Sreenivasa Rao Parcha

Enhanced Proliferation and Osteogenic Differentiation of Human Umbilical Cord Blood Stem Cells by L-Ascorbic Acid, In Vitro

The multilineage potentiality of cord blood stem cells has been experimentally proven in a number of cell based therapies. Umbilical cord blood (UCB) derived mesenchymal stem cells (MSCs), on prolonged exposure with Lascorbic acid have been successfully differentiated in to osteoblasts (bone forming cells) without altering the phenotype of the cells. In this case study, the role of L-ascorbic acid on collagen biosynthesis and mineral deposition in MSCs has been assessed, which are ultimately matured in to an insoluble extra cellular matrix (ECM), giving mechanical strength to the bone cells. Moreover, up to specific concentration of L-ascorbic acid (250μM), proliferation as well as differentiation potential of the cells remains unaltered. Further increase in concentrations of L-ascorbic acid (500 μM) reduced the cell proliferation and subsequently leads to morphological changes in the cultures. This may be due to an immature antioxidant defense system, which can be overcome by treating the cell cultures with antioxidants. Our final results conclude that Lascorbic acid has positive effect on the ostogenic differentiation of cord blood stem cells, and the concentration of ascorbic acid is vital in cell proliferation and differentiation.

Physical and degradation properties of PLGA scaffolds fabricated by salt fusion technique

Tissue engineering scaffolds require a controlled pore size and interconnected pore structures to support the host tissue growth. In the present study, three dimensional (3D) hybrid scaffolds of poly lactic acid (PLA) and poly glycolic acid (PGA) were fabricated using solvent casting/particulate leaching. In this case, partially fused NaCl particles were used as porogen (200-300µ) to improve the overall porosity (≥90%) and internal texture of scaffolds. Differential scanning calorimeter (DSC) analysis of these porous scaffolds revealed a gradual reduction in glass transition temperature (Tg) (from 48°C to 42.5°C) with increase in hydrophilic PGA content. The potential applications of these scaffolds as implants were further tested for their biocompatibility and biodegradability in four simulated body fluid (SBF) types in vitro. Whereas, simulated body fluid (SBF) Type1 with the optimal amount of HCO3− ions was found to be more appropriate and sensible for testing the bioactivity of scaffolds. Among three combinations of polymer scaffolds, sample B with a ratio of 75:25 of PLA: PGA showed greater stability in body fluids (pH 7.2) with an optimum degradation rate (9% to 12% approx). X-ray diffractogram also confirmed a thin layer of hydroxyapatite deposition over sample B with all SBF types in vitro.

STUDIES ON INDUSTRIALLY IMPORTANT ALKALINE PROTEASE PRODUCTION FROM LOCALLY ISOLATED SUPERIOR MICROBIAL STRAIN FROM SOIL MICROORGANISMS

