Microgram Quantities Of Protein Research Articles

Comparison of different extraction solutions for the analysis of allergens in hen’s egg

An important requirement for the correct procedure of allergen analysis in hen’s egg is to obtain complete and unaltered protein extracts. Besides the aim of a quantitative extraction of the allergens from the matrix, it is equally important not to alter their allergenic potential during the extraction process. This paper describes and compares six extraction solutions for the analysis of whole-egg proteins and allergens. These requirements were examined via protein determination according to Bradford [Bradford, M. M. (1976). Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Analytical Biochemistry, 72, 248–254] and Kjeldahl [Meyer, A. H. (2006). Lebensmittelrecht, Verlag C.H. Beck München, Stand: 1. February 2006, § 64, Lebensmittel- und Futtermittelgesetzbuch, Amtliche Sammlung von Untersuchungsmethoden, Nr. L 06.00-7] as well as the EAST-inhibition method. It could be demonstrated that the extraction with a urea solution (8M) led to significant interferences during the protein determination, and substantially reduced the allergenic potential of egg proteins. With all other extraction solutions adequate protein contents could be extracted. The highest protein content was achieved by the extraction with phosphate buffered saline followed by a Tween 20 solution, physiological saline, water, and acetate buffer. The results show that none of these extracts – except for the urea solution (8M) – was altered in its’ allergenic potential.

A Metal-coded Affinity Tag Approach to Quantitative Proteomics

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.

Protease-Activated Receptor-2 Augments Experimental Crescentic Glomerulonephritis

Protease-activated receptor-2 (PAR-2) is a cellular receptor expressed prominently on epithelial, mesangial, and endothelial cells in the kidney and on macrophages. PAR-2 is activated by serine proteases such as trypsin, tryptase, and coagulation factors VIIa and Xa. It induces pleiotropic effects including vasodilatation, increasing plasminogen activator inhibitor (PAI-1) expression, mesangial cell proliferation, and cytokine production by macrophages. The role of PAR-2 in renal inflammation was studied in antiglomerular basement membrane antibody-induced crescentic glomerulonephritis (CGN) using PAR-2-deficient (PAR-2(-/-)) mice and wild-type littermate controls. PAR-2(-/-) mice had reduced crescent formation, proteinuria, and serum creatinine compared with wild-type mice 21 days after initiation of CGN. Glomerular accumulation of CD4(+) T cells and macrophages and the number of proliferating cells in glomeruli were similar in both groups. Glomerular fibrin deposition was significantly reduced in PAR-2(-/-) mice, and this was associated with reduced renal plasminogen activator inhibitor expression and increased renal matrix-metalloprotinase-9 activity. These results demonstrate a proinflammatory role for PAR-2 in CGN that is independent of effects on glomerular leukocyte recruitment and mesangial cell proliferation. PAR-2-mediated augmentation of renal plasminogen activator inhibitor expression and inhibition of matrix-metalloprotinase-9 activity may contribute to increased glomerular fibrin accumulation and glomerular injury in CGN.

Mast Cells Play a Crucial Role in Staphylococcus aureus Peptidoglycan-Induced Diarrhea

Bacterium-induced diarrhea results in 2 to 2.5 million deaths in the world each year. The mechanism needs to be further understood. Staphylococcus aureus infection has a close relation with diarrhea; its cell wall component peptidoglycan (PGN) has strong biological activity on immune cells and possibly plays a role in S. aureus-induced diarrhea. The present study showed that oral PGN-induced diarrhea in mice in a dose-dependent manner. Intestinal epithelial cells absorbed PGN via the intracellular pathway. Intestinal mast cells were activated after PGN gavage. Toll-like receptor (TLR)2 expression was detected in mast cells in the intestine as well as in the murine mast cell line p815 cells. Blocking TLR2 or nucleotide-binding oligomerization domain (NOD)1 with related antibodies or RNA interference abolished PGN-induced p815 cell activation. The mast cell mediator histamine and serotonin had synergistic effects in PGN-induced diarrhea. In summary, oral PGN can induce diarrhea in mice, and TLR2 and NOD1 mediate the PGN-induced mast cell activation that plays a critical role in diarrhea induction. Blockade of TLR2 or NOD1 or treating mice with a mast cell stabilizer can efficiently inhibit PGN-induced-diarrhea, providing potential therapeutic significance.

