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Biological activity of plant derived extracts against cancer tissue
cultures
Tova Cohen, Marina Tevrovski, Michal Maoz
Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9901901, Fax: 972-4-9901738, E-mail: tocohen@braude.ac.il, mmaoz@braude.ac.il Keywords: Tissue cultures, plant extracts, synergism
The general public is interested in naturally derived protectants and drugs. There are diverse arrays of possible candidates; our research is concentrated on Inula viscosa extract and cinnamaldehyde. An antifungal activity of these extracts was proved against wood decay fungi and against dermatophytes, each extract by itself and in various mixtures (synergism). The anticancer activity of Inula viscosa extract and cinnamaldehyde is tested against 3 carcinogenic cell lines: RMA/OVA, BO-7911 and J744. Synergism phenomena of these extracts are also tested. The procedure involves treating cancer cells with different concentrations and then assessing cell death after 4 and 24 hours under 5% CO2 at 37°C. Cytotoxicity was assessed using the XTT test. Preliminary results showed that both extracts caused 50% cell death. The Inula viscosa extract toxicity is concentration dependant. Acknowledgement: This study was supported by a grant from the ORT Braude College
research committee
The 4th ORT Braude Interdisciplinary Research Conference 62 The effect of the Insulin Sensitizing Agents (ISA) on "Protein C" and
on Paraoxonase mRNA expression in rats with fatty liver
Masha Grozovski1, Sigal Korem2, Geni Lipshiz3, Nimer Assy4
1Department of Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9901895, Fax: 972-4-9901839, E-mail: mgrozovs@braude.ac.il 2Department of Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9901891, Fax: 972-49901839, E-mail: sigalc@ort.org.il 3Department of Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-2561526, Fax: 972-49901839, E-mail: jennylipets@walla.com 4Liver Units, Sieff Hospital, Safed Israel, Tel: 972-468284413, Fax: 972-45828442, E-mail: Assy.n@ziv.health.gov.il Keywords: Fatty liver, rats, FED diet, rosiglitazone, metformin, lipid, Paraoxonase, MDA
Non-alcoholic liver disease (NAFLD) is a component of the metabolic syndrome, with a clinical spectrum ranging from fatty liver to steatohepatitis, cirrhosis, and hepatocellular carcinoma. The primary event of NAFLD is the accumulation of TG in hepatocytes due to insulin resistance and therapeutic modalities for this condition are needed. This research focuses on determining whether there is a relationship between NAFLD and Protein C and Paraoxonase levels in quiescent and in regenerating rats fatty liver, and on determining the effect of Insulin Sensitizing Agents (ISA) on hepatic "protein C" and paraoxonase mRNA expression. Methods: Forty-eight Sprague-Dawley rats were treated with a fructose-enriched diet (FED), or
FED with metformin (200mg/Kg/Day), FED with rosiglitazone (3 mg/kg/Day), or with a
combination of metformin (200mg/Kg/Day) and rosiglitazone (3mg/Kg/Day) for a total of 5
weeks. 30% partial hepatectomy (PHX) was performed at the end of week 5 of the experiment.
Protein C and paraoxonase mRNA expressions, liver lipids, MDA, α-tocopherol and hepatic fat
content were measured before and 24 hours after PHX.
Results: At base line, hepatic protein C mRNA expression was significantly higher in rats with
fatty liver than control rats (37.0 ± 0.4 Vs 18.6 ± 1.0, +105%, p<0.01) whereas hepatic
paraoxonase mRNA levels were significantly lower in rats with fatty liver than control rats
(55.0±7.0 Vs 77.0±9.0, - 28%, p<0.005). At 24 hours after PHX , the extent of both hepatic
protein C and paraoxonase mRNA expression increased significantly in rats with fatty liver in
regeneration by +116%, p<0.01, and by +15%, (p<0.01) respectively. During peak liver
regeneration (24 h post PHX), metformin, rosiglitazone, and their combination decreased
hepatic expression of protein C by -50%, -20%, and by -170% (p<0.001) and increased
paraoxonase mRNA level by +51%, +23%, and +50% (p<0.01) respectively. Serum
paraoxonase level correlates with serum protein C (r = -0.2), MDA (r = 0.4), TG (r = -0.23),
and RBP 4 serum level (r = - 0.12) respectively.
Conclusion: In rats with NAFLD hepatic protein C mRNA levels are high at baseline, up-
regulated during liver regeneration and decrease after treatment with (ISA) whereas hepatic
paraoxonase mRNA levels are low at baseline, up regulated during liver regeneration and
increase after treatment with ISA. These findings suggest that protein C and paraoxonase may
be related to the pathogenesis of NAFLD.
Acknowledgement: This study was supported by a grant from the ORT Braude College
research committee.
