Search

Background: Hepatitis B (HB) is a worldwide public health problem affecting around 250 million people. Only about 10% of people with HB are aware that they are infected. Vaccination is crucial to prophylactically controlling HB and the combination of vaccination and HB immune globulin (HBIG) are essential in preventing disease after exposure to the HB virus. Objectives: In this article, a caprylate-chromatography process has been used for the production of HBIG (HBIG-C). The previously used solvent-detergent process produced an HBIG (HBIG-S/D) with an excellent safety profile but allowed the retention of some procoagulant plasma proteins. The present studies were conducted to assess the character and purity of HBIG produced by the caprylate-chromatography process. Design and Methods: Several analytical methods (eg, chromatography, immunoassays, and nephelometry) were used to assess the molecular characteristics, purity and HBIG potency and specific activity of HBIG-C. In addition, testing was conducted to assess the levels of several pro-coagulant factors. HBIG-C was compared with HBIG-S/D and other immunoglobulins manufactured by the S/D process. Results: The analysis of HBIG-C showed that the product was almost entirely IgG (99.3 ± 0.2% by electrophoresis) and that the residual IgA was less than that found in S/D products. The IgG present in HBIG-C was 99.7 ± 0.6% monomers and dimers as measured by size exclusion chromatography. Aggregates and fragments constituted < 1%. The IgG subclass distribution in HBIG-C was in the normal reference range. Coagulation factor impurities and pro-coagulant activity were reduced in HBIG-C compared to IgG prepared by the S/D method. Conclusions: HBIG-C takes advantage of long-established donor selection processes combined with recently improved manufacturing processes to produce a safe and effective HBIG-product. HBIG-C combines high purity with reduced pro-coagulant factors in a product used for post-exposure prophylaxis of HB. [ABSTRACT FROM AUTHOR]

Background and Aim: Penicillinase plays diverse roles, such as inducing drug resistance by hydrolyzing the amide bond of beta-lactam factors, the metabolism of cephalosporin-attached prodrugs, as well as the metabolism of some antibiotics, and renal clearance. The present study aimed to evaluate the optimum conditions for extracting and purifying penicillinase enzyme. The enzyme source was Bacillus licheniformis 6346 obtained from the Iranian Research Organization for Science and Technology. Materials and Methods: B. licheniformis was cultured in a medium containing mineral salts, nitrogen compounds and carbon sources. Following the harvesting and preparation of bacterial suspension, the destruction of the bacterial cell wall and crude cell extraction was completed using a combination of lysozyme and weak ultrasonication. Afterward, enzyme amount was determined utilizing chromatography and electrophoresis. Furthermore, the optimum growth temperature and incubation time were assessed based on the absorbance of samples at 240 nm, enzyme activity, aerification intensity optimization, enzyme production time, and optimum pH. Results: Approximately 8 g of cells were retrieved in one liter of culture. The optimum pH was determined as 6.5. The heat stability evaluation of the enzyme at different times revealed that the enzyme was stable for more than 1 h at 20°C-45°C, while it is inactivated in 10 min at 60°C. Moreover, the best incubation time for this bacterium was obtained as 9 h at 30°C. In terms of aerification intensity, the highest enzyme production rate of 2394 U/mL was achieved in a 250 mL Erlenmeyer flask with a 50 mL culture medium at 200 RPM. More aerification led to the inhibitory effect of oxygen and reduced enzyme activity. Conclusion: Our findings showed that the bacterium B. licheniformis 6346 can be used for producing penicillinase enzyme in optimum conditions (30°C, 200 RPM, and pH: 6.5). [ABSTRACT FROM AUTHOR]

Oversulfated chondroitin sulfate (OSCS), a non-natural sulfated glycosaminoglycan, recognizes as a significant containment in the pharmaceutical heparin, and it could trigger adverse reactions. Chromatography-, electrophoresis-, electrochemistry-, and spectroscopy-related techniques are currently available for accurate and precise analysis of a trace amount of OSCS in heparin. Recently, emerging studies focus on developing colorimetric and fluorescent probes to monitor OSCS containments in heparin. Therefore, this current review aims to describe the sensing principle and procedure of the reported probes that are sensitive and selective toward OSCS in heparin without the interferences of other sulfated glycosaminoglycans. The reported OSCS-specific probes are comprehensively discussed according to the recognition elements of OSCS, including coralyne, AG73 peptides, positively charged tetraphenylethene derivatives, polythiophene polymer, and poly-L-lysine, protamine, superpositively charged green fluorescent proteins, and poly (diallyldimethylammonium chloride). The sensing of OSCS in heparin is generally achieved using, (i) the specific affinity of the recognition element with OSCS and heparin, (ii) heparinase-mediated hydrolysis of heparin, and (iii) OSCS-induced inhibition of heparinase activity. Additionally, coralyne-based DNA probes can detect OSCS in heparin in the presence of Ca2+ ions without the assistance of heparinase. This review will pave the way to design another sensing probe towards other sulfated contaminants, like dermatan sulfate. [ABSTRACT FROM AUTHOR]

