Additive effect of recombinant Mycobacterium tuberculosis ESAT-6 protein and ESAT-6/CFP-10 fusion protein in adhesion of macrophages through fibronectin receptors

Background/purposeTuberculous granulomas are the sites of interaction between the T cells, macrophages, and extracellular matrix (ECM) to control the infection caused by Mycobacterium tuberculosis (M. tuberculosis). A predominant role of RD-1-encoded secretory proteins, early secreted antigenic target-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in the formation of granulomas has recently been emphasized. However, the precise molecular events that induce the formation of these granulomatous structures are yet to be elucidated. Macrophages use integrins to adhere to fibronectin (FN) as a major component of the ECM. The major goal of this study was to investigate whether recombinant M. tuberculosis antigens can modulate integrin-mediated macrophage adhesion.MethodsDifferentiated THP-1 cell line was stimulated with recombinant ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins and evaluated for alterations in the expression levels of α5β1 and α4β1 by semiquantitative real-time polymerase chain reaction. The role of these recombinant antigens in the cytoskeleton rearrangement was determined by adhesion assay and immunofluorescent microscopy.ResultsOur data showed that ESAT-6 and ESAT-6/CFP-10 fusion proteins could induce adhesion of macrophages to FN through α4β1 integrin. An increased expression level of α4β1 integrin in comparison with α5β1 integrin in differentiated THP-1 cells was also observed. Results of immunofluorescence studies showed that recombinant proteins-treated THP-1 cells form well-organized stress fibers and focal contacts containing vinculin compared with untreated THP-1 cells.ConclusionIncreased expression level of α4β1 in differentiated THP-1 cells could suggest the important role of α4β1 integrin in adhesion and focal contact formation of macrophages exposed to M. tuberculosis antigens

Effects of Over-Expression of LOC92912 Gene on Cell Cycle Progression

Background: We had previously identified the genes involvedin squamous cell carcinoma of the head and neck using differentialdisplay and DNA microarray techniques. We also reportedthe first analytical study on a novel human gene calledLOC92912, which was identified by differential display as agene up-regulated in such carcinomas. LOC92912, which is aputative member of the E2 ubiquitin conjugating enzyme family,is located on chromosome 15q and encodes a protein of 375amino acids. In this study, we present the extended analysis ofLOC92912 gene in order to uncover the pathway implicated incancer development or progression. We established a series ofRPMI 2650 cell line permanently over-expressing LOC92912gene, together with their related controls.Methods: LOC92912 gene was cloned in pSG5-expressingvector. In vitro translation assay was performed using pSG5-expressing LOC92912. The construct was used for transientand permanent transfection of LOC92912 gene into RPMI2650 cell line. Cell cycle analysis, clonogenicity, and cellgrowth assay for cells permanently over-expressing LOC92912were performed. Focus-like formation studies, also, wereinvestigated on cells permanently over-expressing LOC92912.Results: We found that RPMI 2650 cells permanently overexpressingLOC92912 show an increase in the number of cellsaccumulated in G0/G1 phase of the cell cycle, a decrease inclonogenicity and cell growth and formation of focus-likestructures. Preliminary data also showed changes in cell shapeand cell-cell adhesion.Conclusion: Our results demonstrated that LOC92912 inducedalterations in the proliferation of cells and might represent aputative novel regulator of cell cycle and some other cellularfunctions. This novel human gene may also represent a newtarget for treatment of cancers

