miR-130b is a potent stimulator of hepatic very-low-density lipoprotein assembly and secretion via marked induction of microsomal triglyceride transfer protein.

miR-130b is a microRNA whose expression is particularly elevated within adipose tissue and in the circulation in diabetic states. Hepatic miR-130b expression has been linked to hepatocellular carcinoma and changes in lipid metabolism. Here, we investigated the role of miR-130b in hepatic lipid homeostasis and lipoprotein export. We observed that overexpression of miR-130b-3p or -5p in HepG2 cells markedly enhanced the secretion of very-low-density lipoprotein (VLDL) particles, enhanced the secretion of [3H]glycerol metabolically labeled triglyceride (TG), and significantly increased the number or the average size of lipid droplets (LDs), respectively. Overexpression of miR-130b also altered the expression of key genes involved in lipid metabolism and in particular markedly increased both mRNA and protein expression levels of microsomal triglyceride transfer protein (MTP). Conversely, the miR-130b inhibitor decreased mRNA levels of MTP and fatty acid synthase (FAS) in HepG2 cells. However, dual-luciferase reporter assays indicated that MTP is not a direct target of miR-130b-3p. miR-130b overexpression did not alter de novo synthesized TG or the stability and secretion of apolipoprotein B 100. Interestingly, knockdown of phosphatase and tensin homolog (PTEN) blocked the upregulation of MTP mRNA induced by miR-130b. Finally, miR-130b-induced stimulation of VLDL secretion was also observed in a second hepatocyte cell culture model, immortalized human hepatocytes, confirming the effects observed in HepG2 cells. Overall, these data suggest a potential role for miR-130b in promoting hepatic VLDL assembly and secretion mediated by marked stimulation of MTP expression and TG mobilization. Thus miR-130b overexpression corrects the defect in VLDL production in HepG2 cells.

The Effect of pH on Globular State of Lipase-3646; an Appropriate Model for Molten Globule Investigations.

Secondary structure content of proteins in molten globule state is relatively constant while the quantity of tertiary structures clearly declines due to alterations in side-chain packing. In the present study, we analyze the MG state of lipase-3646 for the first time. We introduce lipase-3646 as an appropriate model for investigating the properties and behavior of a protein in MG state as well as folding pathway. Applying fluorescence spectroscopy we measured both intrinsic and extrinsic fluorescence of lipase-3646 in a pH range from 1.0 to 12.0. It was found that at pH 3.0 the protein acquires a MG state. Applying far-UV circular dichroism (CD), our analysis on the secondary structure of lipase-3646 revealed a slight change in the MG state intermediate (pH 3.0) compared to the native state (pH 8.5), which this amount of change is common for MG. Measurements in near-UV CD also showed a significant change in the enzyme conformation at pH 3.0 in comparison with the pH 8.5 wherein the protein acquires its native structure. Quenching the fluorescence by applying acrylamide, the amount 23 and 35 M(-1) were measured at pHs 8.5 and 3.0 respectively for stern-volmer constant (KSV). An increase in the enzyme molecular volume in the MG state was confirmed by gel filtration chromatography.

Primary blast-induced traumatic brain injury in rats leads to increased prion protein in plasma: a potential biomarker for blast-induced traumatic brain injury.

Traumatic brain injury (TBI) is deemed the "signature injury" of recent military conflicts in Afghanistan and Iraq, largely because of increased blast exposure. Injuries to the brain can often be misdiagnosed, leading to further complications in the future. Therefore, the use of protein biomarkers for the screening and diagnosis of TBI is urgently needed. In the present study, we have investigated the plasma levels of soluble cellular prion protein (PrPC) as a novel biomarker for the diagnosis of primary blast-induced TBI (bTBI). We hypothesize that the primary blast wave can disrupt the brain and dislodge extracellular localized PrPC, leading to a rise in concentration within the systemic circulation. Adult male Sprague-Dawley rats were exposed to single pulse shockwave overpressures of varying intensities (15-30 psi or 103.4-206.8 kPa] using an advanced blast simulator. Blood plasma was collected 24 h after insult, and PrPC concentration was determined with a modified commercial enzyme-linked immunosorbent assay (ELISA) specific for PrPC. We provide the first report that mean PrPC concentration in primary blast exposed rats (3.97 ng/mL ± 0.13 SE) is significantly increased compared with controls (2.46 ng/mL ± 0.14 SE; two tailed test p < 0.0001). Furthermore, we report a mild positive rank correlation between PrPC concentration and increasing blast intensity (psi) reflecting a plateaued response at higher pressure magnitudes, which may have implications for all military service members exposed to blast events. In conclusion, it appears that plasma levels of PrPC may be a novel biomarker for the detection of primary bTBI.

