Search PubMed⌕ Search

Biomedical subjects

K Mihara

Publications and source records attributed to K Mihara.

At least 163 records · Page 9Linked to original sources
PubMed1993

Neoplastic transformation and characterization of human fibroblasts by treatment with 60Co gamma rays and the human c-Ha-ras oncogene.

Human fibroblasts (KMST-6) immortalized by treatment with 60Co gamma rays were further neoplastically transformed by transfection of the c-Ha-ras oncogene from human lung cancer. The ras-transfected cells formed undifferentiated fibrosarcoma in nude mice. One of the tumors was recultured and a neoplastic human fibroblast line, KMST-6/RAS, was established. To analyze multistep carcinogenesis of human cells, the cellular characteristics of these genetically matched immortalized (KMST-6) and neoplastic (KMST-6/RAS) cell lines were studied in detail. KMST-6/RAS cells showed an increased saturation density, colony formation on confluent monolayers of normal human fibroblasts, proliferation in neomycin-containing medium, anchorage-independent growth, and enhanced expression of the transfected c-Ha-ras oncogene, whereas the immortalized cells did not demonstrate these characteristics. Unexpectedly, growth of KMST-6/RAS cells was serum-dependent, although they were neoplastic. Interestingly, the neoplastic cells did not show the criss-crossing or piling up growth pattern characteristic of transformed rodent fibroblasts.

Animals↗
PubMed1993

Disposition characteristics of protein drugs in the perfused rat kidney.

The renal disposition characteristics of 111In-labeled neocarzinostatin (NCS), soybean trypsin inhibitor (STI), and superoxide dismutase (SOD) were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous and urinary recovery profiles and tissue accumulation of proteins were determined under filtering or nonfiltering conditions. In the nonfiltering kidney perfusion experiment, no significant tissue accumulation was observed, suggesting minimal uptake from the glomerular and peritubular capillary sides. Therefore, tissue recovery corresponded to that with tubular reabsorption after glomerular filtration. The total amount of NCS or STI being filtrated through glomeruli, the sum of tissue and urinary recoveries, was similar to that of inulin, but that of SOD was about half. Similarly, the steady-state distribution volumes (Vd) of NCS and STI obtained by moment analysis of their venous outflow curves were similar to that of inulin, while the Vd value of SOD was significantly lower. These results suggest the restricted passage of SOD through the glomerular and postglomerular capillary wall. The tubular reabsorption ratio of proteins against the total filtrated amount decreased with an increase in the administered dose, suggesting nonlinearity of reabsorption. SOD had the largest reabsorption ratio. Thus, this experimental system is useful for quantitative analysis of renal disposition of proteins.

Animals↗
PubMed1993

An isozyme of microsomal carboxyesterases, carboxyesterase Sec, is secreted from rat liver into the blood.

It is generally believed that liver carboxyesterases are localized exclusively in the endoplasmic reticulum (ER), mostly in the lumen, loosely bound to the inner side of the membrane. A cDNA clone, clone (8-1/2-1) supposed to code for one of the isozymes, carboxyesterase E1, was isolated by Takagi et al. [J. Biochem. 104, 801-806 (1988)]. However, the protein coded by clone (8-1/2-1) had no consensus ER retention signal at its carboxy terminus, and the mechanism of its retention by ER lumen was unclear. When clone (8-1/2-1) was expressed in COS cells in this study, the plasmid-coded protein was secreted into the medium. When the carboxy terminal portion of the clone (8-1/2-1)-coded protein was replaced with the corresponding region of another carboxyesterase, pI 6.1 esterase, which had the HVEL sequence at the carboxy terminus, the chimeric protein was retained in the COS cells. We searched for a secretory form carboxyesterase in rat blood immunochemically using polyclonal antibodies to carboxyesterase E1, and detected a cross-reacting protein with a molecular weight of 68 kDa. The molecular weight was decreased by endoglycosidase F treatment but not by endoglycosidase H treatment, indicating that the protein carries complex type sugar chains. In addition, the cross-reacting protein was labeled with [3H] diisopropylfluorophosphate (DFP), suggesting that the protein has an esterase-type active center serine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Recoverin alters its surface properties depending on both calcium-binding and N-terminal myristoylation.

