Biomedical subjects

S J Fey

Publications and source records attributed to S J Fey.

At least 19 recordsLinked to original sources

PubMed2002

IL-1beta induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis.

AIMS/HYPOTHESIS: Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1beta (IL-1beta) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1beta exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen in IL-1beta exposed Wistar Furth (WF) rat islets. METHODS: The 82 protein spots, that changed expression after IL-1beta exposure, were all re-identified on preparative gels of 200 000 neonatal WF rat islets, cut out and subjected to mass spectrometry for identification. RESULTS: Forty-five different proteins were identified from 51 spots and grouped according to function: (i) energy transduction and redox potentials; (ii) glycolytic and Krebs cycle enzymes; (iii) protein, DNA and RNA synthesis, chaperoning and protein folding; (iv) signal transduction, regulation, differentiation and apoptosis; (v) cellular defence; and (vi) other functions. Comparison of IL-1beta exposed BB-DP and WF islets showed common changes in 14 proteins and several proteins influencing similar pathways, suggesting that similar routes in the two strains lead to beta-cell destruction. CONCLUSION/INTERPRETATION: We demonstrate that proteome analysis is a powerful tool to identify proteins and pathways in BB-DP rat islets exposed to IL-1beta.

Animals↗

PubMed2001

Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry.

Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis. The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis. In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated. A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2. The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1). Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.

Amino Acid Sequence↗

PubMed2001

Phospho-proteomics: evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis.

Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in such proteins is a major challenge in proteomics. In the present study we evaluate the use of enzymatic de-phosphorylation in combination with differential peptide mass mapping for identification of phosphorylated peptides in peptide mixtures derived from in-gel digested phospho-proteins. Phospho-peptides could be identified provided that improved sample preparation methods prior to mass spectrometric analysis were used. An attempt to identify the proteins visualized by [32P] autoradiography in a proteomics study and their phosphorylation sites, demonstrated that protein identification was possible whereas reliable identification of the phospho-peptides requires more protein than normally available in our proteomics studies.

Alkaline Phosphatase↗

PubMed2001

The effects of transcription regulating genes PDR1, pdr1-3 and PDR3 in pleiotropic drug resistance.

Mutations in the yeast PDR1 or PDR3 genes lead to acquisition of resistance towards various unrelated cytotoxic compounds. The broad range and different properties of these compounds indicate the existence of mechanisms which protect cellular targets, neutralise or expel the compounds from the cell. In wild type and pdr mutants, 83 proteins, out of 2706 detected by two-dimensional gel electrophoresis, were differentially expressed. Fifty-three of these could be identified by mass spectrometry. The functions of these 53 proteins fall into several metabolic groups demonstrating that drug resistance phenotype is a mosaic response derived from such diverse functions as stress defence, endocytosis, oxidation and reduction, amino acid synthesis and mitochondrial biogenesis. The patterns of synthesis of the selected proteins clearly demonstrates the complex interaction between Pdr1p and Pdr3p in exerting their regulatory functions. The data also indicate that, in the Saccharomyces cerevisiae pleiotropic drug resistance phenomenon, translational events exert a more decisive effect than transcription in regulating the levels of active forms of the proteins involved.

DNA-Binding Proteins↗

PubMed2001

2D or not 2D. Two-dimensional gel electrophoresis.

2D gel electrophoresis is the technology that everyone loves to hate-it requires manual dexterity and precision to reproduce precisely and is thus not well-suited as a high-throughput technology. Although almost everyone would like to replace it, the resolution and sensitivity it offers are exquisite and unsurpassed if one wants a global view of cellular activity. There have been several recent developments, for example, the detection of low abundance proteins, and the resolution possible with narrow-range IPG gels.

Animals↗

PubMed2001

Proteome analysis of interleukin-1beta--induced changes in protein expression in rat islets of Langerhans.

