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Objectives: We have investigated the effect of lutein on phospholipase A₂ (PLA₂) isozymes., Methods: We measured arachidonic acid release in [³H]arachidonic acid-labelled Raw 264.7 cells and PLA₂ activity using 1-palmitoyl-2-[¹⁴C]arachidonyl phosphatidylcholine ([¹⁴C]AA-PC) and 10-pyrene phosphatidylcholine in vitro., Key Findings: Lutein suppressed the release of arachidonic acid and inhibited Raw 264.7 cell-derived cytosolic Ca²+-dependent PLA₂ (cPLA₂-induced hydrolysis of [¹⁴C]AA-PC in a dose- and time-dependent manner. In contrast, lutein did not affect secretory Ca²+-dependent PLA₂ (sPLA₂)-induced hydrolysis of [¹⁴C]AA-PC. A Dixon plot showed that the inhibition by lutein on cPLA₂ appeared to be competitive with an inhibition constant, K(i) , of 13.6 µm., Conclusions: We suggest that lutein acted as a competitive inhibitor of cPLA₂ but did not affect sPLA₂., (© 2010 The Authors. JPP © 2010 Royal Pharmaceutical Society of Great Britain.)

Antimicrobial peptides (AMPs) are produced by all forms of living organisms and represent a novel class of antibiotics to treat infectious diseases. In this study, 29 AMPs of varying length and characteristics were synthesised chemically and were evaluated for their ability to inhibit the growth of Bacillus globigii, Bacillus anthracis and Burkholderia thailandensis. Amongst the peptides tested, sheep myeloid antimicrobial peptide-29 (SMAP-29) was the most potent, inhibiting both B. globigii and B. anthracis at submicromolar concentrations. However, SMAP-29 was less effective against B. thailandensis (minimum inhibitory concentration of 71 microM). Haemolytic activity and cytotoxicity were determined using human blood cells and human embryonic kidney 293S cells, respectively. Most of the peptides tested showed varying degrees of haemolytic activity and cytotoxicity, with SMAP-29 being highly haemolytic and cytotoxic under the conditions tested. Nevertheless, strategies to reduce toxicity whilst maintaining high antimicrobial activity are worth pursuing in light of the results obtained., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

In the present study we examined the effect of culture media and protein source on the formation of pluripotent primary outgrowths from in vitro produced and in vivo derived porcine embryos as the first step towards the isolation of embryonic stem cells (ESCs). To do this we compared high glucose Dulbeccos Modified Eagles Medium (DMEM) with Minimal Essential Alpha Medium (αMEM) both supplemented with fetal bovine serum (FBS) or serum replacement (SR) in a 2 × 2 factorial design. Culture in DMEM or αMEM supplemented with 10% SR resulted in the establishment of homogenous populations of cells which expressed Oct 4 and Nanog. In contrast culture in either media with FBS resulted in the formation of embryonal outgrowths composed entirely of differentiated cells or a mixture of differentiated cells and putative ESCs which grew poorly and could not be passaged. Using αMEM medium containing 10% SR and culturing in 5% oxygen, putative ESC lines were isolated from in vitro and in vivo derived embryos at efficiencies of 2 and 10% respectively.

Twelve peptides of the general X-SO(2)-D-Ser-Ala-Arg-OH formula (where X = methyl, phenyl, α-tolyl, p-tolyl, 4-methylbenzyl, 1-naphtyl, 2-naphtyl, 4-chlorophenyl, 4-bromophenyl, 2-mesityl, 2,4,6-triisopropylphenyl, 4-acetamidophenyl) were obtained and tested for their effect on the amidolytic activities of urokinase, thrombin, trypsin, plasmin, t-PA and kallikrein. 2,4,6-triisopropylphenyl-SO(2)-D-Ser-Ala-Arg-OH was the most selective inhibitor of urokinase and α-tolyl-SO(2)-D-Ser-Ala-Arg-OH was the most active inhibitor of uPA with K(i) value 24 µM. The compounds were tested for their in vitro antitumour activity in the following human breast cancer cells: standard MCF-7 and estrogen-independent MDA-MB-231. Four of the synthesized peptides showed cytotoxic effects against MDA-MB-231 cell lines in the range from 2.9 to 8.5 µM. The examined compound did not influence to MCF-7 cancer cells. The synthesized peptides were nontoxic to pig's erythrocytes.

Thrombopoietin (TPO) is a humoral growth factor that has been shown to increase platelet activation in response to several agonists. Patients with sepsis have increased circulating TPO levels, which may enhance platelet activation, potentially participating to the pathogenesis of multi-organ failure. Aim of this study was to investigate whether TPO affects myocardial contractility and participates to depress cardiac function during sepsis. We showed the expression of the TPO receptor c-Mpl on myocardial cells and tissue by RT-PCR, immunofluorescence and western blotting. We then evaluated the effect of TPO on the contractile function of rat papillary muscle and isolated heart. TPO did not change myocardial contractility in basal conditions, but, when followed by epinephrine (EPI) stimulation, it blunted the enhancement of contractile force induced by EPI both in papillary muscle and isolated heart. An inhibitor of TPO prevented TPO effect on cardiac inotropy. Treatment of papillary muscle with pharmacological inhibitors of phosphatidylinositol 3-kinase, NO synthase, and guanilyl cyclase abolished TPO effect, indicating NO as the final mediator. We finally studied the role of TPO in the negative inotropic effect exerted by human septic shock (HSS) serum and TPO cooperation with TNF-alpha and IL-1beta. Pre-treatment with the TPO inhibitor prevented the decrease in contractile force induced by HSS serum. Moreover, TPO significantly amplified the negative inotropic effect induced by TNF-alpha and IL-1beta in papillary muscle. In conclusion, TPO negatively modulates cardiac inotropy in vitro and contributes to the myocardial depressing activity of septic shock serum.

The epithelial barrier of the intestine and the gut-associated lymphoid tissue (GALT) protects the host against luminal pathogenic micro-organisms. This is important at weaning, when animals are exposed to infectious agents and stresses. We have developed a rat model of intestinal inflammation post weaning, based on the systemic administration of Staphylococcus aureus enterotoxin B (SEB). Since the inflammatory response obtained is mild, the food intake pattern is not affected, which makes this model useful for studies of nutritional therapies for intestinal inflammatory disease. SEB increased T-lymphocytes in Peyer's patches and the number of activated T-lymphocytes in mesenteric lymph nodes (organized GALT). In the lamina propria, SEB increased activated T-lymphocytes as well as cytotoxic and natural killer-cell populations of the diffuse GALT. It also increased pro-inflammatory cytokines and inflammatory mediators in both Peyer's patches and mucosa. Rats given SEB had higher paracellular permeability to macromolecules, which was associated with a reduction in epithelial tightness. This model was used to examine whether dietary supplementation with spray-dried animal plasma proteins affects intestinal inflammation. Results showed that dietary plasma proteins can attenuate the mucosal immune response in both organized and diffuse GALT and that these effects are mediated by a reduction in the production of pro-inflammatory cytokines.

