Biomedical subjects

Y Hitomi

Publications and source records attributed to Y Hitomi.

At least 37 records · Page 2Linked to original sources

PubMed1999

The role of Ets family transcription factor PU.1 in hematopoietic cell differentiation, proliferation and apoptosis.

The PU.1 gene encodes an Ets family transcription factor which controls expression of many B cell- and macrophage-specific genes. Expression of the gene is critical for development of lymphoid and myeloid cell lineages, since PU.1-deficient mice exhibit defects in the development of these cell lineages. The PU.1 gene is identical to the Spi-1 gene isolated from common proviral integration sites in Friend virus-induced murine erythroleukemia (MEL), and deregulated expression of the gene is believed to be an essential step of the disease. We recently demonstrated that overexpression of PU.1 inhibits erythroid differentiation of MEL cells induced with the differentiating agent DMSO. We also noticed unexpectedly that overexpression of PU.1 together with DMSO induces marked growth arrest and apoptosis in MEL cells, supporting the notion that some oncogenes induce growth inhibition and apoptosis rather than cell proliferation and transformation under specific circumstances as shown with the c-myc gene. In this review, the role of PU.1 in hematopoietic cell differentiation, proliferation and apoptosis is described and the possible molecular mechanisms of PU.1-induced effects in MEL cells are discussed.

Animals↗

PubMed1999

The protective effect of cyclosporine A on anti-Fas antibody-induced hepatitis in mice.

The effect of cyclosporine A (CsA) on anti-Fas antibody-induced hepatitis was studied. The administration of anti-Fas antibody (250 microg/kg) to mice elevated plasma alanine aminotransferase (ALT) activity at 3 hr. This anti-Fas antibody-induced elevation of ALT was inhibited by treatment with CsA (10, 30 and 100 mg/kg) in a dose-dependent manner. Anti-Fas antibody administration elevated CPP32-like protease activity at 3 hr in mouse liver, and this elevation of CPP32-like activity was inhibited by treatment with CsA. The present results show that CsA treatment inhibits the anti-Fas antibody-induced apoptotic process of hepatitis, at least in part, by affecting a reaction upstream of CPP32-like protease activation.

Alanine Transaminase↗

PubMed1999

Increased growth of Bifidobacterium and Eubacterium by germinated barley foodstuff, accompanied by enhanced butyrate production in healthy volunteers.

Germinated barley foodstuff (GBF) derived from the aleurone and scutellum fractions of germinated barley mainly consists of low-lignified hemicellulose and glutamine-rich protein. GBF improves the proliferation of intestinal epithelial cells and defecation, through the bacterial production of short chain fatty acids (SCFA), especially butyrate. In this study we investigated the mechanism of production of butyrate by microflora in humans and in vitro. Daily administration of 9 g GBF for 14 successive days significantly increased fecal butyrate content. Fecal Bifidobacterium and Eubacterium were also significantly increased by GBF administration in healthy volunteers. Ten anaerobic micro-organisms selected from intestinal microflora were cultured in vitro in the medium containing GBF as a sole carbon source (GBF medium). After a 3-day incubation, 7 strains (Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Bacteroides ovatus, Clostridium butyricum, and Eubacterium limosum) lowered the medium pH producing SCFA. Eubacterium grown together with Bifidobacterium in GBF medium efficiently produced butyrate. On the other hand, GBF changed the intestinal microflora and increased probiotics such as Bifidobacterium in the intestinal tract. As a result, butyrate was produced by the mutual action of Eubacterium and Bifidobacterium. This butyrate is considered to enhance the proliferation of colonic epithelial cells.

Adult↗

PubMed1998

Suppression of the 5' splice site mutation in the Nagase analbuminemic rat with mutated U1snRNA.

