Subtilisin proprotein convertase-6 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation, a balance of factors regulates the invasive properties of the embryo and the anti-invasive properties of uterine decidua. Although antiproteinases such as the metalloproteinase inhibitor TIMP-3 are thought to play critical roles in preventing the overaggressive invasion of trophoblasts, the mechanism of antiproteinase regulation is unknown. Recently, the prohormone convertase SPC-6 has been found to be co-expressed in embryo-proximal decidua in association with TIMP-3. As members of this serine proteinase family are known to activate latent TGFbeta family members which regulate decidual TIMP-3 levels, we sought to characterize the expression of SPC-6 during pregnancy and artificial decidualization. In this study, we demonstrate that the zone of SPC-6 gene expression exhibits a great degree of temporal and spatial overlap with TIMP-3 gene expression in uterine decidua from E5.5 through to E8.5. Like TIMP-3, we demonstrate that SPC-6 expression is induced during the decidual cell response using an in vivo model of artificial decidualization. Both the secreted and membrane bound forms of SPC-6 are expressed throughout the period of decidualization, suggesting that SPC-6 may play multiple roles during this developmental period. This is confirmed by our observation of the movement of SPC-6 expression to the presumptive placental region, as TIMP-3 expression regresses at the implantation site.     

The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein-like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf.     

Chronic ethanol feeding impairs endothelin-1-stimulated glucose uptake via decreased G alpha 11 expression in rat adipocytes

Chronic ethanol feeding decreases insulin-stimulated glucose uptake in rat adipocytes. Here, we show that chronic ethanol also decreases endothelin-stimulated glucose uptake. Endothelin-1 increased uptake of 2-deoxyglucose 2.4-fold in adipocytes isolated from pair-fed rats. However, in adipocytes isolated from rats that had consumed a diet containing 35% ethanol for 4 wk, endothelin-1 did not increase glucose uptake. Although endothelin-1 increased GLUT4 quantity at the plasma membrane in adipocytes from pair-fed rats, there was no increase in GLUT4 after chronic ethanol feeding. Loss of endothelin-1-stimulated glucose uptake after ethanol feeding was associated with a specific decrease in the quantity of Galpha11 in plasma membranes, with no change in Galphaq quantity. Activation of proline-rich tyrosine kinase 2 (PYK2), a downstream target of Galphaq/11 that is required for endothelin-1-stimulated GLUT4 translocation in 3T3-L1 adipocytes, was also suppressed after chronic ethanol feeding. In contrast, activation of p38 MAPK by endothelin-1 was not affected by chronic ethanol exposure. These data demonstrate that chronic ethanol feeding suppresses endothelin-1-stimulated glucose uptake and suggest that decreased expression of Galpha11 coupled to impaired endothelin-1-dependent activation of PYK2 contributes to this response.     

Low Resistance to First and Second Line Anti-Tuberculosis Drugs among Treatment Naive Pulmonary Tuberculosis Patients in Southwestern Uganda

BackgroundThere are limited data on region-specific drug susceptibility of tuberculosis (TB) in Uganda. We performed resistance testing on specimens collected from treatment-naive patients with pulmonary TB in Southwestern Uganda for first and second line anti-TB drugs. We sought to provide data to guide regional recommendations for empiric TB therapy.MethodsArchived isolates, obtained from patients at Mbarara Regional Referral Hospital from February 2009 to February 2013, were tested for resistance to isoniazid and rifampicin using the MTBDRplus and Xpert MTB/RIF assays. A subset of randomly selected isolates was tested for second line agents, including fluoroquinolones (FQs), aminoglycosides, cyclic peptides, and ethambutol using the MTBDRsl assay. We performed confirmatory testing for FQ resistance using repeated MTBDRsl, the Mycobacteria growth indicator tube (MGIT) assay, and sequencing of the gyrA and gyrB genes.ResultsWe tested isolates from 190 patients. The cohort had a median age of 33 years (IQR 26-43), 69% (131/190) were male, and the HIV prevalence was 42% (80/190). No isolates (0/190) were rifampicin-resistant and only 1/190 (0.5%) was isoniazid-resistant. Among 92 isolates tested for second-line drug resistance, 71 (77%) had interpretable results, of which none were resistant to aminoglycosides, cyclic peptides or ethambutol. Although 7 (10%) initially tested as resistant to FQs by the MTBDRsl assay, they were confirmed as susceptible by repeat MTBDRsl testing as well as by MGIT and gyrase gene sequencingConclusionWe found no MDR-TB and no resistance to ethambutol, FQs, or injectable anti-TB drugs in treatment naïve patients with pulmonary TB in Southwestern Uganda. Standard treatment guidelines for susceptible TB should be adequate for most patients with TB in this population. Where possible, molecular susceptibility testing methods should be routinely validated by culture methods.     

