Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A

Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. We conclude that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis.     

Retinal Vascular Caliber Is Associated with Cardiovascular Biomarkers of Oxidative Stress and Inflammation: The POLA Study

Purpose

Retinal vascular caliber has been linked with increased cardiovascular risk and is predictive of cardiovascular pathology, including stroke and coronary heart disease. Oxidative stress, as well as inflammatory mechanisms, plays a major role in the pathogenesis and progression of atherosclerosis, plaque rupture and vascular thrombotic propensity. The purpose of this study is to explore the relationship between retinal vascular calibers and biomarkers of oxidative stress and inflammation, in subjects free of cardiovascular pathology.

Patients and Methods

Cross-sectional analysis from a community-dwelling cohort comprising 1224 individuals aged 60 years and over, without a history of coronary or peripheral artery disease or stroke. Retinal vascular caliber was measured from fundus photographs using semi-automated standardized imaging software. Oxidative stress was evaluated using plasma superoxide dismutase 2 and glutathione peroxidase (GPx-3) activities, and inflammatory state was assessed using plasma high sensitivity C-reactive protein (hsCRP) and orosomucoid.

Results

In a multivariate model controlling for cardiovascular risk factors, larger retinal arteriolar caliber was independently related to higher level of GPx-3 activity (p = 0.003) whereas larger venular caliber was associated with higher levels of hsCRP (p = 0.0001) and orosomucoid (p = 0.01).

Conclusion

In the present study, biomarkers of oxidative stress regulation and inflammation were independently associated with retinal vascular calibers. This suggests that an assessment of retinal vessels may offer early and non-invasive detection of subclinical vascular pathology.     

Differing Epidemiological Dynamics of Influenza B Virus Lineages in Guangzhou,Southern China, 2009-2010

The epidemiological and evolutionary dynamics of the two cocirculating lineages of influenza B virus, Victoria and Yamagata, are poorly understood, especially in tropical or subtropical areas of Southeast Asia. We performed a phylogenetic analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of influenza B viruses isolated in Guangzhou, a southern Chinese city, during 2009 to 2010 and compared the demographic and clinical features of infected patients. We identified multiple viral introductions of Victoria strains from both Chinese and international sources, which formed two phylogenetically and antigenically distinct clades (Victoria 1 and 2), some of which persisted between seasons. We identified one dominant Yamagata introduction from outside China during 2009. Our phylogenetic analysis reveals the occurrence of reassortment events among the Victoria and Yamagata lineages and also within the Victoria lineage. We found no significant difference in clinical severity by influenza B lineage, with the exceptions that (i) the Yamagata lineage infected older people than either Victoria lineage and (ii) fewer upper respiratory tract infections were caused by the Victoria 2 than the Victoria 1 clade. Overall, our study reveals the complex epidemiological dynamics of different influenza B lineages within a single geographic locality and has implications for vaccination policy in southern China.     

Caffeine fostering of mycoparasitic fungi against phytopathogens

Caffeine (1,3,7-trimethixanthine) is a typical purine alkaloid produced in more than 80 plant species. Its biological role is considered to strengthen plant''s defense capabilities, directly as a toxicant to biotic attackers (allelopathy) and indirectly as an activator of defense system (priming). Caffeine is actively secreted into rhizosphere through primary root, and possibly affects the structure of microbe community nearby. The fungal community in coffee plant rhizosphere is enriched with particular species, including Trichoderma family, a mycoparasite that attacks and kills phytopathogens by coiling and destroying their hyphae. In the present study, the caffeine response of 8 filamentous fungi, 4 mycoparasitic Trichoderma, and 4 prey phytopathogens, was examined. Results showed that allelopathic effect of caffeine on fungal growth and development was differential, being stronger on pathogens than on Trichoderma species. Upon confronting, the prey immediately ceased the growth, whereas the predator continued to grow, indicating active mycoparasitism to have occurred. Caffeine enhanced mycoparasitism up to 1.7-fold. Caffeine thus functions in a double-track manner against fungal pathogens: first by direct suppression of growth and development, and second by assisting their natural enemy. These observations suggest that caffeine is a powerful weapon in the arms race between plants and pathogens by fostering enemy''s enemy, and we propose the idea of "caffeine fostering" as the third role of caffeine.     

