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Biomedical subjects

T Hase

Publications and source records attributed to T Hase.

At least 109 records · Page 6Linked to original sources
PubMed1993

Intramitochondrial protein sorting. Isolation and characterization of the yeast MSP1 gene which belongs to a novel family of putative ATPases.

Replacement of the presequence of yeast cytochrome c1 by the amino-terminal 61 residues of MAS70, a yeast mitochondrial outer membrane protein, resulted in exclusive localization of the fusion protein (termed the 61-mC1 protein) to the outer membrane. When a cytochrome c1-deficient yeast strain was transformed with a plasmid encoding the fusion protein, the cells could not grow on nonfermentable carbon sources such as glycerol. We isolated a novel yeast gene MSP1 (mitochondrial sorting of proteins) whose overexpression causes mislocalization of the 61mC1 fusion protein to the inner membrane, probably via the intermembrane space, and thereby allows the host cells to grow on glycerol. The predicted MSP1 protein (MSP1) is a hydrophilic 40-kDa polypeptide containing a putative membrane-spanning domain near the amino terminus. Further sequence analyses revealed that MSP1 is a member of a novel family of putative ATPases which share a highly conserved domain of about 185 amino acid residues, including a consensus motif for a nucleotide binding site. MSP1 was found to be an intrinsic mitochondrial outer membrane protein of an apparent molecular mass of 40 kDa with a large domain facing to the cytosol. The MSP1 gene is not essential for the cell growth either on fermentable or nonfermentable carbon sources.

Adenosine Triphosphatases↗
PubMed1993

Genistein, a dietary-derived inhibitor of in vitro angiogenesis.

Consumption of a plant-based diet can prevent the development and progression of chronic diseases that are associated with extensive neovascularization; however, little is known about the mechanisms. To determine whether prevention might be associated with dietary-derived angiogenesis inhibitors, we have fractionated urine of healthy human subjects consuming a plant-based diet and examined the fractions for their abilities to inhibit the proliferation of vascular endothelial cells. Using gas chromatography-mass spectrometry, we showed that one of the most potent fractions contained several isoflavonoids, which we subsequently synthesized. Of all synthetic compounds, the isoflavonoid genistein was the most potent and inhibited endothelial cell proliferation and in vitro angiogenesis at concentrations giving half-maximal inhibition of 5 and 150 microM, respectively. As we have previously demonstrated, genistein concentrations in urine of subjects consuming a plant-based diet are in the micromolar range, while those of subjects consuming a traditional Western diet are lower by a factor of > 30. The high excretion of genistein in urine of vegetarians and our present results suggest that genistein may contribute to the preventive effect of a plant-based diet on chronic diseases, including solid tumors, by inhibiting neovascularization. Thus, genistein may represent a member of a new class of dietary-derived anti-angiogenic compounds.

Adrenal Cortex↗
PubMed1993

Comparison of replication rates and pathogenicities between the SA14 parent and SA14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons.

The replication rates and pathogenicities of the SA 14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis (JE) virus in neurons of the mouse brain following intracerebral inoculation were compared. All the mice inoculated with the SA 14 parent strain died within one week postinoculation (p.i.), whereas all the mice inoculated with the SA 14-14-2 vaccine strains survived without showing any signs of central nervous system (CNS) involvement. The virus titers of the mouse brains inoculated with the SA 14 strain reached progressively higher levels until day 5 when the animals died. On the other hand, the virus titers of the mouse brains inoculated with the SA 14-14-2 strain persisted at low levels for several days and could not be detected after 10 days. In the routine electron microscopical study, a majority of neurons in the mouse brains inoculated with the SA 14 strain contained virions and showed characteristic cytopathological changes in connection with viral replication. In the brains inoculated with the SA 14-14-2 strain, however, we failed to find neurons containing virions or showing characteristic cytopathological changes. In the alkaline phosphatase immunostaining of paraffin-embedded sections, a majority of neurons in the brains of mice inoculated with the SA 14 strain stained positively on day 5 p.i., but only a small number of neurons in scattered small foci stained positively in the brains inoculated with the SA 14-14-2 strain. The immunogold staining of Vibratome sections also revealed the identical patterns; moreover, electron microscopical examination of the immunopositive foci of the brain inoculated with the vaccine strain revealed neurons that contained virions in dilated cisternae of rough endoplasmic reticulum (RER), indicating that the SA 14-14-2 strain also replicated, albeit poorly, in neurons. The present results showed that upon intracerebral inoculation into mice the SA 14 parent strain of JE virus grew vigorously in a large number of neurons, killing the animals, while the SA 14-14-2 vaccine strain grew poorly only in a small number of neurons without causing mortality. Possible mechanisms involved in the alteration of pathogenicity between the SA 14 parent virus and the SA 14-14-2 vaccine virus are discussed.

