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Objective— The platelet storage lesion accelerates platelet clearance after transfusion, but the underlying molecular mechanism remains elusive. Although inhibiting sheddase activity hampers clearance of platelets with storage lesion, the target platelet protein responsible for ectodomain shedding–induced clearance is not definitively identified. Monoclonal antibody 5G6 was developed recently to bind specifically human platelet receptor glycoprotein (GP)Ibα and inhibit its shedding but not shedding of other receptors. Here, the role of GPIbα shedding in platelet clearance after transfusion was addressed. Approach and Results— Both human leukoreduced apheresis-derived platelets and transgenic mouse platelets expressing human GPIbα were stored at room temperature in the presence and absence of 5G6 Fab fragment. At various time points, aliquots of stored platelets were analyzed and compared. 5G6 Fab inhibited GPIbα shedding in both platelets during storage and preserved higher level of GPIbα on the platelet surface. Compared with age-matched control platelets, 5G6 Fab–stored platelets exhibited similar levels of platelet activation, degranulation, and agonist-induced aggregation. 5G6 Fab–stored human GPIbα platelets exhibited significantly higher post-transfusion recovery and in vivo hemostatic function in recipient mice than control platelets. Consistently, 5G6 Fab–stored, 8-day-old human platelets produced similar improvement in post-transfusion recovery in immunodeficient mice and in ex vivo thrombus formation over collagen under shear flow. Conclusions— Specific inhibition of GPIbα shedding in the stored platelets improves post-transfusion platelet recovery and hemostatic function, providing clear evidence for GPIbα shedding as a cause of platelet clearance. These results suggest that specific inhibition of GPIbα shedding may be used to optimize platelet storage conditions.

UNLABELLED ESSENTIALS: Many anti-glycoprotein (GP)Ibα antibodies induce platelet clearance in a dimer-dependent manner. Characterization of monoclonal antibodies that bind the mechanosensitive domain (MSD) of GPIbα. An anti-MSD antibody binds two copies of GPIbα in platelets but does not induce platelet clearance. The prevailing clustering model of GPIbα signaling is incorrect or needs revision. BACKGROUND The mechanism of platelet clearance is not clear. Many antibodies binding the membrane-distal ligand-binding domain of glycoprotein (GP)Ibα induce rapid clearance of platelets and acute thrombocytopenia, which requires the bifurcated antibody structure. It was thought that binding of these antibodies induced lateral dimerization or clustering of GPIbα in the plasma membrane, which leads to downstream signaling and platelet clearance. However, many antibodies targeting GPIbβ and GPIX, which are associated with GPIbα in the GPIb-IX complex, do not induce platelet clearance, which is in contradiction to the clustering model. OBJECTIVES To test whether dimerization or clustering of GPIbα is sufficient to transmit the signal that leads to platelet clearance. METHODS We have recently raised several mAbs targeting the mechanosensitive domain (MSD) of GPIbα. Binding of these anti-MSD antibodies was characterized with biochemical methods. Their ability to stimulate platelets and induce platelet clearance in mice was assessed. RESULTS AND CONCLUSION Infusion of anti-MSD antibodies does not cause thrombocytopenia in mice. These antibodies show no detectable effects on platelet activation and aggregation in vitro. Further biochemical investigation showed that the anti-MSD antibody 3D1 binds two copies of GPIbα on the platelet surface. Therefore, lateral dimerization of GPIbα induced by antibody binding is not sufficient to initiate GPIb-IX signaling and induce platelet clearance. Our results suggest that a factor other than or in addition to clustering of GPIbα is required to induce platelet clearance.

Summary Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ∼98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca2+ transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies., Graphical Abstract, Highlights • Molecular beacon (MB)-based method was developed to purify ventricular CMs from ESCs • Ventricular CM-specific MBs targeting Irx4 mRNA were successfully generated • About 98% of the CMs sorted via Irx4-MB displayed ventricular CM-like phenotypes • Irx4-MB-based purified CMs continuously maintained ventricular CM characteristics, In this article, Yoon and colleagues demonstrate that vCMs can be purified from differentiating ESCs using MBs targeting mRNA encoding Irx4, a transcription factor specific for vCMs. Purified IRX4-MB-positive cells displayed vCM-like action potentials and continuously maintained their vCM characteristics. This study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes.

The goal of this study is to develop an integrated, mathematical–experimental approach for understanding the interactions between the immune system and the effects of trastuzumab on breast cancer that overexpresses the human epidermal growth factor receptor 2 (HER2+). A system of coupled, ordinary differential equations was constructed to describe the temporal changes in tumour growth, along with intratumoural changes in the immune response, vascularity, necrosis and hypoxia. The mathematical model is calibrated with serially acquired experimental data of tumour volume, vascularity, necrosis and hypoxia obtained from either imaging or histology from a murine model of HER2+ breast cancer. Sensitivity analysis shows that model components are sensitive for 12 of 13 parameters, but accounting for uncertainty in the parameter values, model simulations still agree with the experimental data. Given theinitial conditions, the mathematical model predicts an increase in the immune infiltrates over time in the treated animals. Immunofluorescent staining results are presented that validate this prediction by showing an increased co-staining of CD11c and F4/80 (proteins expressed by dendritic cells and/or macrophages) in the total tissue for the treated tumours compared to the controls ($p < 0.03$). We posit that the proposed mathematical–experimental approach can be used to elucidate driving interactions between the trastuzumab-induced responses in the tumour and the immune system that drive the stabilization of vasculature while simultaneously decreasing tumour growth—conclusions revealed by the mathematical model that were not deducible from the experimental data alone.

