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Biomedical subjects

T Funahashi

Publications and source records attributed to T Funahashi.

At least 91 records · Page 5Linked to original sources
PubMed1995

Marked hypocholesterolemia in a case with adrenal adenoma--enhanced catabolism of low density lipoprotein (LDL) via the LDL receptors of tumor cells.

A 16-yr-old girl was hospitalized because of amenorrhea and virilism, and was diagnosed with an adrenal tumor on the right side. Her serum androgen levels were markedly elevated, and severe hypocholesterolemia (total cholesterol, 0.59 mmol/L) was observed. After resection of the tumor, her serum cholesterol level dramatically rose to normal, suggesting a role of this tumor in her marked hypocholesterolemia. To investigate the mechanism of hypocholesterolemia in this case, we examined the effects of dehydroepiandrosterone and dehydroepiandrosterone sulfate on the low density lipoprotein (LDL) receptor activity of fibroblasts. These hormones did not have any effect on LDL receptor activity. Northern blot analysis demonstrated that the LDL receptor messenger ribonucleic acid level of this tumor tissue was increased about 8-fold compared with that of normal adrenal cortex. The LDL receptor activity of the cultured cells established from this tumor was 2-fold higher than that of Hep G2 cells. Furthermore, the LDL receptor activity could not be down-regulated by an excessive dose of 25-hydroxycholesterol. These results suggest that increased LDL receptor activity and unrestricted uptake of LDL by the adrenal tumor may have caused the marked hypocholesterolemia in this patient.

Adenoma↗
PubMed1995

Tissue-specific, developmental and nutritional regulation of the gene encoding the catalytic subunit of the rat apolipoprotein B mRNA editing enzyme: functional role in the modulation of apoB mRNA editing.

Apolipoprotein B (apoB) mRNA editing, a posttranscriptional site-specific cytidine deamination reaction, is mediated by a protein complex of which the catalytic component (REPR) has recently been cloned. REPR mRNA was demonstrated by RNase protection at highest abundance in small intestine and colon but the transcript was detectable in all tissues examined including kidney, spleen, lung, liver, and ovary. ApoB mRNA was found predominantly in the liver and small intestine but low levels were detected in all adult tissues examined and found to be variably (29-86% TAA) edited. In addition, S100 extracts prepared from spleen and kidney were competent to edit an apoB RNA template in vitro, suggesting that the entire apoB mRNA editing complex is present and functionally active in these tissues. In situ hybridization demonstrated REPR mRNA to be distributed along the entire villus-crypt axis, while apoB mRNA distribution did not extend into the crypts. In the liver, both apoB and REPR mRNA were detected in all cells of the hepatic lobule without an apparent gradient of expression. REPR mRNA was found in the red pulp of the spleen and in the superficial crypt cells of the colon. This distribution of REPR mRNA was recapitulated by immunocytochemical localization of the protein within these tissues. Finally, the developmental and nutritional modulation of REPR was examined in relation to endogenous apoB mRNA editing. Small intestinal apoB mRNA editing was found to undergo a developmentally regulated increase beginning at gestational day 20, preceding a developmental increase in REPR mRNA abundance. Additionally, hepatic and kidney apoB mRNA editing both revealed a temporal dissociation from alterations in REPR mRNA abundance. By contrast, adult rats subjected to fasting and refeeding a high carbohydrate diet, demonstrated concordant modulation of endogenous apoB mRNA editing and REPR mRNA abundance (r = 0.92, P < 0.001). Taken together, the data demonstrate that REPR and other components of the rat apoB mRNA editing complex are widely distributed and undergo distinct developmental and metabolic regulation that interact to regulate apoB mRNA editing in a tissue-specific manner.

APOBEC-1 Deaminase↗
PubMed1994

Molecular cloning of a human small intestinal apolipoprotein B mRNA editing protein.

