Search PubMed⌕ Search

Biomedical subjects

J Fujimoto

Publications and source records attributed to J Fujimoto.

At least 271 records · Page 15Linked to original sources
PubMed1992

Protein kinase-C activation during thyrotropin-stimulated proliferation of rat FRTL-5 thyroid cells.

We have reported previously that TSH and insulin-like growth factor-I (IGF-I) synergistically stimulate DNA synthesis and elevate the 1,2-diacylglycerol (1,2-DG) content of FRTL-5 thyroid cells and have suggested that protein kinase-C (PKC) may mediate the growth-promoting effects of these hormones. We now present evidence that the effects of TSH on 1,2-DG content are associated with commensurate changes in PKC activity. We measured 1,2-DG content and PKC activity in TSH-deprived growth-arrested cells when TSH was readded. Cells were maintained in medium containing a high dose of insulin (which interacts with IGF-I receptors) and no TSH. When cells incubated in the absence of TSH were reexposed to TSH for 24 h, the 1,2-DG content increased to 234 +/- 22% of the control value, and the ratio of PKC activity in membrane and cytosol fractions of cell homogenates, an index of the state of activation of PKC in situ, increased to 323 +/- 42% of the control value. In cells growing under the influence of TSH in medium containing a high dose of insulin, we found that PKC activity varied during growth. Total cellular PKC activity (3.2 +/- 0.1 nmol/min.micrograms DNA) and the ratio of membrane/cytosol PKC activity (0.24 +/- 0.002) were high during exponential proliferation and fell progressively to 1.1 +/- 0.08 nmol/min.micrograms DNA and 0.12 and fell progressively to 1.1 +/- 0.08 nmol/min.micrograms DNA and 0.12 +/- 0.01, respectively, as cells attained confluence. The specific activity of membrane-associated PKC was 3.0 +/- 0.37 nmol/mg.min in early exponential growth and declined to 0.72 +/- 0.14 nmol/mg.min as cell proliferation ceased. The 1,2-DG content also varied during growth, with a peak occurring during exponential growth, followed by a decline as cells attained a confluent state. These data are consistent with the hypothesis that the growth-promoting effects of TSH in FRTL-5 cells are mediated, at least in part, by 1,2-DG activation of PKC. Since we have demonstrated previously that the effect of TSH to elevate 1,2-DG is, in turn, mediated by cAMP, this represents a special example of the interaction of these two signal transduction systems in regulation of cell proliferation.

Animals↗
PubMed1992

Mechanism of regulation of thyrotropin-releasing hormone receptor messenger ribonucleic acid in stably transfected rat pituitary cells.

We showed previously that the level of TRH receptor (TRH-R) mRNA in rat pituitary GH3 cells is down-regulated by TRH. Here, we study the mechanism of regulation of TRH-R mRNA in a line of GH3 cells that are stably transfected with mouse pituitary TRH-R cDNA (GH-mTRHR-1 cells). GH-mTRHR-1 cells were found to have 2.4 times the number of TRH-Rs and to stimulate a 2.5-fold greater increase in inositol phosphates in response to TRH than the parent cell line and to show TRH-induced down-regulation of TRH-R number. GH-mTRHR-1 cells contained 26 +/- 1.6 molecules of mouse TRH-R mRNA/cell and 230 +/- 31 molecules of mRNA for the neomycin resistance gene (NEO) with which it was cotransfected. In GH-mTRHR-1 cells, TRH caused a dose-dependent transient decrease in mouse TRH-R mRNA, with a nadir to 20% of control levels after 6 h. In contrast, TRH did not affect NEO mRNA or glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA, an endogenous gene product. TRH stimulated the rate of transcription of mouse TRH-R DNA by approximately 2-fold, but did not affect total poly(A) RNA synthesis. Most importantly, TRH caused a 4-fold increase in the rate of degradation of mouse TRH-R mRNA, but did not affect degradation of GAPDH mRNA. The half-lives of mouse TRH-R and GAPDH mRNAs were 3 and more than 20 h in control cells and 0.75 and more than 20 h in cells treated with 1 microM TRH for 1.5 h, respectively. These data show that the predominant effect of TRH on mouse TRH-R mRNA in GH-mTRHR-1 cells is to enhance the rate of its degradation. We suggest, therefore, that down-regulation of TRH-R mRNA caused by TRH in the parent GH3 cell line is secondary to increased TRH-R mRNA degradation.

