The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs

We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth.     

Structural Insights into the Inhibition of Zika Virus NS2B-NS3 Protease by a Small-Molecule Inhibitor

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Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles

Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.     

Genetic variants at 14q24.1 and breast cancer susceptibility: a fine-mapping study in Chinese women

A single nucleotide polymorphism (SNP) rs999737 at 14q24.1 was identified as a susceptibility marker of breast cancer in a genome-wide association study of the European population, which was also confirmed by some of the following studies in populations of European descent. However, rs999737 is very rare or nonpolymorphic in non-Europeans including Chinese, and the role of other genetic variants at 14q24.1 has not been evaluated in populations of non-European descent. In this study, we first selected 21 common tagging SNPs (minor allele frequency [MAF] >0.05 in the Chinese population) by searching the Hapmap database, covering a linage disequilibrium region of more than 70?Kb at 14q24.1, and then conducted a two-stage study (stage I: 878 cases and 900 controls; stage II: 914 cases and 967 controls) to investigate the associations between these tagging SNPs and risk of breast cancer in a Chinese population. In stage I, two SNPs (rs2842346 and rs17828907) were identified to be significantly associated with breast cancer risk (p=0.030 and 0.027 for genotype distributions, respectively). However, no significant associations were found between these two SNPs and breast cancer risk in either stage II or the combined dataset. These findings suggest that common variants at 14q24.1 might not be associated with the risk of breast cancer in the Chinese population, which will need the replication in additional larger studies.     

Expression of Tumor-Associated Macrophage in Progression of Human Glioma

The aim of this study is to investigate the expression of tumor-associated macrophages (TAMs) M1, M2 phenotypic in human glioma tissues, and to explore the clinical significance and prognostic value of TAMs in glioma patients. A total of 50 glioma samples were obtained from patients diagnosed in our hospital from 2007 to 2010. Clinical follow-up was conducted via return visits and telephone interviews after discharge. Progression free survival (PFS) was calculated based on tumor progression by MRI and CT examination from the primary operation. Overall survival (OS) time was calculated from the initial surgical operation date to end date of follow-up or death. Kaplan–Meier methodology was used to evaluate the survival of patients and log-rank test for comparing differences between groups. The expression levels of CD16 and CD206 were investigated in the 4 μm serial paraffin sections by immunohistochemistry. M1-type macrophages filtrated in all the grades of glioma samples, and the lower expression level was associated with high grade glioma. A negative correlation was found between WHO pathological grades and the expression of M1-type macrophages by Spearman correlation analysis. M2-type macrophages filtrated in all the grades of glioma samples with the higher expression level associated with high grade glioma. A positive correlation was found between WHO pathological grades and the expression of M2-type macrophages by Spearman correlation analysis. The PFS and OS among patients with high levels of M1-type macrophages (CD16+++) were significantly higher than those with less expression. The PFS and OS among patients with high levels of M2-type macrophages (CD206+++) were significantly lower than those with low expression. M1-type macrophages may inhibit the tumor growth and improve the therapeutic outcome of glioma patients. M2 ratios are associated with tumor proliferation and poor prognosis. TAMs phenotypes of glioma samples are the potential biomarkers in assessing the degree of malignancy, tumor invasion, and patient prognosis in clinic.     

Homology modeling and function of trehalose synthase from Pseudomonas putida P06

Trehalose is a non-reducing disaccharide that has wide applications in the food industry and pharmaceutical manufacturing. Trehalose synthase (TreS) from Pseudomonas putida P06 catalyzes the reversible interconversion of maltose and trehalose and may have applications in the food industry. However, the catalytic mechanism of TreS is not well understood. Here, we investigated the structural characteristics of this enzyme by homology modeling. The highly conserved Asp294 residue was identified to be critical for catalytic activity. In addition, flexible docking studies of the enzyme–substrate system were performed to predict the interactions between TreS and its substrate, maltose. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the biochemical characterization of the relevant mutants generated by site-directed mutagenesis.     

iTRAQ-based proteomics analysis of autophagy-mediated immune responses against the vascular fungal pathogen Verticillium dahliae in Arabidopsis

The mechanisms underlying the functional link between autophagy and plant innate immunity remain largely unknown. In this study, we investigated the autophagy-mediated plant defense responses against Verticillium dahliae (V. dahliae) infection by comparative proteomics and cellular analyses. An assessment of the autophagy activity and disease development showed that autophagic processes were tightly related to the tolerance of Arabidopsis plant to Verticillium wilt. An isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics analysis was performed, and we identified a total of 780 differentially accumulated proteins (DAPs) between wild-type and mutant atg10-1 Arabidopsis plants upon V. dahliae infection, of which, 193 ATG8-family-interacting proteins were identified in silico and their associations with autophagy were verified for several selected proteins. Three important aspects of autophagy-mediated defense against V. dahliae infection were revealed: 1) autophagy is required for the activation of upstream defense responses; 2) autophagy-mediated mitochondrial degradation (mitophagy) occurs and is an important player in the defense process; and 3) autophagy promotes the transdifferentiation of perivascular cells and the formation of xylem hyperplasia, which are crucial for protection against this vascular disease. Together, our results provide several novel insights for understanding the functional association between autophagy and plant immune responses.     

Localization of nitric oxide synthase in canine ileocolonic and pyloric sphincters

The distribution of neurons containing NADPH-diaphorase (NADPH-d) activity and nitric oxide synthase-like immunoreactivity (NOS-LI) in the canine pyloric and ileocolonic sphincters was studied. Cells within the myenteric and submucosal ganglia were positive for NADPH-d. These cells generally had the morphology of Dogiel type-I enteric neurons, however, there was some diversity in the morphology of NADPH-d-positive neurons in the myenteric plexus of the pylorus. Intramuscular ganglia were observed in both sphincters, and NADPH-d was found in a sub-population of neurons within these ganglia. Dual staining with an antiserum raised against nitric oxide synthase (NOS) demonstrated that almost all cells with NOS-LI were also NADPH-d positive. Varicose fibers within ganglia and within the circular and longitudinal muscle layers also possed NOS-LI and NADPH-d activity. Dual staining with anti-VIP antibodies showed that some of the NADPH-d-positive cells in the myenteric and submucosal ganglia also contained VIP-LI, but all VIP-LI-positive cells did not express NADPH-d activity. These data are consistent with recent physiological studies suggesting that nitric oxide serves as an inhibitory neurotransmitter in the pyloric and ileocolonic sphincters. The data also suggest that VIP is expressed in a sub-population of NADPH-d-positive neurons and may therefore act as a co-transmitter in enteric inhibitory neurotransmission to these specialized muscular regions.