Fifteen-day-old rats were fed a powdered diet containing or lacking CsA for days. Analysis was performed by micro-computed tomography muCT and light microscopy to examine histomorphometric changes in rat tibiae on days 8, 16, and 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 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 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.
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. 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.
Summary 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.
A case-control study comparing subjects taking calcium channel blockers 72 with vs. Summary 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. 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.
Summary 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 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.
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 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm Furthermore, significant reduction of fluorescence intensity in the cells stained with 2. These results indicate that the Agilent bioanalyzer is potentially useful for monitoring DeltaPsi m in cell assays.
Summary 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. 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. Summary 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. 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. 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.
The N-terminal half of this region Lys -Phe 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 -Phe 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. On the basis of these results, possible roles of the conformational changes of the LC1 in the transport activity are discussed. 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.
Summary 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. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.
Summary 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.
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.
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. 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.
Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNA fragments of and bp. Although relatively good quantification of this fragment was observed with a DNA concentration of 1. 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 pUC PvuII digest was treated with endonuclease KpnI on the microchip for 10 min, sequential analysis showed that the bp fragment completely disappeared and two peaks corresponding to the and bp fragments appeared. This analysis was performed within 4 min, and the peaks were estimated as and 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. Summary 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 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. 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. 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.
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. Of these, 24 clones were found to code for mitochondrial proteins. 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.
BAT showed a value similar to that of brain, but this value was only about 3. Summary 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. Summary 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. 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.
Summary The thermogenic function of brown adipose tissue BAT is known to be mainly regulated by a signal transduction cascade via beta-adrenoceptor. 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. 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. 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.
Summary 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. Summary 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.
Summary 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. AH cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP lysis. Intranuclear plasmids were amplified by quantitative PCR.
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. 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. Summary 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. Summary 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.
Northern analysis revealed that the RNA that was added to the reaction mixture promptly underwent degradation. Furthermore, the effect of continuous exchange of reaction mixture was also evaluated by measurement of the amount of synthesized A-FABP.
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.
As a result, amino acids at positions 45, and were found to show strict isoform dependency regardless of species differences. 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 and Ser in the first and second loops facing cytosol, respectively, and Leu 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 eosinmaleimide 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. Acta , 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.
We also examined the steady-state transcript levels and expression of these derivatives in whole yeast cells. The phosphate transport inhibitor pyridoxal 5'-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.
Of the isolated clones, structural features of two of them, and , were studied in detail. Summary 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.
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. 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 fold over that of dish-cultured AH cells. However, such marked changes in the transcript levels of HKII and GLUT1 were not observed when AH 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. 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.
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. Summary 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. However, the transcript levels of the three VDAC isoforms in AH 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.
Summary 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.
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. The data also indicate that Bcl-xL and Bax possess an ability to regulate mitochondrial membrane permeability independently of other Bcl-2 family members.
Summary 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.
Possible roles of these proteins in energy metabolism in BAT were discussed. Growth of the transformant was very low in glycerol medium, and a little amount of bhAAC was detected in the mitochondrial membrane. 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. 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.
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. Summary We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH 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. By Northern analysis, we confirmed the dominant expression of this isoform in heart and skeletal muscle.
Summary 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.
Thus, palmitate is a classical uncoupler of oxidative phosphorylation. 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. Summary In a previous study, we found that the steady state transcript level of type II hexokinase was specifically and remarkably elevated in rat hepatoma AH 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. Summary 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.
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.
Summary Two cDNA clones encoding rat mitochondrial adenine nucleotide translocator were isolated from libraries constructed from mRNAs of heart and liver. A genomic clone encoding rat ANT1 was also isolated and characterized.
Summary 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.
We identified two enhancer elements; the first one is located 2. 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.
Summary 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.
Furthermore, regarding evacuees in a school shelter where a daily list is available, a logistic regression analysis is performed to explain evacuees' decisions on whether or not to stay in the shelter on the basis of variables such as gender, age, and family situation. The regression analysis for the first scale reveals that the decreasing trend in the number of evacuees in shelters in Yamada Town has been slow compared to those in other affected coastal municipalities.
The study reveals that progress in the construction of emergency temporary housing is the factor with the greatest impact on an evacuee's decision to leave a shelter. Regarding the second scale, the geographical distribution of the number of shelter residents in Yamada Town is analyzed on the basis of the scale of a district and a small area.
The analysis reveals that regional differences in shelter entry rate reflect social network, topographical features, and developmental process of the settlement. Regarding the third scale, the relationship between distance from the shelter and entry rate of affected households is analyzed by small area using the rosters of Yamada Minami Elementary School and Orikasa Elementary School evacuation shelters.
A significant correlation is found between average road distance from Yamada Minami Elementary School and affected household entry rate, and it is observed that many residents were from areas located within 1 km from the school.
On the other hand, no significant correlation is found between average road distance from Orikasa Elementary school and affected household entry rate. Regarding the Orikasa Elementary School shelter, almost daily entry and exit records could be obtained for the period from April 9, to August 3, Using this record, age and family composition of withdrawers during this period could be identified.
A logistic regression analysis was performed with gender, age group, marital status, and family type as explanatory variables. Reverso for Windows It's free Download our free app. Join Reverso, it's free and fast!
Register Login. These examples may contain rude words based on your search. These examples may contain colloquial words based on your search. See examples translated by mogul Noun 6 examples with alignment.
See examples translated by bump 16 examples with alignment. See examples translated by hump 7 examples with alignment. See examples translated by Cobb Noun 91 examples with alignment. See examples containing Cob 8 examples with alignment. Hiedayama Course 1 of non compacted snow coverage was full mogul slope, and in the center there was a large mogul lane.
And then a few weeks later, we found a bump. Look: that huge hump in the middle already have girls in school. Miss Lisa : Is the wire diameter variable?
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