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Biomedical subjects

K Mihara

Publications and source records attributed to K Mihara.

At least 181 records · Page 10Linked to original sources
PubMed1991

Systematic analysis of stop-transfer sequence for microsomal membrane.

Co-translational protein translocation across the endoplasmic reticulum membrane is interrupted by particular amino acid sequences which are called stop-transfer sequences. Since the stop-transfer process should reflect the character of the protein translocation machinery, systematic examination on the structural requirements for stop-transfer sequences should give information about the translocation process. By the manipulation of the cDNA of interleukin 2, a typical secretory protein, the middle portion of the molecule was replaced with systematically constructed hydrophobic stretches, and two positively or negatively charged amino acid residues were introduced just behind the hydrophobic stretches. The modified proteins were synthesized with an in vitro transcription-translation system in the presence of dog pancreas rough microsomes, and their topologies in the membrane were examined with proteinase K digestion. The efficiency of stop-translocation depended on the hydrophobicity and the length of the inserted stretch. The segments followed by positively charged residues interrupted the translocation more efficiently than those with negatively charged residues. We observed that more than 19 alanine residues were required for efficient stop-translocation, whereas only 9 leucine residues were sufficient. We suggest that the positively charged residues following the hydrophobic stretches promote stop-translocation of the peptides through the channel.

Amino Acid Sequence↗
PubMed1991

Cytosolic and mitochondrial surface factor-independent import of a synthetic peptide into mitochondria.

We chemically synthesized a peptide, 11 beta-45, which was composed of 45 amino acid residues including the whole extension peptide and some of the mature portion of bovine cytochrome P-450(11 beta) precursor. 11 beta-45 was imported into mitochondria in vitro depending on the mitochondrial membrane potential, but its import did not require extramitochondrial ATP. Although cytosolic protein factors in the high speed supernatant of reticulocyte lysate are known to stimulate the import of various precursor proteins into mitochondria, the import of 11 beta-45 was not stimulated by cytosolic factors in reticulocyte lysate. The import of the peptide did not require mitochondrial surface protein components because its import was not affected by trypsin treatment of mitochondria. On the other hand, trypsin treatment of mitoplasts resulted in a great reduction in the import of the peptide, indicating that 11 beta-45 interacts during the import process with some protein components located inside mitochondria. These observations indicated that the peptide 11 beta-45 was imported via the potential-dependent pathway as in the case of precursor proteins, but skipped the interactions with cytosolic factors and mitochondrial surface components normally required for the import of precursor proteins.

Adenosine Triphosphate↗
PubMed1991

Hepatic disposition characteristics of electrically charged macromolecules in rat in vivo and in the perfused liver.

The effect of electric charge on the hepatic disposition of macromolecules was studied in the rat. Charged derivatives of dextran (T-70) and bovine serum albumin (BSA), mitomycin C-dextran conjugates (MMC-D), and lactosaminated BSA (Lac-BSA) were employed as model macromolecules. After intravenous injection, cationic macromolecules were rapidly eliminated from plasma because of their extensive hepatic uptake, while anionic and neutral macromolecules were slowly eliminated. Cationic macromolecules were recovered from parenchymal and nonparenchymal hepatic cells at a cellular uptake (per unit cell number) ratio of 1.4-3.2, while that of Lac-BSA was 14. During liver perfusion using a single-pass constant infusion mode, cationic macromolecules were continuously extracted by the liver, with extraction ratios at steady-state (Ess) ranging between 0.03 and 0.54, whereas anionic and neutral macromolecules were almost completely recovered in the outflow at steady state. The Ess for cationized BSA (Cat-BSA) and cationic MMC-Dcat were concentration dependent and decreased at low temperatures and in the presence of colchicine and cytochalasin B. The possible participation of the internalization process in the uptake of cationic macromolecules by hepatocytes was suggested.

Animals↗
PubMed1990

The amino-terminal structures that determine topological orientation of cytochrome P-450 in microsomal membrane.

