Murat Kasap

Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties, differentiation potential and immunophenotypic markers

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can differentiate into many lineages. Although the growing interest in BM-MSCs has led to a number of characterization studies, some important biochemical and immunohistochemical properties are still lacking. In this study, morphological and immunophenotypic properties of BM-MSCs were examined in detail. Differentiation potential and growth kinetics of adult rat BM-MSCs were also determined. Immunohistochemistry and RT-PCR results indicated that BM-MSCs expressed myogenic (desmin, myogenin, myosin IIa, and α-SMA), neurogenic (γ-enolase, MAP2a,b, c-fos, nestin, GFAP and beta III tubulin), and osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, BMP-2, BMP-4 and type I collagen) markers without stimulation towards differentiation. These expression patterns indicated why these cells can easily differentiate into multiple lineages both in vitro and in vivo. Ultrastructural characteristics of rBM-MSCs showed more developed and metabolically active cells.

Characterization of ESBL (SHV-12) producing clinical isolate of Enterobacter aerogenes from a tertiary care hospital in Nigeria

BackgroundWe studied the beta-lactamases of an E. aerogenes isolate recovered from the blood of a two-year-old patient. The isolate demonstrated a disk-diffusion phenotype typical for an AmpC-ESBL co-producer.MethodsMicrobiology studies were performed according to standard protocols. The resistance gene was identified by transconjugation and cloning experiments.ResultsBy transconjugation only a narrow spectrum beta-lactamase (TEM-1) encoded on a small plasmid was transmitted. The ESBL was cloned and expressed in an E. coli host. Sequence analysis of the recombinant plasmid revealed blaSHV-12 associated to the insertion sequence, IS26.ConclusionThis is the first study demonstrated the occurrence of SHV-12 in Nigeria.

Emergence and spread of carbapenem-resistant Acinetobacter baumannii in a tertiary care hospital in Turkey

An intensive care unit (ICU)-based OXA-23-producing multiple-drug resistant Acinetobacter baumannii (MDRAB) outbreak was detected between October 2005 and October 2006. A total of 47 patients were infected/colonized with the outbreak strain. Clinical data were available from 37 patients. The all-cause mortality rate among the patients exposed to the epidemic strain was 35% (13/37). The outbreak strain and the resistance determinants were characterized both by microbiological methods and by molecular techniques. Cloning and sequencing experiments identified ISAbaI-associated bla(oxa-23) on the chromosome. Screening of imipenem-resistant Acinetobacter isolated from the ICU during the outbreak period with PCR identified 97 isolates as positive for the ISAbaI-bla(oxa-23) structure. Pulsed-field gel electrophoresis and plasmid analyses with selected nonrepetitive isolates revealed the clonality. Disk diffusion on cloxacillin-supplemented agar media and the real-time PCR experiments showed that outbreak isolates are overexpressing the ampC enzyme. This study highlights the occurrence of OXA-23-producing and ampC-overexpressing MDRAB in ICUs.

Phenotypic and Proteomic Characteristics of Human Dental Pulp Derived Mesenchymal Stem Cells from a Natal, an Exfoliated Deciduous, and an Impacted Third Molar Tooth

The level of heterogeneity among the isolated stem cells makes them less valuable for clinical use. The purpose of this study was to understand the level of heterogeneity among human dental pulp derived mesenchymal stem cells by using basic cell biology and proteomic approaches. The cells were isolated from a natal (NDPSCs), an exfoliated deciduous (stem cells from human exfoliated deciduous (SHED)), and an impacted third molar (DPSCs) tooth of three different donors. All three stem cells displayed similar features related to morphology, proliferation rates, expression of various cell surface markers, and differentiation potentials into adipocytes, osteocytes, and chondrocytes. Furthermore, using 2DE approach coupled with MALDI-TOF/TOF, we have generated a common 2DE profile for all three stem cells. We found that 62.3 ± 7% of the protein spots were conserved among the three mesenchymal stem cell lines. Sixty-one of these conserved spots were identified by MALDI-TOF/TOF analysis. Classification of the identified proteins based on biological function revealed that structurally important proteins and proteins that are involved in protein folding machinery are predominantly expressed by all three stem cell lines. Some of these proteins may hold importance in understanding specific properties of human dental pulp derived mesenchymal stem cells.

