Biomedical subjects

Hiroshi Terada

Publications and source records attributed to Hiroshi Terada.

At least 19 recordsLinked to original sources

PubMed2006

The structure of the second cytosolic loop of the yeast mitochondrial ADP/ATP carrier AAC2 is dependent on the conformational state.

To detect structural changes in the second cytosolic loop of the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae AAC2, we prepared 20 single cysteine mutants by replacing each amino acid in the S213 to L232 region. All single cysteine mutants were fully functional, because they could restore growth on glycerol of a yeast strain lacking functional ADP/ATP carriers. First, these single-Cys mutants were treated with carboxyatractyloside to lock the carrier in the cytosolic state or with bongkrekic acid to generate the matrix state, and then with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) to probe accessibility. The amino acid residues S213C, L214C, F231C and L232C were not labeled, indicating that these 4 residues must have been buried in the membrane, whereas the region between residues K215 and S230 is accessible to labeling and must, therefore, have protruded into the aqueous phase. Residue L218C showed strong resistance against EMA labeling regardless of the state of the carrier, but the reason for such behavior is unclear. On the contrary, the labeling of the residues between F227C and S230C was strongly dependent on the state of the carrier. Thus, the C-terminal region of the second cytosolic loop in AAC2 changes its environment when the carrier cycles between the matrix and cytosolic state.

Amino Acid Substitution↗

PubMed2006

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin.

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.

Animals↗

PubMed2006

VDAC1, having a shorter N-terminus than VDAC2 but showing the same migration in an SDS-polyacrylamide gel, is the predominant form expressed in mitochondria of various tissues.

The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.

Amino Acid Sequence↗

PubMed2006

Roles of adjoining Asp and Cys residues of first matrix-facing loop in transport activity of yeast and bovine mitochondrial ADP/ATP carriers.

The mitochondrial ADP/ATP carrier (AAC) transports substrate by interconversion of its conformation between m- and c-states. The 1st loop facing the matrix (LM1) is extruded into the matrix in the m-state and is suggested to intrude into the mitochondrial membrane on conversion to the c-state conformation [Hashimoto, M., Majima, E., Goto, S., Shinohara, Y., and Terada, H. (1999) Biochemistry 38, 1050-1056]. To elucidate the mechanism of the translocation of LM1, we examined the effects of site-directed mutagenesis of two adjoining residues, Cys56 and Asp55 in the bovine type 1 AAC and Cys73 and Asp72 in the yeast type 2 AAC, on the substrate transport activity. We found that (i) replacement of the Cys by bulky and hydrophilic residues was unfavorable for efficient transport activity, (ii) the carboxyl groups of the Asp residues of the bovine and yeast AACs were essential and strictly position-specific, and (iii) hence, the mutation to Glu showed transport activity comparable to that of the native AACs. Based on these results, we discussed the functional role of LM1 in the transport activity of AAC.

Adenosine Diphosphate↗

PubMed2006

Divergence paresis without positional headache: an unusual presentation of cerebrospinal fluid hypovolemia after spinal anesthesia.

We report a rare complication of spinal anesthesia-divergence paresis-which is characterized by an acquired horizontal diplopia at distance without evidence for abducens palsy. A 64-yr-old man underwent prostatectomy under spinal anesthesia with 2.5 mL of dibucaine hydrochloride 0.3% injected through a 20-gauge cutting-tip spinal needle. Seventeen days after the operation, the patient noticed horizontal diplopia for distant objects. Although cranial magnetic resonance imaging demonstrated diffuse pachymeningeal gadolinium enhancement and subdural effusion, characteristic findings of cerebrospinal fluid hypovolemia, the patient had no positional headache. Gadolinium-enhanced magnetic resonance imaging may be useful when a patient develops neurologic symptoms after dural puncture.

Anesthesia, Spinal↗

PubMed2005

Inherent potential for production of tumor necrosis factor-alpha by human intestinal macrophages.

BACKGROUND AND AIMS: Tumor necrosis factor (TNF) production by the macrophages in intestines appears to play a critical role in the pathogenesis of Crohn's disease (CD). However, it is reported that resident intestinal macrophages (both colonic and small-bowel) do not produce TNF after lipopolysaccharide (LPS) stimulation. It has not yet been proven whether or not intestinal macrophages have an inherent potential to produce TNF. The purpose of this study is to answer this question. MATERIALS AND METHODS: Colonic macrophages were isolated from lamina propria of human large intestine and stimulated with a variety of substances: LPS, a lipid A derivative (ONO-4007), killed Streptococcus bacterial body (OK-432), phorbol 12-myristate 13-acetate, and lectins (pokeweed mitogen and Sarcophaga lectin). RESULTS: Colonic macrophages were phenotypically negative for CD14 and positive for CD68 and produced very little TNF in response to LPS, as reported previously. Of the substances tested, only Sarcophaga lectin, which is a defense protein of fleshflies (Sarcophaga peregrina), induced TNF production by the intestinal macrophages. In addition, when the colonic macrophages were cultured on immunoglobulin-A-coated dishes, their characteristic response to LPS was altered, and they produced TNF at a level 6.6 times higher than when on collagen-coated dishes. CONCLUSION: Colonic macrophages have an inherent ability to produce TNF. Activation of colonic macrophages by unknown substances may contribute to the induction of TNF production, which causes the intestinal inflammation of CD.