The intracellular molecular events involved in the beta-cell death process are complex but poorly understood. Cytokines, e.g., interleukin (IL)-1beta, may play a crucial role in inducing this process. Protein synthesis is necessary for the deleterious effect of IL-1, and induction of both protective and deleterious proteins has been described. To characterize the rather complex pattern of islet protein expression in rat islets in response to IL-1, we have attempted to identify proteins of altered expression level after IL-1 exposure by 2D gel electrophoresis and mass spectrometry. Of 105 significantly changed (i.e., up- or downregulated or de novo-induced) protein spots, we obtained positive protein identification for 60 protein spots. The 60 identifications corresponded to 57 different proteins. Of these, 10 proteins were present in two to four spots, suggesting that posttranslatory modifications had occurred. In addition, 11 spots contained more than one protein. The proteins could be classified according to their function into the following groups: 1) energy transduction; 2) glycolytic pathway; 3) protein synthesis, chaperones, and protein folding; and 4) signal transduction, regulation, differentiation, and apoptosis. In conclusion, valuable information about the molecular mechanisms involved in cytokine-mediated beta-cell destruction was obtained by this approach.

Animals↗

PubMed2000

Towards higher resolution: two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients.

The rising number of proteome projects leads to new challenges for two-dimensional electrophoresis with immobilized pH gradients and different applications of this technique. Not only wide pH gradients such as 4-12 or 3-12 (Görg et al., Electrophoresis 1999, 20, 712-717) which can give an overview of the total protein expressions of cells are in demand but also overlapping narrow immobilized pH gradients are to be used for more specialized and detailed research and micropreparative separations. The advantage of overlapping narrow pH gradients is the gain in higher resolution by stretching the protein pattern in the first dimension. This simplifies computer-aided image analysis and protein identification (e.g., by mass spectrometry). In this study the protein patterns of yeast cells in pH gradients 4-5, 4.5-5.5, 5-6, 5.5-6.7 and 6-9 are presented and compared to the pH 4-7 and 3-10 gradients. This combination allowed us to reveal a total of 2286 yeast protein spots compared to 755 protein spots in the pH 3-10 gradient.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2000

Cytokine- or chemically derived nitric oxide alters the expression of proteins detected by two-dimensional gel electrophoresis in neonatal rat islets of Langerhans.

Interleukin-1beta (IL-1beta) treatment of neonatal rat islets for 24 h induces changes in the expression of 105 of 2,200 proteins, as determined previously by two-dimensional (2D) gel electrophoresis. Nitric oxide (NO) has been implicated as one of the mediators of IL-1beta effects in insulin-containing cell lines and rat islets. The aims of this study were 1) to determine the involvement of NO in IL-1beta-induced alterations in protein expression and 2) to investigate the effects of chemically generated NO on protein expression by 2D gel electrophoresis of neonatal rat islet samples. IL-1beta-induced NO production was prevented by incubation of islets in arginine-free medium supplemented with the arginine analog NG-nitro-L-arginine. [35S]methionine-labeled islet proteins were separated using 2D gel electrophoresis and analyzed using the BioImage computer program. Analysis revealed that the expression levels of 23 protein spots of the 105 protein spots, altered by prior treatment with IL-1beta (60 U/ml) alone, were significantly affected (P < 0.01 [n = 4] and P < 0.05 [n = 19]) when NO production was prevented. The effects of chemically generated NO were investigated by exposing islets to the NO donor GSNO (100 micromol/l) for 24 h before labeling with [35S]methionine and 2D gel electrophoresis. Computer-based analysis identified alterations in the expression of 19 of a total of 1,600 detectable proteins in GSNO-treated islets (P < 0.01). We conclude 1) that the expression of up to 42 proteins is altered by cytokine-induced or chemically generated NO in the precise experimental conditions chosen and 2) that the majority of proteins altered by prior treatment with IL-1beta may be the result of NO-independent IL-1beta-mediated regulation of gene expression. This study demonstrates that the combination of 2D gel electrophoresis and mass spectrometry is a powerful tool in the identification of beta-cell proteins involved in the response to toxic mediators.

Animals↗

PubMed2000

Islet protein expression changes during diabetes development in islet syngrafts in BB-DP rats and during rejection of BB-DP islet allografts.

