Microsomal Membranes Research Articles

Anti-inflammatory and Hypolipidemic Effect of Novel Conjugates with Trolox and Other Antioxidant Acids.

A series of esters and amides, incorporating an antioxidant residue, such as trolox or caffeic acid, and various moieties with different biological activities, were synthesised. The obtained compounds demonstrated considerable anti-inflammatory, radical scavenging and antioxidant action. Thus, they could reduce carrageenan-induced rat paw oedema by 31-60% at 150 μmol/kg and inhibit rat microsomal membrane lipid peroxidation with IC50 values as low as 1.4 μM, which is much lower than that of trolox. Most of them could also inhibit soybean lipoxygenase. The thiomorpholine derivatives decreased significantly all lipidemic indices of Triton-induced hyperlipidemia in rats. The most active, the caffeic acid derivative (6), decreases triglycerides, total cholesterol and low density lipoprotein, in the plasma of hyperlipidemic rats, by 70%, 67%, and 73%, respectively, at 150 μmol/kg (i.p.). The synthesised compounds, designed to exhibit two or more pharmacological actions, may be considered useful in the study of agents addressed to conditions involving inflammation and oxidative stress.

PIP1 aquaporins, sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm3) and “heavy” (≥1.146 g/cm3) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.

Novel LC/MS/MS and High-Throughput Mass Spectrometric Assays for Monoacylglycerol Acyltransferase Inhibitors

Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography–tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.

Differences in the Association of BH3-Only Proteins to Biological Membranes

Apoptosis, a prevalent mechanism of programmed cell death, is regulated by the Bcl-2 protein family. The balance between pro- and anti-apoptotic Bcl-2 members in the mitochondrial outer membrane (MOM) protects or triggers MOM permeabilization. Bcl-2 homology-3 (BH3)-only proteins participate in this process activating pro-apoptotic effectors and promoting permeabilization of the MOM. The membrane association of BH3-only proteins is controversial due to the lack of a canonical carboxyl-terminal (C-terminal) transmembrane (TM) domain. We used an in vitro transcription/translation system to study the insertion capacity of these hydrophobic C-terminal regions of the BH3-members Bik, Bim, Noxa, Puma, Bmf and Hrk into microsomal membranes, and an Escherichia coli complementation assay to validate our results in bacterial cells. Furthermore, we have fused these poorly hydrophobic regions to the GFP as C-terminal tails to investigate subcellular sorting in mammalian cells. Our approaches provide additional tools for testable descriptions of membrane integration, allowing further refinement for the understanding of the core mechanisms of the regulation of membrane permeabilization by Bcl-2 family members.

Trehalose‐based hydrogel potentially useful for the skin burn treatment

ABSTRACTThe purpose of this work is the preparation of trehalose hydrogel for dermatological applications, particularly, for skin burn treatment. Trehalose is a disaccharide compound containing hydroxyl groups in its structure susceptible to derivatization. The obtained hydrogel was loaded once with hyaluronic acid, a fundamental component of connective tissue, and again with silver nitrate, an ingredient with antibacterial properties. The trehalose hydrogel was characterized by FTIR spectrometry and 1H‐NMR. The hydrogel swelling characteristics and release studies of entrapped hyaluronic acid or silver nitrate were evaluated at two pH values: pH 4.5 mimicking physiological conditions and pH 6.5 simulating the burned skin condition. The antioxidant activity of not loaded hydrogel in inhibiting the lipid peroxidation was performed in rat‐liver microsomal membranes, induced in vitro by tert‐butyl hydroperoxide (tert‐BOOH). The results confirm the possibility that the trehalose hydrogel can find use in the treatment of various skin lesions, particularly in burn therapy. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 44755.

Localization of the Ethylene-Receptor Signaling Complex to the Endoplasmic Reticulum: Analysis by Two-Phase Partitioning and Density-Gradient Centrifugation.

Ethylene receptors and other elements of the ethylene-signal transduction pathway localize to membranes of the endoplasmic reticulum (ER). New players in the ethylene signaling pathway continue to be discovered and so it is important to have methods by which to diagnose their cellular localization. Two methods for microsome isolation and fractionation are described here that can assist in determining if a protein localizes to the ER: aqueous two-phase partitioning and equilibrium density-gradient centrifugation. Two-phase partitioning serves to purify plasma membrane away from other cellular membranes and can thus discriminate whether a protein is localized to the plasma membrane or not. Equilibrium density-gradient centrifugation is particularly useful for resolving if a protein is localized to the ER. Ribosomes are associated with the rough ER in the presence of Mg2+ but are stripped away when Mg2+is removed from the medium, resulting in a reduction in the ER membrane density and a diagnostic shift in migration when analyzed by equilibrium density-gradient centrifugation. A method for growing plants in liquid culture is also provided because these microsomal membranes exhibit consistent fractionation by both two-phase partitioning and density-gradient centrifugation.

