Stimulation of phospholipid hydrolysis and arachidonic acid mobilization in human uterine decidua cells by phorbol ester.

Vasopressin and oxytocin both stimulated inositol phosphate accumulation in isolated uterine decidua cells. Pretreatment of cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) prevented this agonist-induced phosphoinositide hydrolysis. TPA (0.1 microM) alone had no effect on basal inositol phosphate accumulation, but stimulated phosphoinositide deacylation, as indicated by a 2-fold increase in lysophosphatidylinositol and glycerophosphoinositol. TPA also stimulated a dose-related release of arachidonic acid from decidua-cell phospholipid [phosphatidylcholine (PC) much greater than phosphatidylinositol (PI) greater than phosphatidylethanolamine]. The phorbol ester 4 beta-phorbol 12,13-diacetate (PDA) at 0.1 microM had no effect on arachidonic acid mobilization. The TPA-stimulated increase in arachidonic acid release was apparent by 2 1/2 min (116% of control), maximal after 20 min (283% of control), and remained around this value (306% of control) after 120 min incubation. TPA also stimulated significant increases in 1,2-diacylglycerol and monoacylglycerol production at 20 and 120 min. Although the temporal increases in arachidonic acid and monoacylglycerol accumulation in the presence of TPA continued up to 120 min, that of 1,2-diacylglycerol declined after 20 min. In decidua cells prelabelled with [3H]choline, TPA also stimulated a significant decrease in radiolabelled PC after 20 min, which was accompanied by an increased release of water-soluble metabolites into the medium. Most of the radioactivity in the extracellular pool was associated with choline, whereas the main cellular water-soluble metabolite was phosphorylcholine. TPA stimulated extracellular choline accumulation to 183% and 351% of basal release after 5 and 20 min respectively and cellular phosphorylcholine production to 136% of basal values after 20 min. These results are consistent with a model in which protein kinase C activation by TPA leads to arachidonic acid mobilization from decidua-cell phospholipid by a mechanism involving phospholipase A-mediated PI hydrolysis and phospholipase C-mediated PC hydrolysis, coupled with further hydrolysis of the 1,2-diacylglycerol product.     

Phosphatidylinositol hydrolysis in isolated guinea-pig islets of Langerhans.

Previous studies have reported an increased turnover of phospholipid in isolated islets of Langerhans in response to raised glucose concentrations. The present investigation was thus undertaken to determine the nature of any phospholipases that may be implicated in this phenomenon by employing various radiolabelled exogenous phospholipids. Hydrolysis of 1-acyl-2-[14C]arachidonoylglycerophosphoinositol by a sonicated preparation of islets optimally released radiolabelled lysophosphatidylinositol, arachidonic acid and 1,2-diacylglycerol at pH 5,7 and 9 respectively. This indicates the presence of a phospholipase A1 and a phospholipase C. However, the lack of any labelled lysophosphatidylinositol production when 2-acyl-1-[14C]stearoylglycerophosphoinositol was hydrolysed argues against a role for phospholipase A2 in the release of arachidonic acid. Phospholipase C activity as measured by phosphatidyl-myo-[3H]inositol hydrolysis was optimal around pH8, required Ca2+ for activity and was predominantly cytosolic in origin. The time course of phosphatidylinositol hydrolysis at pH 6 indicated a precursor-product relationship for 1,2-diacylglycerol and arachidonic acid respectively. The release of these two products when phosphatidylinositol was hydrolysed by either islet or acinar tissue was similar. However, phospholipase A1 activity was 20-fold higher in acinar tissue. Substrate specificity studies with islet tissue revealed that arachidonic acid release from phosphatidylethanolamine and phosphatidylcholine was only 8% and 2.5% respectively of that from phosphatidylinositol. Diacylglycerol lipase was also demonstrated in islet tissue being predominantly membrane bound and stimulated by Ca2+. The availability of non-esterified arachidonic acid in islet cells could be regulated by changes in the activity of a phosphatidylinositol-specific phospholipase C acting in concert with a diacylglycerol lipase.     

