Sodium Dodecyl Sulfate Gel Electrophoresis Research Articles

Immunogenicity assay of KatG protein from Mycobacterium tuberculosis in mice: preliminary screening of TB vaccine

the tuberculosis (tB) disease is still widely found even though BCg vaccine given to many people. Ineffectiveness of the BCG vaccine is one of causes that make the difficulties in preventing TB transmission. objective of the research was to determine the immunogenicity of katg protein of M. tuberculosis clinical iso-late L19 in mice. the katg protein as antigen was prepared by expression of the katg gene of M. tuberculosis clinical isolate L19 in Escherichia coli BL21 using pColdII-DNA vector. After purification by affinity chromatography, the katg was vaccinated to mice to detect its immunogenicity. the expression of katg in e.coli BL21 could result in katg protein with molecular weight 80 kda in sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The pure KatG protein could significantly stimulate the immune response of mice by triggering the antibodies production of Igg1, Igg2a, Igg2b, Igg2c, Igg3, and IgM. the highest antibody level was obtained when the mice were vaccinated by KatG L19 with the dose of 45 μg/ml. Of the antibodies, the IgG2c isotype was dominantly produced in the blood serum. the katg protein exhibited a high immunogenici ty in mice, so it is possible to develop as a vaccine candidate for tB. a clinical test should be performed in a future to ensure its safety as a therapeutic protein.

Characterization of the T-cell response to polypropylene mesh in women with complications

Polypropylene mesh is used widely for surgical treatment of pelvic organ prolapse and stress urinary incontinence. Although these surgeries demonstrate favorable functional and anatomic outcomes, their use has been limited by complications, the 2 most common being exposure and pain. Growing evidence suggests that T lymphocytes play a critical role in the regulation of the host response to biomaterials. The purpose of this study was to define and characterize the T-cell response and to correlate the response to collagen deposition in fibrotic capsules in mesh tissue complexes that are removed for the complications of pain vs exposure. Patients who were scheduled to undergo a surgical excision of mesh for pain or exposure at Magee-Women's Hospital were offered enrollment. Forty-two mesh-vagina tissue complexes were removed for the primary complaint of exposure (n=24) vs pain (n=18). Twenty-one patients agreed to have an additional vaginal biopsy away from the site of mesh that served as control tissue. T cells were examined via immunofluorescent labeling for cell surface markers CD4+ (Th), CD8+ (cytotoxic) and foxp3 (T-regulatory cell). Frozen sections were stained with hematoxylin-eosin for gross morphologic condition and picrosirius red for collagen fiber analysis. Interrupted sodium-dodecyl sulfate gel electrophoresis was used to quantify the content of collagens type I and III, and the collagen III/I ratio. Transforming growth factor-β and connective tissue growth factor, which are implicated in the development of fibrosis, were measured via enzyme-linked immunosorbent assays. Data were analyzed with the Student's t tests, mixed effects linear regression, and Spearman's correlation coefficients. Demographic data were not different between groups, except for body mass index, which was 31.7 kg/m2 for the exposure group and 28.2 kg/m2 for pain (P=.04). Tissue complexes demonstrated a marked, but highly localized, foreign body response. We consistently observed a teardrop-shaped fibroma that encapsulated mesh fibers in both pain and exposure groups, with the T cells localized within the tip of this configuration away from the mesh-tissue interface. All 3 T-cell populations were significantly increased relative to control: CD4+ T helper (P<.001), foxp3+ T regulatory (P<.001), and CD8+ cytotoxic T cell (P=.034) in the exposure group. In the pain group, only T-helper (P<.001) and T-regulatory cells (P<.001) were increased, with cytotoxic T cells (P=.520) not different from control. Picrosirius red staining showed a greater area of green (thin) fibers in the exposure group (P=.025) and red (thick) fibers in the pain group (P<.001). The ratio of area green/(yellow+ orange+ red) that represented thin vs thick fibers was significantly greater in the exposure group (P=.005). Analysis of collagen showed that collagen type I was increased by 35% in samples with mesh complications (exposure and pain) when compared with control samples (P=.043). Strong correlations between the profibrosis cytokine transforming growth factor-β and collagen type I and III were found in patients with pain (r≥0.833; P=.01) but not exposure (P>.7). T cells appear to play a critical role in the long-term host response to mesh and may be a central pathway that leads to complications. The complexity of this response warrants further investigation and has the potential to broaden our understanding of mesh biology and clinical outcomes.