Animals↗
PubMed1992

Posttranscriptional up-regulation of thyrotropin-releasing hormone (TRH) receptor messenger ribonucleic acid by TRH in COS-1 cells transfected with mouse pituitary TRH receptor complementary deoxyribonucleic acid.

We found previously that the level of endogenous TRH receptor (TRH-R) mRNA in pituitary (GH3) cells and the level of mouse TRH-R mRNA in GH3 cells stably transfected with mouse pituitary TRH-R cDNA are down-regulated by TRH. This down-regulation is caused by TRH stimulation of TRH-R mRNA degradation via a mechanism that appears to involve protein kinase-C. In this report we study regulation of TRH-R mRNA in monkey kidney (COS-1) cells transiently transfected with mouse pituitary TRH-R cDNA. In transfected COS-1 cells, TRH and phorbol 12-myristate 13-acetate (PMA) caused increases in the level of TRH-R mRNA. In contrast, TRH caused only a small transient increase in the level of the mRNA for the neomycin resistance gene, which was cotransfected with TRH-R, and did not affect the level of the mRNA for glyceraldehyde phosphate dehydrogenase, an endogenous gene. The increases in TRH-R mRNA caused by TRH and PMA were inhibited to similar extents by H-7 (1-[5-isoquinolinesulfonyl]2-methyl piperazine dihydrochloride), an inhibitor of protein kinases. The effect of TRH was observed in cells transfected with expression vectors in which TRH-R cDNA was controlled by cytomegalovirus or Rous sarcoma virus promoters. There was no effect of TRH or PMA on the rate of transcription of the transfected TRH-R cDNA. In contrast, TRH caused the rate of degradation of TRH-R mRNA to decrease from 8.0% to 5.1%/h. Hence, TRH, most likely via a protein kinase-C-mediated mechanism, up-regulates TRH-R mRNA levels in transfected COS-1 cells by decreasing the rate of TRH-R mRNA degradation. Since TRH and PMA down-regulate TRH-R mRNA in GH3 cells, posttranscriptional regulation of TRH-R mRNA is a cell-type specific process.

Animals↗
PubMed1991

Flow cytometric DNA analysis of hepatocellular carcinoma.

The prognostic value of nuclear DNA content was studied retrospectively using flow cytometry in 203 cases of resected hepatocellular carcinoma. The occurrence of DNA aneuploidy, which was detected in 50% of patients, correlated significantly with tumor size and the presence of vascular invasion or intrahepatic metastasis. Overall, patients with DNA aneuploid tumors had a significantly worse prognosis than those with DNA diploid tumors (P less than 0.001) and, also in subdivided groups by tumor size (P less than 0.01). Among DNA aneuploid patients, the survival times were significantly shorter for patients with a low DNA index (less than 1.5) than for those with a high DNA index (greater than or equal to 1.5) (P less than 0.05). In a Cox multivariate analysis, nuclear DNA content provided significant prognostic value (P = 0.008), as did vascular invasion (P = 0.001) and intrahepatic metastasis (P = 0.005). These results indicated that nuclear DNA content has an important prognostic value in hepatocellular carcinoma.

Carcinoma, Hepatocellular↗
PubMed1991

Evidence for heterogeneous groups of neuronal differentiation of Ewing's sarcoma.

