Regulation of the interaction between PIPKI gamma and talin by proline-directed protein kinases

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2beta). Both PtdIns-3-P and PI3K-C2beta are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2beta as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2beta-PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.     

Cobalt(II) Oxidation by the Marine Manganese(II)-Oxidizing Bacillus sp. Strain SG-1

The geochemical cycling of cobalt (Co) has often been considered to be controlled by the scavenging and oxidation of Co(II) on the surface of manganese [Mn(III,IV)] oxides or manganates. Because Mn(II) oxidation in the environment is often catalyzed by bacteria, we have investigated the ability of Mn(II)-oxidizing bacteria to bind and oxidize Co(II) in the absence of Mn(II) to determine whether some Mn(II)-oxidizing bacteria also oxidize Co(II) independently of Mn oxidation. We used the marine Bacillus sp. strain SG-1, which produces mature spores that oxidize Mn(II), apparently due to a protein in their spore coats (R.A. Rosson and K. H. Nealson, J. Bacteriol. 151:1027-1034, 1982; J. P. M. de Vrind et al., Appl. Environ. Microbiol. 52:1096-1100, 1986). A method to measure Co(II) oxidation using radioactive ⁵⁷Co as a tracer and treatments with nonradioactive (cold) Co(II) and ascorbate to discriminate bound Co from oxidized Co was developed. SG-1 spores were found to oxidize Co(II) over a wide range of pH, temperature, and Co(II) concentration. Leucoberbelin blue, a reagent that reacts with Mn(III,IV) oxides forming a blue color, was found to also react with Co(III) oxides and was used to verify the presence of oxidized Co in the absence of added Mn(II). Co(II) oxidation occurred optimally around pH 8 and between 55 and 65°C. SG-1 spores oxidized Co(II) at all Co(II) concentrations tested from the trace levels found in seawater to 100 mM. Co(II) oxidation was found to follow Michaelis-Menten kinetics. An Eadie-Hofstee plot of the data suggests that SG-1 spores have two oxidation systems, a high-affinity-low-rate system (K_m, 3.3 × 10^-8 M; V_max, 1.7 × 10^-15 M · spore^-1 · h^-1) and a low-affinity-high-rate system (K_m, 5.2 × 10^-6 M; V_max, 8.9 × 10^-15 M · spore^-1 · h^-1). SG-1 spores did not oxidize Co(II) in the absence of oxygen, also indicating that oxidation was not due to abiological Co(II) oxidation on the surface of preformed Mn(III,IV) oxides. These results suggest that some microorganisms may directly oxidize Co(II) and such biological activities may exert some control on the behavior of Co in nature. SG-1 spores may also have useful applications in metal removal, recovery, and immobilization processes.     

Photosynthetic eukaryotes unite: endosymbiosis connects the dots

The photosynthetic organelle of algae and plants (the plastid) traces its origin to a primary endosymbiotic event in which a previously non-photosynthetic protist engulfed and enslaved a cyanobacterium. This eukaryote then gave rise to the red, green and glaucophyte algae. However, many algal lineages, such as the chlorophyll c-containing chromists, have a more complicated evolutionary history involving a secondary endosymbiotic event, in which a protist engulfed an existing eukaryotic alga (in this case, a red alga). Chromists such as diatoms and kelps then rose to great importance in aquatic habitats. Another algal group, the dinoflagellates, has undergone tertiary (engulfment of a secondary plastid) and even quaternary endosymbioses. In this review, we examine algal diversity and show endosymbiosis to be a major force in algal evolution. This area of research has advanced rapidly and long-standing issues such as the chromalveolate hypothesis and the extent of endosymbiotic gene transfer have recently been clarified.     

Structure and function of a vimentin-associated matrix adhesion in endothelial cells

The alpha4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the alpha4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by alphav and beta3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the alpha4 laminin subunit G domain in an alphavbeta3-integrin-dependent manner. The alphavbeta3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the alphavbeta3 integrin-positive focal contacts. We have investigated the function of alpha4-laminin and alphavbeta3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.     

