Acrylamide Gel Electrophoresis Research Articles

Purification and Characterization of Thermostable Alkaline Xylanase by Aspergillus terreus from Elephant Dung

Aims: Purification and characterization of xylanase by Aspergillus terreus isolated from elephant dung.
 Study Design: Xylanase enzyme produced by A. terreus was extracted from the fermented solid medium using solid state fermentation and purified using chromatographic techniques. The purified enzyme was characterized for physio-chemical and kinetic properties.
 Place and Duration of Study: Experiments were performed at the School of Biotechnology, Devi Ahilya University, Indore, INDIA and Maharaja Ranjit Singh College of Professional Sciences, Indore, INDIA, between June, 2019 and September, 2021.
 Methodology: The enzyme was extracted and purified using ammonium sulphate precipitation, ion exchange chromatography using DEAE cellulose and gel filtration chromatography (GFC) using Sephadex G-200. The molecular weight of the purified enzyme was determined using native poly acrylamide gel electrophoresis (Native PAGE) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme was characterized to determine pH and temperature optima. Thermostability, pH stability and substrate kinetics were studied for purified xylanase. Effect of several metal ions and detergents were also studied.
 Results: The alkali-thermo xylanase of Aspergillus terreus was purified to 14.6-fold with a 39.48% recovery through IEC on DEAE-cellulose and GFC using Sephadex G-200. The purified enzyme had a specific activity, 111.6 U/mg. The SDS-PAGE revealed that the enzyme was monomeric with a molecular weight of 87 kDa. The optimum pH and temperature were 8 and 60⁰C, respectively. The Km and Vmax values of the partially purified xylanase were found to be 0.016 mg/l and 102.64 U/mg protein, respectively with birch wood xylan as substrate.
 Conclusion: The enzyme was active over a wide pH range of 6-11 and temperature range of 40⁰C to 80ºC, indicating its alkali tolerant and thermostable characteristics which is the requirement of different industries. Several ions namely Zn2+ enhanced xylanase activity by 187% followed by Ca2+ (129%), Mg2+ (123%), NH4+ (146%) and Fe3+ (118%) at 10 mM concentration. However, Cu2+ and Hg2+ completely inhibited xylanase activity. This research provides the basis for application of xylanase and improves its biotechnological potential in mainly in paper pulp industry, biofuel generation and in bread baking.

ASSESSMENT OF NUTRITIONAL RELEVANCE OF DIPLAZIUM ESCULENTUM BY QUALITATIVE PHYTOCHEMICAL AND OVERALL PROTEIN PROFILING

Diplazium esculentum is one of the very well-liked edible ferns, a common pteridophyte. Very few reports are published about the plant from India. Pteridophytes are acquainted for their biological and medicinal properties for long time but very fewevidences are seen about wetland pteridophytes. The present work focuses on assessment of nutritional relevance of D. esculentum collected from Alappuzha District of Kerala in India, by qualitative phytochemical and overall protein profiling. Chloroform and methanolic extracts of leaves of D. esculentum are used for both assays. The results showed the presence of vital phytoconstituents on qualitative analyses that included Alkaloids, Saponins, Flavonoids, Steroids, Phenols, Cardiac glycosides and Carbohydrates. On protein profiling by Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS PAGE), 5 distinct bands corresponding to molecular weights ~250 kDa, ~130 kDa, ~100 KDa, ~55 KDa and ~15 KDa which showed nutritional importance of the fern, Diplazium esculentum. The future perspectives of study may include the quantitative estimation of phytoconstituents and proteins that paves the way for the incredible health benefits of the fern for humans.

