Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Gels Research Articles

A New Algorithm for Detecting GPN Protein Expression and Overexpression of IDC and ILC Her2+ Subtypes on Polyacrylamide Gels Associated with Breast Cancer

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is used to identify protein presence, absence, or overexpression and usually, their interpretation is visual. Some published methods can localize the position of proteins using image analysis on images of SDS-PAGE gels. However, they cannot automatically determine a particular protein band’s concentration or molecular weight. In this article, a new methodology to identify the number of samples present in an SDS-PAGE gel and the molecular weight of the recombinant protein is developed. SDS-PAGE images of different concentrations of pure GPN protein were created to produce homogeneous gels. Then, these images were analyzed using the developed methodology called Image Profile Based on Binarized Image Segmentation (IPBBIS). It is based on detecting the maximum intensity values of the analyzed bands and produces the segmentation of images filtered by a binary mask. The IPBBIS was developed to identify the number of samples in an SDS-PAGE gel and the molecular weight of the recombinant protein of interest, with a margin of error of 3.35%. An accuracy of 0.9850521 was achieved for homogeneous gels and 0.91736 for heterogeneous gels of low quality.

The Blood Group Rhc Protein from Human Erythrocyte Membranes as an Immunogen for Producing Antibodies in Mice

Background: Various methods have been used to isolate red blood cell (RBC) membrane antigens. In this regard, obtaining the antigen and preserving its structure is of special importance. However, limited studies have been conducted to purify cellular membrane antigens such as Rh proteins.
 Materials and Methods: In this experimental study, Rhc antigens of the RBC membrane was purified. Here, the RBC membrane was solubilized through the lysis buffer. Next, dialysis and affinity chromatography were performed using polyclonal anti-human RhCcEe antibody to isolate Rhc/e antigens from the RBCs with the following blood group characteristics: Rhc+, RhC-, Rhe+, and RhE-. The purified proteins were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dot blot methods. The immunization process was performed in Balb/c mice using the Rhc antigen as an immunogen. After the last injection, the mouse serum was used to titrate antibodies.
 Results: Protein bands of the purified antigen were observed in the silver-stained SDS-PAGE gel (region of 25-35 kDa). The OD405nm = 0.56 ± 0.05 results showed the reactivity with Rhc antibody. The specificity of the purified protein was evaluated using the dot blot assay. The anti-sera titration was greater than 1/10,000 against Rhc-coated microwells. Rh antigens can be isolated from the RBC membrane using the non-ionic NP-40 detergent and affinity chromatography.
 Conclusion: The Rh antigen can be isolated from the RBC membrane with proper purity by solubilization with the non-ionic NP-40 detergent and purification by affinity chromatography. It seems that the membrane antigen maintains its antigenicity and structure. As a result, it can be detected by blood group-specific antibodies used in the hemagglutination method. Purified antigens may be used to generate antibodies or to study the protein structure.

Optimization strategy for purification and isolation of active lectin from Colocasia esculenta L. Schott rhizome

Lectins are non-enzymatic proteins that attach to carbohydrates and exhibit diverse biological activities and are found in Colocasia esculenta rhizomes. This study determined the optimal conditions for purifying and isolating lectin from C. esculenta rhizome. The methods involved the preparation of crude extracts with variations of salting in, salting in followed by salting out, and precipitation with phosphate buffer saline (PBS), then all extracts were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purification and isolation were carried out using ion exchange chromatography with pH variations of 8.3, 7.2, and 9.6. The isolates were evaluated using the bicinchoninic acid method, SDS-PAGE, and hemagglutination test. The results of SDS-PAGE showed that the best extraction method involved the use of PBS as indicated by the presence of a prominent band at ~12.5kDa. During pH optimization for purification, the best isolate was obtained at pH 9.6. This was observed by the appearance of a single band with ~12.5kDa MW on 12% SDS-PAGE gel and occurrence of an agglutination reaction at the hemagglutination stage. The total protein content of lectins ranged from 1284.83 to 2947.33 μg/mL. The C. esculenta rhizome contains lectins as active protein compounds. The best isolation condition is by extraction with PBS solvent and purification using ion exchange chromatography at pH 9.6.

