Differential Display Analysis Research Articles

Methylcytosine-binding protein VIM1 decreases As(III) accumulation by epigenetic downregulation of the As(III) importer NIP3;1 in Arabidopsis

Arsenic (As) is a highly toxic metal that contaminates water and land and enters humans through plants. In the present study, using a differential display analysis, we observed that VARIANT IN METHYLATION ( VIM1; a DNA methylation modulator) was induced by As(III) in As(III)-tolerant NtCyc07 -expressing transgenic Arabidopsis. Therefore, the roles of VIM1 and DNA methylation in As(III) tolerance and accumulation were further investigated by expressing VIM1 in yeast and Arabidopsis. AtVIM1 -expressing yeast showed approximately 2-fold higher growth rate with 40% lower accumulation of As(III), compared with control. Similarly, overexpression of AtVIM1 in Arabidopsis increased the plant biomass by ~ 1.7-fold and decreased As(III) accumulation by up to 23%. In contrast, vim1 knockout mutant of Arabidopsis was As(III)-sensitive and showed more severe growth retardation and higher As(III) accumulation than control. Furthermore, the decreased expression of the As(III) importer NIP3;1 was responsible for the attenuated accumulation of As(III) in VIM1 -overexpressing Arabidopsis. Interestingly, methylation levels at three CpG sites of the NIP3;1 promoter in transgenic plants were 6–7-fold higher than those in control plants, suggesting that NIP3;1 expression was decreased due to enhanced promoter methylation by VIM1. Taken together, these findings suggest that VIM1 enhances As(III) tolerance by reducing As(III) accumulation through enhancing DNA methylation at the NIP3;1 (an As(III) importer) promoter. These results can be applied to generate “As(III) phytoremediators” and “less As(III)-accumulating crops” using gene editing and overexpression, respectively. AtVIM1-expressing Arabidopsis displays lower As(III) accumulation and root-to-shoot transport. • VIM1 expression is highly induced by As(III) in Arabidopsis root. • VIM1 enhances As(III) tolerance by mitigating As(III) accumulation in Arabidopsis. • VIM1-mediated methylation of NIP3;1 promoter causes lower As(III) level. • As (III) importer NIP3;1 is repressed via VIM1-mediated promoter methylation. • VIM1 is a potential target gene for phytoremediation improvement.

Silencing of Tropomyosin 1 suppresses the proliferation, invasion and metastasis of oral squamous cell carcinoma in vitro

We focused on the expression of Tropomyosin 1 (TPM1) in oral squamous cell carcinoma (OSCC) on the basis of proteomic differential display analysis data. TPM1 is an actin binding and microfilament-associated protein, which is thought to affect the tumor behavior of various malignancies. In this study, we examined the role of TPM1 in proliferation, invasion and metastasis of OSCC cells in vitro as we tried to understand its usefulness as a potential biomarker for OSCC. Human OSCC cell lines were transfected by TPM1-siRNA. MTT assay, cell migration assay and wound healing assay were carried out to understand the characteristics of the TPM1-siRNA-transfected cells. Our data showed that suppression of TPM1 increased the proliferative ability, wound healing ability, and migratory ability of OSCC cells. These findings suggest that low expression of TPM1 could contribute to cancer cell invasion and metastasis in OSCC patients, and that TPM1 may have potential as a useful biomarker for OSCC. Availability of data and materialsAll data generated or analyzed during the present study are included in this published article.

