Sodium Dodecyl Sulfate Gel Research Articles

Studying the Stability of S-Layer Protein of Lactobacillus Acidophilus ATCC 4356 in Simulated Gastrointestinal Fluids Using SDS-PAGE and Circular Dichroism.

Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are now recognized as one of the most common outermost cell envelope components of prokaryotic organisms. The surface layer protein of Lactobacillus acidophilus ATCC4356 is composed of a single species of protein of apparent molecular weight of 43-46 KDa. Considering the Lactobacillus acidophilus ATCC4356 having the S-layer is stable in harsh gastrointestinal (GI) conditions, a protective role against destructive GI factors which has been proposed for these nanostructures. It opens interesting perspectives in the using and development of this S-layer as a protective coat for oral administration of unstable drug nanocarriers. To achieve this goal, it is necessary to study the in-vitro stability of the S-layers in the simulated gastrointestinal fluids (SGIF). This study was planned to evaluate the in-vitro stability of the extracted S-layer protein of Lactobacillus acidophilus ATCC4356 in SGIF using it as a protective coat in oral drug delivery. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy were used to study the stability of the S-layer protein incubated in SGIF. Both the SDS-PAGE and CD spectra results showed that Lactobacillus acidophilus ATCC4356 S-layer protein is stable in simulated gastric fluid (SGF) with pH = 2 up to 5 min. It is stable in SGF pH = 3.2 and above it, with and without pepsin. It is also stable in all the simulated intestinal fluids. This S-layer is also stable in all of the simulated intestinal fluids.

Natural proteins: Sources, isolation, characterization and applications

Worldwide, plant protein contributes substantially as a food resource because it contains essential amino acids for meeting human physiological requirements. However, many versatile plant proteins are used as medicinal agents as they are produced by using molecular tools of biotechnology. Proteins can be obtained from plants, animals and microorganism cells. The abundant economical proteins can be obtained from plant seeds. These natural proteins are obtained by isolation procedures depending on the physicochemical properties of proteins. Isolation and purification of single protein from cells containing mixtures of unrelated proteins is achievable due to the physical and chemical attributes of proteins. The following characteristics are unique to each protein: Amino acid composition, sequence, subunit structures, size, shape, net charge, isoelectric point, solubility, heat stability and hydrophobicity. Based on these properties, various methods of isolation exist, like salting out and isoionic precipitation. Purification of proteins is quiet challenging and, therefore, several approaches like sodium dodecyl sulfate gel electrophoresis and chromatography are available. Characterization of proteins can be performed by mass spectrometry/liquid chromatography-mass spectrometry (LC-MS). The amino acid sequence of a protein can be detected by using tandem mass spectrometry. In this article, a review has been made on the sources, isolation, purification and characterization of natural proteins.

Purification, properties and cDNA cloning of glutamate decarboxylase in germinated faba bean (Vicia faba L.)

Gamma-aminobutyric acid (GABA) is a non-protein amino acid with bioactive functions in humans. In this work, glutamate decarboxylase (EC 4.1.1.15, GAD) which is key in the GABA bioformation was purified from 5-day germinated faba beans and characterized. A single band was observed at 58kDa using sodium dodecyl sulphate gel electrophoresis. GAD optimal activity was at pH 6.0 at 40°C with a Km value for glutamic acid (Glu) of 2.63mM. The enzyme was inhibited significantly by Cu2+, Fe3+, Mg2+, Ba2+, aminoxyacetate, EGTA, Na2EDTA, l-cysteine and beta-mercaptoethanol; and activated at low Ca2+ 0.2mM. Using RT-PCR, the GAD cDNA was sequenced which indicated 1787bp long, containing a 1527bp open reading frame (ORF) that encoded 509 amino-acid peptides with a calculated molecular weight of 57.74kDa and a pI of 5.41 (GenBank accession number: JX444699).