Antigens, CD↗

PubMed2005

No high-mass protostars in the silhouette young stellar object M17-SO1.

The birth of very massive stars is not well understood, in contrast to the formation process of low-mass stars like our Sun. It is not even clear that massive stars can form as single entities; rather, they might form through the mergers of smaller ones born in tight groups. The recent claim of the discovery of a massive protostar in M17 (a nearby giant ionized region) forming through the same mechanism as low-mass stars has therefore generated considerable interest. Here we show that this protostar has an intermediate mass of only 2.5 to 8 solar masses (M(o), contrary to the earlier claim of 20M(o) (ref. 8). The surrounding circumstellar envelope contains only 0.09M(o) and a much more extended local molecular cloud has 4-9M(o).

Journal Article↗

PubMed2005

Essential role of citrate export from mitochondria at early differentiation stage of 3T3-L1 cells for their effective differentiation into fat cells, as revealed by studies using specific inhibitors of mitochondrial di- and tricarboxylate carriers.

1,2,3-Benzenetricarboxylate (BTA) and n-butylmalonate (BM), specific inhibitors of the mitochondrial tricarboxylate and dicarboxylate carrier, respectively, have been used to study the contribution of citrate export from mitochondria to the accumulation of fat in 3T3-L1 cells. Continuous treatment of the cells with BTA or BM for 5 days after the induction of differentiation caused a significant reduction in fat accumulation in the cells in an inhibitor concentration-dependent manner. These inhibitory effects of BTA and BM were not due to their side effects on DNA replication, since similar inhibition of fat accumulation was not observed with ordinary inhibitors of DNA replication. A similar reduction in fat accumulation was also observed when the cells were treated with BTA or BM for only 2 days just after induction of differentiation. However, interestingly, treatment of the cells with an inhibitor starting 2 days after the induction did not result in reduced fat accumulation. Furthermore, Northern analysis clearly indicated that transcript levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and adipose-type fatty acid binding protein (A-FABP) were well correlated with the levels of fat accumulation. These results clearly indicate the essential role of citrate export from the mitochondrial matrix to the cytosol at the early differentiation stage of 3T3-L1 cells for their effective differentiation into fat cells.

3T3 Cells↗

PubMed2005

Cysteine labeling studies detect conformational changes in region 106-132 of the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae.

To know the structural and functional features of the cytosolic-facing first loop (LC1) including its surrounding region of the mitochondrial ADP/ATP carrier (AAC), we prepared 27 mutants, in which each amino acid residue between residues 106 and 132 of the yeast type 2 AAC (yAAC2) was replaced by a cysteine residue. For mutant preparation, we used a Cys-less AAC mutant, in which all four intrinsic cysteine residues were substituted with alanine residues, as a template [Hatanaka, T., Kihira, Y., Shinohara, Y., Majima, E., and Terada, H. (2001) Biochem. Biophys. Res. Commun. 286, 936-942]. From the labeling intensities of the membrane-impermeable SH-reagent eosin-5-maleimide (EMA), sequence Lys(108)-Phe(127) was suggested to constitute the LC1. The N-terminal half of this region (Lys(108)-Phe(115)) was suggested to change its location from the cytosol to a region close to the membrane on conversion from the c-state to the m-state in association with disruption or unwinding of its alpha-helical structure, whereas the C-terminal half region (Gly(116)-Phe(127)) was considered to extrude essentially into the cytosol, while keeping its alpha-helical structure. Hence, the conformation of m-state LC1 is greatly different from that of c-state LC1. Possibly the LC1 changes its location between the membranous region and the cytosol during ADP/ATP transport. Lys(108) in the LC1 of the yAAC2 was found to be associated with binding of the transport substrates, and its -NH(3)(+) moiety, to be of importance for the transport function. On the basis of these results, possible roles of the conformational changes of the LC1 in the transport activity are discussed.

Animals↗

PubMed2005

Two critical factors affecting the release of mitochondrial cytochrome C as revealed by studies using N,N'-dicyclohexylcarbodiimide as an atypical inducer of permeability transition.

