Biomedical subjects

K Mihara

Publications and source records attributed to K Mihara.

At least 73 records · Page 4Linked to original sources

PubMed1998

Improved high-performance liquid chromatographic analysis of teniposide in human plasma.

A simple and practical high-performance liquid chromatographic analysis has been developed for measuring teniposide (VM26) in human plasma. The present analytical method has improved extraction efficiency from human plasma, therefore allowing determination of VM26 in a clinical setting using ultraviolet detection alone. Furthermore, sample preparation was simplified and shortened through use of a one-step extraction procedure. VM26 and internal standard (ibuprofen) were extracted from human plasma (0.5 ml) with ethyl acetate. A phenyl muBondapak column eluted with a mobile phase, consisting of acetonitrile-distilled water-acetic acid (30:68:2, v/v/v) was used for separation, and quantitation was achieved with a UV monitor set at 240 nm. Average extraction efficiency was 96.8+/-6.6% for VM26 between 1 and 25 microg/ml, and 91.4+/-4.3% for internal standard, with both intra- and inter-day coefficients of variation being less than 10%. The detection limit with a 100-microl injection was estimated at 0.2 microg/ml with a signal-to-noise ratio of 3 for VM26 in human plasma. The stability data of VM26 in plasma, standard and stock solutions were also obtained. The present method was found to be an alternative to the previously reported method with an electrochemical detection, and can be easily applied to routine clinical pharmacokinetic studies of VM26.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Ubiquitous presence of cellular proteins that specifically bind to the 3' terminal region of hepatitis C virus.

The 3' terminal region (3'-X tail) of hepatitis C virus (HCV) genomic RNA forms a stable stem-loop structure. The 3'-X tail consists of 98 nucleotides (nt) that are highly conserved among the HCV strains and supposed to function as a cis-acting region for replication of negative strand RNA and/or viral encapsidation. In the present study, by UV cross-linking assay we found two kinds of cellular proteins of approximately 87 and 130 kDa, which specifically bind to the full-length 3'-X tail (nt 1 to 98), but not the 3'- or 5'-truncated 3'-X tail, consisting of nt 1 to 50 or nt 51 to 98, respectively. These proteins were detected in human cell lines such as hepatic tumor cell lines and a T-lymphocyte cell line and also in a human embryonic lung fibroblast cell strain. In addition, human hepatocellular carcinoma tissues expressed these proteins regardless of infection or uninfection of HCV. Furthermore, these proteins were also detected in normal human tissues derived from the lung, heart, kidney, stomach, intestine, and colon. Thus, these cellular proteins, which are ubiquitously present in human tissues, might be involved in viral replication and/or encapsidation.

Binding Sites↗

PubMed1998

Membrane topology of NADPH-cytochrome P450 reductase on the endoplasmic reticulum.

Topology of the membrane-anchoring segment of mouse NADPH-cytochrome P450 reductase in the endoplasmic reticulum membrane was elucidated. An N-glycosylation site was generated in the amino-terminal hydrophilic sequence of the reductase, and the mutated protein was expressed in a cell-free system in the presence of microsomal vesicles. The in vitro synthesized reductase protein was integrated into the microsomal membrane and N-glycosylated depending on the presence of signal recognition particles. We conclude that the amino-terminal membrane-anchoring segment of the reductase is a type I signal-anchor sequence which shows amino-terminus-lumen and carboxy-terminus-cytoplasm topology.

Amino Acid Sequence↗

PubMed1998

Cyclin E overexpression responsible for growth of human hepatic tumors with p21WAF1/CIP1/SDI1.

We examined a relationship between p21WAF1/CIP1/SDI1 and cell-cycle-related proteins in 12 human liver tumor cell lines (JHH-1, -2, -4, -5, -6, -7; HLE; HuH-7; Hep3B; PLC/PRF/5; HuH-6; HepG2). Seven (JHH-1, -2, -5, -6, -7; Hep3B; HepG2) out of eight cell lines having p21WAF1/CIP1/SDI1 protein overexpressed cyclin E protein, although one of them (JHH-5) overexpressed a reduced size of cyclin E. The rest (HuH-6) of the 8 cell lines with p21WAF1/CIP1/SDI1 showed a decreased expression of cyclin E. Four cell lines (JHH-4; HLE; HuH-7; PLC/PRF/5) deficient of p21WAF1/CIP1/SDI1 protein did not overexpress cyclin E protein. As to expression of the other cell-cycle-related proteins, cyclin A, cyclin D1, CDK2 or CDK4, no significant difference was detected among the 12 cell lines. These findings indicate that the human liver tumor cell lines which have the p21WAF1/CIP1/SDI1-inducible barriers of the cell cycle progression can go through the G1/S checkpoint by overexpressing cyclin E.

