Sodium Dodecyl Sulfate Electrophoresis Research Articles

Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography

The purpose of this study was to develop a protocol for the purification of acetylcholinesterase (AChE, acetylcholine acetylhydrolase, E.C.3.1.1.7) enzyme and to extend a purification method for further enzyme characterization. A further aim was to study whether the edrophonium's pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission. The purification of a soluble AChE from sheep liver using affinity chromatography on Concanavalin A-Sepharose 4B and edrophonium-Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine (ACh) and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanisms. It was purified 842-fold with a specific activity of 21U/mg protein. Sodium dodecyl sulfate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.04kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5kDa. Based on the molecular weight obtained by gel filtration, the purified AChE was assumed to be a tetrameric form.

Recent advances in electrophoretic techniques for the characterization of protein biomolecules: A poker of aces

The four classical modes of electrophoresis of protein molecules (sodium dodecyl sulphate electrophoresis, SDS-PAGE, isoelectric focusing, IEF, and immobilized pH gradients, IPGs, two-dimensional maps, 2D, and capillary electrophoresis, CE) are here reviewed, with special emphasis on recent innovations. Thus, in the case of SDS-PAGE, a novel method, consisting in focusing SDS-protein micelles against a gradient of cationic charges grafted onto a polyacrylamide gel is presented. In the case of IEF, the recent decoding of the structure, polydispersity, molecular mass distribution and buffering properties of the soluble carrier ampholyte buffers are here discussed. In regard to two dimensional mapping, recent instrumentation for performing 2D maps in horizontal, large gel slabs (up to 30 cm × 40 cm) and in a radial format for the SDS dimension is here evaluated. Finally, in the case of CE, three major applications are presented: a thorough study of capillary IEF and of all experimental variables, a method of importance in screening of rDNA products; the possibility of running proteins and peptide separations in very acidic, amphoteric, isoelectric buffers in absence of any capillary coating; finally, the possibility of producing a facile, user friendly, covalent coating of the wall silanols via bonding of quaternarized piperazines endowed with an iodinated tail. In acidic, volatile buffers, such protein/peptide runs can be directly interfaced with mass spectrometry instrumentation.

Use of capillary electrophoresis–sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process

Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis–sodium dodecyl sulfate (nrCE–SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was used during the CE–SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE–SDS versus SDS–PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins.

Clinical features of severe acquired ADAMTS13 deficiency in thrombotic thrombocytopenic purpura: the Korean TTP registry experience

The clinical significance of ADAMTS13 activity for response to treatment, mortality rate, recurrence, and prognosis is unclear. Therefore, we investigated the characteristics of severe ADAMTS13 deficiency and evaluated its prognostic features in Thrombotic thrombocytopenic purpura (TTP). The Korean TTP Registry includes 66 patients from 13 teaching hospitals in Korea who received the diagnosis of TTP from January 2005 to December 2008. Blood samples obtained upon admission were sent for ADAMTS13 analysis (multimer analysis by sodium dodecyl sulfate electrophoresis) to a central laboratory along with patient clinical information. Patients with severe ADAMTS13 deficiency had lower serum creatinine levels (P=0.001) than patients with non-severe ADAMTS13 deficiency. Although severe ADAMTS13 deficiency was associated with better response rate (75 vs. 53%, P=0.145), remission rate (81 vs. 61%, P=0.209), and mortality rate (19 vs. 31%, P=0.508) than non-severe ADAMTS13 deficiency, treatment outcomes did not differ significantly between groups. After adjusting for clinical and laboratory features, multivariate analysis did not reveal any independent risk factors for TTP-associated mortality. Patients with severe ADAMTS13 deficiency had lower serum creatinine levels at presentation, but severe ADAMTS13 activity deficiency at TTP diagnosis does not appear to have prognostic significance.