We previously showed that the amino-terminal region of P-450 is responsible not only for targeting to endoplasmic reticulum (ER) membrane but also for stable anchoring to the membrane. In the present study, we introduced several mutations or deletions into the signal-anchor region of the chimeric proteins in which the amino-terminal regions of two forms of cytochrome P-450 were fused to the mature portion of interleukin 2. The amino-terminal acidic amino acid residues were replaced with basic amino acid residues or the hydrophobic core sequences were partially deleted, and these mutant proteins were assayed in vitro for their capacity to be inserted into or translocated across the ER membrane. The proteins that received the former manipulations were processed and the IL-2 portion was translocated across the membrane. In one case, the processing did not occur, thereby enabling the chimeric protein to anchor on the luminal side of the ER. Those that received the latter manipulation were also processed and the IL-2 portion translocated across the ER. These results strongly suggest that the signal-anchor function is determined both by the amino-terminal charged amino acid residues and by the length of the hydrophobic stretch.

Amino Acid Sequence↗
PubMed1990

Structural requirements for interruption of protein translocation across rough endoplasmic reticulum membrane.

Co-translational translocation of proteins across the membrane of rough endoplasmic reticulum (ER) is interrupted by particular amino acid sequences, which are functionally termed "stop-transfer sequence." We analyzed the structural requirements for the interruption of the peptide translocation. By the manipulation of the cDNA of interleukin 2 (IL2), which passes through ER membrane co-translationally, the middle portion of the IL2 molecule was replaced with systematically altered hydrophobic segments, leucine, alanine, or leucine/alanine mixed clusters. Furthermore, charged amino acid residues were introduced just downstream of the hydrophobic segments. These modified IL2 peptides were synthesized with wheat germ cell-free system in the presence of rough microsomes and the topology of the peptides in the microsomes was assessed by post-translational digestion with proteinase K. We obtained the following results. (i) Each modified protein was processed to the mature form but the extent of stop-translocation varied widely. The ratio of the stopped to the translocated products increased as the length and hydrophobicity of the inserted segment increased. (ii) Shorter hydrophobic segments than naturally occurring native transmembrane segment promoted stop-translocation. (iii) Proteins with hydrophobic segments followed by positive charges were more efficiently stop-translocated than those having negative charges. (iv) If the hydrophobicity of the segment was sufficiently high, the positive charges after the segment were not essential for stop-translocation. We also suggest that the stop-transfer process includes protein-protein interaction between the hydrophobic segment and translocation channel.

Amino Acid Sequence↗
PubMed1990

Structure and regulation of rat liver microsomal stearoyl-CoA desaturase gene.

The cosmid library of rat genomic DNA was screened with the coding region of microsomal stearoyl-CoA desaturase (SCD) cDNA as the probe. Two independent clones having 36 kb (SCDI) and 31 kb (SCDII) of inserts were isolated. Southern mapping analyses revealed that SCDI codes for SCD and SCDII for an SCD-homologue, and that the genes differ markedly in the upstream regulatory regions. Nucleotide sequence analysis of SCDI revealed that the SCD gene is organized with 6 exons and 5 introns spanning about 15 kb, thus coding an mRNA with an open reading frame of 1,074 bp of the desaturase. Transcription starts at 114 by upstream of the translation initiation codon, and 32 bp upstream of the transcription initiation site there is a TATA box. A sequence similar to Fat Specific Element 2 (FSE2), the negative regulatory element of adipocyte differentiation, was present at 45 bp upstream of the TATA box. The tissue distribution and induction due to dietary manipulation of SCDI and SCDII mRNAs as revealed by Northern hybridization were markedly different from each other.

Adipose Tissue↗
PubMed1990

Trehalose levels and survival ratio of freeze-tolerant versus freeze-sensitive yeasts.

Five freeze-tolerant yeast strains suitable for frozen dough were compared with ordinary commercial bakers' yeast. Kluyveromyces thermotolerans FRI 501 cells showed high survival ability after freezing when their resting cells were fermented for 0 to 180 min in modified liquid medium, and they grew to log and stationary phases. Among the freeze-tolerant strains of Saccharomyces cerevisiae, FRI 413 and FRI 869 showed higher surviving and trehalose-accumulating abilities than other S. cerevisiae strains, but were affected by a prolonged prefermentation period and by growth phases. The freeze tolerance of the yeasts was, to some extent, associated with the basal amount of intracellular trehalose after rapid degradation at the onset of the prefermentation period. In the freeze-sensitive yeasts, the degree of hydrolysis of trehalose may thus be affected by the kind of saccharide, unlike in freeze-tolerant yeasts.

