Gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri: cloning and expression in Escherichia coli.

A library of cloned Spiroplasma citri genomic sequences was constructed by incorporating HindIII digestion fragments into the plasmid vector pBR328. Immunological screening allowed the identification of a recombinant plasmid containing the gene for spiralin, the major membrane protein of S. citri. The spiralin produced by the Escherichia coli transformant was characterized by immunological detection with monoclonal antibody after Western blotting of two-dimensional (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide) electrophoresis gels and by partial proteolytic mapping. The gene for spiralin occurred within a 6.5-kilobase-pair cloned DNA fragment. Spiralin in E. coli was produced regardless of the orientation of the insert within the pBR328 vector. A spiroplasmal DNA sequence which acted as a promoter in E. coli was cloned along with the structural spiralin gene which is expressed in E. coli from that sequence.     

Statin Therapy Is Associated with Improved Survival in Patients with Non-Serous-Papillary Epithelial Ovarian Cancer: A Retrospective Cohort Analysis

Aim

To determine whether statin use is associated with improved epithelial ovarian cancer (OvCa) survival.

Methods

This is a single-institution retrospective cohort review of patients treated for OvCa between 1992 and 2013. Inclusion criteria were International Federation of Gynecology and Obstetrics (FIGO) stage I–IV OvCa. The primary exposures analyzed were hyperlipidemia and statin use. The primary outcomes were progression-free survival (PFS) and disease-specific survival (DSS).

Results

442 patients met inclusion criteria. The cohort was divided into three groups: patients with hyperlipidemia who used statins (n = 68), patients with hyperlipidemia who did not use statins (n = 28), and patients without hyperlipidemia (n = 346). OvCa outcomes were evaluated. When we analyzed the entire cohort, we found no significant differences in PFS or DSS among the groups. The median PFS for hyperlipidemics using statins, hyperlipidemics not using statins, and non-hyperlipidemics was 21.7, 13.6, and 14.7 months, respectively (p = 0.69). Median DSS for hyperlipidemics using statins, hyperlipidemics not using statins, and non-hyperlipidemics was 44.2, 75.7, and 41.5 months, respectively (p = 0.43). These findings did not change after controlling for confounders. However, a secondary analysis revealed that, among patients with non-serous-papillary subtypes of OvCa, statin use was associated with a decrease in hazards of both disease recurrence (adjusted HR = 0.23, p = 0.02) and disease-specific death (adjusted HR = 0.23, p = 0.04). To augment the findings in the retrospective cohort, the histology-specific effects of statins were also evaluated in vitro using proliferation assays. Here, statin treatment of cell lines resulted in a variable level of cytotoxicity.

Conclusion

Statin use among patients with non-serous-papillary OvCa was associated with improvement in both PFS and DSS.     

Repair of DNA-protein cross-links in mammalian cells

DNA-protein cross-links are generated by both endogenous and exogenous DNA damaging agents, as intermediates during normal DNA metabolism, and during abortive base excision repair. Cross-links are relatively common lesions that are lethal when they block progression of DNA polymerases. DNA-protein cross-links may be broadly categorized into four groups by the DNA and protein chemistries near the cross-link and by the source of the cross-link: DNA-protein cross-links may be found (1) in nicked DNA at the 3' end of one strand (topo I), (2) in nicked DNA at the 5' end of one strand (pol beta), (3) at the 5' ends of both strands adjacent to nicks in close proximity (topo II; Spo 11), and (4) in one strand of duplex DNA (UV irradiation; bifunctional carcinogens and chemotherapeutic agents). Repair mechanisms are reasonably well-defined for groups 1 and 3, and suggested for groups 2 and 4. Our work is focused on the recognition and removal of DNA-protein cross-links in duplex DNA (group 4).     