Blotting, Northern↗

PubMed1998

Effects of various factors including the CYP2D6 genotype and coadministration of flunitrazepam on the steady-state plasma concentrations of bromperidol and its reduced metabolite.

The effects of various factors, including the cytochrome P450 (CYP) 2D6 genotype and the coadministration of flunitrazepam, on the steady-state plasma concentrations (Css) of bromperidol and its reduced metabolite were studied in 62 schizophrenic inpatients receiving bromperidol 12 mg/day. By use of allele-specific PCR analysis, the wild type allele (CYP2D6*1A) and four mutated alleles causing either absent (CYP2D6*3, CYP2D6*4 and CYP2D6*5) or decreased (CYP2D6*10) CYP2D6 activity were identified. The means (ranges) of the Css of bromperidol and reduced bromperidol corrected to the median body weight were 7.2 (1.3-17.4) and 2.2 (0.4-8.9) ng/ml, respectively. Neither the Css of bromperidol nor that of reduced bromperidol significantly differed among the patients with no (n = 28), one (n = 30) and two mutated alleles (n = 4). The patients coadministered with flunitrazepam (n = 52) had significantly (P < 0.05) higher Css of bromperidol, but not reduced bromperidol, than those not (n = 10). Age, sex and smoking had no significant effects on the Css of these compounds. The present study thus suggests that the polymorphic CYP2D6 is not involved in the metabolism of bromperidol and reduced bromperidol to a major extent. The coadministration of flunitrazepam inhibits the metabolism of bromperidol, but age, sex and smoking do not affect it.

Adolescent↗

PubMed1998

Correlation between steady-state plasma concentrations of mianserin and trazodone in depressed patients.

OBJECTIVE: The correlations between steady-state plasma concentrations of mianserin and its active metabolite desmethylmianserin and those of trazodone and its active metabolite m-chlorophenylpiperazine (m-CPP) were examined in 19 depressed patients. METHODS: Ten patients received first mianserin (30 mg per day) and second trazodone (150 mg per day), while 9 patients received these treatments in the opposite sequence, with at least 2-week intervals between the two phases. Blood was sampled at steady state, 1-3 weeks after initiation of each treatment. Plasma concentrations of mianserin, the separate enantiomers S(+)- and R(-)-mianserin, desmethylmianserin, trazodone and m-CPP were measured by means of high-performance liquid chromatography. RESULTS: There was a significant correlation between steady-state plasma concentrations of trazodone and total mianserin (r = 0.59) or S(+)-mianserin (r = 0.57), but not R(-)-mianserin (r = 0.33). CONCLUSION: The present study thus suggests that the metabolic capacity of mianserin, especially the more active S(+)-enantiomer, and that of trazodone correlate to each other. This finding supports the previous suggestions that cytochrome P4502D6 is involved in the metabolism of mianserin and trazodone.

Antidepressive Agents, Second-Generation↗

PubMed1998

Forced transmembrane orientation of hydrophilic polypeptide segments in multispanning membrane proteins.

In a current model of integration of multispanning membrane proteins into the endoplasmic reticulum, it is proposed that the transmembrane segments show alternating translocation initiation and stop-transfer functions. Here, we present evidence for a mode of cotranslational insertion in which an internal signal-anchor sequence with Nexo/Ccyt topology confers a transmembrane disposition onto a preceding hydrophilic segment, resulting in a topology where the hydrophilic segment apparently can slip back and forth across the membrane. Our results demonstrate that hydrophobicity is not, as hitherto thought, an absolute requirement for the formation of a transmembrane segment, and suggest that integral membrane proteins may contain hydrophilic transmembrane segments with a considerable freedom to move in relation to the membrane.