Clinical Characteristics of Thrombotic Thrombocytopenic Purpura with Severe ADAMTS13 Deficiency

AbstractAbstract 4666The clinical significance of ADAMTS13 activity for response to treatment, mortality rate, recurrence, and prognosis is unclear. Therefore, we investigated the characteristics of severe ADAMTS13 deficiency and evaluated its clinical significance in Thrombotic thrombocytopenic purpura (TTP). The Korean TTP Registry includes 66 patients from 13 teaching hospitals in Korea who received the diagnosis of TTP from January 2005 to December 2008. Blood samples obtained upon admission were sent for ADAMTS13 analysis (multimer analysis by sodium dodecyl sulfate electrophoresis and/or ELISA) to a central laboratory along with patient clinical information. After 6 months, patient data regarding treatment, response, and prognosis were collected on standardized report forms. Patients with severe ADAMTS13 deficiency had lower serum creatinine levels (P=0.001) and WBC counts (P=0.050) than patients with non-severe ADAMTS13 deficiency. Although severe ADAMTS13 deficiency was associated with better response rate (75% vs 53%, P=0.145), remission rate (81% vs 61%, P=0.209), and mortality rate (19% vs 31%, P=0.508) than non-severe ADAMTS13 deficiency, treatment outcomes did not differ significantly between groups. After adjusting for clinical and laboratory features, multivariate analysis did not reveal any independent risk factors for TTP-associated mortality. Patients with severe ADAMTS13 deficient had lower serum creatinine levels and WBC counts at presentation but Severe ADAMTS13 activity deficiency at TTP diagnosis does not appear to have prognostic significance. Disclosures:No relevant conflicts of interest to declare.

Simultaneous Presence of PrtH and PrtH2 Proteinases in Lactobacillus helveticus Strains Improves Breakdown of the Pure α s1 -Casein

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified α(s1)- and β-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure β-casein was observed for all strains, the hydrolysis kinetics of α(s1)-casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from α(s1)-casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.

A fast preparative method for detection of recombinant erythropoietin in blood samples

While firstand second-generation recombinant erythropoietins (EPOs) are detectable in urine, we showed that analysis in blood is more appropriate for the third-generation, continuous erythropoietin receptor activator (CERA), due to its poor excretion in urine. However, isoelectric focusing (IEF) of EPO in serum or plasma is not as simple as in urine since it requires an initial preparative step by immunoaffinity chromatography. That is why enzyme-linked immunosorbent assay (ELISA) and IEF are used as screening and confirmation tests for CERA detection, respectively. Due to the observation of false negative results of ELISA, we have developed a fast method for preparing plasma or serum samples, making IEF as easy to use in blood as in urine. While preparation by immunoaffinity is kept for confirmation analysis, this convenient method is proposed for screening analysis. In addition to CERA, Darbepoetin alfa is very easily identified in blood due to its typical IEF profile. Identification of first-generation EPO drugs will require criteria specific for blood. In comparison with urine, EPO analyses in blood appear beneficial in terms of sensitivity, stability during physical exercise, and prevention of sample adulteration at collection. Anti-doping control of erythropoietin (EPO) is based on the differentiation of natural endogenous from recombinant hormones by their isoelectric profiles.[1] SDS (sodium dodecyl sulfate) electrophoresis is a useful complementary method.[2] Both methods were developed for urine samples. While firstand second-generation recombinant EPOs are detectable in urine, we showed that analysis in blood is more appropriate for the thirdgeneration, continuous erythropoietin receptor activator (CERA), due to its poor excretion in urine.[3] However, isoelectric focusing (IEF) of EPO in serum or plasma is not as simple as in urine since it requires an initial preparative step by immunoaffinity chromatography.[4] We thus developed a convenient method (more than 20 samples can be treated in parallel) for preparing plasma or serum samples, making IEF as easy to use in blood as in urine.

