Expression and Purification of Membrane Proteins in Different Hosts

International Journal of Peptide Research and Therapeutics - Membrane proteins play important functions, such as cellular communication and transferring materials in the cell. Many membrane...     

Pierce into the Native Structure of Ata,a Trimeric Autotransporter of Acinetobacter baumannii ATCC 17978

International Journal of Peptide Research and Therapeutics - Acinetobacter baumannii is an important pathogen responsible for nosocomial infections worldwide. Trimeric autotransporters, the...     

Immune response variations to Salmonella enterica serovar Typhi recombinant porin proteins in mice

ObjectivesTyphoid fever is caused by Salmonella enterica serovar Typhi. OmpC, OmpF and OmpA, the three major outer membrane proteins (OMPs), could serve as vaccine candidates.MethodsThe porins antigenicity was predicted in silico. The OMP genes were amplified, cloned and expressed. Sero-reactivities of the recombinant proteins purified by denaturing method were assayed by ELISA. BALB/c mice were immunized with the recombinant porins followed by bacterial challenge.ResultsBacterial challenge of the animal model brought about antibody triggering efficacy of the antigen in OmpF > OmpC > OmpA order. Experimental findings validated the in silico results. None of the antigens had synergic or antagonistic effects on each other from immune system induction points of view. Despite their high immunogenicity, none of the antigens was protective. However, administration of two or three antigens simultaneously resulted in retardation of lethal effect. Porins, in addition to their specific functions, share common functions. Hence, they can compensate for each other's functions.ConclusionsThe produced antibodies could not eliminate the pathogenicity by blockade of one or some of the antigens. Porin antigens are not suitable vaccine candidates alone or in denatured forms. Native forms of the antigens maybe studied for protective immunogenicity.     

Nuclear Localization of the Mitochondrial Factor HIGD1A during Metabolic Stress

Cellular stress responses are frequently governed by the subcellular localization of critical effector proteins. Apoptosis-inducing Factor (AIF) or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), for example, can translocate from mitochondria to the nucleus, where they modulate apoptotic death pathways. Hypoxia-inducible gene domain 1A (HIGD1A) is a mitochondrial protein regulated by Hypoxia-inducible Factor-1α (HIF1α). Here we show that while HIGD1A resides in mitochondria during physiological hypoxia, severe metabolic stress, such as glucose starvation coupled with hypoxia, in addition to DNA damage induced by etoposide, triggers its nuclear accumulation. We show that nuclear localization of HIGD1A overlaps with that of AIF, and is dependent on the presence of BAX and BAK. Furthermore, we show that AIF and HIGD1A physically interact. Additionally, we demonstrate that nuclear HIGD1A is a potential marker of metabolic stress in vivo, frequently observed in diverse pathological states such as myocardial infarction, hypoxic-ischemic encephalopathy (HIE), and different types of cancer. In summary, we demonstrate a novel nuclear localization of HIGD1A that is commonly observed in human disease processes in vivo.     

Generation and Characterization of an Immortalized Human Esophageal Myofibroblast Line

Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.     

Validated HPLC method for the determination of gabapentin in human plasma using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene and its application to a pharmacokinetic study

A rapid, sensitive and accurate high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of gabapentin in human plasma. Gabapentin was quantified using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene following protein precipitation of plasma with acetonitrile. Amlodipine was used as internal standard. The chromatographic separation was carried out on a Nova-Pak C(18) column using a mixture of 50 mM NaH(2)PO(4) (pH=2.5)-acetonitrile (30:70, v/v) as mobile phase with UV detection at 360 nm. The flow rate was set at 1.5 ml/min. The method was linear over the range of 0.05-5 microg/ml of gabapentin in plasma (r(2)>0.999). The within-day and between-day precision values were in the range of 2-5%. The limit of quantification of the method was 0.05 microg/ml. The method was successfully used to study the pharmacokinetics of gabapentin in healthy volunteers.     