Evaluation of commercially available styrene-co-maleic acid polymers for the extraction of membrane proteins from spinach chloroplast thylakoids

Solubilization of membrane proteins by poly(styrene-co-maleic acid) salts (pSMA-S) has significant potential for membrane protein studies. This approach provides an opportunity to overcome many disadvantages associated with a traditional detergent-based technique including protein denaturation and displacement of boundary lipids which may offer both structural and functional stability to membrane proteins. Thylakoid membranes (TMs) from photosynthetic organisms are well studied protein-rich membranes that host several multi-subunit protein complexes associated with oxygenic photosynthesis. These protein complexes are important for applied photosynthesis and by being extracted and purified they can be used in the near future for direct energy conversion. In this study, we used spinach TMs isolated from purified intact chloroplasts to systematically test the solubilization efficacy of 12 commercially available styrene-maleic acid (SMA) copolymers that vary in size, styrene-to-maleic acid molar ratio, and type of ester group. The efficacy of these pSMA-S to solubilize protein-containing biomembranes was evaluated via quantification of protein and chlorophyll content in the resulting SMA Lipid Particles (SMALPs). In addition, the extracted polymer-lipid-protein complexes were studied by low temperature fluorescence, sodium dodecyl sulfate and clear native polyacrylamide gel electrophoresis (SDS- and CN-PAGE), and immunoblot analysis. Our results indicate considerable variability in the solubilization efficacy of commercially available pSMA-S with at least 5 polymer formulations being able to efficiently extract membrane proteins from TMs. These 5 SMA copolymers may also be effective in extraction of membrane proteins from other biomembranes.

Direct recovery of Bacillus subtilis xylanase from fermentation broth with an alcohol/salt aqueous biphasic system

Xylanase enzyme degrades linear polysaccharide β-1,4 xylan and the hemicellulose of the plant cell wall. There is a growing demand in finding a cost-effective alternative for industrial scale production of xylanase with high purity for pharmaceutical applications. In this study, an alcohol/salt aqueous biphasic system (ABS) was adopted to recover xylanase from the Bacillus subtilis fermentation broth. The effects of several ABS parameters such as types and concentrations of alcohols and salts (i.e., sulphate, phosphate, and citrate), amount of crude loading and pH of the system on the recovery of xylanase were investigated. Partition coefficient of xylanase (KE), selectivity (S) and yield (YT) of xylanase in top phase of the ABS were measured. Highest KE (6.58±0.05) and selectivity (4.84±0.33) were recorded in an ABS of pH 8 composed of 26% (w/w) 1-propanol, 18% (w/w) ammonium sulphate. High YT of 71.88%±0.15 and a purification fold (PFT) of 5.74±0.33 were recorded with this optimum recovery of xylanase using alcohol/salt ABS. The purity of xylanase recovered was then qualitatively verified with sodium dodecyl sulphate (SDS) gel electrophoresis. The SDS profile revealed the purified xylanase was successfully obtained in the top phase of the one-step 1-propanol/sulphate ABS with a distinct single band.

Exploration of the antibacterial proteins in pearl oyster Pinctada fucata induced by bacterial inoculation

The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 1012 colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion.

Amylase Zymography.

Amylase zymography was carried out for the detection of amylases produced by a Geobacillus stearothermophilus strain isolated from the Thermal Center "Las Trincheras" in Venezuela. Zymography is an electrophoretic technique used to study hydrolases by means of thin gels containing copolymerized-specific substrates, under nonreducing conditions. In this study, 0.1% starch was incorporated into the gel as substrate. The formation of clear zones against a dark background in the gel stained with iodine indicated the presence of amylolytic activity. The thermophilic bacteria released several extracellular amylases to a selective growth medium supplemented with 1% soluble starch at 55 °C after 40 h incubation. The amylolytic enzymes showed an optimum temperature of 60 °C and an optimum pH at 6.0. The amylases were partially purified by cold acetone precipitation followed by two chromatographic techniques. These purified amylases showed different molecular masses which were determined by sodium dodecyl sulfate gel electrophoresis and confirmed by zymography.

Characterization of airag collected in Ulaanbaatar, Mongolia with emphasis on isolated lactic acid bacteria.