Deletion of a terminal residue disrupts oligomerization of a transmembrane α-helixThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

In studies of the structural biology of membrane proteins, the success of strategies based on the "divide and conquer" approach, where peptides are used to model the individual transmembrane (TM) alpha-helices of membrane proteins, depends on the correct identification of the membrane-embedded TM alpha-helix amino acid sequence within the full-length protein. In the present work, we examine the effects of excluding or including TM boundary residues on the intrinsic properties of a Lys-tagged TM2 alpha-helix of myelin proteolipid protein (PLP), of parent sequence KKKK-66AFQYVIYGTASFFFLYGALLLAEG89-KKKK along with analogs containing an additional wild type Phe-90, Phe-90 and Tyr-91, and of a hydrophobic mutant Leu-90. Using protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer in the membrane-mimetic detergent sodium dodecylsulfate (SDS), we demonstrate that the removal of a single amino acid from the C-terminus of this TM segment is enough to change its intrinsic properties, with TM2 66-89 displaying only a monomeric form, but with dimers arising for the other 3 peptides. A novel use of trifluoroethanol (TFE) as a maximal helix-supporting solvent demonstrated that peptides containing residues at positions 90 and (or) 90-91 displayed significantly increased helical content vs. the TM2 parent peptide. The findings suggest that deletion of critical C-terminal residue(s) tends to reposition the helix terminus toward the membrane-aqueous interface. Our overall results emphasize the potential influence of boundary residues on TM properties when using peptides as models for TM alpha-helices, and may implicate a role for these residues in membrane protein folding and assembly.

Electrophoresis today and tomorrow: Helping biologists' dreams come true

Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two-dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High-throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser-induced fluorescence detection of labeled DNA strands have been of key importance for the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH-driven mobilization, provides efficient separations and on-line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single-cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single-molecule detection.

Analysis of monoclonal antibody product heterogeneity resulting from alternate cleavage sites of signal peptide

Signal peptides used in biosynthesis of proteins are cleaved at a very specific site by signal peptidase during posttranslational translocation of cytoplasmic proteins across the membrane. In some cases, however, there can be cleavage at nonspecific sites, giving rise to heterogeneity in the mature protein, which manifests itself as either elongation or truncation of the N terminus of the mature protein. When used as biopharmaceutical therapeutics, such heterogeneities may be a cause for concern, depending on the nature of the heterogeneity. This article describes the determination of such heterogeneity by peptide mapping in both the heavy chain and the light chain (LC) of a Chinese hamster ovary (CHO) cell-expressed monoclonal antibody (mAb). The peptide map method described here was capable of detecting the extended N-terminal peptides at levels as low as 1% relative to the peak area of the intact N-terminal peptide. The LC of a mAb product was truncated at its N termini by two amino acid residues at approximately 3–4% levels, resulting from alternate signal peptide cleavage. This article describes the quantitation of this truncation by liquid chromatography–mass spectrometry (LC–MS) peptide mapping. Also described is analysis and characterization of LC truncation by reduced and denatured capillary electrophoresis in sodium dodecyl sulfate (CE–SDS). The truncated mAb, which was devoid of the two N-terminal amino acids, was engineered and shown to migrate as the “pre-LC” peak in reduced CE–SDS assay. The amount of the pre-LC peak recovered from the CE–SDS assay was shown to correlate with the amount of truncated peptide observed from the reduced and alkylated peptide map of the engineered mAb.

Oligomerization of Transmembrane Alpha-Helices Modulated By C-terminal Boundary Residues

In studies of the structural biology of membrane proteins, the success of strategies based on the “divide-and-conquer” approach, where peptides are used to model the individual transmembrane (TM) α-helices of membrane proteins, depends upon the correct identification of the membrane-embedded TM α-helix amino acid sequence within the full-length protein. In the present work, we examine the effects of excluding or including TM boundary residues on the intrinsic properties of the TM2 α-helix of myelin proteolipid protein (PLP). Using protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer in the membrane-mimetic detergent sodium dodecylsulfate (SDS) to study parent sequence KKKK-61AFQYVIYGTASFFFLYGALL-LAEG89-KKKK - along with analogs containing an additional wild type Phe-90, Phe-90 and Tyr-91, and a hydrophobic mutant Leu-90 - we demonstrate that the removal of a single amino acid from the C-terminus of this TM segment is sufficient to change its intrinsic properties, with TM2 61-89 displaying only a monomeric form, but with principally dimers arising for the other three peptides. The findings suggest that deletion of critical C-terminal residue(s) tends to re-position the helix terminus toward the membrane-aqueous interface, and emphasize the potential influence of boundary residues on TM properties when utilizing peptides as models for TM α-helices. These finding may implicate a role for such residues in membrane protein folding and assembly.

