Differential protein expression profile in gastrointestinal stromal tumors

Summary. Gastrointestinal stromal tumors (GISTs) arise from the interstitial cells of Cajal through gain of function mutations of the oncogene KIT. Imatinib offers the first effective treatment for patients with GISTs, but the therapeutic outcome strongly depends on the type of KIT mutation. We used ProteinChip technology to investigate whether GISTs with different KIT mutations express different proteins. In total, 154 proteins were significantly differentially expressed in GISTs with exon 9 KIT mutation compared to GISTs with exon 11 KIT mutation.     

Differential proteomic analysis of proteins in wheat spikes induced by Fusarium graminearum

Scab, caused by Fusarium graminearum, is a serious spike disease in wheat. To identify proteins in resistant wheat cultivar Wangshuibai induced by F. graminearum infection, proteins extracted from spikes 6, 12 and 24 h after inoculation were separated by 2-DE. Thirty protein spots showing 3-fold change in abundance when compared with treatment without inoculation were characterized by MALDI-TOF MS and matched to proteins by querying the mass spectra in protein databases or the Triticeae EST translation database. Based on their volume profiles, these proteins were classified into four categories. The first one fell off rapidly at the initial inoculation and then rose at 12 or 24 hai, the second one decreased considerably after inoculation and remained at low level, the third one rose at the initial inoculation and then declined at 12 or 24 hai, the forth one showed steady increase after inoculation and maintained at a high level. Many of the proteins identified in the first two categories are related to carbon metabolism and photosynthesis. While most of proteins identified in the last two categories are related to stress defense of plants, indicating that proteins associated with the defense reactions were activated or translated shortly after inoculation.     

Proteomics analysis of mature seed of four peanut cultivars using two-dimensional gel electrophoresis reveals distinct differential expression of storage, anti-nutritional, and allergenic proteins

Profiles of total seed proteins isolated from mature seeds of four peanut cultivars, New Mexico Valencia C (NM Valencia C), Tamspan 90, Georgia Green, and NC-7, were studied using two-dimensional gel electrophoresis coupled with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC–MS/MS). Two-dimensional gels stained with silver nitrate revealed a total of 457, 516, 556, and 530 protein spots in NM Valencia C, Tamspan 90, Georgia Green, and NC-7, respectively. Twenty abundant protein spots showing differences in relative abundance among these cultivars were analyzed by nESI-LC–MS/MS, resulting in identification of 14 non-redundant proteins. The majority of these proteins belonged to the globulin fraction consisting of arachin (glycinin and Arah3/4) and conarachin seed storage proteins as well as other allergen proteins. The expression of some of these identified protein spots was cultivar-specific. For example, allergen Arah3/Arah4 and conarachin protein spots were only detected in Tamspan 90 and NC-7, whereas the Gly1 protein spot was detected only in NM Valencia C and NC-7. Moreover, a galactose-binding lectin protein spot with anti-nutritive properties was only present in Tamspan 90. Other proteins showing differences in relative abundance among the four cultivars included 13-lipoxygenase, fructose-biphosphate aldolase, and glyceraldehyde 3-phosphate dehydrogenase. Together, these results suggest that identified proteins might serve as potential markers for cultivar differentiation and may be associated with underlying sensory and nutritional traits of peanut cultivars.     

Redox regulation of heat shock protein expression in aging and neurodegenerative disorders associated with oxidative stress: A nutritional approach

Summary. Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Alzheimers disease (AD) is a progressive disorder with cognitive and memory decline, speech loss, personality changes and synapse loss. Many approaches have been undertaken to understand AD, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to AD pathogenesis.Brains of AD patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress.Recently, the involvement of the heme oxygenase (HO) pathway in anti-degenerative mechanisms operating in AD has received considerable attention, as it has been demonstrated that the expression of HO is closely related to that of amyloid precursor protein (APP). HO induction occurs together with the induction of other HSPs during various physiopathological conditions. The vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, products of HO-catalyzed reaction, represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response.Increasing interest has been focused on identifying dietary compounds that can inhibit, retard or reverse the multi-stage pathophysiological events underlying AD pathology. Alzheimers disease, in fact, involves a chronic inflammatory response associated with both brain injury and -amyloid associated pathology. All of the above evidence suggests that stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms. Spice and herbs contain phenolic substances with potent antioxidative and chemopreventive properties, and it is generally assumed that the phenol moiety is responsible for the antioxidant activity. In particular, curcumin, a powerful antioxidant derived from the curry spice turmeric, has emerged as a strong inducer of the heat shock response. In light of this finding, curcumin supplementation has been recently considered as an alternative, nutritional approach to reduce oxidative damage and amyloid pathology associated with AD. Here we review the importance of the heme oxygenase pathway in brain stress tolerance and its significance as an antidegenerative mechanism potentially important in AD pathogenesis. These findings have offered new perspectives in medicine and pharmacology, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms such as the heat shock response, through nutritional antioxidants or pharmacological compounds, represents an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. Consistent with this notion, maintenance or recovery of the activity of vitagenes, such as the HO gene, conceivably may delay the aging process and decrease the occurrence of age-related neurodegenerative diseases.     