The development of insulator-based dielectrophoresis chromatography is proposed here as a novel hybrid technique that capitalizes on the simplicity of insulator-based dielectrophoresis (iDEP) and the well-known chromatographic theory. Chromatographic parameters are employed to characterize dielectrophoretic separation of particles with particles being eluted from the system as enriched particle peaks. By varying the characteristics of the insulating posts, it was possible to manipulate the interactions of the particles with the insulating post array which acted as the stationary phase. The present work studied how the characteristics of the particles affected the particle retention. Different types of particles have distinct interactions with the post array; these interactions depend on particle properties (size, electrical charge, and polarizability). This work includes mathematical modeling with COMSOL and extensive experimentation. Particles ranging from 1 to 10 μm in diameter were tested for retention time and eluted as peaks in the iDEP chromatography devices. Separation results were reported in the form of dielectropherograms including the estimation of retention time (tR), separation efficiency (N/meter), and separation resolution (Rs). Two full separations were demonstrated: a separation by charge between two types of particles of similar size (~ 10 μm) with different electrical surface charges and a separation by size between 2- and 5-μm particles with similar surface charge (difference in ζP of 4 mV). The achieved separation resolutions were Rs = 1.8 and Rs = 3.5, respectively. This is the first study on DEP chromatography to assess performance in terms of resolution and separation efficiency, demonstrating the unique potential of iDEP chromatography. [ABSTRACT FROM AUTHOR]

The benefits of Eurycoma longifolia (Tongkat Ali, TA) for its aphrodisiac capabilities are well known and many products are marketed worldwide. Due to its popularity, the plant is being abused for promoting fake products. Therefore, there is a need for better testing of the markers required by authorities and responsible manufacturers. A low-molecular-weight protein has been studied for developing it as a testing marker. Two dimensional electrophoresis (2DE) (four spots were observed) was used for positive detection of proteins in an aqueous extract of TA root and the pronounced separation of a Coomassie-stained spot, subsequently referred to as Marker A. Consecutive chromatographic separations of the aqueous extract of TA led to the isolation of pure protein from Marker A. When this marker was used to test 46 TA-based products randomly selected from markets worldwide, in 20 of them, the results were found to be comparable to those obtained using the organic eurycomanone marker. The ranking of products from the highest quantity to the lowest observed to be ordered differently if compared for both markers. This is an expected outcome because Marker A was measured for its protein content and eurycomanone for its organic molecule. Marker A detection using 2DE is shown to be a useful tool to test products supplemented with E. longifolia root. [ABSTRACT FROM AUTHOR]

N-lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N-glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography–mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for N-glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit, N-glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of <5% for peaks with >0.5% relative area. [ABSTRACT FROM AUTHOR]

Characterization and evaluation of immunotherapeutic potential of ovotransferrin PC2 (OTf PC2) were performed in this study. The ovoprotein was obtained from egg white from hens immunized with bacterial antigens, pathogenic for humans. For the negative control samples, OTf was extracted from eggs collected from Specific Pathogen-Free (SPF) hens and purified by affinity chromatography on Protein G-agarose column with two eluting peaks: I, representing ovalbumin, and II, ovotransferrin. The final apo-OTf form was reached by successive precipitation with ammonium sulfate and citric acid and the holo-OTf form by saturating the apo-form with FeCl3. Multiple OTf PC2 samples were analyzed through Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and, based on the molecular marker migration model, the ovotransferrin (76.5 kDa) and ovalbumin (45 kDa) were detected. The agglutination reaction exhibited statistically significant high specificity of the multiple OTf PC2, by reacting with the antigens used for hens’ immunization. Following ELISA, it was established that OTf PC2 from hyperimmune eggs has specificity for all antigens; the antibody titer was high, indicating that OTf PC2 possesses immunological properties similar to immunoglobulin Y (Ig Y). This study suggests that OTf PC2 immunological activity may play a crucial role in the prevention and treatment of infections resistant to antibiotics and OTf PC2 can also act as a valuable nutraceutical. [ABSTRACT FROM AUTHOR]

