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

A Seat Belt Injury Causing a Large Breast Hematoma: A Case Report

Seat belts with shoulder restraints have decreased the frequency of life-threatening severe chest trauma caused by car accidents. However, the introduction of seat belt legislation has led to an increase in a specific pattern of blunt trauma known as seat belt syndrome, which includes rib, clavicle, spine, and sternum fractures, as well as rupture of hollow pelvic and abdominal viscera, mesenteric tears, and major vessel injuries. The shoulder restraint part of the three-point seat belt commonly rests near or over the female and male breast. A 54-year-old female presented to our emergency department complaining of swelling and pain in her left breast immediately after a traffic accident. The patient had used a seat belt with a shoulder restraint. Bruising was noted along her chest where there had been seat belt contact. Her breast hematoma was most likely caused by breast tissue compression between her rib and the seat belt. Contrast-enhanced computed tomography demonstrated a sizable breast hematoma with active arterial contrast material extravasation, as well as multiple left rib fractures. The patient was conservatively treated with analgesic and anti-inflammatory drugs. Complete resolution was achieved, and her breast returned to its normal appearance. Although endovascular treatment and surgical hemostasis have been proposed for the treatment of breast injuries with active bleeding, conservative treatment such as compression hemostasis may be feasible

Spatial variability in recruitment of benthos near drilling sites in the Iheya North hydrothermal field in the Okinawa Trough

Due to increasing anthropogenic impacts on deep-sea hydrothermal vent ecosystems, it is essential to understand population structure and maintenance through larval recruitment and recovery of vent faunas after disturbances. In this study, we quantified vent animal recruitment in the Okinawa Trough, in the western Pacific Ocean. This is the first study to investigate recruitment patterns at a man-made hydrothermal vent. Colonization plates were deployed at three sites. Site 1 manifested new hydrothermal shimmering with small chimneys, white bacterial mats, and some alvinocaridid shrimp that arrived after drilling. Site 2 showed no evidence of newly arrived foundation species after drilling, and Site 3 had pre-existing animal communities in the vicinity of the new vent. Twenty-two months after deployment, colonization plates were retrieved and recruited animals were inventoried. Species composition and abundance differed among sites, but relatively high similarity in species composition was observed at Sites 1 and 3, though not at Site 2. Newly established communities on the plates at Sites 1 and 2 (no pre-existing fauna) showed lower species richness and abundance than at Site 3. Differences in abundance and size-frequency distributions of major recruits on the plates (i.e. Lepetodrilus mix, Bathymodiolus spp.) suggest the importance of reproductive and early life-history characteristics in spatial variability of recruitment. Lepetodrilus mix populations established on the plates at Site 1 showed high genetic connectivity. These results illustrate the importance of localized recruitment, which may have a significant impact on sustainability of vent faunal populations, despite the existence of regional metapopulations

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

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier

Bongkrekic acid (BKA), isolated from Burkholderia cocovenenans, is known to specifically inhibit the mitochondrial ADP/ATP carrier. However, the manner of its interaction with the carrier remains elusive. In the present study, we tested the inhibitory effects of 17 bongkrekic acid analogues, derived from the intermediates obtained during its total synthesis, on the mitochondrial ATP/ATP carrier. Rough screening of these chemicals, done by measuring their inhibitory effects on the mitochondrial ATP synthesis, revealed that 4 of them, KH-1, 7, 16, and 17, had moderate inhibitory effects. Further characterization of the actions of these 4 analogues on mitochondrial function showed that KH-16 had moderate; KH-1 and KH-17, weak; and KH-7, negligible side effects of both permeabilization of the mitochondrial inner membrane and inhibition of the electron transport, indicating that only KH-7 had a specific inhibitory effect on the mitochondrial ADP/ATP carrier. Although the parental bongkrekic acid showed a strong pH dependency of its action, the inhibitory effect of KH-7 was almost insensitive to the pH of the reaction medium, indicating the importance of the 3 carboxyl groups of BKA for its pH- dependent action. A direct inhibitory effect of KH-7 on the mitochondrial ADP/ATP carrier was also clearly demonstrated

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

Effects of employment of distinct strategies to capture antibody on antibody delivery into cultured cells

The characteristics of antibody delivery into cultured HeLa cells were examined by using two delivery systems. Both systems used a cell-penetrating peptide as a tool for intrusion of an antibody into the cells, but either a “protein A derivative” or “hydrophobic motif” was employed to capture the antibody. When we examined the uptake of the Alexa Fluor-labeled antibody by use of these two systems, both systems were found to effectively deliver the antibody into the cultured cells. However, when we compared the amount of antibody delivered by these systems with the amount of transferrin uptake, the former was 10 times smaller than the latter. The lower efficiency of antibody delivery than transferrin uptake seemed to be attributable to the involvement of the antibody delivery reagent, which failed to catch the antibody molecule. This interpretation was validated by an experiment using a larger amount of antibody, and the amount of antibody delivered by the “protein A derivative” system under this condition was determined to be 13 ng proteins/105 cells. The antibody delivery achieved by the “protein A derivative” or “hydrophobic motif” showed two differences, i.e., a difference in intracellular distribution of the delivered antibody molecules and a difference in the fluorescence spectrum observed with cellular lysates. Possible reasons for these differences between the two delivery systems are discussed

Utility of syntenic relationships of VDAC1 pseudogenes for not only an understanding of the phylogenetic divergence history of rodents, but also ascertaining possible pseudogene candidates as genuine pseudogenes

Rodent and human genomes were screened to identify pseudogenes of the type 1 voltage-dependent anion channel (VDAC1) in mitochondria. In addition to the 16 pseudogenes of rat VDAC1 identified in our recent study, 15 and 13 sequences were identified as pseudogenes of VDAC1 in mouse and human genome, respectively; and 4, 2, and 1 sequences, showing lower similarities with the VDAC1 sequence, were identified as “possible pseudogene candidates” in rat, mouse, and human, respectively. No syntenic combination was observed between rodent and human pseudogenes, but 2 and 1 possible pseudogene candidates of VDAC1 of rat and mouse, respectively, were found to have syntenic counterparts in mouse and rat genome, respectively; and these syntenic counterparts were genuine VDAC1 pseudogenes. Therefore, syntenic combinations of pseudogenes of VDAC1 were useful not only for a better understanding of the phylogenetic divergence history of rodents but also for ascertaining possible pseudogene candidates as genuine pseudogenes