Bioinformatics Predictions,Expression, Purification and Structural Analysis of the PE38KDEL-scfv Immunotoxin Against EPHA2 Receptor

International Journal of Peptide Research and Therapeutics - Various strategies have been proposed for treatment of cancer, including common therapies such as surgery, chemotherapy, radiation...     

Loss of DJ‐1 protein stability and cytoprotective function by Parkinson's disease‐associated proline‐158 deletion

DJ‐1 is a ubiquitous protein regulating cellular viability. Recessive mutations in the PARK7/DJ‐1 gene are linked to Parkinson's disease (PD). Although the most dramatic L166P point mutation practically eliminates DJ‐1 protein and function, the effects of other PD‐linked mutations are subtler. Here, we investigated two recently described PD‐associated DJ‐1 point mutations, the A179T substitution and the P158Δ in‐frame deletion. [A179T]DJ‐1 protein was as stable as wild‐type [wt]DJ‐1, but the P158Δ mutant protein was less stable. In accord with the notion that dimer formation is essential for DJ‐1 protein stability, [P158Δ]DJ‐1 was impaired in dimer formation. Similar to our previous findings for [M26I]DJ‐1, [P158Δ]DJ‐1 bound aberrantly to apoptosis signal‐regulating kinase 1. Thus, the PD‐associated P158Δ mutation destabilizes DJ‐1 protein and function. As there is also evidence for an involvement of DJ‐1 in multiple system atrophy, a PD‐related α‐synucleinopathy characterized by oligodendroglial cytoplasmic inclusions, we studied an oligodendroglial cell line stably expressing α‐synuclein. α‐Synuclein aggregate dependent microtubule retraction upon co‐transfection with tubulin polymerization‐promoting protein p25α was ameliorated by [wt]DJ‐1. In contrast, DJ‐1 mutants including P158Δ failed to protect in this system, where we found evidence of apoptosis signal‐regulating kinase 1 (ASK1) involvement. In conclusion, the P158Δ point mutation may contribute to neurodegeneration by protein destabilization and hence loss of DJ‐1 function.     

Evaluation of glycolamide esters of indomethacin as potential cyclooxygenase-2 (COX-2) inhibitors

A number of novel indomethacin glycolamide esters were synthesized and tested for their cyclooxygenase (COX-1 and COX-2) inhibition properties in vitro. Many of these compounds proved to be selective COX-2 inhibitors, and subtle structural changes in the substituents on the glycolamide ester moiety altered the inhibitory properties as well as potencies significantly. Their in vitro data were rationalized through molecular modeling studies. Few of them displayed anti-inflammatory activity in vivo. Compound 32, [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid 2-morpholin-4-yl-2-oxo ethyl ester, was identified as a promising compound in this class and its good anti-inflammatory activity was demonstrated in the in vivo model.     

An in silico chimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant

Enteric infections resulting in diarrheal diseases remain as major global health problems. Among bacteria, enterotoxigenic Escherichia coli (ETEC) causes the largest number of diarrheal cases. There is a great interest in developing an effective ETEC vaccine. An ETEC vaccine could focus on virulence factors present in ETEC pathogens and nontoxic Heat-labile B subunit (LTB). Chimeric proteins carrying epitopes, or adjuvant sequences increase the possibility of eliciting a broad cellular or humoral immune response. In-silico tools are highly suited to study, design and evaluate vaccine strategies. Colonization factors are among the virulence factor studied in the present work employing bioinformatic tools. A synthetic chimeric gene, encoding CfaB, CstH, CotA, and LTB was designed. Modeling was done to predict the 3D structure of protein. This model was validated using Ramachandran plot statistics. The predicted B-cell epitopes were mapped on the surface of the model. Validation result showed that 97.2% residues lie in favored or additional allowed region of Ramachandran plot. VaxiJen analysis of the protein showed high antigenicity. Linear and conformational B-cell epitopes were identified. The identified T-cell epitopes are apt to bind MHC molecules. The epitopes in the chimeric protein are likely to induce both the B-cell and T-cell mediated immune responses.     