Staphylococcus aureus is a leading cause of both nosocomial and community-acquired infection. Biofilm formation at the site of infection reduces antimicrobial susceptibility and can lead to chronic infection. During biofilm formation, a subset of cells liberate cytoplasmic proteins and DNA, which are repurposed to form the extracellular matrix that binds the remaining cells together in large clusters. Using a strain that forms robust biofilms in vitro during growth under glucose supplementation, we carried out a genome-wide screen for genes involved in the release of extracellular DNA (eDNA). A high-density transposon insertion library was grown under biofilm-inducing conditions, and the relative frequency of insertions was compared between genomic DNA (gDNA) collected from cells in the biofilm and eDNA from the matrix. Transposon insertions into genes encoding functions necessary for eDNA release were identified by reduced representation in the eDNA. On direct testing, mutants of some of these genes exhibited markedly reduced levels of eDNA and a concomitant reduction in cell clustering. Among the genes with robust mutant phenotypes were gdpP, which encodes a phosphodiesterase that degrades the second messenger cyclic-di-AMP, and xdrA, the gene for a transcription factor that, as revealed by RNA-sequencing analysis, influences the expression of multiple genes, including many involved in cell wall homeostasis. Finally, we report that growth in biofilm-inducing medium lowers cyclic-di-AMP levels and does so in a manner that depends on the gdpP phosphodiesterase gene.

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease characterized by autoantibody-induced platelet clearance. Some ITP patients are refractory to standard immunosuppressive treatments such as intravenous immunoglobulin (IVIg). These patients often have autoantibodies that target the ligand-binding domain (LBD) of glycoprotein Ibα (GPIbα), a major subunit of the platelet mechanoreceptor complex GPIb-IX. However, the molecular mechanism of this Fc-independent platelet clearance is not clear. Here, we report that many anti-LBD monoclonal antibodies such as 6B4, but not AK2, activated GPIb-IX in a shear-dependent manner and induced IVIg-resistant platelet clearance in mice. Single-molecule optical tweezer measurements of antibodies pulling on full-length GPIb-IX demonstrated that the unbinding force needed to dissociate 6B4 from the LBD far exceeds the force required to unfold the juxtamembrane mechanosensory domain (MSD) in GPIbα, unlike the AK2-LBD unbinding force. Binding of 6B4, not AK2, induced shear-dependent unfolding of the MSD on the platelet, as evidenced by increased exposure of a linear sequence therein. Imaging flow cytometry and aggregometry measurements of platelets and LBD-coated platelet-mimetic beads revealed that 6B4 can sustain crosslinking of platelets under shear, whereas 6B4 Fab and AK2 cannot. These results suggest a novel mechanism by which anti-LBD antibodies can exert a pulling force on GPIb-IX via platelet crosslinking, activating GPIb-IX by unfolding its MSD and inducing Fc-independent platelet clearance.

Essentials The mechanism for the auto-inhibition of von Willebrand factor (VWF) remains unclear. Hydrogen exchange of two VWF A1 fragments with disparate activities was measured and compared. Discontinuous residues flanking A1 form a structural module that blocks A1 binding to the platelet. Our results suggest a potentially unified model of VWF activation. Click to hear an ISTH Academy presentation on the domain architecture of VWF and activation by elongational flow by Dr Springer SUMMARY: Background How von Willebrand factor (VWF) senses and responds to shear flow remains unclear. In the absence of shear flow, VWF or its fragments can be induced to bind spontaneously to platelet GPIbα. Objectives To elucidate the auto-inhibition mechanism of VWF. Methods Hydrogen-deuterium exchange (HDX) of two recombinant VWF fragments expressed from baby hamster kidney cells were measured and compared. Results The shortA1 protein contains VWF residues 1261-1472 and binds GPIbα with a significantly higher affinity than the longA1 protein that contains VWF residues 1238-1472. Both proteins contain the VWF A1 domain (residues 1272-1458). Many residues in longA1, particularly those in the N- and C-terminal sequences flanking the A1 domain, and in helix α1, loops α1β2 and β3α2, demonstrated markedly reduced HDX compared with their counterparts in shortA1. The HDX-protected region in longA1 overlaps with the GPIbα-binding interface and is clustered with type 2B von Willebrand disease (VWD) mutations. Additional comparison with the HDX of denatured longA1 and ristocetin-bound longA1 indicates the N- and C-terminal sequences flanking the A1 domain form cooperatively an integrated autoinhibitory module (AIM) that interacts with the HDX-protected region. Binding of ristocetin to the C-terminal part of the AIM desorbs the AIM from A1 and enables longA1 binding to GPIbα. Conclusion The discontinuous AIM binds the A1 domain and prevents it from binding to GPIbα, which has significant implications for the pathogenesis of type 2B VWD and the shear-induced activation of VWF activity.

Enteric bacteria, such as Escherichia coli , are exposed to a variety of stresses in the nonhost environment. The development of biofilms provides E. coli with resistance to environmental insults, such as desiccation and bleach. We found that biofilm formation, specifically production of the matrix components curli and cellulose, protected E. coli against killing by the soil-dwelling nematode Caenorhabditis elegans and the predatory bacterium Myxococcus xanthus . Additionally, matrix-encased bacteria at the air-biofilm interface exhibited ∼40-fold-increased survival after C. elegans and M. xanthus killing compared to the non-matrix-encased cells that populate the interior of the biofilm. To determine if nonhost Enterobacteriaceae reservoirs supported biofilm formation, we grew E. coli on media composed of pig dung or commonly contaminated foods, such as beef, chicken, and spinach. Each of these medium types provided a nutritional environment that supported matrix production and biofilm formation. Altogether, we showed that common, nonhost reservoirs of E. coli supported the formation of biofilms that subsequently protected E. coli against predation.