Microgram-scale protein structure determination by NMR

Using conventional triple-resonance nuclear magnetic resonance (NMR) experiments with a 1 mm triple-resonance microcoil NMR probe, we determined near complete resonance assignments and three-dimensional (3D) structure of the 68-residue Methanosarcina mazei TRAM protein using only 72 mug (6 microl, 1.4 mM) of protein. This first example of a complete solution NMR structure determined using microgram quantities of protein demonstrates the utility of microcoil-probe NMR technologies for protein samples that can be produced in only limited quantities.

Trans geometric isomers of EPA decrease LXRα-induced cellular triacylglycerol via suppression of SREBP-1c and PGC-1β

Dietary mono- or di-trans fatty acids with chain lengths of 18-22 increase the risk of cardiovascular diseases because they increase LDL cholesterol and decrease HDL cholesterol in the plasma. However, the effects of trans isomers of PUFAs on lipid metabolism remain unknown. Dietary PUFAs, especially eicosapentaenoic acid (EPA) in marine oils, improve serum lipid profiles by suppressing liver X receptor alpha (LXRalpha) activity in the liver. In this study, we compared the effects of trans geometric isomers of eicosapentaenoic acid (TEPA) on triacylglycerol synthesis induced by a synthetic LXRalpha agonist (T0901317) with the effects of EPA in HepG2 cells. TEPA significantly decreased the amount of cellular triacylglycerol and the expression of mRNAs encoding fatty acid synthase, stearoyl-CoA desaturase-1, and glycerol-3-phosphate acyltransferase induced by T0901317 compared with EPA. However, there was no significant difference between the suppressive effect of TEPA or EPA on the expression of sterol-regulatory element binding protein-1c (SREBP-1c) induced by T0901317. We found that TEPA, but not EPA, decreased the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1beta (PGC-1beta), which is a coactivator of both LXRalpha and SREBP-1. These results suggest that the hypolipidemic effect of TEPA can be attributed to a decrease not only in SREBP-1 but also in PGC-1beta expression.

Infiltration of Inflammatory Cells Plays an Important Role in Matrix Metalloproteinase Expression and Activation in the Heart during Sepsis

Septicemia is an emerging pathological condition involving, among other effects, refractory hypotension and heart dysfunction. Here we have investigated the contribution of resident nonmyocytic cells to heart alterations after lipopolysaccharide administration. These cells contributed to the rapid infiltration of additional inflammatory cells that enhance the onset of heart disease through the release of inflammatory mediators. Early activation of resident monocytic cells played a relevant role on the infiltration process, mainly of major histocompatibility complex class II- and CD11b-positive cells. This infiltration was significantly impaired in animals lacking the nitric-oxide synthase-2 (NOS-2) gene or after pharmacological in-hibition of NOS-2 or cylooxygenase-2, suggesting a significant contribution of nitric oxide and prostanoids to the infiltration process. Under these conditions, the expression of NOS-2 and cylooxygenase-2 in the whole organ was attenuated because cardiomyocytes failed to express these enzymes. However, cardiomyocytes expressed and activated matrix metalloproteinase-9 through mechanisms regulated, at least in part, by nitric oxide and prostaglandins in an additive way. These results directly link the inflammatory response in the heart and extracellular matrix remodeling by the matrix metalloproteinases released by the cardiomyocytes, suggesting that activation and recruitment of inflammatory cells to the heart is a major early event in cardiac dysfunction promoted by septicemia.

Identification of Nicotinamide N-Methyltransferase as a Novel Tumor Marker for Renal Clear Cell Carcinoma

To explore the involvement of enzymes of drug metabolism in renal cell carcinoma we analyzed the gene expression profiles of tumor and nontumor tissues from the same patient by DNA macroarray. The enzyme nicotinamide N-methyltransferase was selected for further evaluation. Nicotinamide N-methyltransferase mRNA expression was investigated in paired tissue samples from cancerous and noncancerous parts of the kidneys of 30 patients with clear cell renal cell carcinoma who underwent tumor nephrectomy. Measurements were performed by semiquantitative reverse transcriptase-polymerase chain reaction and quantitative real-time polymerase chain reaction. Paired tissue samples were also obtained from 1 patient with chromophobe renal cell carcinoma and from another with oncocytoma to compare the specificity of changes in nicotinamide N-methyltransferase expression among tumors that are related to different renal epithelial cell types. Western blot analysis and catalytic activity assay were also performed to study nicotinamide N-methyltransferase expression. Expression correlated with tumor characteristics. A marked increased expression in tumor tissue was found for nicotinamide N-methyltransferase, which is an enzyme involved in the biotransformation of many drugs and xenobiotic compounds. Differential gene expression measurements in tumor vs normal tissue revealed up-regulation in all clear cell renal cell carcinomas at between 3 and 294-fold (mean 41). In contrast, in chromophobe renal cell carcinoma and oncocytoma nicotinamide N-methyltransferase expression did not increase. In addition, nicotinamide N-methyltransferase expression significantly correlated inversely with tumor size. Our results indicate that a marked nicotinamide N-methyltransferase increase is a peculiar feature of clear cell renal cell carcinoma. Additional studies may establish the role of nicotinamide N-methyltransferase in tumor formation and progression.