Simulating the fidelity and the three Mg mechanism of pol η and clarifying the validity of transition state theory in enzyme catalysis

Pol η belongs to the important Y family of DNA polymerases that can catalyze translesion synthesis across sites of damaged DNA. This activity involves the reduced fidelity of Pol η for 8‐oxo‐7,8‐dhyedro‐2′‐deoxoguanosin(8‐oxoG). The fundamental interest in Pol η has grown recently with the demonstration of the importance of a 3rd Mg2+ ion. The current work explores both the fidelity of Pol η and the role of the 3rd metal ion, by using empirical valence bond (EVB) simulations. The simulations reproduce the observed trend in fidelity and shed a new light on the role of the 3rd metal ion. It is found that this ion does not lead to a major catalytic effect, but most probably plays an important role in reducing the product release barrier. Furthermore, it is concluded, in contrast to some implications, that the effect of this metal does not violate transition state theory, and the evaluation of the catalytic effect must conserve the molecular composition upon moving from the reactant to the transition state. Proteins 2017; 85:1446–1453. © 2017 Wiley Periodicals, Inc.     

Expression and identification of a minor extracellular fibrinolytic enzyme (Vpr) from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KCTC 3014

Previously, three extracellular proteases, Vpr, PepT, and subtilisin were identified from Bacillus subtilis KCTC 3014. To confirm the activity of Vpr, two recombinant Vpr proteins, full Vpr with TTG (pGST-fTTG-Vpr) and full Vpr with ATG (pGST-fATG-Vpr) as an initiation codon were expressed using a pGEX-2T vector encoding glutathione S-transferase (GST) in Escherichia coli. Vpr was produced in two forms, occurring as four spots on a 2-DE gel, 68 and 75 kDa proteins with similar pI values (4.0 ∼ 4.5). Activity was detected in a fibrin zymography at the expected molecular size of 68 kDa (mature form) processed from full Vpr. However, the recombinant 75 kDa of GST-fVpr did not exhibit activity. Replacement of the TTG codon with ATG led to 1.9-fold increased enzyme activity in 68 kDa. Interestingly, the expression of GSTVpr resulted in the proteolytic degradation of the protein and no GST fusion Vpr protein was detected.     

Implication of Progranulin and C1q/TNF-Related Protein-3 (CTRP3) on Inflammation and Atherosclerosis in Subjects with or without Metabolic Syndrome

Objective

Progranulin and C1q/TNF-related protein-3 (CTRP3) were recently discovered as novel adipokines which may link obesity with altered regulation of glucose metabolism, chronic inflammation and insulin resistance.

Research Design and Methods

We examined circulating progranulin and CTRP3 concentrations in 127 subjects with (n = 44) or without metabolic syndrome (n = 83). Furthermore, we evaluated the relationship of progranulin and CTRP3 levels with inflammatory markers and cardiometabolic risk factors, including high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), estimated glomerular filtration rate (eGFR), and adiponectin serum concentrations, as well as carotid intima-media thickness (CIMT).

Results

Circulating progranulin levels are significantly related with inflammatory markers, hsCRP (r = 0.30, P = 0.001) and IL-6 (r = 0.30, P = 0.001), whereas CTRP3 concentrations exhibit a significant association with cardiometabolic risk factors, including waist circumference (r = −0.21), diastolic blood pressure (r = −0.21), fasting glucose (r = −0.20), triglyceride (r = −0.34), total cholesterol (r = −0.25), eGFR (r = 0.39) and adiponectin (r = 0.26) levels. Serum progranulin concentrations were higher in patients with metabolic syndrome than those of the control group (199.55 [179.33, 215.53] vs. 185.10 [160.30, 204.90], P = 0.051) and the number of metabolic syndrome components had a significant positive correlation with progranulin levels (r = 0.227, P = 0.010). In multiple regression analysis, IL-6 and triglyceride levels were significant predictors of serum progranulin levels (R ² = 0.251). Furthermore, serum progranulin level was an independent predictor for increased CIMT in subjects without metabolic syndrome after adjusting for other cardiovascular risk factors (R ² = 0.365).

Conclusions

Serum progranulin levels are significantly associated with systemic inflammatory markers and were an independent predictor for atherosclerosis in subjects without metabolic syndrome.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01668888","term_id":"NCT01668888"}}NCT01668888     

A small interfering RNA targeting coxsackievirus B3 protects permissive HeLa cells from viral challenge

We examined the ability of small interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3). The incorporation of siRNAs dramatically decreased cell death in permissive HeLa cells in parallel with a reduction in viral replication. Three of four siRNAs had potent anti-CVB3 activity. The present study thus demonstrates that the antiviral effect is due to the downregulation of viral replication. In addition, an effective CVB3-specific siRNA had similar antiviral effects in other related enteroviruses possessing sequence homology in the targeted region. Because the CVB3-specific siRNA is effective against other enteroviruses, siRNAs have potential for a universal antienterovirus strategy.