The Assembly of Bacteriophage Functional Enzymatic Models in Association with E. coli Proteins’ Profiles

Introduction: The invasion of bacteriophage on the associated host bacterium depends on their receptors’ orientation that adsorb them to cell surface. During phage replication a valuable number of proteins acts as lytic enzymes for host puncher at the beginning of the infection and other for burst after lytic cycle compilation. Accordingly, the proteomic relationship among phage and bacterium proteins could easily be studied by their protein profiles analysis. Objective: To detect bacteriophages functional enzymes during lytic cycle. Methods: The isolation and identification of Escherichia coli and their parasitic T7 phage group was done using bacterial culture and common plaque assay techniques. The investigations and protein-protein interactions’ assays were inveterate by proteins profile of phage and bacterium using Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) to find out their molecular weights, where the scaled location of each mobile band was compared to the standards of identified proteins weights in the molecular ladder. Thereafter, Protein model’s assembly and bands migration was done by computer analytical software. Results: Mobilization of the phage’ proteins inside the Two Dimensions (2D) gel ranged between 60 and 12 kDa where a model of 4 main bands with molecular weights of (46, 35, 24 and 14 kDa) is corresponded to the host ones, where pure 9 bands with molecular weight ranged between 96-24 kDa. The computational model analysis showed common shared molecular masses of 47, 34 and 16 kDa on plot area of the phage and the bacterium. Model interpretation confirmed that proteins ranged from 47.7 to 34.3 kDa resembles 43.3% of whole phage’s proteins that assembled the capsid head and the coil, while the molecular weight mass of 22.5 formed the tail’s proteins. The lytic enzymes’ molecular weight was ranged between 18-14 kDa according to the function of the enzyme. The study revealed that the 34 kDa band has the common shared peak between T7 phage group and associated Escherichia coli host. Conclusion: Functional models of analysed proteins during phage assembly, ensures lytic enzymes are built in the capsid head and the lysozyme in the tail, they facilitate the enzymatic decay for bacterial host. This enzymatic function is related to the lytic cycle of the bacteriophages and their phenomenon in employing the bacterial DNA in proteins manufacturing during their replication inside host.

Genetic diversity in the natural population of threatened fruit- bearing plant species Monotheca buxifolia (Falc.) A.D.

Monotheca buxifolia is an ethnomedicinally and economically important threatened fruit bearing plant species in Malakand Division Pakistan. The genetic diversity among the 92 various genotypes of Monotheca buxifolia was carried out using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) method. A considerable amount of inter districts genetic diversity (66.70%) was observed among the genotypes of M. buxifolia. Protein profiling was conducted on 12% gel electrophoresis. A total of 6 protein bands were observed in M. buxifolia genotypes. SDS-PAGE practice is a convenient scheme for the examination of both genetic diversity and relationship. Particularly, L-4 and L-5 were monomorphic in the inter districts Monotheca buxifolia genotypes and was recognized as species specific. The remaining other loci were polymorphic. In this investigation, the high inter and intra- districts specific diversity was observed demonstrating SDS-PAGE is an authoritative procedure for categorizing the genetically diverse germplasms in M. buxifolia. The findings from this study could be useful in the identification and selection of suitable M. buxifolia genotypes for future conservation programmes. Today, there is still a need to examine the genetic diversity and protect genetic resources, in particular wild species, for possible benefits in plant conservation programmes. To the best of our knowledge, this is the first ever report that addresses genetic variability in M. buxifolia.

Hydrolysis of Peptide Bonds in Protein Micelles Promoted by a Zirconium(IV)-Substituted Polyoxometalate as an Artificial Protease.