Melatonin and human mitochondrial diseases.

Mitochondrial dysfunction is one of the main causative factors in a wide variety of complications such as neurodegenerative disorders, ischemia/reperfusion, aging process, and septic shock. Decrease in respiratory complex activity, increase in free radical production, increase in mitochondrial synthase activity, increase in nitric oxide production, and impair in electron transport system and/or mitochondrial permeability are considered as the main factors responsible for mitochondrial dysfunction. Melatonin, the pineal gland hormone, is selectively taken up by mitochondria and acts as a powerful antioxidant, regulating the mitochondrial bioenergetic function. Melatonin increases the permeability of membranes and is the stimulator of antioxidant enzymes including superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase. It also acts as an inhibitor of lipoxygenase. Melatonin can cause resistance to oxidation damage by fixing the microsomal membranes. Melatonin has been shown to retard aging and inhibit neurodegenerative disorders, ischemia/reperfusion, septic shock, diabetes, cancer, and other complications related to oxidative stress. The purpose of the current study, other than introducing melatonin, was to present the recent findings on clinical effects in diseases related to mitochondrial dysfunction including diabetes, cancer, gastrointestinal diseases, and diseases related to brain function.

Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment.

The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

Novel polyfunctional esters of ibuprofen and ketoprofen with hypolipidemic, lipoxygenase inhibitory and enhanced anti-inflammatory activity

A series of ibuprofen and ketoprofen esters were designed and synthesised, incorporating an antioxidant moiety, such as quercetin and salicylic alcohol, as well as anti-inflammatory or neuroprotective components. They showed greatly increased activity against carrageenan-induced inflammation, compared with the parent drugs. They could also reduce significantly total cholesterol and triglyceride levels in the plasma of Triton-induced hyperilipidemic rats. Most of them inhibited rat microsomal membrane lipid peroxidation. They also acquire lipoxygenase inhibitory activity and at least two of them could inhibit cyclooxygenases-1/2. 2-Hydroxy-5-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenyl 2-(4-isobutylphenyl)propanoate (7), the ester of ibuprofen with quercetin, is the most potent derivative. It is an effective antioxidant, inhibits cyclooxygenases 1/2 and lipoxygenase activity and reduces carrageenan induced paw oedema by 61%. It also decreases triglyceride and total cholesterol levels by 69 and 48%, respectively. The results of this study indicate that proper molecular modifications of non steroidal anti-inflammatory drugs may offer a useful route to potent anti-inflammatory agents, which, in addition, acquire a series of biological properties able to prevent or restore a number of pathological changes in multicausal conditions like the metabolic syndrome.

Plasma membrane ATPase and H+ transport activities of microsomal membranes from wheat roots under Ni deficiency conditions as affected by exogenous histidine

ATPase activity and ATP-dependent H+ transport have been analyzed in membrane vesicles isolated from a winter wheat cultivar (Triticum aestivum cv. Back Cross Rushan) which was exposed to different concentrations of nickel (Ni) and histidine (His). Seedlings were grown in a modified Johnson nutrient solution and then exposed to the uptake solution containing 10μM Ni to which either 100μM His or no amino acid was added. Control plants were transplanted to nutrient solutions free of Ni and His. According to our results, using Ni alone or together with His enhanced the activity of plasma membrane (PM) H+-ATPase and resulted in a higher pH gradient across the plasma membrane. The highest initial rate of H+-pumping by PM H+-ATPase also achieved in the Ni alone treatment which was roughly 2.2 times higher than the control treatment. His applied together with Ni to the nutrient solution resulted in higher Ni uptake but lower H+-ATPase activity. Our results suggest that the (PM) H+-ATPase in wheat roots plays an important role in Ni uptake but using His in combination with Ni can moderate the pump activity. Undissociated Ni-His may directely taken up via His transporters which seems to be less dependent on ATPase activity.

Drug Distribution. Part 1. Models to Predict Membrane Partitioning.