Mammalian small intestinal apolipoprotein B (apo B) mRNA undergoes posttranscriptional cytidine deamination with the production of an in frame stop codon and the translation of apo B48. We have isolated a cDNA from human jejunum which mediates in vitro editing of a synthetic apo B RNA template upon complementation with chicken intestinal S100 extracts. The cDNA specifies a 236 residue protein which is 69% identical to the apo B mRNA editing protein (REPR) cloned from rat small intestine [Teng, B., Burant, C. F. and Davidson, N. O. (1993) Science 260, 1816-1819] and which, by analogy, is referred to as HEPR. HEPR does not contain the carboxyl-terminus leucine zipper motif identified in REPR but contains consensus phosphorylation sites as well as the conserved histidine and both cysteine residues identified as a Zn2+ binding motif in other cytidine deaminases. The distribution of HEPR mRNA was predominantly confined to the adult small intestine with lower levels detectable by reverse-transcription polymerase chain reaction amplification in the stomach, colon and testis. These differences in the structure and distribution of the human as compared to the rat apo B mRNA editing protein suggest an important evolutionary adaptation in the mechanisms restricting apo B48 production to the small intestine.

APOBEC-1 Deaminase↗
PubMed1994

Complementation of apolipoprotein B mRNA editing by human liver accompanied by secretion of apolipoprotein B48.

Mammalian small intestine secretes a truncated apolipoprotein B (apoB48) species as a result of tissue-specific post-transcriptional RNA editing. The human liver, by contrast, contains only unedited apoB mRNA and secretes only apoB100. We have recently isolated a cDNA clone from rat small intestine which encodes an apoB mRNA editing protein, REPR (Teng, B., Burant, C.F., and Davidson, N.O. (1993) Science 260, 1816-1819). The current study demonstrates that homogenates of Xenopus oocytes expressing REPR confer editing ability upon S100 extracts prepared from human liver when tested on a synthetic apoB RNA template in vitro. Transfection of REPR into HepG2 cells resulted in editing of endogenous apoB mRNA and the appearance of an apoB48-like protein in the media. Extracts prepared from these transfected cells edit mammalian apoB RNA templates when incubated alone and with enhanced efficiency in the presence of chicken intestinal S100 extracts. The results suggest that human liver expresses factor(s) which are critical to apoB mRNA editing and which allow functional complementation of REPR in vivo.

APOBEC-1 Deaminase↗
PubMed1994

Proportionality of protease activities in malignant cells to their metastatic potentials.

It has been suggested that the activities of type IV collagenase and/or ectopeptidases possessed by malignant cells are related to their metastatic potentials. In the present study, we examined the activities of three aminopeptidases, two serine proteases, as well as type IV collagenase, in three kinds of cell lines of malignant cells. The activities of the aminopeptidases and serine proteases, rather than of type IV collagenase, were found to be proportionate to the metastatic potentials of those cell lines. Such activities of aminopeptidases were effectively suppressed by the addition of low molecular weight inhibitors.

Animals↗
PubMed1994

Suppression of splenic enzyme activities by administration of aminopeptidase N (CD13) inhibitors: relationship between actions in vivo and in vitro.

The enzymatic changes in murine spleen caused by the administration for 20 successive days of various inhibitors of aminopeptidase N (leucocyte antigen CD13) have been compared. When compared with the control (saline), most of the inhibitors significantly suppressed splenic enzyme activities including those of ectoenzymes. A multivariate study indicated that the in vivo effects of the inhibitors were closely related to their inhibitory actions in vitro.

Amino Acids↗
PubMed1994

REPR and complementation factor(s) interact to modulate rat apolipoprotein B mRNA editing in response to alterations in cellular cholesterol flux.