Conjugated linoleic acid and hepatic lipogenesis in mouse: role of the mitochondrial citrate carrier

Conjugated linoleic acid (CLA) is able to reduce adiposity by affecting lipid metabolism. In particular, CLA administration to mice reduces body fat mass with a concomitant lipid accumulation in the liver. We investigated the effects of CLA on the activity of the mitochondrial citrate carrier (CIC), which is implicated in hepatic lipogenesis. The transport activity of the CIC, measured both in intact mitochondria and in the proteoliposomes, progressively increased with the duration of CLA feeding. An increase in the CIC activity of approximately 1.7-fold was found in 16 week CLA-treated mice with respect to control animals. A kinetic analysis showed a 1.6-fold increase in the V(max) of citrate transport but no change in the K(m) value. Western blot experiments revealed an increase of approximately 1.7-fold in the expression of CIC after CLA treatment. A strict correlation between the increase in CIC activity and the stimulation of the cytosolic lipogenic enzymes was also found. These data indicate that the CIC may play a role in the onset of hepatic steatosis in CLA-fed mice by supplying the carbon source for de novo fatty acid synthesis.

Identification of Proteomic Signatures of Exposure to Marine Pollutants in Mussels (Mytilus edulis)

Bivalves and especially mussels are very good indicators of marine and estuarine pollution, and so they have been widely used in biomonitoring programs all around the world. However, traditional single parameter biomarkers face the problem of high sensitivity to biotic and abiotic factors. In our study, digestive gland peroxisome-enriched fractions of Mytilus edulis (L., 1758) were analyzed by DIGE and MS. We identified several proteomic signatures associated with the exposure to several marine pollutants (diallyl phthalate, PBDE-47, and bisphenol-A). Animals collected from North Atlantic Sea were exposed to the contaminants independently under controlled laboratory conditions. One hundred and eleven spots showed a significant increase or decrease in protein abundance in the two-dimensional electrophoresis maps from the groups exposed to pollutants. We obtained a unique protein expression signature of exposure to each of those chemical compounds. Moreover a set of proteins composed a proteomic signature in common to the three independent exposures. It is remarkable that the principal component analysis of these spots showed a discernible separation between groups, and so did the hierarchical clustering into four classes. The 14 proteins identified by MS participate in alpha- and beta-oxidation pathways, xenobiotic and amino acid metabolism, cell signaling, oxyradical metabolism, peroxisomal assembly, respiration, and the cytoskeleton. Our results suggest that proteomic signatures could become a valuable tool to monitor the presence of pollutants in field experiments where a mixture of pollutants is often present. Further studies on the identified proteins could provide crucial information to understand possible mechanisms of toxicity of single xenobiotics or mixtures of them in marine ecosystems.

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

The small intestinal epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and differentiation. The intestinal epithelium constitutes a permeability barrier regulating the vectorial transport of ions, water, and solutes. Morphological changes during cell differentiation, as well as changes in the activity of brush-border enzymes and the expression of transport proteins, are well established. However, little is known about the arachidonic acid (AA) cascade underlying epithelial cell differentiation or its role in the development of epithelial barrier function. The main purpose of this study was to examine the activity of the high-molecular-weight phospholipases A(2) (PLA(2)) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA(2) activity, AA release, COX-2 expression, prostaglandin E(2) (PGE(2)) production, and paracellular permeability were studied in preconfluent, confluent, and differentiated Caco-2 cell cultures. Our results show that Caco-2 differentiation induces a decrease in both calcium-independent PLA(2) activity and COX-2 expression and, consequently, a decrease in AA release and PGE(2) synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE(2) to differentiated cells, at concentrations similar to those detected in nondifferentiated cultures, induces the disruption of epithelial barrier function. These results suggest that AA release by calcium-independent PLA(2), COX-2 expression, and subsequent PGE(2) release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.

Warm Ischemia-induced Alterations in Oxidative and Inflammatory Proteins in Hepatic Kupffer Cells in Rats

The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 +/- 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 +/- 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 +/- 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 +/- 0.19; p < 0.001) and catalase (1.23 +/- 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options.