Downregulation of tropomyosin-1 in squamous cell carcinoma of esophagus, the role of Ras signaling and methylation.

Tropomyosins (TMs) are a family of cytoskeletal proteins that bind to and stabilize actin microfilaments. Non-muscle cells express multiple isoforms of TMs including three high molecular weight (HMW) isoforms: TM1, TM2, and TM3. While reports have indicated downregulation of TMs in transformed cells and several human cancers, nevertheless, little is known about the underlying mechanism of TMs suppression. In present study the expression of HMW TMs was investigated in squamous cell carcinoma of esophagus (SCCE), relative to primary cell cultures of normal esophagus by western blotting and real-time RT-PCR. Our results showed that TM1, TM2, and TM3 were significantly downregulated in cell line of SCCE. Moreover, mRNA level of TPM1 and TPM2 were markedly decreased by 93% and 96%, in tumor cell line relative to esophagus normal epithelial cells. Therefore, downregulation of TMs could play an important role in tumorigenesis of esophageal cancer. To asses the mechanism of TM downregulation in esophageal cancer, the role of Ras dependent signaling and promoter hypermethylation were investigated. We found that inhibition of two Ras effectory downstream pathways; MEK/ERK and PI3K/Akt leads to significant increased expression of TM1 protein and both TPM1 and TPM2 mRNAs. In addition, methyltransferase inhibition significantly upregulated TM1, suggesting the prominent contribution of promoter hypermethylation in TM1 downregulation in esophageal cancer. These data indicate that downregulation of HMW TMs occurs basically in SCCE and the activation of MEK/ERK and PI3K/Akt pathways as well as the epigenetic mechanism of promoter hypermethylation play important role in TM1 suppression in SCCE.

Proteomics of a new esophageal cancer cell line established from Persian patient.

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study.

Protein-x of hepatitis B virus in interaction with CCAAT/enhancer-binding protein α (C/EBPα)--an in silico analysis approach.

BACKGROUND: Even though many functions of protein-x from the Hepatitis B virus (HBV) have been revealed, the nature of protein-x is yet unknown. This protein is well-known for its transactivation activity through interaction with several cellular transcription factors, it is also known as an oncogene. In this work, we have presented computational approaches to design a model to show the structure of protein-x and its respective binding sites associated with the CCAAT/enhancer-binding protein α (C/EBPα). C/EBPα belongs to the bZip family of transcription factors, which activates transcription of several genes through its binding sites in liver and fat cells. The C/EBPα has been shown to bind and modulate enhancer I and the enhancer II/core promoter of HBV. In this study using the bioinformatics tools we tried to present a reliable model for the protein-x interaction with C/EBPα. RESULTS: The amino acid sequence of protein-x was extracted from UniProt [UniProt:Q80IU5] and the x-ray crystal structure of the partial CCAAT-enhancer α [PDB:1NWQ] was retrieved from the Protein Data Bank (PDB). Similarity search for protein-x was carried out by psi-blast and bl2seq using NCBI [GenBank: BAC65106.1] and Local Meta-Threading-Server (LOMETS) was used as a threading server for determining the maximum tertiary structure similarities. Advanced MODELLER was implemented to design a comparative model, however, due to the lack of a suitable template, Quark was used for ab initio tertiary structure prediction.The PDB-blast search indicated a maximum of 23% sequence identity and 33% similarity with crystal structure of the porcine reproductive and respiratory syndrome virus leader protease Nsp1α [PDB:3IFU]. This meant that protein-x does not have a suitable template to predict its tertiary structure using comparative modeling tools, therefore we used QUARK as an ab initio 3D prediction approach. Docking results from the ab initio tertiary structure of protein-x and crystal structure of the C/EBPα- DNA region [PDB:1NWQ] illustrated the protein-binding site interactions. Indeed, the N-terminal part of 1NWQ has a high affinity for certain regions in protein-x (e.g. from Ala76 to Ser101 and Thr105 to Glu125). CONCLUSION: In this study, we predicted the structure of protein-x of HBV in interaction with C/EBPα. The docking results showed that protein-x has an interaction synergy with C/EBPα. However, despite previous experimental data, protein-x was found to interact with DNA. This can lead to a better understanding of the function of protein-x and may provide an opportunity to use it as a therapeutic target.

