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

AbstractTo characterize energy metabolism in rat brown adipose tissue (BAT), we carried out differential screening of a cDNA library of BAT with a cDNA probe of white adipose tissue (WAT) and isolated one cDNA clone. It contained a single open reading frame of 2,316 bases which encodes a protein of 88.2 kDa. The predicted amino acid sequence showed the highest homology (62.6%) with that of rat carnitine palmitoyltransferase I (CPTI). The transcript corresponding to this cDNA was found to be abundantly expressed in BAT and heart. Therefore, the isolated clone is concluded to encode a CPTI like protein expressed in BAT and heart

ASSOCIATION OF CHANGES IN SPATIOTEMPORAL VARIABLES AT EACH STEP WITH 100-M SPRINT PERFORMANCE IN PREADOLESCENT SPRINTERS

The purpose of this study was to investigate the association of acceleration and changes in spatiotemporal variables at each step with 100-m sprint performance in preadolescent sprinters. Twenty-six boys performed 100-m sprints, and their spatiotemporal variables were measured at each step. Acceleration was negatively correlated with the 100-m sprint time from the 1st to 21st step. The rates of change in step frequency were positively correlated with acceleration at the 2nd and 3rd step. Posterior to 3rd step, rates of change in step length were positively correlated with acceleration. The results suggest that the acceleration caused by increase in step frequency and step length up to reaching to the maximal sprint velocity is effective for improving the 100-m sprint time

DIFFERENCE IN ACCELERATION PATTERNS IN TWO START TECHNIQUES: CROUCH AND STANDING STARTS

The purpose of this study was to investigate the difference in the acceleration patterns between the crouch and standing starts. Ten male sprinters performed two maximal effort 60-m sprints from each of two start techniques. Step-to-step spatiotemporal variables and ground reaction forces over the 50-m distance were measured using 54 force platforms. The current results showed that, when compared variables at each step, the crouch start showed shorter block clearing time, higher running speed through higher step frequency during the second half of the acceleration phase, and more horizontally oriented ground reaction force than standing start. These findings suggest that change in start techniques may alter the acceleration pattern through changes in block clearance time, step frequency and force application technique

Effects of cold exposure on metabolites in brown adipose tissue of rats

Brown adipose tissue (BAT) plays an important role in regulation of energy expenditure while adapting to a cold environment. BAT thermogenesis depends on uncoupling protein 1 (UCP1), which is expressed in the inner mitochondrial membranes of BAT. Gene expression profiles induced by cold exposure in BAT have been studied, but the metabolomic biological pathway that contributes to the activation of thermogenesis in BAT remains unclear. In this study, we comprehensively compared the relative levels of metabolites between the BAT of rats kept at room temperature (22 °C) and of those exposed to a cold temperature (4 °C) for 48 h using capillary electrophoresis (CE) time-of-flight mass spectrometry (TOFMS) and liquid chromatography (LC)-TOFMS. We identified 218 metabolites (137 cations and 81 anions) by CE-TOFMS and detected 81 metabolites (47 positive and 34 negative) by LC-TOFMS in BAT. We found that cold exposure highly influenced the BAT metabolome. We showed that the cold environment lead to lower levels of glycolysis and gluconeogenesis intermediates and higher levels of the tricarboxylic acid (TCA) cycle metabolites, fatty acids, and acyl-carnitine metabolites than control conditions in the BAT of rats. These results indicate that glycolysis and β-oxidation of fatty acids in BAT are positive biological pathways that contribute to the activation of thermogenesis by cold exposure, thereby facilitating the generation of heat by UCP1. These data provide useful information for understanding the basal metabolic functions of BAT thermogenesis in rats in response to cold exposure

STEP-TO-STEP ANALYSIS OF ANTEROPOSTERIOR GROUND REACTION FORCE DURING 110 M HURDLE

The purpose of this study was to examine acceleration and deceleration profiles throughout the hurdle sprint until the 5th hurdle in terms of step-to-step ground reaction force. Four male collegiate hurdlers (performance range: 13.73–14.27 s) performed two maximal effort 60 m hurdle sprint. Ground reaction forces from the start to the 50 m mark was measured using 54 force platforms. The braking and propulsive impulse, the running speed and the amount of change of the speed at each step were calculated. The current results demonstrate that the force application profiles were different among four steps in one interval of a hurdle sprint, indicating that the role of each step in one interval is different. Moreover, the roles of steps in each interval likely does not change across the four intervals