The 4th ORT Braude Interdisciplinary Research Conference 63 Molecular modelling of gelatin and chondroitin sulphate interactions
in aqueous medium
Dafna Knani1, Maya Shilo1, Sivan Zagreson1
1Department of Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9901875, Fax: 972-4-9901839, E-mail: dknani@braude.ac.il Keywords: chondroitin sulphate, gelatin, molecular modeling
Gelatin and chondroitin sulphate tend to form microspheres which can be used for encapsulation of water insoluble bioactive materials, especially drugs*. The microspheres are formed by complex coacervation - a spontaneous phase separation process that occurs, under certain conditions, when oppositely charged polyelectrolytes are mixed in aqueous medium. The formation and size of the microspheres depend on various parameters, such as reactant concentration, pH and temperature. In the present study, we have investigated the interactions between gelatin (positively charged) and chondroitin sulphate (CS) (negatively charged) using computational methods. Simulation of gelatin, chondroitin sulphate and water, and their binary mixtures in the condensed phase was performed using "Amorphous Cell" module (by Accelrys) at various temperatures and molar ratios. CS was constructed as an oligomer of 5 repeating units and was assigned an electric charge of (-10). Gelatin was constructed as a typical segment of 9-amino acids peptide, and was assigned an electric charge of (+2). Molecular dynamics (MD) simulations of both charged molecules and of water molecules have been performed, and cohesive energy density (CED) was obtained. CED is used to compute solubility parameter and Flory-Huggins interaction parameter. The interaction parameter, defined as (ΔEmix/V)/RT, is used as a quantitative measure for miscibility (negative values of χ) or immiscibility, i.e. phase separation (positive values of χ). χ was calculated for the binary mixtures gelatin-chondroitin sulphate (1:1 , 1:5 and 5:1 molar ratios), chondroitin sulphate–water and gelatin–water at 3040K (310C). An additional analysis tool is Pair Correlation Function, which is a measure of the probability that a defined atom is located in a spherical shell at a distance r from another defined atom. The pair correlation function gives an insight as to how the atoms pack in an amorphous structure and is calculated for various pairs of atoms of molecules, such as between a sulfur atom of sulphate group or a carbon atom of carboxylate group and a nitrogen atom of amino groups of gelatin. *Azhari R. and Leong K., “Controlled release of pharmaceutically active substances from coacervate microcapsules”, US patent US5759582, issued June 1998. The 4th ORT Braude Interdisciplinary Research Conference 64 Conductivity of an alkaline glucose fuel cell
Lea Mor1, Zeev Rubin2
1Department of Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9901757, Fax: 972-4-9901839, E-mail: leamor@ort.org.il 2Physics Unit, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9953206, E-mail: zrubin@braude.ac.il Keywords: Fuel cell, conductivity
In recent years, appreciable efforts have been invested in studying and reducing fuel cell degradation. Such degradation is customarily demonstrated through increased bulk and/or electrodes resistance. In an alkaline glucose fuel cell, changes with glucose and KOH concentrations may increase the bulk resistance with time due to unfavourable chemical reactions,. The KOH, glucose and intermediate reactions products were monitored using titration and HPLC analysis of the control solution and fuel cell solution. The relative contribution of the bulk and the electrodes to resistance was verified by measuring separately the solution conductivity and the electrical resistance. The solution conductivity was measured for control solutions having various glucose and KOH concentrations, as well as for the bulk solution in the fuel cell, operated in open or closed modes. The electrical resistance of the fuel cell was measured for both open and closed modes. Both the solution conductivity and the electrical resistance were measured over several days of operating the fuel cell. The results show the contribution of glucose to the resistivity of the glucose alkaline fuel cell. Conductivity increases linearly with KOH concentration (as expected) and decreases both in the fuel cell and in the control solutions with glucose concentration. The resistivity is linear with glucose concentration, and decreases over time. The HPLC analysis pointed out differences in the chemical reactions’ intermediated form in the control solution and fuel cell solution. The solution conductivity was interpreted using a model for collisions of ions and molecules in solutions under the influence of an electrical field. Using this model, the conductivity could be expressed as a function of the concentration, electrical charge and effective size of the various species in solution. Both the HPLC analysis and the theoretical representation emphasized the effect of the fuel cell electrodes on the chemical reaction conducted in the fuel cell. Acknowledgement: This study was supported by a grant from the ORT Braude College
research committee.