Determination of deferiprone in urine and serum using a terbium‐sensitized luminescence method

Optimized conditions, validation and practical applications of a new, rapid and specific fluorometric method for the determination of deferiprone (DFP) in urine and serum samples are reported. The proposed method, which is based on the formation of a luminescent complex with Tb³⁺ ion, is evaluated in terms of linearity, accuracy, precision, stability, recovery and limits of detection (LOD) and quantification (LOQ). Under optimum conditions (pH 7.5, [Tb³⁺] = 3 × 10^–4 mol/L, temperature 0 °C and excitation wavelength 295 nm), the relative intensities at 545 nm are linear, with the concentration of DFP in the range 0.072–13 mmol/L for urine and serum samples. The LOD and LOQ, respectively, are calculated to be 0.014 and 0.045 mmol/L for urine and 0.022 and 0.072 mmol/L for serum samples. The intra‐day and inter‐day values for the precision and accuracy of the proposed method are all < 5%, and the recovery of the method is in the range 97.1–103.8%. The method was applied to human urine and serum samples collected from patients receiving DFP. The results indicated that the method can be successfully applied to the determination of DFP in human urine and serum samples collected for clinical or biopharmaceutical investigations in which simple, rapid, cheap and specific determination methods facilitate and speed up the analytical procedure. Copyright © 2011 John Wiley & Sons, Ltd.     

Human Serum Protein Adsorption onto Synthesis Nano-Hydroxyapatite

Adsorption of human serum proteins (Albumin and total protein) onto high purity synthesis nano-hydroxyapatite (HA), Ca₁₀(PO₄)₆(OH)₂, has been studied in a wide temperature range by UV–visible spectrophotometer. Adsorption isotherm is basically important to describe how solutes interact with adsorbent, and is critical in optimizing the use of adsorbent. In the present study, the experimental results were fitted to the Langmuir, Freundlich, Temkin and Dubinin-Radushkevich (DR) models to obtain the characteristic parameters of each model and square of the correlation coefficients (R²). According to the results, the DR isotherm model had the best agreement with the experimental data. The effect of temperature on adsorption of human serum proteins (HSP) onto the synthesized nano-HA was studied. The experimental results indicated that temperature increase generally causes an increase in the adsorption of HSP onto the nano-HA. This is basically due to the effect of temperature on the HSP activity and its diffusion rate on HA surfaces.     

Role of the 5' TAR stem--loop and the U5-AUG duplex in dimerization of HIV-1 genomic RNA

The HIV-1 genome consists of two identical RNAs that are linked together through noncovalent interactions involving nucleotides from the 5' untranslated region (5' UTR) of each RNA strand. The 5' UTR is the most conserved part of the HIV-1 RNA genome, and its 335 nucleotide residues form regulatory motifs that mediate multiple essential steps in the viral replication cycle. Here, studying the effect of selected mutations both singly and together with mutations disabling SL1 (SL1 is a 5' UTR stem-loop containing a palindrome called the dimerization initiation site), we have done a rather systematic survey of the 5' UTR requirements for full genomic RNA dimerization in grown-up (i.e., predominantly >/=10 h old) HIV-1 viruses produced by transfected human and simian cells. We have identified a role for the 5' transactivation response element (5' TAR) and a contribution of a long-distance base pairing between a sequence located at the beginning of the U5 region and nucleotides surrounding the AUG Gag initiation codon. The resulting intra- or intermolecular duplex is called the U5-AUG duplex. The other regions of the 5' UTR have been shown to play no systematic role in genomic RNA dimerization, except for a sequence located around the 3' end of a large stem-loop enclosing the primer binding site, and the well-documented SL1. Our data are consistent with a direct role for the 5' TAR in genomic RNA dimerization (possibly via a palindrome encompassing the apical loop of the 5' TAR).     