Isolation and characterization of stem cells from pancreatic islet: pluripotency, differentiation potential and ultrastructural characteristics

BACKGROUND AIMS Stem cells (SC) in different locations have individual characteristics. Important questions to be answered include how these specialties are generated, what the mechanism underlying their generation is, and what their biologic and clinical merits are. A basic approach to answering these questions is to make comparisons between the differences and similarities among the various SC types. They may focus on aspects of biologic marker discovery, capacity of proliferation and differentiation, along with other characteristics. The aim of this study was to characterize in detail the SC isolated from pancreatic islet (PI) and compare their properties with bone marrow (BM)-derived mesenchymal stromal cells (MSC) of the rat. METHODS Immunophenotypic characteristics, proliferation capacities, telomerase activities, pluripotent-related gene expressions, ultrastructure and the potential for multilineage differentiation of PI SC and BM MSC were studied. RESULTS We found that PI SC expressed markers of embryonic SC (Oct-4, Sox-2 and Rex-1) and had a high proliferation capacity, proven also by high telomerase activities. Surprisingly, markers belonging to differentiated cells were expressed by these cells in a constitutive manner. PI SC ultrastructure showed more developed and metabolically active cells. CONCLUSIONS The immunocytochemical identification of both PI SC and BM MSC was demonstrated to be typical MSC. Without stimulation of differentiation markers of adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells in these SC, the expression of those markers might explain their multilineage differentiation potential. We suggest that, by reason of the respectively high telomerase activity in PI SC, they could be better candidates than BM MSC for cell replacement therapy of type 1 diabetes.

Genotyping and Phylogenetic Analysis of Giardia duodenalis Isolates from Turkish Children

Background Giardiasis is caused by the intestinal protozoan parasite Giardia duodenalis (synonyms: G. lamblia, G. intestinalis), which is one of the most frequent parasites that infect Turkish children. However, molecular characterization of G. duodenalis in Turkey is relatively scarce. The present work aimed at genotyping G. duodenalis isolates from Turkey using molecular techniques. Material/Methods In the present study, 145 fecal samples from children were collected to search for the presence of Giardia by microscopy and PCR screening. PCR generated a 384 bp fragment for β-giardin. The PCR products were sequenced and the sequences were subjected to phylogenetic analysis by using PHYLIP. Results Based on the phylogenetic analysis of the sequences, assemblage A, B, and mixed subtypes were determined. Of 22 isolates, 11 were identified as assemblage A (50%), 7 were assemblage B (31.8%), and 4 were assemblage AB (18.2%). Association between G. duodenalis assemblages and the epidemiological data was analyzed. No correlation was found between symptoms and infection with specific assemblages (P>0.05), but we found statistically significant association between age and the assemblage AB (P=0.001). Conclusions The association between G. duodenalis and the epidemiologic data were analyzed. Since assemblage A is the more prevalent subgroup compared with assemblage B, this subgroup might be responsible for common Giardia infections in Turkey. This is the first study that included a detailed phylogenetic analysis of Giardia strains from Turkey.

OXA-162, a novel variant of OXA-48 displays extended hydrolytic activity towards imipenem, meropenem and doripenem

Context: Isolation and characterization of OXA-162, a novel variant of OXA-48. Objectives: Klebsiella pneumoniae isolate with decreased susceptibility to carbapenems was recovered from a Turkish patient. This study aimed at characterizing the carbapenem resistance determinants of this isolate. Materials and methods: Antibiotic susceptibility tests, analytic isoelectric focusing (IEF), cloning and sequencing were performed. Cloned β-lactamase was purified by means of preparative gel electrophoresis and the kinetic constants were determined under initial rate conditions. Results: The identified blaOXA-162 gene was located on a ca. 45-kb plasmid carrying a transposon consisted of two IS1999-2 elements. OXA-162 differed from OXA-48 by a single amino acid substitution (Thr213Ala) which increased the catalytic efficiency (kcat/KM) of OXA-162 towards imipenem and meropenem. Also this substitution caused a gain of hydrolysis ability towards doripenem. Analysis of OXA-162 model implied that the amino acid change might generate an extension in the opening of the substrate entry site and might cause extended hydrolytic activity towards imipenem, meropenem and doripenem. Discussion and conclusion: OXA-162, a derivative of OXA-48 has enhanced catalytic properties.