Interleukin 1beta (IL-1) is cytotoxic to rat pancreatic beta-cells in vitro, and increased expression of IL-1 mRNA is found in the islets of Langerhans during development of diabetes in BB/Wor/Mol-BB2 (BB-DP) rats and NOD mice. It has been proposed that IL-1 induces a race between protective and deleterious proteins in the beta-cells during development of diabetes, and that heat shock proteins 70 and 90, and manganese superoxide dismutase, all inducible by IL-1 are potentially protective proteins. We have established a database of approximately 2000 neonatal rat-islet proteins by two-dimensional gel (2-D gel) electrophoresis of [35S]-methionine labelled neonatal Wistar Furth rat islets. In these IL-1 was shown to up- or down-regulate the islet-expression level of 99, and to induce de novo synthesis of 6 proteins. The identity of most of the IL-1 induced proteins is unknown and under study. In this study we wished to investigate if changes in protein expression induced in vitro by IL-1 stimulation of islets are also seen in vivo during spontaneous development of diabetes in BB-DP rats, and during islet allograft rejection. Two-hundred neonatal BB-DP rat islets were grafted under the kidney capsule of either 30-day-old BB-DP rats killed at onset of diabetes or of 30-day-old Wistar Kyoto (WK) rats, killed 12 days after grafting. Proteins in excised islet-grafts and in vitro IL-1 exposed isolated neonatal BB-DP rat islets were labelled with [35S]-methionine, and processed for 2-D gel electrophoresis. Fluorographs of the gels were analysed by computer. A total of 1815 proteins were found in 3 of 3 12.5% polyacrylamide gels. Interleukin-1 was found to change expression level of 82 of these proteins (22 up- and 60 down-regulated) in neonatal BB-DP rat islets in vitro. Of these 82 proteins 33 (4 up- and 29 down-regulated) also changed level of expression during disease occurrence in syngeneic islet grafts from diabetic BB-DP rats, and 29 (4 up- and 25 down-regulated) during rejection of BB-DP islets grafted to WK rats. Changes in the expression level of 14 (3 up- and 11 down-regulated) of the 82 proteins altered by IL-1 in vitro were only found in syngeneic islet grafts in diabetic BB-DP rats, and changes in the expression level of 8 (2 up- and 6 down-regulated) of these 82 proteins expression were only found in BB-DP islet allografts in WK recipients. Identification of these proteins may be important in understanding the mechanisms of islet destruction during development of insulin-dependent diabetes mellitus and during islet allograft rejection.

Animals↗

PubMed1999

Two-dimensional gel analysis of human endometrial proteins: characterization of proteins with increased expression in hyperplasia and adenocarcinoma.

In the search for new markers of human endometrial hyperplasia and adenocarcinoma the method of quantitative two-dimensional gel electrophoresis was applied to study the protein expression profiles of metabolically [(35)S]-methionine-labelled proteins of endometrial explants. Approximately 1700 protein spots were resolved by the two-dimensional gel electrophoresis, and the expression pattern of each of these proteins was assessed for increased expression during hyperplasia or adenocarcinoma. In total, six protein spots showed increased expression in hyperplasia, 19 in carcinoma, and eight in both hyperplasia and carcinoma. Twelve of these 33 differentially expressed proteins were identified by peptide mass mapping combined with sequence database searching. Among the identified proteins were proteins involved in cellular transport and chaperoning, i.e. heat shock protein 27 kDa protein, heat shock 70 kDa protein, heat shock cognate 71 kDa protein, and serotransferrin. Other identified proteins were: regulatory chain protein of cAMP-dependent protein kinase, prohibitin, and heterogeneous nuclear ribonucleoprotein A2/B1. Finally we identified proteins associated with the cytoskeleton, vimentin and tropomyosin isoform 3, and the glycolytic pathway, alpha enolase, and phosphoglycerate kinase. The remaining unidentified proteins were either not contained in the database and must be assumed to be novel proteins, or were present in too low amounts to allow characterization.

Adenocarcinoma↗

PubMed1998

Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification.

Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross-references demonstrated that there is no obvious pattern by which the mobility of a protein in one gel system can be used to predict its mobility in the other. Thus, as laboratories adopt the immobilized pH gradient-based 2-D gel systems, the only reliable means of translating the data gained with the carrier ampholyte-based gel system is to positively identify the proteins in both 2-D systems.

Ampholyte Mixtures↗

PubMed1997

Proteome analysis of Saccharomyces cerevisiae: a methodological outline.

Proteome analysis offers a unique means of identifying important proteins, characterizing their modifications and beginning to describe their function. This is achieved through the combination of two technologies: protein separation and selection by two-dimensional gel electrophoresis, and protein identification and characterization by mass spectrometry. This methodological outline sketches the strengths and weaknesses of the two central technologies used, and provides both practical tips and the theoretical background for their utilization. One application of these technologies is illustrated by the characterization of genes, revealed by sequencing, but which have no--or only weak homology--to any other known genes. Other applications, for example the identification of protein markers for particular human diseases, are only referred to. The aim of the article is thus to provide the basis for a sound understanding of the full potential and limitations of proteome analysis.