BackgroundAirag, alcoholic sour-tasting beverage, has been traditionally prepared by Mongolian nomads who naturally ferment fresh mares’ milk. Biochemical and microbiological compositions of airag samples collected in Ulaanbaatar, Mongolia and physiological characteristics of isolated lactic acid bacteria were investigated.MethodsProtein composition and biochemical composition were determined using sodium dodecyl sulfate-gel electrophoresis and high performance liquid chromatography, respectively. Lactic acid bacteria were identified based on nucleotide sequence of 16S rRNA gene. Carbohydrate fermentation, acid survival, bile resistance and acid production in skim milk culture were determined.ResultsEquine whey proteins were present in airag samples more than caseins. The airag samples contained 0.10–3.36 % lactose, 1.44–2.33 % ethyl alcohol, 1.08–1.62 % lactic acid and 0.12–0.22 % acetic acid. Lactobacillus (L.) helveticus were major lactic acid bacteria consisting of 9 isolates among total 18 isolates of lactic acid bacteria. L. helveticus survived strongly in PBS, pH 3.0 but did not grow in MRS broth containing 0.1 % oxgall. A couple of L. helveticus isolates lowered pH of skim milk culture to less than 4.0 and produced acid up to more than 1.0 %.ConclusionHighly variable biochemical compositions of the airag samples indicated inconsistent quality due to natural fermentation. Airag with low lactose content should be favorable for nutrition, considering that mares’ milk with high lactose content has strong laxative effect. The isolates of L. helveticus which produced acid actively in skim milk culture might have a major role in production of airag.

Aplikasi Metode SDS-PAGE (Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis) untuk Mengidentifikasi Sumber Asal Gelatin pada Kapsul Keras

Gelatin as the main ingredient of capsules is still a problem for a moslem. Most of gelatin production remains largely derived from non-halal materials. One of gelatin source is came from collagen of the skin and bones of bovine or pork. The main of study is determine the source of gelatin used in hard capsules by using SDS-PAGE (Sodium Dodecyl Sulphate Gel electrophoresis Poly Acrylamide) method. In the early stages, optimization of standards bovine and pork gelatin were hydrolyzed by pepsin at pH 4.5 and 60°C for 1 hour, 2 hours, and 3 hours. Gelatin hydrolyzateswere analyzed by SDS-PAGE to determine the optimal hydrolysis time. Identification of gelatin hydrolyzate fragments were carried by molecular weight. Hydrolysis time optimization throught applied to identify the source of hard gelatin capsules in the samples obtained from market and compared with the simulation of hard gelatin capsules. The results showed there were of specific bands of bovine gelatin with a molecular weight of 11,4 kDa; 34 kDa; 47kDa and specific bands of pork gelatin with a molecular weight of 24.7 kDa; 28 kDa; and 60 kDa. Similar results were obtained on a sample of hard capsules with bands of protein fragments that were identical to bovine gelatinstandard. Based on the results,each of the samples were tested contain of bovine gelatin respectively. DOI :http://dx.doi.org/10.15408/jkv.v0i0.3150

Effect of quorum sensing signals produced by seaweed-associated bacteria on carpospore liberation from Gracilaria dura.

Epiphytic and endophytic bacteria associated with green macroalgae Ulva (U. fasciata and U. lactuca) and red macroalgae Gracilaria (G. corticata and G. dura) have been identified from three different seasons to evaluate the effect of quorum sensing (QS) molecules on carpospores liberation from Gracilaria dura. The bacterial isolates belonging to the orders Bacillales, Pseudomonadales, Alteromonadales, and Vibrionales were present in all seasons, whereas Actinomycetales and Enterobacteriales were confined to pre-monsoon and post-monsoon seasons, respectively. Among all the Gram-negative bacteria, seven isolates were found to produce different types of N-acyl homoserine lactones (AHLs). Interestingly, Shewanella algae produced five types of AHL: C4-HSL, HC4-HSL, C6-HSL, 3-oxo-C6-HSL, and 3-oxo-C12-HSL. Subsequently, the AHLs producing bacterial isolates were screened for carpospore liberation from G. dura and these isolates were found to positively induce carpospore liberation over the control. Also, observed that carpospore liberation increased significantly in C4- and C6-HSL treated cystocarps. Sodium dodecyl sulfate and native polyacrylamide gel electrophoresis of the total protein of the C4- and C6-HSL treated cystocarps showed two specific peptide bands of different molecular weights (50 kDa and 60 kDa) as compared to the control, confirming their indirect effect on carpospore liberation.

