Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K _d) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.     

Exosomes and their importance in metastasis,diagnosis, and therapy of colorectal cancer

Extracellular vesicles are known as actual intermediaries of intercellular communications, such as biological signals and cargo transfer between different cells. A variety of cells release the exosomes as nanovesicular bodies. Exosomes contain different compounds such as several types of nucleic acids and proteins. In this study, we focused on exosomes in colorectal cancer as good tools that can be involved in various cancer-related processes. Furthermore, we summarize the advantages and disadvantages of exosome extraction methods and review related studies on the role of exosomes in colorectal cancer. Finally, we focus on reports available on relations between mesenchymal stem cell–derived exosomes and colorectal cancer. Several cancer-related processes such as cancer progression, metastasis, and drug resistance of colorectal cancer are related to the cargoes of exosomes. A variety of molecules, especially proteins, microRNAs, and long noncoding RNAs, play important roles in these processes. The microenvironment features, such as hypoxia, also have very important effects on the properties of the origin cell–derived exosomes. On the other hand, exosomes derived from colorectal cancer cells also interfere with cancer chemoresistance. Furthermore, today it is known that exosomes and their contents can likely be very effective in noninvasive colorectal cancer diagnosis and therapy. Thus, exosomes, and especially their cargoes, play different key roles in various aspects of basic and clinical research related to both progression and therapy of colorectal cancer.     

Molecular study of two distinct phytoplasma species associated with streak yellows of date palm in Iran

A disease with symptoms similar to palm lethal yellowing was noticed in the early 2013 in Khuzestan Province (Iran) in date palm (Phoenix dactylifera). Infected trees displaying symptoms of streak yellows and varied in the incidence and severity of yellowing. A study was initiated to determine whether phytoplasma was the causal agent. Polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods using universal phytoplasma primers pairs R16mF1/mR1 and M1/M2 were employed to detect putative phytoplasma(s) associated with date palm trees. Nested PCR using universal primers revealed that 40 out of 53 trees were positive for phytoplasma while asymptomatic date palms from another location (controls) tested negative. RFLP analyses and DNA sequencing of 16S rDNA indicated that the presence of two different phytoplasmas most closely related to clover proliferation (CP) phytoplasma (group 16SrVI) and ash yellows (AY) phytoplasma (group 16SrVII). Sequence analysis confirmed that palm streak yellows phytoplasmas in each group were uniform and to be phylogenetically closest to “CandidatusP. fraxini” (MF374755) and “Ca. P. trifolii” isolate Rus‐CP361Fc1 (KX773529). Result of RFLP analysis of secA gene of positive samples using TruI and TaqI endonuclease is in agreement with rDNA analysis. On this basis, both strains were classified as members of subgroups 16SrVI‐A and 16SrVII‐A. This is the first report of a phytoplasma related to CP and AY phytoplasma causing date palm yellows disease symptoms.     

Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4(+) T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast, equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.     

Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis

The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-beta) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN-beta promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.     

Modulation of hematology changes by polymorphism of glutathione S-transferase M1 and T1

Workers in the petroleum distribution trades experience relatively low-level exposures to gasoline vapors whose consequences have not been fully elucidated. The purpose of this study was to investigate changes in the hematological parameters among filling station workers who were occupationally exposed to gasoline. The target group for the study consisted of 41 workers from eight filling stations of Shiraz (south of Iran). The control group consisted of 27 healthy subjects matched for age and sex from general population. The complete blood count analysis was done in one laboratory. Using PCR-based method, the genotypes of glutathione S-transferase T1 (GSTT1) and M1 (GSTM1) were determined. Workers were divided into three exposure groups according to employment history: duration less than 1 year, 1-5 years, and more than 5 years. Comparison was performed using Kruskal-Wallis test. In the individuals with the presence of both GSTT1 and GSTM1 functional alleles, comparison between four exposure groups revealed no significant difference for studied hematological variables. There were statistically significant differences between study groups, with only one functional allele, either GSTT1 or GSTM1, for relative number of lymphocytes (chi(2)=9.147, df=3, P=0.027) and neutrophils (chi(2)=9.951, df=3, and P=0.019), and absolute number of lymphocytes (chi(2)=9.135, df=3, and P=0.028), and RBC (chi(2)=10.586, df=3, and P=0.014). These findings could indicate the possible protective effect of concurrent presence of GSTM1 and GSTT1 enzymes on the hematopoietic system of filling station workers.     

Screening for recombinant glutathione transferases active with monochlorobimane

A rapid and facile colony assay has been developed for catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferases (GST), expressed in Escherichia coli. The basis of the method is the conjugation of glutathione (GSH) with the fluorogenic substrate monochlorobimane (MCB). This screening method makes it possible to isolate and characterize one recombinant clone that is active with MCB among thousands of inactive variants. Colonies containing GSTs that catalyze the conjugation of GSH with MCB display fluorescence under long-wavelength UV light. The fluorescence is visible instantly. One rat and 11 human GSTs representing four distinct enzyme classes were studied, and all except human GST T1-1 gave rise to fluorescent colonies. The colony assay based on MCB can consequently be broadly applied for identifying active GSTs both after subcloning of wild-type enzymes and in the screening of mutant libraries. Populations of bacteria expressing GSTs can also be analyzed by flow cytometry.     

Theoretical studies of biliverdin: energetics of the reduction pathways to bilirubin

Geometries and energies of formation of bilirubin formed by reduction of biliverdin via three meso carbon sites, the , and positions, have been calculated using semiempirical methods. It has been shown that -bilirubin with a ridge-tile conformation forms six intramolecular hydrogen bonds and is the most stable of the three above mentioned positions by at least 22 kcal mol^–1. Reduction pathways for -, - and -bilirubin formations from biliverdin are studied in detail. The roles of loss of conjugation and hydrogen bond formations in stability of different conformers have been discussed. -Bilirubin was fully optimized by using ab initio methods. Fine refinements of calculated results show excellent agreement with experimental results. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0078-9.Electronic Supplementary Material available.