Low resolution crystal structure of hagfish insulin

Insulin from the Atlantic hagfish, Myxine glutinosa, crystallizes in space group P4₁2₁2 with a monomer in the asymmetric unit. The application of the Rossmann &; Blow (1962) rotation function, utilizing the known 2-zinc pig insulin crystal structure, has established the existence of an insulin dimer containing a crystallographic 2-fold axis. The position of the hagfish insulin molecule in the unit cell has been determined and a set of calculated phases derived. These are compared to phases found from isomorphous replacement studies. A 6 Å resolution electron density map has been calculated which shows the A and B chains are folded in a similar way to pig insulin and that the monomers are similarly organized into dimers.     

Alterations in mRNA levels, expression, and function of GTP-binding regulatory proteins in adipocytes from obese mice (C57BL/6J-ob/ob)

Messenger RNA levels for the alpha subunit of G-proteins expressed in adipocytes of lean and obese (ob/ob) mice were compared with relative levels of the encoded proteins. Using both toxin labeling and Western blots, expression of Gs alpha, Gi alpha-1, and Gi alpha-3 was decreased by approximately 2-fold in adipocytes of obese mice, while levels of Gi alpha-2 did not differ between the phenotypes. The decreases in Gi alpha-1 and Gs alpha in the obese mouse were attributed to decreased mRNA levels for these proteins. Similar mRNA levels for Gi alpha-3 were noted in both phenotypes, but Gi alpha-2 message was increased 2-fold in the obese mouse. Inhibitory regulation of adipocyte adenylylcyclase through G-proteins was evaluated by comparing the ability of R-PIA to inhibit isoproterenol-stimulated responses between the phenotypes. In spite of the decrease in Gi alpha-1 and Gi alpha-3 in adipocytes from obese mice, R-PIA inhibited adenylylcyclase, cAMP-dependent protein kinase, and lipolysis in similar fashion in both phenotypes. The GTP analog, Gpp(NH)p also inhibited forskolin-stimulated adenylylcyclase in a comparable manner, but the magnitude of the inhibition was slightly less in adipocyte membranes from obese mice. In contrast, the decrease in expression of Gs alpha was translated into substantially poorer activation of isoproterenol-stimulated responses in the obese mouse. The concentration of isoproterenol producing half-maximal activation of adenylylcyclase, protein kinase, and lipolysis did not differ between the phenotypes, but the maximal responses were much lower in cells from obese mice. Similar lipolytic potential in isolated adipocytes from each phenotype and similar total forskolin-stimulated cyclase activity in adipocyte membranes from each phenotype suggest that decreased expression of Gs alpha may contribute to the characteristic alteration in mobilization of triglycerides noted in adipocytes from obese mice.     

Epithelial to mesenchymal transition (EMT) of feto-maternal reproductive tissues generates inflammation: a detrimental factor for preterm birth

Human pregnancy is a delicate and complex process where multiorgan interactions between two independent systems, the mother, and her fetus, maintain pregnancy. Intercellular interactions that can define homeostasis at the various cellular level between the two systems allow uninterrupted fetal growth and development until delivery. Interactions are needed for tissue remodeling during pregnancy at both fetal and maternal tissue layers. One of the mechanisms that help tissue remodeling is via cellular transitions where epithelial cells undergo a cyclic transition from epithelial to mesenchymal (EMT) and back from mesenchymal to epithelial (MET). Two major pregnancy-associated tissue systems that use EMT, and MET are the fetal membrane (amniochorion) amnion epithelial layer and cervical epithelial cells and will be reviewed here. EMT is often associated with localized inflammation, and it is a well-balanced process to facilitate tissue remodeling. Cyclic transition processes are important because a terminal state or the static state of EMT can cause accumulation of proinflammatory mesenchymal cells in the matrix regions of these tissues and increase localized inflammation that can cause tissue damage. Interactions that determine homeostasis are often controlled by both endocrine and paracrine mediators. Pregnancy maintenance hormone progesterone and its receptors are critical for maintaining the balance between EMT and MET. Increased intrauterine oxidative stress at term can force a static (terminal) EMT and increase inflammation that are physiologic processes that destabilize homeostasis that maintain pregnancy to promote labor and delivery of the fetus. However, conditions that can produce an untimely increase in EMT and inflammation can be pathologic. These tissue damages are often associated with adverse pregnancy complications such as preterm prelabor rupture of the membranes (pPROM) and spontaneous preterm birth (PTB). Therefore, an understanding of the biomolecular processes that maintain cyclic EMT-MET is critical to reducing the risk of pPROM and PTB. Extracellular vesicles (exosomes of 40-160 nm) that can carry various cargo are involved in cellular transitions as paracrine mediators. Exosomes can carry a variety of biomolecules as cargo. Studies specifically using exosomes from cells undergone EMT can carry a pro-inflammatory cargo and in a paracrine fashion can modify the neighboring tissue environment to cause enhancement of uterine inflammation.     

Adenosine and dopamine receptor interactions in striatum and caffeine-induced behavioral activation

This review will examine how dopamine, a monoamine neurotransmitter, and adenosine, a neuromodulator, regulate behavioral activation, primarily as reflected by locomotor activity, in rodents. Complex interactions among 2 major types of adenosine receptors (A1AR and A2AAR) and 2 dopamine receptors (D1R and D2R) occur due to physical interactions that alter their ligand-binding properties and subsequent effects on common postreceptor signaling molecules. The output from these interactions in striatum modulates neurotransmission and subsequently influences spontaneous locomotor activity. Caffeine is a nonselective adenosine receptor antagonist that blocks 2 major types of adenosine receptors, A1AR and A2AAR, in the brain. Pharmacologic manipulation of these receptors with drugs such as caffeine offers potential therapeutic benefit for treatment of Parkinson disease.     