Animals↗
PubMed1993

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus.

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.

Animals↗
PubMed1993

Morphogenesis of the protein secretory system in PC12 cells infected with Japanese encephalitis virus.

Infection of PC12 cells with Japanese encephalitis (JE) virus caused marked proliferation of the protein secretory system. Accordingly, in this study the morphogenesis of the secretory organelles, i.e., rough endoplasmic reticulum (RER) and the Golgi apparatus, in JE virus-infected PC12 cells was analyzed by electron microscopical observation. Starting 24 h postinoculation (p.i.), a structure that represented nascent RER appeared in the cytoplasm in the form of rows of ribosomes which surrounded membrane-unbounded, electron-lucent lacunae in a reticular, honey-comb pattern (reticular RER). Although the reticular RER lacked membrane components, its lacunae contained progeny virions, indicating that the rows of ribosomes synthesized the viral proteins and discharged them into the lacunae for the viral assembly. The reticular RER apparently transformed into the familiar lamellar RER during the RER morphogenesis as the lacunae coalesced to form flat cisternae and RER membrane assembled to border the cisternae. These findings indicated that the proliferating RER was the site of not only active protein synthesis but also active membrane biogenesis. The proliferating RER released a large number of membrane vesicles including virion-carrying vesicles into the cytoplasm. These vesicles congregated in the juxtanuclear region, especially around the centrioles, and fused to existing Golgi complexes for enlargement or fused among themselves to form new Golgi complexes. The present study, therefore, indicated that (a) nascent RER was formed by polysomes that arranged themselves in rows of ribosomes without participation of a preexisting membrane framework of endoplasmic reticulum (ER), (b) membrane components of RER were assembled de novo within the structure during the RER morphogenesis, and (c) RER released membrane vesicles that moved to the Golgi apparatus and contributed to the morphogenesis of the Golgi apparatus. Possible causative mechanisms involved in the proliferation of the secretory system in JE virus-infected PC12 cells are discussed.

Animals↗
PubMed1993

Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens.

Isoflavonoid phytoestrogens and lignans in plants are known to be constituents of animal and human food and recently they have been found in human urine and other biological materials. These compounds have received increasing attention because of their interesting biological properties and possible role in human cancer and other diseases. The present study demonstrates that the main mammalian lignan enterolactone (trans-2,3-bis[(3-hydroxyphenyl)methyl]-butyrolactone) and some other diphenols are moderate or weak inhibitors of human estrogen synthetase (aromatase) and that this lignan binds to or near the substrate region of the active site of the P-450 enzyme. The inhibition is competitive with respect to testosterone and androstenedione, and the lignan affinity is 1/75-1/300 that of these natural substrates. It is suggested that the high concentration of lignans in vegetarians, by inhibiting aromatase in peripheral and/or cancer cells and lowering estrogen levels, may play a protective role as antipromotional compounds during growth of estrogen-dependent cancers.

4-Butyrolactone↗
PubMed1993

Acidic regions of cytochrome c1 are essential for ubiquinol-cytochrome c reductase activity in yeast cells lacking the acidic QCR6 protein.

It has been suggested that the two acidic regions around residue 70 and residue 170 in yeast cytochrome c1, a subunit of ubiquinol-cytochrome c reductase (complex III), interact with cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex III, enhances this interaction. In order to determine the roles of the acidic regions of cytochrome c1 more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified cytochrome c1 to complement the respiration deficiency of yeast cells lacking only cytochrome c1 or both cytochrome c1 and the QCR6 protein. The mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) was still functional in the cytochrome c1-deficient strain. Mutant cytochrome c1 with the deletion of the second acidic region (delta 168-179) caused a decrease in the complementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex III. Mutant cytochrome c1 with altered charge distribution in the acidic regions (Asp170Asp171-->Asn170Asn171 or Asp170Asp171-->Asn170Lys171) made the cytochrome c1-deficient cells respiration-competent. On the other hand, mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) or altered charge distribution in the second region (Asp170Asp171-->Asn170Lys171) did not restore the respiration deficiency of the cells lacking not only cytochrome c1 but also the QCR6 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Anti-Trichophyton mentagrophytes activity and percutaneous permeation of butenafine in guinea pigs.

