Protein expression changes in a cell system of beta-cell maturation reflect an acquired sensitivity to IL-1beta.

AIM/HYPOTHESIS: Type 1 diabetes mellitus (T1DM) is caused by specific destruction of the pancreatic beta cells in the islets of Langerhans. Increased sensitivity to cytokines, in particular to interleukin-1beta (IL-1beta) seems to be an acquired trait during beta-cell maturation. In response to cytokines both protective and deleterious mechanisms are induced in beta cells, and when the deleterious prevail, T1DM develops. The aims of this study were to identify perturbation in protein patterns (PiPP) associated with beta-cell maturation, and compare these changes to previous analyses of IL-1beta exposed rat islets. For this purpose, proteome analyses were carried out using a cell-line, which matures from a glucagon-producing pre-beta-cell phenotype (NHI-glu) to an insulin-producing beta-cell phenotype (NHI-ins). We have previously shown that this maturation is accompanied by acquired sensitivity to the toxic effects of IL-1beta. METHODS: 2D-gel electrophoresis was used to separate the proteins and MALDI-MS and database searches were performed to identify the proteins. RESULTS: During beta-cell maturation 135 protein spots out of 2239 detectable changed expression levels. Of these, 74 were down-regulated, 44 up-regulated, 16 were suppressed and 1 was expressed de novo. Using MALDI-MS, positive identification was obtained for 93 out of the 135 protein-spots revealing 97 different proteins. Of these, 22 proteins were in common with changes identified in previous proteome analysis of perturbation in protein pattern in IL-1beta exposed rat islets. Several of the proteins were present in more than one spot suggesting post-translational modification. CONCLUSION/INTERPRETATION: Several proteins and protein modifications were identified that could be critically involved in beta-cell maturation, insulin-gene expression and the acquired IL-1beta sensitivity.

Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans.

Streptococcus mutans is a component of the dental plaque biofilm and a major causal agent of dental caries. Log-phase cells of the organism are known to induce an acid tolerance response (ATR) at sub-lethal pH values ( approximately 5.5) that enhances survival at lower pH values such as those encountered in caries lesions. In this study, we have employed a rod biofilm chemostat system to demonstrate that, while planktonic cells induced a strong ATR at pH 5.5, biofilm cells were inherently more acid resistant than such cells in spite of a negligible induction of an ATR. Since these results suggested that surface growth itself triggered an ATR in biofilm cells, we were interested in comparing the effects of a pH change from 7.5 to 5.5 on protein synthesis by the two cell types. For this, cells were pulse labeled with [(14)C]-amino acids following the pH change to pH 5.5, the proteins extracted and separated by two-dimensional (2D) electrophoresis followed by autoradiography and computer-assisted image analysis. A comparison between the cells incubated at pH 5.5 and the control biofilm cells revealed 23 novel proteins that were absent in the control cells, and 126 proteins with an altered relative rate of synthesis. While the number of changes in protein expression in the biofilm cells was within the same range as for planktonic cells, the magnitude of their change was significantly less in biofilm cells, supporting the observation that acidification of biofilm cells induced a negligible ATR. Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregulation of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment. On the other hand, the glycolytic enzymes in control biofilm cells were significantly less downregulated and key enzymes, such as lactate dehydrogenase were upregulated during pH 5.5 incubation, suggesting that the enhanced acid resistance of biofilm cells is associated with the maintenance of pH homeostasis by H+ extrusion via membrane ATPase and increased lactate efflux.

Intrauterine programming of fetal islet gene expression in rats--effects of maternal protein restriction during gestation revealed by proteome analysis.

AIMS/HYPOTHESIS: Fetal undernutrition can result in intrauterine growth restriction and increased incidence of Type 2 diabetes mellitus. Intrauterine malnutrition in form of an isocaloric low-protein diet given to female rats throughout gestation decreases islet-cell proliferation, islet size and pancreatic insulin content, while increasing the apoptotic rate and sensitivity to nitrogen oxide and interleukin-1beta. Hence, the influence of a low-protein diet on the development of beta-cells and islets could also be of interest for the pathogenesis of Type 1 and Type 2 diabetes mellitus. We hypothesise that the effects of a low-protein diet in utero are caused by intrauterine programming of beta-cell gene expression. METHODS: Pregnant Wistar rats were fed a low-protein diet (8% protein) or a control diet (20% protein) throughout gestation. At day 21.5 of gestation fetal pancreata were removed, digested and cultured for 7 days. Neoformed islets were collected and analysed by proteome analysis comprising 2-dimensional gel electrophoresis and mass spectrometry. RESULTS: A total of 2810 different protein spots were identified, 70 of which were changed due to the low-protein diet. From 45 of the changed protein spots, identification was obtained by mass spectrometry (64% success rate). Proteins induced by the low-protein diet were grouped according to their biological functions, e.g. cell cycle and differentiation, protein synthesis and chaperoning. CONCLUSIONS/INTERPRETATION: Our study offers a possible explanation of the alterations induced by a low-protein diet in islets. It shows that in Wistar rats the intrauterine milieu could program islet gene expression in ways unfavourable for the future of the progeny. This could be important for our understanding of the development of Type 1 and Type 2 diabetes mellitus.