Databases, Factual↗

PubMed1997

Interleukin-1beta induced changes in the protein expression of rat islets: a computerized database.

Insulin-dependent diabetes mellitus is caused by an autoimmune destruction of the beta-cells in the islets of Langerhans. The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to beta-cells in isolated pancreatic rat islets. The antigen(s) triggering the immune response as well as the intracellular mechanisms of action of interleukin 1-mediated beta-cell cytotoxicity are unknown. However, previous studies have found an association of beta-cell destruction with alterations in protein synthesis. Thus, two-dimensional (2-D) gel electrophoresis of pancreatic islet proteins may be an important tool facilitating studies of the molecular pathogenesis of insulin-dependent diabetes mellitus. 2-D gel electrophoresis of islet proteins may lead to (i) the determination of qualitative and quantitative changes in specific islet proteins induced by cytokines, (ii) the determination of the effects of agents modulating cytokine action, and (iii) the identification of primary islet protein antigen(s) initiating the immune destruction of the beta-cells. Therefore, the aim of this study was to create databases (DB) of all reproducibly detectable protein spots on 10% and 15% acrylamide 2-D gels of neonatal rat islets (10% and 15% DB), labeled under standardized culture conditions. 1235 and 557 spots were present in 5 of 5 gels in the 15% isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) DB, respectively, whereas 995 and 378 spots were present in 5 of 5 gels in the 10% IEF and NEPHGE DB, respectively, yielding a reproducibility of spot detection between 75.2% and 91.7%. In both DBs, the average coefficient of variation of the percentage of integrated optical density (CV% of %IOD) for spots present in all gels was between 42.4% and 45.7%. When the same sample was analyzed in consecutive sets of gels on different days (interassay analysis), the average CV% of %IOD was 35.5%-36.1%. When the same sample was analyzed repeatedly in one set of gels (intra-assay analysis), the average CV% of %IOD was 30.2% in the IEF gels, while the average CV% of %IOD was 45.7% in the NEPHGE gels. Addition of interleukin-1beta (IL-1beta) to the cultures resulted in statistically significant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2-D gel protein databases of neonatal rat islets of Langerhans and demonstrate its usage to identify proteins altered in expression by IL-1beta.

Animals↗

PubMed1995

Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two-dimensional gel electrophoresis.

An interlaboratory comparison was conducted on the positional and quantitative reproducibility of yeast proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using isoelectric focusing with immobilized pH gradient (pH 4-7) in the first dimension. The basic experimental set-up was as follows: one laboratory prepared and distributed a [35S]methionine-labeled total yeast protein extract (Göteborg, Sweden), another laboratory prepared the IPG strips to be used by all labs in this study (Munich, Germany), the third laboratory (Aarhus, Denmark) circulated the protocols and coordinated the modest attempts to unify them. Samples were run horizontally in the first dimension and vertically in the second. The gels were sent to Göteborg for processing by phosphoimager technology and computerized image analysis (PDQuest), and the 2-D PAGE resolved proteins were located and quantified automatically. A subset of 470 spots was manually matched in all gels out of an average of 1328 resolved proteins. The positional interlaboratory comparison revealed great pattern reproducibility, the correlation coefficient in no case being less than 0.9994. In absolute terms an average deviation of 2.8 mm (x-position) and 1.8 mm (y-position) were obtained for all nine gels (three gels per lab). The interlaboratory comparison of protein quantitation displayed higher variability, and the best correlation coefficient generated was 0.975. An average standard deviation of 34.5% was calculated for protein quantitation including all three labs, a value slightly higher than the intralaboratory variation (range 20-28%). Thus, despite differences in protocols, chemicals and equipment, the immobilized pH gradient technology gave extremely high positional and quantitative reproducibility. This will greatly facilitate the exchange of data and the establishment of multi-user image-based 2-D gel databases.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1995

Two-dimensional gel analysis of human endometrial proteins: cyclic changes in the expression of specific proteins during the normal menstrual cycle.