We have investigated the capability of differentiation of Ewing's sarcoma (ES) towards a neuronal direction through the establishment of four extraosseous ES cell lines and by in vitro stimulation with dibutyryladenosine cyclic monophosphate (db-cAMP) of eight ES lines. All except one of the lines expressed the molecule defined by 5C11, the antibody specifically reactive with ES. Two ES lines expressed a 200 kilodalton (kD) neurofilament protein (NFP) although their original tumours were negative for NFP. Elongation of cytoplasmic processes and increased NFP expression were observed after db-cAMP treatment of these lines and microtubules in the cytoplasmic processes were ultrastructurally demonstrated. Six lines were NFP negative, but three lines changed their morphology after induction of 200 kD NFP expression by db-cAMP treatment. The other three showed no definitive differentiation after db-cAMP treatment. Chromosomal analysis of the new ES lines showed the typical t(11;22) in one line and a +der(22) in two lines. No correlation was observed between the chromosomal abnormality and the differentiation capability. We conclude that ES is a heterogeneous group of tumours with respect to capability of differentiation into the neuronal lineage, but it is clearly distinguished from peripheral primitive neuroectodermal tumours by its 5C11 reactivity.

Adolescent↗
PubMed1991

Monoclonal antibody against bone marrow stromal cells. Its production and characterization.

Bone marrow stromal cells play an essential role in the proliferation and differentiation of hematopoietic stem cells (1, 2). As a means of analyzing of the bone marrow microenvironment immunohistochemically, we attempted to produce a rat monoclonal antibody against the murine preadipocyte line H-1 derived from long-term bone marrow culture (LTBMC) of C57BL/6 mice (3, 4). A newly established monoclonal antibody, designated R4-A9, was obtained from a hybridoma prepared by fusion of Y.B2/3.0Ag20(YO) rat myeloma cells with spleen cells of LEW rats immunized with H-1 cells. The immunofluorescence of live H-1 cells showed that the antigen reacting with this antibody was strongly expressed on the cell surface. The specificity of R4-A9 was assessed immunohistochemically on frozen sections of various tissues from normal adult mice. R4-A9 demonstrated specificity for hematopoietic stroma in bone marrow and spleen. No staining was observed in thymus, lymph nodes or other tissues examined, with the exception of Leydig cells in the testis and the endothelium of small arteries in several organs. Detailed immunohistochemical observations at both the light microscopy and electron microscopy level showed that R4-A9 selectively reacted with the sinusoidal endothelium, perisinusoidal adventitial cells (5) (adventitial reticular cells (6] and intersinusoidal reticular cells (5) and the reticular cells of the splenic red pulp. These findings indicate that reticular cells and the endothelium of the bone marrow possess the common cell surface molecules recognized by R4-A9. SDS-PAGE analysis showed that R4-A9-immunoprecipitated proteins had a molecular mass of 100 kDa under reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Purification of human pancreatic lipase and the influence of bicarbonate on lipase activity.

Pancreatic lipase was purified from human pancreatic juice by ion exchange chromatography and HPLC molecular sieve chromatography. The molecular weight was measured as 50 Kda by SDS-PAGE, as 47 Kda by HPLC and was calculated as 51 Kda following amino acid analysis. The isoelectric point of the purified lipase was 7.4 and maximal enzyme activity occurred at pH 9.5 in glycine buffer (0.1 mol/L) containing deoxycholate (19 mmol/L), colipase (3 mg/L) and triolein (0.3 mmol/L). However, the addition of bicarbonate at a final concentration of 0.1 mol/L decreased the pH for maximal enzyme activity and increased the lipase activity significantly. At higher concentrations of bicarbonate the lipase activity decreased. These results suggest that bicarbonate is an important regulator of lipase activity, perhaps related to an effect on the detergent properties of deoxycholate.

Amino Acids↗
PubMed1991

Evidence for dual regulation by protein kinases A and C of thyrotropin-releasing hormone receptor mRNA in GH3 cells.

We showed previously that TRH down-regulates TRH receptor (TRH-R) mRNA in GH3 cells by a mechanism that appears to be mediated by protein kinase C. Here we show that vasoactive intestinal peptide (VIP) down-regulates TRH-R mRNA and present evidence that this action is mediated by protein kinase A. In GH3 cells, VIP caused a time- and concentration-dependent decrease in TRH-R mRNA. This VIP effect was simulated by 8-(4-chlorophenylthio)-cAMP, forskolin, cholera toxin and 1-methyl-3-isobutylxanthine. When cells were incubated with agents that elevate cAMP and TRH or phorbol 12-myristate 13-acetate, the decrease in TRH-R mRNA was greater than with either agent alone. When cells were pre-incubated with H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, the effects of VIP, TRH and phorbol 12-myristate 13-acetate were inhibited. We suggest that VIP, via protein kinase A, and TRH, via protein kinase C, dually regulate TRH-R mRNA.