Animals↗

PubMed1998

Enantioselective tissue distribution of the basic drugs disopyramide, flecainide and verapamil in rats: role of plasma protein and tissue phosphatidylserine binding.

PURPOSE: The stereoselective distribution of three basic drugs, disopyramide (DP), flecainide (FLC) and verapamil (VP), was studied to clarify the relationship between the tissue-to-unbound plasma concentration ratio (Kpf) and drug lipophilicity and binding to phosphatidylserine phs), which are possible factors determining the tissue distribution of these drug enantiomers. METHODS: The drug enantiomer or racemate was administered to rats by intravenous constant infusion. Their concentrations in plasma and tissues were determined using enantioselective high-performance liquid chromatography. Plasma protein binding, and buffer-octanol and buffer-hexane containing PhS partition coefficients were also determined. RESULTS: The stereoselectivity of the tissue-to-plasma concentration ratio (Kp) was partly associated with that of serum protein binding. However, the Kpf value of R(+)-VP in the lung was significantly higher than that of S(-)-VP. A linear correlation was observed between the Kpf values of these drug enantiomers in brain, heart, lung and muscle, and their buffer-hexane containing PhS partition coefficients. The in vitro data for the binding of these drugs to PhS suggest that stereoselective binding of VP to PhS may correspond to its stereoselective tissue binding. CONCLUSIONS: Our findings provide some evidence for a role of tissue PhS in the tissue distribution of basic drugs with respect to stereoselectivity of drug enantiomers distribution.

Animals↗

PubMed1998

Identification of potential regulatory elements for the transport of Emp24p.

To examine the possibility of active recycling of Emp24p between the endoplasmic reticulum (ER) and the Golgi, we sought to identify transport signal(s) in the carboxyl-terminal region of Emp24p. Reporter molecules were constructed by replacing parts of a control invertase-Wbp1p chimera with those of Emp24p, and their transport rates were assessed. The transport of the reporter was found to be accelerated by the presence of the cytoplasmic domain of Emp24p. Mutational analyses revealed that the two carboxyl-terminal residues, leucine and valine (LV), were necessary and sufficient to accelerate the transport. The acceleration was sequence specific, and the terminal valine appeared to be more important. The LV residues accelerated not only the overall transport to the vacuole but also the ER to cis-Golgi transport, suggesting its function in the ER export. Hence the LV residues are a novel anterograde transport signal. The double-phenylalanine residues did not affect the transport by itself but attenuated the effect of the anterograde transport signal. On the other hand, the transmembrane domain significantly slowed down the ER to cis-Golgi transport and effectively counteracted the anterograde transport signal at this step. It may also take part in the retrieval of the protein, because the overall transport to the vacuole was more evidently slowed down. Consistently, the mutation of a conserved glutamine residue in the transmembrane domain further slowed down the transport in a step after arriving at the cis-Golgi. Taken together, the existence of the anterograde transport signal and the elements that regulate its function support the active recycling of Emp24p.

Amino Acid Sequence↗

PubMed1998

Identification of the protein import components of the rat mitochondrial inner membrane, rTIM17, rTIM23, and rTIM44.

We cloned rat liver mitochondrial 18.1, 21.9, and 51.0 kDa proteins with a significant structural homology to the components of the translocase of the yeast mitochondrial inner membrane, Tim17, Tim23, and Tim44. The 18.1 and 21.9 kDa proteins were synthesized as mature forms having four potential transmembrane segments and localized to the mitochondrial inner membrane. The 51.0 kDa protein is a precursor having a presequence of approximately 6 kDa which is cleaved during import into the mitochondria. The mature 45 kDa protein is located in the matrix, both in a soluble form and in a membrane-bound, alkali-extractable form. Immunofluorescence microscopy confirmed the location of all three proteins in the mitochondria. Antibodies against the 21.9 kDa protein, but not those against the 18.1 and 51.0 kDa proteins, inhibited the precursor import into the mitoplasts in vitro. Immunoprecipitation indicated that all three proteins interacted with the protein in transit to the matrix. Immunoprecipitation also revealed that the 18.1 kDa protein formed a complex with the 21.9 kDa protein and the 45 kDa protein with mHsp70; the latter complex was dissociated in an ATP- or ADP-dependent manner and the reaction was impeded by AMP-PNP or inorganic phosphate. These assays thus demonstrated the 18.1, 21.9, and 45 kDa proteins to be the translocator components of the rat mitochondrial inner membrane and, therefore, the functional homologues of Tim17, Tim23, and Tim44, respectively.

