Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.     

Effects of endurance training on three superoxide dismutase isoenzymes in human plasma

The effects of endurance training and acute exhaustive exercise on plasma levels of three superoxidedismutase (SOD) isoenzymes and the ability of superoxide generation in neutrophils were studied. Eighteen healthy male students, aged 17-22 years, who volunteered for this study, underwent three months of endurance training in swimming or running. Before and after the training course, they performed acute exercise and blood samples were collected before and after this exercise. The endurance training significantly increased maximal oxygen uptake (VO2max) in all subjects. Neither the endurance training nor the acute exercise affected the plasma CuZn-SOD level. Acute exercise after the training, but not before the training, increased both the plasma Mn-SOD and extracellular SOD (EC-SOD) levels by 33.6 and 33.5%, respectively. The training decreased the EC-SOD level at rest by 22.2%. Acute exercise after the training, but not before the training, increased the plasma lipid peroxide level, suggesting higher oxidative stress in trained subjects during exhaustive exercise. The ability of neutrophils to generate superoxide was increased by the acute exercise, but induction of the superoxide was suppressed after training. These results indicate that EC-SOD levels were changed in a different manner from the CuZn-SOD and Mn-SOD: it was decreased by training but was increased by acute exercise, suggesting that endurance training increases the reserve of EC-SOD in tissues. The results also suggest the possibility of plasma EC-SOD assay as a new index of endurance training.     

The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis

We have identified a new Arabidopsis mutant, yore-yore (yre), which has small trichomes and glossy stems. Adhesion between epidermal cells was observed in the organs of the yre shoot. The cloned YRE had high homology to plant genes involved in epicuticular wax synthesis, such as ECERIFERUM1 (CER1) and maize GLOSSY1. The phenotype of transgenic plants harboring double-stranded RNA interference (dsRNAi) YRE was quite similar to that of the yre mutant. The amount of epicuticular wax extracted from leaves and stems of yre-1 was approximately one-sixth of that from the wild type. YRE promoter::GUS and in situ hybridization revealed that YRE was specifically expressed in cells of the L1 layer of the shoot apical meristem and young leaves, stems, siliques, and lateral root primordia. Strong expression was detected in developing trichomes. The trichome structure of cer1 was normal, whereas that of the yre cer1 double mutant was heavily deformed, indicating that epicuticular wax is required for normal growth of trichomes. Double mutants of yre and trichome-morphology mutants, glabra2 (gl2) and transparent testa glabra1 (ttg1), showed that the phenotype of the trichome structure was additive, suggesting that the wax-requiring pathway is distinct from the trichome development pathway controlled by GL2 and TTG1.     

Phosphohexose isomerase/autocrine motility factor/neuroleukin/maturation factor is a multifunctional phosphoprotein

Phosphohexose isomerase (PHI) is a member of the ectoenzyme/exoenzyme family and plays a key role in both glycolysis and gluconeogenesis pathways. Upon secretion PHI acts as a cytokine with tumor autocrine motility factor (AMF), neuroleukin (NLK) and maturation factor (MF) functions. Signaling is initiated by its binding to a cell surface 78 kDa glycoprotein (gp78). However, since PHI protein is a 'leaderless' secretory protein, released from cells via a non-classical route(s), we questioned whether the molecule undergoes post-translation modification while retaining proper folding and maintaining intact enzymatic and motogenic activities. To address this, we have generated, expressed and isolated a recombinant human AMF (rhAMF). The rhAMF retained the biological activities of the native AMF, i.e., catalyzes phosphohexose isomerization and stimulated cell motility. Additionally, we show here that human PHI is phosphorylated at serine 185 by casein kinase II (CK II) and we provide experimental evidence suggesting that this phosphorylation is associated with secretion, thus providing insights for elucidating the intracellular signal transmission of cell response to stimulation by AMF/NLK/MF.     

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-alpha and interleukin-1beta contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.     

Induction of Conjugation by Methyl Cellulose in Paramecium

ABSTRACT An improved method has been developed for the induction of selfing conjugation in Paramecium. Methyl cellulose induces selfing conjugation simply and efficiently in all species examined. Induction of conjugation by methyl cellulose was characterized in P. caudatum , where it occurred only in sexually mature, mating reactive cells. Conjugation produced sexual offspring and the time course of nuclear processes was substantially the same as that in natural conjugation between cells of complementary mating types. The method is useful for genetic studies in a wide variety of Paramecium species including P. caudatum, P. tetraurelia, P. multimicronucleatum and P. bursaria.     