Comparison of Traditional and Industrial Sausages Baranjski Kulen and Kulenova Seka Using Comprehensive Proteome, Peptidome and Metabolome Techniques

SUMMARYResearch backgroundBaranjski kulen is one of the most popular fermented meat sausages originating from Croatia. It has protected geographical indication, and is traditionally produced in the Baranja region of Croatia. Kulenova seka is a fermented sausage very similar to baranjski kulen, but it has a different calibre and consequently, a shorter time of production. In recent decades, due to the high demand and popularity of these products, industrially produced baranjski kulen and kulenova seka have become available on the market. This work aims to identify specific characteristics of traditional and industrial sausages baranjski kulen and kulenova seka on proteome, peptidome and metabolome level, which could potentially lead to better optimization of the industrial production process in order to obtain an equivalent to the traditional product.Experimental approachProtein profiles of baranjski kulen and kulenova seka (traditional and industrial) were analysed using two-dimensional gel electrophoresis followed by differential display analysis and protein identification by mass spectrometry. Peptidomic profile was analysed via liquid chromatography-tandem mass spectrometry. Furthermore, aroma profiles were investigated via headspace solid phase microextraction and gas chromatography-mass spectrometry.Results and conclusionsThe major identified characteristics of each product were: industrial baranjski kulen: specific degradation of myosin-1 and titin, overabundance of stress-related proteins and increased phenylalanine degradation; traditional baranjski kulen: decreased concentration of phenylalanine and overabundance of fructose-bisphosphate aldolase A and carbonic anhydrase 3; industrial kulenova seka - specific myosin-4 and haemoglobin subunit alpha degradation process; traditional kulenova seka - overabundance of dihydropyrimidine dehydrogenase [NADP(+)] and myosin light chain 1/3, skeletal muscle isoform, degradation of albumin and myoglobin, decreased concentrations of almost all free amino acids and increased amounts of smoke-derived volatile compounds. Presented results showed that potential product type-specific quality markers for each sausage could be found.Novelty and scientific contributionIn this preliminary communication, the first insights into protein degradation processes and generation of peptides, free amino acids and aroma compounds of industrial and traditional baranjski kulen and kulenova seka are presented. Although further research is needed to draw general conclusions, the specific profile of proteins, peptides, amino acids and volatile compounds represents the first step in the industrial production of sausages that meet the characteristics of traditional flavour.

The Expression Levels of Vinculin in Pancreatic Cancer Tissues Significantly Correlates With Patient Survival.

Proteomics is an approach that can detect differentially expressed proteins between cancerous and non-cancerous tissue samples. Previously, we found that vinculin was predominantly expressed in pancreatic cancerous tissues compared to adjacent non-cancerous tissues by performing proteomic differential display analysis. However, the clinicopathological significance of vinculin in pancreatic cancer has not yet been documented. The GEPIA2 and the Human Protein Atlas databases were used to analyze vinculin expression levels in cancerous tissue samples and investigate whether its expression level is clinically associated with patient survival. Vinculin mRNA expression levels were solely increased in pancreatic cancer tissues, and increased expression was inversely related to patient survival. Higher levels of vinculin protein were found in pancreatic cancer tissues. In contrast, faint staining of vinculin was observed throughout the normal pancreatic tissues. Vinculin may be an unfavorable prognostic indicator for patients with pancreatic cancer.

Identification of key genes and functions of circulating tumor cells in multiple cancers through bioinformatic analysis

BackgroundCirculating tumor cells (CTCs) play a key role in cancer progression, especially metastasis, due to the rarity and heterogeneity of CTCs, fewer researches have been conducted on them at the molecular level. However, through the Gene Expression Omnibus (GEO) database, this kind of minority researches can be well integrated, the gene expression differences between CTCs and primary tumors can be identified, and molecular targets for CTCs can be found.MethodsWe analyzed 7 sets of gene chips (GSE82198, GSE99394, GSE31023, GSE65505, GSE67982, GSE76250, GSE50746) obtained by GEO. Analysis of differentially expressed genes (DEGs) between CTCs and corresponding primary tumors by NetworkAnalyst. Metascape tool for Gene Ontology (GO) / Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differential genes and visual display. Cytoscape performs protein-protein interaction (PPI) analysis and obtains the hub genes. Renal cancer patients’ clinical specimens to verify the correctness of enrichment results. Prognostic analysis of hub genes in kidney cancer patients using the Kaplan–Meier plotter survival analysis tool.ResultsWe obtained a total of 589 DEGs. The GO / KEGG enrichment results indicate that the DEGs are mainly concentrated in cell adhesion, epithelial-mesenchymal transition (EMT), and apoptosis. Renal cancer clinical specimens suggest that CTCs have epithelial and mesenchymal types. At the same time, PSMC2 can be used as a poor prognostic indicator for renal cancer patients.ConclusionsIn summary, our study suggests that compared with primary tumors, CTCs mainly change cell adhesion, EMT, and apoptosis. PSMC2 can be used as a poor prognostic factor.

