An African Ancestry-Specific Allele of CTLA4 Confers Protection against Rheumatoid Arthritis in African Americans

Cytotoxic T-lymphocyte associated protein 4 (CTLA4) is a negative regulator of T-cell proliferation. Polymorphisms in CTLA4 have been inconsistently associated with susceptibility to rheumatoid arthritis (RA) in populations of European ancestry but have not been examined in African Americans. The prevalence of RA in most populations of European and Asian ancestry is ~1.0%; RA is purportedly less common in black Africans, with little known about its prevalence in African Americans. We sought to determine if CTLA4 polymorphisms are associated with RA in African Americans. We performed a 2-stage analysis of 12 haplotype tagging single nucleotide polymorphisms (SNPs) across CTLA4 in a total of 505 African American RA patients and 712 African American controls using Illumina and TaqMan platforms. The minor allele (G) of the rs231778 SNP was 0.054 in RA patients, compared to 0.209 in controls (4.462×10⁻²⁶, Fisher's exact). The presence of the G allele was associated with a substantially reduced odds ratio (OR) of having RA (AG+GG genotypes vs. AA genotype, OR 0.19, 95% CI: 0.13–0.26, p=2.4×10⁻²⁸, Fisher's exact), suggesting a protective effect. This SNP is polymorphic in the African population (minor allele frequency [MAF] 0.09 in the Yoruba population), but is very rare in other groups (MAF=0.002 in 530 Caucasians genotyped for this study). Markers associated with RA in populations of European ancestry (rs3087243 [+60C/T] and rs231775 [+49A/G]) were not replicated in African Americans. We found no confounding of association for rs231778 after stratifying for the HLA-DRB1 shared epitope, presence of anti-cyclic citrullinated peptide antibody, or degree of admixture from the European population. An African ancestry-specific genetic variant of CTLA4 appears to be associated with protection from RA in African Americans. This finding may explain, in part, the relatively low prevalence of RA in black African populations.     

Sequencing, cloning and expression of a β-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4

Abstract We report here the isolation of a Renibacterium salmoninarum DNA sequence capable of transforming a non-invasive Escherichia coli strain into a microorganism able to enter the fish cell line, CHSE-214. Immunofluorescence and electron microscopy techniques were used to assess the acquired invasive phenotype by HB101 E. coli cells, upon transformation with pPMV-189. This plasmid carries a 2282-bp R. salmoninarum DNA segment. The invasive phenotype is qonserved upon deletion of approximately 1000 bp at the 3' end of the insert. The remaining segment contains an ORF region encoding a putative protein of about 30 kDa.     

Proteomic Identification of DNA-PK Involvement within the RET Signaling Pathway

Constitutive activation of the Rearranged during Transfection (RET) proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC). The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.     

Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock

Interferon Regulatory Factor (IRF) 6, a member of the IRF family, is essential for epidermal and orofacial embryonic development. Irf6 is strongly expressed in keratinocytes, in which it regulates epidermal proliferation, differentiation, and migration. A recent role for Irf6 in Toll-like receptor 2-dependent chemokine gene expression was also reported in an epithelial cell line. However, a function for Irf6 in innate immune cells was not previously reported. In the present study, we investigated the expression and function of Irf6 in bone marrow-derived neutrophils and macrophages. We show here, using a conditional knockout of Irf6 in lysosymeM expressing cells, that Irf6 is required for resistance to LPS-induced endotoxic shock. In addition, Irf6-deficient bone marrow-derived neutrophils exhibited increased chemotactic index and velocity compared with wild-type cells in vitro. TLR4-specific KC and IL6 secretions were upregulated in Irf6-deficient bone marrow-derived macrophages in vitro. These cells also exhibited an increased level of phosphorylated IkBa. Collectively, our findings suggest a role for Irf6 in the resistance to endotoxic shock due to NFk-B-mediated alteration of cytokine production.     