Emergence and Characterization of Unusual DS-1-Like G1P[8] Rotavirus Strains in Children with Diarrhea in Thailand

The emergence and rapid spread of unusual DS-1-like G1P[8] rotaviruses in Japan have been recently reported. During rotavirus surveillance in Thailand, three DS-1-like G1P[8] strains (RVA/Human-wt/THA/PCB-180/2013/G1P[8], RVA/Human-wt/THA/SKT-109/2013/G1P[8], and RVA/Human-wt/THA/SSKT-41/2013/G1P[8]) were identified in stool specimens from hospitalized children with severe diarrhea. In this study, we sequenced and characterized the complete genomes of strains PCB-180, SKT-109, and SSKT-41. On whole genomic analysis, all three strains exhibited a unique genotype constellation including both genogroup 1 and 2 genes: G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. This novel genotype constellation is shared with Japanese DS-1-like G1P[8] strains. Phylogenetic analysis revealed that the G/P genes of strains PCB-180, SKT-109, and SSKT-41 appeared to have originated from human Wa-like G1P[8] strains. On the other hand, the non-G/P genes of the three strains were assumed to have originated from human DS-1-like strains. Thus, strains PCB-180, SKT-109, and SSKT-41 appeared to be derived through reassortment event(s) between Wa-like G1P[8] and DS-1-like human rotaviruses. Furthermore, strains PCB-180, SKT-109, and SSKT-41 were found to have the 11-segment genome almost indistinguishable from one another in their nucleotide sequences and phylogenetic lineages, indicating the derivation of the three strains from a common origin. Moreover, all the 11 genes of the three strains were closely related to those of Japanese DS-1-like G1P[8] strains. Therefore, DS-1-like G1P[8] strains that have emerged in Thailand and Japan were assumed to have originated from a recent common ancestor. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like G1P[8] strains that have emerged in an area other than Japan. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] rotaviruses.     

Clinical Report on the First Prototype of a Photoacoustic Tomography System with Dual Illumination for Breast Cancer Imaging

Photoacoustic tomography is a recently developed imaging modality that can provide high spatial-resolution images of hemoglobin distribution in tissues such as the breast. Because breast cancer is an angiogenesis-dependent type of malignancy, we evaluated the clinical acceptability of breast tissue images produced using our first prototype photoacoustic mammography (PAM) system in patients with known cancer. Post-excisionally, histological sections of the tumors were stained immunohistochemically (IHC) for CD31 (an endothelial marker) and carbonic anhydrase IX (CAIX) (a marker of hypoxia). Whole-slide scanning and image analyses were used to evaluate the tumor microvessel distribution pattern and to calculate the total vascular perimeter (TVP)/area for each lesion. In this clinical study, 42 lesions were primarily scanned using PAM preoperatively, three of which were reported to be benign and were excluded from statistical analysis. Images were produced for 29 out of 39 cancers (visibility rate = 74.4%) at the median depth of 26.5 (3.25–51.2) mm. Age, menopausal status, body mass index, history of neoadjuvant treatment, clinical stage and histological tumor angiogenesis markers did not seem to affect the visibility. The oxygen saturation level in all of the measured lesions was lower than in the subcutaneous counterpart vessels (Wilcoxon test, p value<0.001), as well as in the counterpart contralateral normal breast region of interest (ROI) (Wilcoxon test, p value = 0.001). Although the oxygen saturation level was not statistically significant between CAIX-positive vs. -negative cases, lesional TVP/area showed a positive correlation with the oxygen saturation level only in the group that had received therapy before PAM. In conclusion, the vascular and oxygenation data obtained by PAM have great potential for identifying functional features of breast tumors.     

Abnormal Localization of STK17A in Bile Canaliculi in Liver Allografts: An Early Sign of Chronic Rejection

The biological significance of STK17A, a serine/threonine kinase, in the liver is not known. We analyzed STK17A expression in HepG2 cells and human liver tissue. Accordingly, we investigated whether STK17A could help in identifying earlier changes during the evolution of chronic rejection (CR) after liver transplantation. RT-PCR and immunofluorescence were used to analyze STK17A expression in HepG2 cells. Antibody microarray was performed using human liver samples from CR and healthy donors. Immunohistochemistry was used to verify the clinical utility of STK17A on sequential biopsies for the subsequent development of CR. A novel short isoform of STK17A was found in HepG2 cells. STK17A was localized in the nuclei and bile canaliculi in HepG2 cells and human livers. Microarray of STK17A revealed its decrease in failed liver allografts by CR. During the evolution of CR, the staining pattern of bile canalicular STK17A gradually changed from diffuse linear to focal intermittent. The focal intermittent staining pattern was observed before the definite diagnosis of CR. In conclusion, the present study was the first to find localization of STK17A in normal bile canaliculi. Abnormal expression and localization of STK17A were associated with CR of liver allografts since the early stage of the rejection process.