Apolipoprotein B (apoB) mRNA editing is a post-transcriptional cytidine deamination involving several protein factor(s), one of which has recently been cloned. We have examined the effects of alterations in cellular cholesterol flux in the rat liver and small intestine as a means of dissecting the physiologic mechanisms regulating apoB mRNA editing, both in vivo and in isolated S-100 extracts. Hepatic cholesteryl ester accumulation was produced by feeding rats a high cholesterol diet, alone, or in combination with either ethinyl estradiol treatment, or after induction of hypothyroidism. Endogenous hepatic apoB mRNA editing decreased in parallel with the increase in cellular cholesteryl ester content (r = -0.948, P < 0.001). None of these conditions altered endogenous intestinal apoB mRNA editing. Hepatic S-100 extracts demonstrated decreased in vitro apoB RNA editing activity, in parallel with the changes observed in vivo. By contrast, the activity of intestinal S-100 extracts demonstrated a paradoxical increase in hypothyroid rats and a similar, paradoxical decrease in hyperthyroid rats, when compared to controls. Hepatic REPR mRNA, quantitated by RNase protection assay, showed a 25-50% decrease in cholesterol-fed rats. The editing activity of hepatic S-100 extracts prepared from cholesterol-fed, hypothyroid rats was restored to control levels with REPR supplementation but not with chicken intestinal S-100 extracts, suggesting that changes in REPR, but not complementation activity, may play a critical role in the regulation of apoB mRNA editing in rat liver. By contrast, the editing activity of intestinal S-100 extracts prepared from hyperthyroid animals was unaltered by supplementation with REPR, but was restored to control levels after the addition of chicken intestinal S-100 extracts. Taken together, the data suggest that tissue-specific factors regulate apoB mRNA editing in the rat and that the complex interplay of REPR and complementation factor(s) may be modulated in response to alterations in cholesterol flux, in vivo.

APOBEC-1 Deaminase↗
PubMed1993

The p27 catalytic subunit of the apolipoprotein B mRNA editing enzyme is a cytidine deaminase.

The messenger RNA for apolipoprotein B undergoes a discrete and specific C to U editing of nucleotide 6666. This generates a stop translation codon and defines the carboxyl terminus of apolipoprotein B48. A 27-kDa rat intestinal protein that does not itself edit apolipoprotein B mRNA, but confers editing activity on chick intestinal extracts that do not have intrinsic editing activity, has recently been identified and its cDNA cloned (Teng, B., Burant, C. F., and Davidson, N. O. (1993) Science 260, 1816-1819). Here we show that p27 is homologous in the zinc coordinating region of the active site to cytidine deaminases from Escherichia coli, Bacillus subtilis, yeast, and man and to deoxycytidylate deaminases from T2 and T4 bacteriophages and man. p27 expressed in Xenopus laevis oocyte extracts has cytidine deaminase activity and specifically confers editing activity on chick intestinal extracts. The homologous E. coli cytidine deaminase does not confer editing activity. The zinc-specific chelating agent o-phenanthroline abolishes p27 activity and site-specific apolipoprotein B mRNA editing in rat enterocyte editing extracts. We conclude that p27 is the catalytic subunit of the apolipoprotein B mRNA editing enzyme and is a zinc-containing cytidine deaminase.

APOBEC-1 Deaminase↗
PubMed1993

Extralysosomal degradation of high-density lipoproteins in a human hepatoma cell line, Mahlavu.

Lipoprotein metabolism has been investigated in a novel human hepatoma cell line, Mahlavu, which has been reported to possess the characteristics of hepatocytes. Low-density lipoprotein (LDL) was degraded by Mahlavu cells. LDL taken up by the cells suppressed intracellular cholesterol biosynthesis and promoted cholesterol esterification in a manner similar to that of HepG2 cells. High-density lipoprotein (HDL) was also degraded by Mahlavu cells, whereas it was not degraded by fibroblasts. We compared the mode of intracellular metabolism of HDL to that of LDL. In contrast to the LDL receptor pathway, the degradation of HDL was not inhibited by 100 microM chloroquine added to the medium, indicating that the degradation may not occur in lysosomes. Cholesterol taken up as HDL-cholesterol by the Mahlavu cells had no effect on the intracellular biosynthesis of cholesterol nor on cholesterol esterification. The conditioned media in which Mahlavu cells had been cultured did not promote the degradation of HDL, suggesting that HDL is degraded intracellularly. These data suggest that HDL is taken up and degraded by the liver cells in contrast to extrahepatic peripheral cells such as fibroblasts and macrophages in which the degradation of HDL does not occur. The results indicate that HDL-associated cholesterol may be processed via a pathway different from that of LDL metabolism and that the degradation of HDL occurs extralysosomally.