Animals↗
PubMed1991

Thyrotropin-releasing hormone (TRH) and phorbol myristate acetate decrease TRH receptor messenger RNA in rat pituitary GH3 cells: evidence that protein kinase-C mediates the TRH effect.

In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

[Autotransfusion in hepatectomy for hepatocellular carcinoma].

Experimental data of autotransfusion in hepatectomy for hepatocellular carcinoma and clinical data from forty-eight patients were analyzed. By a density gradient medium, cancer cells banded at a lower density (1.056 g/ml: mean) than red blood cells (1.105 g/ml: mean). The centrifuge autotransfusor separated the red blood cells from cancer cells and all sections of cell blocks of the blood harvested intraoperatively in 20 cases revealed no malignant cells. The average requirement of bank blood was significantly reduced in these patients than in patients without autotransfusion during one year before, 1573 ml to 478 ml (whole blood of concentrated red blood cells) (p less than 0.05). These findings indicate that autotransfusion in hepatectomy for hepatocellular carcinoma is safe and practical.

Blood Transfusion, Autologous↗
PubMed1991

[Treatment of hepatocellular carcinoma from the aspect of nuclear DNA analysis].

Flow cytometric DNA analysis was done on 302 resected hepatocellular carcinoma and 30 specimens by fine needle aspiration biopsy. Patients with DNA aneuploid tumors had a significant worse prognosis than those with DNA diploid tumors in subdivided groups by curability (p less than 0.01). The recurrence after hepatectomy occurred frequently in DNA aneuploid or non-curative resected group. In a cox multivariate analysis, nuclear DNA content provided significant prognostic value (p = 0.008) as did vascular invasion and intrahepatic metastasis. For the multiple tumors of hepatocellular carcinoma, it is helpful to get the DNA ploidy pattern to diagnose tumors with intrahepatic metastasis or multicentric origin. Nuclear DNA analysis by fine needle aspiration biopsy was useful to determine the treatment method of hepatocellular carcinoma.

Carcinoma, Hepatocellular↗
PubMed1991

Clonal analysis of transient myeloproliferative disorder in Down's syndrome.

Three Down's syndrome patients with transient myeloproliferative disorder were studied for clonality of the proliferating blast cells using the X chromosome-linked polymorphic gene phosphoglycerate kinase, immunoglobulin heavy chain (IgH) gene and T-cell antigen receptor (TCR) (beta, gamma, delta) genes. None of the three cases showed rearrangements of IgH, TCR beta, gamma, or delta genes, indicating the non-lymphoid nature of the proliferating blast cells. The X chromosome inactivation pattern showed that the cells in the blast population in all of the three cases of transient myeloproliferative disorder were clonal. These data suggest that at least some of this disorder can be due to a spontaneously regressing clone of malignant cells.

Adolescent↗
PubMed1991

A case of mixed lineage acute non-lymphocytic leukemia with t(5;12)(p13;p13).

A two-year-old boy had mixed lineage acute non-lymphocytic leukemia (ANLL) with a 46,XY, t(5;12)(p13;p13) karyotype. He was admitted to the hospital with fever and petechiae. Morphological and cytochemical characteristics showed the blasts to meet the standard French-American-British criteria for M1, but surface marker analysis showed the blasts to express both myeloid (CD33: 91.3%) and T-cell (CD2: 82.3%, CD7: 97.9%) antigens. The boy was treated with an ANLL protocol, and successfully brought to a remission which has continued for more than 30 months.

Antigens, Differentiation↗
PubMed1990

Prognostic factors after hepatectomy for hepatocellular carcinomas. A univariate and multivariate analysis.