Biophysical Analyses of Synthetic Amyloid-β(1−42) Aggregates before and after Covalent Cross-Linking. Implications for Deducing the Structure of Endogenous Amyloid-β Oligomers

A neuropathological hallmark of Alzheimer's disease (AD) is the presence of large numbers of senile plaques in the brain. These deposits are rich in fibrils that are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that soluble Abeta aggregates as well as fibrils are important in the etiology of AD. Low levels of endogenous soluble Abeta aggregates make them difficult to characterize, but several species in extracts of AD brains have been detected by gel electrophoresis in sodium dodecyl sulfate (SDS) and immunoblotting. Individual Abeta oligomers ranging in size from dimers through dodecamers of 4 kDa monomeric Abeta have been resolved in other laboratories as discrete species by size exclusion chromatography (SEC). In an effort to reconstitute soluble Abeta aggregates in vitro that resemble the endogenous soluble Abeta aggregates, we previously found that monomeric Abeta(1-42) rapidly forms soluble oligomers in the presence of dilute SDS micelles. Here we extend this work in two directions. First, we contrast the size and secondary structure of these oligomers with those of synthetic Abeta(1-42) fibrils. SEC and multiangle light scattering were used to obtain a molecular mass of 150 kDa for the isolated oligomers. The oligomers partially dissociated to monomers through nonamers when incubated with SDS, but in contrast to endogenous oligomers, we saw no evidence of these discrete species prior to SDS treatment. One hypothesis to explain this difference is that endogenous oligomers are stabilized by covalent cross-linking induced by unknown cellular agents. To explore this hypothesis, optimal mass spectrometry (MS) analysis procedures need to be developed for Abeta cross-linked in vitro. In our second series of studies, we began this process by treating monomeric and aggregated Abeta(1-42) with three cross-linking agents: transglutaminase, glutaraldehyde, and Cu(II) with peroxide. We compared the efficiency of covalent cross-linking with these agents, the effect of cross-linking on peptide secondary structure, the stability of the cross-linked structures to thermal unfolding, and the sites of peptide cross-linking obtained from proteolysis and MS.

Biochemical characterization of digestive amylase of wheat bug, Eurygaster maura (Hemiptera: Scutelleridae)

Biochemical characterization of
-amylase in the midgut and salivary glands of Eurygaster maura was conducted. Results showed that
-amylase activities were present in the salivary glands and gut. The activity of
-amylase in the midgut and in the salivary glands was 0.098 and 0.057 U/ml, respectively. The pH of salivary glands and the gut was determined to be in the range of 5- 5.5 (for the salivary glands) and in the range of 6-6.5 (for the gut), using staining indicator. The optimum pH and temperature for salivary glands and midgut amylase activity was 6-7 and 35-40oC, respectively. The stability of amylase was highest in the acidic pH (4-5). Ethylenediamine tetraacetic acid (EDTA), urea, sodium dodecyl sulfate (SDS) and Mg 2+ inhibited the enzyme activity but, NaCl and KCl enhanced enzyme activity. Based on linear regression analysis of reciprocal starch concentration versus reciprocal amylase activity K m and V max were 0.11% and 0.04 mM maltose/min for midgut amylase and 0.298% and 0.071 mM maltose/min for salivary amylase, respectively. Sodium dodecyl sulfate electrophoresis (SDS-PAGE) showed that both midgut and salivary glands contain isozymes.