Physical training in obese women. Effects of muscle morphology, biochemistry and function

Peripheral adaptations to 3 months of physical endurance training without food restrictions were studied in skeletal muscles of 14, middle-aged, physically untrained, obese women. In comparison to aged-matched controls of normal weight, the obese group showed significantly lower isometric endurance. In the obese group, physical training resulted in a significant increase of maximal isometric and isokinetic strength. Isokinetic but not isometric endurance also increased after training. The isometric strength of obese women showed a positive correlation with the percentage of FTb fibres. The training (50 min/day, 3 days/w) did not result in any change in body weight, body fat, and the number and weight of fat cells. The 20% increase of VO2 max after training was found to be significantly correlated with the increase in the number of capillaries around muscle fibres. The relative percentage of FTa fibres, the number of capillaries per fibre as well as the activities of citrate synthase, 3-hydroxy-acyl-CoA-dehydrogenase, and hexokinase showed a significant increase after training. The concentrations of glucose during OGTT showed a trend to decrease with a significant decrease at the end glucose curve (120-min value). The concentration of insulin and C peptide and the insulin removal did not change after training. The changes in the concentration of glucose during OGTT was significantly correlated with the increase in muscle capillarization and of dynamic endurance.     

Nascent HDL formation by hepatocytes is reduced by the concerted action of serum amyloid A and endothelial lipase

Inflammation is associated with significant decreases in plasma HDL-cholesterol (HDL-C) and apoA-I levels. Endothelial lipase (EL) is known to be an important determinant of HDL-C in mice and in humans and is upregulated during inflammation. In this study, we investigated whether serum amyloid A (SAA), an HDL apolipoprotein highly induced during inflammation, alters the ability of EL to metabolize HDL. We determined that EL hydrolyzes SAA-enriched HDL in vitro without liberating lipid-free apoA-I. Coexpression of SAA and EL in mice by adenoviral vector produced a significantly greater reduction in HDL-C and apoA-I than a corresponding level of expression of either SAA or EL alone. The loss of HDL occurred without any evidence of HDL remodeling to smaller particles that would be expected to have more rapid turnover. Studies with primary hepatocytes demonstrated that coexpression of SAA and EL markedly impeded ABCA1-mediated lipidation of apoA-I to form nascent HDL. Our findings suggest that a reduction in nascent HDL formation may be partly responsible for reduced HDL-C during inflammation when both EL and SAA are known to be upregulated.     