We examined anti-Trichophyton mentagrophytes activity, cutaneous penetration, and skin localization of butenafine, a novel benzylamine antifungal agent. The following results were obtained. (i) In the guinea pig dorsal skin trichophytosis model, butenafine produced complete eradication of fungi from infected sites. Clotrimazole was active when animals were infected with 10(4) or 10(5) cells but was almost inactive when the inoculum size was 10(6) cells. (ii) The MICs of butenafine and clotrimazole against arthrospores of T. mentagrophytes KD-04 were 0.025 and 0.39 microgram/ml, respectively. (iii) When 0.2 ml of a 1% 14C-butenafine solution was applied for 23 h/day for 7 days, high radioactivity corresponding to 250 to 500 micrograms of butenafine per g of skin in the epidermis, including the horny layer, was observed. (iv) Butenafine penetrates through transepidermal and transfollicular routes. The excellent therapeutic efficacy of butenafine on experimental dermatophytosis may be attributed to its low MIC and good penetration and distribution in the horny layer and hair follicles, where fungi reside.

Animals↗
PubMed1993

Quantitative determination of lignans and isoflavonoids in plasma of omnivorous and vegetarian women by isotope dilution gas chromatography-mass spectrometry.

The first quantitative method for the determination of both lignans and isoflavonoid phytoestrogens in plasma is presented. Using ion-exchange chromatography the diphenols are separated into two fractions 1) the biologically "active" fraction containing the free compounds + mono- and disulfates and 2) the biologically "inactive" fraction containing the mono- and diglucuronides and the sulfoglucuronides. After hydrolysis the fractions are further purified by solid phase extraction and ion exchange chromatography. Losses during the complete procedure are corrected for using radioactive estrogen conjugates during the first steps and later by adding deuterated internal standards of all compounds measured (matairesinol, enterodiol, enterolactone, daidzein, O-desmethylangolensin, equol, and genistein). The final determination is carried out by isotope dilution gas chromatography-mass spectrometry in the selected ion monitoring mode (GC/MS/SIM). The diphenols may be measured at concentrations as low as 0.2 to 1.0 nmol/l. Results of plasma analyses of all compounds in 27 pre- and postmenopausal omnivorous and vegetarian women are presented for the first time. The most important findings are that the free+sulfate fraction is low for genistein (3.8% of total), but as much as 21-25% of enterolactone and enterodiol occurs in this fraction. A good correlation between plasma and urine values was found. Total concentrations of individual compounds vary greatly between the subjects (from pmol/l to mumol/l), the vegetarians having higher values, particularly one vegan subject. The highest total enterolactone concentration value exceeded 1 mumol/l. It is concluded that a highly specific method for the assay of 3 lignans and 4 isoflavonoids in plasma has been developed. This method will be useful in future studies of lignan and isoflavonoid metabolism.

Diet, Vegetarian↗
PubMed1992

Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.

Amino Acid Sequence↗
PubMed1992

Cytopathology of PC12 cells infected with Japanese encephalitis virus.

Infection of a clonal rat pheochromocytoma cell line, PC12, with Japanese encephalitis (JE) virus produced successively higher titers of virus in the culture fluid during the 72-h experimental period. In electron microscopical observation, JE virus entered PC12 cells by direct penetration through the plasma membrane at 2 min postinoculation (p.i.) and caused marked cellular hypertrophy and extensive proliferation of the cellular secretory system including rough endoplasmic reticulum (RER) and Golgi complexes starting 24 h p.i. The proliferating RER of the virally infected cells contained progeny virions and characteristic endoplasmic reticulum vesicles in its cisternae, and the proliferating Golgi complexes contained virions in their saccules. These findings indicated that the proliferation of the cellular secretory system occurred in association with viral replication and maturation in the system. Seventy-two hours p.i., the cellular secretory system of infected PC12 cells showed degenerative changes with vesiculation, disorganization, and dispersion of the Golgi complexes and fragmentation, focal cystic dilation, and dissolution of the RER in the same manner as those seen in the secretory system of JE-virus-infected neurons in the mouse brain. Thus, JE-virus-infected PC12 cells seem to be a suitable neurogenic cell line for the study of the pathogenic mechanism of JE virus. At the same time, the virally infected cells seem to offer an interesting cell model for the study of the morphogenesis of the cellular secretory system.

Animals↗
PubMed1992

Dietary phytoestrogens and cancer: in vitro and in vivo studies.