The current study determines the prognostic factors after hepatectomy for hepatocellular carcinomas. The 295 patients who underwent hepatectomy from 1973 through 1987 were included for a univariate and a Cox multivariate analysis. The favoring conditions were determined as follows. The essential requirements are (1) the absence of tumor thrombi; (2) no intrahepatic metastasis, but even when present, it should be close to the main tumor and removed with a massive resection; and (3) retention rate of indocyanine green dye (ICG) at 15 minutes should be within 14 +/- 4.2% (M +/- SD) to allow that resection. The desired requirement is that the tumor size should preferably be less than 5 cm; a wider free margin from tumors (greater than or equal to 1 cm) is recommended, but not determining factor. The eligible patients, having no thrombi, no intrahepatic metastasis, a tumor size of 5 cm or less, negative surgical margin (greater than or equal to 1 cm), had achieved a 5-year survival of 78%. In conclusion, resection therapy is the first option for patients with those requirements.

Adult↗
PubMed1990

Characterization of the common acute lymphoblastic leukaemia antigen (CD10) as an activation molecule on mature human B cells.

Distinct expression pattern of CD10 molecules during B cell activation was analysed using in vivo and in vitro systems. By two-colour flowcytometrical analysis, CD10 was found to be expressed at a specific stage of in vivo activating B cells. The expression of CD10 during B cell activation appeared to be unique from that of other activation-related B cell antigens including L29, MA6, OKT9 and OKT10. Although the expression of CD10 was associated with that of the activation-related B cell antigens, CD10+ B cells could be separated in the distinct fractions to those expressing other activation-related B cell antigens when fractionated by cell gravity. In particular, certain CD10+ B cells were detected positive for the resting B cell antigen, L30. In vitro studies revealed that CD10+ B cells arose from CD10- B cells at an early step of B cell activation, and disappeared lately when activated by Staphylococcus aureus Cowan I. Collectively, CD10 was an antigen transiently expressed at an early phase of B cell activation process. Expression of CD10 and other antigens on Burkitt's lymphomas (15 cases) was studied next. All cases were CD10+, and 87% (13 cases) were also L30+. In addition, six of CD10+ L30+ cases were L29+. This observation suggested that Burkitt's lymphomas were phenotypically similar to the B cells at an early phase of activation, those expressing CD10 and L30, simultaneously. The present study has dissected a precise expression pattern of CD10 on mature B cell activation in vitro and in vivo, and could be implicated for the histogenesis of one of the poorly characterized B cell lymphoma, namely Burkitt's lymphoma.

Antigens, CD↗
PubMed1990

Establishment of specific monoclonal antibodies against recombinant human granulocyte colony-stimulating factor (hG-CSF) and their application for immunoperoxidase staining of paraffin-embedded sections.

Detection of granulocyte colony-stimulating factor (G-CSF), one of the substances responsible for proliferation and differentiation of granulocytes, has been performed up to the present by use of the granulocyte colony-formation assay, because of the lack of a specific anti-G-CSF antibody. This has prevented the advancement of biological investigations of cell dynamics linked to G-CSF, e.g., cell localization of G-CSF and its pathophysiological changes. In the present work, two monoclonal antibodies (MAb), 1E7 and 4A6, against recombinant human G-CSF (rhG-CSF) were developed by cell hybridization between NS-1 myeloma cells and splenocytes from a mouse immunized with rhG-CSF. 1E7 and 4A6 were shown to be reactive with hG-CSF but not with other CSF (hGM-CSF, hIL-3, and mouse GM-CSF) by Western blot analysis. An immunoperoxidase staining method using these MAb was then established. This method was applicable to frozen sections, paraffin-embedded sections, and cells fixed with 4% paraformaldehyde. Positive staining for G-CSF was observed in tumor cells secreting G-CSF and also in Chinese hamster ovary (CHO) cells transfected with hG-CSF cDNA. However, no staining was seen in tumor cells secreting no G-CSF, untransfected CHO cells, lung fibroblasts, or bone marrow stromal cells after short periods of culture. These results confirmed the immunospecificity of MAb 1E7 and 4A6 and the validity of their application to immunohistochemistry using paraffin-embedded sections.

Animals↗