Chemical Genetics Identify eIF2α Kinase Heme Regulated Inhibitor as Anti-Cancer Target

Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2·GTP·Met-tRNAi translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemia. The chemical modifiers of the eIF2·GTP·Met-tRNAi ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining if this complex can be pharmacologically targeted for therapeutic purposes. Using a cell based assay, we identified N,N’-diarylureas as novel inhibitors of the ternary complex abundance. Direct functional-genetics and biochemical evidence demonstrated that the N,N’-diarylureas activate heme regulated inhibitor kinase, thereby phosphorylate eIF2α and reduce abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N’-diarylureas are potent and specific tools for studying the role eIF2·GTP·Met-tRNAi ternary complex in the pathobiology of human disorders

Comprehensive identification of erythrocyte membrane protein deficiency by 2D gel electrophoresis based proteomic analysis in hereditary elliptocytosis and spherocytosis

Purpose Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1DE) may reveal qualitative or quantitative defects in red blood cell (RBC) membrane proteins, two-dimensional gel electrophoresis (2DE) can be used for determination of the protein changes caused by the disease process in a relatively high-throughput manner, because it permits an analysis of thousands of modified or unmodified proteins simultaneously. The principal aim of this study was to compare hereditary elliptocytosis (HE), hereditary spherocytosis (HS), and control RBC membrane protein profiles and identify proteins as a clinical marker by the sensitive methods. Experimental design RBC membrane proteins of HE, HS, and control groups were compared by 2DE and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis was obtained using peptide mass fingerprint for protein identification. Results Twenty proteins were identified with peptide mass fingerprint analysis and different expression levels were found in HE, HS, and controls for some proteins that includes three biomarker proteins (ankyrin, spectrin, band 3) that may have prognostic or predictive importance. Conclusions and clinical relevance 2DE of RBC proteins is a potentially valuable method for studying heritable disorders such as HE and HS. By identifying a deficiency in membrane proteins associated with the RBC cytoskeleton, the diagnosis of HE and HS can be confirmed.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK)This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK)

Identification of Meat Species by Using Molecular and Spectroscopic Techniques

Meat is one of the main nutrition source in the human diet with its excellent protein, vitamin and mineral contents. Despite its advantages, being high-priced makes meat products open to adulteration. Meat products are mixed food types which can contain different species of meat. However, mixing two or more types of meats is not always allowed by laws. On the other hand, replacement high quality meats with cheaper meat types are a cost lowering way for the producers. The commonly consumed meat types differ from country to country, but generally economical, ethnic and religion concerns are in the foreground. In this case, species identification techniques are gaining importance. Although some techniques depending on DNA or spectroscopy have been developed for many years, choosing the best method for species identification is still among the controversial issues today. Thus, the currently used methods and promising techniques in this area were discussed in this review

Biology of stem cells in human umbilical cord stroma: In situ and in vitro surveys

Cells in the umbilical cord stroma have gained attention in recent years; however, differentiation to certain lineages in humans has been demonstrated in few studies. Unlike bone marrow MSCs, human umbilical cord stroma cells (HUC-SCs) are far from being well characterized. This study attempts to describe proliferation, structural, and differentiation properties of these cells to account for their exceptional nature in many aspects. Cellular dynamics, cellular structure, and the degree of transformations during expansion and differentiation into mesenchymal and neuronal lineages were examined in vitro over a 10-month period. Comparisons with human bone marrow MSCs regarding differentiation were performed. HUCSCs in culture revealed two distinct cell populations, type 1 and type 2 cells, that possessed differential vimentin and cytokeratin filaments. Corresponding cells were encountered in cord sections displaying region-specific localization. alpha-Smooth muscle actin and desmin filaments, which were evident in cord sections, diminished through passages. No difference was noted regarding type 1 and type 2 cells in differentiation to chondrogenic, adipogenic, and osteogenic lineages, whereas a preferential differentiation was noted in neuronal lineage. Relative success was achieved by production of chondrocytic spheres and osteogenic monolayers, whereas adipocytes were immature compared with bone marrow MSCs. The presence of neuronal markers suggests that they transform into a certain state of maturity under neurogenic induction. Conclusively, HUCSCs retain their original phenotype in culture without spontaneous differentiation, have a limited lifespan, and bear multipotent stem cell characteristics. Given these characteristics, they may be generally considered progenitor cells if manipulated under appropriate conditions and deserve further study to be potentially used in cell-based therapies

Tbx3 represses PTEN and is over-expressed in head and neck squamous cell carcinoma