A method to obtain purified free light chain monomers and dimers from urine samples of patients with multiple myeloma

Antibody light chains are synthesized in excess by plasma cells, and this excess can be secreted into biological fluids as dimers or monomers in various proportions. Structural differences between monomers or dimers of free light chains (FLC) can affect their biological functions and possibly their pathogenicity. They also may exhibit differential immune reactivity, perhaps explaining discrepant quantifications when measured by different immunoreagents. Having purified FLC monomers and dimers available can be useful for studying their properties. Here we propose a simple preparatory procedure to purify FLC monomers and dimers from urine samples of patients with plasma cell disorders. Two representative urine samples containing lambda or kappa FLC were loaded into a nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The gel strips containing separate monomers and dimers were excised, electroeluted, and the FLC recovered. The FLC were recovered from SDS-PAGE gel in sufficient amounts to be quantified by UV and two automated nephelometric assays immunochemical. The procedure was found to be simple, reproducible, and with a high yield, thus offering the opportunity to compare different assays. Not all urine samples are suitable for this procedure, but this approach allows for the purification of FLC monomers and dimers from many selected urine samples which maintain their oligomeric organization.

Design and Production of Arginine Deiminase-Azurin Recombinant Fusion Protein from Pseudomonas aeruginosa and its Confirmation by Western Blot

Background: Pseudomonas aeruginosa is a common Gram-negative, rod-shaped bacterium that has a unique genome that allowed the bacteria to produce various enzymes and proteins. Azurin and arginine deiminase are low molecular weight proteins that produced by P. aeruginosa. These proteins can be used alone or in combination together in order to become effective in cancer therapy or inhibiting of metastasis. This study aimed to design, express and purify the Azurin and Arginine Deiminease recombinant fusion protein.Materials and Methods: The sequences of Azurin and arginine deiminase from P. aeruginosa were studied for synthesis in a pET28a vector. The recombinant plasmid was transfected into the E.coli BL-21 strain and expression was induced by isopropyl-β-D-thio galactopyranoside (IPTG). The fusion protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Purification of the recombinant product was performed by the Ni-NTA chromatography column, obtained product analysis with SDS-PAGE and Western blot technique.Results: Cloning was confirmed by observing bands from the enzyme digestion. The protein band with a molecular weight of 65 kDa on the SDS-PAGE gel was an indication of the correct expression of the protein. The single-band of this recombinant protein was confirmed by the western blot technique.Conclusion: In this study, due to the successful production of arginine deiminase-azurin fusion protein, and considering the separate anti-cancer properties of these compounds, which have been reported in previous studies, it is suggested that immunological assessments and effects of this fusion protein in different cancerous cell line be investigated.

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Salivary tannin-binding proteins: A foraging advantage for goats?

Tannins are a key chemical defense that plants use against mammalian herbivores. To cope with tannins, many herbivores have evolved salivary tannin-binding proteins that precipitate tannins in forage items, and thus minimize their deleterious effects. In the past, one specific type of salivary tannin-binding protein was identified (proline-rich proteins), and has been found in a range of mammalian species. However, there are other proteins in the saliva of mammalian herbivores that also have a high tannin-binding affinity, which may allow for tolerance of low-quality food resources. Domestic goats (Capra hircus) are able to survive on low quality, tannin-rich food items. However, it has been reported that they do not have proline-rich proteins in their saliva. Thus, the question that arises is, do they have other tannin-binding proteins in their saliva, or do they rely on other physiological mechanisms (e.g. gut microflora) to negate the negative effects of tannins? We explored whether goats had tannin-binding proteins in the saliva by using a series of laboratory assays. As a starting point, we tested for the presence of proline-rich proteins in goat saliva using two different approaches: (1) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using Laemmli's (1970) destaining method, and (2) comparative SDS-PAGE gels using Beeley et al. (1991) method for staining and destaining to probe for proline-rich proteins. We found, as in previous studies, that goats did not have proline-rich proteins, but that they did have other proteins in their saliva that have a high tannin-binding affinity. To test for the tannin-binding affinity of goat saliva, we then performed an inhibition assay based on a modified radial diffusion assay. For the inhibition assay, we mixed goat saliva with three tannic acid solutions as well as extracts from seven common South African tree species. To determine whether goat saliva could significantly reduce the amount of tannins left to bind after interacting with the tannin-rich solutions, we used a Wilcoxon test. Our inhibition assays revealed that goat saliva is able to significantly bind to and precipitate tannic acid and tannins in common South African tree species. The presence of these other as yet unclassified proteins, coupled with other physiological mechanisms, helps explain how goats survive on tannin-rich foods, and why they are so successful in areas dominated by highly defended food items.