Carcinoma, Hepatocellular↗
PubMed1993

Frequency of intron 14 splicing defect of cholesteryl ester transfer protein gene in the Japanese general population--relation between the mutation and hyperalphalipoproteinemia.

Cholesteryl ester transfer protein (CETP) deficiency, which has been found only in Japan, is characterized by marked hyperalphalipoproteinemia (HALP) and abnormalities of both low density and high density lipoproteins. We have reported that this deficiency is commonly associated with a G-->A mutation at the intron 14 splice donor site of the CETP gene (Yamashita et al., Biochem. Biophys. Res. Commun., 170 (1990) 1346-1351). In the current study, we determined the frequency of this mutation in Japanese subjects by using polymerase chain reaction. A single primer-template mismatch of one base pair from the CETP gene mutation permitted the introduction of a cleavage site for Nde I in mutant alleles but not in normal ones. Out of 171 patients with marked HALP whose serum HDL-cholesterol was more than 100 mg/dl, 6 (3.5%) subjects were homozygous and 48 (28.1%) were heterozygous for this mutation. Furthermore, in unrelated 512 healthy Japanese subjects, 5 (0.98%) were identified as heterozygotes. Relative allelic frequency of A at the intron 14 splice donor site was 0.0049 and the frequency of homozygous CETP deficiency was estimated to be approximately 1/42,000. These results demonstrate that this common mutation may be frequent in the Japanese population. Although HALP is very heterogenous, this mutation could be one of the major causes of marked HALP.

Alleles↗
PubMed1993

Low-density lipoprotein receptor mutation that deletes exons 2 and 3 by Alu-Alu recombination.

A deletion mutant in the low density lipoprotein receptor gene of a Japanese patient with heterozygous familial hypercholesterolemia was analyzed. Genomic Southern blotting showed abnormal size restriction fragments with BamHI (7.8 kb), EcoRI (3.8 kb), BglII (17 kb), KpnI (> 23 kb), EcoRV (13 kb), and XbaI (14 kb). The abnormal EcoRI fragment, 3.8 kb, was cloned into lambda phage vector, and the deleted region of 10 kb including exons 2 and 3 was identified. The nucleotide sequence around the deletion joint was determined. The sequence of the eight nucleotides in the deletion-joint region of the mutant gene was identical to the corresponding sequences of both introns 1 and 3 of the normal gene. The deletion seemed to occur by an unequal recombination between the Alu-like sequences in the same direction in introns 1 and 3.

Base Sequence↗
PubMed1993

Marked reduction of acyl-CoA synthetase activity and mRNA in intra-abdominal visceral fat by physical exercise.

Several reports have suggested that the reduction of intra-abdominal visceral fat after physical exercise is more prominent than that of subcutaneous fat. We compared some parameters in mesenteric and subcutaneous fats between sedentary and exercised rats (treadmill running; 10-20 m/min, 60 min/day, 7 days). Tissue weight and cell volume were decreased in mesenteric fat by the exercise. The exercise reduced activity and mRNA levels of acyl-CoA synthetase (ACS; 67 and 26% of those of the sedentary group, respectively), mRNA levels of lipoprotein lipase (LPL; 49% of those of the sedentary group), and GLUT-4 (38% of those of the sedentary group) in the mesenteric fat. In contrast, all of these parameters did not change significantly in the subcutaneous fat. Gastrocnemius muscle was heavier in exercised rats. ACS activity was elevated in the gastrocnemius muscle of the exercised rats (137% of those of sedentary group), although mRNA levels of ACS, LPL, and GLUT-4 did not change in the muscle by the exercise. These observations suggest that mesenteric fat may contribute to switching of distribution of plasma energy flux, including lipid and glucose, from fat tissue to muscle in physical exercise.