In the present study, 50 microbial strains were isolated from the soil samples from different regions of Andhra Pradesh. Among the isolates hyper producing strain namely, Bacillus Clausii was selected for alkaline protease production. The protease production efficiency of the organisms was measured with different environmental and nutritional parameters. The optimum fermentation conditions of production were temperature 400C, pH 8 and time 32H. Fructose, peptone and Copper sulphate as good nutritional sources for producing higher yields of the enzyme. KeywordsAlkaline protease, Screening, Bacillus sp. Introduction Proteases are proteolytic enzymes that catalyze the breakdown of proteins by hydrolysis of peptide bonds. Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. Proteases represent one of the three largest groups of industrially important enzymes [4]. Bacteria are the most dominant group of alkaline protease Bacillus being the most relatively prominent and serve as an ideal source of these enzymes biotechnological importance [6, 14] because of their rapid growth and limited space required for their cultivation [1]. Among different types Acidic, neutral and alkaline proteases, alkaline protease plays very important role as most commonly used industrial enzyme in view of their activity and stability at alkaline pH. They are used in detergent formulations, food, pharmaceuticals, and leather, in film industry, medical use and by waste processing companies [2, [5-6], [8-9], 12, 15]. Multiple application of these enzymes stimulated interest to discover them with novel properties and considerable advancement of basic research into these enzymes. Proteases can be produced from wide diverse sources such as plants, animals and microorganisms. Protease can be produced by all microorganisms that produce a substantial amount of extracellular protease have been exploited commercially. Microbial proteases play a crucial role in numerous pathogenic processes mainly responsible for degradation of elastin, collagen, proteoglycons and also proteins that function in vivo host defence. Identification and characterization of microbial protease are prerequisite for understanding their role in pathogenesis. MATERIALS AND METHODS Isolation and Enzyme Production Soil samples from different regions of Andrapradesh were collected.1gm of soil sample was taken in 250ml conical flask containing 100 ml of sterilized water and contents were mixed well in an orbital shaker to get homogeneous suspension. The suspension is serially diluted 107 times and using streak plate technique the diluted samples are transferred to petri-dishes containing sterile skim milk agar medium. After inoculation the plates were incubated at 370C for 48 hours. After incubation bacterial colonies appearing over skim milk agar medium were identified based on colony characteristics and their identities were confirmed through Gram staining methods and by a series of biochemical tests as prescribed by Bergley manual. For enzyme production, strain was cultured in 250 ml of Erlenmeyer flask containing 100 ml culture medium, which consists of 10.0 g of glucose, 5.0 peptone g, 5.0 g yeast extract, 5.0 g K2HPO4, 0.1 g MgSO4.7H2O.The inoculated medium was placed in a thermostatic orbital shaker for 48 hrs at 37°C and 120 rpm. The culture was centrifuged at 10,000 rpm for 10 min to obtain crude enzyme. Studies on industrially important alkaline protease production from locally isolated superior microbial strain from soil microorganisms 103 International Journal of Biotechnology Applications ISSN: 0975–2943 & E-ISSN: 0975–9123, Vol. 3, Issue 3, 2011 Protease assay Alkaline protease activity was determined with a modification of the method [6].1ml of suitable diluted enzyme solution was added to 5 ml 0.5% (w/v) casein solution (dissolved in 50 mM Tris-HCL buffer with pH of 8 ) and incubated at 550C for 5 min. The reaction was terminated with 5 ml of 10% (w/v) trichloroacetic acid and the mixture filtrated through a filter paper. The filtrate absorbance was determined using Lowry method and extrapolated against a tyrosine standard curve. One unit of alkaline protease activity is defined as the amount of enzyme required to liberate 1 mg of tyrosine per minute under the experimental conditions. Optimization of production parameters for higher enzyme yield Effect of Nutritional parameters Nutritional parameters include nitrogen source, carbon source and metal ions. Firstly the respective nitrogen sources soya, malt extract, peptone, yeast extract, ammonium sulphate, ammonium chloride were added as a sole source of nitrogen (0.5%, w/v). The different carbon sources sodium acetate, sodium citrate, glucose, maltose, sucrose, starch and fructose were added as a sole source of carbon (1%, w/v). Different metal ions (Fe, Cu, Zn, Ag, Li and ca) were added as metal ion source (0.1% w/v). The enzyme activity was monitored for all different nutritional parameters after 24 h growth at 37 C. Effect of Environmental parameters Environmental parameters include pH (7-10), temperature (37-45) and time (0-72 H). The enzyme activity was monitored for all the above parameters in the respective ranges. Results and Discussion Isolation of bacteria Bacteria producing alkaline protease were isolated from soil by serial dilution techniques. Superior microbial strain having high productivity is selected from zone of hydrolysis assay. The isolated bacteria having maximum zone of hydrolysis “Fig. (1)” were identified through a series of biochemical tests (Table 1) as Bacillus sp for further confirmation the sample was sent to IMTECH Chandigarh and the biochemical characterization results were shown in Table 1. Effect of Nutritional parameters The effect of different nutritional parameter on enzyme production was represented graphically “Fig. (2 a, b, c)” below. Among different carbon sources tested fructose has significant positive impact on both growth as well as higher enzyme yields. Maltose and glucose also had positive impact comparatively where as starch, sucrose had very less impact and sodium citrate, sodium acetate almost had no significant impact on both growth as well as enzyme yield. Organic carbon source have been found to be better carbon source for growth and protease yield [3, 10]. Among different nitrogen sources organic nitrogen sources showed significantly increased activity where as inorganic nitrogen sources showed very low almost negligible activity. The negative effect of inorganic nitrogen sources on protease production by Bacillus sp. has been observed in earlier investigations [15]. The repression of protease biosynthesis may be ascribed to the release of ammonia from these inorganic nitrogen sources. Among the various metal ions tested Ag, Cu, Li and Zn showed high enzyme activity and maximum activity was observed using Cu and Ag ions. Where as the activity was comparatively low for Ca and Fe ions. Effect of Environmental parameters The enzyme production was observed between temperature (37-450C) and pH 7.0-10.0 “Fig. (3 a, b )”. The highest enzyme activity was observed at 400C and also comparatively high activity was also observed at 370C, decrease in enzyme activity is observed with increase in temperature above 40 0C. The highest enzyme activity was obtained at pH 9 and comparatively higher enzyme activity is also observed at 8. However, further increase in pH was not favourable on enzyme production. It was reported that proteases secreted by Bacillus sp. presented activity at a wide range of pH (7.0 to 11.0) and temperature (30°C to 60°C) [6-8]. The growth curve of the bacillus species “Fig. (3 c)” was observed and activity at every 4h intervals and activity along with growth increase in the exponential phase and maximum activity is observed in the stationary phase at 32 h, The production of an extra cellular proteolytic enzyme during the stationary phase of growth is a characteristic of many bacterial species [11, 13] and the activity decreased along with growth in the decline phase. Conclusion Bacillus clausii can be used profitably for large scale production of alkaline protease to meet the present day demand of the industrial sector. Fructose, peptone and Cu best nutritional parameters for an enhanced enzyme production with optimum range of pH (8-9) and temperature (37-400C). Hence, further analysis on these medium components with reference to their specific role in the induced production of the enzyme is a very much necessary for motivation of further studies. References [1] Arulmani M., Aparanjini K., Vasanthi K., Arumugam P., Arivuchelvi M.and Kalaichelvan P.T. (2007) World J. Microbiol. Biotechnol., 23, 475-481 [2] Banerjee C.U. Sani R.K., Azmi W., and Soni R.(1999) Proc. Biochem., 35, 213-219. [3] Berhor R.W. and Novelli C.D.(1963) Arch. Biochem. Biophys., 103, 94-97. Swapna Vadlamani, Sreenivasa Rao Parcha 104 Bioinfo Publications [4] Chu W.H (2007) Journal of Industrial Microbiology Biotechnology, 34, 241-245. [5] Dias D.R., VilelaDM., Silvestre M.P.C., Schwan R.F. (2008) World Journal of Microbiology and Biotechnology, 24, 2027-2034. [6] Gupta R., Beg Q.K., andLorenz P. (2002) Applied Microbiology and Biotechnology, 59, 15-32. [7] Horikoshi K. (1999) Microbiology and Molecular Biology Reviews ,63, 735-750. [8] Joo H.S., Kumar C.G., Park G.C., Kim K.T., Paik S.R. and Chang C.S. (2002) Process Biochemistry, 38, 155-159. [9] Joo H.S., Kumar C.G., Park G.C., Paik S.R. and Chang C.S. (2003) Journal of Applied Microbiology, P5, 267-272. [10] Kitada M. and Horikoshi R. (1976)Can. J.Microbiol., 54, 383-385. [11] Mandelstam J. (1960) Bacteriol. Rev., 24, 289 293. [12] Naja M.F. and Deobagkar D. (2005)Electron. J. Biotechnol., 8, 198-207. [13] Pollock M.R. (1962)The Bacteria Dunsalus, Acad. Press, New York(1962). [14] Ramakrishna D.P.N., Gopi N.R. and Rajagopal S.V. (2010) Intl. J. B

