Inhibition of platelets and tumor cell adhesion by the disintegrin domain of human ADAM9 to collagen I under dynamic flow conditions

This work aimed to investigate the role of the disintegrin domain of the human ADAM9 (ADAM9D) on the adhesion of breast tumor cells and platelets to collagen I, in a dynamic flow assay to simulate in vivo shear conditions. Recombinant ADAM9D was able to support tumor cell adhesion through binding to the β1 integrin subunit and also to inhibit the invasion through matrigel in vitro. In a dynamic flow assay ADAM9D inhibited about 75% and 65% of MDA-MB-231 tumor cells and platelet adhesion to collagen I, respectively. In addition, it was demonstrated that αVβ3 integrin is new interacting partner for ADAM9D. In conclusion, these results suggest a role for the disintegrin domain of ADAM9 in the metastatic process. Also, ADAM9D may be a tool for investigating the role of ADAMs in metastasis and cancer progression and for the design of selective inhibitors against the adhesion and extravasation of cancer cells.     

Contribution of the Periplasmic Chaperone Skp to Efficient Presentation of the Autotransporter IcsA on the Surface of Shigella flexneri

IcsA is an outer membrane protein in the autotransporter family that is required for Shigella flexneri pathogenesis. Following its secretion through the Sec translocon, IcsA is incorporated into the outer membrane in a process that depends on YaeT, a component of an outer membrane β-barrel insertion machinery. We investigated the role of the periplasmic chaperone Skp in IcsA maturation. Skp is required for the presentation of the mature amino terminus (alpha-domain) of IcsA on the bacterial surface and contributes to cell-to-cell spread of S. flexneri in cell culture. A mutation in skp does not prevent the insertion of the β-barrel into the outer membrane, suggesting that the primary role of Skp is the folding of the IcsA alpha-domain. In addition, the requirement for skp can be partially bypassed by disrupting icsP, an ortholog of Escherichia coli ompT, which encodes the protease that processes IcsA between the mature amino terminus and the β-barrel outer membrane anchor. These findings are consistent with a model in which Skp plays a critical role in the chaperoning of the alpha-domain of IcsA during transit through the periplasm.Type V secretion apparatuses (also called autotransporters) consist of an extensive class of large, outer membrane proteins of gram-negative bacteria, typically virulence factors, found in all subdivisions of proteobacteria (28). Although originally designated as “autotransporters” because they were thought to mediate their own insertion into and translocation across the outer membrane, more recent evidence suggests that autotransporter secretion and insertion requires the aid of accessory factors (21, 29). Secretion involves the insertion of the carboxy-terminal β-barrel domain into the outer membrane and translocation of the mature passenger (alpha) domain across the outer membrane (Fig. ​(Fig.1).1). Whether these two events occur sequentially or simultaneously is unclear. Analysis of crystal structures indicates that the carboxy-terminal end of the passenger domain is present within the central pore of the β-barrel (4, 27). Several studies provide evidence that at least some autotransporters are partially folded in the periplasm (7, 20), and one of these studies provides strong evidence that the passenger domain may be partially or fully incorporated into the β-barrel prior to incorporation of the mature protein into the outer membrane (20).Open in a separate windowFIG. 1.Schematic of the autotransporter IcsA. (A) Linear diagram showing the signal peptide (SP), alpha-domain (IcsA_53-757), and carboxy-terminal β-barrel domain. (B) IcsA in the outer membrane. The carboxy-terminal β-barrel is inserted into the outer membrane, and the mature amino-terminal alpha-domain is exposed on the bacterial surface. N′, mature amino terminus; C, carboxyl terminus; OM, outer membrane; arrow, proposed site of cleavage between residues 757 and 758 by IcsP.Shigella flexneri is a gram-negative human pathogen which, upon passage through the lower digestive tract, gains entry into colonic epithelial cells. Once S. flexneri is intracellular, it spreads to adjacent cells by secreting IcsA, a surface-associated autotransporter that is required for the polymerization of host cell actin on the bacterial surface. Actin polymerization occurs at a single pole of the bacterium and is required for infection of adjacent cells and disease pathogenesis (5, 24, 33).IcsA is encoded on a large virulence plasmid. The full-length protein is approximately 120 kDa and has three assigned functional and structural domains (25): an atypical Sec secretion signal (IcsA_1-52), the alpha-domain (IcsA_53-757), which is exposed on the bacterial surface and contains sequences that are required for actin polymerization, and the beta-domain (IcsA_758-1102), which forms a β-barrel structure in the outer membrane (Fig. ​(Fig.1A)1A) (21, 25). In vivo, a fraction of IcsA molecules are proteolytically processed at the junction between the alpha- and beta-domains by the protease IcsP (SopA), a protein which is also encoded on the virulence plasmid (14, 34). IcsA_53-757 is found in the supernatant of liquid cultures, while mature full-length IcsA (IcsA_53-1102), IcsA_758-1102 (14, 34), and some IcsA_53-757 (this work) remain cell associated. IcsA, like other autotransporters, is secreted at the bacterial pole (22), the site at which actin tail assembly occurs. As it is for other β-barrel-containing outer membrane proteins, insertion of IcsA and other autotransporters into the outer membrane requires the outer membrane insertase YaeT (BamA, Omp85) (21).Skp, DegP, and SurA are periplasmic chaperones that, like YaeT, appear to function in the targeting and/or insertion of outer membrane proteins (35). Evidence based on synthetic phenotypes suggests that during outer membrane protein insertion Skp and DegP act in one pathway and that SurA acts in a distinct but parallel pathway (35).We investigated the role of the periplasmic chaperone Skp in the folding and secretion of IcsA in S. flexneri. We found that in the absence of skp, IcsA is inefficiently presented on the surface of S. flexneri, leading to a cellular spread defect. Surprisingly, the protein was still efficiently cleaved by the outer membrane protease IcsP, as wild-type levels of IcsA_53-757 were detected in the culture supernatants. We found that introduction of the icsP mutation into the skp strain background led to an increase in the levels of full-length IcsA presented on the bacterial cell surface of the skp mutant, and we present models that could explain our results.     

