Usefulness of the 5' region of the cDNA encoding acidic ribosomal phosphoprotein P0 conserved among rats, mice, and humans as a standard probe for gene expression analysis in different tissues and animal species.

Housekeeping genes are often used as internal standards for gene expression analysis. When steady-state transcript levels of 4 typically used housekeeping genes, i.e., beta-actin, glyceraldehyde 3-phosphate dehydrogenase, cyclophilin, and acidic ribosomal phosphoprotein P0 (36B4), were evaluated in various rat tissues, the 36B4 gene seemed to be the most suitable as a standard to compare the expression levels of genes among different tissues. Next, for possible quantitative comparison of the expression level of this gene among different animal species, we compared the nucleotide sequence of the cDNA of 36B4 among rats, mice, and humans. As a result, highly conserved regions showing more than 97.5% identities were observed in the 5' portion of its open reading frame. When samples of synthesized mRNA encoding rat, mouse, and human 36B4 were hybridized with the entire cDNA encoding rat 36B4 as a probe, hybridization signals of mRNAs of mouse and human 36B4 were much weaker than those of mRNA encoding rat 36B4. However, when they were hybridized with an oligonucleotide probe corresponding to the highly conserved regions, they showed similar signal intensities. Thus, these highly conserved regions of the cDNA encoding 36B4 were concluded to be an effective standard for use in gene expression analysis.


Regulation of Calprotectin Expression by IL-1α and TGF-β in Human Gingival Keratinocytes

Calprotectin, a heterodimer of S100A8 and S100A9 with antimicrobial properties, is expressed in gingival keratinocytes and plays an important role in innate immunity. Because calprotectin expression is localized in the spinous cell layer of the gingival epithelium, we hypothesized that the expression of calprotectin in keratinocytes is related to the differentiation stage. The aim of the present study was to investigate the relationship between calprotectin expression and keratinocyte differentiation using some factors that regulated its differentiation. Normal human gingival keratinocytes were isolated from gingival tissues obtained at the extraction of wisdom teeth, and were cultured in serum-free keratinocyte medium supplemented with interleukin-1alpha or calcium, which promote keratinocyte differentiation, and transforming frowth factor-beta (TGF-beta) or retinoic acid, which suppress its differentiation. The expression of S100A8/A9 mRNA and the production of calprotectin in normal human gingival keratinocytes were examined by northern blotting and enzyme-linked immunosorbent assay, respectively. The expression of cytokeratin 14, involucrin and filaggrin (marker proteins of keratinocyte differentiation) was investigated by immunohistochemical staining, and the DNA-binding activity of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor, was examined by electrophoretic mobility shift assay. The expression of S100A8/A9 mRNA and the production of calprotectin were increased by interleukin-1alpha and calcium, but decreased by TGF-beta. RA inhibited the expression of S100A8/A9 and keratinocyte differentiation, which were induced by interleukin-1alpha. C/EBPalpha DNA-binding activity in normal human gingival keratinocytes was enhanced by interleukin-1alpha and calcium, but suppressed by TGF-beta. The present study suggests that calprotectin expression is related to keratinocyte differentiation and that C/EBPalpha is a regulator of calprotectin expression in keratinocytes.


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.


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.


Norepinephrine Stimulates Calprotectin Expression in Human Monocytic Cells

Calprotectin is composed of two proteins, S100A8 and S100A9, which are S100 family members, and is detected in gingival crevicular fluid and gingival tissue with inflammation. The release and production of calprotectin are regulated by lipopolysaccharides of periodontopathic bacteria and cytokines. Emotional or psychological stress, a risk factor of periodontal disease, is transmitted by stress modulators including norepinephrine and cortisol. The aim of the present study was to investigate the effect of stress on calprotectin expression using norepinephrine and cortisol. U-937 cells, a human monocytic cell line, were incubated with norepinephrine in the presence or absence of beta- or alpha-adrenergic receptor antagonists, or with cortisol. The expression of S100A8/S100A9 mRNAs was examined by northern blotting and the amount of calprotectin was measured by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha (CCAAT enhancing binding protein), a transcription factor, was examined by electrophoretic mobility shift assay. Norepinephrine stimulated the expression of S100A8/S100A9 mRNAs via beta-adrenergic receptors in U-937 cells and significantly increased calprotectin production to about 3.6-fold that of the control. However, cortisol had no effect on calprotectin expression at the mRNA and protein levels. Norepinephrine elevated C/EBPalpha DNA binding activity, but cortisol did not increase the activity. Norepinephrine, a stress modulator, stimulated calprotectin expression in human monocytic cells. Calprotectin expression may be regulated by stress in addition to inflammatory factors.


High-turnover osteoporosis is induced by cyclosporin A in rats.