Measurement of Serum Acetaminophen–Protein Adducts in Patients With Acute Liver Failure

Acetaminophen toxicity is the most common cause of acute liver failure (ALF) in the United States and Great Britain, but may be underrecognized in certain settings. Acetaminophen-protein adducts are specific biomarkers of drug-related toxicity in animal models and can be measured in tissue or blood samples. Measurement of serum adducts might improve diagnostic accuracy in acute liver failure (ALF) patients. We measured serum acetaminophen-protein adducts using high-pressure liquid chromatography with electrochemical detection in coded sera of 66 patients with ALF collected prospectively at 24 US tertiary referral centers. Samples were included from 20 patients with well-characterized acetaminophen-related acute liver failure, 10 patients with ALF owing to other well-defined causes, 36 patients with ALF of indeterminate etiology, and 15 additional patients without ALF but with known acetaminophen overdose and minimal or no biochemical liver injury. Acetaminophen-protein adducts were detected in serum in 100% of known acetaminophen ALF patients and in none of the ALF patients with other defined causes, yielding a sensitivity and specificity of 100%. In daily serial samples, serum adducts decreased in parallel with aminotransferase levels. Seven of 36 (19%) indeterminate cases demonstrated adducts in serum suggesting that acetaminophen toxicity caused or contributed to ALF in these patients. Low adduct levels were present in 2 of 15 patients with acetaminophen overdose without significant liver injury. Measurement of serum acetaminophen-protein adducts reliably identified acetaminophen toxicity, and may be a useful diagnostic test for cases lacking historical data or other clinical information.

Targeting the signaling pathway of acylation stimulating protein

Acylation stimulating protein (ASP; C3adesArg) stimulates triglyceride synthesis (TGS) and glucose transport in preadipocytes/adipocytes through C5L2, a G-protein-coupled receptor. Here, ASP signaling is compared with insulin in 3T3-L1 cells. ASP stimulation is not Galpha(s) or Galpha(i) mediated (pertussis and cholera toxin insensitive), suggesting G(alphaq) as a candidate. Phospholipase C (PLC) is required, because the Ca(2+) chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester and the PLC inhibitor U73122 decreased ASP stimulation of TGS by 93.1% (P < 0.0.001) and 86.1% (P < 0.004), respectively. Wortmannin and LY294002 blocked ASP effect by 69% (P < 0.001) and 116.1% (P < 0.003), respectively, supporting phosphatidylinositol 3-kinase (PI3K) involvement. ASP induced rapid, transient Akt phosphorylation (maximal, 5 min; basal, 45 min), which was blocked by Akt inhibition, resembling treatment by insulin. Downstream of PI3K, mamalian target of rapaycin (mTOR) is required for insulin but not ASP action. By contrast, both ASP and insulin activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK(1/2)) pathway, with rapid, pronounced increases in ERK(1/2) phosphorylation, effects partially blocked by PD98059 (64.7% and 65.9% inhibition, respectively; P < 0.001). Time-dependent (maximal, 30 min) transient calcium-dependent phospholipase A(2) (cPLA(2))(-Ser505) phosphorylation (by MAPK/ERK(1/2)) was demonstrated by Western blot analysis. ASP signaling involves sequential activation of PI3K and PLC, with downstream activation of protein kinase C, Akt, MAPK/ERK(1/2), and cPLA(2), all of which leads to an effective and prolonged stimulation of TGS.

Proteomic Profiling of Accessory Structures from the Mouse Sperm Flagellum

The flagellum of a mammalian spermatozoon consists of an axoneme surrounded in distinct regions by accessory structures known as the fibrous sheath, outer dense fibers, and the mitochondrial sheath. Although the characterization of individual proteins has provided clues about the roles of these accessory structures, a more complete understanding of flagellar function requires the identification of all the polypeptides in these assemblies. Epididymal mouse sperm were treated with SDS to dislodge sperm heads and to extract the axoneme and membranous elements. The remaining flagellar accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal Coomassie Blue staining showed a highly reproducible pattern of >200 spots. Individual spots were picked, digested with trypsin, and identified by mass spectrometry and peptide microsequencing. Approximately 50 individual proteins were identified that could be assigned to five general categories: 1) proteins previously reported to localize to the accessory structures, e.g. ODF2 in the outer dense fibers, the sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath, and glutathione peroxidase in the mitochondrial sheath, validating this proteomic approach; 2) proteins that had not been shown to localize to any accessory structure but would be predicted to be present, e.g. glycolytic enzymes; 3) proteins known to be part of the flagellum but not localized to a specific site, e.g. adenylate kinase; 4) proteins not expected to be part of the accessory structures based on their previously reported locations, e.g. tektins; and 5) unknown proteins for which no information is available to make a determination as to location. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This proteomic analysis identified a number of unexpected and novel proteins in the accessory structures of the mammalian flagellum.

