Haemonchus contortus: prokaryotic expression and enzyme activity of recombinant HcSTK, a serine/threonine protein kinase

Members of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily are important regulators of the cytoskeleton, and their characterization can provide insights into a number of critical processes relating to the development and survival of an organism. We previously investigated the mRNA expression for and organization of a gene (hcstk) representing HcSTK, an STK from the parasitic nematode Haemonchus contortus. In the present study, a recombinant form of HcSTK was expressed and characterized. Affinity-purified anti-HcSTK antibodies reacted with native HcSTK in protein homogenates extracted from third-stage larvae (L3) of H. contortus and were also used to immunolocalize the protein around the nuclei of ovarian and intestinal tissues of adult H. contortus. The enzyme activity of the recombinant HcSTK protein was also demonstrated. The findings show that recombinant HcSTK is a functional protein kinase, with activity directed to KXGS motifs, consistent with other members of the PAR-1/MARK STK subfamily.     

A Non-Synonymous Single Nucleotide Polymorphism in an OPRM1 Splice Variant Is Associated with Fentanyl-Induced Emesis in Women Undergoing Minor Gynaecological Surgery

Background

Fentanyl-induced emesis (FIE) is a distressing adverse effect in the postoperative setting. The genetic basis of FIE remains largely unknown, therefore, we examined whether it was associated with specific genetic variants of OPRM1, the gene encoding the main receptor target of fentanyl.

Methods

In this prospective case-control study, 193 women undergoing gynaecological surgery under a standardized anaesthetic with a low emetogenic risk were enrolled. Inclusion and exclusion criteria were designed to select extreme phenotypes as well as to ensure that most major confounders for FIE were either excluded or present in all patients. To control for unforeseen intra- and postoperative confounders for FIE, only 161 patients were further analysed, out of which 10 were categorized as having FIE, defined by the presence of at least one of three symptoms: nausea, vomiting or retching that was likely to be fentanyl-related. To identify SNPs relevant to FIE in our population, DNA from 40 controls and 10 cases was sequenced at the following OPRM1 regions: 3 kbp of the promoter, main and alternative exons as well as 2 kbp of the 3′ downstream region. The genotype of the significant SNP was further determined in the remaining 111 controls.

Results

The incidence of FIE was 6.2%. Initial sequencing of 10 cases and 40 controls identified 25 SNPs. Only rs540825, a non-synonymous SNP in the splice variant, MOR1X, showed a significant association with FIE post-Bonferroni correction. This SNP was further examined in the remaining 111 controls which confirmed its significant association with FIE (p = 0.019 post-Bonferroni, OR: 5.6, 95% CI: 1.42–21.91).

Conclusions

This is the first report of an association between the occurrence of FIE in Chinese women undergoing gynaecological surgery and an OPRM1 splice variant SNP, rs540825.     

Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells.

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 microm and 0.5-1.5 microm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.     

Structural and Functional Divergence within the Dim1/KsgA Family of rRNA Methyltransferases

The enzymes of the KsgA/Dim1 family are universally distributed throughout all phylogeny; however, structural and functional differences are known to exist. The well-characterized function of these enzymes is to dimethylate two adjacent adenosines of the small ribosomal subunit in the normal course of ribosome maturation, and the structures of KsgA from Escherichia coli and Dim1 from Homo sapiens and Plasmodium falciparum have been determined. To this point, no examples of archaeal structures have been reported. Here, we report the structure of Dim1 from the thermophilic archaeon Methanocaldococcus jannaschii. While it shares obvious similarities with the bacterial and eukaryotic orthologs, notable structural differences exist among the three members, particularly in the C-terminal domain. Previous work showed that eukaryotic and archaeal Dim1 were able to robustly complement for KsgA in E. coli. Here, we repeated similar experiments to test for complementarity of archaeal Dim1 and bacterial KsgA in Saccharomyces cerevisiae. However, neither the bacterial nor the archaeal ortholog could complement for the eukaryotic Dim1. This might be related to the secondary, non-methyltransferase function that Dim1 is known to play in eukaryotic ribosomal maturation. To further delineate regions of the eukaryotic Dim1 critical to its function, we created and tested KsgA/Dim1 chimeras. Of the chimeras, only one constructed with the N-terminal domain from eukaryotic Dim1 and the C-terminal domain from archaeal Dim1 was able to complement, suggesting that eukaryotic-specific Dim1 function resides in the N-terminal domain also, where few structural differences are observed between members of the KsgA/Dim1 family. Future work is required to identify those determinants directly responsible for Dim1 function in ribosome biogenesis. Finally, we have conclusively established that none of the methyl groups are critically important to growth in yeast under standard conditions at a variety of temperatures.     