Affinity adsorption of bovine hyaluronidase with ligands targeting to active site

Four affinity ligands were designed from 6-chloromethyluracil and 2-aminobenzimidazole and simulated for the interaction with bovine hyaluronidase-1. Regarding sequence alignment, bovine hyaluronidase-1 precursor showed circa 83.6% similarity with human hyaluronidase-1. Regarding structural modeling and molecular docking, bovine hyaluronidase-1 interacted with ligands in the active site. Using epichlorohydrin, 1,3-propanediamine and cyanuric chloride as spacers, 6-chloromethyluracil and 2-aminobenzimidazole were composed to Sepharose beads. The modified Sepharose beads were then subjected to adsorption analysis with bovine hyaluronidase. After one step of affinity adsorption, the samples extracted from bovine testes were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and activity assay. As calculated, the densities of four ligands on sorbents (entitled as L-1, L-2, L-3 and L-4) were 37.7 ± 2.3, 36.4 ± 3.2, 42.4 ± 4.2 and 33.7 ± 2.3 μmol/g wet gel; the theoretical maximum adsorption (Qmax) of bovine hyaluronidase on the four sorbents were 63.6 ± 1.6, 72.0 ± 0.7, 111.0 ± 4.1 and 121.7 ± 2.3 mg/g wet gel, respectively; the dissociation constants (Kd) of the four sorbents were 18.5 ± 0.8, 48.1 ± 4.3, 35.0 ± 3.0, 40.6 ± 2.7 μg/g wet gel, respectively. After optimization, the proteins captured by sorbents attaching 2-aminobenzimidazole based ligands (L-3 and L-4) revealed the main single band at approximately 50 kDa, and the purities were about 85.2 and 96.4%; the bioactivity recoveries were 83.5 and 89.4%. In addition, the bands on SDS-PAGE gel were also extracted and confirmed with linear trap quadropole mass spectrometry (LTQ-MS) analysis.

Seminal plasma and sperm proteome of ring-tailed coatis (Nasua nasua, Linnaeus, 1766)

Ring-tailed coati is listed as a species of least concern in the International Union for Conservation of Nature (IUCN) Red List, however, there has been a sharp decline in their population. The present study was conducted to evaluate the major proteins of both seminal plasma and sperm in ring-tailed coatis. Semen sample was collected from three adult coatis and evaluated for their morphological characteristics. Further, the sample was centrifuged to separate spermatozoa from seminal plasma, and then stored in liquid nitrogen. The seminal plasma and sperm proteins were subjected to one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by mass spectrometry. Gene ontology and protein networks were analyzed using bioinformatics tools. Based on sperm concentration and average protein content of the semen, the concentration of protein/spermatozoon was found to be 104.69 ± 44.43 μg. The analysis of SDS-PAGE gels showed 20.3 ± 3.1 and 17 ± 2 protein bands/lane for seminal plasma and sperm, respectively. In-gel protein digestion and peptide analysis by mass spectrometry revealed 238 and 246 proteins in the seminal plasma and sperm, respectively. The gene ontology analysis revealed that the proteins of seminal plasma mainly participated in cellular (35%) and regulatory (21%) processes. According to their cellular localization, seminal plasma proteins were categorized as structural (18%), extracellular (17%), and nuclear (14%) proteins with molecular functions, such as catalytic activity (43%) and binding (43%). The sperm proteins were also involved in cellular (38%) and regulatory (23%) processes, and mainly categorized as extracellular (17%), nuclear (13%), and cytoplasmic (10%) proteins. The major molecular functions of the sperm proteins were catalytic activity (44%) and binding (42%). These results indicated that the seminal plasma of ring-tailed coati has an array of proteins that can potentially modulate several sperm functions, from sperm protection to oocyte binding. However, further studies are necessary to interpret the roles of these major seminal plasma proteins in coatis.