Identification of common outer membrane proteins of Porphyromonas gingivalis by shotgun proteomics

P. gingivalis is considered a major pathogen for periodontitis. In the process of identifying candidate antigens for a vaccine, two shotgun proteomics workflows by differentsolubilization and digestion methods were investigated in identifying outer membrane proteins (OMPs) of P. gingivalis. The first approach utilized the direct organic/detergent solubilization and separation in the studies of P. gingivalis membrane proteome. The second approach primarily used one-dimensional (1D) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of enriched membrane protein fractions, which relies on the solubilizing efficiency of the SDS and separation gel for OMPs. All of them followed by protein digestion and two-dimensional liquid chromatography-tandem mass spectrometry (2-DLC-MS/MS) to identify proteins. It was found that the second approach has been successfully applied to a Gram-positive pathogen for the first time, and the adaptation to Gram-negative P. gingivalis in our study yielded an even better result than the first approach. Thirty four OMPs were discovered by 1D-SDS-PAGE/2-DLC-MS/MS from four strains of P. gingivalis. Nine of them were shared by all the four serotypes. The method described here presents a system that can rapidly identify the outer membrane proteome of Gram-negative bacteria, which may prove valuable in vaccine development. Key words: Porphyromonas gingivalis, outer membrane protein, shotgun, proteomics, periodontitis.

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1–7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.

High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling

Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry, and increasingly becoming the method of choice that meets the requirements of the standards of International Conference of Harmonization (ICH). Automated single channel capillary electrophoresis systems are usually equipped with UV absorbance and/or laser-induced fluorescent (LIF) detection options offering general applicability and high detection sensitivity, respectively; however, with limited throughput. This shortcoming is addressed by the use of multicapillary gel electrophoresis (mCGE) systems with LED-induced fluorescent detection (LED-IF), also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of biotherapeutics, especially monoclonal antibodies (mAb). The methodology we report in this paper is readily applicable for rapid purity assessment and subunit characterization of IgG molecules including detection of non-glycosylated heavy chains (NGHC) and separation of possible subunit variations such as truncated light chains (Pre-LC) or alternative splice variants. Covalent fluorophore derivatization and the mCGE analysis of the labeled IgG samples with multi-capillary gel electrophoresis are thoroughly described. Reducing and non-reducing conditions were both applied with and without peptide N-glycosidase F mediated deglycosylation.

Epigenetic modifiers enhance the synergistic cytotoxicity of combined nucleoside analog-DNA alkylating agents in lymphoma cell lines

Hematopoietic stem cell transplantation is used for treatment of lymphoma. In an attempt to design an efficacious and safe prehematopoietic stem cell transplantation conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M), and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used ~IC(10) drug concentrations (57 μM B, 1 μM M and 0.02 μM G), which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 μM B, 0.7 μM M and 0.01 μM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (0.6 μM) to this combination resulted in a marked (~65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ataxia telangiectasia mutated-CHK2 pathway, phosphorylation of KRAB-associated protein-1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2'-deoxycytidine to (BMG+suberoylanilide hydroxamic acid) further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by suberoylanilide hydroxamic acid and 5-aza-2'-deoxycytidine further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using (BMG+suberoylanilide hydroxamic acid) as pre-hematopoietic stem cell transplantation conditioning for lymphoma.

RECOVERY AND UTILIZATION OF PROTEIN DERIVED FROM SURIMI WASH‐WATER

ABSTRACTSurimi processors are committed to improve utilization of seafood resources, increase productivity and reduce organic matter discharged into the environment. The object of this study was to recover protein from pollock surimi processing wash‐water using membrane filtration and characterize properties of the recovered material. A pilot unit equipped with membrane elements concentrated protein from the surimi wash‐water. Membrane concentrate and control surimi samples were analyzed for proximate composition, lipid oxidation, color, sodium dodecyl sulfate gel electrophoresis, amino acids and minerals. Membrane concentrate, membrane concentrate plus surimi and control surimi were monitored for 180 days of storage at −20C. The membrane concentrate had significantly higher moisture and lipid, but lower protein content than surimi. As determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, membrane concentrate proteins displayed a greater amount of lower molar mass molecules compared with surimi. The amino acid profile was comparable to control surimi and the recovered membrane concentrate proteins had similar nutritional values to that of surimi. The results indicate that the addition of 5% membrane concentrate to surimi will not adversely affect the storage at −20C and that the recovered wash‐water protein could be used to obtain a fish protein ingredient or added back at a low percentage to surimi products.PRACTICAL APPLICATIONSIn order to increase productivity and improve utilization of seafood resources, surimi processors are looking into alternative technologies to recover proteins and other material from the wastewater. Membrane filtration is a promising option for the concentration of wastewater. This study was conducted to determine the recovery and characterize the material recovered from surimi wash‐water using a commercial membrane filtration unit. It was demonstrated that the recoverable material is nutritionally similar to the final surimi product and that the overall yield can be increased using membrane technology. In addition to the benefit of recovering protein, the membrane filtration can reduce the amount of material in the waste stream.

