Effects of Co-chemotherapy Ethyl Acetate Fraction of Eurycoma Longifolia Jack Roots and Doxorubicin Against Apoptosis Through Expression P53 Mutant and Bcl-2

Background : It was found mutations of p53 gene in Breast cancer. Mutant p53 protein caused a decrease in cell apoptosis mechanisms through increased expression of Bcl-2. Breast cancer therapy is commonly used chemotherapy using Doxorubicin. However, effectiveness of the use of this chemotherapeutic agent is limited due to the emergence of side effects and toxic to normal cells. Therefore, it is necessary to develop new drugs for combination of chemotherapy. Eurycoma longifolia Jack roots has the potential as co-chemotherapy of breast cancer and it is not toxic to normal cells. Method : Rats were divided into 5 groups. Each group consisted of four female white Sprague Dawley rats. Group 1 (Normal), group 2 (DMBA 20 mg/kgB.W), group 3 (DMBA +Doxorubicin 1.12 mg/kgB.W), group 4 (DMBA +fractions 100 mg/kgB.W), group 5 (DMBA+Doxorubicin +fractions). All the rats were sacrificed at weeks 16 and to be taken their breast tissue. Immunohistochemistry was performed using a mouse monoclonal antibody mutant (BioGenex) and Bcl-2 (BIOSS). Results : expression of mutant p53 percentage obtained for group I (9.35 ± 0.32)%, II (21.65 ± 1.60)%, III (10.72 ± 2.52)%, IV (11.63 ± 3.39)%, V (12.72 ± 3.44)%, While the percentage of Bcl-2 expression obtained for I (20.62 ± 10.09)%, II (52.83 ± 3.61)%, III (24.38 ± 3.54)%, IV (38.01 ± 6.25)%, V (27.99 ± 4.27)%. The data was statistically tested by Kruskal Wallis test (p< 0.005). Conclussion : Co-chemotherapy of E. longifolia Jack roots and Doxorubicin can stimulate apoptosis through decreased in the expression of mutant p53 protein and Bcl-2 in breast tissue of rats induced by DMBA

Structure of the C-terminal domain of the Prokaryotic Sodium Channel Orthologue NsvBa

Crystallographic and electrophysiological studies have recently provided insight into the structure, function and drug binding of prokaryotic sodium channels. These channels exhibit significant sequence identities, especially in their transmembrane regions, with human voltage-gated sodium channels. However, rather than being single polypeptides with four homologous domains, they are tetramers of single domain polypeptides, with a C-terminal domain (CTD) composed of an inter-subunit four helix coiled-coil. The structures of the CTDs differ between orthologues. In NavBh and NavMs, the C-termini form a disordered region adjacent to the final transmembrane helix, followed by a coiled-coil region, as demonstrated by synchrotron radiation circular dichroism (SRCD) and double electron-electron resonance electron paramagnetic resonance spectroscopic measurements. In contrast, in the crystal structure of the NavAe orthologue, the entire C-terminus is comprised of a helical region followed by a coiled-coil. In this study we have examined the CTD of the NsvBa from Bacillus alcalophilus, which unlike other orthologues is predicted by different methods to have different types of structures: either a disordered adjacent to the transmembrane region, followed by a helical coiled-coil, or a fully helical CTD. To discriminate between the two possible structures we have used SRCD spectroscopy to experimentally determine the secondary structure of the C-terminus of this orthologue and used the results as the basis for modelling the transition between open and closed conformations of the channel

Differential lipid dependence of the function of bacterial sodium channels

The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative 22Na+ uptake into proteoliposomes containing a 3:1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different “guest” glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains

Mutagenesis of the NaChBac sodium channel discloses a functional role for a conserved S6 asparagine

Asparagine is conserved in the S6 transmembrane segments of all voltage-gated sodium, calcium, and TRP channels identified to date. A broad spectrum of channelopathies including cardiac arrhythmias, epilepsy, muscle diseases, and pain disorders is associated with its mutation. To investigate its effects on sodium channel functional properties, we mutated the simple prokaryotic sodium channel NaChBac. Electrophysiological characterization of the N225D mutant reveals that this conservative substitution shifts the voltage-dependence of inactivation by 25 mV to more hyperpolarized potentials. The mutant also displays greater thermostability, as determined by synchrotron radiation circular dichroism spectroscopy studies of purified channels. Based on our analyses of high-resolution structures of NaChBac homologues, we suggest that the side-chain amine group of asparagine 225 forms one or more hydrogen bonds with different channel elements and that these interactions are important for normal channel function. The N225D mutation eliminates these hydrogen bonds and the structural consequences involve an enhanced channel inactivation

NaChBac: The Long Lost Sodium Channel Ancestor

In excitable cells, the main mediators of sodium conductance across membranes are voltage-gated sodium channels (Na(V)s). Eukaryotic Na(V)s are essential elements in neuronal signaling and muscular contraction and in humans have been causally related to a variety of neurological and cardiovascular channelopathies. They are complex heavily glycosylated intrinsic membrane proteins present in only trace quantities that have proven to be challenging objects of study. However, in recent years, a number of simpler prokaryotic sodium channels have been identified, with NaChBac from Bacillus halodurans being the most well-characterized to date. The availability of a bacterial Na(V) that is amenable to heterologous expression and functional characterization in both bacterial and mammalian systems has provided new opportunities for structure--function studies. This review describes features of NaChBac as an exemplar of this class of bacterial channels, compares prokaryotic and eukaryotic Na(V)s with respect to their structural organization, pharmacological profiling, and functional kinetics, and discusses how voltage-gated ion channels may have evolved to deal with the complex functional demands of higher organisms

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228 tr. ; 19 cm

Design, production and characterisation of a thermally-stable mutant of the bacterial voltage gated sodium channel Nachbac

NaChBac from B. halodurans is a bacterial homologue of the eukaryotic voltage-gated sodium channels which has been expressed and purified from E. coli. We have previously shown (Nurani et al (2008) Biochemistry 31:8114-8121) that this membrane protein,, purified from E. coli, forms a mostly helical, tetrameric detergent-solubilisable protein that is capable of binding the drug mibefradil and inducing sodium flux when reconstituted into vesicles. The tetrameric quaternary structure of NaChBac differentiates it from the single-chain eukaryotic sodium channels. The aim of the present study was to produce a more thermally-stable version of this ion channel which would be suitable for a wide range of structural and functional studies. Using molecular modelling techniques, we have designed a mutant, G219S, which incorporates a serine instead of a glycine at the proposed site which is proposed to form the hinge which enables opening and closing of the channel. The aim was to reduce flexibility and “lock” the protein in a single state. Mutant protein was cloned, expressed and purified from E. coli and compared with the wild type protein isolated in the same manner. Whilst it had a similar secondary structure, thermal melting curves monitored by circular dichroism spectroscopy indicated that the mutant was considerably more stable than the wild type protein, although it is still capable of binding mibefradil. Thus the protein produced had the properties as designed and is a particularly suitable candidate for new structural, functional and drug binding studies

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220 tr. ; 19 cm

Kẻ đánh cắp thần linh

220 tr. ; 19 cm