Objective: Lung inflammation is present in patients with systemic sclerosis (SSc) and interstitial lung disease (ILD), but the mechanisms linking inflammatory and fibrotic processes in ILD are unknown. Our aim was to investigate whether alveolar inflammation, reflected by increased alveolar concentration of exhaled nitric oxide (C(A)NO), is related to the ability of serum from patients with SSc to induce pulmonary fibroblast proliferation (PFP) and myofibroblast conversion., Methods: C(A)NO was measured in all subjects (37 patients with SSc and 10 healthy controls) whose sera were used to stimulate PFP (assessed by BrdU labeling index) and myofibroblast conversion (detected by alpha-smooth muscle actin expression). The PFP index in patients with SSc was compared to control values, and between patients with SSc who had elevated (> 4.3 ppb) and normal ( 4.3 ppb; n = 25, median 1.1, range 0.98-1.23) than in patients with SSc who had normal C(A)NO ( 4.3 ppb than in patients whose C(A)NO was

Natural populations often show genetic variation in pathogen resistance, which is paradoxal because natural selection is expected to erode genetic variation in fitness-related traits. Several different factors have been suggested to maintain such variation, but their relative importance is still poorly understood. Here we examined if environmental heterogeneity and genetic trade-offs could contribute to the maintenance of genetic variation in immune function of a freshwater snail Lymnaea stagnalis. We assessed the immunocompetence of snails originating from different families and maintained in different feeding treatments (ad libitum feeding, no food) by measuring the density of circulating hemocytes, phenoloxidase activity, and antibacterial activity of snail hemolymph. Food limitation reduced snail immune function, and we found significant among-family variation in hemocyte concentration and PO activity, but not in antibacterial activity. Interestingly, food availability modified the family-level variation observed in PO activity so that the relative immunocompetence of different snail families changed over environmental conditions (G x E interaction). We found no evidence for genetic trade-offs between snail growth and immune defense nor among immune traits. Thus, our findings support the idea that environmental heterogeneity may promote maintenance of genetic variation in immune defense, but also suggest that different immune traits might not respond similarly to environmental variation.

Melanospheres, the melanoma cells that grow as nonadherent colonies and that show in vitro self-renewing capacity and multipotency, were selected from melanoma specimens or from melanoma cell lines. Melanospheres were highly tumorigenic, and intradermal injections in severe combined immunodeficient (SCID) mice of as few as 100 cells generated tumors that maintained tumorigenic potential into subsequent recipients. Primary and serially transplanted xenografts recapitulated the phenotypic features of the original melanoma of the patient. Melanoma cells cultured in the presence of fetal calf serum (FCS) were also tumorigenic in SCID mice, although with lower efficiency; these xenografts showed a homogeneous phenotype for the expression of melanoma-associated markers, Melan-A/Mart-1, HMB45, and MITF, and contained cells with features of fully differentiated cells. Melanospheres were heterogeneous for the expression of stem cell markers and showed a significantly enhanced expression of the Nanog and Oct3/4 transcription factors when compared with adherent melanoma cells. No direct and unique correlation between any of the examined stem cell markers and in vivo tumorigenicity was found. Taken together, our data provide further evidence on the heterogeneous nature of human melanomas and show that melanospheres and their corresponding tumors, which are generated in vivo in immunocompromised mice, represent a model to investigate melanoma biology.

The burgeoning obesity and diabetes epidemics threaten health worldwide, yet the molecular mechanisms underlying these phenomena are incompletely understood. Recently, attention has focused on the potential contributions of environmental pollutants that act as endocrine disrupting chemicals (EDCs) in the pathogenesis of metabolic diseases. Because glucocorticoid signaling is central to adipocyte differentiation, the ability of EDCs to stimulate the glucocorticoid receptor (GR) and drive adipogenesis was assessed in the 3T3-L1 cell line. Various EDCs were screened for glucocorticoid-like activity using a luciferase reporter construct, and four (bisphenol A (BPA), dicyclohexyl phthalate (DCHP), endrin, and tolylfluanid (TF)) were shown to significantly stimulate GR without significant activation of the peroxisome proliferator-activated receptor-gamma. 3T3-L1 preadipocytes were then treated with EDCs and a weak differentiation cocktail containing dehydrocorticosterone (DHC) in place of the synthetic dexamethasone. The capacity of these compounds to promote adipogenesis was assessed by quantitative oil red O staining and immunoblotting for adipocyte-specific proteins. The four EDCs increased lipid accumulation in the differentiating adipocytes and also upregulated the expression of adipocytic proteins. Interestingly, proadipogenic effects were observed at picomolar concentrations for several of the EDCs. Because there was no detectable adipogenesis when the preadipocytes were treated with compounds alone, the EDCs are likely promoting adipocyte differentiation by synergizing with agents present in the differentiation cocktail. Thus, EDCs are able to promote adipogenesis through the activation of the GR, further implicating these compounds in the rising rates of obesity and diabetes.

The ICH S6 guidance was developed to describe a rational science-based flexible approach to the preclinical evaluation for biotechnology-derived pharmaceutical products. It also suggested that some of the principles described may be suitable for plasma-derived therapeutics. Some of the specific concerns unique to protein-based therapeutics include complexity in structure and potential immunogenicity. S6 has been interpreted by some industry and regulatory authorities, often due to lack of experience with these types of products, as encouraging a broader or more conventional toxicology program similar to that normally conducted for small molecules. The guidance does encourage important and necessary preclinical evaluations but also recognizes the limitations of studies in non-relevant animal species because they are without pharmacological interaction with the biologic. In addition, studies of human proteins are often limited in useful chronic, reproductive and carcinogenic toxicity evaluations by the immunological response in animals. Thus the safety evaluation of biopharmaceuticals and plasma derivatives in animals has limitations that cannot be adequately addressed by the use of testing paradigms used for small molecule pharmaceuticals. S6 focuses evaluations on well-designed studies in relevant species for reasonable time periods to make the best use of available resources and enable clinical trials., (2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.)

The inhibition of dihydrofolate reductase (DHFR) by antifolates is a common practice both in cell culture and in chemotherapy. Surprisingly, antifolate resistance was also observed in cultured murine myeloma cells (SP2/0) in the presence of human plasma (HP); thus, we used a proteomic approach to identify novel plasma biomarker(s) for this condition. In contrast to the in vitro antifolate response, metabolic enzymes and translation machinery proteins were found to be up-regulated in the presence of HP. The antifolate resistance inherent in HP may be explained by a simultaneous promotion of cell proliferation and the maintenance of DNA integrity. Furthermore, the factor(s) was found to be extrinsic, heat stable and very small in size. Adenine, a supplemented additive in erythrocyte preservation, was subsequently identified as the contributing factor and exogenous addition in cultures reversed the cytotoxicity induced by antifolates. Importantly, adenine-containing blood components, which may provide enhanced survival to otherwise sensitive antifolate-targeted cells, showed a dose-dependent adverse effect in transfusion recipients receiving antifolate (methotrexate) medications. These findings not only highlight a previously unnoticed role of adenine, but also emphasize a novel mechanistic link between transfusion and subsequently reduced survival in patients taking methotrexate.

In many bacterial species, the translational GTPase TypA acts as a global stress- and virulence regulator and also mediates resistance to the antimicrobial peptide BPI. On the chromosome of M. tuberculosis, typA is located next to narGHJI, which plays a role in adaptation of the pathogen to various environmental conditions. Here, we show that Mycobacterium tuberculosis is sensitive to P2, a derivative of BPI. Using a typA mutant of M. tuberculosis, we found this phenotype to be independent of TypA. We further tested typA expression in M. tuberculosis under defined stress conditions, such as oxygen- and nutrient depletion, low pH, heat shock, antibiotic stress and the presence of P2, and found that typA expression remains unaffected by any of these conditions. Analysis of growth and whole-genome expression revealed similar growth kinetics and gene expression profiles of the wild type and the mutant under normal growth conditions as well as under stress conditions. Our results suggest that in contrast to the findings in other bacteria, TypA does not act as a global stress- and virulence regulator in M. tuberculosis.