The development of artificial proteases is challenging, but important for many applications in modern proteomics and biotechnology. The hydrolysis of hydrophobic or unstructured proteins is particularly difficult due to their poor solubility, which often requires the presence of surfactants. Herein, it is shown that a zirconium(IV)-substituted Keggin polyoxometalate (POM), (Et2 NH2 )10 [Zr(α-PW11 O39 )2 ] (1), is able to selectively hydrolyze β-casein, which is an intrinsically unstructured protein at pH 7.4 and 60 °C. Four surfactants (sodium dodecyl sulfate (SDS), N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZW3-12), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and polyethylene glycol tert-octylphenyl ether (TX-100)), which differ in the nature of their polar groups, were investigated for their role in influencing the selectivity and efficiency of protein hydrolysis. Under experimental conditions, β-casein forms micellar structures in which the hydrophilic part of the protein is water accessible and able to interact with 1. Identical fragmentation patterns of β-casein in the presence of 1 were observed through SDS poly(acrylamide) gel electrophoresis both in the presence and absence of surfactants, but the rate of hydrolysis varied, depending on the nature of surfactant. Whereas TX-100 surfactant, which has a neutral polar head, caused only a slight decrease in the hydrolysis rate, stronger inhibition was observed in the presence surfactants with charges in their polar heads (CHAPS, ZW3-12, SDS). These results were consistent with those of tryptophan fluorescencequenching studies, which showed that the binding between β-casein and 1 decreased with increasing repulsion between the POM and the polar heads of the surfactants. In all cases, the micellar structure of β-casein was not significantly affected by the presence of POM or surfactants, as indicated by circular dichroism spectroscopy.

Changes in High-Density Lipoproteins Related to Outcomes in Patients with Acute Stroke.

Modifications in high-density lipoprotein (HDL) particle sizes and HDL-binding proteins have been reported in stroke patients. We evaluated whether the lipoprotein profile, HDL composition and functionality were altered in stroke patients according to their clinical outcome using the modified Rankin Score at 3 months. Plasma samples were obtained from stroke patients treated with intravenous thrombolysis. Levels of cardiovascular and inflammatory markers in plasma were measured using the Human CVD Panel 1 (Milliplex® MAP). Lipoprotein subfractions from plasma were quantified by non-denaturing acrylamide gel electrophoresis, using the Lipoprint®-System (Quantimetrix®), and HDLs were isolated by ultracentrifugation. Relative amounts of paraoxonase-1 (PON1) and alpha-1 anti-trypsin (AAT) in the isolated HDLs were determined by Western blot. HDL anti-inflammatory function was evaluated in human blood–brain barrier endothelial cells stimulated with 100 ng/mL TNFα, and HDL antioxidant function was evaluated via their capacity to limit copper-induced low-density lipoprotein oxidation. Stroke patients with unfavorable outcomes had a lower proportion of small-sized HDLs and increased plasma levels of E-selectin (SELE) and the intercellular adhesion molecule 1 (ICAM1). HDLs from patients with unfavorable outcomes had lower levels of PON1 and displayed a blunted capacity to reduce the expression of SELE, interleukin 8 (IL8) and the monocyte chemoattractant protein-1 (MCP1) mRNA induced by TNFα in endothelial cells. These HDLs also had a reduced antioxidant capacity relative to HDLs from healthy donors. In conclusion, an increased ratio of large/small HDLs with impaired anti-inflammatory and antioxidant capacities was associated with unfavorable outcomes in stroke patients. Alteration of HDL functionality was mainly associated with a low amount of PON1 and high amount of AAT.

Genetic diversity in threatened plant species Alnus nitida (Spach.) Endel

Alnus nitida (Spach) Endl. is an ethnobotanically important threatened plant species. The genetic diversity among the 50 different genotypes of Alnus nitida was carried out using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) characterization. A considerable amount of genetic diversity (90%) was observed among the genotypes of A. nitida. The protein characterization was carried out on 12% gel electrophoresis. A total of 10 protein bands were detected in A. nitida genotypes. SDS-PAGE procedure is a useful method for the investigation of both genetic diversity and phylogenetic relationship. Especially, B-5 was monomorphic in A. nitida genotypes and was considered as species specific. All other bands/loci were polymorphic. These polymorphic bands displayed 12, 16, 72, 88, 2, 44, 84, 54 and 12 percent variation respectively. In the present examination, the high intra-specific diversity was observed representing SDS-PAGE is a powerful tool for determining the genetically diverse germplasms in A. nitida. The results obtained by this study could be helpful in the identification and selection of desired genotypes of Alnus nitida for conservation programmes in future. Today, there is still a need to assess genetic variation and protect genetic resources, especially of wild species for prospective benefits in plant conservation programmes.