To investigate antidiabetic efficacy of the extract of field grown and in vitro raised leaves of Solanum xanthocarpum (S. xanthocarpum) against alloxan induced diabetic rats.The antidiabetic activity of the crude methanol extracts of the field grown and in vitro raised leaves of S. xanthocarpum at different concentrations (100-200 mg/kg bw) was tested against alloxan induced diabetic rats. The antidiabetic efficacy was validated through various biochemical parameters and the antioxidant effect was also determined. The phytochemical analyses of field grown S. xanthocarpum and in vitro rasied S. xanthocarpum leaves were done by estimating their chlorophyll, carotenoids, total sugar, protein, amino acid and minerals contents.The results revealed that the methanol extracts of both the leaves (field grown and in vitro raised) of S. xanthocarpum was efficient anti hyperglycemic agents at a concentration of 200 mg/kg bw and posses potent antioxidant activity. However, the extracts of in vitro rasied S. xanthocarpum raised leaves exhibit higher efficacy than the field grown leaves in all tested concentrations. Proximal composition and mineral analysis of S. xanthocarpum revealed higher concentration of contents in in vitro rasied S. xanthocarpum than field grown S. xanthocarpum.From the results it can be concluded that the leaves extracts of S. xanthocarpum can be a potential candidate in treating the hyperglycemic conditions and suits to be an agent to reduce oxidative stress.

Antipathogenic therapy is an outcome of the quorum-sensing inhibition (QSI) mechanism, which targets autoinducer-dependent virulent gene expression in bacterial pathogens. N-acyl homoserine lactone (AHL) acts as a key regulator in the production of virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1 and violacein pigment production in Chromobacterium violaceum. In the present study, the marine bacterial strain SS4 showed potential QSI activity in a concentration-dependent manner (0.5-2 mg/ml) against the AHL-mediated violacein production in C. violaceum (33-86%) and biofilm formation (33-88%), total protease (20-65%), LasA protease (59-68%), LasB elastase (36-68%), pyocyanin (17-86%) and pyoverdin productions in PAO1. The light and confocal laser scanning microscopic analyses confirmed the reduction of the biofilm-forming ability of PAO1 when treated with SS4 extract. Furthermore, the antibiofilm potential was confirmed through static biofilm ring assay, in which ethyl acetate extract of SS4 showed concentration-dependent reduction in the biofilm-forming ability of PAO1. Thus, the result of this study clearly reveals the antipathogenic and antibiofilm properties of the bacterial isolate SS4. Through 16S rDNA analysis, the strain SS4 was identified as Bacillus sp. (GenBank Accession Number: GU471751).

Background: To find out an alternative strategy to antibiotic usage against bacterial infection. Materials and Methods: The purpose of this study is to describe the quorum-sensing (QS) inhibitory activity of edible plants and fruits against N-acyl-homoserine lactone (AHL)-mediated violacein production in Chromobacterium violaceum and virulence factor expression in Pseudomonas aeruginosa PAO1. Results: Aqueous extracts of Ananas comosus (Bromeliaceae), Musa paradiciaca (Musaceae), Manilkara zapota (Sapotaceae) and Ocimum sanctum (Lamiaceae) were prepared and anti-QS activity of each extract was tested against AHL-mediated phenotypic expressions of C. violaceum and PAO1. Most of these extracts showed significant reduction in AHL-mediated violacein production in C. violaceum as well as pyocyanin pigment, staphylolytic protease, elastase production and biofilm formation in PAO1. However, these extracts were not inhibitory to bacterial growth, revealing that the QS inhibition by the extracts is not related to static or killing effects on the bacteria. Conclusions: The present study identified the anti-QS activity of A. comosus, M. paradiciaca, M. zapota and O. sanctum. An AHL-inactivating compound from these plant sources can be used as an alternative to antibiotic compounds to prevent AHL-mediated bacterial infection in higher organisms.

Mechanisms by which blood cells sense shear stress are poorly characterized. In platelets, glycoprotein (GP)Ib–IX receptor complex has been long suggested to be a shear sensor and receptor. Recently, a relatively unstable and mechanosensitive domain in the GPIbα subunit of GPIb–IX was identified. Here we show that binding of its ligand, von Willebrand factor, under physiological shear stress induces unfolding of this mechanosensory domain (MSD) on the platelet surface. The unfolded MSD, particularly the juxtamembrane ‘Trigger' sequence therein, leads to intracellular signalling and rapid platelet clearance. These results illustrate the initial molecular event underlying platelet shear sensing and provide a mechanism linking GPIb–IX to platelet clearance. Our results have implications on the mechanism of platelet activation, and on the pathophysiology of von Willebrand disease and related thrombocytopenic disorders. The mechanosensation via receptor unfolding may be applicable for many other cell adhesion receptors., The platelets detect and respond to shear stress generated by blood flow. Here the authors show that the binding of the soluble von Willebrand factor to its receptor GPIba under physiological shear stress induces receptor's domain unfolding on the platelet and signalling into the platelet, leading to platelets clearance.

Activated protein C (APC) is an important modulator of vascular function that has antithrombotic and anti-inflammatory properties. Studies in humans have shown modulation of endotoxin-induced hypotension by recombinant human APC, drotrecogin alfa (activated), however, the mechanism for this effect is unclear. We have found that APC suppresses the induction of the potent vasoactive peptide adrenomedullin (ADM) and could downregulate lipopolysaccharide (LPS)-induced ADM messenger RNA (mRNA) and nitrite levels in cell culture. This effect was dependent on signaling through protease-activated receptor 1. Addition of 1400W, an irreversible inducible nitric oxide synthase (iNOS) inhibitor, inhibited LPS-induced ADM mRNA, suggesting that ADM induction is NO mediated. Furthermore, in a rat model of endotoxemia, APC (100 microg/kg, i.v.) prevented LPS (10 mg/kg, i.v.)-induced hypotension, and suppressed ADM mRNA and protein expression. APC also inhibited iNOS mRNA and protein levels along with reduction in NO by-products (NOx). We also observed a significant reduction in iNOS-positive leukocytes adhering to vascular endothelium after APC treatment. Moreover, we found that APC inhibited the expression of interferon-gamma (IFN-gamma), a potent activator of iNOS. In a human study of LPS-induced hypotension, APC reduced the upregulation of plasma ADM levels, coincident with protection against the hypotensive response. Overall, we demonstrate that APC blocks the induction of ADM, likely mediated by IFN-gamma and iNOS, and suggests a mechanism that may account for ameliorating LPS-induced hypotension. Furthermore, our data provide a new understanding for the role of APC in modulating vascular response to insult.

