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M Hitomi

Publications and source records attributed to M Hitomi.

At least 37 records · Page 2Linked to original sources
PubMed1996

A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA.

An enormous variety of primary and secondary mRNA structures are compatible with export from the nucleus to the cytoplasm. Therefore, there seems to be a mechanism for RNA export which is independent of sequence recognition. There nevertheless is likely to be some relatively uniform mechanism which allows transcripts to be packaged as ribonucleoprotein particles, to gain access to the periphery of the nucleus and ultimately to translocate across nuclear pores. To study these events, we and others have generated temperature-sensitive recessive mRNA transport (mtr) mutants of Saccharomyces cerevisiae which accumulate poly(A)+ RNA in the nucleus at 37 degrees C. Several of the corresponding genes have been cloned. Upon depletion of one of these proteins, Mtr4p, conspicuous amounts of nuclear poly(A)+ RNA accumulate in association with the nucleolus. Corresponding dense material is also seen by electron microscopy. MTR4 is essential for growth and encodes a novel nuclear protein with a size of approximately 120 kDa. Mtr4p shares characteristic motifs with DEAD-box RNA helicases and associates with RNA. It therefore may well affect RNA conformation. It shows extensive homology to a human predicted gene product and the yeast antiviral protein Ski2p. Critical residues of Mtr4p, including the mtr4-1 point mutation, have been identified. Mtr4p may serve as a chaperone which translocates or normalizes the structure of mRNAs in preparation for export.

Amino Acid Sequence↗
PubMed1995

Adenoviral E1B-55kDa protein inhibits yeast mRNA export and perturbs nuclear structure.

The mechanisms of export of RNA from the nucleus are poorly understood; however, several viral proteins modulate nucleocytoplasmic transport of mRNA. Among these are the adenoviral proteins E1B-55kDa and E4-34kDa. Late in infection, these proteins inhibit export of host transcripts and promote export of viral mRNA. To investigate the mechanism by which these proteins act, we have expressed them in Saccharomyces cerevisiae. Overexpression of either or both proteins has no obvious effect on cell growth. By contrast, overexpression of E1B-55kDa bearing a nuclear localization signal (NLS) dramatically inhibits cell growth. In this situation, the NLS-E1B-55kDa protein is localized to the nuclear periphery, fibrous material is seen in the nucleoplasm, and poly(A)+ RNA accumulates in the nucleus. Simultaneous overexpression of E4-34kDa bearing or lacking an NLS does not modify these effects. We discuss the mechanisms of selective mRNA transport.

Adenoviridae↗
PubMed1995

Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae.

Synthesis of mRNA and rRNA occur in the chromatin-rich nucleoplasm and the nucleolus, respectively. Nevertheless, we here report that a Saccharomyces cerevisiae gene, MTR3, previously implicated in mRNA transport, codes for a novel essential 28-kDa nucleolar protein. Moreover, in mtr3-1 the accumulated polyA+ RNA actually colocalizes with nucleolar antigens, the nucleolus becomes somewhat disorganized, and rRNA synthesis and processing are inhibited. A strain with a ts conditional mutation in RNA polymerase I also shows nucleolar accumulation of polyA+ RNA, whereas strains with mutations in the nucleolar protein Nop1p do not. Thus, in several mutant backgrounds, when mRNA cannot be exported i concentrates in the nucleolus. mRNA may normally encounter nucleolar components before export and proteins such as Mtr3p may be critical for export of both mRNA and ribosomal subunits.

Amino Acid Sequence↗
PubMed1995

A yeast protein that bidirectionally affects nucleocytoplasmic transport.

We have identified a temperature-sensitive mutant of Saccharomyces cerevisiae (npl3) that accumulates polyadenylated RNA in the nucleus at 37 degrees C, as judged by in situ hybridization. The strong nuclear signal is not simply due to increased cytoplasmic turnover of mRNA, as reincubation at 37 degrees C with an RNA polymerase inhibitor shows no diminution in the in situ signal. Over several hours at 37 degrees C, the average poly(A) tail length increases and a characteristic ultrastructural alteration of the nucleoplasm occurs. Cloning and sequencing indicate that the corresponding gene is NPL3/NOP3, which codes for a nucleolar/nuclear protein implicated in protein import into the nucleus (Bossie et al. (1992). Mol. Biol. Cell 3, 875-893) and in rRNA maturation (Russell and Tollervey (1992). J. Cell Biol. 119, 737-747). NPL3 includes bipartite RNA recognition motifs (RRM) and a Gly-Arg repeat domain, as in several nucleolar proteins. A point mutation adjacent to one of the RRM has been identified in the ts copy of the gene. Although this protein is not concentrated in nuclear pores, NPL3 is implicated in both import and export from the nucleus. Judging from the site of the npl3 mutation and since the block in RNA export can be detected prior to an obvious nuclear import defect in npl3, the defect in RNA export may be primary. Since other mutants that interrupt RNA export do not block protein import, the NPL3 protein itself appears to be implicated in protein import.

