No apparent effects of the buccal cavity attaching parasite,Salmincola sp. (Copepoda: Lernaeopodidae), on a stream salmonid: a mark-recapture study

Ichthyological Research - We evaluated the effects of a buccal cavity attaching Salmincola sp. on white-spotted charr Salvelinus leucomaenis by mark-recapture method to compare body condition,...     

p90 RSK-1 associates with and inhibits neuronal nitric oxide synthase

Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain.     

Inhibition of neuronal nitric-oxide synthase by phosphorylation at Threonine1296 in NG108-15 neuronal cells

We demonstrate that neuronal nitric-oxide synthase (nNOS) is directly inhibited through the phosphorylation of Thr(1296) in NG108-15 neuronal cells. Treatment of NG108-15 cells expressing nNOS with calyculin A, an inhibitor of protein phosphatase 1 and 2A, revealed a dose-dependent inhibition of nNOS enzyme activity with concomitant phosphorylation of Thr(1296) residue. Cells expressing a phosphorylation-deficient mutant in which Thr(1296) was changed to Ala proved resistant to phosphorylation and suppression of NOS activity. Mimicking phosphorylation mutant of nNOS in which Thr(1296) is changed to Asp showed a significant decrease in nNOS enzyme activity, being competitive with NADPH, relative to the wild-type enzyme. These data suggest that phosphorylation of nNOS at Thr(1296) may involve the attenuation of nitric oxide production in neuronal cells through the decrease of NADPH-binding to the enzyme.     

Electroporation as an efficient method of gene transfer

Gene transfer by electroporation is an indispensable method for the study of developmental biology, especially for the study using chick embryos. Here we briefly review the principles of the method, and its application to chick embryos. Methods of transient misexpression and long-term misexpression by retrovirus vector or transposon system, and knockdown by small interference RNA are reviewed.     

Simultaneous measurement of F2-isoprostane, hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples

Oxidative stress induced by various oxidants in a random and destructive manner is considered to play an important role in the pathophysiology of a number of human disorders and diseases. It is important to assess the oxidative injury in vivo accurately and inclusively. We have developed an improved method for the measurement of in vivo lipid peroxidation by using a single plasma or liver sample, where total 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)), total hydroxyoctadecadienoic acids (tHODEs), total hydroxyeicosatetraenoic acids (tHETEs), and total 7-hydroxycholesterol (t7-OHCh), as well as their parent molecules linoleic acid (t18:2) and cholesterol (tCh), are determined by LC-MS/MS (for t8-iso-PGF(2alpha), tHODE, and tHETE) and GC-MS (for t7-OHCh, t18:2, and tCh) analyses. The plasma and liver samples from human are reduced with sodium borohydride and saponified by potassium hydroxide after the addition of heavy isotopic standards. After extraction by chloroform/ethyl acetate (CHCl(3)/CH(3)COOC(2)H(5), 4:1), they are analyzed without any further sample processing. We applied this method to hepatitis C virus-infected patients (n=8, plasma and liver), hepatitis B virus-infected patients (n=2, plasma and liver), and controls (virus free, n=8, plasma and liver). It was found that in the plasma of patients and controls, the concentrations of oxidized lipids decreased in the following order: tHODE tHETE t7-OHCh > t8-iso-PGF(2alpha). As expected, the virus clearly increased these concentrations. The ratio of stereoisomers of HODE [(E,E)-HODE/(E,Z)-HODE], which reflects the antioxidant capacity in vivo, can also be determined by this method. A significant decrease in the stereoisomer ratio for the liver of patients was observed, indicating liver dysfunction. t8-iso-PGF(2alpha), tHODE, tHETE, and t7-OHCh are measured satisfactorily and inclusively by the current method from biological fluids and tissues, and they can account for a large portion of oxidized lipids in vivo.     

An Application of Wastewater Treatment in a Cold Environment and Stable Lipase Production of Antarctic Basidiomycetous Yeast Mrakia blollopis

Milk fat curdle in sewage is one of the refractory materials for active sludge treatment under low temperature conditions. For the purpose of solving this problem by using a bio-remediation agent, we screened Antarctic yeasts and isolated SK-4 strain from algal mat of sediments of Naga-ike, a lake in Skarvsnes, East Antarctica. The yeast strain showed high nucleotide sequence homologies (>99.6%) to Mrakia blollopis CBS8921^T in ITS and D1/D2 sequences and had two unique characteristics when applied on an active sludge; i.e., it showed a potential to use various carbon sources and to grow under vitamin-free conditions. Indeed, it showed a biochemical oxygen demand (BOD) removal rate that was 1.25-fold higher than that of the control. We considered that the improved BOD removal rate by applying SK-4 strain was based on its lipase activity and characteristics. Finally, we purified the lipase from SK-4 and found that the enzyme was quite stable under wide ranges of temperatures and pH, even in the presence of various metal ions and organic solvents. SK-4, therefore, is a promising bio-remediation agent for cleaning up unwanted milk fat curdles from dairy milk wastewater under low temperature conditions.     

