Effects of Major Ingredients in Cattle Milk on Enzyme Kinetics of Recombinant β-galactosidase (BGalP) Expressed in Pichia pastoris

Background and objective: β-galactosidase enzymes hydrolyze lactose into glucose and galactose for production of lactose free dairy products. However, different ions and fat content in milk may act as the inhibitor or activator for β-galactosidase enzymes. A cold-active β-galactosidase enzyme (BGalP), originally from Planococcus sp. L4, was previously expressed in Pichia pastoris to perform lactose hydrolysis in the refrigerated milk. In this study, the effects of milks major ingredients were evaluated on the enzymatic kinetics to confirm its capacity for hydrolyzing milk lactose.Material and methods: The activity was determined in different concentrations of NaCl, KCl, MgCl2, and CaCl2 as well as in the milk with low, medium or high-fat content. In these experiments ortho-Nitrophenyl β-galactoside was used as the substrate. Additionally, glucose was measured as the product after incubation of milk with BGalP enzyme for 24 h at room temperature.Results and conclusion: This study demonstrated that ions and fat content did not adversely affect the enzyme activity in the concentration corresponding to the milk contents. Ca (27.5-32.5 mM), Cl (25.3-30.9 mM), Na (15.2-39.1 mM) and Mg (3.75-5.83 mM) had no inhibitory effects, but KCl decreased the enzyme activity. Since Cl existed in MgCl2, and CaCl2 exerted no inhibitory effects, it can be concluded that inhibitory effects of KCl resulted from potassium rather than chloride. The results indicate that BGalP enzyme was not inhibited by milks major ingredients and has the potential to be used for the production of lactose-free dairy products.Conflict of interest: The authors declare no conflict of interest

Interleukin-6 as A Prognostic Biomarker in Perinatal Asphyxia

Abstract Objective Early diagnosis is considered as a priority for prevention and treatment of asphyxia-related complications. The main aim of the present study was to evaluate the prognostic value of interleukin-6 (IL-6) and hypoxic ischemic encephalopathy grade in prediction mortality and developmental status of neonates affected by prenatal asphyxia. Materials & Method The cohort study was conducted on 38 term asphyxiated infants at Ghaem hospital, Mashhad, Iran, during 2013-2017. The HIE grade and serum IL-6 levels were determined at the time of birth. The developmental status was determined using the Denver II test at the end of two-year follow-up. Results  HIE grade 3 resulted in 83% mortality rate and developmental delay in all the survivors. The average IL-6 level was 2.7 ng/ml in the control group (not affected HIE) which increased up to 29, 175 and 136 ng/ml in those with HIE grades 1, 2 and 3, respectively. Roc curve analysis revealed the cut-off levels 24 pg/ml to predict the developmental delay with sensitivity and specificity of 96 and 92%, respectively. Conclusion  The IL-6 level and HIE grade are the potential prognostic biomarkers for determination of mortality and morbidity rate in the asphyxiated neonate

Differences in growth promotion, drug response and intracellular protein trafficking of FLT3 mutants

Objective(s): Mutant forms FMS-like tyrosine kinase-3 (FLT3), are reported in 25% of childhood acute lymphoid leukemia (ALL) and 30% of acute myeloid leukemia (AML) patients. In this study, drug response, growth promoting, and protein trafficking of FLT3 wild-type was compared with two active mutants (Internal Tandem Duplication (ITD)) and D835Y. Materials and Methods: FLT3 was expressed on factor-dependent cells (FDC-P1) using retroviral transduction. The inhibitory effects of CEP701, imatinib, dasatinib, PKC412 and sunitinib were studied on cell proliferation and FLT3 tyrosine phosphorylation. Total expression and proportion of intracellular and surface FLT3 was also determined. Results: FDC-P1 cells became factor-independent after expression of human FLT3 mutants (ITD and D835Y). FDC-P1 cells expressing FLT3-ITD grow 3 to 4 times faster than those expressing FLT3-D835Y. FD-FLT3-ITD cells were three times more resistant to sunitinib than the FD-FLT3-WT cells. The Geo means for surface FLT3 expression in FD-FLT3-ITD and -D835Y were 65 and 70% less than the FD-FLT3-WT cells. About 40% of expressed FLT3 was detected as intracellular in FD-FLT3-D835Y cell compared to 4 and 4.5% in FD-FLT3-WT and -ITD cells. Conclusion: Retention of D835Y FLT3 mutant protein may cause altered signaling, endoplasmic reticulum stress and activation of apoptotic signaling pathways leading to lower proliferation rate in FD-FLT3-D835Y than the FLT3-WT and ITD mutant., these may also also contribute, along with the preferential affinity, to the increased sensitivity of D835Y of CEP701 and PKC412. Studying these genetic variations can help determining the prognosis and designing a therapeutic plan for the patients with FLT3 mutations