Silver Nanoparticle Trypsin Corona Formation and the Impacts on Enzymatic Potency

Herein, the protein corona formation on the spherical metal nanoparticles was studied to investigate the possible effects of silver nanoparticles (AgNPs) on the protein activity and conformation. The digestion capability of trypsin was monitored on the human serum albumin (HSA) at standard enzymatic hydrolysis conditions in the absence and presence of different concentrations of AgNPs. So the ratio of enzyme:HSA, the duration and the temperature of nanoparticle treatment were evaluated. The activity of treated trypsin molecules, in the form of hard (HC) and soft corona (SC) were studied using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE), and nano liquid chromatography electrospray (ion trap) mass spectrometry (nano LC-ESI/MS, LC-ESI/MS). In addition the characteristics of silver nanoparticles and the formation of HSA corona on the nanoparticle surface were monitored using ultra-violet/visible spectroscopy (UV-Vis), Dynamic light scattering (DLS) and fluorescence spectrophotometry. The results demonstrated that not only the corona formation but also the AgNPs/Trypsin interaction, decreases the hydrolysis potency of trypsin. Furthermore, the encountering of the AgNPs/HSA could influence both nanoparticles and HSA molecule features. Accompanied with fluorescence study, that the HSA secondary structure. Also LC-ESI/MS data revealed the most affected HSA triptics have α-helix structure.

Isolation and characterization of partially furified bacteriocin of <i>Bacillus cereus</i> (CF1) soil isolate

Bacteriocins are proteinaceous antimicrobial substances produce by bacteria against closely related bacterial species. However their broad spectrum inhibitory activity has been observed against other microorganisms such as fungi. The aim of this study was to isolate and determine physical, chemical and biological characteristics of bacteriocin from B. cereus of soil origin. A Gram-positive spore forming bacilli coded as CF1 was isolated by pour plating technique from the soil sample of the cereal farm land of Kura local government area, Kano State Nigeria and identified as Bacillus cereus based on cultural, microscopic and biochemical characteristics. Bacteriocin was isolated from this B. cereus and partially purified by solvent method (cold acetone) and had broad spectrum inhibitory activity with MIC of 64 AU/ ml. This bacteriocin was thermo stable up to 121°C for 15 minutes and its activity was maintained at wider pH value of 2-12. Although, the bacteriocin isolated by this B. cereus was not affected by some chemical such as Urea, EDTA and Tween 80, other chemical such as SDS and trypsin enzyme deactivated its activity. It is therefore recommended that further studies be carryout on this bacteriocin to purify the bacteriocin using Poly Acryl Amide Gel Electrophoresis PAGE and to elucidate its sequence, to determine its molecular conformation and antibacterial activity on multi drug resistant pathogens.
 Key words: Bacteriocin, Bacillus cereus, Broad spectrum, Tween 80 and Sodium Dodecyl Sulphate.

Biochemical and immunological characterization of haemolysin produced by <i>Pseudomonas aeruginosa</i> PAO1 isolated from burn wounds