Amino Acid Sequence↗
PubMed1995

Effects of spinach leaf protein concentrate on the serum cholesterol and amino acid concentrations in rats fed a cholesterol-free diet.

The effects of spinach leaf protein concentrate (SPPC) on serum and liver lipid concentrations and on serum free amino acid concentrations were examined in rats fed a cholesterol-free diet containing 2 and 10% fats. The serum total cholesterol, triacylglycerol and phospholipid concentrations in the rats fed an SPPC diet containing 2% corn oil were significantly lower than those of the rats fed a corresponding casein diet. When 10% corn oil or lard was used, the serum cholesterol-lowering effect of the SPPC became insignificant, but the serum and liver triacylglycerol concentrations were kept at significantly lower levels. Both the amounts of fecal neutral steroids and bile acids were significantly higher in the rats fed the SPPC than those of the casein-fed rats. The concentrations of serum threonine, serine, glutamine, glycine, cystine, and isoleucine were significantly higher in the rats fed the SPPC diet containing 2% corn oil compared with those of the control rats, but when the dietary fat was raised to 10%, only glycine showed a higher serum concentration. These results indicate that the SPPC has a stronger cholesterol-lowering effect at a lower dietary fat level, 2%, and the activity is partly due to the inhibition of intestinal absorption of cholesterol and bile acid, and partly due to an increase in the concentration of some of the serum amino acids.

Amino Acids↗
PubMed1994

Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants.

To understand the mechanisms of mRNA transport in eukaryotes, we have isolated Saccharomyces cerevisiae temperature-sensitive (ts) mutants which accumulate poly(A)+ RNA in the nucleus at the restrictive temperature. A total of 21 recessive mutants were isolated and classified into 16 complementation groups. Backcrossed mRNA transport-defective strains from each complementation group have been analyzed. A strain which is ts for heat shock transcription factor was also analyzed since it also shows nuclear accumulation of poly(A)+ RNA at 37 degrees C. At 37 degrees C the mRNA of each mutant is characterized by atypically long polyA tails. Unlike ts pre-mRNA splicing mutants, these strains do not interrupt splicing of pre-mRNA at 37 degrees C; however four strains accumulate oversized RNA polymerase II transcripts. Some show inhibition of rRNA processing and a further subset of these strains is also characterized by inhibition of tRNA maturation. Several strains accumulate nuclear proteins in the cytoplasm when incubated at semipermissive temperature. Remarkably, many strains exhibit nucleolar fragmentation or enlargement at the restrictive temperature. Most strains show dramatic ultrastructural alterations of the nucleoplasm or nuclear membrane. Distinct mutants accumulate poly(A)+ RNA in characteristic patterns in the nucleus.

Biological Transport↗
PubMed1994

Nuclear mRNA accumulation causes nucleolar fragmentation in yeast mtr2 mutant.

We have identified a set of genes that affect mRNA transport (mtr) from the nucleus to the cytoplasm of Saccharomyces cerevisiae. One of these genes, MTR2, has been cloned and shown to encode a novel 21-kDa nuclear protein that is essential for vegetative growth. MTR2 shows limited homology to a protein implicated in plasmid DNA transfer in Escherichia coli. PolyA+RNA accumulates within the nucleus of mtr2-1 in two to three foci at 37 degrees C. mRNA, tRNA, and rRNA synthesis continue as do pre-mRNA splicing, tRNA processing, and rRNA export at 37 degrees C. Under these conditions the polyA tail length increases, and protein synthesis is progressively inhibited. Nucleolar antigens also redistribute to two to three nuclear foci at 37 degrees C, and this redistribution depends on ongoing transcription by RNA polymerase II. Surprisingly, these foci coincide with the sites of polyA+RNA accumulation. Comparable colocalization and dependance on RNA polymerase II transcription is seen for the mtr1-1 mutant. The disorganization of the nucleolus thus depends on mRNA accumulation in these mutants. We discuss the possible functions of MTR2 and the yeast nucleolus in mRNA export.

Amino Acid Sequence↗
PubMed1992

Intestinal fatty acid-binding protein as a sensitive marker of intestinal ischemia.