Design of antimicrobial compounds based on peptide structures

New antimicrobial compounds are of major importance because of the growing problem of bacterial resistance and antimicrobial peptides have been gaining a lot of interest. Their mechanism of action, however, is often obscure. Here a set of non-peptidic compounds with antimicrobial activity are presented that have been designed based on criteria derived from three-dimensional structures of antimicrobial peptides. Even though only a small set of compounds has been designed, the activity immediately matches that of the original peptides, supporting the proposed criteria for activity, i.e. not the peptidic nature of antimicrobial peptides is responsible for their activity but rather the proper arrangement of the relevant functional groups.     

Broad-scale latitudinal patterns of genetic diversity among native European and introduced house sparrow (Passer domesticus) populations

Introduced species offer unique opportunities to study evolution in new environments, and some provide opportunities for understanding the mechanisms underlying macroecological patterns. We sought to determine how introduction history impacted genetic diversity and differentiation of the house sparrow (Passer domesticus), one of the most broadly distributed bird species. We screened eight microsatellite loci in 316 individuals from 16 locations in the native and introduced ranges. Significant population structure occurred between native than introduced house sparrows. Introduced house sparrows were distinguished into one North American group and a highly differentiated Kenyan group. Genetic differentiation estimates identified a high magnitude of differentiation between Kenya and all other populations, but demonstrated that European and North American samples were differentiated too. Our results support previous claims that introduced North American populations likely had few source populations, and indicate house sparrows established populations after introduction. Genetic diversity also differed among native, introduced North American, and Kenyan populations with Kenyan birds being least diverse. In some cases, house sparrow populations appeared to maintain or recover genetic diversity relatively rapidly after range expansion (<50 years; Mexico and Panama), but in others (Kenya) the effect of introduction persisted over the same period. In both native and introduced populations, genetic diversity exhibited large-scale geographic patterns, increasing towards the equator. Such patterns of genetic diversity are concordant with two previously described models of genetic diversity, the latitudinal model and the species diversity model.     

Dasatinib, imatinib and staurosporine capture compounds - Complementary tools for the profiling of kinases by Capture Compound Mass Spectrometry (CCMS)

Capture Compound Mass Spectrometry (CCMS) is a platform technology for the functional isolation of subproteomes. Here we report the synthesis of two new kinase Capture Compounds (CCs) based on the tyrosine-kinase specific inhibitors dasatinib and imatinib and compare their interaction profiles to that of our previously reported staurosporine-CCs. CCs are tri-functional molecules: they comprise a sorting function (e.g. the small molecule or drug of interest) which interacts with target proteins, a photo-activatable reactivity function to covalently trap the interacting proteins, and a sorting function to isolate the CC-protein conjugates from complex biological samples for protein identification by liquid chromatography/mass spectrometry (LC-MS/MS). We present data of CCMS experiments from human HepG2 cells and compare the profiles of the kinases isolated with dasatinib, imatinib and staurosporine CC, respectively. Dasatinib and imatinib have a more selective kinase binding profile than staurosporine. Moreover, the new CCs allow isolation and identification of additional kinases, complementing the staurosporine CC. The family of kinase CCs will be a valuable tool for the proteomic profiling of this important protein class. Besides sets of expected kinases we identified additional specific interactors; these off-targets may be of relevance in the view of the pharmacological profile of dasatinib and imatinib.     

An evaluation of the in vitro cytotoxicities of 50 chemicals by using an electrical current exclusion method versus the neutral red uptake and MTT assays

According to the 2001 National Institutes of Health guidance document on using in vitro data to estimate in vivo starting doses for acute toxicity, the performance of the electrical current exclusion method (ECE) was studied for its suitability as an in vitro cytotoxicity test. In a comparative study, two established in vitro assays based on the quantification of metabolic processes necessary for cell proliferation or organelle integrity (the MTT/WST-8 [WST-8] assay and the neutral red uptake [NRU] assay), and two cytoplasm membrane integrity assays (the trypan blue exclusion [TB] and ECE methods), were performed. IC50 values were evaluated for 50 chemicals ranging from low to high toxicity, 46 of which are listed in Halles Registry of Cytotoxicity (RC). A high correlation was found between the IC50 values obtained in this study and the IC50 data published in the RC. The assay sensitivity was highest for the ECE method, and decreased from the WST-8 assay to the NRU assay to the TB assay. The consistent results of the ECE method are based on technical standardisation, high counting rate, and the ability to combine cell viability and cell volume analysis for detection of the first signs of cell necrosis and damage of the cytoplasmic membrane caused by cytotoxic agents.     