Nagase analbuminemic rats (NAR) are deficient in serum albumin due to skipping of the albumin exon H caused by a mutation in the intron HI. This mutation deletes nucleotides from +5 to +11 in the 5' splice site region, where it interacts with U1snRNA. To determine whether the mutation can be suppressed by the compensatory base substitution in U1snRNA, we constructed mutated U1snRNA genes with various degrees of complementarity to the mutated 5' splice site. Several mutated U1snRNA genes activated the mutated 5' splice site of the intron HI, when cotransfected with the albumin minigene derived from NAR. In vivo activity of these mutated U1snRNAs correlated well with the predicted thermodynamic stability. Since mutation in the 5' splice site is one of common causes of genetic defects in human (5), these data indicate that NAR is a good model system to examine the possibility of gene therapy using a mutated U1snRNA.

Animals↗

PubMed1998

Germinated barley foodstuff improves constipation induced by loperamide in rats.

The effects of germinated barley foodstuff (GBF) derived from the aleurone and scutellum fractions of germinated barley low-lignified hemicellulose were examined in Sprague-Dawley rats with constipation induced by loperamide by addition to the diet (2 mg/kg body weight). Bowel movements were higher in the GBF-fed rats than in the cellulose-fed rats used as a control. Fecal water content was also higher in the GBF-fed rats. The concentration of short chain fatty acids in cecal content, especially butyrate, was significantly higher in the GBF-fed rats than in the cellulose-fed rats. These findings suggested that GBF helps normalize defecation not only in diarrhea but also constipation.

Animals↗

PubMed1998

Germinated barley foodstuff increases fecal volume and butyrate production at relatively low doses and relieves constipation in humans.

Germinated barley foodstuff (GBF), derived from the aleurone layer, scutellum and germ of germinated barley, contains a large quantity of fermentable dietary fibers, especially hemicellulose. GBF was given to 9 healthy volunteers in a dose of 9 g of GBF per day for 10 consecutive days, and subsequently 18 g of GBF for another 10 days. As a control, no GBF was given for 3 days before administration of GBF (control period). Fecal weight, water content and short chain fatty acid content were measured before and during the last 3 days of each experimental period. Feeding of GBF significantly increased the fecal butyrate content as well as fecal weight at both dose-levels (9 and 18 g/day), compared with those during the control period. Daily administration of 9 g GBF induced the maximum level of defecation in humans. Relatively mild but chronic constipated volunteers (n = 16) were administered 9 g of GBF daily for 14 days. In this experiment, the condition of defecation (frequency, volume) was estimated by a questionnaire survey. GBF significantly improved defecation within a short period without severe adverse effects. No major abnormalities in laboratory findings were found in hematologic and urinary analyses. In conclusion, daily administration of 9 g GBF was effective for improving defecation in healthy but constipated humans. GBF is a highly safe and effective foodstuff for improving defecation.

Adult↗

PubMed1997

Augmentation of ouabain sensitivity of rat liver Na/K-ATPase by in vivo adenovirus-mediated expression of the Na/K-ATPase alpha2 subunit.

These are the first experiments to study the effect of in vivo expression of the Na/K-ATPase alpha2 subunit which serves as a receptor for cardiac glycosides. The alpha2 subunit is not normally expressed in rat liver, so hepatocytes which lack endogenous alpha2 protein are a logical first target to study the effects of alpha2 expression on membrane Na/K-ATPase activity. At 3 days after alpha2 adenovirus vector infusion, Wistar rat livers contained alpha2 DNA, alpha2 mRNA, and alpha2 protein. Rat liver membrane ouabain binding activity and the sensitivity of Na/K-ATPase activity to ouabain significantly increased. Total membrane Na/K-ATPase was regulated at a constant level while expressed alpha2 activity represented 10% of the total active Na/K-ATPase sites in alpha2 transduced rat liver. These studies are the first to establish a paradigm in which an endogenous drug receptor is expressed to alter cellular pharmacologic sensitivity.

Adenoviruses, Human↗

PubMed1997

Evidence for monoclonal expansion of epithelial cells in ovarian endometrial cysts.

