Plasma BDNF and PDGF-AA levels are associated with high TCD velocity and stroke in children with sickle cell anemia

Sickle cell anemia (SCA) associated cerebrovascular disease includes vascular remodeling, abnormal cerebral blood flow (CBF) and infarction. We studied the relationships between plasma brain derived neurotropic factor (BDNF), platelet derived growth factors (PDGF-AA and -AB/BB) and high trans-cranial Doppler (TCD) velocity, an indication of CBF velocity. Baseline plasma samples from 39 children (19 SCA with abnormal/high TCD [SATCD], 13 SCA with normal TCD [SNTCD] and 7 healthy non-SCA), were assayed for BDNF, PDGF-AA and -AB/BB plus 11 other cytokines. The sensitivity, specificity and usefulness of these biomarkers for stroke prediction was investigated. All subject groups were of similar age and gender distribution. Mean BDNF was significantly higher among SATCD than SNTCD (p=0.004) as was mean PDGF-AA (p=0.001). Similarly, mean PDGF-AA was higher among SCA subjects who developed stroke than those who did not (p=0.012). Elevated BDNF and PDGF-AA were good predictors of the presence of abnormally high CBF velocity and were both associated with severity of anemia. Elevated PDGF-AA predicted risk for stroke development. Stroke incidence and high TCD velocity were associated with elevated BDNF and PDGF-AA. These findings suggest a role for BDNF and PDGF-AA in the patho-physiological mechanism of cerebrovascular disease in SCA.     

A microplate-based evaluation of complex denaturation pathways: structural stability of Escherichia coli transketolase

We have previously developed a rapid microplate-based approach for measuring the denaturation curves by intrinsic tryptophan fluorescence for simple monomeric and two-state unfolding proteins. Here we demonstrate that it can accurately resolve the multiple conformational transitions that occur during the denaturation of a complex multimeric and cofactor associated protein. We have also analyzed the effect of two active-site mutations, D381A and Y440A upon the denaturation pathway of transketolase using intrinsic fluorescence measurements, and we compare the results from classical and microplate-based instrumentation. This work shows that the rapid assay is able to identify changes in the denaturation pathway, due to mutations or removal of cofactors, which affect the stability of the native and intermediate states. This would be of significant benefit for the directed evolution of protein stability, optimizing enzyme stability under biocatalytic process conditions, and also for engineering specific transitions in protein unfolding pathways.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

NM23-H2, an estrogen receptor beta-associated protein, shows diminished expression with progression of atherosclerosis

While estrogen receptor (ER) profile plays an important role in response to estrogens, receptor coregulators act as critical determinants of signaling. Although the clinical effects of ovarian hormones on various normal and pathological processes are an active area of research, the exact signaling effects on, for example, the vessel wall, are incompletely understood. Hence, we sought to discover proteins that associate with ERbeta, the isoform that shows upregulated mRNA expression after arterial injury. Using a yeast two-hybrid screen we identified NM23-H2, a multifaceted metastasis suppressor candidate protein, as an ERbeta-associated protein. Although NM23-H2 was immunodetected in arteries from young subjects (27 +/- 6 yr, 14 men and 6 women) with benign intimal hyperplasia, expression was diminished in fatty streaks/atheromas and altogether absent in advanced atherosclerotic lesions. Both nm23-H2 mRNA and protein were expressed by vascular cells in vitro. Treatment with 17beta-estradiol and an ERbeta-selective agonist, diarylpropionitrile, increased protein expression of NM23-H2; an effect that was not seen with an ERalpha-selective agonist, propylpyrazole-triol. Estrogen also prompted nuclear localization of NM23-H2 protein in human coronary smooth muscle cells (SMCs). An in vitro mimic of inflammation decreased the expression of NM23-H2 in SMCs, which was restored on addition of estrogen and dependent on the estrogen receptor. In summary, we report the novel association of NM23-H2 with ERbeta and show for the first time its expression in vascular cells and demonstrate regulation of its expression and localization by estrogen. In that the abundance of NM23-H2 diminishes with both the advancement of atherosclerosis and inflammation, this ERbeta-associated protein may play an important role in mediating the vasculoprotective effects of estrogens.     

Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.     

Human RAD18 interacts with ubiquitylated chromatin components and facilitates RAD9 recruitment to DNA double strand breaks

RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2.     

Actin is not an essential component in the mechanism of calcium-triggered vesicle fusion

Actin has been suggested as an essential component in the membrane fusion stage of exocytosis. In some model systems disruption of the actin filament network associated with exocytotic membranes results in a decrease in secretion. Here we analyze the fast Ca2+-triggered membrane fusion steps of regulated exocytosis using a stage-specific preparation of native secretory vesicles (SV) to directly test whether actin plays an essential role in this mechanism. Although present on secretory vesicles, selective pharmacological inhibition of actin did not affect the Ca2+-sensitivity, extent, or kinetics of membrane fusion, nor did the addition of exogenous actin or an anti-actin antibody. There was also no discernable affect on inter-vesicle contact (docking). Overall, the results do not support a direct role for actin in the fast, Ca2+-triggered steps of regulated membrane fusion. It would appear that actin acts elsewhere within the exocytotic cycle.     

Diet-induced obesity in rats leads to a decrease in sperm motility

Background  

Obesity is rapidly becoming a worldwide epidemic that affects children and adults. Some studies have shown a relationship between obesity and infertility, but until now it remains controversial. Thus, the aim of the present study was to investigate the effect of high-fat diet-induced obesity on male reproductive parameters.