Staphylococcus aureus is a human commensal that colonizes the skin. While it is normally innocuous, it has strong associations with atopic dermatitis pathogenesis and has become the leading cause of skin and soft tissue infections in the United States. The factors that dictate the role of S. aureus in disease are still being determined. In this work, we utilized primary keratinocyte culture and an epidermal murine colonization model to investigate the role of S. aureus phenol-soluble modulins (PSMs) in proinflammatory cytokine release and inflammation induction. We demonstrated that many species of Staphylococcus are capable of causing release of interleukin 18 (IL-18) from keratinocytes and that S. aureus PSMs are necessary and sufficient to stimulate IL-18 release from keratinocytes independently of caspase 1. Further, after 7 days of epicutaneous exposure to wild-type S. aureus , but not S. aureus Δ psm , we saw dramatic changes in gross pathology, as well as systemic release of proinflammatory cytokines. This work demonstrates the importance of PSM peptides in S. aureus -mediated inflammatory cytokine release from keratinocytes in vitro and in vivo and further implicates PSMs as important contributors to pathogenesis.

The Drosophila ovary is a model system for examining the genetic control of epithelial morphogenesis. The somatic follicle cells form a polarized epithelium surrounding the 16-cell germ line cyst. The integrity of this epithelium is essential for the successful completion of oogenesis. Reciprocal signaling between germ line and somatic cells establishes embryonic and eggshell polarity. The follicle cells are responsible for shaping the egg and secreting the eggshell. Follicle cells at the boundary between the nurse cells and the oocyte migrate centripetally to cover the anterior end of the oocyte and secrete the operculum. Dorsal anterior main body follicle cells undergo elaborate patterning to produce the dorsal appendages. We have examined the expression of the Toll-like receptor, 18-wheeler (18w), in the ovary and find it to be restricted to subpopulations of follicle cells. Females carrying loss-of-function 18w mutant clones in their ovaries show delayed follicle cell migrations. The eggs laid by such females also show morphological defects in egg shape and dorsal appendage morphology. We propose that the 18W protein plays an adhesive or signaling role in regions of the epithelium engaged in cell migration. Developmental Dynamics 235:1953–1961, 2006. © 2006 Wiley-Liss, Inc.

Many bacteria produce cytolytic toxins that target host cells or other competing microbes. It is well known that environmental factors control toxin expression, however, recent work suggests that some bacteria manipulate the fold of these protein toxins to control their function. The β-sheet rich amyloid fold is a highly stable ordered aggregate that many toxins form in response to specific environmental conditions. When in the amyloid state, toxins become inert, losing the cytolytic activity they display in the soluble form. Emerging evidence suggest that some amyloids function as toxin storage systems until they are again needed, while other bacteria utilize amyloids as a structural matrix component of biofilms. This amyloid matrix component facilitates resistance to biofilm disruptive challenges. The bacterial amyloids discussed in this review reveal an elegant system where changes in protein fold and solubility dictate the function of proteins in response to the environment.

Staphylococcus aureus virulence is coordinated through the Agr quorum-sensing system to produce an array of secreted molecules. One important class of secreted virulence factors is the phenol-soluble modulins (PSMs). PSMs are small-peptide toxins that have recently been characterized for their roles in infection, biofilm development, and subversion of the host immune system. In this work, we demonstrate that the signal peptide of the S. aureus quorum-sensing signal, AgrD, shares structural and functional similarities with the PSM family of toxins. The efficacy of this peptide (termed N-AgrD) beyond AgrD propeptide trafficking has never been described before. We observe that N-AgrD, like the PSMs, is found in the amyloid fibrils of S. aureus biofilms and is capable of forming and seeding amyloid fibrils in vitro . N-AgrD displays cytolytic and proinflammatory properties that are abrogated after fibril formation. These data suggest that the N-AgrD leader peptide affects S. aureus biology in a manner similar to that described previously for the PSM peptide toxins. Taken together, our findings suggest that peptide cleavage products can affect cellular function beyond their canonical roles and may represent a class of virulence factors warranting further exploration.

The biocide triclosan is used in many personal care products, including toothpastes, soaps, clothing, and medical equipment. Consequently, it is present as a contaminant in the environment and has been detected in some human fluids, including serum, urine, and milk. Staphylococcus aureus is an opportunistic pathogen that colonizes the noses and throats of approximately 30% of the population. Colonization with S. aureus is known to be a risk factor for several types of infection. Here we demonstrate that triclosan is commonly found in the nasal secretions of healthy adults and the presence of triclosan trends positively with nasal colonization by S. aureus. We demonstrate that triclosan can promote the binding of S. aureus to host proteins such as collagen, fibronectin, and keratin, as well as inanimate surfaces such as plastic and glass. Lastly, triclosan-exposed rats are more susceptible to nasal colonization with S. aureus. These data reveal a novel factor that influences the ability of S. aureus to bind surfaces and alters S. aureus nasal colonization., IMPORTANCE Triclosan has been used as a biocide for over 40 years, but the broader effects that it has on the human microbiome have not been investigated. We demonstrate that triclosan is present in nasal secretions of a large portion of a test population and its presence correlates with Staphylococcus aureus nasal colonization. Triclosan also promotes the binding of S. aureus to human proteins and increases the susceptibility of rats to nasal colonization by S. aureus. These findings are significant because S. aureus colonization is a known risk factor for the development of several types of infections. Our data demonstrate the unintended consequences of unregulated triclosan use and contribute to the growing body of research demonstrating inadvertent effects of triclosan on the environment and human health.