Ovarian endometrial cysts, one of the typical manifestations of endometriosis, are generated by the retention of cyclic hemorrhages and are classified as tumor-like lesions rather than neoplasms. Clonality analysis provides important information about the histogenesis and progression of neoplastic diseases. As it is generally accepted that most neoplasms are monoclonal in origin, however, the clonality of endometrial cysts remains uncertain. Using the human androgen receptor gene (HUMARA) as an X-linked polymorphic marker, we examined the clonal status of epithelial cells in endometrial cysts. We separated 21 fresh epithelial cell samples from 11 endometrial cysts and found that all were monoclonal in the methylation pattern of the HUMARA alleles. Moreover, in each of the five cysts from which epithelial cells were sampled from multiple and distant areas, the methylation patterns of all samples from a single cyst were identical. These data indicate that endometrial cysts are monoclonal in origin and suggest their neoplastic potentiality.

Adult↗

PubMed1996

Suppression of Lck protooncogene expression in murine somatic cell hybrids between T lymphoma cells and fibroblasts.

Somatic cell hybrids were obtained by cell fusions between Lck-positive EL4 mouse T lymphoma cells and Lck-negative B82 mouse fibroblasts of S194 mouse plasmacytoma cells to examine negative control of lck gene expression in the resulting hybrids. Western blot analysis using a monoclonal antibody against the Lck protein showed a marked decrease in p56lck expression in B82 x EL4 (BEL) hybrids. In contrast to BEL hybrids, the level of p56lck was not changed significantly in S194 x EL4 (SEL) hybrids and was approximately one-half of that seen in EL4 cells. Diminished expression of the Lck protein in BEL hybrids paralleled downregulation of lck mRNA, which was exclusively transcribed from the distal promoter in EL4 cells. It is unlikely that the suppression was simply a consequence of chromosome segregation critical for lck gene expression, since BEL hybrids retained the EL4-derived lck gene and most of the chromosomes from both parental cells. The results from treatment of BEL hybrids with actinomycin D or cycloheximide suggested that suppression of lck gene expression in the hybrids might not be due to posttranscriptional control. DNA methylation status in the lck distal promoter and the coding regions did not appear to correlate with the expression of the gene. Our results suggest that negative control of lck gene expression differs between fibroblasts and B cells, in that lck gene expression in T cells can be shut down by transfer of a putative repressor factor or factors in fibroblasts but not in B cells.

Animals↗

PubMed1996

Complete correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome type I following transient in vivo adenovirus-mediated expression of human bilirubin UDP-glucuronosyltransferase.

Recombinant adenoviral vectors are useful for the in vivo expression of genes in hepatocytes. Adenoviral vectors deleted in E1a, E1b, and E3b were constructed and used to study in vivo expression of the major human bilirubin UDP-glucuronosyltransferase isoform (HUG Br1) under the transcriptional control of the cytomegalovirus (CMV) immediate-early promoter-enhancer (H5.010CMV hugBr1). As a control, a recombinant adenoviral vector containing the beta-galactosidase reporter gene driven by the CMV promoter-enhancer was employed (H5. 010CMVlacZ). Recombinant virus was expanded following exposure to E1 transcomplementing (293) cells and concentrated to t titer of approximately 10(13) particles per milliliter. A rat model for Crigler-Najjar syndrome type I deficient in HUG Br1 (ie the Gunn rat) was injected with 5 X 10(9) plaque-forming units (p.f.u.) via the portal vein of either H5.010CMVhugBr1 or H5. 010CMVlacZ. Rats from each set were killed at 3 days, 11 days and 22 days after infusion. Liver total cellular DNA, RNA and protein were analyzed for the transgene and the transgene product at the specified times. Analysis of livers by Southern blot hybridization demonstrates sequence-specific hybridization to adenoviral vector DNA, and Northern blot hybridization demonstrates sequence-specific hybridization to transgene-derived RNA. DNA levels peak at approximately one copy number at 3 days and decline over 22 days. RNA and Western blot analyses demonstrate overexpression of message and protein at 3 days, declining over 22 days. In virto functional assay for bilirubin glucuronosyl-transferase activity demonstrates overexpression of bilirubin UDP-glucurosyltransferase function. In situ hybridization of frozen sections to detect expressed mRNA using beta-galactosidasederived 35S-labeled riboprobes demonstrates adenovirus-derived transgene expression in hepatocytes. Significant drops in serum bilirubin levels were noted following expression of HUG Br1 but not beta-galactosidase. The drop in serum bilirubin correlates with the appearance of bilirubin glucuronides in bile. In summary, recombinant adenoviral vectors were used to demonstrate in vivo complementation of the genetic defect in Gunn rat livers with the HUG Br1 cDNA leading to a resolution of hyperbilirubinemia lasting approximately 7 weeks. These studies suggest that delivery of the HUG Br1 cDNA might provide a reasonable therapeutic benefit for Crigler-Najjar syndrome type I patients, as safe and efficacious gene delivery systems are developed.