Differential expression of proteins induced by lead in the Dwarf Sunflower Helianthus annuus

The Dwarf Sunflower (Helianthus annuus) is a hyperaccumulator of toxic metals including cadmium (Cd), mercury (Hg), nickel (Ni), and lead (Pb). In order to identify stress response to Pb, plants were exposed to a mixture of 30 mg/l of three ions, Cd, Cr, and Ni, with and without Pb. Soluble proteins were resolved by two-dimensional gel electrophoresis. Four proteins were differentially expressed and very abundant in the leaf samples after plants were exposed to all these four metals. The first protein spot contained two proteins: chitinase and a chloroplast drought-induced stress protein CDSP-34. The second spot contained a thaumatin-like protein. Two proteins in spot 3 were identified as heat-shock cognate 70-1 and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Several peptides were identified in spot 4 but none could be matched to any sequence in the NCBI database.     

Proteomics for the identification of specifically oxidized proteins in brain: Technology and application to the study of neurodegenerative disorders

Summary. Proteomics offers the opportunity elucidate the complex protein interactions of cellular systems by studying the products of genes, i.e., proteins, and their structure, function and localization. The purpose of proteomics is to explain the information contained in the genome sequences in order to provide clues on cellular events, especially related to disease.Our proteomic approach has made possible the identification of specifically oxidized proteins in Alzheimers disease (AD) brain, providing for the first time evidence on how oxidative stress plays a crucial role in AD-related neurodegeneration. This represents an example of the use of proteomics to solve biological problems related to disease. The field, which is still in its infancy, represents a very promising way to elucidate mechanism of disease at a protein level. However, the techniques that support its development present several limitations and require introduction of new tools and innovation in order to achieve a fast, reliable and sensitive method to understand normal biological processes and their regulation as well as these cellular properties in disease.     

Integrated analysis of DNA methylation and mRNA expression profiling reveals candidate genes associated with cisplatin resistance in non-small cell lung cancer

DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2′-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.     

The reduction of Calpain-10 expression is associated with risk polymorphisms in obese children

Excessive weight gain and obesity are major public health concerns. Childhood obesity is growing at an alarming rate. Polymorphisms in the Calpain-10 gene and the reduced expression of this gene in muscle cells and adipocytes have been associated with an increased risk of type 2 diabetes mellitus in several populations. In the present study, we explored the contribution of Calpain-10 in the development of metabolic impairment in childhood. We evaluated the presence of risk polymorphisms in the CAPN10 gene (SNP-44, SNP-43, InDel-19 and SNP-63) and the associated changes in the Calpain-10 mRNA levels in a pediatric population. A total of 161 Mexican children between 4 and 18 years old were included in this study. This population was classified into three groups according to international growth references: healthy weight (HW), overweight (OW) and obese (OB). Association studies of the anthropometric data, clinical values, genotyping and expression assays showed a decrease in the Calpain-10 mRNA and protein expression in the OW and OB groups with respect to the HW group. This decrease in the Calpain-10 mRNA expression was more evident in individuals homozygous for SNP-44 (T/T) and InDel-19 (3/3), alone (p < 0.001 and p = 0.015, respectively) or in combination (p = 0.017). These polymorphisms were also associated with elevated BMI, weight percentiles, z-scores, waist circumferences, fasting glucose levels and beta cell functions in the OW and OB groups (p < 0.05). Moreover, our results indicate a statistically significant decrease in the expression of the 75-kDa Calpain-10 isoform in the OW+OB group. The presence of polymorphisms and alterations in the expression of the CAPN10 gene at early ages might result in metabolic impairment in adulthood and should be further investigated.     

Identification and expression of an autosomal paralogue of ribosomal protein S4, X-linked,in mice: Potential involvement of testis-specific ribosomal proteins in translation and spermatogenesis

Recent studies have revealed heterogeneity in the structure of eukaryotic cytoplasmic ribosomes, including a difference in protein composition. It has been proposed that this heterogeneity, or the specialized ribosome, contributes to tissue development and homeostasis through selective mRNA translation, although this remains largely unclear. Our previous proteomic survey of rodent ribosomes found the testis-specific ribosomal proteins L10-like and L39-like, which are paralogues of the X-linked ribosomal proteins L10 and L39, respectively. We have hypothesized that the rodent testis provides a good model for examining the possible functional importance of ribosome heterogeneity. In the present study, a new paralogue of X-linked ribosomal protein S4 has been identified in the mouse testis. The gene encoding this paralogue was autosomal, intronless and expressed predominantly in the testis. It appeared that this paralogue was included in polysomes as a component of the ribosome. Although these properties were similar to those of the ribosomal proteins L10-like and L39-like, this S4 paralogue and L10-like showed partially different expression patterns in spermatogenic cells. These findings are discussed in relation to the unique evolution of genes encoding a paralogue of ribosomal protein S4 in mammals and to the significance of testis-specific paralogues of ribosomal proteins in active ribosomes during spermatogenesis.     

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48 h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1 nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5 nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.