Abstract Background Despite advances in diagnostic and treatment strategies, head and neck squamous cell cancer (HNSCC) constitutes one of the worst cancer types in terms of prognosis. PTEN is one of the tumour suppressors whose expression and/or activity have been found to be reduced in HNSCC, with rather low rates of mutations within the PTEN gene (6-8%). We reasoned that low expression levels of PTEN might be due to a transcriptional repression governed by an oncogene. Tbx2 and Tbx3, both of which are transcriptional repressors, have been found to be amplified or over-expressed in various cancer types. Thus, we hypothesize that Tbx3 may be over expressed in HNSCC and may repress PTEN, thus leading to cancer formation and/or progression. Methods Using immunohistochemistry and quantitative PCR (qPCR), protein and mRNA levels of PTEN and Tbx3 were identified in samples excised from cancerous and adjacent normal tissues from 33 patients who were diagnosed with HNSCC. In addition, HeLa and HEK cell lines were transfected with a Tbx3 expressing plasmid and endogenous PTEN mRNA and protein levels were determined via qPCR and flow cytometry. Transcription assays were performed to demonstrate effects of Tbx3 on PTEN promoter activity. Mann–Whitney, Spearman’s Correlation and Wilcoxon signed-rank tests were used to analyze the data. Results We demonstrate that in HNSCC samples, Tbx3 mRNA levels are increased with respect to their normal tissue counterparts (p Conclusions We show that Tbx3 is up-regulated in tissue samples of HNSCC patients and that Tbx3 represses PTEN transcription. Thus, our data not only reveals a new mechanism that may be important in cancer formation, but also suggests that Tbx3 can be used as a potential biomarker in cancer.</p

Comparative evaluation of various miniplate systems for the repair of mandibular corpus fractures

Miniplates have been used during the last decade to facilitate stability between bony fragments in the maxillofacial region and are currently the preferred surgical method for the fixation of fractures and osteotomies. The aim of this study was to evaluate and compare the biomechanical behaviors of six different types of miniplates used to reconstruct mandibular body fractures: Group 1 (straight, 2 holes, 12.0 ram spacing), Group 2 (straight, 4 holes, 9.0 spacing), Group 3 (straight, 6 holes, 9.0 mm spacing), Group 4 (L-shaped, 4 holes, 9.0 mm spacing, right hand plate), Group 5 (Y-shaped, 5 holes, 12.0 mm spacing), and Group 6 (double Y-shaped, 6 holes, 9.0 mm spacing). Thirty bovine hemimandibles and a custom-made 3-point biomechanical test frame mounted on a Shimadzu universal test machine were used to evaluate the six different miniplate systems. Results revealed that Group 1 (straight, 2 holes, 12.0 mm spacing) and Group 4 (9.0 mm spacing, right hand plate) had the lowest biomechanical stability, whereas Group 6 (6 holes, 9.0 ram spacing) had the highest biomechanical stability. Group 6 also provided statistically greater resistance to displacement than Group 1 and Group 4

Application of Fourier Transform Infrared Spectroscopy to Biomolecular Profiling of Cultured Fibroblast Cells From Gaucher Disease Patients: A Preliminary Investigation

Background. Gaucher disease (GD) is defined as an autosomal recessive disorder resulting from the deficiency of glucocerebrosidase (E.C. 3.2.1.45). Glucocerebrosidase is responsible for the degradation of glucosylceramide into ceramide and glucose. The deficiency of this enzyme results in the accumulation of undegraded glucosylceramide, almost exclusively in macrophages. With Fourier transform infrared (FTIR) spectroscopy, the complete molecular diversity of the samples can be studied comparatively and the amount of the particular materials can be determined. Also, the secondary structure ratios of proteins can be determined by analysing the amide peaks. Objectives. The primary aim of this study is to introduce FTIR-ATR spectroscopy technique to GD research for the first time in the literature and to assess its potential as a new molecular method. Material and methods. Primary fibroblast cell cultures obtained from biopsy samples were used, since this material is widely used for the diagnosis of GD. Intact cells were placed onto a FTIR-ATR crystal and dried by purging nitrogen gas. Spectra were recorded in the mid-infrared region between 4500-850 cm(-1) wavenumbers. Each peak in the spectra was assigned to various organic biomolecules by comparing their wavenumbers with the reference values in the literature. A quantitative analysis was performed using peak areas and we also used a hierarchical cluster analysis as a multivariate spectral analysis. Results. We obtained FTIR spectra of fibroblast samples and assigned the biomolecule origins of the peaks. We observed individual heterogeneity in FTIR spectra of GD fibroblast samples, confirming the well-known phenotypic heterogeneity in GD at the molecular level. Significant alterations in protein, lipid and carbohydrate levels related to the enzyme replacement therapy were also observed, which is also supported by cluster analysis. Conclusions. Our results showed that the application of FTIR spectroscopy to GD research deserves more attention and detailed studies with an increased sample size in order to evaluate its potential in the diagnosis and follow-up of GD patients.WoSScopu

Proteomic Characterization of the Venom of Five Bombus (Thoracobombus) Species

Venomous animals use venom, a complex biofluid composed of unique mixtures of proteins and peptides, to act on vital systems of the prey or predator. In bees, venom is solely used for defense against predators. However, the venom composition of bumble bees (Bombus sp.) is largely unknown. The Thoracobombus subgenus of Bombus sp. is a diverse subgenus represented by 14 members across Turkey. In this study, we sought out to proteomically characterize the venom of five Thoracobombus species by using bottom-up proteomic techniques. We have obtained two-dimensional polyacrylamide gel (2D-PAGE) images of each species’ venom sample. We have subsequently identified the protein spots by using matrix assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS). We have identified 47 proteins for Bombus humilis, 32 for B. pascuorum, 60 for B. ruderarius, 39 for B. sylvarum, and 35 for B. zonatus. Moreover, we illustrated that intensities of 2DE protein spots corresponding to putative venom toxins vary in a species-specific manner. Our analyses provide the primary proteomic characterization of five bumble bee species’ venom composition.PubMedWoSScopu