Objective: To investigate the capacity of ADAM15, a disintegrin metalloproteinase that is up-regulated in osteoarthritic (OA) cartilage, to protect chondrocytes against apoptosis induced by growth factor deprivation and genotoxic stress., Methods: Caspase 3/7 activity was determined in primary OA and ADAM15-transfected T/C28a4 chondrocytes upon exposure to the DNA-damaging agent camptothecin or serum withdrawal. Camptothecin-induced cytotoxicity was determined by measuring cellular ATP content. (Anti-)apoptotic proteins were analyzed by immunoblotting, and levels of messenger RNA (mRNA) for X-linked inhibitor of apoptosis (XIAP) were determined using real-time polymerase chain reaction. RNA interference was applied for down-regulation of ADAM15 and XIAP expression. Immunohistochemistry analysis of normal and OA cartilage samples was performed using XIAP- and ADAM15-specific antibodies., Results: ADAM15-transfected chondrocytes cultured on a collagen matrix displayed significantly reduced caspase 3/7 activity upon serum or intermittent matrix withdrawal, compared with vector-transfected control cells. Apoptosis induction by camptothecin exposure also led to significantly elevated caspase 3/7 activity and reduced cell viability of the vector-transfected compared with ADAM15-transfected chondrocytes. Increased levels of activated caspase 3 and cleaved poly(ADP-ribose) polymerase were detected in the vector controls. XIAP, an inhibitor of activated caspase 3, was significantly up-regulated ( approximately 3-fold) at the protein and mRNA levels in ADAM15-transfected chondrocytes upon camptothecin treatment. Specific down-regulation of either ADAM15 or XIAP in OA chondrocytes led to significant sensitization to camptothecin-induced caspase 3/7 activity. Immunohistochemical analysis revealed low to moderate XIAP expression in normal specimens and markedly increased XIAP staining, colocalizing with ADAM15, in OA cartilage., Conclusion: ADAM15 conveys antiapoptotic properties to OA chondrocytes that might sustain their potential to better resist the influence of death-inducing stimuli under pathophysiologic conditions.

Collagen-platelet (PL)-rich plasma composites have shown in vivo potential to stimulate anterior cruciate ligament (ACL) healing at early time points in large animal models. However, little is known about the cellular mechanisms by which the plasma component of these composites may stimulate healing. We hypothesized that the components of PL-rich plasma (PRP), namely the PLs and PL-poor plasma (PPP), would independently significantly influence ACL cell viability and metabolic activity, including collagen gene expression. To test this hypothesis, ACL cells were cultured in a collagen type I hydrogel with PLs, PPP, or the combination of the two (PRP) for 14 days. The inclusion of PLs, PPP, and PRP all significantly reduced the rate of cell apoptosis and enhanced the metabolic activity of fibroblasts in the collagen hydrogel. PLs promoted fibroblast-mediated collagen scaffold contraction, whereas PPP inhibited this contraction. PPP and PRP both promoted cell elongation and the formation of wavy fibrous structure in the scaffolds. The addition of only PLs or only plasma proteins did not significantly enhance gene expression of collagen types I and III but the combination, as PRP, did. Our findings suggest that the addition of both PLs and plasma proteins to collagen hydrogel may be useful in stimulating ACL healing by enhancing ACL cell viability, metabolic activity, and collagen synthesis.

Hypertrophic "light" and "dark" chondrocytes have been reported as morphologically distinct cell types in growth cartilage during endochondral ossification in many species, but functional differences between the two cell types have not been described. The aim of the current study was to develop a pellet culture system using chondrocytes isolated from epiphyseal cartilage of neonatal mice and rats, for the study of functional differences between these two cell types. Hypertrophic chondrocytes resembling those described in vivo were observed by light and electron microscopy in sections of pellets treated with triiodothyronine, 1% fetal calf or mouse serum, 10% fetal calf serum or 1.7MPa centrifugal pressure at day 14, and in pellets cultured with insulin or 0.1% fetal calf or mouse serum at day 21. A mixed population of light and dark chondrocytes was found in all conditions leading to induction of chondrocyte hypertrophy. This rodent culture system allows the differentiation of light and dark chondrocytes under various conditions in vitro and will be useful for future studies on tissue engineering and mechanisms of chondrocyte hypertrophy., (Copyright 2010 Elsevier Ltd. All rights reserved.)

There is mounting evidence that tumors are initiated by a rare subset of cells called cancer stem cells (CSCs). CSCs are generally quiescent, self-renew, form tumors at low numbers, and give rise to the heterogeneous cell types found within a tumor. CSCs isolated from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Here we show that vitronectin is the component of human serum driving stem cell differentiation through an integrin alpha V beta 3-dependent mechanism. CSCs cultured on vitronectin result in downregulation of stem cell genes, modulation of differentiation markers, and loss of beta-catenin nuclear localization. Blocking integrin alpha V beta 3 inhibits differentiation and subsequently tumor formation. Thus, CSCs must be engaged by one or more extracellular signals to differentiate and initiate tumor formation, defining a new axis for future novel therapies aimed at both the extrinsic and intracellular pathways.

Activation of the serine/threonine kinase Akt is associated with aggressive clinical behavior of prostate cancer. We found that the human prostate cancer cell lines LNCaP and PC-3 express predominantly Akt1 and Akt2. Selective down-regulation of Akt1, but not Akt2, by short-hairpin RNA reduced the viability of prostate cancer cells. In addition, structurally different Akt inhibitors were cytotoxic for the prostate cancer cells, confirming that the Akt pathway is indispensable for their viability. We have purified the tetracyclic triterpenoids 3-oxo-tirucallic acid, 3-alpha-acetoxy-tirucallic acid, and 3-beta-acetoxy-tirucallic acid from the oleogum resin of Boswellia carterii to chemical homogeneity. The acetoxy-derivatives in particular potently inhibited the activities of human recombinant Akt1 and Akt2 and of constitutively active Akt immunoprecipitated from PC-3 cells, whereas inhibitor of nuclear factor-kappaB kinases remained unaffected. Docking data indicated that these tetracyclic triterpenoids form hydrogen bonds within the phosphatidylinositol binding pocket of the Akt pleckstrin homology domain. Accordingly, 3-beta-acetoxy-tirucallic acid did not inhibit the activity of Akt1 lacking the pleckstrin homology domain. In the prostate cancer cell lines investigated, these compounds inhibited the phosphorylation of cellular Akt and the Akt signaling pathways, including glycogen synthase kinase-3beta and BAD phosphorylation, nuclear accumulation of p65, the androgen receptor, beta-catenin, and c-Myc. These events culminated in the induction of apoptosis in prostate cancer, but not in nontumorigenic cells. The tirucallic acid derivatives inhibited proliferation and induced apoptosis in tumors xenografted onto chick chorioallantoic membranes and decreased the growth of pre-established prostate tumors in nude mice without overt systemic toxicity. Thus, tirucallic acid derivatives represent a new class of Akt inhibitors with antitumor properties.

The consequences of cell stress induced by misfolded proteins are an important contributor to many human diseases. One such disease is epidermolysis bullosa simplex (EBS), caused by mutations in the structural proteins (keratins K5 or K14) of the proliferative compartment of the epidermis (basal keratinocyte layer), leading to cell fragility and blistering. In severe EBS, the mutation is associated with aggregates of nonfilamentous keratin protein, and cell lines carrying such mutations show a constitutively activated stress response. Analysis of the cellular mechanisms leading to cell breakdown on physical stress may point the way to mutation-independent therapeutic approaches to these incurable genetic disorders. We therefore subjected EBS cell lines, immortalized from patients with EBS, to an oscillating mechanical stress in assays designed to mimic the physical trauma that leads to cell breakdown in vivo. These experiments show that mechanical stress activates extracellular signal-regulated kinase (ERK) signaling in these cells, and that the keratin mutant cells also show a resistance to apoptosis following mechanical stress that is reversed by inhibiting ERK. The consequences of constitutive expression of large amounts of defective structural protein in a tissue cell must be properly understood for the development of safe and effective therapies for these disorders.