Plasma myeloperoxidase activity and apolipoprotein A-1 expression in chronic hepatitis B patients.

BACKGROUND: Hepatitis B virus initiates a complicated cascade process leading to chronic hepatitis B, cirrhosis, and hepatocellular carcinoma. In inflammatory situations, myeloperoxidase is released in plasma and binds to apolipoprotein A-1 in high-density lipoproteins. This study aims to evaluate the level of plasma myeloperoxidase as well as the pattern of plasma proteins in patients with chronic hepatitis B. METHODS: Included in this study were 30 male subjects: 19 chronic hepatitis B patients, 6 HBV-related cirrhotic patients, and 5 healthy controls. Plasma myeloperoxidase was measured using enzyme-linked immunosorbent assay. Proteomic analysis of plasma proteins was performed by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. One way ANOVA was used for data analysis. RESULTS: Mean plasma myeloperoxidase levels were higher in patients with liver cirrhosis (65.5±12.5; P=0.007) and chronic hepatitis B (53.7±10.6; P=0.18) when compared with healthy subjects (45±7.6). Moreover, a positive correlation was found between plasma myeloperoxidase levels and hepatic fibrosis stage (r=0.53, P=0.002; r=0.63, P=0.000). Proteomic analysis showed an altered plasma protein pattern in progressive hepatitis B and down-regulation of the major apolipoprotein A-1 along with the appearance of a variety of spots noted to be apolipoprotein A-1isoforms with different molecular masses. CONCLUSION: In this study, progressive liver injury due to HBV infection correlated with higher plasma myeloperoxidase and an altered plasma apolipoprotein A-1pattern.

Proteomic analysis of a spring wheat cultivar in response to prolonged cold stress.

Cold represents one of the major abiotic factors influencing plant growth and development worldwide. We analysed the long-term responsiveness of an Iranian spring wheat (cv. Kohdasht) to cold from a proteomic point of view, in order to unravel the molecular mechanisms helping a cold-sensitive cultivar to survive exposure to suboptimal temperatures. Plants were grown at 20 or 4°C until entering the reproductive stage and a cross-comparison on the leaf proteomes was performed. Quantitative analyses on protein alterations occurring upon low-temperature exposure showed a reinforcement in ascorbate recycling (dehydroascorbate reductase, ascorbate peroxidase) and protein processing (proteasome subunit, cysteine proteinase), as well as the accumulation of the enzyme devoted to tetrapyrrole resynthesis (glutamate semialdehyde aminomutase). In contrast, among proteins down-regulated after cold stress, we could identify some key Krebs cycle enzymes (isocitrate dehydrogenase, malate dehydrogenase), together with many photosynthesis-related proteins (oxygen-evolving complex proteins, ATP synthase subunits, ferredoxin NADPH oxidoreductase and some Calvin cycle enzymes). Physiological and biochemical parameters (such as shoot apex dissection, chlorophyll, proline and sugar content determination) sustained proteomics findings allowing the present research to contribute to the current knowledge on these long-term responses, which may be crucial to stress adaptation under field conditions.

Salt stress induced differential proteome and metabolome response in the shoots of Aeluropus lagopoides (Poaceae), a halophyte C(4) plant.