Proteomics Characterization of Extracellular Space Components in the Human Aorta

The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.Vascular cells, in particular vascular smooth muscle cells, produce and maintain a complex meshwork of ECM.¹ The ECM is not only the scaffold for the anchorage and mobility of residing cells but also absorbs and transduces the shear and strain forces of the blood flow. It is primarily composed of elastin, collagen, proteoglycans, and glycoproteins. The elastin fibers and type I and III fibrillar collagens form a rigid network of highly cross-linked interstitial matrix. They offer elasticity (elastin) and tensile strength (collagens). Proteoglycans, because of their negative charge, attract water and confer resistance to compression. Finally, glycoproteins participate in matrix organization and are essential for cell attachment.The vascular ECM also serves as a substrate for the binding and retention of secreted, soluble proteins of vascular cells as well as molecules coming from the circulation, including lipoproteins, growth factors, cytokines, proteases, and protease inhibitors. These components are invariably associated with ECM proteins, especially proteoglycans. Together they comprise the vascular extracellular environment and are pivotal for disease processes, such as atherosclerosis and aneurysm formation (1).Although proteomics has been previously applied to vascular tissues, only one study has specifically targeted the extracellular vascular environment (2). This study was focused on the isolation of intimal proteoglycans from human carotid arteries. Moreover, most proteomics studies use whole tissue lysates, which are rich in cellular proteins that inevitably mask the identification of the less abundant proteins of the vascular extracellular environment (3–5). Thus, the composition of the vascular ECM and its associated proteins remains poorly defined. In the present study, we used morphologically normal human aortic samples to develop a method for the extraction of proteins present in the extracellular environment, including ECM proteins and proteins attached to the ECM. We had three specific aims: first, to reduce the contamination with cellular proteins, thereby increasing the chance of identifying scarce extracellular proteins; second, to efficiently solubilize and deglycosylate ECM proteins to improve their analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS); and third, to interface the nanoflow LC system to a recently developed injection device, which splits the flow from the analytical column, to allow the reanalysis of the same sample during a single LC-MS/MS run (RePlay, Advion).Our methodology provides a detailed overview of the aortic ECM and its associated proteins, many reported for the first time in proteomics analysis of the vasculature. Most importantly, this method could be adapted for use with other tissues to further our understanding of the composition of extracellular environment and ECM turnover under various disease conditions.     

Progenitor cells in vascular disease

Stem cell research has the potential to provide solutions to many chronic diseases via the field of regeneration therapy. In vascular biology, endothelial progenitor cells (EPCs) have been identified as contributing to angiogenesis and hence have therapeutic potential to revascularise ischaemic tissues. EPCs have also been shown to endothelialise vascular grafts and therefore may contribute to endothelial maintenance. EPC number has been shown to be reduced in patients with cardiovascular disease, leading to speculation that atherosclerosis may be caused by a consumptive loss of endothelial repair capacity. Animal experiments have shown that EPCs reendothelialise injured vessels and that this reduces neointimal formation, confirming that EPCs have an atheroprotective effect. Smooth muscle cell accumulation in the neointimal space is characteristic of many forms of atherosclerosis, however the source of these cells is now thought to be from smooth muscle progenitor cells (SMPCs) rather than the adjacent media. There is evidence for the presence of SMPCs in the adventitia of animals and that SMPCs circulate in human blood. There is also data to support SMPCs contributing to neointimal formation but their origin remains unknown. This article will review the roles of EPCs and SMPCs in the development of vascular disease by examining experimental data from in vitro studies, animal models of atherosclerosis and clinical studies.     

Dietary fish oil alters cardiomyocyte Ca2+ dynamics and antioxidant status

The n-3 polyunsaturated fatty acids (PUFAs) found in fish oil (FO) have been shown to protect against reperfusion arrhythmias, a manifestation of reperfusion injury, which is believed to be induced by the formation of reactive oxygen species (ROS) and intracellular calcium (Ca2+) overload. Adult rats fed a diet supplemented with 10% FO had a higher proportion of myocardial n-3 PUFAs and increased expression of antioxidant enzymes compared with the saturated fat (SF)-supplemented group. Addition of hydrogen peroxide (H2O2) to cardiomyocytes isolated from rats in the SF-supplemented group increased the proportions of cardiomyocytes contracting in an asynchronous manner, increased the rate of Ca2+ influx, and increased the diastolic and systolic [Ca2+]i compared with the FO group. H2O2 exposure increased the membrane fluidity of cardiomyocytes from the FO group. These results demonstrate that dietary FO supplementation is associated with a reduction in the susceptibility of myocytes to ROS-induced injury and this may be related to membrane incorporation of n-3 PUFAs, increased antioxidant defenses, changes in cardiomyocyte membrane fluidity, and the ability to prevent rises in cellular Ca2+ in response to ROS.     

Oxidative stress promotes polarization of human T cell differentiation toward a T helper 2 phenotype

These studies were conducted to determine the effects of oxidative stress on human T cell differentiation and polarization into Th1 or Th2 phenotypes. Highly purified naive CD4+ T cells were isolated from PBMC of healthy, nonatopic donors. CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAb in the presence or absence of oxidative stress as supplied by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which generates a low level of superoxide anion. Increases in cellular superoxide were observed by exposure to DMNQ. Exposure of unpolarized CD4+ T cells to IL-12 or IL-4 resulted in a Th1 or Th2 phenotype, respectively. T cells stimulated in the absence of polarizing cytokines secreted modest amounts of IFN-gamma and TNF-alpha. Cells stimulated in the continuous presence of 5 microM DMNQ, displayed a marked up-regulation in Th2 cytokines, including IL-4, IL-5, and IL-13, but not the Th1 cytokine IFN-gamma. Th2 responses were blunted by concomitant exposure to thiol antioxidants. Long-term exposure of T cells to DMNQ resulted in growth of cells expressing CCR4, and a decrease in cells expressing CXCR3, indicating phenotypic conversion to Th2 cells. These results suggest that oxidative stress favors a Th2-polarizing condition.