Cyclosporin A (CsA) is used widely as an immunosuppressive agent, but it induces osteoporosis as a prominent side effect. To elucidate the mechanisms involved in CsA-induced osteoporosis, the effects of CsA on bone metabolism were investigated in a rat experimental model. Fifteen-day-old rats were fed a powdered diet containing or lacking CsA for 8-30 days. Analysis was performed by micro-computed tomography (muCT) and light microscopy to examine histomorphometric changes in rat tibiae on days 8, 16, and 30. Plasma parathyroid hormone (PTH) and osteocalcin (OCN) levels were determined by enzyme-linked immunosorbent assay (ELISA) on days 8, 16, and 30. The expression of OCN, osteopontin (OPN), and cathepsin K mRNAs in tibial bone marrow was examined by Northern blot analysis on days 8 and 16. Although no significant differences were observed in tibial length during the experimental periods, or in histomorphometric parameters on day 8, an apparent decrease in bone volume was observed in the CsA-treated group after day 16. Histologic analysis showed that the number of osteoblasts and osteoclasts on the surface of trabecular bone in the CsA-treated group had increased significantly on day 16. Plasma PTH and OCN levels in CsA-treated rats were significantly higher than those in control animals on day 8. Northern blot analysis revealed that the CsA-treated group showed an increase in the expression of OCN, OPN, and cathepsin K mRNAs on day 8 compared with the controls. These findings suggest that bone resorption in CsA-treated rats is induced by high-turnover osteoporosis and that bone remodeling activity may be activated by PTH.


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.


Alpha 2 integrin +807 polymorphism in drug-induced gingival overgrowth

Alpha2 integrin on fibroblasts is reported to play an important role in the induction of drug-induced gingival overgrowth, which is characterized by excessive accumulation of type I collagen in gingival connective tissue. Silent polymorphism 807 T/C within the alpha2 integrin gene is associated with high/low alpha2 integrin expression. The aim of this study was to test the hypothesis that expression of alpha2 integrin 807 T/C polymorphism correlates with drug-induced gingival overgrowth. A case-control study comparing 136 subjects taking calcium channel blockers (72 with vs. 64 without drug-induced gingival overgrowth) demonstrated that the frequency of the +807 C allele was significantly higher in the case group than in the controls (odds ratio, 3.61; 95% confidence interval, 2.14 - 6.10; P < 0.05). The present findings suggest that the alpha2 +807 C allele is one of the genetic risk factors for drug-induced gingival overgrowth.


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.

Journal of Bioenergetics and Biomembranes


Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry

The mitochondrial membrane potential (DeltaPsi(m)) is an important indicator of the energetic state of both the mitochondria and the cells. To develop a sensitive, convenient, and rapid method for the measurement of DeltaPsi(m), we carried out cell fluorescence assays using the Agilent 2100 bioanalyzer system which, unlike the conventional flow cytometry, is based on microfluidic technology employing fluorescence detection with a 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)) fluorescent probe. The use of DiOC(6)(3) in the fluorometer was shown to be feasible for monitoring variations in DeltaPsi(m) in the mitochondria isolated from rat liver and treated with rotenone, succinate, ADP, and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Flow cytometry analysis showed severe reduction of fluorescence intensity in Jurkat cells after treatment with 1.0 and 10 microM FCCP. However, fluorescence microscopy demonstrated obvious accumulation of fluorescence in the mitochondria and induction of diffuse cytoplasmic fluorescence not localized to the mitochondria in these cells. The dose response range of DiOC(6)(3) in the Agilent 2100 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm-2.0 microM. Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 microM DiOC(6)(3) was observed after treatment with 10 microM FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring DeltaPsi(m) in cell assays.


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.


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 tricarbodylate 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.


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.


Twisting of the second transmembrane a-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.


Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing PDGF receptor in the muscle, but does not affect blood glucose levels

Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached approximately 60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.


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.


Synthesis of CdTe Quantum Dots using a Heterogeneous Process at Low Temperature and their Optical and Structural Properties

Applied Physics. A, Materials Science & Processing


Usefulness of microchip electrophoresis for reliable analyses of non-satndard DNA samples and subsequent on-chip enzymatic digestion

The Hitachi SV1100 utilizes capillary electrophoresis on a microchip that is capable of rapidly sizing DNA fragments. Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNA fragments of 100 and 800 bp. The range of DNA sizing for this microchip is between 100 and 800 bp, and accuracy in sizing of a 322 bp DNA fragment of a pUC118 PvuII digest was observed, independent of DNA concentration. Although relatively good quantification of this fragment was observed with a DNA concentration of 1.83 ng.microL(-1), error increased in a dose-dependent manner. Furthermore, the feasibility of sequential analysis with this microchip was shown by the reproducibility of successive electrophoreses of the internal control in one channel. When the pUC118 PvuII digest was treated with endonuclease KpnI on the microchip for 10 min, sequential analysis showed that the 322 bp fragment completely disappeared and two peaks corresponding to the 130 and 192 bp fragments appeared. This analysis was performed within 4 min, and the peaks were estimated as 127 and 183 bp, respectively. These results indicate the potential of on-microchip endonuclease treatment of plasmid DNA with sequential analysis, offering high resolution in a short time.