EVALUATION OF REGENERATIVE THERAPY USING CELL SHEET THROUGH CEMENTUM PROTEIN-1 EXPRESSION ON MACACA NEMESTRINA

Objective: The main objective of periodontal therapy is tissue regeneration. Previous studies have identified the potential of mesenchymal stem cells to improve major periodontal defect reconstruction in bone tissue engineering. Cell sheet technology (CST), in which a cell culture is obtained from a material coated with a temperature-sensitive substrate, has been developed for the reconstruction of various tissues, including periodontal tissue. Cementum protein-1 (CEMP-1) is a 50-kDa protein that plays a crucial role in cementogenesis by enhancing the combining of cells formed by cell cementoblast. Evaluate periodontal tissue regeneration with CST, and the application of chitosan, chitosan cell sheet or arginineglycineaspartic acid (RGD)-modified chitosan cell sheet on Macaca nemestrina one-wall defect model.Methods: The CEMP-1 was analyzed expression in a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis assay.Results: CEMP-1 expression in gingival crevicular fluid was observed using the SDS-PAGE method every week for 3 weeks. Protein band expressions on SDS-PAGE gel were identified at around 50 kDa with different thicknesses between groups. The chitosan, chitosan cell sheet, and RGD-modified chitosan cell sheet groups showed protein bands of CEMP-1 between 50 and 70 kDa at weeks 1, 2, and 3; weeks 2 and 3; and weeks 1 and 2, respectively.Conclusion: Our results demonstrated that the application of chitosan and RGD-modified chitosan cell sheets could enhance bone regeneration, as evidenced by CEMP-1 protein expression levels.

Fractionation and identification of the allergic proteins in Aspergillus species.

Background and Purpose:Allergy is an undesired immune response to non-pathogenic agents. However, some opportunistic microorganisms such as fungi can also cause allergy. Among those fungi, hyphae form of Aspergillus strains including A. fumigatus, A. flavus, and A. niger could be mentioned. In this study, we aimed to separate allergic proteins from Aspergillus strains and determine their identity.Materials and Methods:Standard species of Aspergillus strains were cultivated in optimized conditions and the mycelium was separated by centrifugation. The fungal cells were lysed through physical methods such as freeze-thawing and grinding to prepare a suitable protein extract. The protein concentration was measured by Bradford method and the electrophoretic pattern of the extract was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were fractionated by ammonium sulfate precipitation and anion exchange chromatography using fast protein liquid chromatography (FPLC) system. The IgE immunoreactivity of the sensitized patients and controls was studied using the fractionated proteins by enzyme-linked immunosorbent assay (ELISA). Following SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes and the strips were blotted with allergic patients' and controls' sera. The immunoreactive bands were excised from colloidal coomassie-stained SDS-PAGE gels and studied by mass spectroscopy methods.Results:Among the studied species, A. fumigatus showed stronger IgE reactivity and more IgE reactive protein bands than others did. The proteins with higher molecular weights showed stronger immunoreactivity in Western blotting. Receiver operating characteristic curve analysis demonstrated a correlation between the results of the applied ELISA methods. One of the most prominent IgE-reactive proteins was confirmed to be 45 kDa mycelia catalase. Conclusion:Our findings confirmed that high molecular weight proteins might play a major role in allergy and IgE reactivity to Aspergillus species. Moreover, the results showed that precipitation and chromatographic methods are applicable for fractionation of fungal proteins such as mycelial catalase.