The 4th ORT Braude Interdisciplinary Research Conference 65 Tissue engineering of skeletal muscle tissue on hybrid scaffolds
Rosa Azhari1, Ehud Kroll2, Eyal Zussman3
1Department of Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9901750, Fax: 972-4-9901839, E-mail: razhari@braude.ac.ill 2Department of Mechanical Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9901756, Fax: 972-4-9901886, E-mail: ekroll@braude.ac.il 3Faculty of Mechanical Engineering, Technion - Israel Institute of Technology, Technion City, Haifa 32000, Israel, Tel: 972-4-8292836, Fax: 972-4-8324533, E-mail: meeyal@tx.technion.ac.il Keywords: Tissue engineering, scaffold, electrospinning, skeletal muscle
Tissue engineering (TE) aims at producing bioartificial tissue to replace damaged or disfunctioning tissue and minimize the need for donor-originated tissue. One of the major obstacles in tissue engineering is the sensitivity of the cells to the growth environment which can induce major differences between tissues originating from the same cells grown under different biochemical or mechanical environments. In common TE practices the microenvironment is determined by the composition and structure of the scaffold on which the cells are seeded, the medium used, biochemical factors in the medium, the mechanical environment, the bioreactor used and the parameters of its operation. In the current research we study the effect of different scaffolds and mechanical environments on skeletal muscle tissue production. Electrospun, hybrid micro-fibrous scaffolds have been produced, combining a synthetic polymer, polycaprolactone (PCL), with natural connective tissue components, chondroitin sulfate and gelatin. Scaffolds made of randomly oriented PCL fibers (~1μm diameter), aligned PCL fibers and hybrid components had ultimate tensile strengths of 3, 12 and 20 MPa respectively, and maximal elongations of 170%, 63% and 70% respectively. Skeletal muscle cell (C2 myoblasts) affinities and tissue expression were higher on hybrid scaffolds when compared to PCL ones. A computer-controlled stretching machine was built to induce various accurate, quantitative stretching patterns on the cell–scaffold constructs while measuring cell growth, tissue expression, tissue organization and mechanical properties of the skeletal muscle tissue obtained. Acknowledgement: This study was supported by a grant from the ORT Braude College
research committee.
The 4th ORT Braude Interdisciplinary Research Conference 66 Production of immuno-stimulating microspheres by complex
coacervation
Rosa Azhari1, Ditza Levin2, Ariel Doron3
1Department of Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9901750, Fax: 972-4-9901839, E-mail: razhari@braude.ac.ill 2Department of Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel, Tel: 972-4-9901924, Fax: 972-4-9901839, E-mail: dlevin@braude.ac.il 3Department of Biotechnology Engineering, ORT Braude College, P.O. Box 78, Karmiel 21982, Israel. Keywords: Vaccine, microspheres, complex coacervation, hyaluronic acid
New potent and tailor-made adjuvants for antigen (Ag) delivery are widely studied in order to facilitate the development of new vaccines to infectious diseases, cancer immunotherapy and control of autoimmunity and allergy. In previous studies we have shown that microspheres (MS), produced by complex coacervation of chondroitin sulfate (CS) and gelatin (Ge), have adjuvant properties and can induce long term humoral and cellular responses following oral or parenteral administration. The MS efficiently deliver the encapsulated Ag to Dendritic cells (DC) and macrophages (MQ), which present it to CD4 Th cells. B cells are poor antigen presenting cells (APC) of the encapsulated Ag. The encapsulation also mediates non- conventional presentation of exogenous Ag to CD8 T cells by MQ and, to a lesser extent, by DC. The MS target the Ag to these two APC, In-vivo. Hyaluronic acid (HyA) has been shown to have immuno-stimulating effects, dependent on its molecular weight. In the current study, microspheres were produced by complex coacervation of gelatin with hyaluronic acid fragments of different sizes. The presentation of antigen (OVA), encapsulated in HyA-Ge MS, to an OVA-specific hybridoma (BO-97-11), using a line of dendritic cells (DC2.4) as APC, was studied and compared to that of soluble OVA and OVA encapsulated in CS-Ge MS. Three fragment-size distributions were obtained by incubation of HyA with 50u/ml hyaluronidase (Hyase) for 1 hr (HyA 1), 20u/ml for 5 hrs (HyA 2) and 5u/ml for 1 hr (HyA 3). The effect of initial concentration of HyA and Ge on MS production was studied. No microspheres were produced under the current conditions with the HyA1 fragments. The amount of coacervate produced with HyA2 or HyA3 varied with the initial concentrations of HyA and Ge. Microspheres ranging in size from 1-10μm were obtained in the complex coacervation process. At the conditions chosen for production of MS for antigen presentation, MS obtained were very uniform with diameters of 0.5-2μm (HyA2-Ge MS) and 1-4μm (HyA3-Ge MS). OVA loading was more than 50% of MS dry weight. Incubation of an OVA-specific CD4 T hybridoma (BO-97-11) with OVA, encapsulated within HyA-Ge MS, in the presence of APC (DC2.4 dendritic cells) induced IL-2 secretion, which indicates successful presentation of the antigen to the hybridoma cells. Stimulation was dependent on HA fragment size distribution. Acknowledgement: This study was supported by a grant from the ORT Braude College
research committee.
The 4th ORT Braude Interdisciplinary Research Conference 67

Source: http://conferences.braude.ac.il/brdresearch/brdresearch04/files/BiotechnologyEngr.pdf

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