An assessment of relative transcriptional availability from nonviral vectors

To design better delivery systems that enhance transfection efficiency of nonviral vectors, we need to improve our understanding of the mechanisms governing both the amounts of plasmid delivered to the nucleus and gene expression. What is needed is a measure of transcriptional availability (TA): the average level of gene expression per plasmid delivered to the nucleus over the course of an experiment. We describe a method to measure TA and demonstrate its application. The chloramphenicol acetyltransferase reporter gene was transfected into NIH/3T3 cells using either cationic liposomes (TFL-3; O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl) diethanolamine chloride (DC-6-14), dioleoylphosphatidylethanolamine (DOPE) and cholesterol, molar ratio 1/0.75/0.75) or cationic polymer (PEI; polyethylenimine). The time courses of both nuclear delivery of plasmids and reporter gene expression were measured for 4 h thereafter. For the conditions used, time courses of gene expression and plasmid nuclear delivery for the two vectors were different. To understand the origins of those differences, we applied a simple pharmacokinetic model, used the data to estimate the values of the model parameters, and interpret differences in estimated parameter values. The rate constant of delivery of plasmids into the nucleus for the TFL-3 vector was twice that of the PEI vector, whereas rate constant of elimination of plasmids in the nucleus for the PEI vector was four times that for the TFL-3 vector. The gene expression rate constant for the TFL-3 vector was estimated to be seven times larger than that of the PEI vector for the conditions used. The pharmacokinetically determined average exposure of a nucleus to plasmid was about 17 times larger for the TFL-3 vector, relative to the PEI vector. That greater exposure resulted in increased relative gene expression. Overall, the TA from the TFL-3 vector was about 13 times greater than from the PEI vector. The experimental design combined with the adoption of pharmacokinetic concepts and principles provide a method to measure TA along with detailed insights into the mechanisms governing gene delivery and expression.


Comparison of gene expression profiles between white and brown adipose tissues of rat by microarray analysis

To characterize the energy metabolism in brown adipose tissue (BAT), the differences in gene expression profiles between BAT and white adipose tissue (WAT) were analyzed using a high-density cDNA microarray. RNAs isolated from two adipose tissues were hybridized to an Agilent rat cDNA Microarray that contained about 14,500 cDNA probe sets. The expression levels of 499 cDNA/ESTs were found to be at least 5-fold higher or lower in BAT than in WAT. Consistent with our previous findings, high expression levels of genes encoding uncoupling protein 1, muscle-type carnitine palmitoyltransferase and some other proteins involved in energy metabolism in BAT were found. Most of the genes encoding mitochondrial proteins, such as subunits of ATP synthase, cytochrome c oxidase, and NADH dehydrogenase, were highly expressed, reflecting possible differences in the cellular content of mitochondria between BAT and WAT. However, the expression levels of several genes encoding mitochondrial protein, such as liver mitochondrial aldehyde dehydrogenase and dicarboxylate carrier, were remarkably lower in BAT. These results may give important clues to understand the unique energy metabolism in BAT.


Three-way effect of cyanine dye on the structure and function of mitochondria

Previously, we found that the cationic cyanine dye tri-S-C_4(5) uncoupled mitochondrial oxidative phosphorylation by acting as an inducer of the mitochondrial permeability transition (PT). In the present study, the actions of cyanine dyes such as tri-S-C_4(5) and tri-S-C_7(5) on the mitochondrial structures and functions were further characterized. In the presence of inorganic phosphate (Pi), cyanine dyes were found to accelerate mitochondrial oxygen consumption that was partially sensitive to the PT inhibitor cyclosporin A (CsA). However, not only the CsA-sensitive but also CsA-insensitive acceleration of mitochondrial respiration was induced by cyanine dyes; and both of them were found to be associated with the release of mitochondrial cytochrome c. On the contrary, in the absence of Pi, moderate acceleration of respiration was induced by cyanine dyes, but this respiratory effect was not associated with the iniduction of swelling or the release of mitochondrial cytochrome c. Thus, cyanine dyes were concluded to have 3 different effects on the mitochondrial functions depending on the Pi status.


PCR-select subtraction for characterization of messages differentially expressed in brown compared with white adipose tissue.

To understand the energy metabolism occurring in brown adipose tissue (BAT), we subtracted the messages expressed in white adipose tissue (WAT) from those in BAT. Thereby we succeeded in identifying 37 cDNA clones as being significantly expressed in BAT but not in WAT. Of these, 24 clones were found to code for mitochondrial proteins. Since BAT is well known to have a higher mitochondrial content than WAT, these results would seem to reflect simply the differences in mitochondrial content between BAT and WAT. To examine this possibility, we next measured the amount of mitochondrial DNA (mtDNA) in various rat tissues. As a result, the mtDNA copy number per cell was found to be markedly different among the tissues analyzed, and the highest value of about 5.3x10(4) copies per cell was observed with the rat brain. BAT showed a value similar to that of brain, but this value was only about 3.5-fold higher than that for WAT. Since observed differences in mitochondrial content between BAT and WAT was smaller than those observed with transcript levels of proteins, we conclude that the observed differences in the transcript levels of certain proteins between BAT and WAT reflect the functional differences between BAT and WAT, and do not reflect the differences in mitochondrial content between BAT and WAT.