PRODUCTION OF POLYCLONAL ANTIBODY TO THE COAT PROTEIN OF CITRUS TRISTEZA VIRUS IN CHICKEN EGGS

Citrus tristeza virus (CTV) is one of the most destructive diseases in many citrus growing areas of Indonesia. Effective strategies for controlling CTV depend on diagnostic procedure namely enzyme-linked immunosorbent assay (ELISA). Study aimed to purify the CTV antigen and produced its polyclonal antibody. Virion of the severe CTV isolate designated UPM/ T-002 was concentrated by polyethylene glycol (PEG) precipitation combined with low speed centrifugation. Semipurified antigen was further purified by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). The specific coat protein (CP) band of CTV with molecular weight of 25 kD was excised and eluted using elution buffer containing 0.25 M Tris-HCl pH 6.8 + 0.1% SDS, then used as antigen for injection into 6-month-old female of White Leghorn chicken. Results, showed than the specific polyclonal antibody raised against the 25-kDa CP had a titer of approximately 104, gave low background reaction with healthy plant sap and reacted specifically with CTV isolates. The reaction was equally strong for a severe, a moderate, a mild, and a symptomless isolate, suggesting a broad reaction range of this antibody toward different CTV isolates. Optimal virus titer can be obtained since virus loss during purification could be minimized and the highly purified antigen as an immunogen could be obtained by cutting out the CP band from SDS-PAGE gels. Large amount of highly titer of CTV antibody can be produced in chicken egg. The simplicity of the procedure makes it economically acceptable and technically adoptable because the antibody can be produced in basic laboratory.

PRODUCTION OF POLYCLONAL ANTIBODY TO THE COAT PROTEIN OF CITRUS TRISTEZA VIRUS IN CHICKEN EGGS

Citrus tristeza virus (CTV) is one of the most destructive diseases in many citrus growing areas of Indonesia. Effective strategies for controlling CTV depend on diagnostic procedure namely enzyme-linked immunosorbent assay (ELISA). Study aimed to purify the CTV antigen and produced its polyclonal antibody. Virion of the severe CTV isolate designated UPM/ T-002 was concentrated by polyethylene glycol (PEG) precipitation combined with low speed centrifugation. Semipurified antigen was further purified by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). The specific coat protein (CP) band of CTV with molecular weight of 25 kD was excised and eluted using elution buffer containing 0.25 M Tris-HCl pH 6.8 + 0.1% SDS, then used as antigen for injection into 6-month-old female of White Leghorn chicken. Results, showed than the specific polyclonal antibody raised against the 25-kDa CP had a titer of approximately 104, gave low background reaction with healthy plant sap and reacted specifically with CTV isolates. The reaction was equally strong for a severe, a moderate, a mild, and a symptomless isolate, suggesting a broad reaction range of this antibody toward different CTV isolates. Optimal virus titer can be obtained since virus loss during purification could be minimized and the highly purified antigen as an immunogen could be obtained by cutting out the CP band from SDS-PAGE gels. Large amount of highly titer of CTV antibody can be produced in chicken egg. The simplicity of the procedure makes it economically acceptable and technically adoptable because the antibody can be produced in basic laboratory.

The Structural Proteins of Bdellovibrio bacteriovorus Bacteriophage MAC-1

In the present investigation the structural proteins associated with MAC-1 bacteriophage have been characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); tandem mass spectrometry of protein bands from SDS-PAGE gel; from the open reading frames (ORFs) deduced from MAC-1 genome sequence and amino acid sequence homology searches from the Uniprot database (up000002418). Results have led to the identification of at least three structural proteins associated with MAC-1 phage genome. They are: capsid protein (~55,000-daltons); spike protein (~22,000-daltons) and a low molecular weight DNA binding protein (~4000-dal- tons). In addition, two other minor proteins were tentatively identified as replicative and scaffold proteins based on two to three unique peptides from mass spectrometry data. However, other proteins coded (ORFs) by phage genome remain to be identified.

Differential sensitivity of lipegfilgrastim and pegfilgrastim to neutrophil elastase correlates with differences in clinical pharmacokinetic profile.