Single acute stress-induced progesterone and ovariectomy alter cardiomyocyte contractile function in female rats.

AimTo assess how ovarian-derived sex hormones (in particular progesterone) modify the effects of single acute stress on the mechanical and biochemical properties of left ventricular cardiomyocytes in the rat.MethodsNon-ovariectomized (control, n = 8) and ovariectomized (OVX, n = 8) female rats were kept under normal conditions or were exposed to stress (control-S, n = 8 and OVX-S, n = 8). Serum progesterone levels were measured using a chemiluminescent immunoassay. Left ventricular myocardial samples were used for isometric force measurements and protein analysis. Ca2+-dependent active force (Factive), Ca2+-independent passive force (Fpassive), and Ca2+-sensitivity of force production were determined in single, mechanically isolated, permeabilized cardiomyocytes. Stress- and ovariectomy-induced alterations in myofilament proteins (myosin-binding protein C [MyBP-C], troponin I [TnI], and titin) were analyzed by sodium dodecyl sulfate gel electrophoresis using protein and phosphoprotein stainings.ResultsSerum progesterone levels were significantly increased in stressed rats (control-S, 35.6 ± 4.8 ng/mL and OVX-S, 21.9 ± 4.0 ng/mL) compared to control (10 ± 2.9 ng/mL) and OVX (2.8 ± 0.5 ng/mL) groups. Factive was higher in the OVX groups (OVX, 25.9 ± 3.4 kN/m2 and OVX-S, 26.3 ± 3.0 kN/m2) than in control groups (control, 16.4 ± 1.2 kN/m2 and control-S, 14.4 ± 0.9 kN/m2). Regarding the potential molecular mechanisms, Factive correlated with MyBP-C phosphorylation, while myofilament Ca2+-sensitivity inversely correlated with serum progesterone levels when the mean values were plotted for all animal groups. Fpassive was unaffected by any treatment.Conclusion Stress increases ovary-independent synthesis and release of progesterone, which may regulate Ca2+-sensitivity of force production in left ventricular cardiomyocytes. Stress and female hormones differently alter Ca2+-dependent cardiomyocyte contractile force production, which may have pathophysiological importance during stress conditions affecting postmenopausal women.

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases (n=43) were tested against 8 antimicrobial agents and typed by outer membrane protein (OMP) pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin (MICs, ≥16 μg mL−1) and sulfamonomethoxine (MICs, ≥64 μg mL−1), but susceptible to norfloxacin (MICs, ≤0.5 μg mL−1). There was a high incidence of resistance to erythromycin (90.70%) and tylosin (93.02%), while a low incidences of resistance to ciprofloxacin (2.33%), enrofloxacin (2.33%) and florfenicol (4.65%). Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

Purification and characterisation of thermo-alkaline pectinase enzyme from Hylocereus polyrhizus

The thermo-alkaline pectinase enzyme from Hylocereus polyrhizus was purified 232.3-fold with a 73.3 % recovery through ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. Ion exchange chromatography combined with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme was monomeric with a molecular weight of 34.2 kDa. The pectinase exhibited broad specificity towards polygalacturonic acid, arabinan, oat spelt xylan, and pNP-α-glucopyranoside. The optimum pH and temperature were 8.0 and 75 °C, respectively. This enzyme was stable over a wide pH range (3.0–11.0) and at relatively high temperature (85 °C for 1 h). The Km and Vmax values of pectinase towards polygalacturonic acid were 2.7 mg/ml and 34.30 U/mg proteins, respectively. In addition, the enzyme activity was inhibited by Ni2+, Al3+, and Fe2+ and was increased in the presence of Ca2+ and Mg2+ by 120 and 112 %, respectively. The purified pectinase demonstrated robust stability in response to surfactants and oxidising agents. EDTA, which is a powerful chelating agent, did not exert any significant effect on the enzyme stability. Thus, enzymes with these unique properties may be widely used in different types of industries and biotechnological applications.

Purification and characterization of detergent-compatible protease from Aspergillus terreus gr.