Determination of the serum level of intestinal fatty acid-binding protein has been used to detect rat intestinal ischemia following ligation or 30-min occlusion of the superior mesenteric artery. The normal values were under the minimal detectable level of less than 2 ng/ml in all the 10 rats. The serum fatty acid-binding protein level increased rapidly, to 340.7 +/- 54.6, 438.5 +/- 40.1, 388.1 +/- 37.4, and 292.2 +/- 95.7 ng/ml (P less than 0.01) at 1, 2, 4, and 8 hr after ligation, respectively. It also increased, to 347.2 +/- 127.7 ng/ml (P less than 0.01) at 1 hr, after a 30-min transient occlusion and then returned to a normal level. Histological studies showed destruction of the villi, disappearance of the mucosa, and transmural necrosis with the progress of time after ligation, while no remarkable morphological change was observed following 30-min transient occlusion. These observations strongly suggest that the intestinal fatty acid-binding protein is a useful biochemical marker for intestinal ischemia, particularly in the early reversible phase.

Alkaline Phosphatase↗
PubMed1991

Ontogenic appearance of three fatty acid binding proteins in the rat stomach.

With the use of specific antibodies against three structurally different fatty acid binding proteins (FABPs), viz, liver FABP (L-FABP), heart FABP (H-FABP), and intestinal FABP (I-FABP), the localization and relative amount of the immunoreactive proteins were determined by immunoblotting and immuno-cytochemistry in the gastric epithelium of rats during prenatal and postnatal development. H-FABP immunoreactivity was first detected at embryonic day 20 (E20), with predominant localization in the parietal cells, whereas I-FABP immunoreactivity was detected at the day of birth in the surface mucous cells. Both immunoreactivities were continuously localized in the same cell types with increasing intensity into adulthood. In contrast, the immunoreactivity for L-FABP showed remarkable changes in intensity and localization during development of the rat stomach. It was first detected in the surface mucous cells of E19. In the first 2 weeks of postnatal life, i.e., the suckling period, L-FABP immunoreactivity reached a peak in intensity and was localized not only in the surface mucous cells, but also in some of the parietal cells, brush cells, and endocrine D cells. In the following few weeks of weaning, the reactivity of surface mucous cells and parietal cells disappeared, leaving only a small amount of total L-FABP immunoreactivity in the adult stomach, which was localized exclusively in the brush cells and D cells. These results revealed that the appearance of the three types of FABPs in the rat stomach is specific to cell types and developmental stages.

Animals↗
PubMed1990

Glutathione-protein mixed disulfide decreases the affinity of rat liver fatty acid-binding protein for unsaturated fatty acid.

.16 +/- 0.062% of the fatty acid-binding protein purified from 50 mM N-ethylmaleimide-treated rat liver (L-FABP) was determined as a form S-thiolated by glutathione (L-FABP-SSG). L-FABP-SSG, which was prepared in vitro through thiol-disulfide exchange reaction, showed more acidic pI (approximately 5.0) than the pI (approximately 7.0) of reduced L-FABP. S-thiolation of L-FABP by glutathione decreased the affinity of the protein for unsaturated fatty acids without changing the equimolar maximum binding. The changes in Kd were from 0.63 +/- 0.054 microM to 1.03 +/- 0.14 microM for oleic acid, from 0.63 +/- 0.028 microM to 0.97 +/- 0.12 microM for linoleic acid and from 0.85 +/- 0.050 microM to 1.45 +/- 0.024 microM for arachidonic acid. This modification did not alter the affinity nor the maximum binding for saturated fatty acids, which were determined to be Kd of approximately 1.0 microM for palmitic acid and approximately 0.9 microM for stearic acids, and equimolar maximum binding for both fatty acids. The binding affinity of L-FABP for unsaturated fatty acid may be regulated by redox state of the liver.

Amino Acids↗
PubMed1989

Purification and characterization of a fatty-acid-binding protein from the gastric mucosa of rats. Possible identity with heart fatty-acid-binding protein and its parietal cell localization.

Fatty acid-binding protein (FABP) was purified from rat gastric mucosa by successive Sephadex G-75 chromatography, DEAE-cellulose chromatography and HPLC on an RP-2 (Merck) reversed-phase column. The purified stomach FABP migrated as a single band corresponding to an apparent molecular mass of 15 kDa on SDS/PAGE. Stomach FABP appeared to be identical with rat heart FABP, as judged from its electrophoretic mobility, amino acid composition and tryptic peptide map. In addition, the amino acid sequences of two selected tryptic peptides coincided completely with the rat heart FABP sequence deduced from that of cDNA. Stomach FABP showed immunochemical identity with rat heart FABP when tested with an antiserum against rat heart FABP. Immunohistochemically, stomach FABP was specifically stained with anti-(rat heart FABP) serum in parietal cells of the gastric mucosa. The results suggested that the primary structure of stomach FABP is identical with that of rat heart FABP, and showed that stomach FABP is localized in parietal cells of the gastric mucosa.

Amino Acid Sequence↗
PubMed1989

Rat heart fatty acid-binding protein. Evidence that supports the amino acid sequence predicted from the cDNA.

