Relaxin-3 and receptors in the human and rhesus brain and reproductive tissues

Evidence suggests that relaxin-3 may have biological functions in the reproductive and central nervous systems. To date, however, relaxin-3 biodistribution has only been investigated in the mouse, rat, pig and teleost fish. Characterizing relaxin-3 gene structure, expression patterns, and function in non-human primates and humans is critical to delineating its biological significance. Experiments were performed to clone the rhesus macaque orthologues of the relaxin-3 peptide hormone and its cognitive receptors (RXFP1 and RXFP4). An investigation of rhesus relaxin-3 bioactivity and RXFP1 binding properties was also performed. Next we sought to investigate relaxin-3 immunoreactivity in human and rhesus macaque tissues. Immunohistofluorescence staining for relaxin-3 in the brain, testis, and prostate indicated predominant immunostaining in the ventral and dorsal tegmental nuclei, interstitial space surrounding the seminiferous tubules, and prostatic stromal cells, respectively. Further, in studies designed towards exploring biological functions, we observed neuroprotective actions of rhesus relaxin-3 on human neuronal cell cultures. Taken together, this study broadens the significance of relaxin-3 as a peptide involved in both neuronal cell function and reproductive tissues in primates.     

Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms,and the phylogenetic pattern of salinity preference in heterotrophic stramenopiles

Recent culture‐based studies demonstrate the distinctiveness of the microbial eukaryote biota of very hypersaline environments. In contrast, microscopy‐based faunistic studies suggest that the biota of habitats of more moderate hypersalinity (60–150‰) overlaps substantially with that of marine environments, but this has barely been studied with modern techniques. To investigate the diversity and salinity tolerance range of these organisms, eight cultures of heterotrophic stramenopiles were established from (or from nearby) moderately hypersaline locations. These isolates represent five independent groups; Groups A, B and C are bicosoecids; Groups D and E belong to Placididea. One isolate (Group A) is a strain of the widespread marine species Cafeteria roenbergensis, and cannot grow above 100‰ salinity. The other isolates – Groups B–E – can all grow at 150–175‰ salinities and are probably moderate halophiles. Groups B–E all represent previously unsequenced species or even genera, although Group B is the sister group of the borderline extreme halophile Halocafeteria. The high level of novelty en countered suggests that moderately hypersaline environments may harbour a heterotrophic stramenopile biota distinct from that of marine environments. Interestingly, our new isolates are all most closely related to marine or halophilic forms, and our phylogenies show large clades defined by saline/non‐saline habitats within bicosoecids, placidomonads and related lineages. In particular, most freshwater/soil bicosoecids form one well‐supported clade. The sole major exception is Bicosoeca, which is intermixed with marine environmental sequences originally referred to as ‘MAST‐13’, which are from brackish water, not typical seawater. It seems that the freshwater/marine barrier has been crossed very few times in the evolutionary history of these heterotrophic stramenopile flagellates.     

Coincident regulation of PKCdelta in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PKC (protein kinase C)d plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCd. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCd with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCd is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCd. Both thrombin and PMA induce recruitment of PKCd to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCd, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCd is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity.     

The Role of Climate in Settlement and Landscape Change in the North Atlantic Islands: An Assessment of Cumulative Deviations in High-Resolution Proxy Climate Records

In order to assess possible contributions of climate change to the human ecology of the North Atlantic islands we evaluate the utility of cumulative deviations from the mean, calculated for the Greenland ice core storm frequency proxy (GISP2 Na⁺) and sea ice proxy (GISP2 chloride excess). Our aim is to identify episodes of unpredictable change in the context of long-term trends of cultural and environmental development. Key changes are identified in the proxy climate records in 975 and 980 ad, 1025 and 1040 ad, 1180 ad, 1425 and 1450 ad, and 1520 and 1525 ad. Some of these changes are consistent with those inferred from new studies of the palaeoecological record of the Faroes. This indicates that the cumulative deviation measure could give greatest prominence to the most important climate changes affecting landscapes and settlement (such as the changes of 1425 and 1450 ad and their immediate aftermath), rather than extreme events, such as great single storms.     

A robust, target-driven, cell-based assay for checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1), a serine/threonine kinase, plays an important role in DNA damage checkpoint control and is an attractive target for cancer treatment. To develop a Chk1-specific cell-based assay, stable clones were established in which Chk1 kinase domain fused at its N-terminus with p53 through 4 tandem repeats of Gly-Gly-Gly-Gly-Ser was expressed in an inducible manner. Chk1 kinase specificity of the phosphorylation of fused p53 was confirmed by the experiments with a kinase-inactive Chk1. Only in the presence of an inducer molecule was phosphorylation of p53 at Ser-15 in the stable clones induced. Furthermore, its assay performance proved acceptable for high-throughput screening applications, judging from the Z' factor values (> 0.77). Finally, the cell-based assay thus established yielded structure-activity relationship data for a small set of test inhibitors of Chk1 within cells. Collectively, these results demonstrate that the established cell-based assay provides a novel and highly sensitive cellular platform for Chk1 inhibitor discovery.     

Characterisation of the genetic diversity of <Emphasis Type="Italic">Brucella</Emphasis> by multilocus sequencing

Background  

Brucella species include economically important zoonotic pathogens that can infect a wide range of animals. There are currently six classically recognised species of Brucella although, as yet unnamed, isolates from various marine mammal species have been reported. In order to investigate genetic relationships within the group and identify potential diagnostic markers we have sequenced multiple genetic loci from a large sample of Brucella isolates representing the known diversity of the genus.     

Enantiomers of naringenin as pleiotropic,stereoselective inhibitors of cytochrome P450 isoforms

Interactions between naringenin and the cytochrome P450 (CYP) system have been of interest since the first demonstration that grapefruit juice reduced CYP3A activity. The effects of naringenin on other CYP isoforms have been less investigated. In addition, it is well known that interactions with enzymes are often stereospecific, but due to the lack of readily available pure naringenin enantiomers, the enantioselectivity of its effects has not been characterized. We isolated pure naringenin enantiomers by chiral high‐performance liquid chromatography and tested the ability of (R)‐,(S)‐ and rac‐naringenin to inhibit several important drug‐metabolizing CYP isoforms using recombinant enzymes and pooled human liver microsomes. Naringenin was able to inhibit CYP19, CYP2C9, and CYP2C19 with IC₅₀ values below 5 μM. No appreciable inhibition of CYP2B6 or CYP2D6 was observed at concentrations up to 10 μM. Whereas (S)‐naringenin was 2‐fold more potent as an inhibitor of CYP19 and CYP2C19 than (R)‐naringenin, (R)‐naringenin was 2‐fold more potent for CYP2C9 and CYP3A. Chiral flavanones like naringenin are difficult to separate into their enantiomeric forms, but enantioselective effects may be observed that ultimately impact clinical effects. Inhibition of specific drug metabolizing enzymes by naringenin observed in vitro may be exploited to understand pharmacokinetic changes seen in vivo. Chirality, 2011. © 2011 Wiley‐Liss, Inc.