Platelet activating factor induces transient blood‐brain barrier opening to facilitate edaravone penetration into the brain

The blood–brain barrier (BBB) greatly limits the efficacy of many neuroprotective drugs' delivery to the brain, so improving drug penetration through the BBB has been an important focus of research. Here we report that platelet activating factor (PAF) transiently opened BBB and facilitated neuroprotectant edaravone penetration into the brain. Intravenous infusion with PAF induced a transient BBB opening in rats, reflected by increased Evans blue leakage and mild edema formation, which ceased within 6 h. Furthermore, rat regional cerebral blood flow (rCBF) declined acutely during PAF infusion, but recovered slowly. More importantly, this transient BBB opening significantly increased the penetration of edaravone into the brain, evidenced by increased edaravone concentrations in tissue interstitial fluid collected by microdialysis and analyzed by Ultra‐performance liquid chromatograph combined with a hybrid quadrupole time‐of‐flight mass spectrometer (UPLC‐MS/MS). Similarly, incubation of rat brain microvessel endothelial cells monolayer with 1 μM PAF for 1 h significantly increased monolayer permeability to ¹²⁵I‐albumin, which recovered 1 h after PAF elimination. However, PAF incubation with rat brain microvessel endothelial cells for 1 h did not cause detectable cytotoxicity, and did not regulate intercellular adhesion molecule‐1, matrix‐metalloproteinase‐9 and P‐glycoprotein expression. In conclusion, PAF could induce transient and reversible BBB opening through abrupt rCBF decline, which significantly improved edaravone penetration into the brain.

     

Clue to a New Deafness Gene: A Large Chinese Nonsyndromic Hearing Loss Family Linked to DFNA4

Hereditary hearing loss is one of the most common neurosensory defects in humans.Approximately 70％ of cases are nonsyndromic and could be inherited in autosomal dominant,autosomal recessive,mitochondrial,X-linked,and Y-linked manners (Wang et al.,2004;Alford,2011).The autosomal dominant type,comprising 15％-20％ of nonsyndromic hearing loss,is monogenic and genetically heterogeneous.Since the first dominant deafness locus (DFNA1) was identified in 1992,a total of 64 DFNA loci have been mapped (DFNA1-DFNA64),and 27 corresponding genes have been identified (http://hereditaryhearingloss.org).Previous studies have revealed that one deafness locus can be linked to more than one gene (Bayazit and Yilmaz,2006),and the question "one locus,how many genes?" was first raised about a decade ago (Van-Hauwe et al.,1999).So far,several loci,including DFNA2 and DFNA3,have been shown to be related to one or more genes,showing high genetic heterogeneity in hereditary hearing loss (Grifa et al.,1999;Goldstein and Lalwani,2002;Yan et al.,2011).     

The Role of Physical Activity in Harm Reduction among Betel Quid Chewers from a Prospective Cohort of 419,378 Individuals

ObjectiveTo assess the benefits of regular exercise in reducing harms associated with betel quid (BQ) chewing.MethodsThe study cohort, 419,378 individuals, participated in a medical screening program between 1994 and 2008, with 38,324 male and 1,495 female chewers, who consumed 5–15 quids of BQ a day. Physical activity of each individual, based on “MET-hour/week”, was classified as “inactive” or “active”, where activity started from a daily 15 minutes/day or more of brisk walking (≥3.75 MET-hour/week). Hazard ratios for mortality and remaining years in life expectancy were calculated.ResultsNearly one fifth (18.7%) of men, but only 0.7% of women were chewers. Chewers had a 10-fold increase in oral cancer risk; and a 2-3-fold increase in mortality from lung, esophagus and liver cancer, cardiovascular disease, and diabetes, with doubling of all-cause mortality. More than half of chewers were physically inactive (59%). Physical activity was beneficial for chewers, with a reduction of all-cause mortality by 19%. Inactive chewers had their lifespan shortened by 6.3 years, compared to non-chewers, but being active, chewers improved their health by gaining 2.5 years. The improvement, however, fell short of offsetting the harms from chewing.ConclusionsChewers had serious health consequences, but being physically active, chewers could mitigate some of these adverse effects, and extend life expectancy by 2.5 years and reduce mortality by one fifth. Encouraging exercise, in addition to quitting chewing, remains the best advice for 1.5 million chewers in Taiwan.     