Adenoviridae↗

PubMed1995

Laminin-rich extracellular matrix maintains high level of hepatocyte nuclear factor 4 in rat hepatocyte culture.

Laminin-rich extracellular matrix, EHS-gel, has been demonstrated to keep a high level of liver-specific gene expression in cultured rat hepatocytes. To obtain information about the effect of EHS-gel on liver-specific functions, gene expression of liver-enriched transcription factors in rat hepatocytes was investigated. The apolipoprotein A-I and albumin mRNA levels were higher in hepatocytes cultured on EHS-gel than in those cultured on type I collagen (TIC). The levels of mRNA for HNF-4, C/EBP alpha and C/EBP beta were also higher on EHS-gel than on TIC. The level of HNF-4 mRNA in hepatocytes on EHS-gel was almost comparable to that in liver. The HNF-3 alpha mRNA level was lower on EHS-gel than on TIC. C/EBP beta mRNA was induced by dexamethasone in both EHS-gel and TIC. The induction of C/EBP alpha and HNF-4 by dexamethasone was observed only on TIC. These data suggest that EHS-gel leads hepatocytes to keep the phenotypic expression through high expression of liver-enriched transcription factors, such as HNF-4.

Animals↗

PubMed1995

Role of intron I in expression of the human factor IX gene.

The first intron (intron I) of the human factor IX gene, which has been previously suggested of having an expression-augmenting activity, was systematically studied for its potential enhancer activity. When tested with the chloramphenicol acetyltransferase expression vector with a minimal factor IX promoter, subregions of intron I showed only marginal enhancing activities (1.7-1.9-fold enhancement at the highest). Smaller subregions encompassing nucleotides 5660-6350 of the intron sequence even showed some weak negative regulatory activities (approximately 50% suppression at the highest), while a cytomegalovirus enhancer sequence, which was used as the positive control, had a 7-fold enhancement. A set of three factor IX minigene expression vectors with the same factor IX promoter were then constructed: p-416FIXc which contained the factor IX cDNA, p-416FIXm1 which contained the factor IX cDNA with a largely truncated intron I, and p-416FIXm2 which contained the factor IX cDNA with the intron I sequence further truncated. The p-416FIXm1 and p-416FIXm2 constructs showed 7-9-fold higher expression activities than p-416FIXc. The elevated factor IX antigen levels agreed well with the grossly elevated factor IX clotting activity and mRNA levels. These results indicate that the expression enhancing activity of intron I is not due to specific enhancer elements present in the intron subsequences, but is due to functional splicing sequences present in the precursor mRNAs produced from the minigene constructs containing intron I. By being efficiently assembled into spliceosome complexes, transcripts with splicing sequences may be better protected in the nucleus from random degradations than those without such sequences.

Animals↗

PubMed1995

Differential expression of the T cell receptor/CD3 genes and their lymphoid-specific transcription factor genes in murine T cell x fibroblast and T cell x B cell hybrids.