Background: Infection of burn wounds by multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) is a leading cause of morbidity and mortality and remains one of the most challenging concerns for the burns unit. The aim of this study is purify and characterize the haemolysin produced by multidrug resistant P. aeruginosa PAO1 isolated from burn wounds. Methods: Isolation and identification of P. aeruginosa from burns was done by standard bacteriological methods. P. aeruginosa PAO1 was identified by PCR amplification and sequencing of the 16S rRNA gene. The haemolysin of P. aeruginosa PAO1 was purified by 70% ammonium sulphate precipitation followed by gel filtration on Sephadex G-100, and separation by SDS-Poly Acrylamide Gel Electrophoresis. In vivo toxicity of the purified haemolysin was determined by intraperitoneal injection of Swiss albino mice, and in vitro toxin-antitoxin neutralization test was performed as previously described. Results: The pure haemolysin had a molecular weight of 37 kDa, with maximum activity at 25°C for 30 minutes and stable within pH range of 4-9 (maximum activity at pH 7). The haemolysin was activated by Ca2+, Fe3+ and Cu2+. Intraperitoneal injection of mice with 0.5ml of haemolysin (128 HU/ml) caused 100% mortality while 0.5 and 0.1 ml of haemolytic titer (64 HU/ml) of the heated haemolysin (toxoid) caused 50% and 0% mortality respectively. In vitro toxin-antitoxin neutralization test revealed that anti-haemolysin antitoxin was present in the serum of the mice that were previously vaccinated with heated toxin. Conclusion: This study concluded that haemolysin can be a potential vaccine component for prevention of haemolysis caused by multidrug resistant P. aeruginosa in burn patients.Keywords: haemolysin, Pseudomonas aeruginosa, multidrug resistant organism

Alteration of protein profile in cerebral cortex of rats exposed to bisphenol a: a proteomics study

Bisphenol A (BPA) is one of the most widely used chemicals in plastic industry, which enters the human body through occupational and food contact. We studied the protein changes in rat cerebral cortex to evaluate the neurotoxicity of BPA. Twenty-four adult male rats were randomly selected and divided into four groups and each group respectively received 0, 0.5, 5 and 50 mg/kg of BPA for 4 weeks orally. To determine the oxidative status, reduced glutathione content and the level of malondialdehyde were measured in brain cortical tissue. The proteins of each sample extracted and separated on a two-dimensional acrylamide gel electrophoresis. From the obtained protein map, the 10 most altered protein spots were used for mass spectroscopy analysis. The lipid peroxidation in both doses of 0.5 and 5 mg/kg was significantly higher than the control group, but the glutathione content had no significant difference between the groups. Based on the results of the MS data analysis by the MASCOT database search engine, 10 proteins with altered intensity were identified as pyruvate kinase, alpha-enolase, aconitate hydratase, creatine kinase B-type, phosphatidylethanolamine-binding protein 1, 14-3-3 protein eta, guanine nucleotide-binding protein subunit beta-1, dihydropyrimidinase-related protein 2, glutamine synthetase and the neurofilament light polypeptide. There are several reports suggesting that the increase or decrease in the level and activity of these 10 proteins, similar to those observed in this study, is related to some neurological and psychosocial disorders including neurodegenerative diseases, schizophrenia, depression, epilepsy and some brain tumors.

