments. None of these growth factors influenced the rate of development from the two-cell stage to the blastocyst stage when added to the culture medium. These effects were further examined on measuring the incorporation of tritiated thymidine and leucine, providing indices of the synthesis of DNA and protein, respectively. A concentration of only 0.1 ng/ml of TGF-alpha, which shares a cell surface receptor with EGF, stimulated the synthesis of both DNA and protein. EGF at a concentration of 10 ng/ml stimulated the synthesis of DNA and protein by blastocysts. To explore autocrine effects of TGF-alpha on the rate of blastocoel expansion, TGF-alpha antisense oligodeoxynucleotides was used to reduce expression of the TGF-alpha gene. TGF-alpha at a concentration of 0.1 ng/ml stimulates the rate of blastocoel expansion in early cavitating mouse blastocysts. In contrast, TGF-alpha antisense oligonucleotides significantly reduced the rate of expansion. CONCLUSIONS: Our present observations suggest that TGF-alpha/EGF and the EGFR may be involved in regulating embryonic development. In particular, TGF-alpha may serve as an autocrine factor in the regulation of embryonic development.

Animals↗

PubMed1997

Effect of acid-base disturbances on aldosterone secretion in man.

The present study was carried out to elucidate whether changes in blood pH induced by administration of alkalinizing and acidifying agents influence aldosterone secretion in man. Since aldosterone secretion is known to be regulated by various factors such as the renin-angiotensin system, adrenocorticotropic hormone (ACTH), and serum potassium concentration, these indices were simultaneously measured during manipulation. Oral administration of tris hydroxymethyl aminomethane (THAM) resulted in a moderate but not significant decrease in serum aldosterone together with an increase in blood pH, whereas plasma renin activity (PRA) remained unchanged and serum potassium was elevated. ACTH and cortisol decreased significantly. Following CaCO3 ingestion, blood pH, serum potassium concentration, and hormones did not change significantly compared with before ingestion. NH4Cl ingestion showed a significant fall of blood pH and serum cortisol, on the other hand, aldosterone increased significantly compared with CaCO3 ingestion. Following NaHCO3 ingestion, serum aldosterone decreased with an increase in blood pH compared with before ingestion, while serum cortisol and PRA did not change significantly. The overall results of experiments using THAM, CaCO3, NaHCO3, and NH4Cl led to the conclusion that under acid-base disturbances a change in aldosterone can not be induced by either renin-angiotensin or ACTH. We suggest that under acid-base disturbances blood pH may be related, at least, in part to changes in aldosterone in humans.

Acid-Base Imbalance↗

PubMed1997

Cystic coccygeal medullary vestige presenting as a sacrococcygeal mass: a case report and MRI findings.

The case of a 10-month-old boy with a cystic coccygeal medullary vestige is presented. Although the MRI findings of this lesion resemble those of sacrococcygeal teratomas, the presence of a cystic component located at the tip of the coccyx and associated sinus formation may help in diagnosing it. Cystic coccygeal medullary vestige should be entertained in the differential diagnosis of coccygeal cystic lesions.

Cysts↗

PubMed1997

Short inverted repeats function as hotspots of intermolecular recombination giving rise to oligomers of deleted plastid DNAs (ptDNAs).

We have determined the nucleotide sequences around the junction points of oligomeric-deleted ptDNAs possessing a head-to-head or tail-to-tail configuration from long-term cultured cell lines and albino plants. It was shown that DNA rearrangement occurred by direct fusion of deleted ptDNAs in an inverted orientation, which was linked by an asymmetrical sequence of 254-698 bp derived from either of the ptDNAs joined. It is notable that inverted repeats of 7-14 bp flank the asymmetrical sequences at each of the junction points. These features of the DNA sequence around the junction points are commonly observed in oligomeric ptDNA with a large-scale deletion regardless of the cell lines employed. It is suggested that the short inverted repeats are involved in the intermolecular recombination of ptDNA.