Etude d'un nouveau gène humain surexprimé dans les tumeurs de l'hypopharynx

Notre laboratoire a réalisé un criblage des gènes impliqués dans les tumeurs des voies aéro-digestives supérieures par les techniques de differential display (DD) et de micropuces à ADN. Dans cette étude, nous présentons la première caractérisation d un nouveau gène humain, LOC92912, identifié par DD comme surexprimé dans les tumeurs. LOC92912, un membre potentiel de la famille des enzymes E2 de conjugaison de l ubiquitine, code pour une protéine de 375 acides aminés contenant des domaines RWD, coiles-coil et E2. Des analyses bioinformatiques montrent qu il existe des protéines apparentées dans des espèces aussi diverses que le ver et l homme. La forte conservation de la séquence de LOC92012 entre les espèces, particulièrement au niveau du domaine putatif catalytique C-terminal, suggère une fonction importante de cette protéine. LOC92912 est surexprimé dans environ 85% des échantillons de tumeurs. Il est exprimé dans les masses tumorales et dans les épithéliums invasifs, avec une localisation subcellulaire cytoplasmique. Nous avons développé un anticorps polyclonal de lapin qui détecte spécifiquement LOC92912, endogène et transfecté, à la taille attendue (~43kDa). Pour comprendre quelles voies de signalisation sont affectées dans les tissus cancéreux surexprimant ce gène, nous avons établi des clones stables RPMI2650 surexprimant LOC92912. Dans ce système de surexpression, certaines fonctions normales de la cellule sont affectées. Nous avons observé une augmentation de la phase G0/G1 du cycle cellulaire, la formation de structures en foci en culture, une diminution de la clonogénicité et de la croissance cellulaire et une capacité de migration accrue en chambre de Boyden. Des données préliminaires montrent également des changements de la morphologie cellulaire et de l adhésion intercellulaire. Pour mieux comprendre les fonctions de LOC92912, nous avons identifié les partenaires potentiels d interaction par immunopurification de la protéine flaggée suivie par une analyse en spectrométrie de masse MALDI Peptide Mass Fingerprinting . L actine et des protéines liant l actine ont été identifiées comme partenaires potentiels d interaction, suggérant des fonctions de LOC92912 liées au cytosquelette. Ce nouveau gène humain pourrait représenter une nouvelle cible pour la thérapie anticancéreuse. Cette étude constitue une base solide qui devrait encourager les scientifiques et cliniciens à s intéresser à ce gène.Previous work, performed in our laboratory, screened for genes involved in head and neck squamous cell carcinoma (HNSCC) using differential display (DD) and DNA microarrays. In this study, we present the first analytical analysis on a novel human gene, LOC92912, identified by DD as a gene upregulated in HNSCC. LOC92912, which is a putative member of the E2 ubiquitin conjugating enzyme family, encodes a protein of 375 amino acids containing a RWD domain, a coiled-coil and an E2 domain. Bioinformatics analysis revealed that there are related proteins in organisms as diverse as humans and worms. The striking conservation of LOC92912 sequence homology among species, particularly in the predicted catalytic domain of the carboxy terminal half of the protein, suggests that it has an important catalytic function. LOC92912 is upregulated in about 85% of tumor samples. It is expressed in tumor masses and in invasive epithelium, and is located in the cytoplasm of cells. We raised a rabbit polyclonal antibody that specifically detects the transfected and endogenous LOC92912 polypeptide of the expected size (~43kDa). To understand which pathways are affected in cancer tissues that overexpress our gene, we established RPMI 2650 stable transfectant over-expressing LOC92912, aimed at the identification of the signatures that can be subsequently validated for their implication in cancer. In this over-expressing system some normal functions of the cells were altered. We observed: an increase in the G0/G1 phase of the cell cycle, formation of focus-like structures in the culture, a decrease in clonogenicity and cell growth and more migration in Boyden chamber. Preliminary data also showed changes in cell shape and cell-to-cell adhesion. To gain insights into LOC92912 functions, we identified potential interacting partners by immunoaffinity purification of the flag tagged protein followed by MALDI Peptide Mass Fingerprinting (PMF) mass spectrometry. Actin and 6 actin-binding proteins were unambiguously identified as potential interacting partners, suggesting that LOC92912 s functions may be linked with the cytoskeleton. This novel human gene may represent a new target for cancer therapeutics. This study provides a solid basis that should encourage scientists and clinicians to become interested in this gene.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Urtica Dioica Distillate Regenerates Pancreatic Beta Cells in Streptozotocin-Induced Diabetic Rats

Background: Urtica dioica is known as an anti-hyperglycemic plant. Urtica dioica distillate (UD) is a traditional Iranian drink, locally known as “aragh gazaneh”. In spite of its widespread consumption in Iran, according to traditional Iranian medicine, there is no scientific report on the usefulness of UD for diabetic patients. This survey was designed to evaluate its protective effects for the recovery from diabetes by determining the serum insulin, blood glucose, volume of pancreatic islets, and the number and volume of β-cells in diabetic rats. Methods: A total of 48 Sprague-Dawley male rats (200-250 g) were randomly distributed into 6 groups (n=8), including non-diabetic plus distilled water (DW), non-diabetic plus UD, diabetic plus DW, diabetic plus UD, diabetic plus insulin, and diabetic plus glibenclamide. DW, UD, and glibenclamide were administered via intragastric gavage and insulin was injected subcutaneously. After four weeks of experiments, blood samples were collected for serum insulin and blood glucose assay. Pancreas was also evaluated using stereological method. The SPSS software was used for statistical analysis. Kruskal-Wallis, repeated measurements, and Mann-Whitney U test were applied for comparisons between the groups. Results: The treatment of diabetic rats with UD reduced the blood glucose dramatically (P<0.001) and increased serum insulin levels significantly (P=0.03) in comparison to the diabetic plus DW rats. Treatment with UD did not affect the mean β-cell volumes in the diabetic rats when compared to the diabetic plus DW rats, but the islet volumes and β-cell numbers were significantly recovered. Conclusion: UD treatment in diabetic rats improves hyperglycemia by partially restoring plasma insulin levels. The data suggest that UD prevents islet atrophy and/or regenerate pancreatic β-cells

Macrophage Immune Response Suppression by Recombinant Mycobacterium tuberculosis Antigens, the ESAT-6, CFP-10, and ESAT-6/CFP-10 Fusion Proteins