Identification of differentially expressed transcripts at critical developmental stages in sorghum [Sorghum bicolor (L.) Moench] in relation to grain yield heterosis.

Evaluation of a set of 10 F1 hybrids along with their female (27A and 7A) and male parents (C 43, RS 673, RS 627, CB 26, and CB 29) for grain yield and its component traits revealed that grain yield/plant followed by panicle weight, primary branches/panicle, and 100-seed weight exhibited high levels of heterosis. Eight hybrids exhibited 50% or more mid-parent heterosis for grain yield/plant, of which, one hybrid (27A × RS673) recorded heterobeltiosis above 50% (73.61%). Differential display analysis generated about 2995 reproducible transcripts, which were categorized as UPF1-expressed in any one of the parents and F1 (10.53-14.76%), BPnF1-expressed in both parents but not in F1 (4.56-11.44%), UPnF1-expressed in either of the parents and not in F1 (17.95-27.40%), F1nBP-expressed only in F1 but not in either of the parents (14.39-20.54%), and UET-expressed in both parents and F1 (34.52-42.43%). A comparison between high and low heterotic hybrids revealed that the proportions of UPF1 and F1nBP transcript patterns were much higher in the former (21.31% and 45.24%) as compared to the latter (16.67% and 32.14%) at the booting and flowering stage, respectively, indicating the role of over-dominance and dominance in the manifestation of grain yield heterosis. Significant positive correlations were observed for differential transcript patterns with mid-parent and better-parent heterosis for the components of grain yield such as primary branches (0.63 and 0.61 at p < 0.01) and 100-seed weight (0.64 and 0.52 at p < 0.01). Cloning and sequence analysis of 16 transcripts that were differentially expressed in hybrids and their parental lines revealed that they code for genes involved in basic cellular processes, cellulose biosynthesis, and assimilate partitioning between various organs and allocation between various pathways, pyrimidine, and polyamine biosynthesis, enhancing ATP production and regulation of plant growth and development.

Inhibición del crecimiento y modificación genética de Phytophthora capsici usando quitosano de bajo grado de polimerización

Phytophthora blight of peppers, caused by oomycete Phytophthora capsici, currently causes economic losses in crops such as peppers, tomatoes, eggplant and cucurbits. In this work, we evaluated the effect of chitosan with low degree of polymerization (LDP) on growth and gene expression of P. capsici cultures. LDP chitosan inhibited 88% of P. capsici mycelial growth at concentrations up to 0,4 mg/l, whereas at concentrations higher than 1,6 mg/l it completely inhibit growth. Gel mobility shift assays demonstrated that chitosan interacts with DNA and RNA of the fungus at concentrations ranging from 2 to 4mg/l for DNA and 0,5 to 3mg/l for RNA. The differential display analysis of RT-PCR-amplification products of P. capsici messenger RNA revealed changes in gene expression profiles after the chitosan treatment. Bioinformatic analysis of sequences from selected differentially-expressed bands showed the gene regulation of elements involved in chitin synthesis and carbohydrate-binding proteins.