Osteoblast differentiation of umbilical cord blood-derived mesenchymal stem cells and enhanced cell adhesion by fibronectin

Mononuclear cells isolated from human umbilical cord blood were differentiated in to spindle shaped fibrous mesenchymal stem cells (MSCs) in DMEM with 10% fetal bovine serum. These progenitor cells were further examined for their ability to differentiate into osteoblasts by culturing them in osteogenic differentiation media (Stem Pro®). Calcium mineralization assay, alizarin red S, von kossa staining confirmed that MSCs from cord blood were capable of mineralization when they were cultured in osteogenic medium. The adhesion studies shown that MSCs have greater affinity towards fibronectin (FN) coated dishes, compared to albumin (Alb) coated dishes due to CD29and CD44 surface receptors.

Combination of ERG9 Repression and Enzyme Fusion Technology for Improved Production of Amorphadiene in Saccharomyces cerevisiae

The yeast strain (Saccharomyces cerevisiae) MTCC 3157 was selected for combinatorial biosynthesis of plant sesquiterpene amorpha-4,11-diene. Our main objective was to overproduce amorpha 4-11-diene, which is a key precursor molecule of artemisinin (antimalarial drug) produced naturally in plant Artemisia annua through mevalonate pathway. Farnesyl diphosphate (FPP) is a common intermediate metabolite of a variety of compounds in the mevalonate pathway of yeast and leads to the production of ergosterols, dolichol and ubiquinone, and so forth. In our studies, FPP converted to amorphadiene (AD) by expressing heterologous amorphadiene synthase (ADS) in yeast. First, ERG9 (squalane synthase) promoter of yeast was replaced with repressible methionine (MET3) promoter by using bipartite gene fusion method. Further to overcome the loss of the intermediate FPP through competitive pathways in yeast, fusion protein technology was adopted and farnesyldiphosphate synthase (FPPS) of yeast has been coupled with amorphadiene synthase (ADS) of plant origin (Artemisia annua L.) where amorphadiene production was improved by 2-fold (11.2 mg/L) and 4-fold (25.02 mg/L) in yeast strains YCF-002 and YCF-005 compared with control strain YCF-AD (5.5 mg/L), respectively.