Silver ion induces a cyclosporine a-insensitive permeability transition in rat liver mitochondria and release of apoptogenic cytochrome C

Various reagents are known to open the mitochondrial permeability pore (PTP) and induce a permeability transition (PT), releasing apoptogenic proteins from the intermembrane space and triggering apoptosis. In this study, we examined the effect of Ag(+), a known cytotoxic sulfhydryl-reactive heavy metal, on isolated rat liver mitochondria. The following results were obtained: (1) Upon addition, Ag(+) instantly induced mitochondrial swelling and acceleration of respiration. (2) Cyclosporine A, a specific inhibitor of classical PT, was ineffective against the effect of Ag(+), indicating that silver ions induced non-classic PT. (3) Sulfhydryl reagents such as reduced glutathione completely inhibited the effects of Ag(+) on the mitochondria. (4) Experimental results using polyethylene glycol indicated that Ag(+) induced opening of a pore in the inner mitochondrial membrane, which could be PTP of another open state or a distinct pore. (5) Electron microscopic analysis of mitochondria treated with Ag(+) showed a novel mitochondrial configuration that was apparently different from that of normal mitochondria or Ca(2+)-treated mitochondria. (6) Ag(+) also induced the release of apoptogenic cytochrome c in a CsA-insensitive but GSH-sensitive manner. These results suggest that Ag(+) promotes a nonclassical permeability increase in the mitochondrial inner membrane that is clearly distinguishable from the classical PT and releases apoptogenic cytochrome c in a classical PT-independent manner.


The gene encoding muscle-type carnitine palmitoyltransferase I: comparison of the 5'-upstream region of human and rodent genes

Muscle-type carnitine palmitoyltransferase I (M-CPTI) is the key enzyme for fatty acid beta-oxidation in heart and skeletal muscles and in adipose tissue. So far, M-CPTI mRNA has been detected in white adipocytes from epididymal fat pads of rats and humans, but not in mouse adipocytes. To characterize the gene expression of M-CPTI in mice, we isolated the genomic DNA encoding mouse M-CPTI and determined its transcription initiation site. As a result, the mouse M-CPTI gene seemed to have multiple initiation sites, as in the case of the rat and human genes. Furthermore, the conserved nucleotide sequence of the response element for peroxisome proliferators was shown to exist in the upstream of the mouse gene as in that of the rat and human genes. From these observations, we suggest that the anomalous expression of M-CPTI in mouse adipocytes reported previously may be regulated by factors other than peroxisome proliferators. Previously, we reported that there were transcripts containing regions of both CK/EK-beta and M-CPTI genes in humans. In this study, we found that such transcripts also exist in rodents and that the amounts of the transcripts containing regions of both of these genes did not depend on the expression level of CK/EK-beta.


Expression profiles of three isoforms of inositol 1,4,5-trisphosphate receptor in brown adipose tissue of the rat.

The thermogenic function of brown adipose tissue (BAT) is known to be mainly regulated by a signal transduction cascade via beta-adrenoceptor. However, recent studies indicated that the alpha-adrenoceptor and its downstream signal transduction cascade, causing elevation of the cellular Ca(2+) level, are also important for the regulation of this function of BAT. In the present study, expression profiles of 3 isoforms of the inositol 1,4,5-trisphosphate (IP(3)) receptor, known as one of the major components of the machinery regulating the intracellular Ca(2+) concentration in the BAT of rats, were investigated by Northern analysis. Of these three IP(3) receptor isoforms, the type 2 one was found to be the most abundant of the three in BAT. Furthermore, when rats were exposed to the cold, under which condition the thermogenic function of BAT is known to be stimulated, the expression levels of types 1 and 2 isoforms of IP(3) receptor were remarkably elevated. The results of Western analysis supported the predominant expression of the type 2 isoform in BAT. However, different from the results of Northern analysis, the expression levels of types 1 and 2 isoforms of IP(3) receptor protein in BAT were not influenced by exposure of the animals to the cold.


Cationic liposome-mediated gene delivery:Biophysical study and mechanism of internalization Arch

To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/-) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/-) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex-cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex-cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.


Lipoplex size determines lipofection efficiency with or without serum.

In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.


Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin

To examine whether valinomycin induces a mitochondrial permeability transition (PT), we investigated its effects on mitochondrial functions under various conditions. The acceleration of mitochondrial respiration and swelling, induced by valinomycin, were found to be insensitive to inhibitors of the ordinary PT, indicating that valinomycin does not induce the ordinary PT. Results of experiments using mitochondria isolated from transgenic mice expressing human bcl-2 also supported this conclusion. Furthermore, evidence for induction of PT pores by valinomycin was not obtained by either electron microscopic analysis of mitochondrial configurations or by measurement of the permeability of the inner mitochondrial membrane by use of polyethylene glycol. However, valinomycin did induce a significant release of cytochrome c, and thus it may be a nice tool to study the processes of mitochondrial cytochrome c release.


Cell-free protein synthesis on a microchip

We evaluated the expression of various proteins by using a cell-free protein synthesis system with a rapid translation system, a microtube, and a microchip technique. Protein expression was successfully achieved with a microfabricated reaction chamber on a plastic chip. Proteins were expressed effectively by use of expression vectors for T7 RNA polymerase (pET) instead of a plasmid for in vitro expression vector, which is recommended by the manufacture of the rapid translation system. Expression of the proteins depended on the type of proteins chosen. Two mammalian proteins were synthesized simultaneously with two expression vectors of pET species. Effective application of the cell-free protein synthesis system will enable miniaturization of protein synthesis and mediation between the transcriptome and proteome.