The solution structure and calcium-dependent structural changes of recoverin, a 23 kDa calcium binding protein of vertebrate photoreceptors, have been studied by small-angle X-ray scattering and CD, as well as the effect of N-terminal myristoylation. The CD spectrum is not affected by N-terminal myristoylation, but strongly affected by Ca2+, indicating that N-terminal myristoylation alone does not cause a conformational change. The major conformational change in recoverin induced by Ca2+ is characterized as a decrease in the alpha-helical content of the protein and an increase in global size upon removal of Ca2+. In the presence of Ca2+, unmyristoylated recoverin is monomeric and globular in solution, while N-terminal myristoylation brings about aggregation. In the absence of Ca2+, unmyristoylated recoverin tends to aggregate, while myristoylated recoverin becomes monomeric and globular. These observations indicate that recoverin changes its surface properties depending on both calcium binding and N-terminal myristoylation. Melittin interacts non-specifically only with the myristoylated recoverin in the absence of Ca2+. This may be indicative of the properties of the interaction between recoverin and its normal physiological target enzyme.

Calcium↗
PubMed1993

Detection of a novel 9-kDa endoplasmic reticulum membrane protein in mammalian cells by chemical cross-linking with translocating nascent peptides.

It is widely accepted that a proteinous channel participates in the co-translational translocation of proteins across the endoplasmic reticulum (ER) membrane. Truncated mRNAs encoding N-terminal 70, 75, 88, and 110 amino acids of interleukin 2 were translated with wheat germ cell-free system in the presence of rough microsomal membrane (RM), and integral membrane proteins were probed with the translocating nascent peptides by using a cross-linking reagent (DSS). Two membrane proteins, 9 kDa (Cp9, cross-linking partner 9 k) and 39 kDa (Cp39, cross-linking partner 39 k), were cross-linked with the 75 amino acids nascent peptide. When NEM-treated RM was used for the translocation reaction, neither Cp9 nor Cp39 proteins were cross-linked. When the translation products were treated with puromycin before the cross-linking, both proteins were not cross-linked. The cross-linked products of Cp9 and Cp39 were not extracted by alkaline extraction of the membrane, not sensitive to endoglycosidase H, and did not bind to Con A-Sepharose. These results indicate that both of the cross-linking partners were nonglycosylated integral membrane proteins. Cp39 was cross-linked with the 70, 88, and 110 amino acid nascent peptides as well as the 75 amino acid peptide, whereas Cp9 reacted only with the nascent peptides consisting of 70 and 75 amino acid residues. Even after the digitonin treatment of the RM with the translocating intermediates, the cross-linked products with Cp9 and Cp39 were detected. Cp9 and Cp39 seem to be tightly associated with the ribosome-nascent peptide complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Importance of the proline-rich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome P-450s.

A proline-rich region is present following the signal-anchor sequence in the amino-terminal portion of all known microsomal cytochrome P-450s. To assess the functional significance of the proline residues in this region, we systematically altered these residues of cytochrome P450(M1) (P450 2C11); one, two, and three proline residues out of the five in the region were exchanged for alanine residues. The wild-type and the mutated proteins were expressed in the fission yeast Schizosaccharomyces pombe under the control of nmt1 promoter. The wild-type and the mutated proteins were all highly expressed in the yeast cells (5-9% of the total membrane protein). The expressed wild-type P450(M1) showed a typical carbon monoxide difference spectrum of P-450 and the activity of testosterone hydroxylation, whereas all the mutated proteins constructed in the present study showed no characteristic P-450 spectrum, suggesting that the substitution of the proline residues in this region resulted in a defect of proper heme incorporation. Furthermore, the mutated proteins in which more than one proline residues had been exchanged were more sensitive to trypsin digestion than the wild type. From these results, we propose that the proline residues in the proline-rich region are crucial for the formation of the correct conformation of microsomal P-450 molecules.

Amino Acid Sequence↗
PubMed1993

Prediction of plasma concentrations of mianserin and desmethylmianserin at steady state from those after an initial dose of mianserin.

The relationships between the plasma concentrations of mianserin and desmethylmianserin at 18 h after initial dosing and those at steady state were studied in 19 depressed patients receiving 30 mg of mianserin at bedtime. Significant linear relationships were observed for mianserin, desmethylmianserin, and mianserin plus desmethylmianserin. The present study thus suggests that the plasma concentrations of these compounds after an initial dose of mianserin can be used for the prediction of an optimal dose.

Adult↗
PubMed1993

Disposition characteristics of model macromolecules in the perfused rat kidney.