Insects express a battery of potent antimicrobial proteins in response to injury and infection. Recent work from several laboratories has demonstrated that this response is neither stereotypic nor completely nonspecific, and that different pathways are responsible for inducing the expression of antifungal and antibacterial peptides. Here we report the cloning of two closely linked attacin genes from Drosophila melanogaster. We compare their protein coding sequences and find the amino acid sequences to be more highly conserved than the nucleotide sequences, suggesting that both genes are expressed. Like other antimicrobial peptides, attacin expression is strongly induced in infected and injured flies. Unlike others, attacin transcription is uniquely sensitive to mutations in the 18-Wheeler receptor protein, and thus may be regulated by a distinct signaling pathway. The number and organization of binding sites for kappaB and other transcription factors in the promoter regions of both attacin genes are consistent with strong and rapid immune induction. We demonstrate that these promoter regions are sufficient to direct beta-galactosidase expression in transformed Drosophila third-instar larval fat body in a bacterially inducible manner. We present a comparison of the promoter regions of the two attacin genes to those cloned from other antimicrobial peptide genes to assist a better understanding of how antimicrobial genes are differentially regulated.

The mechanism of action of 2-chlorpromazine (2-chloro-10-(3-dimethylaminopropyl)-phenothiazine) as a reversal agent for P-glycoprotein-mediated multidrug resistance was investigated using inside out-orientated membrane vesicles prepared from vinblastine-resistant human CCRF-CEM leukaemia cells (VBL1000). 2-Chlorpromazine (10 microM) completely inhibited ATP-dependent P-glycoprotein-mediated vinblastine accumulation in the vesicles. Whereas in the absence of added ligands VBL transport was described by a hyperbolic function of vinblastine concentration, in the presence of 2-chlorpromazine vinblastine transport was a sigmoidal function. 2-Chlorpromazine was shown previously [Syed SK, Christopherson RI and Roufogalis BD (1996) Biochem Mol Biol Int 39: 687-696] to be actively transported into vesicles from multidrug-resistant cells. Colchicine (10 microM) and phenoxybenzamine (10 microM) blocked vinblastine transport but had no effect on 2-chlorpromazine transport into vesicles. The results were consistent with a two-state concerted model in which P-glycoprotein exists in two conformational states, P(A) and P(B), where 2-chlorpromazine is transported by the conformer, P(A), and vinblastine by the conformer, P(B). In the presence of 2-chlorpromazine, the conformer P(A) predominates and vinblastine transport is inhibited. Addition of 2-chlorpromazine during the steady state of vinblastine accumulation blocked uptake and resulted in enhanced vinblastine efflux from the vesicles. The findings were similar when vinblastine was added at the steady state of 2-chlorpromazine transport. We propose a minimal kinetic model whereby in these preloaded vesicles the complex VV.P(A).CC is formed, where two internal binding sites of P-glycoprotein (P(A)) are occupied by vinblastine (V) and the two external sites are occupied by 2-chlorpromazine (C). When the two binding sites on both the inside and outside of P-glycoprotein are saturated with ligands vinblastine is effluxed at a very rapid rate, and vice versa when vesicles are preloaded with 2-chlorpromazine and vinblastine is added outside. These unexpected observations and the concerted model developed provide an alternative mechanism of action for reversal agents that sensitize multidrug-resistant cancer cells to anti-cancer drugs.

Extracellular ATP released from pancreatic β-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic β-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin-secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic β-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.

The mechanism by which chlorpromazine (2-chloro-10-(3-dimethylaminopropyl)-phenothiazine) reverses P-glycoprotein (P-gp2) mediated multidrug resistance was investigated using membrane vesicles prepared from human CCRF-CEM leukaemia cells. Chlorpromazine was transported in an ATP-dependent manner into membrane vesicles prepared from vinblastine resistant (VBL1000) cells but not from drug-sensitive cells. The chlorpromazine uptake was sensitive to osmotic pressure, indicating true transport into the vesicle lumen. The ATP-dependent chlorpromazine uptake was inhibited about 30% by the addition of ammonium chloride, indicating that a pH or electrical gradient could not account for the majority of ATP-dependent chlorpromazine uptake. The results of this study show that chlorpromazine is actively transported my P-glycoprotein and that chemosensitization by phenothiazines may occur by competition of these agents for active transport of anticancer agents by P-glycoprotein.

Aim: The aim of the study was to evaluate the local anesthetic activity of root extracts of the Anacyclus pyrethrum, and to check its effect of interaction at the site of injection in guinea pigs. Materials and Methods: The study sample included thirty guinea pigs each weighing 450–500 g, maintained under standard conditions. The root extracts were prepared using three solvents, in 1% and 2% concentration and injected in guinea pigs. The animals were divided into five groups, six in each group based on the type of extract used along with a control and a standard drug. All the animals received 0.5 ml of intradermal injection of the prepared extract, with 1% concentration in the left and 2% in the right dorsal flank of the animal and were checked for local anesthetic activity by a pinprick test. After 72 h, biopsy was done from the injected site to check for drug interaction. Results: The number of negative response obtained from 2% ethanol extract is more effective when compared to other extracts. Histological samples showed inflammatory changes in 1% aqueous extract in a single animal. Conclusion: Among the test compounds, 2% ethanol showed more significant effect; hence, it is suggested to synthesize more compounds in this series and evaluate their pharmacological actions.