To assess the basis of the different half-lives of long-acting human granulocyte colony-stimulating factor (G-CSF) drugs, the effect of neutrophil elastase on lipegfilgrastim and pegfilgrastim was investigated. Sensitivity to human neutrophil elastase (HNE) was evaluated by incubating the drugs with HNE followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Drugs were also incubated with isolated human neutrophils followed by Western blot analysis. Lipegfilgrastim was more resistant to degradation with HNE or neutrophils than pegfilgrastim and appeared more intact on SDS-PAGE gels and Western blots. Lipegfilgrastim retained more functional activity than pegfilgrastim after incubation with HNE (67% vs ∼ 9%, respectively) or neutrophils (80% vs ∼ 4%, respectively) as assessed in an NFS-60 cell-based [(3) H]-thymidine incorporation assay. The binding and affinity of untreated lipegfilgrastim and pegfilgrastim for G-CSF receptors were evaluated using an NFS-60 competitive G-CSF receptor-binding assay and surface plasmon resonance. Untreated drugs were also evaluated in the functional NFS-60 thymidine incorporation assay. G-CSF receptor binding, receptor affinity, and functional activity were comparable between untreated drugs. The results showed a greater resistance to neutrophil elastase degradation and concomitant retention of functional activity of lipegfilgrastim compared with pegfilgrastim, which potentially explains the clinical observations of a longer half-life of lipegfilgrastim versus pegfilgrastim.

Rapid detection of porins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy. A MALDI-TOF MS-based assay was set up to detect porins in the current study. A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance. Ten strains of Escherichia coli and eight strains of Klebsiella pneumoniae were conducted for both sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF MS analysis. MALDI-TOF/TOF MS analysis was then performed to verify the correspondence of proteins between SDS-PAGE and MALDI-TOF MS. The results indicated that the mass spectrum of ca. 35,000, 37,000, and 38,000-m/z peaks of E. coli ATCC 25922 corresponded to OmpA, OmpC, and OmpF with molecular weight of approximately ca. 38, 40, and 41 kDa in SDS-PAGE gel, respectively. The band of OmpC and OmpF porins were unable to be distinguished by SDS-PAGE, whereas it was easy to be differentiated by MALDI-TOF MS. As for K. pneumoniae isolates, the mass spectrum of ca. 36,000 and 38,600-m/z peaks was observed corresponding to OmpA and OmpK36 with molecular weight of approximately ca. 40 and 42 kDa in SDS-PAGE gel, respectively. Porin OmpK35 was not observed in the current SDS-PAGE, while a 37,000-m/z peak was found in K. pneumoniae ATCC 13883 and carbapenem-susceptible strains by MALDI-TOF MS which was presumed to be the characteristic peak of the OmpK35 porin. Compared with SDS-PAGE, MALDI-TOF MS is able to rapidly identify the porin-deficient strains within half an hour with better sensitivity, less cost, and is easier to operate and has less interference.

Cytochrome c adducts with PCB quinoid metabolites.

Polychlorinated biphenyls (PCBs) are a group of 209 individual congeners widely used as industrial chemicals. PCBs are found as by-products in dye and paint manufacture and are legacy, ubiquitous, and persistent as human and environmental contaminants. PCBs with fewer chlorine atoms may be metabolized to hydroxy- and dihydroxy-metabolites and further oxidized to quinoid metabolites both in vitro and in vivo. Specifically, quinoid metabolites may form adducts on nucleophilic sites within cells. We hypothesized that the PCB-quinones covalently bind to cytochrome c and, thereby, cause defects in the function of cytochrome c. In this study, synthetic PCB quinones, 2-(4'-chlorophenyl)-1,4-benzoquinone (PCB3-pQ), 4-4'-chlorophenyl)-1,2-benzoquinone (PCB3-oQ), 2-(3', 5'-dichlorophenyl)-1,4-benzoquinone, 2-(3',4', 5'-trichlorophenyl)-1,4-benzoquinone, and 2-(4'-chlorophenyl)-3,6-dichloro-1,4-benzoquinone, were incubated with cytochrome c, and adducts were detected by liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to separate the adducted proteins, while trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to identify the amino acid binding sites on cytochrome c. Conformation change of cytochrome c after binding with PCB3-pQ was investigated by SYBYL-X simulation and cytochrome c function was examined. We found that more than one molecule of PCB-quinone may bind to one molecule of cytochrome c. Lysine and glutamic acid were identified as the predominant binding sites. Software simulation showed conformation changes of adducted cytochrome c. Additionally, cross-linking of cytochrome c was observed on the SDS-PAGE gel. Cytochrome c was found to lose its function as electron acceptor after incubation with PCB quinones. These data provide evidence that the covalent binding of PCB quinone metabolites to cytochrome c may be included among the toxic effects of PCBs.