Disaccharides↗
PubMed1989

Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product.

The human retinoblastoma gene (RB1) encodes a protein (Rb) of 105 kilodaltons that can be phosphorylated. Analysis of Rb metabolism has shown that the protein has a half-life of more than 10 hours and is synthesized at all phases of the cell cycle. Newly synthesized Rb is not extensively phosphorylated (it is "underphosphorylated") in cells in the G0 and G1 phases but is phosphorylated at multiple sites at the G1/S boundary and in S phase. HL-60 cells that were induced to terminally differentiate by various chemicals lost their ability to phosphorylate newly synthesized Rb at multiple sites when cell growth was arrested. These findings suggest that underphosphorylated Rb may restrict cell proliferation.

Cell Cycle↗
PubMed1989

Nerve endings in palm skin grafts.

We examined regeneration of Meissner corpuscles in palm skin grafts. In only one of the specimens (split-thickness skin graft from the sole of the foot), numerous regenerated Meissner corpuscles were found in the dermal papilla. This suggests that the nerve pattern of the graft depends on reinnervation from the recipient site and the regeneration of encapsulated nerve endings contained in the graft. It is recommended that skin grafts containing the encapsulated nerve endings be used in digits to obtain a satisfactory recovery of the sensory function.

Adolescent↗
PubMed1989

Genomic organization of the human retinoblastoma gene.

Sequence analysis of the human retinoblastoma gene cDNA revealed the presence of repeated elements in the form of direct repeats, inverted repeats and dyad symmetries. The clustering of the dyad symmetrical elements in some exons, #16 and #17, coincides with the hot spots for structural aberrations of the RB-1 locus previously observed in tumors. The RB-1 gene is divided into at least 27 exons distributed over 200 kbp. Three potential Sp1 binding sites are presented within 600 bp upstream of the translation start site. A DNA fragment containing these Sp1 sites ligated to a promotorless CAT gene can promote its transcription in transfected cell culture.

Base Sequence↗
PubMed1988

Detection of hypermethylation of the c-abl genomic locus in the spleen of juvenile LE rats.

Tumor induction by treatment with polycylic hydrocarbons depends on age and the strains of rats used. Juvenile LE rats are very sensitive to the induction of leukemia and chromosomal breaks by intravenous DMBA injection. We have previously demonstrated a chromosomal translocation in chromosome 3 and 12 in a DMBA induced LE rat leukemia cell K3D. In our present communication we have examined the c-abl expression in the leukemic cell line as well as in the LE rats at different ages. We found that in the leukemic cell K3D the c-abl expression is elevated both at the level of mRNA and protein. In the preleukemic stage, highly elevated expression of c-abl mRNA was detected exclusively in the spleen of the juvenile LE rats. Furthermore this high expression of the c-abl gene correlates well with hypermethylation of possible cytosine residue in the c-abl genomic locus.

Animals↗
PubMed1988

An atypical t(12;15) translocation of chromosomes found in murine plasmacytomas and activation of the c-myc oncogene.

Cells from murine plasmacytomas (MPC) consistently exhibit either the 12;15 or the 6;15 translocation. Recently, we found that cells from 2 MPC (ABPC 45 and ABPC 26) did not exhibit any known translocation. However, these MPC contained one chromosome 15 (15q-) that was shorter than its normal homologue and one 12 (12q+) that was longer than its normal 12 counterpart. Chromosome 15q- contained only one thin D band, corresponding in size to either D1 or D3 band. A minor light band D2 was apparently deleted. Chromosome 12q+ contained a light F1 band that was longer than the normal 12F1. In ABPC 45, the c-myc gene was assigned by in situ hybridization to the F1 region of 12q+ as well as to its original site of 15D2. Moreover, the in situ hybridization experiment assigned the S alpha gene to the F1 regions of both chromosomes 12 and 12q+. These findings indicate that a minor light band D2 of chromosome 15 carrying the c-myc gene was translocated into the major light band F1 of 12, resulting in a longer chromosome 12q+ and a correspondingly shorter chromosome 15q-. This type of chromosomal translocation suggests that ABPC 45 developed through a cytogenetic mechanism different from that of MPC with the typical t(12;15) translocation.

