Aspergillus Fumigatus Endo-Beta-1,4-Mannanazının heterolog ev sahibi organizmalarda ekspresyonu ve analizi.

Extracellular endo-1,4-b-mannanase (EC 3.2.1.78) gene of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was cloned and transformed into Aspergillus sojae (ATCC 11906) and Pichia pastoris GS115. High level of expression was achieved in both expression systems. Attempts to produce heterologous mannanase in Arabidopsis thaliana, suitable for large scale production, were not successful. Comparison of the expression levels of heterologous mannanase reveals that A. sojae is a better expression system than P. pastoris with respect to extracellular mannanase activity. The production of mannanase in A. sojae (AsT1) after 3 days of incubation reached 204 U/ml in YpSs containing 1 % glucose. In P. pastoris (PpT1), highest production was observed after 10 hrs of induction with methanol (61 U/ml). Expressed enzymes were purified and analyzed. Both enzymes have specific activity c. 349 U/mg protein with pH and temperature optimum of c. 4.5 and c. 60 °C for mannanases from AsT1 and c. 5.2-5.6 and c. 45 °C for mannanases from PpT1. A truncated form of mannanase (MAN-S) deleted at amino acids from P291 to P368, which still displayed hydrolytic activity was also isolated and characterized. MAN-S has pH and temperature optimum of c. 6.5-8.0 and c. 60 °C. During incubation of the mannanase on locust bean gum, transglycosylation reactions, in which longer or rare prebiotic oligosaccharides could be produced catalyzed by glycolysis, was detected. The products of hydrolytic activity of the enzyme on various carbohydrates were analyzed by PACE and MALDI-TOF. Accordingly, hexamannose and smaller oligosaccharides were characterized.Ph.D. - Doctoral Progra

Stromal Stem Cells from Parathyroid Glands of Patients with Secondary Hyperparathyroidism Demonstrate Higher Telomerase Activity and Osteogenic Differentiation Ability than Normal Bone Marrow Derived Stromal Stem Cells

Aims: The aim of this study was to isolate and extensively characterize parathyroid gland stem cells (PT-SCs) from secondary hyperparathyroidism cases. For this purpose, proliferation capacity, phenotypic properties, differentiation characteristics and gene expression profiles were analyzed and compared with mesenchymal stem cells from bone marrow (BM-MSCs) of the human. Methods: Stem cells isolated from PT and BM were analyzed by flow cytometry, RTPCR, Real Time-PCR, and immunocytochemistry. Both cell lines were directionally differentiated towards adipogenic, osteogenic and neurogenic cell lineages. Results: The isolated hPT-SCs share similar characteristics of hBM-MSCs by immunophenotypic, histological and molecular analyses. Both cells were shown to differentiate successfully into adipogenic and osteogenic cell lines. Embryonic stem cell markers Pou5F1, Zpf42, FoxD3, Sox2 and Nanog were also expressed beside 5 fold higher telomerase activity in hPT-SCs that could indicate the regenerative ability of the human parathyroid gland. The osteogenic cell markers were expressed by hPT-SCs, which transformed efficiently into osteogenic cell lines, both at the level of genes (BMP2, BMP4, BGLAP, Coll11a1, Runx2, Sparc) and of proteins (BMP2, BMP4, Osteocalcin, Osteonectin, Osteopontin). Higher alkaline phosphatase (ALP) activity indicating osteogenic differentiation was determined in hPT-SCs from secondary hyperparathyroidism patients. Conclusion: PT-SCs might responsible for the calcified parathyroid glands and other ectopic calcifications including the vascular ones, observed in the secondary hyperparathyroidism cases, beside parathyroid hormone-dependent hypercalcemia leading diffusion of calcium phosphate precipitation in tissues

Cloning, expression and characterization of endo-β-1,4-mannanase from aspergillus fumigatus in aspergillus sojae and pichia pastoris

To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo-β-1,4-mannanase (EC 32.1.78) of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase (gpdA) and the alcohol oxidase (AOX1) promoters, respectively. The highest production of mannanase (352 U mL -1)in A. sojae was observed after 6 days of cultivation. In P. pastoris, the highest mannanase production was observed 10 h after induction with methanol (61 U mL-1). The fold increase in mannanase production was estimated as ∼12-fold and ∼2-fold in A. sojae and P. pastoris, respectively, when compared with A. fumigatus. Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities (∼350 U mg-1 protein). Temperature optima were at 60°C and 45°C, and maximum activity was at pH 4.5 and 5.2 for A. sojae and P. pastoris, respectively. The enzyme from P. pastoris was more stable retaining most of the activity up to 50°C, whereas the enzyme from A. sojae rapidly lost activity above 40°C