Tissue partitioning is an important component of drug distribution and half-life. Protein binding and lipid partitioning together determine drug distribution. Two structure-based models to predict partitioning into microsomal membranes are presented. An orientation-based model was developed using a membrane template and atom-based relative free energy functions to select drug conformations and orientations for neutral and basic drugs. The resulting model predicts the correct membrane positions for nine compounds tested, and predicts the membrane partitioning for n = 67 drugs with an average fold-error of 2.4. Next, a more facile descriptor-based model was developed for acids, neutrals and bases. This model considers the partitioning of neutral and ionized species at equilibrium, and can predict membrane partitioning with an average fold-error of 2.0 (n = 92 drugs). Together these models suggest that drug orientation is important for membrane partitioning and that membrane partitioning can be well predicted from physicochemical properties.

α-Tocopheryl linolenate solid lipid nanoparticles for the encapsulation, protection, and release of the omega-3 polyunsaturated fatty acid: in vitro anti-melanoma activity evaluation

The main target of this study was the preparation, characterization and antioxidant activity evaluation of α-tocopheryl linolenate based solid lipid nanoparticles (SLNs-TL), able to incorporate omega-3 α-linolenic acid, useful for the treatment of melanoma, a type of skin cancer. In particular, α-linolenic acid was successfully derivatized with α-tocopherol and the obtained compound was characterized by Fourier transform infrared (FT-IR) and by 1H NMR to confirm the ester linkage. Both the empty SLNs-TL that SLNs-TL-LIN, containing omega-3-linolenic acid, were prepared through the technique of the microemulsion. The nanoparticles were characterized for entrapment efficiency, size and shape. Their antioxidant activity was investigated in rat liver microsomal membranes in inhibiting the lipid peroxidation induced by tert-butyl hydroperoxide (tert-BOOH), which endogenously produces alkoxyl radicals by Fenton reactions. The obtained results indicate that the α-tocopherol, linked by ester bond to α-linolenic acid, maintains an excellent antioxidant activity. The encapsulation efficiency was equal to 77% and the polydispersity index 0.198 indicating a good dimensional distribution. Furthermore, the nanoparticles were tested in vitro for their cytotoxic activity against human melanoma cancer cell line C32. Both empty SLNs-TL and loaded SLNs-TL-LIN showed a high biological activity, being more effective than α-linolenic acid and α-tocopherol. The results indicated that these nanoparticles could provide the delivery and the protection of unstable molecules, such as α-linolenic acid, from degradation induced by mechanisms of oxidative stress.

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031-4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187-3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.

The C-terminal Domains of Apoptotic BH3-only Proteins Mediate Their Insertion into Distinct Biological Membranes

Changes in the equilibrium of pro- and anti-apoptotic members of the B-cell lymphoma-2 (Bcl-2) protein family in the mitochondrial outer membrane (MOM) induce structural changes that commit cells to apoptosis. Bcl-2 homology-3 (BH3)-only proteins participate in this process by either activating pro-apoptotic effectors or inhibiting anti-apoptotic components and by promoting MOM permeabilization. The association of BH3-only proteins with MOMs is necessary for the activation and amplification of death signals; however, the nature of this association remains controversial, as these proteins lack a canonical transmembrane sequence. Here we used an in vitro expression system to study the insertion capacity of hydrophobic C-terminal regions of the BH3-only proteins Bik, Bim, Noxa, Bmf, and Puma into microsomal membranes. An Escherichia coli complementation assay was used to validate the results in a cellular context, and peptide insertions were modeled using molecular dynamics simulations. We also found that some of the C-terminal domains were sufficient to direct green fluorescent protein fusion proteins to specific membranes in human cells, but the domains did not activate apoptosis. Thus, the hydrophobic regions in the C termini of BH3-only members associated in distinct ways with various biological membranes, suggesting that a detailed investigation of the entire process of apoptosis should include studying the membranes as a setting for protein-protein and protein-membrane interactions.

The process of lipid storage in insect oocytes: The involvement of β-chain of ATP synthase in lipophorin-mediated lipid transfer in the chagas’ disease vector Panstrongylus megistus (Hemiptera: Reduviidae)