Animals↗
PubMed1988

Effect of prostaglandin E2 on gamma-interferon and 1,25(OH)2D3 vitamin D3-induced c-myc reduction during HL-60 cell differentiation.

The effect of prostaglandin E2 (PGE2) on the reduction of c-myc expression during the differentiation of the human leukemic cell line, HL-60, was examined. PGE2, a potent inducer of intracellular cyclic AMP (cAMP) in HL-60 cells, augmented monocyte-associated cell surface antigens induced by human gamma-interferon (IFN-gamma) or 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in these cells. The elevation of intracellular cAMP was induced dose-dependently by PGE2, but not by IFN-gamma or 1,25(OH)2D3. Changes were also seen in functional differentiation, such as, the increase of phagocytic capability and superoxide generation. PGE2 also enhanced the reduction of c-myc expression and the down-regulation of transferrin receptor by IFN-gamma or 1,25(OH)2D3, whereas PGE2 alone did not induce these phenotypic changes. These data suggest that IFN-gamma and 1,25(OH)2D3 reduce c-myc expression of HL-60 cells by a mechanism other than the augmentation of intracellular cAMP.

Calcitriol↗
PubMed1988

Both amino- and carboxy-terminal portions are required for insertion of yeast porin into the outer mitochondrial membrane.

Yeast porin, the major outer mitochondrial membrane protein, is synthesized without a cleavable extension peptide and post-translationally inserted into the membrane. When inserted into the membrane, it acquires resistance to externally added trypsin. To locate the sequences responsible for membrane insertion and topogenesis in the primary structure of yeast porin, we constructed several deletion and chimeric mutants of the porin cDNA. These cDNAs were expressed in vitro and the products were assayed for capacity to be correctly inserted into isolated mitochondria. It was thus found that deletion of the segment spanning residues 37-98 did not appreciably impair the insertion competence and the inserted protein became resistant to trypsin. On the other hand, the porin mutant lacking the segment consisting of residues 17-98 did not acquire the trypsin resistance, though it could bind to mitochondria specifically. Deletion of the carboxy-terminal 62 amino acid residues also abolished the capacity to be correctly inserted into mitochondria. We conclude that information required for membrane insertion and intramembranous topogenesis of the porin molecule is stored not only in the amino-terminal region but also in the carboxy-terminal portion.

Alkalies↗
PubMed1988

C-myc expression in lymphocytes of MRL/MP-lpr mice activated by A23187 and TPA.

In order to analyse the molecular mechanism of lymphocyte activation defect in MRL/MP-lpr (MRL/1) mice, c-myc proto-oncogene expression was examined in MRL/1 lymph node cells stimulated by various agents. Since a transient increase of c-myc RNA in early hours is required for lymphocyte activation, detection of c-myc messenger RNA is useful to determine whether or not an appropriate signal is transduced to the nucleus. Stimulation by concanavalin A (Con A) plus 12-O-tetradecanoyl phorbol 13-acetate (TPA) or A23187 plus TPA markedly increased c-myc mRNA expression and cell proliferation, whereas stimulation by Con A alone failed to do so. These results suggest that an abnormality exists in the early signal transduction process, and that it could be bypassed by calcium influx and direct activation of protein kinase C.

Animals↗
PubMed1988

[Successful treatment with tegafur and PSK in a patient with gallbladder cancer].

A 56-year-old man was admitted to Miyazaki Prefectural Hospital with a complaint of pain in the right upper quadrant. The liver was moderately enlarged on palpation with tenderness in the upper abdomen. Ultrasonography and abdominal CT showed gallbladder tumor with multiple space-occupying lesions in the liver. In addition, serum levels of CEA and CA19-9 were markedly elevated. These findings suggested gallbladder cancer with liver metastasis, and he was soon started on tegafur rectally (1,000 mg/day) and PSK orally (3.0 g/day). Two months later, the lesions in the liver almost disappeared, although little change in size of the gallbladder tumor was noted. This condition lasted for 6 months indicating partial response. Subsequently, he died of liver failure 14 months after the start of treatment. Since few antineoplastic agents have been reported to be effective against gallbladder cancer, tegafur and PSK might be a good combination for this disease.

Adenocarcinoma↗