PS1038 FLT3 TESTING IN THE RYDAPT ERA

Background:The approval of Rydapt (midostaurin) heralds the first targeted therapy in acute myeloid leukaemia (AML). Accurate detection of both FLT3 internal tandem duplications (ITD) and tyrosine kinase domain (TKD) variants is required to ensure that patients correctly receive the therapy. Additionally, recent European LeukaemiaNet AML guidelines now require the testing of a number of genetic markers including FLT3 to risk stratify patients.Aims:With the increased importance of FLT3 testing for the diagnosis, prognosis and treatment of AML, the aims of this study were to assess methodological variation and the quality of laboratory testing of FLT3 ITD variants.Methods:The United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI), has been providing external quality assessment (EQA) to laboratories undertaking FLT3 ITD testing by molecular genetic methods since 2011, and became IS0 17043 accredited in 2014. The programme currently has 130 active participants in 36 countries globally. Two lyophilised patient donated or cell line based samples are shipped to laboratories 3 times per annum and participants are asked to test the samples with their in‐house techniques, reporting both qualitative (ITD present/absent) and quantitative (mutation load/allelic ratio) data along with a number of methodological details.Results:Forty samples (including samples both positive and negative for the FLT3 ITD) have been issued to laboratories, in 20 separate distributions, since 2011. Positive samples have featured ITD sizes ranging from 30–126 bp, with mutation loads ranging from 10–90%.The most popular analysis methods used by participants were capillary electrophoresis (70.8%) and agarose gel electrophoresis (20.0%), with a small number of participant using next generation sequencing (NGS), melt curve analysis, acrylamide gel electrophoresis, microfluidic electrophoresis and Sanger sequencing. The majority of laboratories use laboratory developed (in‐house) tests (85.8%), with the remaining participants using commercially available assays.Concordance rates ranged from 83.3% to 100% (average 97.5%). Higher error rates were associated with low mutation load samples and larger ITDs (126 bp). High error rates were associated with gel electrophoresis and NGS based methods. Quantitatively, all laboratories were able to distinguish a high (median mutation load = 92%) and low (median mutation load = 11.7%) mutation load sample with a high degree of concordance (interquartile range = 2.5% and 3.7%, respectively), but variation in calculation methods used were identified.Summary/Conclusion:The quality of FLT3 testing was generally high, although a higher error rate was associated with low mutation load samples and large ITDs when participants used agarose gel or NGS based techniques. Gel electrophoresis is an inherently variable, outdated method and its use should not be encouraged. Standard NGS analysis software has difficulty in detecting large ITDs, as large insertions do not retain sufficient homology in the regions flanking the insertion to permit accurate alignment. Pair‐end based analysis approaches have been developed that can reliably detect larger insertions. The high degree of concordance seen in quantitative data was reassuring but could be improved further by standardising calculation methods.

Evaluation of Genetic Diversity of Seven Species of Solanum in Nigeria Using SDS-PAGE

Seed protein profile of 12 accessions belonging to 7 species of the genus Solanum of the family solanaceae were investigated through poly acrylamide gel electrophoresis. The accessions were selected based on their overlapping morphological associations observed on the field. Occurrence of two protein bands of molecular weights 100 kda and 20 kda in all accession showed interspecific relationships and could be used as generic bands among the genus Solanum. All morphologically similar accessions manifested similarities in their protein bands except S. melongena. Dendrogram based on upgma also revealed the generic status and intraspecific relationships of the accession of S. aethiopicum and S. macrocarpon.

Transcriptional regulation of protein S gene

Background: Protein S (PS) is a known Vitamin K dependent plasma glycoprotein that is produced in many human tissues including the liver. Furthermore, it has an important function in the coagulation cascade as an anticoagulant cofactor when it binds active protein C. The deficiency of PS may lead to the development of deep vein thrombosis. There are many transcription factors which play an important role in the regulation of the transcription of PS. One of these regulatory factors is the liver specific transcription factor hepatocyte nuclear factor 1 (HNF1), a binding site which is located in the 638 base pair promoter area at the 5' end of the PS gene. HNF1 and PS is not well understood and to confirm the importance of HNF1 in regulating expression of PS small interfering ribonucleic acid (siRNA) which will knockout the HNF1 gene expression can be used, and the effect on PS expression monitored. The levels of expression of HNF1 and PS can be determined by real time polymerase chain reaction. Materials and Methods: Preparation of total ribonucleic acid for complementary DNA synthesis, Evaluation the quality of HepG2 ribonucleic acid by agarose gel electrophores is, Real time polymerase chain reaction, Acrylamide gel electrophoresis. Results: HepG2 rRNA bands shows a good quality of RNA expression, Analysis of the total RNA quantity can be determined by spectrophotometric analysis, The best housekeeping genes were UBC and YWHAZ therefore, there has been used as a reference for the primers samples in efficiency test. HNF1 and PROS1 shows a good expression but with low efficiency which is not required to carry further gene knockdown experiment, SYBR green dye is less sensitive than TaqMan because it presents a non specific detection (primer dimers) were all dsDNA products are detected including primer dimers, contaminating DNA, and PCR product from mis annealed primer. Conclusion: The primer pairs are not efficient to carry further gene knockdown experiment and required to design another HNF1 and PROS1 primers. The efficiency of the PCR should be 90%-100% meaning doubling of the amplicon at each cycle. This corresponds to a slope of -3.1 to -3.6 in the Ct vs log template amount standard curve. To obtain accurate and reproducible results, reactions should have efficiency as close to 100% as possible.