Lysyl Oxidase Is Essential for Normal Development and Function of the Respiratory System and for the Integrity of Elastic and Collagen Fibers in Various Tissues

Lysyl oxidases, a family comprising LOX and four LOX-like enzymes, catalyze crosslinking of elastin and collagens. Mouse Lox was recently shown to be crucial for development of the cardiovascular system because null mice died perinatally of aortic aneurysms and cardiovascular dysfunction. We show here that Lox is also essential for development of the respiratory system and the integrity of elastic and collagen fibers in the lungs and skin. The lungs of E18.5 Lox(-/-) embryos showed impaired development of the distal and proximal airways. Elastic fibers in E18.5 Lox(-/-) lungs were markedly less intensely stained and more disperse than in the wild type, especially in the mesenchyme surrounding the distal airways, bronchioles, bronchi, and trachea, and were fragmented in pulmonary arterial walls. The organization of individual collagen fibers into tight bundles was likewise abnormal. Similar elastic and collagen fiber abnormalities were seen in the skin. Lysyl oxidase activity in cultured Lox(-/-) skin fibroblasts and aortic smooth muscle cells was reduced by approximately 80%, indicating that Lox is the main isoenzyme in these cells. LOX abnormalities may thus be critical for the pathogenesis of several common diseases, including pulmonary, skin, and cardiovascular disorders.

Modulation of Differentiation-related Gene 1 Expression by Cell Cycle Blocker Mimosine, Revealed by Proteomic Analysis

L-mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G1 phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.

Proteomic Analysis of Cellular Response to Osmotic Stress in Thick Ascending Limb of Henle’s Loop (TALH) Cells

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.

Mitochondrial dysfunction induces triglyceride accumulation in 3T3-L1 cells: role of fatty acid β-oxidation and glucose

Mitochondrial cytopathy has been associated with modifications of lipid metabolism in various situations, such as the acquisition of an abnormal adipocyte phenotype observed in multiple symmetrical lipomatosis or triglyceride (TG) accumulation in muscles associated with the myoclonic epilepsy with ragged red fibers syndrome. However, the molecular signaling leading to fat metabolism dysregulation in cells with impaired mitochondrial activity is still poorly understood. Here, we found that preadipocytes incubated with inhibitors of mitochondrial respiration such as antimycin A (AA) accumulate TG vesicles but do not acquire specific markers of adipocytes. Although the uptake of TG precursors is not stimulated in 3T3-L1 cells with impaired mitochondrial activity, we found a strong stimulation of glucose uptake in AA-treated cells mediated by calcium and phosphatidylinositol 3-kinase/Akt1/glycogen synthase kinase 3beta, a pathway known to trigger the translocation of glucose transporter 4 to the plasma membrane in response to insulin. TG accumulation in AA-treated cells is mediated by a reduced peroxisome proliferator-activated receptor gamma activity that downregulates muscle carnitine palmitoyl transferase-1 expression and fatty acid beta-oxidation, and by a direct conversion of glucose into TGs accompanied by the activation of carbohydrate-responsive element binding protein, a lipogenic transcription factor. Taken together, these results could explain how mitochondrial impairment leads to the multivesicular phenotype found in some mitochondria-originating diseases associated with a dysfunction in fat metabolism.

Structure-Based Engineering of E. coli Galactokinase as a First Step toward In Vivo Glycorandomization

In vitro glycorandomization is a rapid chemoenzymatic strategy to diversify complex natural product scaffolds. The glycorandomization sugar activation pathway is dependent upon the efficient construction of diverse sugar-1-phosphate libraries. In the context of the previously evolved GalK Y371H "gatekeeper" mutation, the active site M173L mutation described herein presents a kinase with remarkably broadened substrate range to include 28 diverse natural and unnatural sugars. Among these new substrates, 6-azido-6-deoxy-galactose and 6-azido-6-deoxy-glucose present unique chemical probes to assess the utility of an E. coli Y371H/M173L-GalK-overproducing strain to generate unnatural sugar-1-phosphates in vivo. Remarkably, the in vivo conversion of both unnatural sugars rival that demonstrated in vitro. This notable in vivo success stands as the first step toward constructing short sugar-activation pathways in vivo and, ultimately, in vivo natural-product glycorandomization.