Evidence for the presence of full-length PARK2 mRNA and Parkin protein in human blood

Research on Parkinsons disease fails to pinpoint a single gene or a gene product as the causative factor. However, the early onset form of the disease may be caused by mutations in PARK2 gene. Some studies related to the biochemistry or other aspects of the PARK2 gene or its product mostly used cDNA generated from substantia nigra of the mid-brain. This is essentially because the presence of the 1.4kb full-length PARK2 cDNA in human leukocytes is, so far, not demonstrated although some splice variants and short RT-PCR products were reported. In this study, we synthesized a 1.4kb full-length PARK2 cDNA from human leukocytes, cloned and expressed it both in Escherichia coli and in HeLa cells. The presence of Parkin protein was also demonstrated in human serum using Western blotting and MALDI-TOF analysis. The results of this study showed a simple way for routine amplification of PARK2 cDNA from human blood and may become a useful diagnostic tool in the future.

Neonatal hyperbilirubinemia and organic anion transporting polypeptide-2 gene mutations.

The aim of this study was to investigate the genotypic distribution of organic anion transporting polypeptide 2 (OATP-2) gene mutations and the relationship with hyperbilirubinemia of unknown etiology. Polymerase chain reaction, restriction fragment length polymorphism, and agarose gel electrophoresis techniques were used for detection of OATP-2 gene mutations in 155 newborn infants: 37 with unexplained hyperbilirubinemia, 65 with explained hyperbilirubinemia, and 53 without hyperbilirubinemia. In the OATP-2 gene, we identified A→G transitions at nucleotide positions 388 and 411 and observed six polymorphic forms. The 388/411-411 mutation was the most common form (43%) in subjects with hyperbilirubinemia of unknown etiology. Male sex [odds ratio (OR): 3.08] and two polymorphic forms of the OATP-2 gene [the 388/411-411 A→G mutation (OR: 3.6) and the 388-411 mutation (OR: 2.4)] increased the risk of neonatal hyperbilirubinemia. In male infants with the 388 A→G mutation of the OATP-2 gene, the levels of unconjugated bilirubin in plasma were significantly increased compared with those observed in females. The polymorphic forms of 388 nucleotide of the OATP-2 gene were identified as risk factors for hyperbilirubinemia of unknown etiology.

The prominent proteins expressed in healthy gingiva: a pilot exploratory tissue proteomics study

Gingiva is a unique tissue which protects the underlying periodontal tissues from consistent mechanical and bacterial aggressions. Molecular analysis of gingiva is likely to improve our understanding of the underlying biological processes at work. The aim of this preliminary exploratory study is to analyze the proteomic profile of healthy gingiva and to detect prominently expressed proteins. Gingival tissue samples were obtained from periodontally healthy individuals who underwent surgical crown lengthening procedure. After protein isolation, two dimensional gel electrophoresis (2DE) gels were prepared for each sample and only protein spots common to all gels were selected to eliminate the bias caused by the effect of individuals on proteomic profile. Following the 2DE; in-gel tryptic digestion and MALDI-TOF/TOF steps were performed for protein identifications. Forty-seven proteins were successfully identified. The identified proteins were classified based on their classes, molecular functions and involvements in biological processes and metabolic pathways. Among them, 14-3-3 protein sigma, Protein DJ-1, Alpha-enolase, Triosephosphate isomerase, Superoxide dismutase, Peroxiredoxin-1, Protein S100-A9, Galectin-7, Annexin A2/A4, Carbonic anhydrase 1 and chaperone proteins are worthy of attention. The proteomic profile of the gingiva reflected its highly dynamic characteristics. Despite complexity of the gingival tissue proteome, 2DE was an effective approach in studying the common protein expression profile of the gingiva. Considering the significance of gingiva in the formation of periodontal diseases, it is important to generate a detailed proteome map of gingival tissue to set up a bridge between molecular events and the disease formation. This study established an initial proteome map of the gingival tissue from healthy individuals.