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.

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.

Molecular biological characterization of an azole-resistant Candida glabrata isolate.

Two isolates of Candida glabrata, one susceptible and one resistant to azole antifungals, were previously shown to differ in quantity and activity of the cytochrome P-450 14alpha-lanosterol demethylase which is the target for azole antifungals. The resistant isolate also had a lower intracellular level of fluconazole, but not of ketoconazole or itraconazole, than the susceptible isolate. In the present study a 3.7-fold increase in the copy number of the CYP51 gene, encoding the 14alpha-lanosterol demethylase, was found. The amount of CYP51 mRNA transcript in the resistant isolate was eight times greater than it was in the susceptible isolate. Hybridization experiments on chromosomal blots indicated that this increase in copy number was due to duplication of the entire chromosome containing the CYP51 gene. The phenotypic instability of the resistant isolate was demonstrated genotypically: a gradual loss of the duplicated chromosome was seen in successive subcultures of the isolate in fluconazole-free medium and correlated with reversion to susceptibility. The greater abundance of the amplified chromosome induced pronounced differences in the protein patterns of the susceptible and revertant isolates versus that of the resistant isolate, as demonstrated by two-dimensional gel electrophoresis (2D-GE). Densitometry of the 2D-GE product indicated upregulation of at least 25 proteins and downregulation of at least 76 proteins in the resistant isolate.

Identification and characterization of glima 38, a glycosylated islet cell membrane antigen, which together with GAD65 and IA2 marks the early phases of autoimmune response in type 1 diabetes.

Immunoprecipitating IgG autoantibodies to glutamic acid decarboxylase, GAD65, and/or a tyrosine phosphatase, IA2, are present in the majority of individuals experiencing pancreatic beta cell destruction and development of type 1 diabetes. Here we identify a third islet cell autoantigen, a novel 38-kD protein, which is specifically immunoprecipitated with sera from a subset of prediabetic individuals and newly diagnosed type 1 diabetic patients. The 38-kD autoantigen, named glima 38, is an amphiphilic membrane glycoprotein, specifically expressed in islet and neuronal cell lines, and thus shares the neuroendocrine expression patterns of GAD65 and IA2. Removal of N-linked carbohydrates results in a protein of 22,000 Mr. Glima 38 autoantibodies were detected in 16/86 (19%) of newly diagnosed patients, including three very young children, who had a rapid onset of disease, and in 6/44 (14%) of prediabetic individuals up to several years before clinical onset. The cumulative incidence of GAD65 and glima 38 antibodies in these two groups was 83 and 80%, respectively, and the cumulative incidence of GAD65, glima 38, and IA2 antibodies in the same groups was 91 and 84%, respectively. GAD65, IA2, and glima 38 represent three distinct targets of immunoprecipitating IgG autoantibodies associated with beta cell destruction and type 1 diabetes.

Human papilloma virus (HPV) and carcinomas of the head and neck.

Increasing evidence for a causal link between human papilloma virus and carcinomas of the cervix has emerged in recent research. This group of species-specific, epitheliotropic viruses has also been associated with tumours of the head and neck, but the individual reports deal only with relatively small sample numbers. In the present review these reports are considered in relation to the methods employed, and it is concluded that HPV is associated with more than 50% of oral and nasal carcinomas, as well as with carcinomas of the larynx and oesophagus. The clinical relevance and strategies for future work are outlined.

Putative association of mitochondria with a subpopulation of intermediate-sized filaments in cultured human skin fibroblasts.

Cultured cell Triton cytoskeletons enriched in intermediate filaments by extraction in high and low salt show, in addition to known intermediate-filament proteins and actin, a few other tenaciously bound polypeptides. An antibody raised against one of these polypeptides (IEF 24) is shown to react specifically with mitochondria of different cell types. With methanol-acetone fixation, required for the antibody reaction, the characteristically long mitochondria of human skin fibroblasts fragment into strings of beads, as identified by antibody staining in the light microscope and whole-mount electron microscopy. From the colinear organization of these beads, their codistribution with the intermediate-filament network and their retention in salt-extracted cytoskeletons, it is suggested that a subpopulation of the intermediate filaments can serve as an anchorage site for mitochondria.