Mutations in Smooth Muscle Alpha-Actin (ACTA2) Cause Coronary Artery Disease, Stroke, and Moyamoya Disease, Along with Thoracic Aortic Disease

The vascular smooth muscle cell (SMC)-specific isoform of α-actin (ACTA2) is a major component of the contractile apparatus in SMCs located throughout the arterial system. Heterozygous ACTA2 mutations cause familial thoracic aortic aneurysms and dissections (TAAD), but only half of mutation carriers have aortic disease. Linkage analysis and association studies of individuals in 20 families with ACTA2 mutations indicate that mutation carriers can have a diversity of vascular diseases, including premature onset of coronary artery disease (CAD) and premature ischemic strokes (including Moyamoya disease [MMD]), as well as previously defined TAAD. Sequencing of DNA from patients with nonfamilial TAAD and from premature-onset CAD patients independently identified ACTA2 mutations in these patients and premature onset strokes in family members with ACTA2 mutations. Vascular pathology and analysis of explanted SMCs and myofibroblasts from patients harboring ACTA2 suggested that increased proliferation of SMCs contributed to occlusive diseases. These results indicate that heterozygous ACTA2 mutations predispose patients to a variety of diffuse and diverse vascular diseases, including TAAD, premature CAD, ischemic strokes, and MMD. These data demonstrate that diffuse vascular diseases resulting from either occluded or enlarged arteries can be caused by mutations in a single gene and have direct implications for clinical management and research on familial vascular diseases.     

Trace Element Composition of Selected Fertilizers Used in Chile: Phosphorus Fertilizers as a Source of Long-Term Soil Contamination

Anthropogenic activities like agriculture have resulted in increased concentrations of some trace elements of toxicological and environmental concern in soils. Application of fertilizers has been one of the major inputs of these contaminants to agricultural soils in developing countries. Twenty-two fertilizers, including straight nitrogen (N), phosphorus (P), potassium (K), and NK fertilizers and micronutrient sources, were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) for arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn). As expected, the trace element content of fertilizers was highly variable and related to the origin of the material. Phosphorus fertilizers, especially triple superphosphate, presented the highest As, Cd, Cu, Cr, Ni, V, and Zn concentrations. In some of these fertilizers, the Cr, V, and Zn contents reached values greater than 3475 mg kg^?1 of P, and the Cd content (up to 288 mg kg^?1 of P) was several times higher than the regulatory limits of different countries. Some micronutrient sources presented the highest concentrations of Mn and Pb. In the cases of N, K, and NK fertilizers, the trace element concentration was very low, sometimes below the detection limits. In some agricultural systems the input of trace elements such as As and Cd to the soil through P fertilizers application may be higher than the outputs through plant uptake and leaching; therefore the long-term use of these fertilizers may cause the trace element concentration to increase in the plow layer of agricultural soils.     

Structure/Function Analysis of PARP-1 in Oxidative and Nitrosative Stress-Induced Monomeric ADPR Formation

Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2^nd messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1′s structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1′s BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2^nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.     