Cytokines and immunoglobulin g response in donkeys with spontaneous Setaria equine infection

Setaria equina (S. equina) is a filarial worm that exists in peritoneal cavity of equines. This study aimed to evaluate cytokine mediators tumour necrosis factor alpha (TNF-α), interleukin-4 (IL-4) and immunoglobulin G (IgG) responses in spontaneously S. equina infected and non-infected donkeys with emphasis on choosing the best antigen that could be used in diagnosis of such filarial infection. A total of 87 donkeys were examined. Two S. equina antigens: crude somatic S. equina antigen (CSS) and excretory secretory S. equina antigen (ESS) were prepared. They were evaluated in diagnosis of the infection using indirect ELISA and electrophoretically characterised through sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE) and western blotting technique. The results indicated that both TNF-α and IL-4 in the serum of infected donkeys were significantly higher compared with the non-infected group at P<0.05 and P<0.01, respectively. However, the IL-4 level of infected donkeys was significantly higher than that of TNF-a (P<0.01). Apparent prevalence, specificity and positive predictive values (96.55%, 100%, and 100% each) of CSS showed higher diagnostic accuracy than that of ESS. In addition, electrophoretic protein profile and IgG reactivity of CSS antigen via western blot presented a prominent reactive protein band at 28 kDa. It was concluded that the CSS antigen was the best antigen that could be used in serodiagnosis of S. equina infection. The cytokine responses were explored in order to differentiate infected from non-infected donkeys.

Analysis of Protein Glycation Using Phenylboronate Acrylamide Gel Electrophoresis.

Carbohydrate modification of proteins adds complexity and diversity to the proteome. However, undesired carbohydrate modifications also occur in the form of glycation, which have been implicated in diseases such as diabetes, Alzheimer's disease, autoimmune diseases, and cancer. The analysis of glycated proteins is challenging due to their complexity and variability. Numerous analytical techniques have been developed that require expensive specialized equipment and complex data analysis. In this chapter, we describe two easy-to-use electrophoresis-based methods that will enable researchers to detect, identify, and analyze these posttranslational modifications. This new cost-effective methodology will aid the detection of unwanted glycation products in processed foods and may lead to new diagnostics and therapeutics for age-related chronic diseases.

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways.

Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells—primarily eosinophils and mast cells—where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose–acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

Association of the CSN1S1 gene polymorphism with milk composition traits in Jamunapari goat

The genetic polymorphism of the CSN1S1 (casein alpha-s1) locus was investigated in Jamunapari goat breed by Sodium Dodicyle Sulphate – Poly Acrylamide Gel Electrophoresis (SDS-PAGE) and Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP). A total of 122 milk and blood samples were collected. The SDS-PAGE pattern revealed the presence of four variants of CSN1S1 locus namely, A, B, E and F. The A allele of a-s1 – casein was observed in the majority of goats. The genotypic frequency of the AA, BB, BE, AF and FF genotypes of CSN1S1 locus were 0.437, 0.175, 0.250, 0.125 and 0.012, respectively. The PCR-RFLP was performed, six different variant groups of F allele (224)bp, the B/E allelic pair (161+63) bp, A/0 allelic pair (150+63) bp, AD allelic pair (212+150+63) bp, AF allelic pair (224+150+63) bp and BF allelic pair (224+160+63) were obtained after restriction digestion with XmnI. The genotypic frequency of AA, AD, AF, BB, BE, BF and BC genotypes were 0.274, 0.114, 0.147, 0.098, 0.163, 0.081 and 0.163, respectively. The allelic frequency of A, B, C, D, E and F were 0.360, 0.303, 0.081, 0.057, 0.081 and 0.114, respectively. The present study showed that AA genotype had significant effect on total protein content on the milk and AD genotype had significant effect on 90 day milk yield in Jamunapari goat. Thus the CSN1S1 genotype information in Jamunapari could be utilised in selection strategies, in order to select genetic stock for the production of goat milk with higher protein content in marker assisted selection to increase the rate of genetic gain.