Background: Hypothermia-induced changes in endothelial cell (EC) morphology and function after organ storage may influence the initial outcome and development of transplant-associated coronary artery disease., Methods: Human saphenous vein ECs were incubated with saline (NaCl), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) solution, with and without protein additives, at 4 degrees C and 37 degrees C. After 6 hours, ECs were recultivated for 24 and 48 hours with culture medium (reperfusion). Mitochondrial activity, adenosine triphosphate concentration, cell count, and inflammatory responses were analyzed., Results: Cold preservation did not affect the mitochondrial activity of ECs and allowed a complete regeneration of the metabolic turnover after reperfusion. However, under normothermic conditions the metabolism of the cells was influenced by time and type of preservation solution. While both the mitochondrial activity and cell count did not change after treatment with NaCl and culture medium, the metabolic turnover of cells treated with HTK and UW solution significantly increased (twofold) and decreased (twofold, p < 0.05), respectively, after reperfusion. The endothelial reactivity remained unchanged after treatment with NaCl and HTK. The addition of serum proteins significantly improved mitochondrial activity of cells treated with warm NaCl and HTK (p < 0.05). The UW-treated cells burned out through a significant up-regulation of the ATP concentration resulting in a complete metabolic regression after reperfusion and induction of apoptosis., Conclusions: Normothermic preservation in UW prevented regeneration of ECs, while treatment with HKT solution did not irreversibly affect mitochondrial activity of ECs and allowed complete regeneration of metabolism and function. Serum proteins improved the preservation effect of HTK and NaCl., (2010 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objective: To develop an efficient freezing method suitable for large-scale cryopreservation of human embryonic stem cells (hESCs)., Design: Experimental study., Setting: Research institute., Patient(s): None., Intervention(s): Two genetically modified hESC lines, H9-EF1-GFP and CHA-hES3-EF1-GFP, were cryopreserved in cryovials using a combination of two equilibration methods (one-step and stepwise) and two cooling vehicles (cryo-container and program-controlled freezer). After thawing, the survival and differentiation rate were compared among groups., Main Outcome Measure(s): The hESC survival was assessed by alkaline phosphatase staining and differentiation status was determined by flow cytometry using an SSEA-4 antibody., Result(s): In both H9-EF1-GFP and CHA-hES3-EF1-GFP cells, the survival rate was highest in the group using stepwise equilibration and program-controlled freezer, and lowest in the group using one-step equilibration and cryo-container. In the groups using cryo-container, the survival and the frequency of undifferentiated cells in both cell lines was highly improved in a stepwise equilibration compared with one-step. Thawed hESCs were positively stained with pluripotent markers SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The karyotypes and expression of three germ layer markers in both cell lines were not changed after freezing/thawing., Conclusion(s): The stepwise equilibration of Knockout Serum Replacement and cryoprotectant during freezing and thawing resulted in higher survival rates by reducing osmotic damage irrespective of cooling vehicles., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Dimebon, a drug currently being evaluated in multiple Phase III Alzheimer's disease trials, has previously been shown to have effects on isolated mitochondria at muM concentrations. Here the effects of nM concentrations of Dimebon on mitochondrial function were investigated both in primary mouse cortical neurons and human neuroblastoma cells (SH-SY5Y). Under non-stress conditions nM concentrations of Dimebon increased succinate dehydrogenase activity (MTT-assay), mitochondrial membrane potential (DeltaPsim), and cellular ATP levels. Dimebon treatment had no effect on mitochondria DNA content, implying that mitochondrial biogenesis was not induced. Under stress conditions, mitochondria in Dimebon-treated neurons showed increased resistance to elevated intracellular calcium concentrations, thus, maintaining their DeltaPsim throughout the experiment, in contrast to control neurons, which rapidly lost their DeltaPsim. Moreover, we show that serum-starved differentiated SH-SY5Y cells treated with Dimebon had an increased survival rate as compared to untreated cells. In conclusion, these data demonstrate that Dimebon enhances mitochondrial function both in the absence and presence of stress and Dimebon-treated cells are partially protected to maintain cell viability.

Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndrome and type 2 diabetes. Adipocyte dysfunction has been recognized as a link between these conditions. To examine the effect of AOPPs on adipocyte perturbation, 3T3-L1 adipocytes were treated with increased levels of AOPPs as seen in these conditions. Exposure of adipocytes to AOPPs induced overexpression of tumor necrosis factor α and interleukin-6. This inflammatory response was completely blocked by nuclear factor-κB inhibitor SN50. AOPPs challenge also impaired insulin signaling, which was partly prevented by SN50. Treatment with AOPPs triggered endoplasmic reticulum (ER) stress, revealed by phosphorylation of PKR-like eukaryotic initiation factor 2α kinase, eukaryotic translational initiation factor 2α, inositol-requiring enzyme 1 and c-jun N-terminal kinase, and by overexpression of glucose regulated protein 78. AOPPs-induced ER stress was mediated by reactive oxygen species (ROS) generated by activation of NADPH oxidase since it was prevented by NADPH oxidase inhibitors or ROS scavenger. Treating the cells with inhibitors of NADPH oxidase or ER stress could completely abolish AOPPs-induced overexpression of adipocytokines and insulin resistance, suggesting that AOPPs induced adipocyte perturbation probably through induction of ROS-dependent ER stress. Our data identified AOPPs as a class of important mediator of adipocyte perturbation. Accumulation of AOPPs might be involved in adipocyte dysfunction as seen in metabolic syndrome and type 2 diabetes., (Copyright © 2010 S. Karger AG, Basel.)

Spray-dried plasma (SDP) is a complex mixture of active proteins that modulates the immune response of gut-associated lymphoid tissue. We examined whether SDP and Ig concentrate (IC) supplementation could modulate cytokine expression and inflammatory mediators in rats challenged with Staphylococcus aureus enterotoxin B (SEB). Wistar-Lewis rats were fed diets supplemented with SDP (8% wt:wt), IC (1.5% wt:wt), or milk proteins (control diet) from weaning (d 21) to d 34 after birth. On d 32 and 35, the rats were given SEB (0.5 mg/kg; intraperitoneal). Six hours after the second SEB dose, jejunal mucosa and Peyer's patches (PP) from the small intestine were collected. The cytokines interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-6, IL-10, transforming growth factor-beta (TGFbeta), and leukotrienne B(4) (LTB(4)) were analyzed using commercial kits. SEB increased the release of proinflammatory mediators (IFNgamma, TNFalpha, IL-6, and LTB(4)) in PP (P < 0.05) and in the mucosa (P < 0.05). In both tissues, SDP prevented the increase in IFNgamma, IL-6, and LTB(4) induced by SEB (P < 0.05). IC reduced the expression of TNFalpha and LTB(4) in PP and mucosa (P < 0.05). SDP supplementation increased IL-10 and mature TGFbeta concentrations in intestinal mucosa from both inflamed and noninflamed rats. Both SDP and IC increased the mature:total TGFbeta ratio (all P < 0.05). Both supplements were effective at preventing the SEB-induced increase in proinflammatory:antiinflammatory cytokine ratios in PP and mucosa and in serum. The preventive effects of plasma supplements on intestinal inflammation involve modulation of intestinal cytokines, characterized by an increased expression of antiinflammatory cytokines.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a 40-residue cationic peptide originally purified from human blood ultrafiltrate. The native peptide contains two disulfide bonds and is unique regarding its primary structure. Its biological role is not known but a previous study showed that chemically synthesized LEAP-2 exhibited in vitro antimicrobial activities against several Gram-positive bacteria. In order to determine its antimicrobial mode of action, we expressed human recombinant LEAP-2 in Escherichia coli. Circular dichroism spectroscopy and nuclear magnetic resonance analyses showed that the structure of the recombinant peptide was identical to that of the chemically synthesized and oxidized LEAP-2, with two disulfide bonds between Cys residues in relative 1-3 and 2-4 positions. Minimal inhibitory concentration (MIC) of the recombinant human LEAP-2 was determined by a conventional broth dilution assay. It was found to be bactericidal against Bacillus megaterium at a 200microM concentration. Interestingly, the linear LEAP-2 had a greater antimicrobial activity with a MIC value of 12.5microM, which was comparable to that of magainin2. SYTOX Green uptake was used to assess bacterial membrane integrity. Linear LEAP-2 and magainin2 permeabilized B. megaterium membranes with the same efficiency, whereas oxidized LEAP-2 did not induce stain uptake. Binding of the peptides to plasmid DNA was evaluated by gel retardation assays. The DNA-binding efficacy of linear LEAP-2 was three times higher than that of the peptide-containing disulfide bridges. Altogether, these results show that the secondary structure of human LEAP-2 has a profound impact on its antibacterial activity.