Role of mesenchymal stem cells transfected with vascular endothelial growth factor in maintaining renal structure and function in rats with unilateral ureteral obstruction

WOS: 000356908200009PubMed ID: 25542189Objectives: Mesenchymal stem cells hold promise for renal disease treatment. Vascular endothelial growth factor may heal tubule-interstitial fibrosis in unilateral ureteral obstruction by inhibiting epithelial-mesenchymal transition. We investigated the protective effect of vascular endothelial growth factor in transfected mesenchymal stem cells in unilateral ureteral obstruction-induced renal injury in rats. Materials and Methods: Male Wistar Albino rats (32 rats; weight, 250-300 g) were divided into 4 equal groups: group 1, control; group 2, unilateral ureteral obstruction; group 3, unilateral ureteral obstruction and mesenchymal stem cells; and group 4, unilateral ureteral obstruction and vascular endothelial growth factor-transfected mesenchymal stem cells. Vascular endothelial growth factor-transfected mesenchymal stem cells were administered intravenously before onset of unilateral ureteral obstruction. On day 14, the rats were killed and kidneys were retrieved. Tubular necrosis, mono-nuclear cell infiltration, and interstitial fibrosis were evaluated in paraffin blocks. We evaluated green fluorescent protein-positive and vascular endothelial growth factor-positive cells; anti-inflammatory (Prostaglandin E2 Receptor) and interleukin 1 receptor antagonist), proinflammatory/anti-inflammatory (interleukin 6), and proinflammatory (MPO) cytokine expression levels; and levels of nitric oxide; transforming growth factor beta 1, E-cadherin, and hydroxyproline. Results: Green fluorescent protein-positive cells were negative in the renal parenchyma in groups 1 and 2 and positive in groups 3 and 4. Vascular endothelial growth factor levels were significantly higher in group 4. Transforming growth factor 131, nitric oxide, and E-cadherin levels were significantly higher in the unilateral ureteral obstruction than control group; however, in the study groups, these values were not significantly different from the unilateral ureteral obstruction group. In stem cell-transplanted tissue samples, EP3, interleukin 1 receptor antagonist, and interleukin 6 levels were elevated, but MPO expression levels were low. Although there were significant differences for tubular necrosis and fibrosis in group 2, there were significant reductions in tubular injury and fibrosis in groups 3 and 4. Conclusions: Systemic stem cells transplanted into the kidney protected against unilateral ureteral obstruction-induced renal epithelial-mesenchymal transition and renal fibrosis

The Effects of Adipose Tissue-Derived Mesenchymal Stem Cell Transplantation During the Acute and Subacute Phases Following Spinal Cord Injury

AIM: To investigate the effectiveness of rat adipose tissue-derived (rAT) mesenchymal stem cell (MSC) transplantation on the functional restoration and regeneration of spinal cord injury (SCI).MATERIAl and METhODS: Six of 48 Wistar albino rats were sacrificed to obtain MSCs, and the remaining rats were divided randomly into six groups. SCI was performed using the clip compression method. The control and transplantation groups were injected with physiological saline and a rAT-MSC suspension at the injury sites, respectively. Each animal was evaluated using the Basso, Beattie and Bresnahan (BBB) rating system and sacrificed at 28 days post-injury period (p.i.). The regeneration process was evaluated based on immunostaining against ?3-tubulin, BDNF, CNTF, and CNPase. RESulTS: rAT-MSC transplantation into the SCI site substantially improved the tissue regeneration and functional recovery (p < 0.05). However, the rAT-MSC transplantation at 9 days p.i. was not more efficient on functional recovery than the transplantation immediately after injury. The expression of ?3-tubulin, BDNF and CNTF at the injury site indicated the potential for functional regeneration. COnCluSIOn: The adaptive nature of rat-MSCs enabled the remodulation and regeneration of the lesion site, decreasing the importance of transplantation time in the treatment of SC

Bone marrow-derived mesenchymal stem cells co-cultured with pancreatic islets display beta cell plasticity