High resolution two-dimensional (2-D) gel electrophoresis was used to compare the patterns of [35S]methionine-labelled cellular proteins in endometrial tissue from healthy, normally menstruating women. Samples of endometrial tissue were incubated with [35S]methionine for 20 h, and total cell lysates were processed for 2-D gel electrophoresis. Using this technique it was possible to study proteins with iso-electric points (pI) ranging from 3.5 to 11 and relative molecular weights (M(r)) ranging from 10,000 to 300,000 Da. The fluorograms were compared by computer-aided analysis whereby a total of 1095 [35S]-labelled proteins were resolved on the iso-electric focusing gels (IEF, pI 3.5-7) and 488 on the non-equilibrium pH gradient electrophoresis (NEPHGE) gels (pI 6.5-11). Of the proteins on the IEF gels, 125 showed differential expression during the menstrual cycle. Of these, 36 were maximally expressed in proliferative phase endometrium, 26 in the interval phase and 63 in secretory and/or late secretory phase endometrium. Correspondingly, on the NEPHGE gels a total of 61 proteins exhibited cyclical variation, of which 30 were more prominent in proliferative phase, 13 in interval phase and 18 in secretory phase endometrium. This study shows that 2-D gel electrophoresis is eminently suited to the identification of proteins whose expression varies in a cyclical manner during the menstrual cycle. Further investigations should be carried out to isolate and characterize these proteins with the aim of establishing useful markers for specific endometrial phases of the menstrual cycle.

Adult↗

PubMed1995

Human endometrial proteins with cyclic changes in the expression during the normal menstrual cycle: characterization by protein sequence analysis.

Endometrial proteins showing cyclic expression during the normal menstrual cycle were localized on two-dimensional (2-D) electrophoresis gels separating proteins with isoelectric points (pl) ranging from 3.5 to 7 and relative molecular weights ranging from 10 to 300 kDa. Menstrual cycle-related proteins were excised from several 2-D gels, concentrated by one-dimensional (1-D) sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, and cleaved in situ by trypsin. The tryptic fragments were extracted and separated by reverse phase high performance liquid chromatography (RP-HPLC). Finally, the partial amino-terminal amino acid sequence of selected tryptic fragments were determined for each protein. We aimed at characterizing the 21 menstrual cycle-related proteins that were visible on silver-stained 2-D electrophoresis gels. Of the proteins being maximally synthesized in the proliferative phase endometrium, we identified proteins associated mainly with the cytoskeleton: vimentins, keratin, tropomyosin and tubulin, but also proteins such as proliferating cell nuclear antigen and beta-galactoside binding lectin. The partial amino acid sequences for another two proteins did not match any protein sequence in the Protein Identification Resource (PIR) and Swissprot databases. In the group of proteins having maximal synthesis in the secretory phase endometrium, we identified creatine kinase chain B and an isocitrate dehydrogenase-homologous protein, both of which are involved in energy metabolism. However, we also identified the annexin IV precursor, the 14-3-3 protein homologue also called stratifin or the epithelial cell marker protein 1 and the 21K tumour protein. Finally, four of the proteins were present in too low amounts to allow characterization. Interestingly, most of the identified proteins have not previously been described as having a menstrual cycle-related synthesis in the human endometrium. It may be considered that the concentration of some of the cycle-related proteins may be used in clinical situations to reflect specific endometrial phases.

Amino Acid Sequence↗

PubMed1995

Two-dimensional gel electrophoresis of rat islet proteins. Interleukin 1 beta-induced changes in protein expression are reduced by L-arginine depletion and nicotinamide.

Interleukin (IL)-1 beta-mediated damage to beta-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 beta-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 beta has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 beta-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 beta-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 beta, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 beta-, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 beta-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 beta +/- nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial beta-cell destruction in insulin-dependent diabetes mellitus.

Animals↗

PubMed1995

Cloning and expression of cytokine-inducible nitric oxide synthase cDNA from rat islets of Langerhans.

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the beta-cells of interleukin (IL)-1 beta-exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokine-exposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH beta-cells cultured in the absence of cytokines. Addition of IL-1 beta alone or in combination with tumor necrosis factor-alpha induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokine-exposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the L-arginine analogs, N omega-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pI values in the range of 6.8 to 7.0. In conclusion, the IL-1 beta-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.

Amino Acid Oxidoreductases↗