Neutrophil-derived antimicrobial peptides and proteins (AMPs) play an important role in the defense against microbes. Absence of defense is illustrated by neutropenic patients with frequent bacterial and fungal infections. However, the specificity of the antimicrobial effects has not been adequately described. We set out to determine the specific antimicrobial pattern of polypeptides in neutrophils (polymorphonuclear leukocytes, PMNs) against 4 potential human pathogens: Moraxella catarrhalis, Staphylococcus aureus, Haemophilus influenzae and Candida albicans. Protein extracts of human PMNs were separated using high-performance liquid chromatography and fractions were assayed for antimicrobial activity. Fractions displaying antimicrobial activity were separated on SDS-PAGE and characterized using MALDI-MS. Depletion experiments were utilized to determine the contribution of each AMP to the antimicrobial effect. Among the identified AMPs, α-defensins 1-3, azurocidin, LL-37, lysozyme, calprotectin and lactotransferrin were studied in detail. We found a divergent pattern of killing, that is, certain peptides and proteins exhibited selective activity against specific pathogens, while others displayed a broader antimicrobial activity. α-Defensins, LL-37 and calprotectin were active against all species, while lactotransferrin exclusively inhibited growth of S. aureus. Conversely, azurocidin was active against all species except S. aureus. Our observations may shed light on bacterial resistance to AMPs and on the elimination of specific bacterial communities on mucosal surfaces., (Copyright © 2010 S. Karger AG, Basel.)

We examined the inhibitory effect of thyroid blocking antibody (TBAb) on the thyroid stimulating activity of human chorionic gonadotropin (HCG) and equine CG (ECG). Five TBAb positive sera obtained from patients who had been hypothyroid but were currently on T4 treatment. The TSH binding inhibitory immunoglobulin (TBII) activities of the sera were 60-160 IU/L. Inhibition of TSH binding to the TSH receptor (TSHR) [TSH binding inhibition (TBI) activity] of HCG or ECG, and inhibition of TBAb on HCG or ECG-stimulated cAMP production were examined. Both HCG and ECG preparations showed weak TBI activity in the presence of small amounts of protein [bovine serum albumin (BSA)] but were negative in the presence of large amounts of protein [normal human serum (NHS) or BSA]. Four thousand IU/mL of HCG and ECG preparation caused cAMP production similar to 100 microU/mL of bovine (b) TSH. The inhibitory effect of TBAb on cAMP production by this amount of HCG or ECG was then examined. The inhibitory effect of TBAb on cAMP production by HCG and ECG was similar to bTSH, and TBAb positive sera with more than 40 IU/L TBII activity completely blocked cAMP production by HCG, ECG and bTSH. This suggests that common alpha -subunit of both HCG and TSH are involved in the inhibitory effect of TBAb. Previous reports demonstrated that the thyroid stimulating activity of thyroid stimulating antibody (TSAb) was blocked by deglycosylated HCG (competitive antagonist of TSH binding to TSHR). The fact and our present study suggest that TSH, HCG ECG, TSAb and TBAb have a similar binding site (alpha-subunit-mimicking binding site) on the TSH receptor.

Antimicrobial peptides (AMPs) are important potential alternatives to conventional therapies against bacterial infections. rBPI(21) is a 21 kDa peptide based on the N-terminal region of the neutrophil bactericidal/permeability-increasing protein (BPI). This AMP possesses highly selective bactericidal effects on Gram-negative bacteria and have affinity for lipopolysaccharide (LPS) which is believed to be at the origin of its neutralizing effect of the LPS segregated into the bloodstream. We aim at understanding the molecular bases of rBPI(21) bactericidal and LPS neutralization actions, using biomembrane model systems. Using dynamic light scattering spectroscopy we demonstrate that rBPI(21) promotes aggregation of negatively charged large unilamellar vesicles (LUV), even in the absence of LPS, and LPS aggregates, while for zwitterionic phosphatidylcholine (POPC) LUV the size remains unchanged. The peptide also promotes the fusion (or hemifusion) of membranes containing phosphatidylglycerol (POPG). The aggregation and fusion of negatively charged LUV are peptide concentration-dependent until massive aggregation is reached, followed by sample flocculation/precipitation. Concomitantly, there is a progressive change in the zeta-potential of the LUV systems and LPS aggregates. LUV systems composed of phosphatidylglycerol (POPG) and lipid mixtures with POPG have higher zeta-potential variations than in the absence of POPG. The interaction of rBPI(21) with lipid vesicles is followed by leakage, with higher effect in POPG-containing membranes. LPS aggregation can be related with a decreased toxicity, possibly by facilitating its clearance by macrophage phagocytosis and/or blocking of LPS specific receptor recognition. Our data indicate that rBPI(21) mechanism of action at the molecular level involves the interaction with the LPS of the outer membrane of Gram-negative bacteria, followed by internalization and leakage induction through the (hemi)fusion of the bacterial outer and inner membranes, both enriched in phosphatidylglycerol.

Polymorphonuclear leukocytes (PMN) contribute to bacterial clearance by uptake and intracellular killing of microbes. However, antimicrobial polypeptides are released extracellularly where they are enweaved in a chromatin web that traps and eliminates bacteria. In addition, PMN-derived antimicrobial polypeptides direct monocytes and macrophages to the site of infection and activate their antimicrobial armor. Increased expression of Fcgamma receptors as well as opsonization of bacteria by PMN granule proteins support bacterial uptake by macrophages. PMN granule proteins also increase intracellular reactive oxygen species formation in macrophages. Finally, apoptotic PMN transfer parts of their antimicrobial peptides to macrophages, hence increasing killing of intracellular bacteria. Understanding mechanisms by which PMN granule proteins stimulate antimicrobial mechanisms in macrophages may open novel strategies in fighting bacterial infections.

Objective: To investigate whether sphingosine analogues, which activate the ceramide signaling pathway to apoptosis, can cause the death of ectopic (EEC) and eutopic stromal endometriotic cells (EEU), as well as healthy eutopic stromal endometrial cells (HEU)., Design: The EEC, EEU, and HEU isolated from fertile and infertile women with endometriosis were cultured for 48 hours in RPMI medium with 10% fetal calf serum (FCS) and with 2.5-10 microM sphingosine analogues., Setting: A clinic for the treatment of endometriosis and basic research laboratories., Patient(s): Nineteen women with follicular cyst and 16 women with endometriosis., Main Outcome Measure(s): The percentage of proliferating cells was determined by 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis and cell cycle were detected by fluorescence-activated cell sorter (FACS) Calibur flow cytometer., Result(s): The viability of EEC after exposure to 10 microM sphingosine analogues was 59.5% +/- 9.7% for D-sphingosine and 77.65 +/- 9.7% for DL-erythro-sphingosine, the viability of EEU was 69.2% +/- 14.2% and 42.0% +/- 15.5%, whereas the viability of comparative HEU was 9.0% +/- 4.8% and 18.8% +/- 8.3%, respectively. The differences were significant using the Mann-Whitney test. The apoptotic level of the cells treated with 10 microM sphingosine analogues for comparative HEU was 42.8% +/- 7.5% for D-sphingosine and 42.5% +/- 10.5% for DL-erythro-sphingosine, whereas for EEC this was 16.7% +/- 5.5% for D-sphingosine and 14.1% +/- 4.4% for DL-erythro-sphingosine and for EEU this was 14.3% +/- 4.7% and 22.9% +/- 8.9%, respectively., Conclusion(s): Ectopic and eutopic stromal endometrial cells from women with endometriosis have a damaged ceramide-downstream pathway to apoptosis.