A COMPARISON OF PHYSICAL AND RHEOLOGIC PROPERTIES OF ARROWTOOTH FLOUNDER PROTEIN MADE USING THREE DIFFERENT EXTRACTING PROCESSES

ABSTRACT Soluble and insoluble fish protein powders were made from arrowtooth flounder (AF) fillets using three methods, which were heating and fractionation, enzymatic hydrolysis and alkali protein extraction. The AF powders were compared, and physical, chemical and rheologic properties were evaluated. The alkali protein extraction soluble (AES) (89.6%) and enzymatic hydrolysis soluble (EHS) (84.8%) powders had higher protein contents than other protein powders (72.6–77.8%). Both heating and fractionation soluble (HFS) and heating and fractionation insoluble powders were whiter than the other protein powders. HFS and EHS had the highest nitrogen solubility values, and EHS had the highest emulsion stability values. Sodium dodecyl sulfate electrophoresis indicated AES and alkali protein extraction insoluble protein powders had more high molecular weight protein bands, while EHS, enzymatic hydrolysis insoluble and HFS protein powders were substantially hydrolyzed and had an abundance of low molecular weight peptides. The flow and viscoelastic properties of the emulsions prepared with soluble AF were investigated using a parallel plate rheometer. The power law model was used to determine the flow behavior index (n) and consistency index (K). The emulsion containing AES had the highest K value (85.3 Pa·s) and the lowest value was for EHS (27.3 Pa·s). Soluble arrowtooth powders exhibited pseudoplastic behavior and viscoelastic characteristics. PRACTICAL APPLICATIONS There is an abundance of arrowtooth flounder (AF); however, its utilization as human food is challenging because of the proteases that reduce texture during cooking. Methods of processing are needed to convert the fish into more marketable forms. One alternative is to produce purified protein powder and high-protein ingredients for human consumption. Fish provide an excellent source of very digestable and high-quality protein. Isolated fish protein powders have desirable functional properties, such as the ability to hold water and fat, and emulsifying capacities. Protein from AF can be converted into a high-value protein powder food ingredient. The application of these ingredients could include incorporation into muscle tissue products by injection, tumbling and coating.

α1-Antitrypsin Inhibits the Activity of the Matriptase Catalytic Domain In Vitro

Matriptase is a type II transmembrane protease that is characterized by an N-terminal transmembrane and multiple extracellular domains, in addition to the conserved extracellular serine protease catalytic domain. The expression pattern of matriptase suggests that this protease may play broad roles in the biology of surface lining epithelial cells. In this study we report that alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteases, inhibits the catalytic domain of human recombinant matriptase in vitro. Co-incubation of AAT with matriptase (at a molar ratio 1:2) resulted in the formation of heat stable complexes, clearly seen in sodium dodecyl sulfate electrophoresis and Western blots. AAT was found to be a slow, tight-binding inhibitor of the catalytic domain of matriptase with a second order reaction rate constant of 0.31 x 10(3) M(-1)s(-1). Notably, the oxidized form of AAT, which lacks serine protease inhibitor activity, failed to generate matriptase complexes and to inhibit matriptase activity. Since matriptase is involved in a number of physiologic processes, including activation of epithelial sodium channels, our findings offer considerable new insights into new regulatory function of AAT in vivo.

Reaction of fluorogenic reagents with proteins: II: Capillary electrophoresis and laser-induced fluorescence properties of proteins labeled with Chromeo P465

The fluorogenic reagent Chromeo P465 is considered for the analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label α-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4 × 10 −4 cm 2 V −1 s −1. The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1% with the addition of each label. The signal increased linearly from 1.0 nM to 100 nM α-lactalbumin ( r 2 = 0.99), with a 3 σ detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, α-chymotrypsinogen A, and α-lactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank.