Expresión diferencial de genes como respuesta a estrés salino en tomate de árbol (Solanum betaceum)

The Andean “tree tomato” (Solanum betaceum) is an exotic fruit crop endemic to the high Andes, but principally cultivated in Colombia, Peru and Ecuador. The species displays broad agro-ecological adaptability and has proven resilient to different marginality factors, including high soil-salinity. This study presents a preliminary exploration of the genetic mechanisms underlying salinity tolerance in S. betaceum. To this end, we selected two S. betaceum genotypes contrasting in their ability to tolerate high salinity in vitro, and used differential display analysis to compare overall differences in gene expression between salinity-stressed and unstressed (control) plants in both genotypes. Overall, 171 differentially expressed transcripts (DETs) were identified; 30 of which showed homology with candidate genes associated with abiotic stress tolerance in different species. These were ascribed putative roles in stressresponse, photosynthesis, cellular metabolism and cell wall metabolism. Several identified DETs (22 in total) also showed homology to proteins of unknown function. These sequences warrant further research for potentially novel abiotic stress tolerance mechanisms. Despite its inherent limitations, differential display analysis allowed us to identify and validate (via RT-qPCR) 3 salinity-stress induced DETs. Prospectively, expanding our analyses via the validation of additional DETs would likely contribute to the identification of genes which can be used as proxies for a better understanding of the regulatory, metabolic and physiological mechanisms used by S. betaceum to respond and adapt to salinity stress.

Calreticulin is a novel independent prognostic factor for oral squamous cell carcinoma.

We focused on the expression of Calreticulin (CALR) in oral squamous cell carcinoma (OSCC) on the basis of proteomic differential display analysis data. We used QR-32 cells in this study which is a regressive murine fibrosarcoma cell clone; and QRsP-11, a progressive malignant tumor cell clone originated from QR-32. CALR is an endoplasmic reticulum luminal Ca2+-binding chaperone protein, which is thought to affect the tumor behavior of various malignancies. This study was aimed to determine the usefulness of CALR as a prognostic factor in patients with OSCC. We investigated the expression of CALR in tissue samples taken from 111 OSCC patients by immunohistochemistry, and we also analyzed the relationship between CALR expression and patients' clinicopathological characteristics as well as patient survival. Positive immunohistchemical staining of CALR was observed in the cancer cell cytoplasm. Among 111 patients, high expression of CALR was observed in 44 patients (39.6%), whereas low expression was observed in 67 patients (60.4%). Significant association was found between CALR expression and T classification (P=0.0027), N classification (P=0.0219), stage (P=0.0013), and patient outcome (P=0.0014). Log-rank test showed that, there is a significant difference (P<0.0001) in 5-year survival rates between patients showing CALR high-expression (59.1%) and CALR low-expression (86.6%). According to our Multivariate analysis, reduced term survival of patients was correlated to high levels of CALR expression (P<0.0001). Our findings suggest that elevated expression of CALR might play an important role in tumor progression in OSCC, and could be considered as a useful prognostic factor in OSCC patients.

Inside Front Cover: A rapid differential display analysis of nasal swab fingerprints to distinguish allergic from non‐allergic rhinitis subjects by mesoporous silica particles and MALDI‐TOF mass spectrometry

Inside Front Cover: A rapid differential display analysis of nasal swab fingerprints to distinguish allergic from non‐allergic rhinitis subjects by mesoporous silica particles and MALDI‐TOF mass spectrometry

A rapid differential display analysis of nasal swab fingerprints to distinguish allergic from non-allergic rhinitis subjects by mesoporous silica particles and MALDI-TOF mass spectrometry.