Characterization of cellular DGK-θ

In this report we have summarized the data regarding the regulation of DGK-θ by two phospholipids: PtdSer and PtdOH. Our previous data has shown that stimulation of quiescent fibroblasts with a potent mitogen (α-thrombin) leads to an increase in nuclear localized DGK-θ ([Bregoli et al., 2001] and [Bregoli et al., 2002]), as has been seen in neuronal cells ([Tabellini et al., 2003] and [Tabellini et al., 2004]). Furthermore, these previous studies demonstrated that DGK-θ is actually localized to the nuclear matrix ([Bregoli et al., 2002] and [Tabellini et al., 2003]). As we have also previously shown that PtdSer and PtdOH modulate DGK-θ activity ([Tu-Sekine et al., 2006] and [Tu-Sekine et al., 2007]), we examined the phospholipid composition of the nuclear matrix as well as the phospholipid composition of the intact nuclei and non-nuclear membranes. This analysis has revealed that there are phospholipids in the “matrix” of nuclei and the composition of these lipids largely resembles those of the other membranes. The physical form and localization of the matrix-associated lipids has not been established, though the nearly identical percent composition of the non-nuclear membranes and the nuclear matrix lend credence to the hypothesis that at least some of the internal nuclear lipid is derived from invaginations of the cellular membranes through the nuclear interior known as nuclear tubules ([Fricker et al., 1997] and [Lee et al., 2006]).An important finding resulting from this analysis is that the nuclear envelope is enriched in PtdSer relative to the nuclear matrix and to cellular membranes, and that over-expression of DGK-θ reduces both the PtdSer and PtdEth levels of the nuclear envelope. While the mechanism behind these fluctuations is unknown, the data are consistent with a DGK-regulated phosphatidylcholine-dependent phosphatidylserine synthase (PSS-1) (EC 2.7.8.8) activity at the nuclear envelope. PtdSer synthesis in mammalian cells proceeds by headgroup exchange with PtdCho or PtdEth, catalyzed by PSS-1 or PSS-2, respectively. Interestingly, the decrease in both PtdSer and PtdEth at the nuclear membrane mirrors effects observed in the CHO-K1 mutant cell lines M.6.1.1 and PSA-3, which have been shown to be deficient in PSS-1 activity (Vance and Steenbergen, 2005). While there is very little information on the regulation of mammalian PSS enzymes, phosphorylation has been shown to regulate serine exchange activities in rat brain (Kanfer et al., 1988), and we have shown that nuclear DGK-θ regulates the exit of PKC-α from the nucleus (see regulation of PKC-α). To our knowledge, there is currently no reported study of nuclear phosphatidylyserine synthases.One other notable fluctuation in cellular lipids from DGK-θ expression is an increase in plasmalogen-PtdEth in both the NNM and nuclear matrix. While the majority of studies conducted on plasmalogen synthesis agree in that there is an almost complete dependence on the classical CDP-ethanolamine pathway to provide the headgroup for precursor plasmalogen synthesized in the peroxisome, there are several studies utilizing radiolabeled serine that report approximately 20–30% of cellular plasmalogen-PtdEth can be derived from PtdSer in at least some cell models ([Yorek et al., 1985] and [Xu et al., 1991]). Whether nuclear PtdSer-derived PtdEth a precursor for the membrane plasmalogen-PtdEth present in DGK-θ expressing cells is not evident from the data presented here, though one potential mechanism for an increase in flux through this pathway could be an increase in PtdSer-derived PtdEth. At this juncture we can only speculate on the cause of the lipid perturbations, and further work is required to support our hypothesis that DGK-θ impacts PtdSer and plasmalogen metabolism.The physiological role for nuclear DGK-θ has remained a mystery. It has long been recognized that an obvious role for DGKs is to modulate cellular levels of its DAG substrate thereby modulating DAG-sensitive proteins like such as many PKCs (Merida et al., 2008). Our data support this notion as suppression of DGK-θ, either by expression of RhoA or a dominant-negative construct of DGK-θ, leads to a sustained increase in nuclear PKC-α (Fig. 2).In addition to the regulation by PtdSer and PtdOH, we report here that the regulation of nuclear DGK-θ by RhoA is modulated by a Class I PI 3-kinase. Furthermore, we have examined the effect of various inhibitors on the activity of DGK-θ in lysates over-expressing this isoform. This is important as many studies often use pharmacologic inhibitors to determine the role of selected enzymes in signaling pathways. While this approach is useful for comparative analyses, there are no data pertaining to the effect of these inhibitors on DGK-θ. We have found that one well-established PI-PLC inhibitor, U73122, but not its inactive analog U73343, competitively inhibits this isoform with respect to its diacylglycerol (DAG) substrate. These data indicate that studies designed to examine the role of PI-PLC in modulating DGK-θ activity using this inhibitor should be interpreted with caution.     