Study on Osteoblast like Behavior of Umbilical Cord Blood Cells on Various Combinations of PLGA Scaffolds Prepared by Salt Fusion

The osteogenic potential of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) on porous poly lactide-co-glycolide (PLGA) scaffolds have been reported to differentially support osteogenic differentiation based on polymer composition (80:20, 75:25 and 70:30 percent of PLA: PGA, respectively). Along with polymer composition; fused NaCl crystal matrix prior to solvent casting improves the porosity and pore interconnectivity in 3D scaffolds, which has significant impact on cell proliferation. FTIR and XRD studies of PLGA scaffolds also verified the intermolecular interactions, phase distribution and crystallinity in scaffolds. Among three scaffold combinations, sample B (75:25) has showed maximum porosity with optimum water uptake/retention abilities. Impact of polymer composition and porosity on cell proliferation was investigated through MTT assay, where sample B was observed to be supporting better cell proliferation,due to its internal structure. The above results were further confirmed by ALP and Col-I gene expression studies using RT-PCR. Immuno fluorescent studies also revealed the extracellular filamentous actin organization over the scaffolds, where cell adhesion and proliferation was found to be higher with increase in PGA content, which is a hydrophilic polymer.

Development of Petri Net-Based Dynamic Model for Improved Production of Farnesyl Pyrophosphate by Integrating Mevalonate and Methylerythritol Phosphate Pathways in Yeast

In this case study, we designed a farnesyl pyrophosphate (FPP) biosynthetic network using hybrid functional Petri net with extension (HFPNe) which is derived from traditional Petri net theory and allows easy modeling with graphical approach of various types of entities in the networks together. Our main objective is to improve the production of FPP in yeast, which is further converted to amorphadiene (AD), a precursor of artemisinin (antimalarial drug). Natively, mevalonate (MEV) pathway is present in yeast. Methyl erythritol phosphate pathways (MEP) are present only in higher plant plastids and eubacteria, but not present in yeast. IPP and DAMP are common isomeric intermediate in these two pathways, which immediately yields FPP. By integrating these two pathways in yeast, we augmented the FPP synthesis approximately two folds higher (431.16 U/pt) than in MEV pathway alone (259.91 U/pt) by using HFPNe technique. Further enhanced FPP levels converted to AD by amorphadiene synthase gene yielding 436.5 U/pt of AD which approximately two folds higher compared to the AD (258.5 U/pt) synthesized by MEV pathway exclusively. Simulation and validation processes performed using these models are reliable with identified biological information and data.

Sponge Biomass for the Development of Biomedical Products and Their Applications

Advancements in the areas of natural biomaterials are not a new branch of science, and it has existed from many decades. The application of natural materials as biomaterials is recently undergoing a revival in the biomedical engineering. The major limitations of natural biomaterials are due to the uncontrolled degradation that can occur following in vivo implantation and a lot of variabilities in molecular structure associated with animal sourcing. The main applications of natural biomaterials as materials in medicine are, namely, tissue engineering, wound management products, and drug delivery systems. In recent times, a significant number of biomaterials with biomedical significance have been discovered from the sponges. This book chapter is going to enlighten few important outcomes and their applications in the field of biomedical sciences.

Molecular Level Characterization of L-Ascorbic Acid Induced Osteoblasts from Umbilical Cord Blood Source

Mesenchymal stem cells (MSCs) isolated from umbilical card blood were capable of differentiated in to multiple mesenchymal cell lineages. The objective of the current study was to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon differentiation. In our previous studies, MSCs cultured in varying concentrations of L-ascorbic acid (50 μM to 500 μM) had shown the various levels of osteoblast differentiation, as determined by morphology, alkaline phosphatase activity, modulations in osteocalcin mRNA expression, and mineralized extracellular matrix (ECM) deposition holding inorganic hydroxyapatite (HA) and Tricalcium phosphate (TCP). In present study, we noticed a study increase in APase activity up to 250 μM L-ascorbic acid and by end of 16 days, cells gained cuboidal morphology, which is characteristic feature of osteoblasts. The intense sharp difractograms (peaks) between 30°–35° demonstrate the deposition of HA and TCP, which are major inorganic components of bone. The reproducible results of our studies provided a useful model for evaluating the factors responsible for the stepwise progression of cells from undifferentiated precursors to secretary osteoblasts, and eventually terminally differentiated osteocytes.