Quantitative Analysis of Correlation between Nunber of Nuclear Plasmids and Gene Expression Activity after Transfection with Cationic Liposomes

A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed. AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification. Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose. These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.


Effect of Cationic Liposomes in an in Vitro Transcription and Translation System

The effects of cationic liposomes complexed with plasmid DNA on the process of transcription was examined using a recently developed rapid cell free translation system. The findings indicate that the liposome itself inhibited the process when the ratio of DNA/liposome typically used in transfection studies was used.


Requirement of continuous transcription for the synthesis of sufficient amounts of protein by a cell-free rapid translation system

To understand the key processes of cell-free protein synthesis, the synthesis of adipose-type fatty acid binding protein (A-FABP) by a rapid translation system was examined under various conditions. The synthesis of A-FABP was achieved by using an expression vector of A-FABP containing a T7 promoter. However, synthesis of A-FABP was not observed when an RNA fragment corresponding to the open reading frame of A-FABP was used in the reaction instead of the expression vector. Northern analysis revealed that the RNA that was added to the reaction mixture promptly underwent degradation. On the contrary, when the expression vector of A-FABP was employed, a strong RNA signal was observed over the entire incubation period. Thus, a continuous supply of RNA is needed in order to account for its loss via degradation to achieve the synthesis of reasonable amounts of A-FABP. Furthermore, the effect of continuous exchange of reaction mixture was also evaluated by measurement of the amount of synthesized A-FABP.


Close location of the first loop to the third loop of the mitochondrial ADP/ATP carrier deduced from cross-linking catalyzed by copper-o-phenanthroline of the solubilized carrier with Triton X-100


Structural properties of mammalian mitochondrial ADP/ATP carriers: identification of possible amino acids that determine functional differences in its isoforms.

The structural properties of isoforms of the mitochondrial ADP/ATP carrier expressed in mammals were characterized in order to understand their possible functional differences. To accomplish this, the cDNA clone of the bovine type 2 isoform was isolated and characterized. We also extensively explored the rat type 3 isoform, but it was not detected. We next compared the amino acid sequences of the ten ADP/ATP carriers, which are expressed in mammals. As a result, amino acids at positions 45, 147 and 164 were found to show strict isoform dependency regardless of species differences. Thus, they are expected to determine functional differences in the isoforms of the ADP/ATP carrier.


Characterization of loops of the yeast mitochondrial ADP/ATP carrier facing the cytosol by site-directed mutagenesis

To characterize structural features of the regions of the yeast type 2 ADP/ATP carrier (yAAC2) facing the cytosol, we prepared its Cys-less mutant, in which all four cysteine residues were replaced by alanine residues. The Cys-less mutant functioned like native yAAC2, showing that the cysteine residues are not essential. We then prepared cysteine mutants by substituting Ser(21) in the putative N-terminal region, Ala(124) and Ser(222) in the first and second loops facing cytosol, respectively, and Leu(312) in the C-terminal region of the Cys-less mutant for cysteine and examined the labeling of the substituted cysteine residues of the mutants with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) from the cytosol. EMA labeled all the mutants, showing that all regions containing mutated residues faced the cytosolic side. The effects of transport inhibitors on EMA labeling were also examined. From the results, the location and conformation of the region around mutated residues were discussed.

Biochemical and Biophysical Research Communications


Significant effect of the N-terminal region of the mitochondrial ADP/ATP carrier on its efficient expression in yeast mitochondria

The low-level expression of the bovine heart mitochondrial ADP/ATP carrier (bovine type 1 ADP/ATP carrier (bAAC1)) in the yeast mitochondrial membrane is significantly improved by replacement of its N-terminal region with corresponding regions of the yeast type 1 and 2 carriers (yAAC1 and yAAC2) (Hashimoto, M., Shinohara, Y., Majima, E., Hatanaka, T., Yamazaki, N., and Terada, H. (1999) Biochim. Biophys. Acta 1409, 113--124). To understand why the bAAC1 chimeras were highly expressed in yeast mitochondria, we examined the effects of the length and sequence of the N-terminal region extending into the cytosol on the expression of bAAC1 and yAAC2 derivatives in yeast mitochondria. For this, their N-terminal regions were replaced with peptide fragments of various lengths and sequences derived from those of bAAC1, yAAC1, and yAAC2. We found that a specific amino acid sequence and a definite length of the N-terminal region of yAAC2 were required for high expression of bAAC1 and yAAC2 in yeast mitochondria. We also examined the steady-state transcript levels and expression of these derivatives in whole yeast cells. Based on our results, we discuss the role of the N-terminal region in efficient expression of bAAC1 and yAAC2 in yeast mitochondria.


Specific labeling of the bovine heart mitochondrial phosphate carrier with fluorescein 5-isothiocyanate --- Roles of Lys185 and putative adenine nucleotide recognition site in phosphate transport

The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys(185) in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 degrees C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424-432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5'-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys(185). In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.