N,N'-dicyclohexylcarbodiimide (DCCD) was earlier reported to have stimulatory effects on mitochondrial respiration and to induce mitochondrial swelling, when it was added to mitochondrial suspensions. These data seem to imply that DCCD caused the mitochondrial permeability transition (PT), but this possibility had never been investigated. In the present study, effects of DCCD on the mitochondrial structure and function were studied in detail. DCCD was found to induce mitochondrial PT in a cyclosporine A-insensitive manner. Electron microscopic analysis also supported the induction of the mitochondrial PT by DCCD. However, different from many other PT inducers, DCCD failed to cause massive release of mitochondrial cytochrome c. To understand the relationship between the induction of mitochondrial PT and the release of mitochondrial cytochrome c, we compared the actions of DCCD on mitochondrial structure and function with those of Ca2+, known as an ordinary PT inducer. As a result, two parameters considered to be critical for controlling the release of mitochondrial cytochrome c on the induction of PT were mitochondrial volume and the velocity of mitochondrial oxygen consumption.

Animals↗

PubMed2005

Molecular basis of morphological changes in mitochondrial membrane accompanying induction of permeability transition, as revealed by immuno-electron microscopy.

The mitochondrial inner membrane typically shows a condensed structure when examined by electron microscopy. However, this typical structure is known to disappear upon induction of the mitochondrial permeability transition (PT). This change in the appearance of the mitochondrial membrane structure that accompanies the induction of PT is thought to reflect changes in the permeability of inner mitochondrial membrane; however, its molecular basis has remained uncertain. In the present study, changes in membrane status were examined by immuno-electron microscopy using antibodies against the voltage-dependent anion channel (VDAC), beta-subunit of F1-ATPase (F1beta), and cytochrome c (cyt. c). In control mitochondria, antibody against VDAC was observed at the rim of the mitochondria, whereas antibodies against F1beta and cytochrome c bound these molecules inside of the mitochondria. However, in PT-induced mitochondria, all three antibodies were observed at the mitochondrial rim. These results strongly suggest that the inner mitochondrial membrane is shoved to the rim region of mitochondria upon induction of mitochondrial PT.

Animals↗

PubMed2005

Oral clonidine advances spermatogenesis in oligozoospermic patients with spermatogenetic maturation arrest.

BACKGROUND: Growth hormone (GH) plays an important role in the pubertal development of males and females, and influences the male reproductive function after puberty. The purpose of this study was to investigate the cause of GH deficiency in infertile men with spermatogenetic maturation arrest using the clonidine loading test (GH stimulation test). Based on the results of the loading test, we tried to con fi rm whether clonidine might be usable as a male infertility remedy. METHODS: We studied 75 infertile men (55 oligozoospermic and 20 azoospermic men) and 25 normal fertile volunteers. We used the oral clonidine (0.15 mg/m2) loading test to evaluate the GH secretory reserve. Histological diagnoses were performed using testicular biopsy and testicular sperm extraction specimens. RESULTS: The most important finding was that the clonidine loading test caused a good response in 41 out of 55 (74.6%) patients with oligozoospermia. We treated these 41 good-response patients with oral clonidine, and the sperm count improved in 34 (82.9%) cases, increasing to 12-79 million/mL. Spontaneous pregnancy was subsequently achieved by 10 (24.4%) couples. CONCLUSIONS: These findings also suggest that GH is necessary for spermatogenesis, at least for the process of spermatozoa maturation. Oral clonidine therapy improved the sperm count in patients who showed an excessive response to the clonidine loading test.

Administration, Oral↗

PubMed2004

Detection of a deep 3-microm absorption feature in the spectrum of Amalthea (JV).

Near-infrared spectra of Jupiter's small inner satellites Amalthea and Thebe are similar to those of D-type asteroids in the 0.8- to 2.5-micrometer wavelength range. A deep absorption feature is detected at 3 micrometers in the spectra of the trailing side of Amalthea, which is similar to that of the non-ice components of Callisto and can be attributed to hydrous minerals. These surface materials cannot be explained if the satellite formed at its present orbit by accreting from a circumjovian nebula. Amalthea and Thebe may be the remnants of Jupiter's inflowing building blocks that formed in the outer part or outside of the circumjovian nebula.

Ice↗

PubMed2004

Twisting of the second transmembrane alpha-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states.

To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis. Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA). EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively. When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel. Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix. In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier. These residues correspond to those modified with EMA in the yeast carrier in the c-state. Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation. On the basis of the results, the roles of TM2 in the transport function of AAC were discussed.

Amino Acid Sequence↗

PubMed2004

Efficient intracellular delivery of rifampicin to alveolar macrophages using rifampicin-loaded PLGA microspheres: effects of molecular weight and composition of PLGA on release of rifampicin.