Primary glioblastoma (GBM) tumours recur following treatment. This is likely due to intratumour heterogeneity (ITH), which reduces the effectiveness of cancer therapies by enabling therapy-driven tumour evolution: Darwinian selection of genotypically and/or phenotypically distinct cells (subclones) that resist treatment to seed regrowth of an even less treatable recurrence. In recent years, next generation sequencing approaches have facilitated the characterisation of subclonal genomic tumour architecture. We have applied these approaches to a pilot cohort of five paired primary and locally recurrent GBM tumours, alongside high-coverage transcriptomic profiling, in order to begin characterising therapy resistant cells and thereby infer the mechanisms by which they evaded treatment. This approach may also indicate relevant targets in recurrent disease. In this preliminary cohort we see subclonal mutation patterns indicative of neutral tumour evolution but with evidence of selection of certain cell populations during therapy. The expressed, mutated genes in these expanded cell populations are enriched for EGF-EGFR signalling pathways and Wnt signalling and pluripotency, with at least one member of each of these pathways being mutated in an expanded subpopulation per tumour pair.

Enzymatic hydrolysis of synthetic methyl 5-, 9-, and 12-thiastearates in aqueous media withCandida cyclindracea or porcine pancreatic lipases gave the corresponding fatty acids in 70–100% yield. Hydrolysis of the 3- and 4-positional isomers gave only 15–25% of the free thia fatty acids, suggesting discrimination against these isomers by lipases. No lipolysis was achieved with methyl 2-thialaurate under a range of reaction conditions. Esterification of the 3-, 4-, 5-, 9-, and 12-thiastearic acids withn-butanol inn-hexane using Lipozyme (immobilizedRhizomucor miehei) as the biocatalyst gave the corresponding butyl esters in 80–95% yield. Interesterification (acyl exchange) of triolein with methyl 9-thiastearate in the presence of Lipozyme showed the incorporation of 9-thiastearoyl chain at only one of the α-positions of triolein. In the case of methyl 2-thialaurate, no lipase-catalyzed acyl exchange reaction was possible. This study showed that the position of the sulfur atom in thia fatty esters affects the lipase-catalyzed hydrolysis and interesterification reactions.

Incubation of a 1-pyrroline ester [viz. methyl 8-(5-hexyl-1-pyrroline-2-yl)octanoate, 1] with bakers' yeast (Saccharomyces cerevisiae) gave the corresponding free fatty acid (1a, 52%). The C = N bond of the 1-pyrroline was not reduced by the yeast. Complete hydrolysis of compound 1 was successful using lipase of Candida cylindracea (CCL) or Lipolase (Rhizomucor miehei) under stirred or ultrasound condition. Fatty esters containing a pyrrolidine [viz. methyl 8-(cis/trans-5-hexyl-pyrrolidine-2-)octanoate, 2] or N-methyl pyrrolidine [viz. methyl 8-(cis-5-hexyl-N-methyl-pyrrolidine-2-)octanoate, 3] system in the alkyl chain were not hydrolyzed by either CCL or Lipolase, unless conducted in an ultrasonic bath. The hydrolytic activities of the enzymes appeared to be strongly affected by the stereochemistry of the N-heterocyclic ring system. Chemical hydrolysis of compounds 1-3 gave the corresponding fatty acid N-HCl salts.

Enrichment ofcis-5 polyunsaturated fatty acids [20:3(5c,11c,14c), 4.3% and 20:4(5c,11c,14c,17c), 11.3%] fromBiota orientalis seed oil was carried out by lipase-catalyzed selective esterification and hydrolysis reactions. Lipases fromRhizomucor miehei (Lipozyme),Candida cylindracea and porcine pancreas were used. Lipozyme-catalyzed esterification ofBiota fatty acids withn-butanol inn-hexane allowed 20:3 and 20:4 (as fatty acids) to be enriched to a maximum level of 52.9%, and in the presence ofC. cylindracea lipase 61.5% enrichment was achieved. Esterification with pancreatic lipase was poor with low levels of enrichment of 20:3 and 20:4 (22%). A multigram scale esterification of the free fatty acids fromBiota seed oil by repeated treatment of the isolated fatty acid fraction withn-butanol inn-hexane in the presence ofC. cylindracea lipase furnished an enrichment yield of 72.5% of a mixture of 20:3 and 20:4 fatty acids. Urea fractionation of the free fatty acids ofBiota oil gave an initial enriched fraction of 20:3 (9.5%) and 20:4 (25.2%) which, upon treatment withC. cylindracea lipase inn-butanol andn-hexane, gave an enriched fraction of 85.3% of 20:3 and 20:4 fatty acids. Partial hydrolysis of the triglycerides ofBiota oil byC. cylindracea lipase in potassium phosphate buffer at 25°C resulted in a 2.8-fold enrichment ofcis-5 polyunsaturated fatty acids (40.8% of 20:3 and 20:4) as contained in the unhydrolyzed acylglycerol fractions.

Prostaglandins E1 and E2 are synthesized in the intestine and mediate a range of gastrointestinal functions via activation of the prostanoid E type (EP) family of receptors. We examined the potential role of EP receptors in the regulation of gut hormone secretion from L cells. Analysis of mRNA expression in mouse enteroendocrine GLUTag cells demonstrated the abundant expression of EP4 receptor, whereas expression of other EP receptors was much lower. Prostaglandin E1 and E2, nonselective agonists for all EP receptor subtypes, triggered glucagon like peptide 1 (GLP-1) secretion from GLUTag cells, as did the EP4-selective agonists CAY10580 and TCS2510. The effect of EP4 agonists on GLP-1 secretion was blocked by incubation of cells with the EP4-selective antagonist L161,982 or by down-regulating EP4 expression with specific small interfering RNA. Regulation of gut hormone secretion with EP4 agonists was further studied in mice. Administration of EP4 agonists to mice produced a significant elevation of plasma levels of GLP-1, glucagon like peptide 2 (GLP-2) and peptide YY (PYY), whereas gastric inhibitory peptide (GIP) levels were not increased. Thus, our data demonstrate that activation of the EP4 receptor in enteroendocrine L cells triggers secretion of gut hormones.