The possibility of using Aspergillus terreus protease in detergent formulations was investigated. Sodium dodecyl sulfate (SDS) and native polyacrylamide gel electrophoresis indicated that the purified alkaline protease (148.9 U/mg) is a monomeric enzyme with a molecular mass of 16 ± 1 kDa. This was confirmed by liquid chromatography–mass spectrometry. The active enzyme degraded the co-polymerized gelatin. The protease demonstrated excellent stability at pH range 8.0–12.0 with optimum at pH 11.0. It was almost 100 % stable at 50 °C for 24 h, enhanced by Ca2+ and Mg2+, but inhibited by Hg2+, and strongly inhibited by phenylmethyl sulfonyl fluoride. It showed maximum activity against casein followed by gelatin; its Vmax was 12.8 U/ml with its corresponding KM of 5.4 mg/ml. The proteolytic activity was activated by Tween-80, Triton-100 and SDS, and remained unaltered in the presence of H2O2 and NaClO. The enzyme exhibited higher storage stability at 4, 28 and −20 °C. It was stable and compatible to the desired level in the local detergents. The addition of the protease to the Super wheel improved its blood stain removal. The isolated protease can thus be a choice option in detergent industry.

Purification and characterization of alkaline-thermostable protease enzyme from Pitaya (Hylocereus polyrhizus) waste: a potential low cost of the enzyme.

The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus) waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent K m and V max of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0). The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe2+ and Zn2+, while protease activity was increased in the presence of Ca2+ and Mg2+ and Cu2+ by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA). The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications.

High-throughput fluorescence assay for membrane-protein interaction

Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mol% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology domains, an epsin N-terminal homology domain, and a phox homology domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.

A marine phytoplankton (Prymnesium parvum) up-regulates ABC transporters and several other proteins to acclimatize with Fe-limitation

Iron (Fe) is one of the vital limiting factors for phytoplankton in vast regions of the contemporary oceans, notably the high nutrient low chlorophyll regions. Therefore, it is apparent to be acquainted with the Fe uptake strategy of marine phytoplankton under Fe-limited condition. In the present study, marine phytoplankton Prymnesium parvum was grown under Fe-deplete (0.0025μM) and Fe-rich (0.05μM) conditions, and proteomic responses of the organism to Fe conditions were compared. In sodium dodecyl sulfate (SDS) gel electrophoresis, 7 proteins (16, 18, 32, 34, 75, 82, and 116kDa) were highly expressed under Fe-deplete condition, while one protein (23kDa) was highly expressed under Fe-rich condition. These proteins were subjected to 2-dimensional gel electrophoresis (2-D DIGE) to differentiate individual proteins, and were identified by matrix-assisted laser desorption–ionization-time of flight-mass spectrometer (MALDI-TOF-MS) analysis. The results showed that under Fe-deplete condition P. parvum increases the biosynthesis of ATP binding cassette (ABC) transporters, flagellar associated protein (FAP), and Phosphoribosylaminoimidazole-succinocarboxamide synthase. These proteins are assumed to be involved in a number of cellular biochemical processes that facilitate Fe acquisition in phytoplankton. Under Fe-deplete condition, P. parvum increases the synthesis of ribulose biphosphate carboxylase (RuBisCo), malate dehydrogenase, and two Fe-independent oxidative stress response proteins, manganese superoxide dismutase (MnSOD) and Serine threonine kinase (STK). Thus, marine phytoplankton may change their Fe acquisition strategy by altering the biosynthesis of several proteins in order to cope with Fe-limitation.

Protective Humoral Immunity Induced by Lipopolysaccharide Incorporated Liposome in Mice Against Shigella flexneri Infection

This study was conducted to determine the role of lipopolysaccahride-liposome conjugate (LPS-LIP) as a potential vaccine against shigellosis in mice by determining the IgG titer. One hundred stool samples were collected from patients with diarrhea from different hospitals in Baghdad city during the period December 2011-May 2012. Thirty isolates were suspected to be Shigella. Four isolates of Shigella flexneri were obtained after performing some biochemical tests and Api System. Antibiotic sensitivity test was carried out and results revealed that all isolates were resist to some antibiotics used in this study. One isolate was selected for LPS extraction by phenol hot water extraction method. Chemical characterization of the extracted LPS revealed that the carbohydrate content was 2.34 mg/ml, while the protein concentration was 0.52µg/ml. Partial purification of the extracted LPS was carried out by using gel- filtration chromatography on Sephacryl S-200 and results showed that three peaks were obtained and protein, carbohydrate concentration were estimated for each peak. The second peak observed to have the highest carbohydrate content was (25%). and the lowest contaminated protein was (0.001%). The partial purified LPS was analyzed by Sodium Dodecyl Sulphate Gel Electrophoresis (SDS-PAGE) and results reveled that two bands with molecular weights 100 and 150 KDa were present. Band with MW 100 kDa represented LPS. IgG titer was estimated by Elisa technique. A significant increase in IgG titer was recorded in mice treated with conjugate after infection with S. flexneri.