The direct co-culturing effect of rat bone-marrow-derived mesenchymal stem cells (rBM-MSCs) on the pancreatic-islets (PIs) was studied to obtain functional islet cells. MSCs were isolated from rat bone marrow and cultivated under standard conditions. Following their characterization, the rBM-MSCs were directly (with cell-islet contact) co-cultured with recovered PIs together with the single cell cultures of those cell cultures as a control. The effect of direct co-cultures of rBM-MSCs with the PIs of normal rats was investigated using immunophenotypical and functional methods. The change in the amount of insulin secretion was evaluated as an indicator for differentiation of rBM-MSCs. One approache for in vitro differentiation to achieve reprogramming for differentiation into suitable cell types by changing the microenvironment of the cells to provide signals that might activate metabolic pathways is to use co-cultures with the microenvironment of the specific cells of the desired cell type, tissue/organ extracts, extracellular matrix compounds or biologically absorbable materials. Differentiated rBM-MSCs were found to be immunopositive for the specific insulin-producing cell marker, insulin, but not in undifferentiated rBM-MSCs. The functionality tests by ELISA confirmed that insulin secretion of co-cultured MSCs with islets was higher than that of islets. These evidences indicated that PIs could be regarded as critical components of the stem cell niche, such that MSCs can be differentiated into insulin-producing cells (IPCs). Moreover, direct cell-to-cell contact might provide additional and independent support. This approach would circumvent the need for PI-stem cell co-culture and could potentially facilitate the production of functional IPCs for future clinical applications. Copyright. (C) 2010 John Wiley & Sons, Ltd

Recovery of fertility in azoospermia rats after injection of adipose-tissue-derived mesenchymal stem cells: the sperm generation

The recent reports on the treatment of azoospermia patients, in which spermatozoa could not be traced in their testes, are focused more on the potential use of adult stem cells, like mesenchymal stem cells (MSCs). The aim of this study was to demonstrate the potential use of MSCs derived from adipose tissue in the treatment of azoospermia using rat disease models. After left rete testes. After 12 weeks, the testes with cell injection (right testes) were compared to control (left testes) after dimensional and immunohistochemical analyses. Testes treated with MSCs appeared morphologically normal, but they were atrophic in rats without stem cell treatment, in which the seminiferous tubules were empty. Spermatogenesis was detected, not in every but in some tubules of cell-treated testes. GFP(+)/VASA(+) and GFP(+)/SCP1(+) cells in testes indicated the transdifferentiation of MSCs into spermatogenetic cells in the appropriate microenvironment. Rats with cell treatment were mated to show the full recovery of spermatogenesis, and continuous generations were obtained. The expression of GFP was detected in the mesenchymal stem cells derived from adipose tissue and bone marrow and also in the sperms of offspring. In conclusion, MSCs might be studied for the same purpose in humans in future.Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [110S506

The Effects of Adipose Tissue-Derived Mesenchymal Stem Cell Transplantation During the Acute and Subacute Phases Following Spinal Cord Injury

AIM: To investigate the effectiveness of rat adipose tissue-derived (rAT) mesenchymal stem cell (MSC) transplantation on the functional restoration and regeneration of spinal cord injury (SCI).MATERIAl and METhODS: Six of 48 Wistar albino rats were sacrificed to obtain MSCs, and the remaining rats were divided randomly into six groups. SCI was performed using the clip compression method. The control and transplantation groups were injected with physiological saline and a rAT-MSC suspension at the injury sites, respectively. Each animal was evaluated using the Basso, Beattie and Bresnahan (BBB) rating system and sacrificed at 28 days post-injury period (p.i.). The regeneration process was evaluated based on immunostaining against ?3-tubulin, BDNF, CNTF, and CNPase. RESulTS: rAT-MSC transplantation into the SCI site substantially improved the tissue regeneration and functional recovery (p &lt; 0.05). However, the rAT-MSC transplantation at 9 days p.i. was not more efficient on functional recovery than the transplantation immediately after injury. The expression of ?3-tubulin, BDNF and CNTF at the injury site indicated the potential for functional regeneration. COnCluSIOn: The adaptive nature of rat-MSCs enabled the remodulation and regeneration of the lesion site, decreasing the importance of transplantation time in the treatment of SC

ENHANCED beta-MANNANASE PRODUCTION FROM ALTERNATIVE SOURCES BY RECOMBINANT ASPERGILLUS SOJAE

beta-mannanases can degrade galactomannans to mannose and it has been used in various application areas. The aim of this study was to produce the beta-mannanase from carob pod extract including different nitrogen sources. The best operation values for fermentation were determined to be 8% initial sugar concentration with 0.5% yeast extract, 100 r.p.m., and 7% inoculation rate, which yielded the maximum beta-mannanase activity as 423.60 U ml(-1). Effects of nitrogen sources on beta-mannanase activity were also studied and it reached 695.6 U ml(-1) by using 0.5% of ammonium nitrate as the nitrogen source at the determined optimum conditions. Results also showed that meat bone meal and soybean meal could be used as cost effective nitrogen sources based on achieved beta-mannanase activity

Effects of mesenchymal stem cells and VEGF on liver regeneration following major resection

The study aims to determine the effects of mesenchymal stem cell (MSC) therapy and a combination therapy of MSCs transfected with vascular endothelial growth factor (VEGF) for liver regeneration after major resection