Design of cocktail peptide vaccine against Cytomegalovirus infection

Objective(s):Human Cytomegalovirus (HCMV) remains a major morbidity and mortality cause in immuno suppressed patients. Therefore, significant effort has been made towards the development of a vaccine. In this study, the expression of the pp65 and gB fusion peptides and Fc domain of mouse IgG2a as a novel delivery system for selective uptake of antigens by antigen-presenting cells (APCs) in Pichia pastoris yeast system were studied. Materials and Method: In this study, four immune dominant sequences in pp65 protein and 3 immuno dominant sequences in gB protein were selected according to literature review. Peptide linker -GGGGS- was used for construction of fusion peptide. This fusion peptide was cloned in the pPICZαA expression vector and transfected into P. pastoris host cells. Results: Dot blot and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) techniques showed that a high level of pp65-gB-Fc fusion peptide was expressed. Conclusion: This CMV pp65-gB-Fc fusion peptide could be a promising candidate for the development of a novel peptide vaccine

Protective effects of BiP inducer X (BIX) against diabetic cardiomyopathy in rats

Diabetic cardiomyopathy (DC) is associated with impaired endoplasmic reticulum (ER) function, development of ER stress, and induction of cardiac cell apoptosis. Preventive effects of BiP inducer X (BIX) were investigated against DC characteristic changes in a type 2 diabetes rat model. To establish diabetes, a high-fat diet and a single dose of streptozotocin were administered. Then, animals were assigned into following groups: control, BIX, diabetic animals monitored for one, two, and three weeks. Diabetic rats treated with BIX for one, two, and three weeks. Expressions of various ER stress and apoptotic markers were assessed by immunoblotting method. CHOP gene expression was assessed by Real-time PCR. Tissue expression of BiP was evaluated by immunohistochemistry method. Hematoxylin and eosin and Masson's trichrome staining were performed to assess histological changes in the left ventricle. Cardiac cell apoptosis was examined using TUNEL assay. BIX administration suppressed the activation of the ER stress markers and cleavage of pro-caspase 3 in the diabetic rats. Likewise, tissue expression of BiP protein was increased, while CHOP mRNA levels were decreased. These results were accompanied by reducing cardiac fibrosis and myocardial cell apoptosis suggesting protective effects of BIX against the development of DC by decreasing cardiomyocyte apoptosis and fibrosis.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Hybrid promoters directed tBid gene expression to breast cancer cells by transcriptional targeting

Developing cancer gene therapy constructs based on transcriptional targeting of genes to cancer cells is a new and promising modality for treatment of cancer. Introducing truncated Bid (tBid), a recently known member of the Bcl-2 family, eradicates cancer cells efficiently. For transcriptional targeting of tBid, two dual-specificity promoters, combining cancer specific core promoters and response modules, were designed. These two core promoter modules contained cancer specific promoters of MUC1 and Survivin genes accompanied by hypoxia-responsive elements and estrogen responsive elements (microenvironment condition of breast cancer cells) which were employed to achieve a higher and more specific level of tBid expression in breast cancer cells. Correlation of the level of tBid expression in normal and cancer cell lines with promoter activity was measured by RT-PCR after treatment with hypoxia and estrogen. The level of tBid expression under control of new hybrid promoters was compared with its expression under control of cytomegalovirus (CMV) promoter as a control. Our data revealed that the level of tBid expression in breast cancer cells were nearly 11 times more than normal cells because of the cancer specific promoters, although tBid expression under control of CMV promoter was almost the same in normal and cancer cell lines. Increased apoptosis was detected in the transfected breast cancer cell lines by the Caspase-3 activity assay. The application of these promoters may prove to have the advantage of tumor selective gene therapy in breast cancer cells and low-potential toxicity for normal tissues

Expression of biologically active human colony stimulating factor-1 in Pichia pastoris