Background: Macrophage immune responses are affected by the secretory proteins of Mycobacterium tuberculosis (Mtb). This study aimed to examine the immune responses of macrophages to Mtb secretory antigens, namely ESAT-6, CFP-10, and ESAT-6/CFP-10. Methods: THP-1 cells (a human monocytic cell line) were cultured and differentiated to macrophages by phorbol 12-myristate 13-acetate. The cytotoxicity of the recombinant Mtb proteins was assessed using the MTT assay. Two important immune responses of macrophages, namely NO and ROS production, were measured in response to the ESAT-6, CFP-10, and ESAT-6/CFP-10 antigens. The data were analyzed using one-way ANOVA with SPSS, version 16, and considered significant at P<0.05. Results: The results showed that the ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins markedly reduced macrophage immune response. The treatment of the THP-1-differentiated cells with ESAT-6, CFP-10, and ESAT-6/CFP-10 reduced NO and ROS production. The treated THP-1-differentiated cells exhibited less inducible NO synthase activity than did the untreated cells. No toxic effect on macrophage viability was observed for the applied proteins at the different concentrations. Conclusion: It seems that the decline in macrophage immune response is due to the suppression of NO and ROS production pathways without any effect on cell viability

Absenteeism from Theory Classes: Perspective of Medical Students

Introduction: Absenteeism is one of the most important and increasing educational problems of universities in recent years whose consequences have a negative impact on many academic aspects of students. The purpose of this study was to identify and prioritize the factors affecting absenteeism from theory classes from the perspective of basic sciences medical students at Shiraz University of Medical Sciences in 2015. Methods: This descriptive cross-sectional study was conducted on 209 basic science medical students who were selected through convenience sampling (census) from October and February semester students in 2012-13 academic year. Data collection tool was a researcher-made questionnaire. The face validity of the questionnaire was confirmed by educational experts and its reliability was measured through Cronbach’s alpha coefficient (α = 0.8). It included 26 items in five-point Likert scale (1 to 5) that were divided into 4 domains as recommended by medical education experts. The data were analyzed using descriptive statistics. Results: The mean scores of the domains related to students’ views about absenteeism from theory classes were classroom management (4±0.59), individual conditions of class engagement (4±0.48), instructor’s academic ability and teaching method (3±0.58) and time and place of classes (2. 8±0.68). Students agreed with the effect of class management and class engagement on absenteeism, while disagreed with the effect of time and place of class on absenteeism. Conclusion: Results showed that from the viewpoints of students, class management and individual conditions for class engagement could affect medical students’ absenteeism from theory classes. Prioritization of these factors could easily eliminate the weaknesses and enhance instructor-learner communication

Implementation and evolution of the horizontal integration at shiraz medical school

Introduction: General medical education starts with basic sciences which usually continue about 2.5 to 3 years. In this period, the students study basic medical sciences and then start the clinical stage in which they deal with diagnoses, care, and cure of disease. The purpose of this study was to assess the integration of basic sciences period with the clinical period at Shiraz University of Medical Sciences. Methods: The present study is a descriptive one. The sample of the study consisted of all students entered Shiraz University of Medical Sciences in January, 2009, and November, 2009, professors of basic sciences courses, and some clinical professors. To evaluate the integration program, we devised various instruments. The collected data were analyzed, using SPSS software. Result: The findings showed that in spite of the students’ objections new educational methods in the first year of implementation, they felt more satisfied as the drawbacks were removed over time. Conclusion: The assessment of educational curricula is an important step to identify educational problems and promote the students’ learning. This issue can help the curriculum planners to design the educational programs so that students, particularly medical students, will be able to acquire the required knowledge and skill and integrate them for the promotion and maintenance of society’s health

Expression Status of UBE2Q2 in Colorectal Primary Tumors and Cell Lines

Background: Activation of the ubiquitin-proteasome pathway in various malignancies, including colorectal cancer, is established. This pathway mediates the degradation of damaged proteins and regulates growth and stress response. The novel human gene, UBE2Q2, with a putative ubiquitin-conjugating enzyme activity, is reported to be overexpressed in some malignancies. We sought to investigate the expression levels of the UBE2Q2 gene in colorectal cell lines as well as in cancerous and normal tissues from patients with colorectal cancer. Methods: Levels of UBE2Q2 mRNA in cell lines were assessed by Real-Time PCR. Western blotting was employed to investigate the levels of the UBE2Q2 protein in 8 colorectal cell lines and 43 colorectal tumor samples. Results: Expression of UBE2Q2 was observed at the level of both mRNA and protein in colorectal cell lines, HT29/219, LS180, SW742, Caco2, HTC116, SW48, SW480, and SW1116. Increased levels of UBE2Q2 immunoreactivity was observed in the 65.11% (28 out of 43) of the colorectal carcinoma tissues when compared with their corresponding normal tissues. Difference between the mean intensities of UBE2Q2 bands from cancerous and normal tissues was statistically significant at P<0.001 (paired t test). Conclusion: We showed the expression pattern of the novel human gene, UBE2Q2, in 8 colorectal cell lines. Overexpression of UBE2Q2 in the majority of the colorectal carcinoma samples denotes that it may have implications for the pathogenesis of colorectal cancer