Mycoplasmalike organisms (MLOs) and distinguish them from other MLOs.

DNA of 10 lines of rice yellow dwarf (RYD) mycoplasmalike organisms (MLOs) from Japan, the Phillippines, and Thailand hybridized with four probes containing chromosomal and six probes containing extrachromosomal DNA of a Tochigi (Japan) line of RYD MLO. One chromosomal probe (RYD9) and all six extrachromosomal probes hybridized with various other MLOs (sugarcane white leaf, onion yellows, cineraria witches'-broom, Japanese hornwort witches'-broom, water dropwort wiches'-broom, gentian witches'-broom, udo dwarf, tsuwabuki witches'-broom, pelargonium witches's-broom, peach western-X, and pear decline). DNA from the culturable mollicutes Spiroplasma kunkelii, Spiroplasma citri, Mycoplasma hominis, and Mycoplasma orale did not hybridize with RYD MLO probes. The extrachromosomal DNAs hybridizing with the probes showed variations in electrophoretic behavior.     

Prognostic Significance of Anti-Aminoacyl-tRNA Synthetase Antibodies in Polymyositis/Dermatomyositis-Associated Interstitial Lung Disease: A Retrospective Case Control Study

Background

In polymyositis/dermatomyositis (PM/DM), anti-aminoacyl-tRNA synthetase (ARS) antibodies are closely associated with interstitial lung disease (ILD), a frequent pulmonary complication. However, the clinical significance of anti-ARS antibodies is not well established.

Objective

We aimed to evaluate the clinical significance of anti-ARS antibodies in PM/DM-ILD patients.

Methods

Forty-eight consecutive PM/DM-ILD patients were studied retrospectively. Anti-ARS antibodies were screened by ELISA and confirmed by RNA immunoprecipitation test. Medical records, high-resolution computed tomography images, and surgical lung biopsy specimens were compared between ARS-positive (ARS group) and ARS-negative patients (non-ARS group).

Results

Anti-ARS antibodies were detected in 23 of 48 patients (48%). Radiologically, nonspecific interstitial pneumonia (NSIP) pattern was observed more frequently in the ARS group than in the non-ARS group (73.9% vs. 40%, P = 0.02). Pathologically, NSIP was the most frequent in both groups. Ten-year survival rate was also significantly higher in the ARS group than in the non-ARS group (91.6% vs. 58.7%, P = 0.02). Univariate Cox hazards analysis revealed that the presence of anti-ARS antibodies was associated with better prognosis (HR = 0.34, 95% CI 0.08–0.80; P = 0.01).

Conclusions

The presence of anti-ARS antibodies is a possible prognostic marker in patients with PM/DM-ILD.     

Novel tumorigenic rearrangement,Delta rfp/ret,in a papillary thyroid carcinoma from externally irradiated patient

Molecular analysis of cDNA derived from a papillary thyroid carcinoma (PTC) (follicular variant of papillary thyroid carcinoma on histology) which developed in an externally irradiated patient 4 years after exposure identified a portion of the 5' region, exons 1-3, of the rfp gene juxtaposed upstream of the fragment encoding the tyrosine kinase (TK) domain of the ret gene. The fusion gene, termed Delta rfp/ret, was the result of a balanced chromosomal translocation t(6;10) (p21.3;q11.2) confirmed by interphase FISH painting, with breakpoints occurring in introns 3 and 11 of the rfp and ret genes, respectively. Both Delta rfp/ret and reciprocal ret/rfp chimeric introns had small deletions around breakpoints consistent with presumed misrepair of a radiation-induced double-strand DNA break underlying the rearrangement. No extensive sequence homology was found between the fragments flanking the breakpoints. The fusion protein retained the propensity to form oligomers likely to be mediated by a coiled-coil of the RFP polypeptide as assessed by a yeast two-hybrid system. NIH 3T3 fibroblasts stably transfected with a mammalian expression vector encoding full-length Delta RFP/RET readily gave rise to the tumors in athymic mice suggestive of high transforming potential of the fusion protein. Thus, the Delta rfp/ret rearrangement may be involved in a causative manner in cancerogenesis and provides additional evidence of the role of activated ret oncogene in the development of a subset of papillary thyroid carcinoma.     