A Translation System Reconstituted with Human Factors Proves That Processing of Encephalomyocarditis Virus Proteins 2A and 2B Occurs in the Elongation Phase of Translation without Eukaryotic Release Factors

The genomic RNA of encephalomyocarditis virus (EMCV) encodes a single polyprotein, and the primary scission of the polyprotein occurs between nonstructural proteins 2A and 2B by an unknown mechanism. To gain insight into the mechanism of 2A-2B processing, we first translated the 2A-2B region in vitro with eukaryotic and prokaryotic translation systems. The 2A-2B processing occurred only in the eukaryotic systems, not in the prokaryotic systems, and the unprocessed 2A-2B protein synthesized by a prokaryotic system remained uncleaved when incubated with a eukaryotic cell extract. These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event. To define the translation factors required for 2A-2B processing, we constituted a protein synthesis system with eukaryotic elongation factors 1 and 2, eukaryotic release factors 1 and 3 (eRF1 and eRF3), aminoacyl-tRNA synthetases, tRNAs, ribosome subunits, and a plasmid template that included the hepatitis C virus internal ribosome entry site. We successfully reproduced 2A-2B processing in the reconstituted system even without eRFs. Our results indicate that this unusual event occurs in the elongation phase of translation.     

Comparative Effectiveness of Emergency Resuscitative Thoracotomy versus Closed Chest Compressions among Patients with Critical Blunt Trauma: A Nationwide Cohort Study in Japan

Background

Although emergency resuscitative thoracotomy is performed as a salvage maneuver for critical blunt trauma patients, evidence supporting superior effectiveness of emergency resuscitative thoracotomy compared to conventional closed-chest compressions remains insufficient. The objective of this study was to investigate whether emergency resuscitative thoracotomy at the emergency department or in the operating room was associated with favourable outcomes after blunt trauma and to compare its effectiveness with that of closed-chest compressions.

Methods

This was a retrospective nationwide cohort study. Data were obtained from the Japan Trauma Data Bank for the period between 2004 and 2012. The primary and secondary outcomes were patient survival rates 24 h and 28 d after emergency department arrival. Statistical analyses were performed using multivariable generalized mixed-effects regression analysis. We adjusted for the effects of different hospitals by introducing random intercepts in regression analysis to account for the differential quality of emergency resuscitative thoracotomy at hospitals where patients in cardiac arrest were treated. Sensitivity analyses were performed using propensity score matching.

Results

In total, 1,377 consecutive, critical blunt trauma patients who received cardiopulmonary resuscitation in the emergency department or operating room were included in the study. Of these patients, 484 (35.1%) underwent emergency resuscitative thoracotomy and 893 (64.9%) received closed-chest compressions. Compared to closed-chest compressions, emergency resuscitative thoracotomy was associated with lower survival rate 24 h after emergency department arrival (4.5% vs. 17.5%, respectively, P < 0.001) and 28 d after arrival (1.2% vs. 6.0%, respectively, P < 0.001). Multivariable generalized mixed-effects regression analysis with and without a propensity score-matched dataset revealed that the odds ratio for an unfavorable survival rate after 24 h was lower for emergency resuscitative thoracotomy than for closed-chest compressions (P < 0.001).

Conclusions

Emergency resuscitative thoracotomy was independently associated with decreased odds of a favorable survival rate compared to closed-chest compressions.     

N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.     

Crystal structure of zinc-finger domain of Nanos and its functional implications

Nanos is an RNA-binding protein that is involved in the development and maintenance of germ cells. In combination with Pumilio, Nanos binds to the 3' untranslated region of a messenger RNA and represses its translation. Nanos has two conserved Cys-Cys-His-Cys zinc-finger motifs that are indispensable for its function. In this study, we have determined the crystal structure of the zinc-finger domain of zebrafish Nanos, for the first time revealing that Nanos adopts a novel zinc-finger structure. In addition, Nanos has a conserved basic surface that is directly involved in RNA binding. Our results provide the structural basis for further studies to clarify Nanos function.