The Role of TIR-NBS and TIR-X Proteins in Plant Basal Defense Responses

Toll/interleukin receptor (TIR) domain-containing proteins encoded in the Arabidopsis (Arabidopsis thaliana) genome include the TIR-nucleotide binding site (TN) and TIR-unknown site/domain (TX) families. We investigated the function of these proteins. Transient overexpression of five TX and TN genes in tobacco (Nicotiana benthamiana) induced chlorosis. This induced chlorosis was dependent on ENHANCED DISEASE RESISTANCE1, a dependency conserved in both tobacco and Arabidopsis. Stable overexpression transgenic lines of TX and TN genes in Arabidopsis produced a variety of phenotypes associated with basal innate immune responses; these were correlated with elevated levels of salicylic acid. The TN protein AtTN10 interacted with the chloroplastic protein phosphoglycerate dehydrogenase in a yeast (Saccharomyces cerevisiae) two-hybrid screen; other TX and TN proteins interacted with nucleotide binding-leucine-rich repeat proteins and effector proteins, suggesting that TN proteins might act in guard complexes monitoring pathogen effectors.Innate immunity is a primary defense mechanism in plants that functions to protect against a variety of biotic stresses (Eitas and Dangl, 2010). The plant basal immune system comprises pattern or pathogen recognition receptors that can recognize a variety of plant pathogens by identifying specific pathogen-associated molecular patterns (PAMPs; Tsuda and Katagiri, 2010). This recognition of PAMPs by plant pattern recognition receptors triggers PAMP-triggered immunity or plant basal immunity (Jones and Dangl, 2006; Zipfel, 2008). Well-known PAMPs or microbe-associated molecular patterns recognized by plants include bacterial flagellin, cold shock proteins, and elongation factor Tu. To suppress PAMP-triggered immunity, plant pathogens secrete an array of virulence factors such as type III effector proteins, while plant resistance (R) proteins function to recognize the effector molecules (Römer et al., 2009; Lewis et al., 2010; Tsuda and Katagiri, 2010; Zhang et al., 2012). Specific recognition of a pathogen effector by a plant R protein triggers a second type of immune response called effector-triggered immunity, resulting in an incompatible reaction (Qi et al., 2011; Sohn et al., 2012; Tahir et al., 2012).The most commonly known plant R proteins are the nucleotide-binding (NB) site Leucine-rich repeat (LRR) proteins that plants use to detect effector proteins. The NB is often called NB-ARC because of sequence similarities to the human apoptotic protease-activating factor APAF1 and Caenorhabditis elegans homolog CELL DEATH PROTEIN4 (Lukasik and Takken, 2009). Plant NB-LRR proteins often also have, at the N terminus, a Toll/Interleukin-1 receptor (TIR) or coiled coil (CC) domain (Meyers et al., 2003). In animal TIR proteins, this domain is more commonly located at the C terminus and is linked by a transmembrane domain to an N-terminal LRR domain (Torto et al., 2002). In Drosophila spp. and other microbes, a TIR domain has been shown to play an important role in the activation of antifungal immune responses (Jenkins and Mansell, 2010). Toll-like receptors (TLRs) perform an integral role in the activation of antimicrobial responses in many animals (Radhakrishnan and Splitter, 2010).In plants, two additional TIR-containing protein families, TIR-NB site (TN) and TIR-unknown/random (TX), were identified, which are distinct from the longer TIR-NB-LRR (TNL) R protein homologs (Meyers et al., 2002). TN proteins contain TIR and NBS domains but lack LRRs, while TX proteins lack both NBS and LRR domains, yet often have a small and variable C-terminal domain (Meyers et al., 2002). In the Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col-0) genome, there are 30 TX genes and 21 TN genes (Meyers et al., 2003). The crystal structure of a TIR domain from an Arabidopsis TN protein (At1g72930/{"type":"entrez-protein","attrs":{"text":"NP_177436","term_id":"15218629","term_text":"NP_177436"}}NP_177436) contains a compact globular fold resembling the mammalian (TLR1 and MYELOID DIFFERENTIATION PRIMARY RESPONSE GENE88 [MYD88]) and bacterial TIR domain proteins (Chan et al., 2010). Although plant TIR domains share less than 20% sequence identity with the human TLR domains, the structures of the TIR domain in plants, mammalian TLRs, and bacterial TIR domain proteins have strong similarity (Chan et al., 2010).A high proportion of TX and TN genes were previously reported to be in complex clusters with TNL genes; these clusters were found to be duplicated to multiple locations in the genome (Meyers et al., 2002). The existence of genetically linked pairs or sets of genes such as RESISTANCE TO PERONOSPORA PARASITICA2A (RPP2A)-RPP2B, RESISTANCE TO PSEUDOMONAS SYRINGAE1 (RPS1)-RPS4, LEAF RUST RESISTANCE GENE10 (LR10)-RESISTANCE GENE ANALOGUE2 (RGA2), RICE BLAST RESISTANCE GENE AT PIK LOCUS1 (PIKM-1)-TS-PIKM2-TS, and RICE BLAST RESISTANCE GENE AT PI LOCUS1 (PI5-1)-PI5-2 in the genomes and their role in disease resistance suggests that these linked genes are required to effect a defense response in plants (Eitas and Dangl, 2010). The genomic pairing of the TNL genes with TX or TN genes suggests a role of the tightly linked TN protein in the function of its cognate TNL protein or proteins (and vice versa).The specific direct or indirect interaction between an R gene and a corresponding avirulence (Avr) gene in the characterized pairs of interaction resulted in an immune response in the form of localized programmed cell death, called the hypersensitive response (HR; Burch-Smith et al., 2007; Caplan et al., 2008). The recognition of avirulence proteins from pathogens by the cognate R proteins induces a cascade of changes that increases the levels of salicylic acid (SA), jasmonic acid (JA), phenyl ammonium lyase, and systemin (Liu et al., 2010). The production of several of these biochemical signals is known to trigger multiple convergent ‘R’-gene signaling pathways, leading to programmed cell death and further changes in gene expression patterns (Vlot et al., 2008a, 2008b). Structure function analysis of Arabidopsis R proteins RPS4 (Zhang et al., 2004) and RPP1A (Michael Weaver et al., 2006) have shown that TIR and NBS domains of the proteins without the LRR domain (TNL truncations) could be sufficient to induce HR. Studies using overexpression of plant R genes (particularly the truncated TNL genes) suggest that the TIR and NBS domains by themselves might be sufficient to induce HR and to initiate plant defense responses (Michael Weaver et al., 2006; Swiderski et al., 2009).In this study, we present experimental and computational data that are collectively consistent with a role for Arabidopsis TX and TN proteins in plant defenses. For example, the ability of the TX and TN genes to induce HR responses upon transient expression is dependent on ENHANCED DISEASE RESISTANCE1 (EDS1). This EDS1 dependency in induced HR was demonstrated in both tobacco (Nicotiana benthamiana) and in Arabidopsis. Stable transgenic overexpression in Arabidopsis of TX and TN genes resulted in a variety of phenotypes involved with basal innate immune responses that are dependent on SA. We also demonstrated the interaction of TX and TN proteins with plant pathogenic elicitor proteins and other plant signal transduction proteins.     