Enantioselective synthesis of (S)-2-cyano-2-methylpentanoic acid by nitrilase

The nitrilase gene of Rhodococcus rhodochrous J1 was expressed in Escherichia coli using the expression vector, pKK223-3. The recombinant E. coli JM109 cells hydrolyzed enantioselectively 2-methyl-2-propylmalononitrile to form (S)-2-cyano-2-methylpentanoic acid (CMPA) with 96 % e.e. Under optimized conditions, 80 g (S)-CMPA l^?1 was produced with a molar yield of 97 % at 30 °C after a 24 h without any by-products.     

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.     

The amino acid composition is different between the cytoplasmic and extracellular sides in membrane proteins.

The amino acid composition of transmembrane proteins was analyzed for their three separate portions: the transmembrane apolar, cytoplasmic and extracellular regions. The composition was different between cytoplasmic and extracellular peptides: alanine and arginine residues were preferentially sited on the cytoplasmic side, while the threonine and cysteine/cystine were preferentially sited on the extracellular side. The composition of cytoplasmic and extracellular peptides of membrane proteins corresponded to those of intracellular and extracellular types of soluble proteins, respectively. This difference in composition was independent of the peptide orientation against the membrane. Peptide chains could be correctly assigned as either cytoplasmic or extracellular, solely from an analysis of sequence composition. For single-spanning membrane proteins the predictive accuracy was 90%, whereas for multi-spanning proteins this was 85%.     

Synthesis and purification of a deleted human growth hormone, hGH delta 135-146: sensitivity to plasmin cleavage and in vitro and in vivo bioactivities

Proteolytically cleaved human 22 kDa growth hormone (22K hGH) between the amino acid residues 134 and 150 by plasmin or other proteases in vitro has been reported to be most active in growth promoting activity. In this study a deleted mutant hGH lacking amino acid residues from 135 to 146 and having more sensitivity to plasmin digestion was produced using the inverse polymerase chain reaction method and the Escherichia coli expression system. The mutant, hGH delta 135-146, was folded and purified effectively and found to be more sensitive to plasmin cleavage to form the two-chain form in vitro. The biological activities of this plasmin sensitive hGH delta 135-146 were tested by in vitro cell proliferation assays and in vivo growth promoting assay. In Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146 showed 10-20% less growth promoting activity than 22K hGH, but expressed comparable quantities of IGF-I mRNA to that of 22K hGH. In Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors, hGH delta 135-146 showed equivalent activities to those of 22K hGH at lower concentrations. By the body weight gain test using hypophysectomized rats, a lower dose (2.5 nmol kg-1) of hGH delta 135-146 exhibited an equivalent activity to that of wild type 22K hGH, but a higher dose (25 nmol kg-1) of the mutant showed less growth promoting activity than 22K hGH. These results indicated that the plasmin sensitive recombinant hGH delta 135-146 failed to show higher biological activity than the 22K hGH in vivo, suggesting the unsuccessful formation of the active two-chain form in vivo.     

Factors Associated with Nurses’ Intention to Leave Their Jobs after the Fukushima Daiichi Nuclear Power Plant Accident

We conducted a survey among nurses who were working at the Fukushima Medical University Hospital at the time of the Fukushima Daiichi Nuclear Power Plant accident to clarify the factors associated with their intention to leave their jobs during the radiation emergency. We asked 345 nurses (17 men and 328 women) about their intention to leave their jobs after the accident. We also asked about relevant factors including the participants’ demographic factors, living situation, working status, and knowledge of radiation health effects. We found that living with preschoolers (OR = 1.87, 95%CI: 1.02–3.44, p = 0.042), anxiety about life in Fukushima City after the accident (OR = 5.55, 95%CI: 1.18–26.13, p = 0.030), consideration of evacuation from Fukushima after the accident (OR = 2.42, 95%CI: 1.45–4.06, p = 0.001), consideration of the possible radiation health effects in children (OR = 1.90, 95%CI: 1.02–3.44, p = 0.042), and anxiety about relationships with colleagues in the hospital after the accident (OR = 3.23, p = 0.001) were independently associated with the nurses’ intention to leave their jobs after the accident. On the other hand, the percentage of nurses with knowledge on radiation health effects was relatively low among those who had the intention to leave the job and among those who did not have the intention to leave the job after the accident, with no significant differences between the two groups. Our results suggest the need for an education program for nurses regarding radiation health effects.