Appropriateness Criteria for Bariatric Surgery: Beyond the NIH Guidelines

Careful selection of bariatric patients is critical for successful outcomes. In 1991, the NIH first established patient selection guidelines; however, some surgeons operate on individuals outside of these criteria, i.e., extreme age groups. We developed appropriateness criteria for the spectrum of patient characteristics including age, BMI, and severity of eight obesity‐related comorbidities. Candidate criteria were developed using combinations of patient characteristics including BMI: ≥40 kg/m², 35–39, 32–34, 30–31, <30; age: 12–18, 19–55, 56–64, 65+ years old; and comorbidities: prediabetes, diabetes, hypertension, dyslipidemia, sleep apnea, venous stasis disease, chronic joint pain, and gastroesophageal reflux (plus severity level). Criteria were formally validated on their appropriateness of whether the benefits of surgery clearly outweighed the risks, by an expert panel using the RAND/UCLA modified Delphi method. Nearly all comorbidity severity criteria for patients with BMI ≥40 kg/m² or BMI = 35–39 kg/m² in intermediate age groups were found to be appropriate for surgery. In contrast, patients in the extreme age categories were considered appropriate surgical candidates under fewer conditions, primarily the more severe comorbidities, such as diabetes and hypertension. For patients with a BMI of 32–34, only the most severe category of diabetes (Hgb A1c >9, on maximal medical therapy), is an appropriate criterion for those aged 19–64, whereas many mild to moderate severity comorbidity categories are “inappropriate.” There is overwhelming agreement among the panelists that the current evidence does not support performing bariatric surgery in lower BMI individuals (BMI <32). This is the first development of appropriateness criteria for bariatric surgery that includes severity categories of comorbidities. Only for the most severe degrees of comorbidities were adolescent and elderly patients deemed appropriate for surgery. Patient selection for bariatric procedures should include consideration of both patient age and comorbidity severity.     

Plasma zinc concentrations of mothers and the risk of oral clefts in their children in Utah

BACKGROUND: The role of maternal zinc nutrition in human oral clefts (OCs) is unclear. We measured plasma zinc concentrations (PZn) of case and control mothers to evaluate the associations between PZn and risk of OCs with and without other malformations. METHODS: Case mothers were ascertained by the Utah Birth Defects Network and control mothers were selected from Utah birth certificates by matching for child gender and delivery month and year. Maternal blood was collected >1 year after the last pregnancy. PZn was available for 410 case mothers who were divided into four subgroups: isolated cleft lip with or without cleft palate (CL/P‐I, n = 231), isolated cleft palate (CP‐I, n = 74), CL/P with other malformations (CLP‐M, n = 42), and CP with other malformations (CP‐M, n = 63). PZn was available for 447 control mothers. The mean age of children at blood sampling was 3.7 years for all cases combined and 4.3 years for controls. RESULTS: Mean PZns of all groups were similar, and low PZn (<11.0 μmol/L) was found in 59% of cases and 62% of controls. Risk of OCs did not vary significantly across PZn quartiles for the four subgroups individually and all OC groups combined. CONCLUSIONS: We previously reported that poor maternal zinc status was a risk factor for OCs in the Philippines, where OC prevalence is high and maternal PZn is low. In Utah, however, no such association was found, suggesting that poor maternal zinc status may become a risk factor only when zinc status is highly compromised. Birth Defects Research (Part A), 2009. © 2008 Wiley‐Liss, Inc.     

Nectar chemistry is tailored for both attraction of mutualists and protection from exploiters

Plants produce nectar to attract pollinators in the case of floral nectar (FN) and defenders in the case of extrafloral nectar (EFN). Whereas nectars must function in the context of plant-animal mutualisms, their chemical composition makes them also attractive for non-mutualistic, exploiting organisms: nectar robbers and nectar-infesting microorganisms. We reviewed the chemical composition of both FNs and EFNs and found that nectar composition appears tailored to fulfil these ambivalent roles. Carbohydrates and amino acids usually function in the attraction of mutualists and appear adapted to the physiological needs of the respective mutualists. Volatiles are a further group of compounds that serves in the attractive function of nectars. By contrast, secondary compounds such as alkaloids and phenols serve the protection from nectar robbers, and most nectar proteins that have been characterised to date protect FN and EFN from microbial infestation. Nectar components serve both in attraction and the protection of nectar.Key words: extrafloral nectar, floral nectar, indirect defence, mutualism, pollination     

Interplay of cysteinyl leukotrienes and TGF-β in the activation of hepatic stellate cells from Schistosoma mansoni granulomas

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC₄-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB_4, into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.