Context: Fetuin-A inhibits the insulin receptor in vitro. Higher serum fetuin-A concentrations are associated with type 2 diabetes longitudinally and greater adiposity in cross-sectional analyses. Whether higher fetuin-A concentrations are associated with accumulation of adiposity over time is unknown., Objective: To determine the association of fetuin-A levels with changes in body composition over 5 yr., Study Design: Observational cohort study nested in the Health Aging and Body Composition Study., Predictor: Serum fetuin-A levels., Outcomes: Visceral adipose tissue (VAT), abdominal sc adipose tissue, and thigh muscle area by computed tomography, and waist circumference and body mass index were measured at baseline and again after 5 yr. Percent change and extreme change (>1.5 sds) in each measure were calculated., Results: Over 5 yr, subjects lost body mass in each measure, including 6% decline in VAT. Yet each sd (0.42 g/liter) higher fetuin-A concentration was associated with a 5.5% increase in VAT over 5 yr (95% confidence interval 1.9-9.2%; P = 0.003) in models adjusted for age, sex, race, clinical site, diabetes, physical activity, triglycerides, kidney function, and the baseline VAT score. Similarly, higher fetuin-A concentrations were associated with extreme VAT gain (relative risk 1.70, 95% confidence interval 1.12-2.60, P = 0.01). Fetuin-A concentrations were not statistically significant associated with change in any other measures of body composition (P > 0.20)., Conclusions: Higher fetuin-A concentrations are associated with the accumulation of VAT in well-functioning, community-living older persons. The mechanisms linking fetuin-A, VAT, and insulin resistance remain to be determined.

A review was done with 25 scientific papers published in the past fifteen years with the objective to verify the effects of spray dried plasma added to diets for pigs weaned at different ages on the feed intake, weight gain, intestinal structure, and number of E. coli colonies. Beneficial effects of spray dried plasma were observed during the first week after weaning for weight gain and feed intake of piglets weaned at 14, 21, and 28 days old. The beneficial effects of spray dried plasma decreased with the increase in weaning age. Positive effects of spray dried plasma were observed on the morph-physiological conditions of the small intestine, with higher villi height and crypt depth, when substituted completely for the powdered skim milk in diets for weaned pigs. Also, pigs fed diets with the inclusion of spray dried plasma had decreased E. coli colonies in the small intestine of pigs weaned at 21, 28, and 35 days old. The research also indicated that spray dried plasma can replace antibiotic in diets to pigs weaned at 28 and 35 days old. The article presents some promising patents on feed intake for pigs weaned at different ages.

Introduction: New in vitro models of castration-resistant prostate cancer (CRPC) are urgently required., Methods: Trans-rectal needle biopsies (TRBP) of the prostate were performed for research purposes on progressing CRPC patients who had not received prior treatment to the prostate. Biopsies were immediately digested with collagenase and plated onto collagen-coated flasks with a feeder layer of 3T6 cells and cultured in cytokine-supplemented keratinocyte serum-free medium., Results: Biopsies from 25 patients were collected and one of these, following an initial period of crisis, spontaneously immortalized. A series of cell lines called Bob were then established from a clone that survived CD133-selection followed by 4 weeks under adhesion-independent conditions in methylcellulose. Gains and losses previously described in clinical prostate tumors, most notably loss of 8(p) and gain of 8(q), were identified on comparative genomic hybridization and long-term growth in culture, survival in methylcellulose and invasion through matrigel confirmed the malignant phenotype of Bob. Furthermore, Bob expressed high levels of p53 and markers of early differentiation, including K8, prostatic acid phosphatase and prostate stem cell antigen. There was, however, no in vivo growth and ERG and ETV1 were not rearranged. Growth in serum permitted some differentiation., Conclusion: This is the first spontaneously immortalized prostate cancer cell line to be established from a TRBP of a patient with CRPC. Bob is a novel pre-clinical model for functional studies in CRPC and especially for studying the CRPC "basal" phenotype.

Background: Postoperative organ dysfunction in conventional surgery for abdominal aortic aneurysm (AAA) is associated with a complex inflammatory reaction, with activation of coagulation and fibrinolysis. A prospective,observational study was performed to define the complex plasma proteomic changes after AAA repair and to identify factor(s) that may affect myocardial function in uncomplicated procedures., Methods: Ten patients undergoing infrarenal AAA repair were investigated. Eight subjects subjected to major abdominal surgery served as controls. Hemodynamic changes were continuously monitored by using the pressure recording analytical method technique. The time course of plasma proteins was investigated after induction of anesthesia and at different times after surgery (6 h, 12 h, 24 h, 36 h) by using two-dimensional difference gel electrophoresis, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and Western blot. The effects of plasma on the functional properties of isolated rat ventricular myocytes were also investigated., Results: In AAA patients alone, 18 spots were found to change more than two-fold in expression level, spot identification revealing an increased thrombin generation 6 h after surgery. At the same time cardiac cycle efficiency significantly reduced versus baseline (-0.5 +/- 0.9 vs. 0.18 +/- 0.3 in AAA patients, P < 0.01; 0.4 +/- 0.1 vs. 0.2 +/- 0.3 in control surgery, not significant; P < 0.01 group x time interaction at ANOVA). Plasma obtained 6 h after AAA surgery dose-dependently inhibited contractile function of control rat myocytes (percent shortening fell by 51% with 10% of AAA plasma and was abolished with 20% of AAA plasma, P < 0.001 for both). The inhibitory response was abolished by thrombin antagonism., Conclusions: These findings show for the first time the possible role of thrombin generation within the complex activation of inflammatory response in causing hemodynamic instability in the early postoperative period after AAA surgery.

When titanium is anodized by discharge in NaCl solution, both antimicrobial activity and osteoconductivity are conferred. The viability of adherent micro-organisms and cells on antimicrobial titanium remains uncertain. We hypothesized that a thin peroxidation barrier would efficiently destroy adherent bacteria, whereas adherent osteoblastic cells would be viable, since these cells adhere to the surface indirectly though serum proteins. The efficacy of antimicrobial titanium appears to be based on peroxidation, since peroxidation products were detected in parallel with the destruction of bacterial cell-surface structures. The peroxidation effect of antimicrobial titanium was confined to the surface within narrow limits. The viability of osteoblastic cells on the surface was strongly dependent on the presence of serum protein, whereas that of adherent Streptococcus mutans was not affected by the presence of serum proteins. Therefore, differences in the adherent systems used by bacteria and osteoblastic cells are important determinants of their viability on antimicrobial titanium.

We previously purified acaloleptin A1, A2, and A3, antibacterial peptides that are produced in the larval hemolymph of Acalolepta luxuriosa (Udo longicorn beetle). In this study, we performed cDNA cloning. The cDNA sequence showed a predicted acaloleptin A precursor that consisted of five acaloleptin A isoforms. Four (isoforms 1, 2, 3 and 4) of the five isoforms of the acaloleptin A precursor had high-level sequence identities with each other, but the N-terminal region of isoform 5 differed from those of the other acaloleptin A isoforms. Northern and Western blot analyses showed that acaloleptin A isoforms were mass-produced soon after bacterial inoculation. Finally, we purified isoform 5 from hemolymph of the immunized larvae. Isoform 5, unlike acaloleptin A1, A2 and A3, showed antimicrobial activities against a Gram-positive bacterium, Micrococcus luteus and a fungus, Magnaporthe grisea. These results suggest that the multipeptide structure of the acaloleptin A precursor allows A. luxuriosa high-level production of antibacterial peptides and resistance to a wide range of microorganisms.