HS628 has been developed as a proposed biosimilar product of originator tocilizumab (Actemra®). An extensive physicochemical and biological characterization was conducted to assess similarity between HS628 and originator tocilizumab. The amino acid sequence was shown to be identical between HS628 and originator tocilizumab. The higher order structure was found to be indistinguishable from originator tocilizumab. Concerning purity and heterogeneity, HS628 was demonstrated to have similar posttranslational modifications, charge heterogeneity, size heterogeneity, and glycosylation to originator tocilizumab. Moreover, HS628 exhibited highly similar binding affinity and antiproliferative activity as well as capability of inhibiting STAT3 phosphorylation compared to originator tocilizumab. Taken together, HS628 can be considered as a highly similar molecule to originator tocilizumab in terms of physicochemical and biological properties. [ABSTRACT FROM AUTHOR]

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5–47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. Ea(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG(d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot. [ABSTRACT FROM AUTHOR]

β-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the β-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a “two-step” method comprising ultrafiltration and gel chromatography. The purified β-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5–7.0. The β-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+ and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 μmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, β-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the β-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes. [ABSTRACT FROM AUTHOR]

At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely, Ganoderma lucidum-CDBT1, Ganoderma japonicum, and Lentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, and G. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation from G. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30 μM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced by G. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants, Km, Vmax, and Kcat, determined using 2,2′-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110 μM, 36 μmol/min/mg, and 246 min−1, respectively. The isolated thermostable laccase will be used in future experiments for delignification process. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development. [ABSTRACT FROM AUTHOR]

AU-rich element binding/degradation factor 1 (AUF1) plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE) in the 3′-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM) and a Gln- (Q-) rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1) was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding. [ABSTRACT FROM AUTHOR]

The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16α,17α-epoxypregnenolone produced by Burkholderia cepacia SE-1 were investigated, and a correlative enzyme, hydroxylase, was also studied. The biotransformation products, 7β-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0. [ABSTRACT FROM AUTHOR]

To clone and express the γ-polyglutamic acid (γ-PGA) synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications. [ABSTRACT FROM AUTHOR]

A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilic Geobacillus toebii LBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease from Geobacillus toebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca2+, Mg2+, DTNB, β-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealed Km=1 mg mL−1,  Vmax=217.5 U mL−1, Kcat/Km=99 mg mL−1 S−1, Ea=51.5 kJ mol−1, and ΔG⁎=56.5 kJ mol−1. [ABSTRACT FROM AUTHOR]

Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potential storage buffer due to significant visible precipitate formation. An additional 24 full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Thus, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules. [ABSTRACT FROM AUTHOR]

The application of ionic liquids to chromatography and capillary electrophoresis as modifiers of background electrolytes, chiral selectors, and components of mobile, stationary, and pseudostationary phases in the separation of different classes of organic compounds is discussed; the effect of ionic liquids on the efficiency of separation and selectivity is considered. [ABSTRACT FROM AUTHOR]

Ovotransferrin is the second most abundant protein (~12-13% of the total egg protein) in egg white after ovalbumin. Ovotransferrin is a potent natural antimicrobial agent as it possesses antibacterial, antifungal, and antiviral properties and is also the major metal binding protein found in egg, which makes it an industrially important protein. Ovotransferrin was extracted from egg white using its metal (iron) binding properties. In the present study, eggs from two different sources were used (fresh local eggs from domestic household source and poultry eggs from shops) to compare the results and Response Surface Methodology was used for the experiment design and data analysis. The following extraction conditions were optimized so as to maximize the yield of ovotransferrin from egg white: ethanol % (v/v) and pH and volume (mL) of 25 mM FeCl3/50 mL of egg white. A maximum yield of ~85 ± 2.5% was obtained near the optimum extraction conditions. The yield was calculated based on the theoretical value (934 mg) of ovotransferrin in 100 mL of 1.5x diluted egg white solution. Our results suggest that efficient downstream processing may reduce the cost of overall production process of this promising enzyme, making it a natural and cost-effective alternative to the existing chemically synthesized antimicrobial agents. [ABSTRACT FROM AUTHOR]

Ruta graveolens (the common rue) has been used for various therapeutic purposes, including relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on main drug-metabolizing enzymes , effects of an aqueous extract of the aerial part of rue and its ingredients on cytochrome P450 (P450/CYP), uridine diphosphate (UDP)- glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H):quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg) induced hepatic Cyp1a and Cyp2b activities and protein levels in males and females. This treatment increased hepatic UDP-glucuronosyltransferase activity only in males. However, NAD(P)H:quinone oxidore- ductase activity remained unchanged. Based on the contents of rutin and furanocoumarins of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of furanocoumarins (Fmix) increased Cyp2b activities in males. The mixture of rutin and F mix increased Cyp1a and Cyp2b activities. These results revealed that rutin and Fmix contributed at least in part to the P450 induction by rue [ABSTRACT FROM AUTHOR]