Genetic Manipulation of Palmitoylethanolamide Production and Inactivation in Saccharomyces cerevisiae

Background

Lipids can act as signaling molecules, activating intracellular and membrane-associated receptors to regulate physiological functions. To understand how a newly discovered signaling lipid functions, it is necessary to identify and characterize the enzymes involved in their production and inactivation. The signaling lipid N-palmitoylethanolamine (PEA) is known to activate intracellular and membrane-associated receptors and regulate physiological functions, but little is known about the enzymes involved in its production and inactivation.

Principal Findings

Here we show that Saccharomyces cerevisiae produce and inactivate PEA, suggesting that genetic manipulations of this lower eukaryote may be used to identify the enzymes involved in PEA metabolism. Accordingly, using single gene deletion mutants, we identified yeast genes that control PEA metabolism, including SPO14 (a yeast homologue of the mammalian phospholipase D) that controls PEA production and YJU3 (a yeast homologue of the mammalian monoacylglycerol lipase) that controls PEA inactivation. We also found that PEA metabolism is affected by heterologous expression of two mammalian proteins involved in neurodegenerative diseases, namely huntingtin and α-synuclein.

Significance

Together these findings show that forward and reverse genetics in S. cerevisiae can be used to identify proteins involved in PEA production and inactivation, and suggest that mutated proteins causing neurodegenerative diseases might affect the metabolism of this important signaling lipid.     

L<Subscript>2</Subscript>-norm multiple kernel learning and its application to biomedical data fusion

Background  

This paper introduces the notion of optimizing different norms in the dual problem of support vector machines with multiple kernels. The selection of norms yields different extensions of multiple kernel learning (MKL) such as L _∞, L ₁, and L ₂ MKL. In particular, L ₂ MKL is a novel method that leads to non-sparse optimal kernel coefficients, which is different from the sparse kernel coefficients optimized by the existing L _∞ MKL method. In real biomedical applications, L ₂ MKL may have more advantages over sparse integration method for thoroughly combining complementary information in heterogeneous data sources.     

Characterization of TonB Interactions with the FepA Cork Domain and FecA N-terminal Signaling Domain

The mechanism of TonB dependent siderophore uptake through outer membrane transporters in Gram-negative bacteria is poorly understood. In an effort to expand our knowledge of the interaction between TonB and the outer membrane transporters, we have cloned and expressed the FepA cork domain (11–154) from Salmonella typhimurium and characterized its interaction with the periplasmic C-terminal domain of TonB (103–239) by isotope assisted FTIR and NMR spectroscopy. For comparison we also performed similar experiments using the FecA N-terminal domain (1–96) from Escherichia coli which includes the conserved TonB box. The FepA cork domain was completely unfolded in solution, as observed for the E. coli cork domain previously [Usher et al. (2001) Proc Natl Acad Sci USA 98, 10676–10681]. The FepA cork domain was found to bind to TonB, eliciting essentially the same chemical shift changes in TonB C-terminal domain as was observed in the presence of TonB box peptides. The FecA construct did not cause this same structural change in TonB. The binding of the FepA cork domain to TonB-CTD was found to decrease the amount of ordered secondary structure in TonB-CTD. It is likely that the FecA N-terminal domain interferes with TonB-CTD binding to the TonB box. Binding of the FepA cork domain induces a loss of secondary structure in TonB, possibly exposing TonB surface area for additional intermolecular interactions such as potential homodimerization or additional interactions with the barrel of the outer membrane transporter.