Discriminating different rhinitis cases can sometimes be difficult as the diagnostic criteria used to identify the various subgroups are not always unambiguous. The nasal fluid (NF) highly reflects the pathophysiology of these inflammatory diseases. However, its collection, as nasal lavage fluid, may cause discomfort. Due to the non-invasiveness and rapidity of collection, nasal swab might represent an alternative to overcome these problems and also an ideal source of biomarkers. In this study, we demonstrate that the combined use of mesoporous silica (MPS) with MALDI-TOF MS allows the rapid detection of differential nasal peptide profiles from nasal swabs of healthy (H), allergic rhinitis (AR) and non-allergic rhinitis (NAR) subjects. NF peptides from nasal swabs were captured by the mean of MPS then profiled by MALDI-TOF MS. As a proof-of-principle, we also explored the ability of our platform to discriminate between nasal swabs of patients with AR and NAR, and between these groups and H controls. Four peaks resulted differentially expressed between NAR and AR, two peaks discriminated AR from H while one peak segregated NAR from H group. Therefore, peptides selected and enriched by our platform could form a part of a diagnostic ''rhinomic'' profile of the allergic and non-allergic patients.

Prediction of pregnancy outcome of IVF-ET cycles following endometrial injury in women with previously failed implantation based on endometrial transcriptomics: A preliminary report

Background/aimsTimed administration of endometrial scratching in natural cycles followed by IVF-ET reportedly yields improved clinical pregnancy in women. In the present study, we have examined the issue of endometrial transcriptomic response in natural cycles preceding to the intervention cycles of IVF-ET to explore the differences, if any, in transcript expression profiles between those who conceived and those who did not following endometrial scratching in IVF-ET. MethodsPatients (n=25) undergoing autologous IVF-ET cycles with history of failed IVF-ET/ICSI cycles underwent endometrial scratching and sampling during 12–18 day of untreated cycles preceding to intervention. Each patient was monitored for pregnancy outcome using the routine protocol. Each sample was subjected to whole genome expression array experiment (n=20) followed by exploratory and differential display analysis. ResultsEndometrial whole genome transcriptomics of two groups (pregnancy positive, n=10; pregnancy negative, n=10) revealed that samples mutually clustered primarily based on pregnancy outcome. The secretory maturation of endometrium was seen to involve pathways related to oxidative phosphorylation, ubiquinone metabolism, cellular adhesion, skeletal and motor activities, and transcription and translation regulation. Additionally, genes related to neurogenesis and synaptogenesis, chemokines associated with cell adhesion, and developmental signalling were up-regulated in samples obtained from pregnancy-positive women. Further, patients with affected endometrial expression of genes involved in gynaecological neoplasm and transcriptional dysregulation in natural cycles did not conceive in IVF-ET. ConclusionTranscriptomic expressions in endometrium yielded information with predictive value of pregnancy outcome following endometrial scratching in women with history of repeated implantation failure upon IVF-ET.

Genetic Approaches to Study Plant Responses to Environmental Stresses: An Overview.

The assessment of gene expression levels is an important step toward elucidating gene functions temporally and spatially. Decades ago, typical studies were focusing on a few genes individually, whereas now researchers are able to examine whole genomes at once. The upgrade of throughput levels aided the introduction of systems biology approaches whereby cell functional networks can be scrutinized in their entireties to unravel potential functional interacting components. The birth of systems biology goes hand-in-hand with huge technological advancements and enables a fairly rapid detection of all transcripts in studied biological samples. Even so, earlier technologies that were restricted to probing single genes or a subset of genes still have their place in research laboratories. The objective here is to highlight key approaches used in gene expression analysis in plant responses to environmental stresses, or, more generally, any other condition of interest. Northern blots, RNase protection assays, and qPCR are described for their targeted detection of one or a few transcripts at a once. Differential display and serial analysis of gene expression represent non-targeted methods to evaluate expression changes of a significant number of gene transcripts. Finally, microarrays and RNA-seq (next-generation sequencing) contribute to the ultimate goal of identifying and quantifying all transcripts in a cell under conditions or stages of study. Recent examples of applications as well as principles, advantages, and drawbacks of each method are contrasted. We also suggest replacing the term “Next-Generation Sequencing (NGS)” with another less confusing synonym such as “RNA-seq”, “high throughput sequencing”, or “massively parallel sequencing” to avoid confusion with any future sequencing technologies.

Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display

Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant–pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew.

An analysis of transcripts and enzyme profiles in drought stressed jute (Corchorus capsularis) and rice (Oryza sativa) seedlings treated with CaCl2, hydroxyapatite nano-particle and β-amino butyric acid

In the following work we have identified and isolated transcripts induced upon treatment of jute (JRC 412) seeds with as elicitors through the differential display analysis. We have tried an approach of pre-treatment of jute seeds with CaCl2 (calcium chloride), CaNP (hydroxyapatite nano-particle) and BABA (β-amino butyric acid) as elicitors to study the changes in both biochemical parameters and transcriptomic changes. Rice was chosen as the biological replicate model for the enzymatic changes involved. Several genes including the Late Embryogenesis Abundant protein, Dehydration Responsive Element along with the biochemical markers like proline and peroxidase associated with the priming and Systemic Acquired Resistance of the jute seedlings were found to be up-regulated under the effect of the elicitors. The significance of the isolated differentially expressed genes has been discussed in the light of these findings and there was distinct similarity in the enzyme profile that suggested that biosynthesis and control of proline levels in stressed plants was central to the survival strategy.

Quantitative urinary proteomics using stable isotope labelling by peptide dimethylation in patients with prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed malignancy in men. The current prevalent diagnosis method, prostate-specific antigen (PSA) screening test, has low sensitivity, specificity and is poor at predicting the grade of disease. Thus, new biomarkers are urgently needed to improve the PCa diagnosis and staging for the management of patients. The aim of this study is to investigate the first voided urinary sample after massage for biomarker discovery for PCa. In this work, untargeted metabolomic profiling of the first voided urinary sample after massage from 28 confirmed prostate cancer patients, 20 benign enlarged prostate patients and 6 healthy volunteers was performed using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Single and multiple peptide protein and cross-linking molecules were identified using PEAKS software. Analytical and diagnostic performance was tested using the Student's t test, Benjamini Hochberg correction and the receiver operating characteristic (ROC) curves. Using differential display analysis to compare peptides and cross-linking molecules of urinary samples between patients with benign, enlarged prostate and malignant cancer, we identified multiple peptides derived from osteopontin (SPP1) and prothrombin (F2) that are lower in PCa patients than in benign and enlarged prostate. The diagnosis accuracies of SPP1 and F2 peptides are 0.65-0.77 and 0.68-0.72, respectively. In addition to this, there are significant differences between PCa and benign/enlarged prostate patients in pyridinoline (PYD) and deoxypyridinoline (DPD) (p value = 0.001). Differences also, as shown in the excretion of these molecules for different stages of PCa (p value = 0.04) as the level of DPD and DPD/PYD ratio, were high in patients with locally advanced tumours. The study underscores the importance of proteomics analysis, and our results demonstrate that a urinary-based in depth proteomic approach allows thepotential identification of dysregulated pathways and diagnostic biomarkers.

Integrin-associated protein promotes neuronal differentiation of neural stem/progenitor cells.

Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. During the development of the mammalian nervous system, NSPCs generate neurons and glia sequentially. However, little is known about the mechanism that determines the timing of switch from neurogenesis to gliogenesis. In this study, we established a culture system in which the neurogenic potential of NSPCs is decreased in a time-dependent manner, so that short-term-cultured NSPCs differentiate into more neurons compared with long-term-cultured NSPCs. We found that short-term-cultured NSPCs express high levels of integrin-associated protein form 2 (IAP2; so-called CD47) mRNA using differential display analysis. Moreover, IAP2 overexpression in NSPCs induced neuronal differentiation of NSPCs. These findings reveal a novel mechanism by which IAP2 induces neuronal differentiation of NSPCs.