Efficiency of photodynamic therapy and Avastin in treatment of corneal neovascularization in rabbit's eye

The aim of the present work is to evaluate the effect of photodynamic therapy (PDT) by using argon laser with rose bengal (Herbs) as a photosensitizer when compared to the Avastin for treatment of corneal neovascularization (CNV) in rabbits. Thirty-Nine New Zealand male rabbits aged 2.5 months and weighing 2.5-3 kg are selected from the animal house of research institute of ophthalmology, Giza, Egypt. Three rabbits are used as control, and 36 rabbits are subjected to corneal neovascularization (CNV) induced by 3 silk sutures at mid-stromal depth approximately 1 mm from the limbus. After four days, sutures are removed and slit lamp examinations are performed to determine the area of CNV. Rabbits with CNV are divided into 3 groups: (1) The first group left without any treatment(n=12); (2) The second group (n=12 rabbits) were generally anesthetized and rose bengal(C20H4Cl4I4O5) with a dose of 50 mg/Kg was administered by slow intravenous injection in the marginal ear vein. After 15 minutes, rabbit’s eyes were exposed to argon laser(Quantel-Medical, Vitra, France) in a pulsed mode at 532 nm, spot size of 50 μm, pulse duration of 0.2 ms and a power of 150-200 mW/cm 2.; (3) The third group are treated with subconjunctival injection of 25 mg Avastin (n=12). The rabbits are followed up for 4 weeks and 3 rabbits are selected and sacrificed weekly from each group. The corneas are isolated for determination of protein content, SDS-poly acrylamide gel electrophoresis, total antioxidant capacity (TAC) and total oxidative capacity (TOC). Final results show that avastin is almost as effective as photodynamic therapy.

Onchocerca - infected cattle produce strong antibody responses to excretory-secretory proteins released from adult male Onchocerca ochengi worms

BackgroundThe front line molecules from filarial worms and other nematodes or helminthes are their Excretory-Secretory (ES) products. Their interaction with the host cells, proteins and immune system accounts for the skin and eye pathology or hyposensitivity observed in human onchocerciasis. ES products and adult worms’ crude extracts from Onchocerca ochengi, a filarial nematode that infects the African zebu cattle, were utilized in the present study as a model for studying Onchocerca volvulus that causes river blindness in man.MethodsThe ES products were generated from adult male and female worms in vitro and analyzed with poly acrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) using sera from Onchocerca-infected cattle and humans. The cattle sera were collected from a herd that had been exposed for six years to natural transmission of Onchocerca spp. The expressed reactivity was evaluated and differences analyzed statistically using Kruskal-Wallis rank and Chi-square tests.ResultsThe gel electrophoretic analyses of 156 ES products from O. ochengi female and male worms and of two somatic extracts from three females and 25 males revealed differences in the protein pattern showing pronounced bands at 15, 30–50 and 75 kDa for male ES proteins and 15, 25 and 40–75 kDa for somatic extracts, respectively and less than 100 kDa for female worms. Proteins in the ES products and somatic extracts from female and male Onchocerca ochengi worms were recognized by IgG in sera from both Onchocerca-exposed cattle and humans. Bovine serum antibodies reacted more strongly with proteins in the somatic extracts than with those in the ES products. Interestingly, the reaction was higher with male ES products than with ES products from female worms, suggesting that the males which migrate from one nodule to another are more exposed to the host immune system than the females which remain encapsulated in intradermal nodules.ConclusionsThis study demonstrates that O. ochengi ES products and, in particular, extracts from male filariae may represent a good source of immunogenic proteins and potential vaccine candidates.