Cytokine gene polymorphisms and atopic disease in two European cohorts. (ECRHS-Basel and SAPALDIA)

Background

Avoidance of allergens is still recommended as the first and best way to prevent allergic illnesses and their comorbid diseases. Despite a variety of attempts there has been very limited success in the area of environmental control of allergic disease. Our objective was to identify a non-invasive, non-pharmacological method to reduce indoor allergen loads in atopic persons' homes and public environments. We employed a novel in vivo approach to examine the possibility of using aluminum sulfate to control environmental allergens.

Methods

Fifty skin test reactive patients were simultaneously skin tested with conventional test materials and the actions of the protein/glycoprotein modifier, aluminum sulfate. Common allergens, dog, cat, dust mite, Alternaria, and cockroach were used in the study.

Results

Skin test reactivity was significantly reduced by the modifier aluminum sulfate. Our studies demonstrate that the effects of histamine were not affected by the presence of aluminum sulfate. In fact, skin test reactivity was reduced independent of whether aluminum sulfate was present in the allergen test material or removed prior to testing, indicating that the allergens had in some way been inactivated.

Conclusion

Aluminum sulfate was found to reduce the in vivo allergic reaction cascade induced by skin testing with common allergens. The exact mechanism is not clear but appears to involve the alteration of IgE-binding epitopes on the allergen. Our results indicate that it may be possible to diminish the allergenicity of an environment by application of the active agent aluminum sulfate, thus producing environmental control without complete removal of the allergen.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Root inoculation with a forest soil streptomycete leads to locally and systemically increased resistance against phytopathogens in Norway spruce

Soil streptomycetes are commonly antagonistic against plant pathogens. However, interactions involving increased defense responses in the host plant, leading to suppression of plant disease development, have not yet been detailed. Here, the mechanisms were studied of disease suppression by Streptomyces sp. GB 4-2 against Heterobasidion root and butt rot in Norway spruce (Picea abies) seedlings. GB 4-2 promoted mycelial growth of the phytopathogenic fungus, germination rate of fungal spores, extension of germ tubes and early colonization of outer cortical layers of the plant root. Reduced colonization of the inner cortical cell layers was accompanied by the induction of cell wall appositions, and increased xylem formation in the vascular cylinder emerged after bacterium-fungus coinoculation. Bacterial treatment led to decreased water content in roots and needles and increased photosynthetic yield (F(v)/F(m)) and peroxidase activities in needles. The infection of needles by Botrytis cinerea was reduced by bacterial pretreatment. Complex interactions of GB 4-2 with Norway spruce and Heterobasidion abietinum were discovered. The bacterium promoted the growth of the phytopathogenic fungus but induced plant defense responses. Host responses indicate that GB 4-2 induces both local and systemic defense responses in Norway spruce.     

Fine-scale genetic population structure of southern pine beetle (Coleoptera: Curculionidae) in Mississippi forests

The southern pine beetle, Dendroctonus frontalis Zimmerman, is the most destructive insect pest of pine forests in the southeastern United States, Mexico, and Central America. Southern pine beetle aggressively attacks pine trees, and when in epidemic stages, they are capable of killing even the most healthy pine trees in a short period of time. Despite the amount of destruction caused by the southern pine beetle and the amount of monetary loss faced by the timber industry and recreation, the population genetics of this species has been limited to comparisons among distant geographic locations. This study investigates the fine-scale genetic population structure of the southern pine beetle in Mississippi. Very little genetic differentiation was observed among samples. Bayesian assignment testing failed to detect multiple groups within all samples; estimates of genetic differentiation and genetic distance were very low in magnitude; and a Mantel test did not reveal a significant relationship between genetic distance and geographic distance. These results suggest that management of the southern pine beetle needs to consider the potential movements of individuals within and among national forests and should be focused on a large scale, at least as big as continuously forested areas and possibly even multiple forests. These results further suggest that removal of beetle-infested trees is important.