The sclerostin-bone protein interactome

The secreted glycoprotein, sclerostin alters bone formation. To gain insights into the mechanism of action of sclerostin, we examined the interactions of sclerostin with bone proteins using a sclerostin affinity capture technique. Proteins from decalcified rat bone were captured on a sclerostin-maltose binding protein (MBP) amylose column, or on a MBP amylose column. The columns were extensively washed with low ionic strength buffer, and bound proteins were eluted with buffer containing 1M sodium chloride. Eluted proteins were separated by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by mass spectrometry. Several previously unidentified full-length sclerostin-interacting proteins such as alkaline phosphatase, carbonic anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1, which have established roles in bone formation or resorption processes, were bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin. Other full-length sclerostin-interacting proteins such as casein kinase II and secreted frizzled related protein 4 that modulate Wnt signaling were identified. Several peptides derived from proteins such as Phex, asporin and follistatin that regulate bone metabolism also bound sclerostin. Sclerostin interacts with multiple proteins that alter bone formation and resorption and is likely to function by altering several biologically relevant pathways in bone.

P1-01-16: Detecting a Breast Carcinoma-Deriving B-Cell Response: An Immunoproteomics Biomarker Approach.

Abstract Background: Noninvasive biomarkers for the early detection of breast cancer are crucial due to the fact that the relapse risk of breast cancer is rising with the time point of its detection. Currently, none of the reported molecular biomarkers is established for the clinical use as a diagnostic tool. Previous proteomics-based studies showed the immunogenicity of breast carcinoma and the following B-cell mediated immune response. As a result, several autoantibodies against tumor proteins were detected in the sera of breast cancer patients. However, these putative biomarkers are still lacking of clinically reliable specificity and sensitivity, even of better discrimination of cancer patients when combining different biomarkers. The search for a new antibody biomarker signature remains very important as a potential cancer detection tool. For further investigations we analyzed the antibody pattern in serum samples of diseased patients and healthy controls after incubation with whole protein extract from a native carcinoma and identified the putative tumor-specific immunoreactive antigens. Materials and methods: For our de novo profiling of tumor antigens we used a protein extract from a primary invasive ductal carcinoma. Sera from 20 women, of which 19 were diagnosed with breast carcinoma and one with DCIS (CA), and 20 sera from age-matched healthy donors (CTRL) were obtained and pooled separately. For an optimal separation of tumor antigens a two-dimensional sodium dodecylsulfate gel electrophoresis (2D SDS-PAGE) was applied. Following immunoblots with each serum pool were performed and the immunospecific reactions visualized with a horseradish peroxidase-conjugated anti-IgG antibody. The relevant tumor antigens were identified via Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI TOF-TOF MS). Results: After the incubation of each serum pool with tumor antigens we obtained different extensive antibody profiles, whereas the visualized immunoreactive components varied in the signal intensity. We identified over 10 tumor antigens which reacted with the corresponding autoantibodies in CA or CTRL approach, showing again the complexity of immune response. Besides of already described breast carcinoma related antigens like alpha enolase, which showed immune reaction also with the healthy serum pool, we identified several potential antigens of interest like peroxiredoxin 6 which showed a strong immune response only after the incubation with cancer sera. Discussion: In our study we visualized a complex immune response pattern showing the autoantibody profiles in cancer and healthy sera against tumor-deriving antigens. Also some of identified breast carcinoma antigens were already described; other novel breast carcinoma-related antigens were detected too. Our next step in the intriguing search for cancer antibody biomarkers is the individual screening of sera to confirm the specificity of the tumor-deriving B-cell responses and the validation of these results by using an independent study population. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-16.