Abusive stimulation of excitatory amino acid receptors: a strategy to limit neurotoxicity

Glutamate is an important excitatory amino acid at many central nervous system synapses. After its release from presynaptic nerve terminals, glutamate transiently binds to specific neuronal membrane receptors, which transduce its signal by the generation of intracellular second messengers before being rapidly cleared from the synapse. However, during ischemia, the glutamate concentration at synapses surrounding the focal lesion can be increased for sustained periods of time, resulting in abusive stimulation of glutamate receptors that can eventually be neurotoxic. To develop drugs capable of selectively blocking the pathological effects of glutamate in neurons surrounding ischemic lesions while leaving the physiological actions of glutamate in nonlesioned areas of the brain unaffected, it is essential to delineate glutamate-induced intracellular events that are specific to receptor abuse. This article describes the intracellular sequelae of physiological and pathological glutamate receptor activation and suggests potential targets for such receptor abuse-dependent antagonists (RADAs).     

CXCR7 expression disrupts endothelial cell homeostasis and causes ligand-dependent invasion

The homeostatic function of endothelial cells (EC) is critical for a number of physiological processes including vascular integrity, immunity, and wound healing. Indeed, vascular abnormalities resulting from EC dysfunction contribute to the development and spread of malignancies. The alternative SDF-1/CXCL12 receptor CXCR7 is frequently and specifically highly expressed in tumor-associated vessels. In this study, we investigate whether CXCR7 contributes to vascular dysfunction by specifically examining the effect of CXCR7 expression on EC barrier function and motility. We demonstrate that CXCR7 expression in EC results in redistribution of CD31/PECAM-1 and loss of contact inhibition. Moreover, CXCR7+ EC are deficient in barrier formation. We show that CXCR7-mediated motility has no influence on angiogenesis but contributes to another motile process, the invasion of CXCR7+ EC into ligand-rich niches. These results identify CXCR7 as a novel manipulator of EC barrier function via alteration of PECAM-1 homophilic junctions. As such, aberrant expression of CXCR7 in the vasculature has the potential to disrupt vascular homeostasis and could contribute to vascular dysfunction in cancer systems.     

Olfactory Thresholds of the U.S. Population of Home-Dwelling Older Adults: Development and Validation of a Short,Reliable Measure

Current methods of olfactory sensitivity testing are logistically challenging and therefore infeasible for use in in-home surveys and other field settings. We developed a fast, easy and reliable method of assessing olfactory thresholds, and used it in the first study of olfactory sensitivity in a nationally representative sample of U.S. home-dwelling older adults. We validated our method via computer simulation together with a model estimated from 590 normosmics. Simulated subjects were assigned n-butanol thresholds drawn from the estimated normosmic distribution and based on these and the model, we simulated administration of both the staircase and constant stimuli methods. Our results replicate both the correlation between the two methods and their reliability as previously reported by studies using human subjects. Further simulations evaluated the reliability of different constant stimuli protocols, varying both the range of dilutions and number of stimuli (6–16). Six appropriately chosen dilutions were sufficient for good reliability (0.67) in normosmic subjects. Finally, we applied our method to design a 5-minute, in-home assessment of older adults (National Social Life, Health and Aging Project, or NSHAP), which had comparable reliability (0.56), despite many subjects having estimated thresholds above the strongest dilution. Thus, testing with a fast, 6-item constant stimuli protocol is informative, and permits olfactory testing in previously inaccessible research settings.