The amino acid sequence of rat heart fatty acid-binding protein was re-examined by analysing the tryptic and the chymotryptic peptides, since some discrepancies have been reported between the sequences determined by protein analyses and that deduced from the cDNA analyses. Our result completely agreed with the amino acid sequence predicted from the cDNA analyses, providing evidence for the actual existence of the molecular species predicted from the cDNA.

Amino Acid Sequence↗
PubMed1989

Immunocytochemical localization of hepatic fatty acid binding protein in the rat intestine: effect of fasting.

Localization of hepatic fatty acid binding protein (h-FABP) in the small and large intestines of rats was studied by light and electron microscopic histochemistry using the peroxidase-antiperoxidase (PAP) method. In the small intestine of rats fed ad libitum, an intense FABP immunoreactivity was confined to the absorptive epithelial cells of the villi, but not of the crypts. The lowest margin of the immunoreactive cell sheet was closer to the crypts in the proximal than in the distal portions of the small intestine. In the large intestine, FABP immunoreactivity was present in the surface epithelial cells, with higher intensity in the proximal than in the distal portions of the intestine. After fasting rats for two days, many crypt cells exhibited intense immunoreactivity for h-FABP, resulting in an extension of the lowest margin of the immunoreactive cell sheet deep into the crypts. Such expansion of the immunoreactive cell population was reversed by refeeding the animals. With regard to the intracellular localization of immunoreactivity in the jejunum, the basolateral portion of the cytoplasm exhibited a more intense immunoreaction than the apical portion in the majority of immunoreactive cells lining the villi, whether the animals were fed or fasted. The immunoreactive products appeared in the cytoplasmic matrix without association with any subcellular structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Immunocytochemical localization of hepatic fatty acid binding protein in the liver of fed and fasted rats.

The immunocytochemical localization of fatty acid binding protein (FABP) of liver type was studied at light and electron microscopic levels by the peroxidase-antiperoxidase (PAP) method using a specific polyclonal antibody against FABP in the liver of fed and fasted rats. In the liver of rats fed ad libitum, the intense immunoreactivity was confined to portions of the liver cell cytoplasm adjacent to the glycogen area. After 2-days' fasting, such a focal intracellular localization of the immunoreactivity was abolished, in association with the disappearance of the glycogen area, and was replaced by a diffuse distribution of the immunoreactivity throughout the cytoplasm, with higher intensity at the periphery of the cells. In liver cells exhibiting an overall hypertrophy of smooth endoplasmic reticulum (SER) induced by the treatment of fasted rats with phenobarbital, the peripheral localization of FABP immunoreactivity remained unchanged compared with that obtained in the case of fasting alone, and the immunoreactivity did not occur in association with the proliferated SER in the central cytoplasm. These results suggest that FABP, although cytosolic in nature, changes its localization within the liver cells in response to the general metabolic alterations caused by the starvation, inferring that FABP is intimately involved in the intracellular transport and metabolism of free fatty acids.

Animals↗
PubMed1988

Immunohistochemical distribution of hepatic fatty acid-binding protein in rat and human alimentary tract.

Tissues from rat and human alimentary tract were immunostained with rabbit antibodies to fatty acid-binding protein (FABP) isolated from rat liver, since the precise immunohistochemical localization of the protein in gut has not been determined. The results obtained indicated that FABP immunoreactivity was found almost exclusively in intestinal absorptive cells, the sole exception being its presence in the cytoplasm of a few goblet cells. In small bowel, FABP-positive cells were most often found in the upper and middle segments, and less frequently in the lower to terminal portion. Immunoreactive cells were also found in large bowel of rat and human, but with differing patterns of distribution. In rat, positive cells were found mainly in the lower portion of the large intestine, whereas in human positive cells were present in all portions. Immunoreactive cells were detected in rat and human cecum, in the upper half of human rectum, and in human vermiform appendix. No such cells were found in esophageal and nonmetaplastic gastric mucosa or in pancreatic tissue, whereas they were present in great numbers in metaplastic gastric mucosa. The results of this study therefore suggest that FABP is a useful marker for research into the physiology or pathology of absorptive cells in the gastrointestinal tracts of both species.

Animals↗
PubMed1985

Effects of mononuclear phagocyte system modulating agents on Fc and C3 receptors of adherent cells.

Agents which modulate the mononuclear phagocyte system (MPS) were examined for their effects on Fc and C3 receptors of adherent cells (A-cells) as judged by rosette formation. Dextran sulphate, carrageenan, and immune complexes, known as MPS suppressants, reduced the percentage of receptor-positive A-cells, while levamisole, known as a MPS-activator, increased the percentage in vitro. The changes in the percentage of Fc receptor were parallel to those of the C3 receptor in vitro. The effects of these agents were also examined in vivo.

Animals↗