The disposition characteristics of model macromolecules such as dextran (70 kDa), bovine serum albumin (BSA), and their charged derivatives were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous and urinary recovery patterns and tissue accumulation of radiolabeled compounds were evaluated under filtering or nonfiltering conditions. In the filtering kidney, cationic macromolecules such as diethylaminoethyl-dextran (DEAE-dex) and cationized BSA (cBSA) accumulated in the kidney to a great extent whereas anionic and neutral macromolecules such as BSA, carboxymethyl-dextran (CM-dex), and dextran showed only small uptake. DEAE-dex and cBSA were distributed to both the medulla and cortex regions of the kidney and their recoveries in the kidney decreased as the injected dose increased. Similar tissue uptake was observed in the nonfiltering kidney perfusion system suggesting that they were mainly taken up by the kidney from the renal capillary side based on electrostatic interaction. In addition, the steady-state distribution volumes of cationic macromolecules calculated from venous outflow patterns were larger than those of the intravascular volume estimated from the distribution volumes of neutral and anionic macromolecules, suggesting their reversible interaction with the vascular wall. On the other hand, dextran derivatives with molecular weight distribution were excreted into urine based on glomerular permselectivity; i.e., cationic DEAE-dex and anionic CM-dex showed enhanced and restricted urinary excretion, respectively, compared with neutral dextran. In contrast, no significant excretion was observed for BSA and cBSA. The utility of the isolated rat kidney perfusion experiment for studying the renal disposition of macromolecular drugs was thus demonstrated.

Animals↗
PubMed1993

Immortalization of normal human cells is a multistep process and a rate limiting step of neoplastic transformation of the cells.

Three normal human fibroblast strains were immortalized by treatment with either 4-nitroquinoline 1-oxide (SUSM-1 and OUMS-24F lines) or 60Co gamma rays (KMST-6 line). Immortalization of normal human cells with these agents required repeated treatments. Before immortalization, most of the cells treated with these mutagenic agents showed more or less chromosome aberrations, but immortalized cells appeared very rarely. This suggests that several mutational events are involved in the immortalization process itself. The immortalized cells showed polygonal or epithelial-like morphology, prominent numerical and structural abnormalities of chromosomes, requirement of serum growth factors for their growth, and no tumorigenicity. The immortalized KMST-6 and OUMS-24F were further transformed into neoplastic ones by the ras oncogene, but not by transfection with the mutant p53 gene. Normal human fibroblasts which are mortal were not immortalized nor neoplastically transformed with the ras oncogene or the mutant p53. These findings strongly indicate that the immortalization of human cells is prerequisite for neoplastic transformation of the cells.

4-Nitroquinoline-1-oxide↗
PubMed1993

[Optical density of human macular pigment in spectral sensitivity measurements].

Optical density of the human macular pigment was measured by studying the spectral sensitivities to 1 degree, 200 ms test flashes on a 430 nm monochromatic background in the fovea and 10 degrees outside the fovea. Narrow-band (6 to 10 nm half band width) interference filters were used for the test lights. The difference between the spectral sensitivity in the fovea and outside the fovea coincided with the optical density of the macular pigment estimated previously by psychophysical means. We suggest that this method is useful for investigating the optical density of human macular pigment.

Adult↗
PubMed1992

Malignant transformation of human fibroblasts previously immortalized with 60Co gamma rays.

Normal human fibroblasts obtained from a whole embryo were malignantly transformed with only 60Co gamma rays, but after extensive passage. The fibroblasts were exposed intermittently to 60Co gamma rays, 13 times, 2,800 rads in total, from the primary culture to the 40th passage level. At the 50th passage level the cells became morphologically changed, showing chromosomal abnormalities, and serial passages showed them to be immortalized. They were not tumorigenic on transplantation into nude mice, but became malignant after extensive passage; i.e., at the 547th passage level and 2,800 days after initiation of the culture. Our results indicate that several different genotypic changes are necessary for malignant transformation of human cells over long periods. No mutation at codons 12 and 61 of H-, K- and N-ras was detected in the malignant cells. Thus, this system may be useful to detect other cellular genes that may contribute to the malignant phenotypes of human fibroblasts.

Base Sequence↗
PubMed1992

Functions of signal and signal-anchor sequences are determined by the balance between the hydrophobic segment and the N-terminal charge.

The signal sequence of secretory proteins and the signal-anchor sequence of type II membrane proteins initiate the translocation of the following polypeptide segments, whereas the signal-anchor sequence of cytochrome P-450-type membrane proteins mediates the membrane insertion of the polypeptide via a signal-recognition particle-dependent mechanism but does not lead to the translocation of the following C-terminal sequences. To establish the structural requirements for the function of signal and signal-anchor sequences, we constructed chimeric proteins containing artificial topogenic sequences in which the N-terminal net charge and the length of the hydrophobic segment were systematically altered. Utilizing an in vitro translation-translocation system, we found that hydrophobic segments consisting of 7-10 leucine residues functioned as signal sequences whereas segments with 12-15 leucine residues showed different topogenic functions, behaving as signal sequences or P-450-type signal-anchor sequences, depending on the N-terminal charge. From these observations, we propose that the function of N-terminal topogenic sequences depends on a balance between the N-terminal charge and the length of the following hydrophobic segment.