A proteomic approach was used to identify proteins affected by salt in the halophyte C(4) plant Aeluropus lagopoides (Poaceae) in an attempt to understand the mechanism of salt tolerance. Plants were treated with 450 mM NaCl for 10 days, and proteins were then extracted from the shoots and separated by two-dimensional polyacrylamide gel electrophoresis. A total of 1805 protein spots were detected, of which 39 were up-regulated and 44 were down-regulated by treatment with NaCl. Metabolism-related proteins were up-regulated, whereas photosynthesis-related proteins were down-regulated. Dose-dependence studies showed that the up-regulation continued at NaCl concentrations above 450 mM for defense-related proteins alone. Western blot analysis confirmed the down-regulation of RuBisCO LSU and RuBisCO SSU and severe down-regulation of RuBisCO activase. The activity of glyoxalase I increased with increasing NaCl concentration. Metabolome studies indicated up-regulation of amino acids and down-regulation of tricarboxylic acid cycle-related metabolites. These studies suggest that up-regulation of energy formation, amino acid biosynthesis, C(4) photosynthesis, and detoxification are the main strategies for salt tolerance in A. lagopoides.

Proteome analysis of soybean leaves, hypocotyls and roots under salt stress.

BACKGROUND: Salinity is one of the most widespread agricultural problems in arid and semi-arid regions that makes fields unproductive, and soil salinization is a serious problem in the entire world. To determine the effects of salt stress on soybean seedlings, a proteomic technique was used. RESULTS: Soybean plants were exposed to 0, 20, 40, or 80 mM NaCl for one week. The effect of treatment at 20 mM NaCl on plant growth was not severe, at 80 mM NaCl was lethal, and at 40 mM NaCl was significant but not lethal. Based on these results, proteins were extracted from the leaves, hypocotyls and roots of soybean treated with 40 mM NaCl. Nineteen, 22 and 14 proteins out of 340, 330 and 235 proteins in the leaves, hypocotyls and roots, respectively, were up- and down-regulated by NaCl treatment. In leaves, hypocotyls and roots, metabolism related proteins were mainly down-regulated with NaCl treatment. Glyceraldehyde-3-phosphate dehydrogenase was down-regulated in the leaf/hypocotyls, and fructokinase 2 was down-regulated in the hypocotyls/root with NaCl treatment. Stem 31 kDa glycoprotein precursor was up-regulated in all three organs with NaCl treatment. Glyceraldehyde-3-phosphate dehydrogenase was specifically down-regulated at the RNA and protein levels by salt stress. CONCLUSION: These results suggest that metabolism related proteins play a role in each organ in the adaptation to saline conditions.

The role of mutations in core protein of hepatitis B virus in liver fibrosis.

The core protein of hepatitis B virus encompasses B- and T-cell immunodominant epitopes and subdivided into two domains: the N-terminal and the functional C-terminal consisted phosphorylation sites. Mutations of the core gene may change the conformation of the core protein or cause alteration of important epitopes in the host immune response. In this study twenty nine men (mean age 40 +/- 9 years old) with chronic hepatitis B were recruited for direct sequencing of the core gene. Serum ALT and HBV DNA level were measured at the time of liver biopsy. The effects of core protein mutations on patients' characteristics and subsequently mutations in B cell, T helper and cytotoxic T lymphocyte (CTL) epitopes and also C-terminal domain of core protein on the activity of liver disease was evaluated. Liver fibrosis was significantly increased in patients with core protein mutation (1.0 +/- 0.8 vs 1.9 +/- 1.4 for mean stage of fibrosis P = 0.05). Mutations in CTL epitopes and in phosphorylation sites of C-terminal domain of core protein also were associated with higher liver fibrosis (P = 0.003 and P = 0.04; Fisher's exact test for both). Patients with mutation in C-terminal domain had higher serum ALT (62 +/- 17 vs 36 +/- 12 IU/l, p = 0.02). Patients with mutations in B cell and T helper epitopes did not show significant difference in the clinical features. Our data suggests that core protein mutations in CTL epitopes and C-terminal domain accompanied with higher stage of liver fibrosis may be due to alterations in the function of core protein.

Qualitative analysis of Adenomatous Polyposis Coli promoter: hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker.