We generated cell hybrids between mouse T cell lymphoma EL4 cells and mouse fibroblast B82 cells (BELIII and BELIV) to examine the expression of T cell receptor (TcR)/CD3 genes and their lymphoid-specific transcription factor genes, which are normally detected in EL4 cells. In BELIII and BELIV, expression of the TcR alpha, TcR beta and CD3 delta genes was extinguished, whereas expression of the CD3 epsilon gene was still detected. Expression of the (lymphoid enhancer binding factor 1) LEF-1 gene was extinguished and that of the GATA-3 gene was hardly detected in BELIII and BELIV. Ets-1 gene expression, observed not only in EL4 cells but also in B82 cells, was considerably reduced in BELIII and BELIV. A much higher level of PEBP2 alpha A gene expression was observed in B82 cells than in EL4 cells and was preserved in BELIII and BELIV. To examine whether reduced expression of these genes is also found in T cell x B cell hybrids, we generated an additional cell hybrid between EL4 cells and mouse plasmacytoma S194 cells (SELIII). Marked differences were observed in the expression of the TcR alpha, CD3 delta, LEF-1 and PEBP2 alpha A genes in BEL and SEL hybrids. Expression of the TcR alpha, CD3 delta and LEF-1 genes, which was extinguished in BELIII and BELIV, was detected in SELIII. PEBP2 alpha A gene expression, not detected in S194 cells, was considerably reduced in SELIII. Almost the sum of the chromosomes from the parental cells were retained by, and the presence of every gene was proven, in each cell hybrid. These results suggest that suppression of the expression of lymphoid-specific transcription factor genes may precede that of the TcR/CD3 genes in the cell hybrids, and that the presence of a different trans-acting negative regulatory mechanism(s) suppresses the expression of T cell specific genes in fibroblasts and B cells.

Animals↗

PubMed1995

High efficiency prokaryotic expression and purification of a portion of the hepatitis C core protein and analysis of the immune response to recombinant protein in BALB/c mice.

Hepatitis C virus (HCV) produces chronic persistent liver infection in 1-2% of the U.S. population and is the leading cause of end stage liver disease in patients presenting for liver transplantation at our center. Efforts to cure persistent HCV infection are frequently unsuccessful, so the development of a HCV vaccine is a high priority. HCV envelope proteins are hypervariable so production of a recombinant surface antigen vaccine such as is available for hepatitis B is not likely to confer widespread, high level protective immunity. As the most highly conserved structural protein in the HCV genome, the core protein is one reasonable target for vaccine production. Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression. The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 and 163 amino acids of the core region. Deletion of the 23 amino acids (aa) from aa141-163 led to a marked increase in the efficiency of protein production from < 1 to 3-4 mg/liter for both systems studied. Protein purification was accomplished using affinity chromatography (MBP) or inclusion body isolation (GST) as determined by SDS-PAGE gels and immunotransblot with HCV core protein-specific monoclonal antibody. Finally, the immune response to recombinant protein was assessed in BALB/c mice using a MBP HCV core fusion protein and an ELISA developed using GST HCV core protein as a target. In all mice of this strain, serum anti-HCV core antibody titer increased to 10(-4), two logs above background, following immunization in conjunction with Freund's complete adjuvant. These results represent an encouraging first step toward production of a core protein vaccine. Recombinant core protein is a useful tool to study the immune response to core protein and may be useful to further study the epidemiology and biology of the HCV virus.

3T3 Cells↗

PubMed1995

Sequence analysis of the hepatitis C virus (HCV) core gene suggests the core protein as an appropriate target for HCV vaccine strategies.

Hepatitis C virus (HCV) is a major healthproblem with a prevalence of 1% in the United States population, and a significant percentage of infected patients progress to chronic liver disease and cirrhosis. Interferon therapy has demonstrated that the immune system can be modulated to alter the acute course of the disease, but long-term treatments remain elusive. Prevention of hepatitis C infection is therefore an important strategy to mitigate the impact of this disease. Initial attempts at vaccination have focused on recombinant envelope vaccines, which have shown an ability to protect against very low titre challenges of HCV in chimps. The need for vaccines capable of protecting against higher titre challenges has led to the search for alternative vaccine strategies. The most highly conserved structural protein in the HCV genome is the core protein, and vaccine strategies targeting the core protein have been proposed to increase vaccine efficacy. The variability of HCV core sequences and genotypes in the Ann Arbor patient population are not known, and the present study was undertaken to assess the theoretical feasibility of developing a HCV core vaccine by excluding promiscuous core (C) gene variability as a mechanism of vaccine failure. Results of nucleotide and deduced amino acid sequence analysis from 13 of 14 patients studied reveal a 93% nucleotide and 96.4% amino acid core sequence homology in the C gene regions studied. Genotype analysis revealed four of 14 to be type 1a and nine of 14 to be type 1b with one infection not being sufficiently characterized to determine genotype. These results demonstrate a sufficiently high degree of conservation of HCV core sequences in our patient population to permit design of a vaccine directed against core protein.