Nitrated Alpha-Synuclein and Microglial Neuroregulatory Activities

Microglial neuroinflammatory responses affect the onset and progression of Parkinson's disease (PD). We posit that such neuroinflammatory responses are, in part, mediated by microglial interactions with nitrated and aggregated alpha-synuclein (alpha-syn) released from Lewy bodies as a consequence of dopaminergic neuronal degeneration. As disease progresses, secretions from alpha-syn-activated microglia can engage neighboring glial cells in a cycle of autocrine and paracrine amplification of neurotoxic immune products. Such pathogenic processes affect the balance between a microglial neurotrophic and neurotoxic signature. We now report that microglia secrete both neurotoxic and neuroprotective factors after exposure to nitrated alpha-syn (N-alpha-syn). Proteomic (surface enhanced laser desorption-time of flight, 1D sodium dodecyl sulfate electrophoresis, and liquid chromatography-tandem mass spectrometry) and limited metabolomic profiling demonstrated that N-alpha-syn-activated microglia secrete inflammatory, regulatory, redox-active, enzymatic, and cytoskeletal proteins. Increased extracellular glutamate and cysteine and diminished intracellular glutathione and secreted exosomal proteins were also demonstrated. Increased redox-active proteins suggest regulatory microglial responses to N-alpha-syn. These were linked to discontinuous cystatin expression, cathepsin activity, and nuclear factor-kappa B activation. Inhibition of cathepsin B attenuated, in part, N-alpha-syn microglial neurotoxicity. These data support multifaceted microglia functions in PD-associated neurodegeneration.

Urinary Sodium Dodecyl Sulfate Electrophoresis With Silver Staining: A Noninvasive Diagnostic Tool for Obstructive Uropathy in Children

Obstructive uropathy such as ureteropelvic junction obstruction in the newborn is a major diagnostic and therapeutic dilemma. We investigated whether urinary sodium dodecyl sulfate electrophoresis with polyacrylamide gel electrophoresis with silver staining could be used to discriminate between children requiring and those not requiring pyeloplasty. In a pilot study we analyzed the urine of 18 children (mean age 2.7 years) with grade III or IV hydronephrosis according to the Society for Fetal Urology classification. A total of 44 healthy children were studied as controls. Children with hydronephrosis were followed using ultrasound, (99m)technetium mercaptoacetyltriglycine diuretic renography and voiding cystourethrography. Urine was obtained by spontaneous voiding and studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining using Melzer's modification. After the study period test results were compared to outcomes, ie whether patients required surgery, and to normalization of previously abnormal protein excretion patterns. All but 1 of the healthy controls had a normal electrophoresis assessment. Of 9 patients followed for hydronephrosis 7 had an abnormal electrophoresis result preoperatively. One child had to be operated on twice because of relapse of ureteropelvic junction obstruction. Six children returned to a normal electrophoresis result postoperatively, including the child who was operated on twice. All children with an initially normal electrophoresis assessment displayed persistent normal values, except 1. Children shifting from a normal to an abnormal electrophoresis result underwent surgery after exclusion of urinary tract infection. Sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining seems to be a good predictive test for clinically relevant ureteropelvic junction obstruction. Further studies are being performed to see whether the test can stand against the gold standard, (99m)technetium mercaptoacetyltriglycine diuretic renography.

A comparison of the protein constituents of the major body compartments of the dog roundworm, Toxocara canis

An analysis of the protein profiles of intact worms and isolated tissues of adult male and female Toxocara canis worms was conducted. Soluble proteins recovered from homogenized whole specimens and dissected tissues (body wall, reproductive tract, esophagus and intestine) of T. canis adults from several different canine hosts were separated by size using gradient sodium dodecyl sulfate electrophoresis (SDS-PAGE) and visualized with silver staining. SDS-PAGE profiles of worms from different hosts were found to be virtually identical irrespective of sex or tissue type. Recovered proteins ranged in size from 3.4 to 325 kDa. As expected, variations existed between the protein profiles of different body tissues, with only slight variations between the sexes. The largest number of recovered proteins was present in the female reproductive tract extracts.

Amyloid-β(1−42) Rapidly Forms Protofibrils and Oligomers by Distinct Pathways in Low Concentrations of Sodium Dodecylsulfate

Alzheimer's disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-beta (Abeta) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Abeta species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Abeta are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Abeta, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Abeta aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Abeta aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Abeta(1-40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Abeta(1-42) undergoes an immediate conversion to a predominant beta-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8-14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30-60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Abeta aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Abeta aggregation procedures.