Base Sequence↗

PubMed1997

Establishment and characterization of spontaneous mesothelioma cell lines derived from F344 rats.

Three mesothelioma cell lines (MeET-4, MeET-5, and MeET-6) established from ascitic fluid of F344 rats with spontaneous abdominal mesothelioma have been maintained through at least 60 passages on the DMEM with 10% FBS. Two of original tumours consisted of epithelioid cells growing in a papillary pattern, while one (original tumour of MeET-5) had sarcomatous areas composed of spindle-shaped tumour cells. The cell line originating from MeET-5 showed a constantly beiphasic growth pattern during the repetitive subcloning, while the other two lines retained a monophasic growth pattern. Although the growth pattern was different, the tumour cells in all three lines were positive for vimentin and keratin and ultrastructurally showed an abundant distribution of glycogen granules in the cytoplasm and numerous long microvilli on all surface. The modal chromosome number of cell lines varied from 41 to 71, and abnormal chromosomes were frequently seen. All cell lines established formed colonies on semi-solid medium and could be successfully transplanted, growing tumour masses in syngeneic rats and thus indicating their malignant nature. Cell lines grew even on a medium with a low concentration of FBS. The evidence suggests that they may produce growth factors that enable them to survive unfavourable medium conditions.

Abdominal Neoplasms↗

PubMed1997

Increased interleukin-6 levels in peritoneal fluid of infertile patients with active endometriosis.

OBJECTIVE: Our purpose was to investigate the relationship between the levels of interleukin-6, interleukin-6 soluble receptor, and tumor necrosis factor-alpha in peritoneal fluid and the size and number of active red endometriotic lesions. STUDY DESIGN: In a university hospital 39 women of reproductive age underwent either laparoscopy for infertility workup or laparoscopic surgery for ovarian chocolate cysts. Peritoneal fluid was collected by laparoscopy. Active lesions, such as red flamelike lesions, glandlike lesions, and red vesicles, were scored according to the revised American Fertility Society classification system according to the size and number of active lesions. Peritoneal fluid levels of interleukin-6, interleukin-6 soluble receptor, and tumor necrosis factor-alpha levels were determined by enzyme-linked immunosorbent assays. The relationship between peritoneal fluid concentrations of interleukin-6 and tumor necrosis factor-alpha and the score of active endometriosis was investigated. RESULTS: Peritoneal fluid levels of interleukin-6 and tumor necrosis factor-alpha were significantly higher in patients with endometriosis compared with patients without endometriosis. The concentrations in patients with active endometriosis increased as the size and the number of active lesions increased. Cyclic variations in interleukin-6 concentrations were seen in peritoneal fluid from patients with endometriosis; the concentrations in the secretary phase were significantly higher than those in the proliferative phase. CONCLUSIONS: Increased peritoneal fluid levels of interleukin-6 in patients with active red endometriosis may relate to endometriosis-associated infertility and to the pathogenesis of endometriosis.

Adult↗

PubMed1997

Endoscopic ultrasonography and endoscopy for staging depth of invasion in early gastric cancer: a pilot study.

BACKGROUND: We compared the accuracy of endoscopic ultrasonography (EUS) for staging depth of invasion of early gastric cancer with that of conventional endoscopy. PATIENTS AND METHODS: We assessed the depth of invasion of 108 lesions (104 patients) using EUS with a thin 20 MHz probe and compared the results with those of conventional endoscopy and of histologic examination of endoscopically or surgically resected specimens. RESULTS: The overall accuracy rates for staging depth of invasion for conventional endoscopy and EUS were 72.2% and 64.8%, respectively. Lesions that were classified as limited to the mucosa on both conventional endoscopy and EUS were very likely (92.2%) to be limited to the mucosa on histologic examination. The rates for understaging and overstaging were 16.7% and 11.1%, respectively, for conventional endoscopy; and 7.4% and 24.1%, respectively, for EUS. The highest rate for understaging based on conventional endoscopy occurred for lesions in the gastric body (including cardia, 23.9%). CONCLUSIONS: EUS appears to be useful in combination with conventional endoscopy for staging depth of invasion of early gastric cancer. In particular, the two techniques in tandem may accurately predict that a lesion is limited to the mucosa, and EUS may be useful to overcome the potential for understaging by conventional endoscopy, particularly in the gastric body.