ZEB1 Enhances Transendothelial Migration and Represses the Epithelial Phenotype of Prostate Cancer Cells

Metastatic colonization involves cancer cell lodgment or adherence in the microvasculature and subsequent migration of those cells across the endothelium into a secondary organ site. To study this process further, we analyzed transendothelial migration of human PC-3 prostate cancer cells in vitro. We isolated a subpopulation of cells, TEM4-18, that crossed an endothelial barrier more efficiently, but surprisingly, were less invasive than parental PC-3 cells in other contexts in vitro. Importantly, TEM4-18 cells were more aggressive than PC-3 cells in a murine metastatic colonization model. Microarray and FACS analysis of these cells showed that the expression of many genes previously associated with leukocyte trafficking and cancer cell extravasation were either unchanged or down-regulated. Instead, TEM4-18 cells exhibited characteristic molecular markers of an epithelial-to-mesenchymal transition (EMT), including frank loss of E-cadherin expression and up-regulation of the E-cadherin repressor ZEB1. Silencing ZEB1 in TEM4-18 cells resulted in increased E-cadherin and reduced transendothelial migration. TEM4-18 cells also express N-cadherin, which was found to be necessary, but not sufficient for increased transendothelial migration. Our results extend the role of EMT in metastasis to transendothelial migration and implicate ZEB1 and N-cadherin in this process in prostate cancer cells.     

Synthesis and evaluation of 3-aryl piperidine analogs as potent and efficacious dopamine D4 receptor agonists

A series of 3-aryl piperidine analogs with 2-piperidinoalkylamino or 2-piperidinoalkyloxy fused bicyclic rings were prepared and found to be potent and efficacious human dopamine D4 agonists. The synthesis and structure-activity relationship (SAR) studies that led to the identification of these compounds are discussed.     

Adrenergic control of coronary arteries

This review of adrenergic control of coronary arteries is based on studies conducted on isolated vessels. Both alpha- and beta-adrenergic receptors are present in the coronary vasculature but with different distributions. The large coronary arteries have a larger percentage of alpha receptors, which mediate contraction, whereas the small coronary arteries are equipped almost exclusively with beta receptors, which mediate relaxation. The beta receptors are of the subclass beta 1. Both alpha 1 and alpha 2 receptors are present in the large coronaries. The alpha 2 receptors are prejunctional and inhibit neuronal release of norepinephrine (NE), and are also postjunctional and mediate contraction of the vascular smooth muscle. Evidence is reviewed suggesting that coronary alpha and beta receptors may be different conformational states of the same parent macromolecule in which the alpha state predominates at lower temperatures. Contrary to these results we have observed that whereas the response of the large coronary artery to NE at 39 C is predominantly constriction, it is predominantly dilation at 29 C. The characteristic of the adrenergic receptor is clearly temperature dependent.     

Control of galactosyl-sugar metabolism in relation to rate of germination

The storage sugars stachyose and raffinose (galactosyl derivatives of sucrose) are metabolized early during germination of soybean [ Glycine max (L.) Merr.] seeds. The activities of four enzymes involved in the catabolism of these sugars were monitored in soybean cotyledons and embryonic axes during a 7-day germination period. An increase in enzyme activities correlated with a decline in galactosyl sugars. In embryonic axes, uridine diphosphate glucose (UDPglc)-hexose-l-P uridyltransferase (EC 2.7.7.12), an enzyme characteristic of the Leloir pathway, predominated over galactose-1-phosphate uridyltransferase (EC 2.7.7.10), an enzyme characteristic of the pyrophosphorylase pathway; whereas in cotyledons, the situation was reversed. There were differences between two cultivars. Ransom and Amsoy, in the levels of UDPglc-4-epimerase (EC 5.1.3.2); but not in glucose-1-phosphate uridyltransferase (EC 2 7.7.9). An accelerated aging treatment had a significant effect on the development of embryonic axes, as measured by dry weight. In vitro aging of seeds reduced the rate of growth and resulted in higher levels of galactose-containing sugars and significantly lower levels of UDPglc-hexose-l-P uridyltransferase. Thus, reduced development may be related to inability to mobilize or utilize stored carbon reserves. However, it has not been proved that the reduced enzyme activity is responsible for the effects of accelerated aging on growth and sugar metabolism.     

Mitochondrial mismatch analysis is insensitive to the mutational process

Mismatch distributions are histograms showing the pattern of nucleotide (or restriction) site differences between pairs of individuals in a sample. They can be used to test hypotheses about the history of population size and subdivision (if selective neutrality is assumed) or about selection (if a constant population size is assumed). Previous work has assumed that mutations never strike the same site twice, an assumption that is called the model of infinite sites. Fortunately, the results are surprisingly robust even when this assumption is violated. We show here that (1) confidence regions inferred using the infinite- sites model differ little from those inferred using a model of finite sites with uniform site-specific mutation rates, and (2) even when site- specific mutation rates follow a gamma distribution, confidence regions are little changed until the gamma shape parameter falls well below its plausible range, to roughly 0.01. In addition, we evaluate and reject the proposition that mismatch waves are produced by pooling data from several subdivisions of a structured population.