Amino Acid Sequence↗

PubMed1998

Characterization of the initial steps of precursor import into rat liver mitoplasts.

Mitochondria have two independent protein-import machineries, one in the outer membrane (the Tom system) and the other in the inner membrane (the Tim system). Here, we have characterized the initial steps of precursor import into rat liver mitoplasts. The import reaction was separated into two stages, consisting of precursor binding to the mitoplasts at 0-10 degreesC, and a subsequent chase reaction at 30 degreesC. This assay revealed four distinct precursor-import steps: DeltaPsi-dependent initial binding of the precursor, precursor transfer to the Tim23-Tim17 stage, DeltaPsi-dependent translocation of the presequence across the inner membrane, and the complete translocation of the mature portion of the precursor. Antibodies against the intermembrane space domain of Tim23 inhibited neither the precursor binding nor the subsequent translocation of the presequence across the inner membrane. In contrast, the antibodies inhibited the complete translocation of the mature domain of the precursor across the inner membrane. Immunoprecipitation with anti-Tim23 IgGs revealed that the precursor-Tim23 complex increased with time and temperature after the initial targeting of the precursor to the mitoplasts. These results suggest that the precursor is first targeted to the inner membrane component DeltaPsi-dependently, then transferred to the Tim system consisting of Tim23-Tim17, and finally imported into the matrix.

Amino Acid Sequence↗

PubMed1998

The effect of HLA alleles on response to interferon therapy in patients with chronic hepatitis C.

OBJECTIVE: To compare HLA alleles in the patients with chronic hepatitis C treated with interferon-alpha (IFN-alpha) between patients with response to IFN treatment and nonresponse. METHOD: Sixty-seven Japanese patients with chronic hepatitis C were treated with daily intramuscular administration of IFN-alpha (6 million units) for 2 weeks followed by three times per week for 22 weeks. Viral loads of hepatitis virus C (HCV), HCV genotypes and HLA antigens were determined just before IFN-alpha treatment. Responders to IFN-alpha were defined as normalization of alanine aminotransferase at the end of treatment and during a follow-up period at least longer than 6 months. The patients who could not reach the above response criteria were defined as nonresponders. RESULTS: There were 20 responders and 47 nonresponders to IFN treatment. The low viral load with less than 1 x 10(6) copy/ml (P< 0.05), and type 2a genotype (P< 0.05) were significantly increased in responders. Other clinical and biochemical parameters were not significant. There was no difference in HLA-A and C antigens between responders and nonresponders. In contrast, HLA-B54,DR4 and A24-B54-DR4 haplotype of nonresponders increased compared with responders or controls (Pc < 0.0001, Pc < 0.001, Pc < 0.0001, respectively). At multivariate analysis, viral loads, HLA-B54 and HLA-A24-B54-DR4 haplotype were significant (P=0.0002, P=0.0258, P=0.0378, respectively). CONCLUSION: The low viral load is a good predictor. HLA-B54 and HLA-A24-B54-DR4 haplotype should be predictors for poor response to IFN therapy in patients with chronic hepatitis C.

Alanine Transaminase↗

PubMed1998

Prolactin response to bromperidol treatment in schizophrenic patients.

The prolactin response to an antipsychotic drug, bromperidol was studied in 24 schizophrenic in-patients (13 males. 11 females). Eight patients were given 6 mg/day, 8 were given 18 mg/day. Duration of treatment was 3 weeks. Plasma concentrations of bromperidol and reduced bromperidol were measured by high-performance liquid chromatography, and that of prolactin was measured by radioimmunoassay. Bromperidol treatment significantly (P < 0.01) increased plasma concentration of prolactin each week. The mean +/- S.D. of the delta-prolactin (the mean concentration during treatment minus the pretreatment concentration) was 13.3 +/- 12.4 ng/ml. Multiple regression analysis showed that the delta-prolactin concentration was significantly greater in females than in males (P < 0.05) and correlated to plasma concentrations of bromperidol (P < 0.001) and reduced bromperidol (P < 0.0001). These results suggest that the prolactin response to bromperidol treatment depend on plasma concentrations of both bromperidol and reduced bromperidol and gender, and that reduced bromperidol is involved in the pharmacological effects during bromperidol treatment.