Adult↗

PubMed1997

ret Proto-oncogene product is a useful marker of lineage determination in the development of the enteric nervous system in rats.

Detailed study of developmental changes in the enteric nervous system is necessary to disclose the pathogenesis of Hirschsprung's and allied disease, some of which have hypoplastic ganglia. Therefore experiments were undertaken to study the fate of neural crest cells that develop in the rat gut during ontogeny. A polyclonal antibody against ret proto-oncogene product (c-Ret protein) and various monoclonal antibodies against neural markers (tyrosine hydroxylase, dopamine beta hydroxylase, microtubule-associated protein 5, microtubule-associated protein 2 and 160-kd neurofilaments) were used to identify neural crest-derived cells in rat embryos (10.5 to 15.5 days' gestation) and adult rats using a double immunostaining method. C-Ret protein was an early marker of lineage determination in the development of the enteric nervous system (11.5-day embryo: E 11.5). C-Ret-positive cells transiently coexpressed tyrosine hydroxylase, which also was observed in the vagal crest-derived precursors of enteric neurons (days E 11.5 to E 13.5). These cells also coexpressed other neural markers in the proximal gut. Expression of neural markers migrated to the distal intestine during development. This study found a discrepancy between the time when these markers appeared in the cranial and when they appeared in the caudal intestine. Tyrosine hydroxylase-positive cells did not appear in the postumbilical gut. The formation of the primitive neural network in the entire myenteric plexus at day E 15.5 was demonstrated by c-Ret protein. Other neural markers were lost or bad decreased immunoreactivity throughout the entire intestine of the E 15.5 and adult animals. In conclusion, (1) c-Ret protein is one of the earliest markers of lineage determination in the development of the enteric nervous system, (2) each neural marker is expressed at its own time and differs in spatial developmental lineage, (3) c-Ret protein and other neural markers are transiently expressed by a particular group of neural cells during the embryonic period, (4) there is a subpopulation of cells that has never transiently expressed tyrosine hydroxylase in the postumbilical gut, which may have originated from tissue other than the vagal crest, and (5) the primitive neural network in the myenteric plexus was completed at day E 15.5.

Age Factors↗

PubMed1997

A new hepatic portoenterostomy with division of the ligamentum venosum for treatment of biliary atresia: a preliminary report.

BACKGROUND/PURPOSE: Kasai's operation consists of transection of the fibrous portal cord including the bile duct remnant. However, it is difficult to transect the fibrous cord at the level of the posterior surface of the portal vein, because the portal vein is fixed at the porta hepatis. METHODS: The authors described a new hepatic portoenterostomy to transect the fibrous cord under an appropriate visual field by division of the ligamentum venosum (Arantius' canal). Between February and December 1996, six patients who had biliary atresia underwent this procedure. RESULTS: Jaundice resolved completely (TB < or = 1.0 mg/dL) in all six patients within 40 days. Postoperative cholangitis did not occur and good bile drainage was obtained. CONCLUSIONS: Using this procedure, the portal vein becomes fully mobile after dividing the ligamentum venosum, and the porta hepatis can be widely exposed. The fibrous cord of the porta hepatis can be easily dissected off posteriorly and laterally.

Biliary Atresia↗

PubMed1997

Unique spindle cells in the involved ileal segment of rats with congenital aganglionosis.