Expression of NAD(+)-dependent isocitrate dehydrogenase in brown adipose tissue.

cDNA clones significantly expressed in brown adipose tissue (BAT) but not in white adipose tissue (WAT) of rats were isolated by use of a PCR-select cDNA subtraction kit. Of the isolated clones, structural features of two of them, 2-58 and 2-67, were studied in detail. The results indicated that these clones were cDNAs encoding alpha- and beta-subunits of rat NAD(+)-dependent isocitrate dehydrogenase (NAD(+)-ICDH). Previous biochemical study suggested the importance of NAD(+)-ICDH in metabolism in BAT; however, transcript levels of individual subunits of this enzyme in BAT had never been analyzed. In the present study, using these newly isolated cDNAs, we clearly demonstrate that the expression of three subunits of NAD(+)-ICDH was the most remarkable in BAT among the various tissues analyzed.


Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.


Growth condition-dependent synchronized changes in transcript levels of type II hexokinase and type 1 glucose transporter in tumor cells

Transcript levels of hexokinase (HK) isozymes and glucose transporter (GLUT) isoforms in RNA samples of AH130 cells obtained from dish cultures and ascites were evaluated in a quantitative manner. In AH130 cells cultured in dishes, HKI and HKII were expressed at a similar level, but HKIII and HKIV were not. GLUT1 and GLUT3 were also expressed, and messages of these two isoforms represented 27% and 71%, respectively, of the total GLUT messages. A faint signal of GLUT2 was also observed. On the contrary, in cells grown as ascites, the transcript of HKII was dominant, and its level was about 15-fold over that of dish-cultured AH130 cells. Transcript levels of GLUT1 and GLUT3 were 4.5- and 2-fold, respectively, higher than those in dish-cultured cells. Thus, GLUT1 was more susceptible to changes in culture conditions than GLUT3. Based on these results, we concluded that the change in growth conditions caused synchronized changes in the transcript levels of HKII and GLUT1 in AH130 cells. However, such marked changes in the transcript levels of HKII and GLUT1 were not observed when AH130 cells were cultured in dishes under a hypoxic condition, indicating that the observed changes were not solely attributable to the difference in oxygen concentration between the ascites and cell culture conditions. Accordingly, other factors such as growth factors may be responsible for this difference in levels of HKII and GLUT1 between the two growth conditions.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research


Novel expression of equivocal messages containing both regions of choline/ethanolamine kinase and muscle type carnitine palmitoyltransferase I.

For characterization of the detailed gene structure of human muscle type carnitine palmitoyltransferase I (M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts. As a result, we found a cDNA clone containing a nucleotide sequence unexpected from the reported M-CPTI gene structure in the upstream region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase-beta (CK/EK-beta) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the CK/EK-beta gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of CK/EK-beta and M-CPTI. Furthermore, cDNAs containing both exons of these genes were detected by the polymerase chain reaction using the cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these results, the production and organization of these overlapping transcripts are discussed.


Characterization of porin isoforms expressed in tumor cells.

Mitochondria from malignant tumor cell lines show a higher capability for hexokinase binding than those from normal liver. To explore possible differences in hexokinase binding sites of mitochondria between tumor cells and normal liver, we characterized porin isoforms expressed in tumor cells. Cloning experiments on the three porin isoforms, VDAC1, VDAC2 and VDAC3 from malignant tumor cell line AH130 clearly showed that their primary structures were completely identical to those of the corresponding VDACs of normal liver cells. Possible expression of the fourth porin isoform in AH130 cells was excluded by degenerate primer-based RT-PCR. However, the transcript levels of the three VDAC isoforms in AH130 cells were significantly higher than those in normal liver. These results suggest that the high hexokinase-binding capability of malignant tumor cell mitochondria was not due to any structural difference, but due to a quantitative difference in binding sites.


Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator

Mitochondria play an essential role in apoptosis by releasing apoptogenic molecules such as cytochrome c and AIF, and some caspases, which are all regulated by Bcl-2 family proteins. Pro-apoptotic Bax and Bak have been shown to induce cytochrome c release and loss of membrane potential (Deltapsi) leading to AIF release in the isolated mitochondria. We have previously shown that Bax and Bak open the voltage-dependent anion channel (VDAC) allowing cytochrome c to pass through the channel, and Bcl-xL closes the channel. However, it has been reported that it is adenine nucleotide translocator (ANT) with which Bax/Bcl-xL interacts that modulate the channel activity. Here, we investigated the role of ANT and VDAC in the changes of isolated mitochondria triggered by Bax and by chemicals that induce permeability transition (PT). In rat and yeast mitochondria, Bax did not affect the ADP/ATP exchange activity of ANT. VDAC-deficient but not ANT-deficient yeast mitochondria showed resistance to cytochrome c release, Deltapsi loss, and swelling caused by Bax and PT inducers. Bcl-xL showed similar inhibition of all these changes in ANT-deficient and wild type yeast mitochondria. Furthermore, Bax induces cytochrome c release in wild type yeast cells but not VDAC1-deficient yeast cells. These data indicate that VDAC, but not ANT, is essential for apoptotic mitochondrial changes. The data also indicate that Bcl-xL and Bax possess an ability to regulate mitochondrial membrane permeability independently of other Bcl-2 family members.


Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure.

To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of beta-adrenergic receptor (beta-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of beta(1)-AR and C/EBPbeta in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.

Biochimica et Biophysica Acta (BBA) - Bioenergetics


Irreversible extrusion of the first loop facing the matrix of he bovine mitochondrial ADP/ATP carrier by labeling the Cys56 residue with the SH-reagent methyl methanethiosulfonate


Fluctuation of the first loop facing the matrix of the mitochondrial ADP/ATP carrier deducing from intermolecular cross-linking of Cys56 residues by bi-functional dimaleimides


Expression of the bovine heart mitochondrial ADP/ATP carrier in yeast mitochondria: significantly enhanced expression by replacement of the N-terminal region of the bovine carrier by the corresponding regions of the yeast carriers.

To characterize the transport mechanism mediated by the mammalian mitochondrial ADP/ATP carrier (AAC), we tried to express bovine heart mitochondrial AAC (bhAAC) in Saccharomyces cerevisiae. The open reading frame of the bhAAC was introduced into the haploid strain WB-12, in which intrinsic AAC genes were disrupted. Growth of the transformant was very low in glycerol medium, and a little amount of bhAAC was detected in the mitochondrial membrane. For improvement of bhAAC expression in WB-12, we introduced DNA fragments encoding chimeric bhAACs, in which the N-terminal region of the bhAAC extending into the cytosol was replaced by the corresponding regions of the type 1 and type 2 yeast AAC isoforms (yAAC1 and yAAC2). These transformants grew well, and the amounts of the chimeric bhAACs in their mitochondria were as high as that of yAAC2. The carriers expressed showed essentially the same ADP transport activities as that of AAC in bovine heart mitochondria.


Cationic uncouplers of oxidative phosphorylation are inducers of mitochondrial permeability transition


Quantitative determinations of the steady state transcript levels of hexokinase isozymes and glucose transporter isoforms in normal rat tissues and the malignant tumor cell line AH130.

The steady state transcript levels of the four hexokinase (HK) isozymes and four glucose transporter (GLUT) isoforms were determined quantitatively by Northern analysis of RNA samples from rat tissues using synthetic fragments of the RNAs encoding the HK isozymes and GLUT isoforms. Results showed that the levels of HK isozyme transcripts were low in rat tissues, the level of that most highly expressed, the type I isozyme (HKI), in the brain being 0.025% of the total poly(A)+ RNA. A good correlation was found between the reported HK activities and the total amounts of transcripts encoding all HK isozymes in various tissues, showing that the HK activities in tissues can be estimated from the total amount of transcripts encoding HK isozymes. The proposed associated expressions of HK isozymes and GLUT isoforms in particular tissues were confirmed at their transcript levels. The steady state transcript levels of type II HK and the type 1 GLUT isoform in the malignant tumor cell line AH130 were also determined quantitatively.

Biochimica et Biophysica Acta (BBA) - Biomembranes


Dramatic enhancement of the specific expression of the heart-type fatty acid binding protein in rat brown adipose tissue by cold exposure.


Structural features of the gene encoding human muscle type carnitine palmitoyltransferase I.


Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation

We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.


Synthesis and evaluation of the hybrid molecules possessing DNA-cleaving activity

Bioorganic & Medicinal Chemistry Letters


Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I.

With a cDNA probe encoding rat muscle type carnitine palmitoyltransferase I (CPTI), we isolated cDNA and genomic clones encoding the human homologue and deduced the primary structure of human muscle type CPTI. By Northern analysis, we confirmed the dominant expression of this isoform in heart and skeletal muscle.

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression


Inhibitory effect of Mg2+ on the protonophoric activity of palmitic acid

To discriminate whether fatty acids are uncouplers that cause acceleration of State-4 respiration, associated with a decrease in the protonmotive force, or decouplers that increase respiration without associated decrease in the protonmotive force, we examined the effect of palmitate on functions of rat-liver mitochondria under various conditions. We found that palmitate itself increases State-4 respiration, releases oligomycin-inhibited State-3 respiration, inhibits ATP synthesis and ATP<->Pi exchange reaction, and increases H+ permeability in mitochondrial and model bilayer phospholipid membranes. Thus, palmitate is a classical uncoupler of oxidative phosphorylation. However, these effects were inhibited by Mg2+, due to rapid formation of a stable complex between palmitate and Mg2+.

Biochimica et Biophysica Acta (BBA) - Bioenergetics


Stablilities of the fluorescent SH-reagent eosin-5-maleimide and its adducts with sulfhydryl compounds

The stabilities of the SH-reagent eosin-5-maleimide (EMA) and its adducts with the SH-compounds L-cysteine, N-acetyl-L-cysteine and glutathione (reduced form) were studied under various conditions in comparison with those of the adducts of N-ethylmaleimide (NEM). Studies by reversed-phase high performance liquid chromatography and mass spectrometry showed that EMA was less stable than NEM at neutral and moderately alkaline pH values. EMA formed a succinimide-type adduct with SH-compounds, and then underwent further modification by nucleophilic attack of OH- or an amino group. The succinimide-type adducts with acetylcysteine and glutathione were converted to open-type adducts, in which the succinimide ring was cleaved, whereas the adduct with cysteine was modified to a thiazine-type adduct. Kinetic analyses showed that these open-type and thiazine-type adducts were readily formed and were stable at moderately alkaline pH values such as pH 8.0 or 9.0.