Patterns of co-association of C-reactive protein and nitric oxide in malaria in endemic areas of Iran

In addition to numerous immune factors, C-reactive protein (CRP) and nitric oxide (NO) are believed to be molecules of malaria immunopathology. The objective of this study was to detect CRP and NO inductions by agglutination latex test and Griess microassay respectively in both control and malaria groups from endemic areas of Iran, including Southeastern (SE) (Sistan & Balouchestan, Hormozgan, Kerman) and Northwestern (NW) provinces (Ardabil). The results indicated that CRP and NO are produced in all malaria endemic areas of Iran. In addition, more CRP and NO positive cases were observed amongst malaria patients in comparison with those in control group. A variable co-association of CRP/NO production were detected between control and malaria groups, which depended upon the malaria endemic areas and the type of plasmodia infection. The percentage of CRP/NO positive cases was observed to be lower in NW compare to SE region, which may be due to the different type of plasmodium in the NW (Plasmodium vivax) with SE area (P. vivax, Plasmodium falciparum, mixed infection). The fluctuations in CRP/NO induction may be consistent with genetic background of patients. Although, CRP/NO may play important role in malaria, their actual function and interaction in clinical forms of disease remains unclear.     

The effects of aerobic exercise training on the age-related lipid peroxidation, Schwann cell apoptosis and ultrastructural changes in the sciatic nerve of rats

The potential role of exercise in preventing the age-related spontaneous peripheral neuropathy has not been studied. We examined the effects of long-term aerobic exercise training on lipid peroxidation, Schwann cell (SC) apoptosis and ultrastructural changes in the sciatic nerve of rats during aging. Three groups of 12-week old Wistar rats ran on a treadmill for 6, 9 and 12 months (exercise trained (ET) group, n=10 each) according to an exercise training program targeted at a speed of 22 m/min (at 7 degrees incline), 60 min/day, 6 days/week. Three corresponding groups of untrained rats were used as the controls (sedentary (SED) group). At the end of each period, sciatic nerve biopsies were performed, and processed for biochemical, immunohistochemical and ultrastructural analyses. The results showed that aging was associated with an increased level of nerve malondialdehyde (MDA, marker of lipid peroxidation) and a higher number of SC apoptosis in SED group. The SED group showed irregular nerve fibers with thin myelin sheaths and areas of myelin-axon detachment. However, the ET group had significantly diminished nerve lipid peroxidation and SC apoptosis. In the ET group, nerve fibers had a thick myelin sheath with frequent folding. These findings suggest that aerobic exercise training protects peripheral nerves by attenuating oxidative reactions, and preserving SCs and myelin sheath from pathologic changes, which occur during normal aging.     

The MUC5AC glycoprotein is the primary receptor for Helicobacter pylori in the human stomach

Background and objectives. Helicobacter pylori shows a characteristic tropism for the mucus‐producing gastric epithelium. In infected patients, H. pylori colocalizes in situ with the gastric secretory mucin MUC5AC. The carbohydrate blood‐group antigen Lewis B (LeB) was deemed responsible for the adherence of H. pylori to the gastric surface epithelium. We sought to determine if MUC5AC is the carrier of LeB, and thus if MUC5AC is the underlying gene product functioning as the main receptor for H. pylori in the stomach. Methods. We studied three types of human tissue producing MUC5AC: Barrett's esophagus (BE), normal gastric tissue, and gastric metaplasia of the duodenum (GMD). Tissue sections were immuno‐fluorescently stained for MUC5AC or LeB, and subsequently incubated with one of three strains of Texas red‐labeled H. pylori, one of which was unable to bind to LeB. We determined the colocalization of MUC5AC or LeB with adherent H. pylori. Results. The binding patterns for the two LeB‐binding strains to all tissues were similar, whereas the strain unable to bind to LeB did not bind to any of the tissues. In normal gastric tissue, the LeB‐binding strains always bound to MUC5AC‐ and LeB‐positive epithelial cells. In four nonsecretor patients, colocalization of the LeB‐binding strains was found to MUC5AC‐positive gastric epithelial cells. In BE, the LeB‐binding H. pylori strains colocalized very specifically to MUC5AC‐positive cells. MUC5AC‐producing cells in GMD contained LeB. Yet, LeB‐binding H. pylori not only colocalized to MUC5AC or LeB present in GMD, but also bound to the LeB‐positive brush border of normal duodenal epithelium. Conclusions. Mucin MUC5AC is the most important carrier of the LeB carbohydrate structure in normal gastric tissue and forms the major receptor for H. pylori.