Hemoglobin and Red Blood Cells Catalyze Atom Transfer Radical Polymerization

Hemoglobin (Hb) is a promiscuous protein that not only transports oxygen, but also catalyzes several biotransformations. A novel in vitro catalytic activity of Hb is described. Bovine Hb and human erythrocytes were found to display ATRPase activity, i.e., they catalyzed the polymerization of vinyl monomers under conditions typical for atom transfer radical polymerization (ATRP). N-isopropylacrylamide (NIPAAm), poly(ethylene glycol) methyl ether acrylate (PEGA), and poly(ethylene glycol) methyl ether methacrylate (PEGMA) were polymerized using organobromine initiators and the reducing agent ascorbic acid in acidic aqueous solution. In order to avoid chain transfer from polymer radicals to Hb's cysteine residues, the accessible cysteines were blocked by a reaction with a maleimide. The formation of polymers with bromine chain ends, relatively low polydispersity indices (PDI), first order kinetics and an increase in the molecular weight of poly(PEGA) and poly(PEGMA) upon conversion indicate that control of the polymerization by Hb occurred via reversible atom transfer between the protein and the growing polymer chain. For poly(PEGA) and poly(PEGMA), the reactions proceeded with a good to moderate degree of control. Sodium dodecyl sulfate (SDS) gel electrophoresis, circular dichroism spectroscopy, and time-resolved ultraviolet-visible (UV-vis) spectroscopy revealed that the protein was stable during polymerization, and only underwent minor conformational changes. As Hb and erythrocytes are readily available, environmentally friendly, and nontoxic, their ATRPase activity is a useful tool for synthetic polymer chemistry. Moreover, this novel activity enhances the understanding of Hb's redox chemistry in the presence of organobromine compounds.

Synthesis of Thermostable Amylase Enzyme from &lt;i&gt;Bacillus licheniformis&lt;/i&gt; and it's Optimization to Different Sources

S: - Among the six isolated amylase producing strains,Bacillus licheniformis(B1), the thermostable strain was selected from the soil of a paddy field. Its enzyme productivity and activity were evaluated. The activity of enzyme was calculated as 27.77 IU/ml. Effects of various carbon and organic nitrogen sources, and C/N ratio on enzyme production were examined. Maximum α - amylase production was obtained in medium containing 1% starch. Fructose supported the maximum amylase production among all the sugar studied. Of the organic nitrogen sources tested, peptone was found to be the best organic nitrogen source for excess yield of the enzyme. The optimum C/N ratio was found to be 1:1. The α amylase exhibited activity at a wide pH and temperature range and activity were found to be optimal at pH 6 and 40 °C respectively. The molecular weight of α amylase was calculated by sodium dodecyl sulphate gel electrophoresis and found to be around 29,000 Daltons.

Partial purification, characterization and cDNA cloning of aminoaldehyde dehydrogenase in germinated soybean (Glycine max L.)

γ-Aminobutyric acid (GABA) is a non-proteinous amino acid with some functions for human health. In this study, aminoaldehyde dehydrogenase (EC 1.2.1.19, AMADH) which has been proven to be an essential enzyme responsible for the GABA bioformation via polyamine degradation pathway was purified from 2-day germinated soybeans and characterized. The partially purified enzyme showed a single major band with a relative molecular mass of approximately 53.72 kDa-subunit using sodium dodecyl sulfate gel electrophoresis. AMADH exhibited the optimal activity at pH 8.0 and at 45 °C, respectively. The activity of purified AMADH was significantly inhibited by some heavy metal ions and sulfhydryl reagents. The cDNA of AMADH was sequenced by RT-PCR, which indicated 1,677 bp long and contained a 1,515 bp open reading frame that encoded 504 amino-acid peptides (GenBank accession number: KC478661).