Lipophorin is the main lipoprotein in the hemolymph of insects. During vitellogenesis, lipophorin delivers its hydrophobic cargo to developing oocytes by its binding to non-endocytic receptors at the plasma membrane of the cells. In some species however, lipophorin may also be internalized to some extent, thus maximizing the storage of lipid resources in growing oocytes. The ectopic β chain of ATP synthase (β-ATPase) was recently described as a putative non-endocytic lipophorin receptor in the anterior midgut of the hematophagous insect Panstrongylus megistus. In the present work, females of this species at the vitellogenic stage of the reproductive cycle were employed to investigate the role of β-ATPase in the transfer of lipids to the ovarian tissue. Subcellular fractionation and western blot revealed the presence of β-ATPase in the microsomal membranes of the ovarian tissue, suggesting its localization in the plasma membrane. Immunofluorescence assays showed partial co-localization of β-ATPase and lipophorin in the membrane of oocytes as well as in the basal domain of the follicular epithelial cells. Ligand blotting and co-immunoprecipitation approaches confirmed the interaction between lipophorin and β-ATPase. In vivo experiments with an anti-β-ATPase antibody injected to block such an interaction demonstrated that the antibody significantly impaired the transfer of fatty acids from lipophorin to the oocyte. However, the endocytic pathway of lipophorin was not affected. On the other hand, partial inhibition of ATP synthase activity did not modify the transfer of lipids from lipophorin to oocytes. When the assays were performed at 4°C to diminish endocytosis, the results showed that the antibody interfered with lipophorin binding to the oocyte plasma membrane as well as with the transfer of fatty acids from the lipoprotein to the oocyte. The findings strongly support that β-ATPase plays a role as a docking lipophorin receptor at the ovary of P. megistus, similarly to its function in the midgut of such a vector. In addition, the role of β-ATPase as a docking receptor seems to be independent of the enzymatic ATP synthase activity.

Glycogen Storage Disease 1b: Diagnosis and Workup of a Novel Mutation

Glycogen Storage diseases (Glycogenoses) are a diverse group of disorders, numbering more than 12, resulting from deficiencies of various enzymes & transport proteins in the pathways of glycogen metabolism. GSD 1 is caused by absence or deficiency of glucose-6-phosphatase activity in the liver, kidney or intestinal mucosa. In GSD 1(b), the enzyme which transports Glucose-6-Phosphate across the microsomal membrane is defective, thereby resulting in accumulation of Glycogen. The clinical features of 1a & 1b are similar with fasting hypoglycaemia, hepatomegaly, growth retardation and metabolic abnormalities except for the presence of neutropenia with recurrent gingivitis in GSD 1b. A genetic diagnosis solves this conundrum with the added benefit of antenatal diagnosis of future pregnancies & identification of carrier state in patients. We report the work up of an infant with suspected GSD where a novel mutation with heterozygous carrier state in the parents was diagnosed by genetic testing.J Nepal Paediatr Soc 2016;36(1):85-87.

The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds.

Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca2+-dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser11 in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca2+-dependent Ser11 phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca2+ ions [K0.5(Ca2+) < 0.4 µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser11 phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser451, RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca2+-signaling and the control of photosynthate partitioning during COS development.

Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo.

Insertion of a nascent membrane protein segment by the translocon channel into the bilayer is naturally promoted by high segmental hydrophobicity, but its selection as a transmembrane (TM) segment is complicated by the diverse environments (aqueous vs lipidic) the protein encounters and by the fact that most TM segments contain a substantial amount (∼30%) of polar residues, as required for protein structural stabilization and/or function. To examine the contributions of these factors systematically, we designed and synthesized a peptide library consisting of pairs of compositionally identical, but sequentially different, peptides with 19-residue core sequences varying (i) in Leu positioning (with five or seven Leu residues clustered into a contiguous "block" in the middle of the segment or "scrambled" throughout the sequence) and (ii) in Ser content (0-6 residues). The library was analyzed by a combination of biophysical and biological techniques, including HPLC retention times, circular dichroism measurements of helicity in micelle and phospholipid bilayer media, and relative blue shifts in Trp fluorescence maxima, as well as by the extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum. We found that local blocks of high hydrophobicity heighten the translocon's propensity to insert moderately hydrophilic sequences, until a "threshold hydrophilicity" is surpassed whereby segments no longer insert even in the presence of Leu blocks. This study codifies the prerequisites of apolar/polar content and residue positioning that define nascent TM segments, illustrates the accuracy in their prediction, and highlights how a single disease-causing mutation can tip the balance toward anomalous translocation/insertion.

Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system.

Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers

Diffuse astrocytoma (DA; WHO grade II) is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on ≥2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tumorigenesis, mTOR signaling pathway being a major pathway identified. Comparison of 340 differentially expressed proteins with the transcript data from Grade II diffuse astrocytomas reported earlier, revealed about 190 of the proteins correlate in their trends in expression. Considering progressive and recurrent nature of these tumors, we have mapped the differentially expressed proteins for their secretory potential, integrated the resulting list with similar list of proteins from anaplastic astrocytoma (WHO Grade III) tumors and provide a panel of proteins along with their proteotypic peptides, as a resource that would be useful for investigation as circulatory plasma markers for post-treatment surveillance of DA patients.