Thirty postmenopausal women (11 omnivores, 10 vegetarians and 9 apparently healthy women with surgically removed breast cancer) were investigated with regard to the association of their urinary excretion of estrogens, lignans and isoflavonoids (all diphenols) with plasma sex hormone binding globulin (SHBG). A statistically significant positive correlation between urinary total diphenol excretion and plasma SHBG was found which remained statistically significant after elimination of the confounding effect of body mass determined by body mass index (BMI). Furthermore we found a statistically significant negative correlation between plasma SHBG and urinary excretion of 16 alpha-hydroxyestrone and estriol which also remained significant after eliminating the effect of BMI. Furthermore we observed that enterolactone (Enl) stimulates the synthesis of SHBG by HepG2 liver cancer cells in culture acting synergistically with estradiol and at physiological concentrations. Enl was rapidly conjugated by the liver cells, mainly to its monosulfate. Several lignans and the isoflavonoids daidzein and equol were found to compete with estradiol for binding to the rat uterine type II estrogen binding site (the s.c. bioflavonoid receptor). It is suggested that lignans and isoflavonoids may affect uptake and metabolism of sex hormones by participating in the regulation of plasma SHBG levels and in this way influence their biological activity and that they may inhibit cancer cell growth like some flavonoids by competing with estradiol for the type II estrogen binding sites.

Animals↗
PubMed1992

Identification in human urine of a natural growth inhibitor for cells derived from solid paediatric tumours.

Partially purified urine of healthy human subjects contains several fractions able to inhibit the proliferation of cultured human neuroblastoma cells. One of the most active fractions was further analysed by gas chromatography-mass spectrometry and shown to contain genistein, a substance formed in the human body from precursors obtained by diet. Synthetic genistein was able to inhibit the proliferation of human neuroblastoma cells with a half-maximal effect at 5-10 mumol l-1 concentrations. Genistein displayed similar potencies in inhibiting the proliferation of cells derived from various other solid pediatric tumours. Our results suggest that genistein is a natural antineoplastic agent present in diet and that it could be useful for the therapy of paediatric tumours.

Cell Division↗
PubMed1992

Induction of activated killer cells from human lymphocytes by medullasin (a serine protease in bone marrow cells).

Medullasin, a serine protease found in human bone marrow cells, has been shown to induce activated killer (AK) cells that lyse both natural killer (NK)-sensitive and -resistant cloned target cells from human lymphocytes. In addition to all the tested malignant cell lines, malignant cells obtained from all patients with acute myelocytic leukaemia, chronic lymphocytic leukaemia and lymphoblastic leukaemia were lysed by AK cells induced by medullasin. Maximum induction was achieved when lymphocytes were incubated at 37 degrees for 60 min in the presence of medullasin (20 micrograms/ml). The cytotoxicity of AK cells induced by medullasin treatment (200 micrograms/ml, 37 degrees for 60 min) was greater than that of lymphokine-activated killer (LAK) cells produced by 500 U/ml of interleukin-2 (IL-2). Cytokines such as IL-2 or interferon (IFN) are not considered to be involved in the medullasin induction of AK cells for the following reasons: (1) neither IL-2 nor IFN activity were detected in the supernatant of lymphocytes treated with medullasin; (2) the supernatant of lymphocytes treated with medullasin failed to induce AK cells; and (3) the presence of antibodies against IL-2 or IFN did not influence the effect of the protease. By employing monoclonal antibodies to the surface antigens of lymphocytes and a panning method using plastic dishes coated with anti-mouse IgG goat Fab', progenitor as well as effector cells were found to be CD16-positive cells.

Cell Line↗
PubMed1991

Rolling-circle replication of the plasmid pKYM isolated from a gram-negative bacterium.

Plasmid pKYM isolated from a Gram-negative bacterium encodes a Rep protein that is essential for plasmid replication. A comparison of Rep protein from pKYM to Rep proteins encoded by other plasmids shows that it has homology to Rep proteins of the pUB110 plasmid family from Gram-positive bacteria. These plasmids replicate by a rolling-circle mechanism in which a tyrosine residue in the Rep protein acts as the acceptor for the 5' end of the single-strand break introduced by the Rep protein. A Tyr----Phe substitution in the pKYM Rep protein abolishes its activity. Strand-specific single-stranded circular plasmid DNA can be recovered from the cells carrying pKYM and thus we propose that the plasmid pKYM replicates by a rolling-circle mechanism.

Adenosine Triphosphatases↗
PubMed1991

The primary structure and elastinolytic activity of medullasin (a serine protease of bone marrow).

The amino acid sequence around the carboxyl terminal of medullasin was determined by digesting the protease with carboxypeptidase Y and measuring the rate of release of amino acids from the carboxyl terminal. By considering the structure of the protease's cDNA, we concluded that His-238 is the C-terminal residue of medullasin. Therefore, medullasin is composed of 238 amino acid residues with Ile as the amino terminal and His as the carboxyl terminal. Medullasin is essentially devoid of elastinolytic activity, because it failed to digest orcein-elastin.

Amino Acid Sequence↗