Detection of miRNA in Cell Cultures by Using Microchip Electrophoresis with a Fluorescence-Labeled Riboprobe

The analysis of a microRNA (miRNA), miR-222 isolated from the PC12 cell line, was performed by use of the ribonuclease (RNase) protection assay, cyanine 5 (Cy5)-labeled miR-222 riboprobe, and a Hitachi SV1210 microchip electrophoresis system, which can be used to evaluate the integrity of total RNA. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary-strand RNA. More highly sensitive detection of miRNA by microchip electrophoresis than by conventional method using fluorescence-labeled riboprobe could be obtained in 180 s. An obvious increase in miR-222 expression induced by nerve growth factor in PC12 cells could be observed. These results clearly indicate the potential of microchip electrophoresis for the analysis of miRNA using RNase protection assay with a fluorescence-labeled riboprobe

Biomechanical Comparison of Posterior Fixation Using Spinal Instrumentation and Conventional Posterior Plate Fixation in Unstable Vertical Sacral Fracture

Vertical sacral fracture is one of the most difficult fractures to treat. Posterior fixation using spinal dual rods is a novel method for treating this fracture, but its biomechanical strength has not yet been reported. The aim of this study was to evaluate the biomechanical strength produced by posterior fixation using spinal instrumentation. Sacral fractures were created in eight pelvic bone models and classified into a posterior plate fixation group [P group, n＝4] and a spinal instrumentation group [R group, n＝4]. The biomechanical strength was tested by pushing down on the S1 vertebra from the top. The mean maximum loads were 1,057.4 N and 1,489.4 N in the P and R groups, respectively (p＝0.014). The loads applied to the construct at displacements of 5mm and 7.5mm from the start of the universal testing machine loading were also significantly higher in the R group. The mean stiffness levels in the P and R groups were 88.3N/mm and 119.6N/mm, respectively (p＝0.014). Posterior fixation using spinal instrumentation is biomechanically stronger than conventional posterior plate fixation. This procedure may be the optimal method for treating unstable sacral fracture fixation

Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate “revertants” viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria

Tumor Cell Detection among Leukocytes by Microchip

Background: Accurate detection and analysis of circulating tumor cells plays an important role in the diagnosis and treatment of metastatic cancer treatment. Methods and Findings: A cell microarray chip was used to detect spiked carcinoma cells among leukocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene; and the formation of monolayers of leukocytes in the microchambers was observed. Cultured human T lymphoblastoid leukemia (CCRF-CEM) cells were used to examine the potential of the cell microarray chip for the detection of spiked carcinoma cells. A T lymphoblastoid leukemia suspension was dispersed on the chip surface, followed by 15 min standing to allow the leukocytes to settle down into the microchambers. Approximately 29 leukocytes were found in each microchamber when about 600,000 leukocytes in total were dispersed onto a cell microarray chip. Similarly, when leukocytes isolated from human whole blood were used, approximately 89 leukocytes entered each microchamber when about 1,800,000 leukocytes in total were placed onto the cell microarray chip. After washing the chip surface, PE-labeled anti-cytokeratin monoclonal antibody and APC-labeled anti-CD326 (EpCAM) monoclonal antibody solution were dispersed onto the chip surface and allowed to react for 15 min; and then a microarray scanner was employed to detect any fluorescence-positive cells within 20 min. In the experiments using spiked carcinoma cells (NCI-H1650, 0.01 to 0.0001%), accurate detection of carcinoma cells was achieved with PE-labeled anti-cytokeratin monoclonal antibody. Furthermore, verification of carcinoma cells in the microchambers was performed by double staining with the above monoclonal antibodies. Conclusion: The potential application of the cell microarray chip for the detection of CTCs was shown, thus demonstrating accurate detection by double staining for cytokeratin and EpCAM at the single carcinoma cell level

Insect Adenine Nucleotide Translocases

Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4