Proteins isolated with TRIzol are compatible with two-dimensional electrophoresis and mass spectrometry analyses

TRIzol is used for RNA isolation but also permits protein recovery. We investigated whether proteins prepared with TRIzol were suitable for two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectrometry. Proteins from TRIzol-treated SH-SY5Y cells produced 2-DE spot patterns similar to those from an equivalent untreated sample. Subsequent identification of TRIzol-treated proteins using peptide mass fingerprinting was successful. TRIzol exposure altered neither the mass of myoglobin extracted from sodium dodecyl sulfate (SDS) gels nor the masses of myoglobin peptides produced by in-gel trypsin digestion. These findings suggest that proteins isolated with TRIzol remain amenable to proteomic analyses.

AFM study of Escherichia coli RNA polymerase σ 70 subunit aggregation

The self-assembly of Escherichia coli RNA polymerase σ 70 subunit was investigated using several experimental approaches. A novel rodlike shape was reported for σ 70 subunit aggregates. Atomic force microscopy reveals that these aggregates, or σ 70 polymers, have a straight rodlike shape 5.4 nm in diameter and up to 300 nm in length. Atomic force microscopy data, Congo red binding assay, and sodium dodecyl sulfate gel electrophoresis confirm the amyloid nature of observed aggregates. The process of formation of rodlike structures proceeds spontaneously under nearly physiological conditions. E. coli RNA polymerase σ 70 subunit may be an interesting object for investigation of amyloidosis as well as for biotechnological applications that exploit self-assembled bionanostructures. Polymerization of σ 70 subunit may be a competitive process with its three-dimensional crystallization and association with core RNA polymerase. From the Clinical Editor In this basic science study, the self-assembly of Escherichia coli RNA polymerase σ 70 subunit was investigated using atomic force microscopy and other complementary approaches.

Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes.

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [γ-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.

Segmental allergen challenge alters multimeric structure and function of surfactant protein D in humans.

Surfactant protein D (SP-D), a 43-kD collectin, is synthesized and secreted by airway epithelia as a dodecamer formed by assembly of four trimeric subunits. We have previously shown that the quaternary structure of SP-D can be altered during inflammatory lung injury through its modification by S-nitrosylation, which in turn alters its functional behavior producing a proinflammatory response in effector cells. We hypothesized that alterations in structure and function of SP-D may occur in humans with acute allergic inflammation. Bronchoalveolar lavage (BAL) fluid was collected from 15 nonsmoking patients with mild intermittent allergic asthma before and 24 hours after segmental provocation with saline, allergen, LPS, and mixtures of allergen and LPS. Structural modifications of SP-D were analyzed by native and sodium dodecyl sulfate gel electrophoresis. The multimeric structure of native SP-D was found to be disrupted after provocation with allergen or a mixture of allergen and LPS. Interestingly, under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that 7 of 15 patients with asthma developed an abnormal cross-linked SP-D band after segmental challenge with either allergen or a mixture of allergen with LPS but not LPS alone. Importantly, patients with asthma with cross-linked SP-D demonstrated significantly higher levels of BAL eosinophils, nitrogen oxides, IL-4, IL-5, IL-13, and S-nitrosothiol-SP-D compared with patients without cross-linked SP-D. We conclude that segmental allergen challenge results in changes of SP-D multimeric structure and that these modifications are associated with an altered local inflammatory response in the distal airways.

RNA channelling by the eukaryotic exosome

The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states. In the RNA-bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate-threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome-like complexes from all domains of life, and might have been present in the most recent common ancestor.

Method development and validation of capillary sodium dodecyl sulfate gel electrophoresis for the characterization of a monoclonal antibody

A capillary sodium dodecyl sulfate gel electrophoresis (cSDS) method has been developed and qualified for purity and impurity analysis of monoclonal antibodies. This method was optimized and qualified for the analysis of monoclonal antibody (mAb1) under reduced and non-reduced conditions. Some of the sample preparation parameters including sample buffer pH, incubation temperature and duration, alkylation conditions with iodoacetamide (IAM), and reduction conditions with 2-mercaptoethanol (2-ME) were optimized. It was observed that under slightly acidic conditions (pH 5.5–6.5) the thermally induced fragmentation of non-reduced mAb1 was greatly decreased. As such, a citrate–phosphate buffer at pH 6.5 was used for sample preparation to replace the original Beckman sample buffer (pH 9.0). The optimal sample preparation conditions were found to be as follows: (1) incubation temperature and duration (reduced and non-reduced), 65 °C for 5 min; (2) alkylation condition, 10 μL of 0.25 M IAM; (3) reduction condition, 10 μL of 5-fold diluted 2-ME. The method was qualified by evaluating specificity, accuracy, precision, limit of quantitation (LOQ), and linearity. The method exhibited no interference from sample buffer matrix. The method was found to be linear, accurate, and precise in the range of 0.25–3.0 mg/mL protein concentration. The LOQ of the method was determined to be 0.02 mg/mL for reduced and non-reduced mAb1. In addition, some aspects of sample stability were examined during qualification.