Base Sequence↗
PubMed1992

Characterization of adrenodoxin precursor expressed in Escherichia coli.

The precursor of bovine adrenodoxin (pAd), a mitochondrial protein, was expressed in Escherichia coli. The cloned cDNA of pAd was ligated to an expression vector pET-3d, and silent mutations were introduced into the N-terminal portion of the cDNA in order to increase the expression. The precursor was highly expressed (approximately 20% of the total cell protein) as the inclusion body, and contained an iron-sulfur center as judged from its optical absorption spectra. The inclusion body was solubilized with 7 M urea and pAd was purified in the presence of urea. The purified pAd was efficiently imported into isolated bovine adrenal cortex mitochondria and processed to the mature form. The import reaction required ATP inside the mitochondria in addition to the inner membrane potential, and was strongly inhibited by trypsin treatment of the mitochondria, as in the case of the in vitro translated precursor. It was, however, not dependent on the unfolding activity of the cytosolic factor with extramitochondrial ATP.

Adenosine Triphosphate↗
PubMed1992

Mitochondrial porin can be translocated across both endoplasmic reticulum and mitochondrial membranes.

Mitochondrial porin is a major integral membrane protein of the outer membrane. To assess the stop-transfer sequence in the yeast porin molecule (P), we constructed the following chimeric proteins. (i) The signal sequence of interleukin 2, a secretory protein, was fused to the amino-terminus of porin (SP). (ii) The matrix targeting presequence of cytochrome c oxidase subunit IV was fused to the amino-terminus of porin (CP). (iii) The amino-terminal segment consisting of 42 amino acid residues of "70 kDa protein" of yeast mitochondria, a major membrane protein of the outer membrane, was introduced into the middle portion of interleukin 2 (IL70). These chimeric proteins were expressed with an in vitro transcription-translation system and their integration into microsomal membrane or mitochondrial membranes was examined. When the proteins were synthesized in vitro with wheat germ cell-free system in the presence of rough microsomal membrane (RM), SP was completely translocated across the membrane, processed by the signal peptidase, and glycosylated. The translocation of IL70 molecule across RM was stopped at the introduced amino-terminal segment of 70 kDa protein. The authentic porin did not interact with the microsomal membrane. To assess the interaction with mitochondria, porin and CP were synthesized with the reticulocyte lysate system and subjected to posttranslational import reaction with isolated rat liver mitochondria. The authentic porin was integrated into the outer membrane in an alkali-resistant fashion. CP was imported into the mitochondria and its presequence was cleaved by the processing protease in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Characterization of the cytoplasmic proline-directed protein kinase in proliferative cells and tissues as a heterodimer comprised of p34cdc2 and p58cyclin A.

Site-specific analysis of tyrosine hydroxylase phosphorylation in rat pheochromocytoma led previously to the identification of a novel growth factor-sensitive serine/threonine protein kinase, designated proline-directed protein kinase (PDPK). In this article we describe further the activation, purification, subunit configuration, and biochemical characteristics of this cytoplasmic enzyme system. In human A431 epidermoid carcinoma cells PDPK activity was found to be stimulated by epidermal growth factor in a dose-dependent, time-dependent manner. The PDPK purified from the cytosol of mouse FM3A mammary carcinoma cells exhibited the same chromatographic behavior and biochemical properties as the tyrosine hydroxylase-associated enzyme purified originally from rat pheochromocytoma. The presence of p34cdc2 was ultimately detected in all active fractions of highly purified PDPK by Western blotting and immunoprecipitation; however, it was determined that this catalytic subunit is complexed with a 58-kDa regulatory subunit that is clearly distinct from that of the "growth-associated" M phase-specific histone H1 kinase (i.e. cyclin B). The 58 kDa regulatory subunit of PDPK was identified by direct immunoblotting as a mammalian A-type cyclin. Furthermore, the p58cyclin A subunit of PDPK was found to be phosphorylated on tyrosine residues in vivo and in vitro, the latter of which resulted in a significant increase in PDPK activity. Additional distinctions between this growth factor-sensitive PDPK (p34cdc2-p58cyclin A) and the M phase-specific histone H1 kinase (p34cdc2-p62cyclin B-p13suc1) are identified on the basis of chromatographic behavior, enzyme kinetics, and physicochemical properties. Based on these findings, it is proposed that PDPK represents a unique complex of the p34cdc2 protein kinase which is active in the cytoplasm of proliferative cells, is regulated differently from the M phase-specific histone H1 kinase by phosphorylation reactions, and is modulated selectively by growth factors.

Amino Acid Sequence↗