Proteomic Differential Display Analysis Reveals Decreased Expression of PEA-15 and Vimentin in FABP7-Deficient Astrocytes

Fatty acid binding proteins (FABPs) are intracellular lipid chaperones which mediate the uptake, transportation and signaling roles of fatty acids. Former studies suggest that FABP7 regulates proliferation and differentiation of radial glia and astrocytes and is associated with various central nervous system diseases including glioma and neuropsychiatric diseases. However, the underlying mechanism is poorly understood. To further explore the mechanistic roles of FABP7 on astrocyte function, the proteome of astrocytes cultured from Fabp7 KO mice was compared with wild-type counterparts by two-dimensional gel electrophoresis (2-DE). We found that 16 protein spots showed differential expression intensity on 2-DE gels. Among them, seven spots appeared elevated, and nine spots appeared decreased in Fabp7 KO astrocytes. Selected spots were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis and their protein identity was subsequently revealed using protein databases. Four spots including phosphoprotein enriched in astrocytes 15 (PEA-15), GFAP, vimentin and FABP7 were selected for further confirmation by Western blotting. Consistently, there was a significant decrease in expression of PEA-15 and vimentin, both of which play significant roles in proliferation, differentiation, maturation, and survival of astrocytes. These results suggest the molecular mechanism how FABP7 controls astrocyte function and provide new insight to explain the role of FABP7 in glioma and reactive gliosis.

Activation of the receptor for advanced glycation end products induces nuclear inhibitor of protein phosphatase-1 suppression

The activation of the receptor for advanced glycation end products (RAGE) is involved in the development of diabetic nephropathy. Analysis of protein phosphatase-1 indicated that advanced glycation end products did not affect its expression, but increased its phosphatase activity. Using differential display analysis we previously demonstrated that stimulation of RAGE in podocytes modulates the expression of numerous genes, among others nuclear inhibitor of protein phosphatase-1 (NIPP1). Here we found that silencing of NIPP1 induced podocyte hypertrophy, cell cycle arrest, and significantly increased protein phosphatase-1 activity. NIPP1 downregulation was associated with increased p27(Kip1) protein expression. Reporter assays revealed a transcriptional activation of nuclear factor-κB in podocytes after suppression of NIPP1. The protein level of NIPP1 was also significantly reduced in podocytes of diabetic mice. Blocking the RAGE in vivo by a soluble analog elevated the NIPP1 protein in podocytes of diabetic mice. Thus, activation of the RAGE by advanced glycation end products or other ligands suppresses NIPP1 expression in diabetic nephropathy, contributes to podocyte hypertrophy, and glomerular inflammation.

SerpinA3g participates in the antiadipogenesis and insulin-resistance induced by tumor necrosis factor-α in 3T3-F442A cells

Tumor necrosis factor alpha (TNF-α) is a proven modulator of adipose metabolism, but the mechanisms by which this cytokine affects the development and function of adipose tissue have not been fully elucidated to date. Using differential display analysis, in this study, we demonstrate that gene expression of the serine protease inhibitor A3g (SerpinA3g) is specifically induced in 3T3-F442A preadipocytes by TNF-α but not by other adipogenic inhibitors, such as retinoic acid (RA) or transforming growth factor type beta (TGF-β). The specific induction of SerpinA3g by TNF-α was confirmed by RT-PCR in both preadipose and terminally differentiated 3T3-F442A cells. The knockdown of SerpinA3g using small interfering RNA prevented the antiadipogenesis elicited by TNF-α in 3T3-F442A cells but not the antiadipogenesis induced by RA or TGF-β. SerpinA3g-silenced 3T3-F442A cells also did not display TNF-α-induced insulin resistance. Our results demonstrate that SerpinA3g is specifically induced by TNF-α in 3T3-F442A cells, regardless of their stage of differentiation, and participates in the antiadipogenesis and insulin resistance induced by this cytokine. Our results suggest that SerpinA3g plays a role in the TNF-α modulation of adipose tissue development and metabolism. Additional studies are warranted regarding the mechanisms mediating adipose SerpinA3g effects.