Structure-Function Analysis of CCL28 in the Development of Post-viral Asthma

CCL28 is a human chemokine constitutively expressed by epithelial cells in diverse mucosal tissues and is known to attract a variety of immune cell types including T-cell subsets and eosinophils. Elevated levels of CCL28 have been found in the airways of individuals with asthma, and previous studies have indicated that CCL28 plays a vital role in the acute development of post-viral asthma. Our study builds on this, demonstrating that CCL28 is also important in the chronic post-viral asthma phenotype. In the absence of a viral infection, we also demonstrate that CCL28 is both necessary and sufficient for induction of asthma pathology. Additionally, we present the first effort aimed at elucidating the structural features of CCL28. Chemokines are defined by a conserved tertiary structure composed of a three-stranded β-sheet and a C-terminal α-helix constrained by two disulfide bonds. In addition to the four disulfide bond-forming cysteine residues that define the traditional chemokine fold, CCL28 possesses two additional cysteine residues that form a third disulfide bond. If all disulfide bonds are disrupted, recombinant human CCL28 is no longer able to drive mouse CD4+ T-cell chemotaxis or in vivo airway hyper-reactivity, indicating that the conserved chemokine fold is necessary for its biologic activity. Due to the intimate relationship between CCL28 and asthma pathology, it is clear that CCL28 presents a novel target for the development of alternative asthma therapeutics.     

Conspecific and heterospecific attraction in assessments of functional connectivity

Functional connectivity is known to have an important, positive influence on species persistence. Measurements of functional connectivity traditionally focus on structural attributes of landscapes such as the distance and matrix type between habitat patches as well as on how species interact with those structural attributes. However, we propose that the social behavior of a species, through conspecific and heterospecific attraction, will also impact connectivity by changing how dispersers move with respect to each other and occupied patches. We analyzed functional connectivity patterns using circuit and graph theory for golden-headed lion tamarins (Leontopithecus chrysomelas) in Brazil under three scenarios. In the first scenario, we looked at connectivity without the effects of attraction under varying maximum dispersal distance and ecological movement cost thresholds. In the second scenario, we allowed dispersers to travel over more hostile matrix than they normally would to reach an occupied patch. In the final scenario, we allowed dispersers to move only to occupied patches. We found that, according to the first scenario, range-wide functional landscape connectivity for golden-headed lion tamarins is low at realistic maximum dispersal distance and movement cost thresholds. Incorporating the effects of conspecific or heterospecific attraction would increase functional connectivity, in the case of scenario two, or decrease functional connectivity, in the case of scenario three. Because conspecific/heterospecific attraction can have an impact on movement for some species, this factor should be incorporated in assessments of functional connectivity patterns for social species and others where patch occupancy is likely to influence the movements of dispersers.