Model of cellular mechanotransduction via actin stress fibers

Mechanical stresses due to blood flow regulate vascular endothelial cell structure and function and play a key role in arterial physiology and pathology. In particular, the development of atherosclerosis has been shown to correlate with regions of disturbed blood flow where endothelial cells are round and have a randomly organized cytoskeleton. Thus, deciphering the relation between the mechanical environment, cell structure, and cell function is a key step toward understanding the early development of atherosclerosis. Recent experiments have demonstrated very rapid (\(\sim \)100 ms) and long-distance (\(\sim \)10 \(\upmu \)m) cellular mechanotransduction in which prestressed actin stress fibers play a critical role. Here, we develop a model of mechanical signal transmission within a cell by describing strains in a network of prestressed viscoelastic stress fibers following the application of a force to the cell surface. We find force transmission dynamics that are consistent with experimental results. We also show that the extent of stress fiber alignment and the direction of the applied force relative to this alignment are key determinants of the efficiency of mechanical signal transmission. These results are consistent with the link observed experimentally between cytoskeletal organization, mechanical stress, and cellular responsiveness to stress. Based on these results, we suggest that mechanical strain of actin stress fibers under force constitutes a key link in the mechanotransduction chain.     

Mesostructure of fibrillar bovine serum albumin gels

The mesostructure of bovine serum albumin (BSA) at low pH was investigated. Rheological measurements were performed to determine the critical percolation concentration (c_p). A decreasing c_p with increasing ionic strength was found. Fibrils with a contour length of about 100–300 nm were found using transmission electron microscopy. The measured conversion of monomers into fibrils was independent of ionic strength (0.20–0.30 M). Dilution of BSA samples showed that the aggregation process is reversible and that there exists a critical concentration for the self-assembly of BSA. We explain the decreasing c_p with increasing ionic strength in terms of an adjusted random contact model.     

VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.     

Genes encoding catalytic subunits of protein kinase A and risk of spina bifida

BACKGROUND: PRKACA and PRKACB are genes encoding the cAMP-dependent protein kinase A (PKA) catalytic subunits alpha and beta, respectively. PKA is known to be involved in embryonic development, as it down-regulates the Hedgehog (Hh) signaling pathway, which is critical to normal pattern formation and morphogenesis. The PKA-deficient mouse model, which has only a single catalytic subunit, provided intriguing evidence demonstrating a relationship between decreased PKA activity and risk for posterior neural tube defects (NTDs) in the thoracic to sacral regions of gene-knockout mice. Unlike most other mutant mouse models of NTDs, the PKA-deficient mice develop spina bifida with 100% penetrance. We hypothesized that sequence variations in human genes encoding the catalytic subunits may alter the PKA activity and similarly increase the risk of spina bifida. METHODS: We sequenced the coding regions and the exon/intron boundaries of PRKACA and PRKACB. We also examined 3 common single-nucleotide polymorphisms (SNPs) of these 2 genes by allele discrimination. RESULTS: Five sequence variants in coding region and 2 intronic sequence variants proximal to exons were detected. None of the 3 SNPs examined in the association study appeared to be associated with substantially increased risk for spina bifida. CONCLUSIONS: Our results did not reveal a strong association between these PKA SNPs and spina bifida risk. Nonetheless, it is important to examine the possible gene-gene interactions between PRKACA and PRKACB when evaluating the risk for NTDs, as well as genes encoding regulatory subunits of PKA. In addition, interactions with other genes such as Sonic Hedgehog (SHH) should also be considered for future investigations.     

Production and purification of monoclonal antibodies to Pisum and Avena phytochrome

At least nine monoclonal antibodies against phytochrome from Pisum sativum L. and 20 against phytochrome from Avena sativa L. have been obtained from mouse hybridomas that were produced by fusion of spleen cells with SP 2/O-Ag14 myeloma cells. Hybridomas were selected and cloned in a single step by plating on a semisolid methylcellulose medium. Eight antibodies to Pisum and one to Avena phytochrome were immunopurified from hybridoma medium or ascitic fluid. When necessary, secreted antibodies were verified to be against phytochrome by demonstrating to be against phytochrome by demonstrating immunoadsorption of phytochrome, detected as loss of photoactivity and-or by appearance of the approx. 120,000-dalton phytochrome band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells †

Here we report on the related TBC/RabGAPs EPI64A and EPI64B and show that they function to organize the apical aspect of epithelial cells. EPI64A binds the scaffolding protein EBP50/NHERF1, which itself binds active ezrin in epithelial cell microvilli. Epithelial cells additionally express EPI64B that also localizes to microvilli. However, EPI64B does not bind EBP50 and both proteins are shown to have a microvillar localization domain that spans the RabGAP domains. CRISPR/Cas9 was used to inactivate expression of each protein individually or both in Jeg-3 and Caco2 cells. In Jeg-3 cells, loss of EPI64B resulted in a reduction of apical microvilli, and a further reduction was seen in the double knockout, mostly likely due to misregulation of Rab8 and Rab35. In addition, apical junctions were partially disrupted in cells lacking EPI64A and accentuated in the double knockout. In Caco2 loss of EPI64B resulted in wavy junctions, whereas loss of both EPI64A and EPI64B had a severe phenotype often resulting in cells with a stellate apical morphology. In the knockout cells, the basal region of the cell remained unchanged, so EPI64A and EPI64B specifically localize to and regulate the morphology of the apical domain of polarized epithelial cells.