Biochimica et Biophysica Acta (BBA) - General Subjects


High expression of a novel carnitine palmitoyltransferase I like protein in rat brown adipose tissue and heart: isolation and characterization of its cDNA clone.


Nucleotide sequence of the 5'-flanking region of the rat type II hexokinase gene

In a previous study, we found that the steady state transcript level of type II hexokinase was specifically and remarkably elevated in rat hepatoma AH130 cells. To determine the molecular mechanism of transcriptional control of the type II hexokinase gene, we examined the nucleotide sequence of its 5'-flanking region and analyzed putative transcription factor binding sites. We also examined the type II hexokinase promoter activity by the chloramphenicol acetyltransferase (CAT) assay.


Importance of loops of mitochondrial ADP/ATP carrier for its transport activity deduced from reactivities of its cysteine residues with the sulfhydryl reagent eosin-5-maleimide


Normal differentiation of rat brown adipocytes in primary cultute judged by their expressions of uncoupling protein and the physiological isoform of glucose transporters

We examined the effects of dexamethasone (DEX) on the expressions of key proteins concerned with energy metabolism in brown adipocytes during their differentiation in primary culture. Transcripts of the uncoupling protein (UCP), lipoprotein lipase (LPL) and CCAAT enhancer binding protein alpha (C/EBP alpha) genes were observed in brown adipocytes cultured in the presence of insulin and thyroid hormones but in the absence of DEX. However, the mRNA level of UCP decreased with the culture period after confluence, and significant mRNA encoding type-1 glucose transporter (GLUT1) was detected in brown adipocytes cultured without DEX, whereas type-4 glucose transporter (GLUT4) was predominantly expressed in mature brown adipocytes in vivo. In contrast, DEX added after confluence consistently elevated the mRNA levels of UCP, LPL and C/EBP alpha, and repressed the level of GLUT1 in a manner synchronized with increase in the level of GLUT4. Therefore, it is concluded that DEX as well as insulin and thyroid hormones is essential for differentiation of brown adipose precursor cells into mature cells that are similar to brown adipocytes in vivo.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research


Steady state transcript levels of the type II hexokinase and type 1 glucose transporter in human tumor cell line


Characterization of cysteine residues of mitochondrial ADP/ATP carrier with the SH-reagents eosin 5-maleimide


Isolation and characterization of cDNA clones and a genomic clone encoding rat mitochondrial adenine nucleotide translocator.

Two cDNA clones encoding rat mitochondrial adenine nucleotide translocator were isolated from libraries constructed from mRNAs of heart and liver. These two clones corresponded to the heart-skeletal muscle type (ANT1) and fibroblast type (ANT2), respectively. A genomic clone encoding rat ANT1 was also isolated and characterized.

Biochimica et Biophysica Acta (BBA) - Biomembranes


Evolution of the type II hexokinase gene by duplication and fusion of the glucokinase gene with conservation of its organization


Identification of two enhancer elements in the gene encoding the type 1 glucose transporter from the mouse which are responsive to serum, growth factor, and oncogenes.

The type 1 glucose transporter (GLUT1) gene encodes an integral membrane glycoprotein responsible for facilitating transfer of glucose across plasma membrane and is rapidly activated by serum, growth factors, and by oncogenic transformation. To elucidate the molecular mechanisms of regulation of GLUT1 gene expression, we isolated and characterized the mouse GLUT1 gene. DNA elements regulating transcription of the gene were analyzed in transient expression assays after transfection of NIH/3T3 cells with a low background chloramphenicol acetyltransferase (CAT) vector system pSVOOCAT. We identified two enhancer elements; the first one is located 2.7 kilobases upstream of the cap site of the gene which contains the homologous sequences with two 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs), a serum response element, a cyclic AMP-responsive element (CRE) and three GC boxes, and the second one is located in the second intron of the gene which contains the homologous sequences with two TREs and one CRE. With the promoter alone the transcription of the gene is activated by src, only slightly activated by ras and is not activated by serum and platelet-derived growth factor. When the gene is accompanied by one of these enhancers, the transcription is activated by all these stimuli.


A cluster of four Sp1 binding sites required for efficient expression of the human insulin receptor gene.

Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.


The hydrophobic cationic cyanine dye inhibits oxidative phosphorylation by inhibiting ADP transport, not by electrophoretic transfer, into mitochondria

シアニン色素tri-S-C4(5)はstate3呼吸及びATP合成を阻害した．しかしミトコンドリア逆転小胞に対しては他の疎水カチオンと同様に作用を示さなかった．さらにtri-S-C4(5)はアデニンヌクレオチド交換担体(ANT)によるADP輸送を阻害した．従って，この色素による酸化的リン酸化の阻害は，色素カチオンが電気泳動的にミトコンドリア内部に輸送されることによるのでなく，ANTの阻害が原因であると結論づけた．

Biochemical and Biophysical Research Communications