Association between BMP15 Gene Polymorphism and Reproduction Traits and Its Tissues Expression Characteristics in Chicken

BMP15 (Bone morphogenetic protein 15) is an oocyte-secreted growth factor required for ovarian follicle development and ovulation in mammals, but its effects on reproduction in chickens are unclear. In this study, the association between BMP15 polymorphisms and reproduction traits were analyzed, and its expression characteristics in different tissues were explored in LaiWu Black chickens. Three single nucleotide polymorphisms (SNPs) were identified in four hundred LaiWu Black chickens. One SNP (NC_006091.3:g.1773T>C) located in exon 2 which was significantly associated with egg weight at first egg (EWFE) (P = 0.0389), was novel. Diplotypes based on the three SNPs were found to be significantly associated with egg weight at age of 43W (EW43) (P = 0.0058). The chickens with H3H3 diplotype had their first egg 0.57 days later than chickens with H5H5 diplotype and 1.21 days-3.96 days earlier than the other five diplotype chickens. The egg production at age of 43W (E43), egg production at age of 46W (E46) and egg production at age of 48W (E48) for chickens with H3H3 diplotype were the highest among all the chickens, and the E48 of chickens with H3H3 diplotype had 11.83 eggs higher than chickens with H1H5 diplotype. RT-qPCR results showed that the expression level of BMP15 gene in ovarian follicle was in the order of 4 mm>6 mm -8 mm> 15 mm -19 mm> 23 mm -29 mm > 33 mm -34 mm in diameter. The mRNA level in follicles of 4 mm and 6–8 mm in diameter were significantly higher than that in the other follicles (P<0.01). In the same week, the highest mRNA level was found in the ovary, and it was significantly different from that found in the liver and oviduct (P<0.01). Our results indicate that BMP15 plays a vital role in the development of ovary and follicles, especially in the development of primary follicles. H3H3 may be an potential advantageous molecular marker for improving reproduction traits in chickens.     

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework.     

NF-kappa B regulation by I kappa B kinase-2 in rheumatoid arthritis synoviocytes

IkappaB kinase-1 and IkappaB kinase-2 (IKK1 and IKK2; also called IKKalpha and IKKbeta, respectively) are part of the signal complex that regulates NF-kappaB activity in many cell types, including fibroblast-like synoviocytes (FLS). We determined which of these two kinases is responsible for cytokine-induced NF-kappaB activation in synoviocytes and assessed the functional consequences of IKK1 or IKK2 overexpression and inhibition. FLS were infected with adenovirus constructs encoding either wild-type (wt) IKK1 or IKK2, the dominant negative (dn) mutant of both kinases, or a control construct encoding green fluorescence protein. Analysis of the NF-kappaB pathway revealed that cytokine-induced IKK activation, IkappaB degradation, and NF-kappaB activation was prevented in cells expressing the IKK2 dn mutant, whereas baseline NF-kappaB activity was increased by IKK2 wt. In addition, synthesis of IL-6 and IL-8, as well as expression of ICAM-1 and collagenase, was only increased by IKK2 wt, and their cytokine-induced production was abrogated by IKK2 dn mutant. However, the IKK1 dn mutant did not inhibit cytokine-mediated activation of NF-kappaB or any of the functional assays. These data indicate that IKK2 is the key convergence pathway for cytokine-induced NF-kappaB activation. Furthermore, IKK2 regulates adhesion molecule, matrix metalloproteinase, and cytokine production in FLS.     

Up-Regulation and redistribution of Bax in ultraviolet B-irradiated melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0-30 mJ/cm2). The levels of p53, Bax, and Bcl-2, determined by immunoblotting, revealed a dose-dependent increase in p53 and Bax, but the level of Bcl-2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl-2. These data suggest that the high constitutional level of Bcl-2 may protect melanocytes from UVB-induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.