BACKGROUND: Squamous cell carcinoma of esophagus (SCCE) occurs at a high incidence rate in certain parts of the world. This feature necessitates that different aspects of the disease and in particular genetic characteristics be investigated in such regions. In addition, such investigations might lead to achievement of molecular markers helpful for early detection, successful treatment and follow up of the disease. Adenomatous Polyposis Coli (APC) promoter hypermethylation has been shown to be a suitable marker for both serum and solid tumors of adenocarcinoma of esophagus. We investigated the status of APC promoter hypermethylation in Iranian patients, compared the results with the former studies, and evaluated its applicability as a candidate molecular marker by examining association between survival of SCCE patients and APC promoter methylation. METHODS: For evaluating the status of APC promoter hypermethylation and its association with SCCE, a qualitative methylation specific PCR (MSP) was used. DNA was extracted and digested with an appropriate restriction enzyme, treated with sodium bisulfite in agarose beads and amplified in two-step PCR reaction by applying either methylated or unmethylated promoter specific primers. Universally methylated DNA and methylase treated blood DNA of healthy donors were used as positive controls as well. Survival of patients was followed up for two years after treatment and survival rate of patients with methylated APC promoter was compared with that of unmethylated patients. RESULTS: Assessment of APC promoter methylation revealed that normal tissues were unmethylated, while twenty out of forty five (44.4%) tumor tissues were hypermethylated either in one or both alleles of APC. Among the tissues in which methylation was detected, seven were hypermethylated in both alleles while the other thirteen were hypermethylated in one of the two alleles of APC. Analyzing two-year survival rate of patients with respect to promoter hypermethylation showed a lower rate of survival for patients with methylated APC promoter following their treatment. Further investigation into the association between promoter hypermethylation and tumor differentiation status indicated that patients with well differentiated tumors were more likely to develop promoter hypermethylation. CONCLUSION: Observing similar level of APC promoter hypermethylation in patients with SCCE in this high risk region and comparing it with other parts of the world could support the hypothesis that a common molecular mechanism might be involved in tumorigenesis of SCCE. In addition, the higher rate of two-year survival for patients with unmethylated APC promoter as well as its relationship with tumor differentiation would suggest that this tumor suppressor could be an appropriate candidate molecular marker for evaluating tumor malignancy and predicting survival of patients subsequent to treatment.

Identification of squamous cell carcinoma associated proteins by proteomics and loss of beta tropomyosin expression in esophageal cancer.

AIM: To assess the proteome of normal versus tumor tissue in squamous cell carcinoma of the esophagus (SCCE) in Iranian patients and compare our results with former reports by using proteomics. METHODS: Protein was extracted from normal and tumor tissues. Two dimensional electrophoresis was carried out and spots with differential expression were identified with mass spectrometry. RNA extraction and RT-PCR along with immunodetection were performed. RESULTS: Fourteen proteins were found whose expression levels differed in tumor compared to normal tissues. Mass spectrometric analysis resulted in the identification of beta-tropomyosin (TMbeta), myosin light chain 2 (and its isoform), myosin regulatory light chain 2, peroxyredoxin 2, annexin I and an unknown polypeptide as the down regulated polypeptides in tumor tissue. Heat shock protein 70 (HSP70), TPM4-ALK fusion oncoprotein 2, myosin light polypeptide 6, keratin I, GH16431p and calreticulin were the up-regulated polypeptides found in tumor tissue. Several of these proteins, such as TMbeta, HSP70, annexin I, calreticulin, TPM4-ALK and isoforms of myosins, have been well recognized in tumorigenesis of esophageal or other types of cancers. CONCLUSION: Our study not only supports the involvement of some of the formerly reported proteins in SCCE but also introduces additional proteins found to be lost in SCCE, including TMbeta.

Identification and characterization of salt-inducible polypeptide in Paenibacillus sp., a moderately halophilic bacterium.

In response to salt, Paenibacillus sp. strain XII expresses a 21.4 kDa polypeptide. N-terminal sequencing and sequence homology analysis indicate homology between the N-terminal sequence of the polypeptide and a segment of the N-terminus of the spore coat associated protein CotN of Oceanobacillus iheyensis, an extremely halotolerant bacteria of the deep-sea.