Ethnic differences in key candidate genes for spontaneous preterm birth: TNF-alpha and its receptors

OBJECTIVES: Spontaneous preterm birth (PTB) has a significant ethnic disparity with people of African descent having an almost 2-fold higher incidence than those of European descent in the United States. This disparity may be caused by differences in the distribution of genetic risk factors. The objective of this study is to examine genetic differences between African-Americans and European Americans for single nucleotide polymorphisms (SNPs) in candidate genes for PTB. METHODS: We examined patterns of variation in 19 SNPs in 3 candidate genes for preterm birth: TNF-alpha, TNF-receptor 1 and TNF-receptor 2. Allele, genotype and haplotype frequencies were compared between African-Americans (AA) and European-Americans (EA) in cases and controls separately. Both maternal and fetal genotypes were studied, as it is unclear whether one or both of these are important in the etiology of PTB. RESULTS: The vast majority of the SNPs differed significantly between ethnic groups, although there are only a few suggestive results comparing cases and controls within an ethnic group. For TNF-alpha, four of six SNPs; for TNF-R1, 5/6; and for TNF-R2, 6/7 showed significant differences between ethnic groups in either allele and/or genotype frequency. CONCLUSIONS: Our data demonstrate highly significant genetic differences between ethnic groups in genes that may play a role in the risk of PTB.     

Synthesis,stereochemistry and antimicrobial studies of novel oxime ethers of aza/diazabicycles

Two series of bicyclic oxime ethers viz, 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-one O-benzyloximes 13–24 and 2,4,6,8-tetraaryl-3,7-diazabicyclo[3.3.1]nonan-9-one O-benzyloximes 31–36 were synthesized and stereochemistry was established by their spectral (1D and 2D NMR) and crystal studies. Synthesized oxime ethers were screened for their in vitro antimicrobial activity against a set of pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Escherichia coli and Klebsiella pneumoniae) and fungi (Candida albicans, Candida-51, Rhizopus sp., Aspergillus niger and Aspergillus flavus) by twofold serial dilution method, respectively, using Ciprofloxacin and Amphotericin B as standards. Most of the molecules expressed promising antimicrobial profile against the tested pathogens and even a few compounds 16, 21, 22, 33 and 34 were better than standard drugs.     

Induction of HIV-1 resistance: cell susceptibility to infection is an inverse function of globotriaosyl ceramide levels

To examine the role of the glycosphingolipid (GSL), globotriaosylceramide(Gb₃, CD77, p^k blood group antigen) in HIV-1 infection, we havepharmacologically modulated Gb₃ metabolism in an X4 HIV-1 infectablemonocytic cell line (THP-1) that naturally expresses Gb₃ andin a Gb₃-expressing glioblastoma cell line (U87) transfectedto express both CD4 and CCR5 to permit R5 HIV-1 infection. THP-1and U87 cells were treated with either a competitive inhibitorof -galactosidase A, 1-deoxygalactonojirimycin (DGJ) to induceGb₃ accumulation, or a glucosylceramide synthase inhibitor,phenyl-2-palmitylamino-3-pyrrolidino-1-propanol (P4) to depletecells of Gb₃. HIV susceptibility was determined via measurementof p24^gag antigen production by ELISA. In addition, total cellularGb₃ content was determined using thin layer chromatography followedby Verotoxin1 overlay binding. The cell surface expression ofGb₃ was verified by FACS analysis. We found that DGJ significantlydecreased THP-1 and U87 cell susceptibility to HIV-1_IIIB andHIV-1_BaL infection, respectively, at a concentration of approximately100 µM. In contrast, P4 (2 µM) substantiallyincreased cellular susceptibility to HIV-1 infection. Totalcellular GSL analysis verified increased Gb₃ expression in cellstreated with DGJ and considerable reduction of Gb₃ in P4-treatedcells as compared to controls. These results show a reciprocalrelationship between Gb₃ expression and infection with eitherX4 HIV-1_IIIB or R5 HIV-1_Ba-L. These results support previousstudies that Gb₃ provides resistance to HIV infection. VariableGb₃ expression may provide a natural HIV resistance factor inthe general population, and pharmacological manipulation ofGb₃ levels may provide an approach to induction of HIV resistance.     

Primate-specific evolution of noncoding element insertion into PLA2G4C and human preterm birth

Background

The use of biological annotation such as genes and pathways in the analysis of gene expression data has aided the identification of genes for follow-up studies and suggested functional information to uncharacterized genes. Several studies have applied similar methods to genome wide association studies and identified a number of disease related pathways. However, many questions remain on how to best approach this problem, such as whether there is a need to obtain a score to summarize association evidence at the gene level, and whether a pathway, dominated by just a few highly significant genes, is of interest.

Methods

We evaluated the performance of two pathway-based methods (Random Set, and Binomial approximation to the hypergeometric test) based on their applications to three data sets of Crohn's disease. We consider both the disease status as a phenotype as well as the residuals after conditioning on IL23R, a known Crohn's related gene, as a phenotype.

Results

Our results show that Random Set method has the most power to identify disease related pathways. We confirm previously reported disease related pathways and provide evidence for IL-2 Receptor Beta Chain in T cell Activation and IL-9 signaling as Crohn's disease associated pathways.

Conclusions

Our results highlight the need to apply powerful gene score methods prior to pathway enrichment tests, and that controlling for genes that attain genome wide significance enable further biological insight.