Colony Stimulating Factor-1 (CSF-1) is involved in proliferation, differentiation, and survival of the mononuclear lineage, in development of the female reproductive system and mammary glands during pregnancy and lactation. It is also implicated in the biology of breast cancer and promotion of its metastasis to bones. Therefore, CSF-1 is required for many applications in cellular and molecular biology studies. Commercial products, usually expressed in prokaryotic systems, are costly, with the likelihood of endotoxin contamination and also lack posttranslational modifications. These considerations provide the rationale to express growth factors in eukaryotic systems. In this study, the biologically active and soluble fragment (residues 33–182) of human (CSF-1) was cloned from K562 cell line and expressed in Pichia pastoris. The expression level of the active CSF-1 was about 100 μg/ml of the P. pastoris culture medium. Protein analysis revealed that the expressed CSF-1 appears in three bands with apparent molecular weight of 30, 26 and 20 kDa constituting 44%, 25% and 13% of all proteins in the culture medium, respectively. The expressed protein was partially purified and concentrated (10x) by ultrafiltration, then filter sterilized. The product was confirmed to be biologically active by stimulation of its receptor (FMS) autophosphorylation in THP-1 cells and also growth promotion of factor dependent FDC-P1 cells expressing human wild-type FMS (FD-FMS-WT). Therefore, P. pastoris is a highly efficient and cost-effective expression system for production of endotoxin-free CSF-1 for research and potentially for therapeutic applications

MicroRNA-16 is down-regulated in mutated FLT3 expressing murine myeloid FDC-P1 cells and interacts with Pim-1.

Activating mutations in the receptor tyrosine kinase FLT3 are one of the most frequent somatic mutations in acute myeloid leukemia (AML). Internal tandem duplications of the juxtamembrane region of FLT3 (FLT3/ITD) constitutively activate survival and proliferation pathways, and are associated with a poor prognosis in AML. We suspected that alteration of small non-coding microRNA (miRNA) expression in these leukemia cells is involved in the transformation process and used miRNA microarrays to determine the miRNA signature from total RNA harvested from FLT3/ITD expressing FDC-P1 cells (FD-FLT3/ITD). This revealed that a limited set of miRNAs appeared to be affected by expression of FLT3/ITD compared to the control group consisting of FDC-P1 parental cells transfected with an empty vector (FD-EV). Among differentially expressed miRNAs, we selected miR-16, miR-21 and miR-223 to validate the microarray data by quantitative real-time RT-PCR showing a high degree of correlation. We further analyzed miR-16 expression with FLT3 inhibitors in FLT3/ITD expressing cells. MiR-16 was found to be one of most significantly down-regulated miRNAs in FLT3/ITD expressing cells and was up-regulated upon FLT3 inhibition. The data suggests that miR-16 is acting as a tumour suppressor gene in FLT3/ITD-mediated leukemic transformation. Whilst miR-16 has been reported to target multiple mRNAs, computer models from public bioinformatic resources predicted a potential regulatory mechanism between miR-16 and Pim-1 mRNA. In support of this interaction, miR-16 was shown to suppress Pim-1 reporter gene expression. Further, our data demonstrated that over-expression of miR-16 mimics suppressed Pim-1 expression in FD-FLT3/ITD cells suggesting that increased miR-16 expression contributes to depletion of Pim-1 after FLT3 inhibition and that miR-16 repression may be associated with up-regulated Pim-1 in FLT3/ITD expressing cells

High-Level Expression of a Biologically Active Staphylokinase in Pichia Pastoris

<p>Staphylokinase (SAK) as the third generation thrombolytic molecule is a promising agent for the treatment of thrombosis.</p> <p>SAK variant of SAKфC was expressed in Pichia pastoris strains KM71H and GS115. The codon adaptation index of SAK was improved from 0.75 to 0.89. The expression of recombinant SAK (rSAK) reached to its maximum (310 mg/L of the culture medium) after 48 h stimulation with 3% methanol and remained steady until day 5. The maximum activity of the enzyme was at pH 8.6 and 37°C. It was highly active at temperatures 20-37°C and pH ranges of 6.8-9 (relative residual activity more than 80%). It was determined that rSAK was 73.8% of the total proteins secreted by P. pastoris KM71H into the culture media. The specific activities of rSAK was measured as 9002 U/mg, and 21042 U/mg for the non-purified and purified proteins, respectively. The quantity of the purified protein (>99% purity) was 720 µg/ml with a purification factor of 2.34. Western blot analysis showed two bands of nearly 22 and 18.6 kDa.</p> <p>It was concluded that P. pastoris is a proper host for expression of biologically active and endotoxin-free rSAK due to its high expression and low protein impurity in culture supernatant.</p