Protein gene product (PGP) 9.5 is a newly characterized brain-specific protein originally detected by high-resolution two-dimensional electrophoresis of soluble human brain proteins. Immunoreactivity for PGP9.5 was investigated in the small and large intestine of aganglionic mutant and control rats with light microscopy, electron microscopy, and confocal laser scanning microscopy. Immunoreactive spindle-shaped cells were seen in the inner circular muscle layer of the aganglionic ileal segment. The long axes of these spindle cells paralleled the inner circular muscle fibers. PGP9.5-positive spindle cells were positive for enteric actin, and some of them were also positive for vimentin, a relatively specific marker for the special smooth muscle (pacemaker cells). These cells may prove to be a maldeveloped microenvironment of aganglionic segment. However, it is necessary to investigate expression of PGP9.5 in embryonal tissue with an abnormal environment to test this hypothesis. This study showed PGP9.5-positive pacemaker cells in the aganglionic ileal segment in the first time.

Animals↗

PubMed1997

Obstructive jaundice, an unusual initial manifestation of intraabdominal non-Hodgkin's lymphoma in children: complications of percutaneous transhepatic cholangial drainage.

Obstructive jaundice is an unusual initial manifestation of non-Hodgkin's lymphoma in children. Two pediatric patients with primary intraabdominal non-Hodgkin's lymphoma causing obstructive jaundice are presented. Although percutaneous transhepatic cholangial drainage (PTCD) is frequently used to treat obstructive jaundice, there was no definite rule for management of PTCD during chemotherapy, which may rapidly resolve the obstruction. Biliary peritonitis occurred in both patients soon after removal of the PTCD tube after chemotherapy. It was speculated that secure formation of the fistula for PTCD was impaired because of chemotherapy for a considerably long period. The PTCD tube that was already placed for obstructive jaundice in a non-Hodgkin's lymphoma patient should be maintained during chemotherapy with great care for displacement of the tube. PTCD can result in complications, and is probably unnecessary when lymphoma has been diagnosed before PTCD.

Abdominal Neoplasms↗

PubMed1997

Laparoscopic management of early primary abdominal pregnancy.

BACKGROUND: Abdominal pregnancy is a very rare condition with a high mortality rate. CASE: A 29-year-old woman with a history of primary infertility was admitted because of lower abdominal pain, bloody vaginal discharge, and positive urine pregnancy test. Transvaginal ultrasonography revealed a 27 x 25-mm mass containing a gestational sac-like structure located outside the uterus. At laparoscopy, a clot was revealed including chorionic villi, adherent to the peritoneum in the right vesicouterine pouch. This was removed along with the adherent peritoneum. Postoperative histology revealed invasion of chorionic tissues into peritoneum. CONCLUSION: Early diagnosis of an ectopic pregnancy by transvaginal ultrasonography enabled the laparoscopic management of early abdominal pregnancy.

Adult↗

PubMed1997

Brain-stem auditory evoked potentials in the common marmoset (Callithrix jacchus).

Brain-stem auditory evoked potentials (BAEPs) were recorded in 10 common marmosets (Callithrix jacchus) to investigate the effects of recording electrode configurations, stimulus rate, and stimulus frequency on BAEP wave forms and peak latencies. Tone burst stimulations were used to evaluate the effects of pure tone on BAEP wave forms. Five positive peaks superimposed on positive and negative slow potentials were identified in the BAEP recorded at the linkage between the vertex and the dorsal base of the ear ipsilateral to a monaural stimulus. When the reference electrode was placed at the ipsilateral mastoid or the neck, the amplitudes of positive and negative slow potentials and the incidence of wave I increased. There were no significant changes in peak latencies of BAEP waves with changes in stimulus rate from 5 to 20/s. It was possible to record the BAEPs in response to tone burst stimulations at frequencies extending from 0.5 to 99 kHz. Wave I appeared apparently at high stimulus frequencies; while waves III to V, at low frequencies. Wave II was recorded at frequencies ranging from 0.5 to 99 kHz and comprised a superposition of 2 or 3 potentials.

Acoustic Stimulation↗