Monodispersed PLGA microspheres containing rifampicin (RFP) have been prepared by solvent evaporation method using a Shirasu porous glass (SPG) membrane. The microspheres were spherical and their average diameter was about 2 microm. The loading efficiency of rifampicin was dependent on the molecular weight of PLGA. The higher loading efficiency was obtained by the usage of PLGA with the lower molecular weight, which may be caused by the interaction of the amino groups of rifampicin with the terminal carboxyl groups of PLGA. PLGA with the monomer compositions of 50/50 and 75/25, of lactic acid/glycolic acid, were used in this study. From rifampicin-loaded PLGA microspheres formulated using PLGA with the molecular weight of 20,000, rifampicin was released with almost constant rate for 20 days after the lag phase was observed for the initial 7 days at pH 7.4. On the other hand, from rifampicin-loaded PLGA microspheres formulated using PLGA with the molecular weight of 5000 or 10,000, almost 90% of rifampicin-loaded in the microspheres was released in the initial 10 days. Highly effective delivery of rifampicin to alveolar macrophages was observed by the usage of rifampicin-loaded PLGA microspheres. Almost 19 times higher concentration of rifampicin was found to be incorporated in alveolar macrophages when rifampicin-loaded PLGA microspheres were added to the cell culture medium than when rifampicin solution was added.

Animals↗

PubMed2004

Multiple effects of DiS-C3(5) on mitochondrial structure and function.

3,3'-Dipropyl-2,2'-thiadicarbocyanine iodide [DiS-C(3)(5)], often used as a tracer dye to assess the mitochondrial membrane potential, was investigated in detail regarding its effects on the structure and function of isolated mitochondria. As reported previously, DiS-C(3)(5) had an inhibitory effect on NADH-driven mitochondrial electron transfer. On the contrary, in the presence of inorganic phosphate, DiS-C(3)(5) showed dose-dependent biphasic effects on mitochondria energized by succinate. At higher concentrations, such as 50 micro m, DiS-C(3)(5) accelerated mitochondrial oxygen consumption. Measurements of the permeability of DiS-C(3)(5)-treated mitochondrial membranes to poly(ethylene glycol) and analysis of mitochondrial configuration by transmission electron microscopy revealed that the accelerating effect of DiS-C(3)(5) on mitochondrial oxygen consumption reflects the induction of the mitochondrial permeability transition (PT). When the mitochondrial PT was induced by DiS-C(3)(5), release of mitochondrial cytochrome c was observed, as in the case of the PT induced by Ca(2+). On the contrary, at a low concentration such as 5 micro m, DiS-C(3)(5) showed an inhibitory effect on the latent oxygen consumption by mitochondria. This effect was shown to reflect inhibition of the PT induced by a low concentration of Ca(2+). Furthermore, in the absence of inorganic phosphate, DiS-C(3)(5) caused mitochondrial swelling. Under this condition, DiS-C(3)(5) caused changes in the membrane status of the mitochondria, but did not induce a release of mitochondrial cytochrome c.

Animals↗

PubMed2004

Identification of possible protein machinery involved in the thermogenic function of brown adipose tissue.

Brown adipose tissue (BAT) is believed to function by dissipating excess energy in mammals. It is very important to understand the energy metabolism held in BAT since disorder of its energy-dissipating function may cause obesity or lifestyle-related diseases such as hypertension and diabetes. This function in BAT is mainly attributable to uncoupling protein (UCP), specifically expressed in its mitochondria. This protein consumes excess energy as heat by dissipating the H+ gradient across the inner mitochondrial membrane that is utilized as a driving force for ATP synthesis. In this review article, in addition to providing a brief introduction to the functional properties of BAT and UCP, we also describe and discuss properties of cultured brown adipocytes and the results of our exploratory studies on protein components involved in the energy-dissipating function in BAT.

Adipose Tissue, Brown↗

PubMed2003

Direct radical scavenging by the bisbenzylisoquinoline alkaloid cepharanthine.

Cepharanthine (Ceph) is known as a potent antiperoxidative agent. Recently, we characterized the antiperoxidative effects of Ceph [Biochim. Biophys. Acta 1426 (1999) 133]. However, it was not clear whether the antiperoxidative effect is really due to its direct radical scavenging activity. Therefore, we studied the interaction of Ceph with the hydroxyl radical (*OH) by the electron paramagnetic resonance (EPR) technique. Results showed that Ceph actually scavenged *OH derived by the Fenton reaction. We also found that Ceph radicals were generated on interaction of Ceph with *OH in neutral aqueous solution, but not in acidic solution, consistent with the pH-dependent anti-lipid peroxidation activity of Ceph. Hence, we concluded that anti-lipid peroxidation by Ceph is due to its direct radical scavenging activity.

Alkaloids↗