Alteration of VSMC (vascular smooth-muscle cell) physiology is associated with the development of atherosclerosis and restenosis. We hypothesize that aging up-regulates the expression of p16 INK4a in VSMCs, which may increase the susceptibility of blood vessels to vascular occlusive diseases. Aortic VSMCs were obtained from young and aged mice. Cells from aged mice grew more slowly than those from their younger counterparts. Progression of cell cycle in response to serum stimulation was significantly inhibited in those cells with aging, as determined by FACS after propidium iodide staining. A significant up-regulation of p16 INK4a (2.5-fold, P=0.0012) was found in VSMC from aged animals using gene arrays. The up-regulation of this gene was further confirmed by quantitative RT-PCR (reverse transcription-PCR) and Western-blot experiments. Immunostaining for p16 INK4a confirmed that aortas from aged mice contained more p16 INK4a+ SMA (smooth-muscle cell actin)+ cells than aortas from young animals (26.79+/-2.45 versus 7.06+/-1.44, P=0.00027, n=4). In conclusion, we have shown that aging up-regulates the expression of p16 INK4a in VSMC in both cultures and arteries. The increase in p16 INK4a in the vasculature with aging may modify VSMC's response to post-injury stress and therefore accelerate the development of age-related cardiovascular diseases.

Background: Atherosclerosis is one of the most life-threatening diseases primarily associated with hypercholesterolemia and is characterized by increased serum cholesterol level. Cholesterol originates from both its de novo synthesis within the hepatic cells and its absorption into the intestine in the form of dietary or bile cholesterol. Interventions influencing both of these processes are promising therapeutic options to lower the cholesterol level. Hydroxymethyl glutaryl-CoA reductase inhibitors, commonly known as statins, effectively block the rate determining step in the biosynthesis of cholesterol. Ezetimibe is the first new class of drugs used to treat hypercholesterolemia by inhibition of cholesterol absorption through Niemann Pick C1 Like 1 membrane of enterocytes. Therefore, combination therapy of ezetimibe and statins offers an efficacious new approach for the prevention and treatment of hypercholesterolemia., Objectives: The present review focuses on updates on ezetimibe and patented profile of novel cholesterol absorption inhibitors followed by critical analysis of different targets such as cholesterol esterase inhibitors, bile acid transport inhibitors or phospholipase-A(2) inhibitors, etc.which play an important role in the lipid absorption., Conclusion: The discovery of ezetimibe has opened a new door for the management of hyper-cholesterolemia in combination with statins. There are newer analogues that are under clinical trials, among which darapladib, FM-VP4 and A-002 are promising compounds.

Synergistic effects of ovine-derived cathelicidins SMAP29 and OaBac5mini with the antimicrobials polymyxin B, lysozyme, nisin and lactoferrin were investigated against E. coli O157:H7 and S. aureus 1056 MRSA. Lysozyme showed synergy against E. coli O157:H7 with SMAP29, polymyxin B and lactoferrin. Synergy was also found between SMAP29 and lactoferrin against this host. Against S. aureus 1056 MRSA, lysozyme showed synergy with OaBac5mini, polymyxin B and nisin, while synergy was also found between nisin and OaBac5mini and polymyxin B. Other combinations of the antimicrobials were either additive or non-synergistic.

Objective: Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine involved in the pathogenesis of several rheumatic diseases, including rheumatoid arthritis (RA), associated with systemic bone loss and subchondral bone erosions. TNF-alpha-blocking agents such as infliximab have been successful in treatment of disease-modifying antirheumatic drug-resistant rheumatic diseases. Infliximab therapy in RA also had beneficial effects on local bone destruction and bone mineral density. We assessed effects of infliximab treatment on the bone tissue compartment and cytokine profile expression in vitro., Methods: Osteoblast-like cells were exposed for 24 h to sera of RA patients collected at baseline and after 1 month (T1) and 3 years (T2) of infliximab treatment. Total RNA was extracted, and expression of interleukin 1beta (IL-1beta), IL-6, and osteoprotegerin (OPG) was measured by RT-PCR., Results: IL-1beta gene expression was significantly reduced by the T1 serum, and the same decrease was elicited by the T2 serum. IL-6 downregulation was evident with the T2 serum. OPG was unaffected., Conclusion: The finding of downregulation of inflammatory cytokines was interesting, particularly IL-6, which plays a crucial role in arthritis-related bone loss due to its involvement in osteoclast recruitment and activation. These results may represent a biological explanation and a link for the clinical observation of the beneficial effects of anti-TNF-alpha agents on the progression of rheumatic diseases at the bone level.

Recent evidence suggests that extranuclear action of retinoid receptors is involved in mediating the pleiotropic effects of retinoids. However, whether they reside in the cytoplasm remains elusive. Here, we showed that retinoic acid receptor-gamma (RARgamma) was cytoplasmic in confluent cells, or when cells were released from serum depletion or treated with growth factors. In studying the regulation of RARgamma subcellular localization, we observed that ectopically overexpressed RARgamma was mainly cytoplasmic irrespective of serum concentration and cell density. The cytoplasmic retention of RARgamma was inhibited by ligand retinoic acid (RA). In addition, coexpression of retinoid X receptor-alpha (RXRalpha) resulted in nuclear localization of RARgamma through their heterodimerization. Mutagenesis studies revealed that a C-terminal fragment of RXRalpha potently prevents RA-induced RARgamma nuclear localization and transcriptional function. Furthermore, our results showed that the cytoplasmic retention of RARgamma was due to the presence of its unique N-terminal A/B domain, which was subject to regulation by p38 MAPK-mediated phosphorylation. Deletion or mutation of the N-terminal A/B domain largely impaired its cytoplasmic localization. Together, our data demonstrate that the subcellular localization of RARgamma is regulated by complex interactions among ligand binding, receptor phosphorylation, and receptor dimerizations.

Snake venoms are mixtures of proteins and peptides with different biological activities, many of which are very toxic. Several animals, including the opossum Didelphis aurita, are resistant to snake venoms due to the presence of neutralizing factors in their blood. An antihemorrhagic protein named DM43 was isolated from opossum serum. It inhibits snake venom metalloproteinases through noncovalent complex formation with these enzymes. In this study, we have used DM43 and proteomic techniques to explore snake venom subproteomes. Four crotalid venoms were chromatographed through an affinity column containing immobilized DM43. Bound fractions were analyzed by one- and two-dimensional gel electrophoresis, followed by identification by MALDI-TOF/TOF mass spectrometry. With this approach, we could easily visualize and compare the metalloproteinase compositions of Bothrops atrox, Bothrops jararaca, Bothrops insularis, and Crotalus atrox snake venoms. The important contribution of proteolytic processing to the complexity of this particular subproteome was demonstrated. Fractions not bound to DM43 column were similarly analyzed and were composed mainly of serine proteinases, C-type lectins, C-type lectin-like proteins, l-amino acid oxidases, nerve growth factor, cysteine-rich secretory protein, a few metalloproteinases (and their fragments), and some unidentified spots. Although very few toxin families were represented in the crotalid venoms analyzed, the number of protein spots detected was in the hundreds, indicating an important protein variability in these natural secretions. DM43 affinity chromatography and associated proteomic techniques proved to be useful tools to separate and identify proteins from snake venoms, contributing to a better comprehension of venom heterogeneity.