The GPR119 receptor plays an important role in the secretion of incretin hormones in response to nutrient consumption. We have studied the ability of an array of naturally occurring endocannabinoid-like lipids to activate GPR119 and have identified several lipid receptor agonists. The most potent receptor agonists identified were three N-acylethanolamines: oleoylethanolamine (OEA), palmitoleoylethanolamine, and linoleylethanolamine (LEA), all of which displayed similar potency in activating GPR119. Another lipid, 2-oleoylglycerol (2-OG), also activated GPR119 receptor but with significantly lower potency. Endogenous levels of endocannabinoid-like lipids were measured in intestine in fasted and refed mice. Of the lipid GPR119 agonists studied, the intestinal levels of only OEA, LEA, and 2-OG increased significantly upon refeeding. Intestinal levels of OEA and LEA in the fasted mice were low. In the fed state, OEA levels only moderately increased, whereas LEA levels rose drastically. 2-OG was the most abundant of the three GPR119 agonists in intestine, and its levels were radically elevated in fed mice. Our data suggest that, in lean mice, 2-OG and LEA may serve as physiologically relevant endogenous GPR119 agonists that mediate receptor activation upon nutrient uptake.

International audience; Auxin transport, which is mediated by specialized influx and efflux carriers, plays a major role in many aspects of plant growth and development. AUXIN1 (AUX1) has been demonstrated to encode a high-affinity auxin influx carrier. In Arabidopsis thaliana, AUX1 belongs to a small multigene family comprising four highly conserved genes (i.e., AUX1 and LIKE AUX1 [LAX] genes LAX1, LAX2, and LAX3). We report that all four members of this AUX/LAX family display auxin uptake functions. Despite the conservation of their biochemical function, AUX1, LAX1, and LAX3 have been described to regulate distinct auxin-dependent developmental processes. Here, we report that LAX2 regulates vascular patterning in cotyledons. We also describe how regulatory and coding sequences of AUX/LAX genes have undergone subfunctionalization based on their distinct patterns of spatial expression and the inability of LAX sequences to rescue aux1 mutant phenotypes, respectively. Despite their high sequence similarity at the protein level, transgenic studies reveal that LAX proteins are not correctly targeted in the AUX1 expression domain. Domain swapping studies suggest that the N-terminal half of AUX1 is essential for correct LAX localization. We conclude that Arabidopsis AUX/LAX genes encode a family of auxin influx transporters that perform distinct developmental functions and have evolved distinct regulatory mechanisms.

Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms., Author Summary Most people know Staphylococcus aureus as a highly infectious “Super Bug”, but many are not aware that S. aureus non-infectiously colonizes much of the population in the nose, among other bodily niches. It is not well understood how and why S. aureus is able to adapt and readily switch between the commensal and pathogenic lifestyle within the host; however, the capacity to accumulate into multicellular aggregates is a hallmark associated with both lifestyles. In this work, we demonstrate that S. aureus produces extracellular fibers in multicellular biofilm communities, and that these fibers help the bacterial community to withstand physical stresses. Surprisingly, these fibers consist of small peptides called phenol soluble modulins (PSMs) that have previously been implicated in biofilm disassembly and virulence. We demonstrate that accumulation of PSM peptides into fibers modulates their ability to disperse biofilms. Thus, PSMs fulfill dual and opposing roles that are modulated by amyloid-like aggregation. Staphylococci have never before been shown to produce this kind of extracellular structure, and our findings may have a profound impact on our understanding of S. aureus physiology. We have found that these structures are composed of known virulence factors, which indicates a common pathway between infectious and static lifestyles in the body.

In this study the ability of five novel anti-oestrogens [4-iodotamoxifen, pyrrolidino-4-iodotamoxifen, ethyl bromide tamoxifen (EBTx), ICI 164,384 (ICI 164) and ICI 182,780] to alter drug toxicity to multidrug resistant cell lines have been compared. The effect of these compounds on ATP-dependent vinblastine (VBL) transport was also tested using inside-out vesicles (IOV) prepared from highly P-glycoprotein (Pgp)-expressing CCRF-CEM/VBL1000 cells. The pure anti-oestrogen ICI 164 was most effective, enhancing doxorubicin and VBL toxicity to MCF-7Adr cells 25- and 35-fold, respectively, and was also the best inhibitor of ATP-dependent [3H]VBL accumulation by IOV. Pure anti-oestrogens, tamoxifen and iodotamoxifens completely reversed VBL resistance in the mdr1 transfected lung cancer cell line, S1 1.1 , where resistance relative to wild-type cells was mediated solely by Pgp. The membrane impermeant tamoxifen derivative EBTx did not modify drug resistance, yet was as effective an inhibitor of VBL accumulation by inside-out Pgp-positive vesicles as tamoxifen. This indicates an intracellular role for tamoxifen and its derivatives in the modulation of Pgp-mediated drug resistance.

γ-Linolenic acid (GLA, all-cis 6,9,12-octadecatrienoic acid) has been enriched from fatty acids of borage (Borago officinalis L.) seed oil to 93% from the initial concentration of 20% by lipase-catalyzed selective esterification of the fatty acids withn-butanol in the presence ofn-hexane as solvent. The immobilized fungal lipase preparation, Lipozyme, used as biocatalyst, preferentially esterified palmitic, stearic, oleic and linoleic acids and discriminated against GLA, which was thus concentrated in the unesterified fatty acids fraction. In the absence of hexane, concentrate containing about 70% GLA was obtained. When the reaction conditions, optimized for borage oil fatty acids, were applied to fatty acids of evening primrose (Oenothera biennis L.) oil, concentrates containing 75% GLA were obtained. From both oils, GLA concentrates were prepared efficiently in short reaction times (1–3 h) at 30–60°C. The process can be applied for the production of GLA concentrates for dietetic purposes.