Adult↗

PubMed1998

A new concept in polytopic membrane proteins following from the study of band 3 protein.

In the present communication, we introduce a novel concept in multispanning polytopic membrane proteins revealed by the study of the band 3 protein. The transmembrane domain of such proteins can be divided into three categories, that is, hydrophilic loops connecting transmembrane peptides (category 1), portions embedded by peptide-peptide interactions (category 2), and portions embedded by peptide-lipid interactions (category 3). Category 2 peptides of polytopic membrane proteins were found to stably reside in the lipid bilayer without peptide-lipid interactions that had been thought to be essential for transmembrane segments. Category 3 peptides are equivalent to single-spanning segments of bitopic membrane proteins. Three different experiments, namely proteolytic digestion, chemical modification of the band 3 protein, and cell free transcription and translation, were used to categorize the transmembrane peptides.

Animals↗

PubMed1998

Inhibition of S-warfarin metabolism by nonsteroidal antiinflammatory drugs in human liver microsomes in vitro.

We studied the inhibition of S-warfarin metabolism by nonsteroidal antiinflammatory drugs (NSAIDs) in human liver microsomes in vitro. After screening for potential inhibitors among ten NSAIDs using human recombinant cytochrome P450, inhibition kinetic parameters were estimated using human liver microsomes. Phenylbutazone and bucolome were suggested to increase the unbound steady-state level of S-warfarin about four- and five-fold, respectively, as estimated from these metabolic parameters.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1997

Partial purification and characterization of a gibberellin-binding protein from seedlings of Azukia angularis.

Little is known about mechanisms for the perception of gibberellin (GA), a phytohormone, by plants. A GA-binding protein (GBP) with receptor-like characteristics was partially purified from azuki bean (Azukia angularis) seedlings. Gibberellins that caused much elongation of azuki epicotyl sections showed strong binding affinity for the GBP, and their biosynthetic precursors and inactive metabolites had weak affinity for it. The apparent Kd value for GA4 was calculated to be 7 x 10(-10) M from Scatchard plots. The molecular weight of the GBP in the native state was estimated to be 25,000 by high-performance liquid chromatography with a gel-permeation column. The GBP may be one of the receptor candidates for GAs.

Carrier Proteins↗

PubMed1997

Analysis of the functional domain of the rat liver mitochondrial import receptor Tom20.

Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Deltatom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.

Amino Acid Sequence↗

PubMed1997

Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones.

We have reconstituted the early steps of precursor targeting to mitochondria in a defined and soluble system consisting of the cytosolic domains of the yeast mitochondrial import receptors Tom20 and Tom70, precursor to bovine adrenal adrenodoxin (which has a cleavable targeting signal) and rat liver cytosolic chaperones hsp70 and mitochondrial import-stimulating factor (MSF). The Tom70 domain only bound the precursor in the presence of MSF, yielding a precursor-MSF-Tom70 complex; ATP hydrolysis by MSF released MSF and generated a precursor-Tom70 complex whose formation was inhibited by an excess of a functional presequence peptide, but not by 150 mM NaCl. In the presence of the Tom20 domain, ATP caused transfer of the precursor from the precursor-MSF-Tom70 complex to Tom20. The Tom20 domain alone only bound the precursor in the presence of hsp70; hsp70 itself was not incorporated into the resulting complex. Formation of the Tom20-precursor complex was inhibited by excess presequence peptide or by 150 mM NaCl. Similar results were obtained with the ADP/ATP carrier and porin precursors, which both lack a cleaved targeting signal. Correct targeting of a precursor to mitochondrial import receptors thus requires cytosolic chaperones, irrespective of the presence or absence of a cleavable presequence.

14-3-3 Proteins↗