Objective: The nucleus pulposus (NP) of the intervertebral disc develops from the notochord. Humans and other species in which notochordal cells (NCs) disappear to be replaced by chondrocyte-like mature NP cells (MNPCs) frequently develop disc degeneration, unlike other species that retain NCs. The reasons for NC disappearance are unknown. In humans, the change in cell phenotype (to MNPCs) coincides with changes that decrease nutrient supply to the avascular disc. We undertook this study to test the hypothesis that the consequent nutrient stress could be associated with NC disappearance., Methods: We measured cell densities and metabolic rates in 3-dimensional cultures of porcine NCs and bovine MNPCs, and we determined survival rates under conditions of nutrient deprivation. We used scanning electron microscopy to examine end plate porosity of discs with NCs and those with MNPCs. Nutrient-metabolite profiles and cell viability were calculated as a function of cell density and disc size in a consumption/diffusion mathematical model., Results: NCs were more active metabolically and more susceptible to nutrient deprivation than were MNPCs. Hypoxia increased rates of glycolysis in NCs but not in MNPCs. Higher end plate porosity in discs with NCs suggested greater nutrient supply in keeping with higher nutritional demands. Mathematical simulations and experiments using an analog disc diffusion chamber indicated that a fall in nutrient concentrations resulting from increased diffusion distance during growth and/or a fall in blood supply through end plate changes could instigate NC disappearance., Conclusion: NCs demand more energy and are less resistant to nutritional stress than MNPCs, which may shed light on the fate of NCs in humans. This provides important information about prospective NC tissue engineering approaches.

Background: Human 'new pressor protein' (NPP), related to coagulation beta-factor XIIa (beta-FXIIa), potently releases sympathoadrenal catecholamines in bioassay rats, with concurrent elevation of systolic and diastolic blood pressure (SBP/DBP) and heart rate (HR). Elevated plasma NPP/beta-FXIIa levels in hypertensive anephric pediatric patients on hemodialysis associated with fluid status and blood pressure changes were previously reported, suggesting that NPP/beta-FXIIa contributed to their hypertension., Objective: To investigate the mechanism of action of NPP/beta-FXIIa., Methods: Hemodynamic and sympathoadrenal responses to NPP (20 microL plasma equivalent/rat) or coagulation beta-FXIIa (300 ng/kg intravenously) were measured in rats treated with pentolinium (ganglion blockade [+GB]) and/or captopril (+CAP; angiotensin converting enzyme [ACE] inhibition)., Results: In controls not receiving GB or CAP (-GB-CAP), NPP/beta-FXIIa raised plasma epinephrine (E) sixfold, SBP/DBP by 14/8 mmHg and HR by 15 beats/min. With blockade of the cholinergic pathway to the sympathoadrenal system (+GB), basal E, norepinephrine (NE), SBP, DBP and HR all dropped. However NPP/beta-FXIIa remained capable of raising E 20-fold, NE fourfold, SBP/DBP by 27/11 mmHg and HR by 20 beats/min, suggesting that it acted through a 'noncholinergic' mechanism. With +CAP alone, NPP/beta-FXIIa raised plasma E 18-fold, NE threefold, SBP/ DBP by 29/8 mmHg and HR by 73 beats/min, implicating an ACE-regulated 'peptidergic' mechanism. Combining +GB with +CAP potentiated NPP/beta-FXIIa actions further by raising E 50-fold, NE sevenfold, SBP/DBP by 55/20 mmHg and HR by 87 beats/min, strengthening the efficacy of this alternate pathway., Conclusions: The cardiovascular effects of NPP/beta-FXIIa are considerably mediated by a noncholinergic (peptidergic) ACE-regulated mechanism for sympathoadrenal catecholamine release that is enhanced by +GB and/or +CAP. Under inflammatory procoagulant conditions, endogenously produced NPP/beta-FXIIa may interfere with the antihypertensive effects of ACE inhibition therapy.

Objective: To cultivate human umbilical vein endothelial cells (HUVECs) in the serum of overfatigue rats with the intervention of Tongxinluo superfine powder (TXLSP). By examining the variation of the activity of JNK/c-Jun/HO-1 pathway, the possible mechanisms of vascular endothelial dysfunction under overfatigue conditions and the intervening effect of TXLSP were explored., Methods: The HUVECs were randomly divided into the normal control group, the model group, the SP600125 (a specific antagonist of JNK) group, the TXLSP group and the TXLSP + SP600125 group. The content of carboyhemoglobin (COHb) and the leak rate of lactic dehydrogenase (LDH) in different groups were measured. The mRNA and protein expression of JNK, c-Jun, HO-1 and the phosphorylation level of c-Jun (P-c-Jun) were detected using Western blot and PCR methods., Results: Compared with the normal control group, the COHb level in supernatant was increased significantly in the model group, and the expression of HO-1, JNK, c-Jun mRNA and corresponding proteins and P-c-Jun were also increased remarkably. The increases in these parameters were significantly decreased by SP600125. TXLSP showed remarkable up-regulation on the expression of JNK, c-Jun, P-c-Jun and HO-1 mRNA and their protein expression. Compared with the SP600125 group, the expressions of JNK, c-Jun, P-c-Jun and HO-1 mRNA and its protein in the TXLSP+SP600125 group were significantly increased at different time points (P<0.05, P<0.01)., Conclusions: The vascular endothelial dysfunction under overfatigue conditions is related to the activity of the JNK/c-Jun/HO-1 pathway. One of the mechanisms of TXLSP in improving the vascular endothelial function is to adjust the activity of the JNK/c-Jun/HO-1 pathway at gene and protein levels.

Purpose: The antiviral activity of an established antibacterial CAP37 domain and its extracellular mechanism of action were investigated., Methods: CAP37-derived peptides modified to assess the importance of disulfide bonds were evaluated in cytotoxicity and antiviral assays (direct time kill, dose dependency, and TOTO-1) for adenovirus (Ad) and herpes simplex virus type 1 (HSV-1)., Results: Variable virus, adenovirus serotype-dependent, and dose-dependent inhibition were demonstrated without cytotoxicity. For peptide A (CAP37(20-44)), TOTO-1 dye uptake was demonstrated for Ad5 and HSV-1., Conclusions: Unlike the antibacterial activity of this CAP37 domain, its antiviral activity is not fully dependent upon disulfide bond formation. Viral inhibition appears to result, in part, from disruption of the envelope and/or capsid.

Urokinase-type plasminogen activator (uPA) signaling via its receptor uPAR inhibits late events in HIV-1 replication in acutely infected primary monocyte-derived macrophages (MDMs) and promonocytic U937 cells. Here we show that U937-derived, chronically infected U1 cells stimulated with phorbol 12-myristate 13-acetate (PMA) express integrins, uPA, and soluble uPAR at levels similar to those of MDMs. uPA inhibited HIV expression in U1 cells incubated with either PMA or tumor necrosis factor-alpha (TNF-alpha), but not with other HIV-inductive cytokines or lipopolysaccharide. Of interest, only PMA and TNF-alpha, but not other HIV-inductive stimuli, induced surface expression of the alpha(M) chain CD11b in U1 cells constitutively expressing CD18, the beta(2) chain of the Mac-1 integrin. Like uPA, fibrinogen, a Mac-1 (CD11b/CD18) ligand, and M25, a peptide homologous to a portion of the beta-propeller region of CD11b preventing its association with uPAR, inhibited HIV virion release in PMA-stimulated U1 cells. Both uPAR small-interference RNA (siRNA) and soluble anti-beta(1)/-beta(2) monoclonal antibodies abolished the anti-HIV effects of uPA, whereas CD11b siRNA reversed the anti-HIV effect of M25, but not that induced by uPA. Thus, either uPA/uPAR interaction, Mac-1 activation, or prevention of its association with uPAR triggers a signaling pathway leading to the inefficient release of HIV from monocytic cells.