Amino Acid Sequence↗

PubMed1994

Cell type specific patterns of mRNA splicing in hepatoma cells transfected with the mutated albumin minigene of Nagase analbuminemic rats.

We constructed an SV40-derived expression vector containing a mutated albumin minigene of Nagase analbuminemic rats (NAR), and introduced it into cultured cells. Transient expression of the minigene mRNA was determined by RT-PCR. Three kinds of aberrant mRNAs were expressed by the non-hepatic cells COS-1, and the undifferentiated human hepatoma cells HLE, transfected with the minigene. Their predominant mRNA lacked exon H (delta H), while mRNAs lacking exons H and I, or exons G and H were less abundant. This pattern of the mRNAs was similar to that of albumin mRNAs in the liver of old NAR. In contrast, a differentiated type of human hepatoma cell line, HepG2, expressed only delta H mRNA, like young NAR. These findings indicate that the expression system of the mutated minigene in cultured hepatoma cells is useful for understanding two-exon-skipping of albumin pre-mRNA of NAR.

Alternative Splicing↗

PubMed1994

Cloning and functional expression of poly(ADP-ribose) polymerase cDNA from Sarcophaga peregrina.

A cDNA spanning the entire coding region for poly(ADP-ribose) polymerase (PARP) of Sarcophaga peregrina was isolated and the nucleotide sequence was determined. The longest open reading frame encodes a polypeptide of 996 amino acid residues with a molecular mass of 113,033 Da. The similarities to the human PARP in amino acid sequence were relatively low in the DNA-binding and auto-modification domains, but very high in the C-terminal catalytic domain: identity of amino acids is 34% in the N-terminal DNA-binding domain (residues 1-369), 27% in the auto-modification domain (residues 370-507), and 56% in the C-terminal NAD-binding domain (residues 508-996). Two zinc-fingers (C-X2-C-X28-H-X2-C and C-X2-C-X31-H-X2-C)2 and a basic region in the N-terminal DNA-binding domain recognized in other PARP are conserved. Downstream of the basic region, another cysteine-rich motif (C-X2-C-X13-C-X9-C), a putative zinc-finger, was found to be well conserved in the PARP of Sarcophaga, Drosophila and human. A leucine-zipper motif (L-X6-L-X6-L-X6-L) which was found in the auto-modification domain of Drosophila PARP, is disrupted in the Sarcophaga enzyme: the second leucine is replaced by proline, and the third leucine by valine. Full-length cDNA for Sarcophaga PARP was cloned into an expression plasmid and expressed in Escherichia coli. A lysate of E. coli cells containing expressed protein reacted with antibody against Sarcophaga PARP, and PARP activity was detected. Thus, we conclude that isolated cDNA encodes a functional Sarcophaga PARP cDNA.

Amino Acid Sequence↗

PubMed1994

Autologous blood transfusion for the patient with placenta previa complicated by placenta increta: a case report.

The patient, who was 34 years of age, had previously had a transverse incision of the lower uterine segment cesarean section because of placenta previa. She was admitted to the hospital due to placenta previa again at 27 weeks of gestation in the current pregnancy. Ultrasound examination revealed placenta increta as well as placenta previa. In an attempt to avoid homologous blood transfusion at the time of profuse hemorrhage anticipated to occur during cesarean section, an autologous blood transfusion was planned. Fifteen hundred ml of autologous blood was collected by a leap-frog method during the 8 weeks prior to cesarean section. A cesarean hysterectomy was performed at 37 weeks of gestation because of placenta increta. Blood loss was estimated at 1,830 ml, and 1,500 ml of autologous blood was transfused. A leap-frog method of autologous blood collection for this pregnant woman with risk of massive hemorrhage was simple and beneficial, resulting in the preservation of more than 1,500 ml of autologous blood for transfusion.

Adult↗