Effects ofVibrio harveyion plasma clotting protein (coagulogen) of white shrimpLitopenaeus vannamei

Blood clotting exhibits various important functions, including the prevention of body fluid loss and invasion of pathogens in shrimp. The effects of pathogenic Vibrio harveyi on plasma of white shrimp (Litopenaeus vannamei) in vitro and in vivo were investigated in this study. The clotting protein (coagulogen) in plasma of white shrimp pre-incubated with extracellular products (ECP) of V. harveyi was found apparently decreased and fast-migrated in crossed immunoelectrophoresis (CIE) gels. In addition, the coagulogen had been degraded to many low molecular-weight protein bands in plasma pre-incubated with ECP on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. When pre-challenged with bacterial cells and ECP of V. harveyi, the white shrimp began to die at about 30 and 16 h respectively. Moreover, plasma coagulogen was decreased more obvious in shrimp challenged with ECP than that with bacterial cells as visualized in CIE gels, and total plasma protein in both group of shrimp were all decreased. Haemolymph withdrawn from moribund shrimp pre-challenged with V. harveyi or its ECP was observed unclottable. However, the addition of clotting factors (transglutaminase and/or Ca2+) to these unclottable plasma could apparently promote their re-clotting ability as jelly-like solid observed in microtubes. The recovery of clotting ability of plasma from moribund shrimp was due to the reformation of coagulogen (200 kDa) after adding the two clotting factors as shown on CIE and SDS-PAGE gels. The present results suggest that the infection of V. harveyi in white shrimp may not only degrade coagulogen but also influence the presence of transglutaminase and Ca2+ ion.

MS2 virus inactivation by atmospheric-pressure cold plasma using different gas carriers and power levels.

In this study, airborne MS2 bacteriophages were exposed for subsecond time intervals to atmospheric-pressure cold plasma (APCP) produced using different power levels (20, 24, and 28 W) and gas carriers (ambient air, Ar-O2 [2%, vol/vol], and He-O2 [2%, vol/vol]). In addition, waterborne MS2 viruses were directly subjected to the APCP treatment for up to 3 min. MS2 viruses with and without the APCP exposure were examined by scanning electron microscopy (SEM), reverse transcription-PCR (RT-PCR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Viral inactivation was shown to exhibit linear relationships with the APCP generation power and exposure time (R(2) > 0.95 for all energy levels tested) up to 95% inactivation (1.3-log reduction) after a subsecond airborne exposure at 28 W; about the same inactivation level was achieved for waterborne viruses with an exposure time of less than 1 min. A larger amount of reactive oxygen species (ROS), such as atomic oxygen, in APCP was detected for a higher generation power with Ar-O2 and He-O2 gas carriers. SEM images, SDS-PAGE, and agarose gel analysis of exposed waterborne viruses showed various levels of damage to both surface proteins and their related RNA genes after the APCP exposure, thus leading to the loss of their viability and infectivity.

Pegylated Versus Glycopegylated G-CSFs and Their Biochemical and Physiological Properties