Lipase-catalyzed selective partial hydrolysis of evening primrose (Oenothera biennis L.) seed oil and borage (Borago officinalis L.) seed oil led to an increase in the level of γ-linolenic acid (GLA; 18∶3n−6) in the unhydrolyzed acylglycerols. Thus, in evening primrose oil, the GLA level could be raised from 9.4% in the starting material to 46.5% in the unhydrolyzed acylglycerols by means of a lipase fromCandida cylindracea. Selective hydrolysis of borage oil with Pancreatin led to an increase in the GLA content from 20.4% in the oil to 33.5% in the unhydrolyzed acylglycerols. Partial hydrolysis of borage oil with lipase fromC. cylindracea raised the GLA content of the acylglycerols to 47.8%.

Chitinases (designated as SPCs) were isolated from ‘Shilbilati’ potatoes, a potato prototype cultivated in Bangladesh by affinity chromatography on a chitin column. SPCs agglutinated rat erythrocytes at the minimum concentration of 7 µg/mL and showed toxicity against brine shrimp nauplii with the LC50 value of 20 µg/mL. The chitinases also agglutinated seven bacterial strains among the twelve as studied. Pseudomonas aeruginosa, Bacillus subtilis and Salmonella typhi were the most sensitive towards the SPCs and were agglutinated at 1.2, 2.5 and 5.0 µg/mL protein concentrations respectively. Antibacterial tests demonstrated that SPCs showed inhibitory activity against the pathogenic bacteria Staphylococcus aureus, Bacillus subtilis and Salmonella typhi. Antifungal activity was investigated by the disc diffusion method. Five fungal species (Candida albicans, Aspergillus niger, Fusarium vasinfectum, Aspergillus fumigatus and Aspergillus flavus) and two fungal genus (Penicillium and Mucor sp.) were examined in the assay. SPCs showed antifungal activity against Candida albicans, Fusarium vasinfectum and Penicillium sp.

A study was conducted on some physico-chemical and sensory characteristics of buffalo meat frozen by plate and blast freezing and stored at −15 ± 3°C for a period of 3 months. A marginal increase in pH values and drip losses were observed during the storage period. Drip losses were less in blast frozen samples. WHC, cooking losses thermal shrinkage and WB Shear values indicated inconsistent results, during storage. Similar observations were recorded with regard to tyrosine and TBA values. No significant differences in the physico-chemical characteristics were observed between meat cuts and minced meat. Plate frozen meat samples scored higher for texture, juiciness and aroma. Both the plate and blast frozen meat samples, however, were similar in overall quality according to taste panel results.

Myelin fractions with different degrees of compaction were isolated from bovine brain, and post-translational methylation of membrane-associated proteins was studied. When the purified myelin-basic-protein-specific protein methylase I and S-adenosyl-L-[methyl-14C]methionine were added exogenously, the most compact myelin fraction exhibited higher methyl-accepting activity than the less compact dense fractions. The methylated protein was identified as myelin basic protein (18.4 kDa) exclusively among the several myelin proteins from all membrane fractions, by SDS/PAGE/radioautography of methyl-14C-labelled membrane proteins. The methyl-14C-labelled amino acid residue in the basic protein was identified by h.p.l.c. as NG-methylarginine, indicating the high degree of specificity for the arginine residue as well as the myelin basic protein in the intact myelin membranes. The possibility of a charge alteration of myelin basic protein resulting from its arginine methylation was investigated by using the purified component 1 of myelin basic protein. The methylated component was shown to be less cationic than the unmethylated component by Bio-Rex 70 cation-exchange chromatography, since the former preceded the latter. However, in the presence of the denaturant (guanidinium chloride), the two species were co-eluted, indicating that the charge difference between methylated and unmethylated myelin basic protein can only be shown under the renatured condition.

The protein C (PC) pathway plays an important role in vascular and immune function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. However, the association of acquired PC deficiency with the pathophysiology of lung injury is unclear. We hypothesized that low PC induced by sepsis would associate with increased pulmonary injury and that replacement with activated protein C (APC) would reverse the activation of pathways associated with injury. Using a cecal ligation and puncture (CLP) model of polymicrobial sepsis, we examined the role of acquired PC deficiency on acute lung injury assessed by analyzing changes in pulmonary pathology, chemokine response, inducible nitric-oxide synthase (iNOS), and the angiotensin pathway. Acquired PC deficiency was strongly associated with an increase in lung inflammation and drivers of pulmonary injury, including angiotensin (Ang) II, thymus and activation-regulated chemokine, plasminogen activator inhibitor (PAI)-1, and iNOS. In contrast, the protective factor angiotensin-converting enzyme (ACE)-2 was significantly suppressed in animals with acquired PC deficiency. The endothelial protein C receptor, required for the cytoprotective signaling of APC, was significantly increased post-CLP, suggesting a compensatory up-regulation of the signaling receptor. Treatment of septic animals with APC reduced pulmonary pathology, suppressed the macrophage inflammatory protein family chemokine response, iNOS expression, and PAI-1 activity and up-regulated ACE-2 expression with concomitant reduction in AngII peptide. These data demonstrate a clear link between acquired PC deficiency and pulmonary inflammatory response in the rat sepsis model and provide support for the concept of APC as a replacement therapy in acute lung injury associated with acquired PC deficiency.

Adiponectin/adiponectin receptors (AdipoR) are involved in energy homeostasis and inflammatory pathways. To investigate the role of AdipoR2 in metabolic control, we studied the lipid and glucose metabolic phenotypes in AdipoR2-deficient mice. AdipoR2 deletion diminished high-fat diet-induced dyslipidemia and insulin resistance yet deteriorated glucose homeostasis as high-fat feeding continued, which resulted from the failure of pancreatic β-cells to adequately compensate for the moderate insulin resistance. A defect in the AdipoR2 gene may represent a mechanism underlying the etiology of certain subgroups of type 2 diabetic patients who eventually develop overt diabetes, whereas other obese patients do not.