Abdomen↗
PubMed1992

A point mutation of low-density-lipoprotein receptor causing rapid degradation of the receptor.

The exons of the low-density-lipoprotein-(LDL)-receptor gene from a Japanese patient with homozygous familial hypercholesterolemia were amplified by the polymerase chain reaction (PCR), and their nucleotide sequences were determined. A point mutation from G to C was found in exon 9, which was expected to change Asp at position 412 to His. This amino acid change occurred within the epidermal-growth-factor-precursor homology domain of the LDL receptor, slightly impairing the processing from the precursor to the mature form and causing rapid degradation of the mature form in the fibroblasts of the patient. The mutant LDL-receptor gene transfected into COS-1 cells expressed a LDL-receptor protein with the same properties as the protein expressed in the fibroblasts of the patient; impaired processing and rapid degradation of the synthesized receptor protein. The mutation was identified in family members of the patient by dot-blot hybridization of PCR-amplified DNA with the mutant oligonucleotide. The family members carrying the mutant gene showed higher serum cholesterol levels than the others. However, their cholesterol levels were also greatly influenced by the apolipoprotein-E phenotype.

Base Sequence↗
PubMed1992

Stimulation of the activity and mRNA level of hepatic triacylglycerol lipase by triiodothyronine in HepG2 cells.

We have studied the effects of triiodothyronine (T3) on the production of hepatic triacylglycerol lipase (HTGL) in the human hepatocellular carcinoma cell line, HepG2, by measuring its activity and mRNA levels. The HTGL activity released into the medium by heparin, increased after the addition of T3 in a both time- (27% increase after 24 and 75% increase after 48 h) and dose-dependent manner (maximum activity with over 0.2 micrograms/ml of T3 in the medium). Messenger RNA levels of HTGL in cells incubated with T3 for 24 and 48 h were increased by 33% and 98% compared to those of the control. These results suggest that the production of HTGL may be regulated by thyroid hormone at the level of gene expression.

Blotting, Northern↗
PubMed1992

Marked enhancement of acyl-CoA synthetase activity and mRNA, paralleled to lipoprotein lipase mRNA, in adipose tissues of Zucker obese rats (fa/fa).

To clarify the role of acyl-CoA synthetase in development of obesity, the mRNA levels and activities were studied in Zucker fatty rats (fa/fa). In Zucker fatty rats compared with their lean littermates, marked enhancement of ACS were observed in adipose tissues. Obese/lean rats ratio of ACS activity and mRNA in abdominal subcutaneous fat (3.3- and 3.9-fold, respectively) were greater than in mesenteric fat (2.0- and 2.2-fold). The enhancement of ACS activity and mRNA in the liver of fatty rats (1.2- and 1.8-fold) were less than those in the adipose tissues. There were no enhancement of ACS activities and mRNA levels in heart tissue of the obese rats. LPL mRNA levels were also enhanced in adipose tissue of fatty rats and obese/lean ratio of LPL mRNA was also higher in abdominal subcutaneous fat than mesenteric fat (6.2- vs 3.1-fold). The larger obese/lean rats ratio of LPL and ACS parameters in abdominal subcutaneous fat than mesenteric fat may be related to the observation that the increase of subcutaneous fat weight was larger than that of mesenteric fat weight in fatty rats (21.1- vs 4.9-fold). Integrated enhancement of LPL and ACS gene expression in adipose tissue may play an important role in the development of obesity.

Adipose Tissue↗
PubMed1992

Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase.

A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27,513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M(r) value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M(r) value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.

Amino Acid Sequence↗