BackgroundLipegfilgrastim and pegfilgrastim are long-acting, once-per-cycle recombinant granulocyte colony-stimulating factors (G-CSFs) developed to reduce the duration of neutropenia and the incidence of febrile neutropenia in cancer patients receiving chemotherapy (CTx). Lipegfilgrastim is recombinant methionyl human G-CSF that is modified at its natural O-glycosylation site (threonine134) using a novel glycoPEGylation technology. Pegfilgrastim is a recombinant methionyl human G-CSF with a methoxy-polyethylene glycol-propionaldehyde covalently conjugated to its N-terminus. GlycoPEGylation generates functional PEGylated proteins with increased bioavailability and prolonged duration of action. Previous studies show that lipegfilgrastim has a 60% higher bioavailability and a 30% greater neutrophil response versus pegfilgrastim in healthy volunteers. When exposed to neutrophil elastase, G-CSF is rapidly cleaved and rendered inactive, suggesting that resistance to this degradation may result in an extended half-life. ObjectiveThe objectives of these studies were to characterize lipegfilgrastim versus pegfilgrastim with respect to bioavailability and affinity for the G-CSF receptor, to evaluate the effect of human neutrophil elastase on the degradation of lipegfilgrastim versus pegfilgrastim, and to evaluate the effect of degradation on the activity of these study drugs. MethodsLipegfilgrastim and pegfilgrastim binding to the G-CSF receptor was evaluated using: 1) a NFS-60-cell–based [125I]-G-CSF competitive G-CSF receptor binding assay; and 2) a label-free surface plasmon resonance (SPR) technology by Biacore™ platform using a recombinant human G-CSF receptor. For the competitive binding assay, cocktails of NFS-60 cells, free [125I]-G-CSF, and multiple concentrations of either (unlabeled) lipegfilgrastim or pegfilgrastim were incubated, and the cells were analyzed for bound [125I]-G-CSF. Dose-dependent inhibition of [125I]-G-CSF binding is indicative of the study drug binding to the G-CSF receptor. The SPR study evaluated binding kinetics and overall binding affinity between G-CSF receptor and lipegfilgrastim or pegfilgrastim. Degradation of lipegfilgrastim and pegfilgrastim by purified human neutrophil elastase was evaluated by incubation ± elastase and visualization using Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To evaluate the effect of elastase degradation on lipegfilgrastim or pegfilgrastim activity, samples treated with or without elastase for 15 or 120 minutes were incubated with NFS-60 cells for 20 hours, then pulsed with [3H]-thymidine for 4 hours. The level of active G-CSF–stimulated NFS-60 cell proliferation was measured by [3H]-thymidine incorporation. ResultsIn the cell-based [125I]-G-CSF competitive G-CSF receptor binding assay, G-CSF receptor binding was equivalent between lipegfilgrastim and pegfilgrastim as indicated by inhibition of [125I]-G-CSF binding (0.70±0.09 nM IC50 versus 0.72±0.18 nM IC50 (mean±SD). In the SPR study, the affinity for lipegfilgrastim and pegfilgrastim was 481±84 nM and 516±153 nM (mean±SD), respectively. Lipegfilgrastim treated with purified neutrophil elastase for 15 minutes appeared minimally degraded in SDS-PAGE gels and demonstrated a 67% relative activity in [3H]-thymidine proliferation assays compared with undigested lipegfilgrastim. In contrast, pegfilgrastim treated under the same conditions appeared markedly degraded and demonstrated an ∼9% relative activity compared with undigested pegfilgrastim. After treatment with purified neutrophil elastase for 120 minutes, lipegfilgrastim was noticeably degraded and the protein retained an ∼18% relative proliferative activity compared with undigested lipegfilgrastim, versus pegfilgrastim, which was almost entirely degraded and retained no activity compared with undigested pegfilgrastim. ConclusionsBased on the present studies, there is no apparent difference in G-CSF receptor-binding affinity between lipegfilgrastim and pegfilgrastim. In addition, lipegfilgrastim showed a greater time-dependent resistance to neutrophil elastase degradation and a greater retention of functional activity, which may provide an explanation for the longer in vivo half-life of lipegfilgrastim versus pegfilgrastim. Disclosures:Abdolzade-Bavil:Merckle GmbH (a Teva Pharmaceutical Inc., company): Employment. Cooksey:Teva Pharmaceuticals, Inc: Employment. Scheckermann:Merckle Biotec GmbH (a Teva Pharmaceuticals, Inc. company): Employment. Lammerich:Merckle/ratiopharm/Teva Pharm Industries: Employment. Pukac:Teva Pharmaceuticals, Inc: Employment. Krasney:Teva Pharmaceuticals, Inc: Employment. Allgaier:Merckle GmbH (a Teva Pharmaceuticals, Inc. company): Employment. Shen:Teva Pharmaceuticals, Inc: Employment. Müller:Teva Pharmaceuticals, Inc: Employment. Liu:Teva Pharmaceuticals, Inc: Employment. von Kerczek:2Teva Pharmaceuticals, Inc.: Employment.