Response of Cyprinus carpio to copper exposure: alterations in reduced glutathione, catalase and proteins electrophoretic patterns

This study was conducted to characterize the alterations in reduced glutathione (GSH) level, catalase (CAT) activity and proteins electrophoretic patterns in response to sublethal copper (Cu) exposure in Cyprinus carpio and to determine whether these responses are related to Cu accumulation in gills, chosen as target tissue. Fish were exposed to 0.1 and 1.0mg/l Cu for 10 and 20days. There were increasing level of Cu in the gill with increasing concentrations of metal in the exposure medium, and with increasing duration of exposure. GSH level and CAT activity increased in fish exposed to 1.0mg/l Cu for both exposure periods, while no change was detected at the lower Cu concentration. Electrophoretic patterns of gill proteins by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) consist of 25-, 26-, 30-, 44- and 48-kDA medium molecular weight proteins (MMP) for five bands and 64-, 72-, 90- and 101-kDA high molecular weight proteins (HMP) for four bands in both control and treatment groups. The levels of 25-, 26- and 30-kDA MMP and 72- and 90-kDA HMP increased in response to Cu exposure. The present study demonstrated that Cu caused stress in fish gills and an acclimation with induction of GSH, CAT, MMP and HMP, which were important in the protection against metal damage, was observed.

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.

Activity assay for nisin-like acidic bacteriocins using an optimal pH-conditioned gel matrix

A new zymography for detecting nisin-like acidic bacteriocins was developed using a tricine–sodium dodecyl sulfate (SDS) gel and an acidic gel matrix (pH 4.0). After electrophoresis, proteins in the tricine gel were electrotransferred to an optimal pH-conditioned gel matrix (OP–CGM). The OP–CGM was overlaid with indicator cells (Bacillus cereus) embedded in nutrient broth soft agar (0.8%, w/v). Antibacterial activity shown as a growth inhibition using B. cereus was detected at approximately 3.8kDa. Because nisin is unstable in buffers at pH values over 6.0, the common electrophoretic systems, SDS–polyacrylamide gel electrophoresis and tricine gel, are not suitable for detection of nisin-like acidic bacteriocins.

Human complement activation by smooth and rough Proteus mirabilis lipopolysaccharides

Proteus mirabilis bacilli play an important role in human urinary tract infections, bacteremia, and rheumatoid arthritis. The authors previously studied human complement C3 conversion by smooth-form P. mirabilis O10, O23, O30, and O43 lipopolysaccharides (LPSs) and showed that smooth Proteus LPSs fragmented C3 in a dose- and time-dependent manner. In the present study, one smooth P. mirabilis S1959 and its two polysaccharide-truncated LPSs isolated from an R mutant strain were used to study the C3 conversion. The conversion of C3 to C3c by smooth and rough P. mirabilis LPSs was studied by capture ELISA and crossed immunoelectrophoresis. Proteins isolated from the outer membrane were analyzed by discontinuous sodium dodecyl sulfate gel electrophoresis. The smooth P. mirabilis S1959 (O3) strain was resistant to the bactericidal activity of human serum, in contrast to the Ra and Re mutant strains. The presence of an exposed core oligosaccharide in R110 LPS was not sufficient to protect the strain from serum-dependent killing. In addition to LPS structure, the outer-membrane proteins may also play roles in protecting the smooth P. mirabilis S1959 (O3) strain from the bactericidal action of serum. It was shown that the Ra P. mirabilis R110 and the Re P. mirabilis R45 mutants possess very different OMP compositions from that of the P. mirabilis S 1959 strain. Regardless of the complement resistance of the P. mirabilis strains, the S1959, R110, and R45 LPSs fragmented C3 and induced C3c neo-antigen exposure. The use of complement-deficient human serum allows the conclusion that the Re-type P. mirabilis R45 LPS fragmented C3 by the antibody-independent classical pathway.