Breast cancer is one of the major issues in the field of oncology, reported with a higher prevalence rate in women worldwide. In attempt to reveal the potential biomarkers for breast cancer, the findings of differentially glycosylated haptoglobin and osteonectin in previous study have drawn our attention towards glycoproteins of secretome from the MCF-7 cancer cell line. In the present study, further analyses were performed on the medium of MCF-7 cells by subjecting it to two-dimensional analyses followed by image analysis in contrast to the medium of human mammary epithelial cells (HMEpC) as a negative control. Carboxypeptidase A4 (CPA4), alpha-1-antitrypsin (AAT), haptoglobin (HP), and HSC70 were detected in the medium of MCF-7, while only CPA4 and osteonectin (ON) were detected in HMEpC medium. In addition, CPA4 was detected as upregulated in the MCF-7 medium. Further analysis by lectin showed that CPA4, AAT, HP, and HSC70 were secreted as N-glycan in the medium of MCF-7, with HP also showing differentially N-glycosylated isoforms. For the HMEpC, only CPA4 was detected as N-glycan. No O-glycan was detected in the medium of HMEpC but MCF-7 expressed O-glycosylated CPA4 and HSC70. All these revealed that glycoproteins could be used as glycan-based biomarkers for the prognosis of breast cancer. [ABSTRACT FROM AUTHOR]

The capabilities of physicochemical methods (chromatographic, electrophoretic, potentiometric, and voltammetric) and also field test methods for the determination of the main markers of the urinary stone disease have been considered. The effect of various factors on the processes of stone formation has been discussed. [ABSTRACT FROM AUTHOR]

Background Atherosclerosis is a major cause of cardiac events and mortality in patients suffering from chronic kidney disease (CKD). Moreover, the risk of cardiovascular disease (CVD) development in patients with CKD increases as kidney function declines. Although the close connection between atherosclerosis and kidney dysfunction is undeniable, particular risk factors and specific mechanisms that promote CVD in patients with CKD remain unclear. To gain insight into better recognition of the mechanisms of accelerated atherosclerosis in patients with CKD, we performed a comparative proteomic analysis of blood plasma from patients in various stages of CKD and thus distinct progression of atherosclerosis (n = 90), patients with advanced CVD and normal renal function (n = 30) and healthy volunteers (n = 30). Methods Plasma samples were depleted using affinity chromatography and divided into three fractions: high-abundant, low-abundant and low-molecular weight proteins. The first two fractions were analyzed by two-dimensional gel electrophoresis and mass spectrometry, the last one has been subjected to direct MS/MS analysis. A proteomic profiles for highabundant, low-abundant and low-molecular weight proteins fractions were obtained. Differential accumulated proteins were confirmed by selected reaction monitoring analysis (SRM). The Gene Ontology (GO) function and the interaction networks of differentially expressed proteins were then analyzed. Results Forty-nine proteins (13 high- and 36 low-molecular mass) showed differences in accumulation levels. For eleven of them differential expression were confirmed by selected reaction monitoring analysis. Bioinformatic analysis showed that identified differential proteins were related to three different processes: the blood coagulation cascade, the transport, binding and metabolism of lipoproteins and inflammatory processes. Conclusions Obtained data provide an additional line of evidence that different molecular mechanisms are involved in the development of CKD- and CVD-related atherosclerosis. The abundance of some anti-atherogenic factors revealed in patients with CKD suggests that these factors are not associated with the reduction of atherosclerosis progression in CKD that is typically observed in "classical" CVD. Moreover, obtained data also suggest that mechanism of CVD acceleration may be different in initial and advanced stages of CKD. Undoubtedly, in advanced stages of CKD inflammation is highly pronounced. [ABSTRACT FROM AUTHOR]

Background: Traditional drug discovery approaches are mainly relied on the observed phenotypic changes following administration of a plant extract, drug candidate or natural product. Recently, target-based approaches are becoming more popular. The present study aimed to identify the cellular targets of crocin, the bioactive dietary carotenoid present in saffron, using an affinity-based method. Methods: Heart, kidney and brain tissues of BALB/c mice were homogenized and extracted for the experiments. Target deconvolution was carried out by first passing cell lysate through an affinity column prepared by covalently attaching crocin to agarose beads. Isolated proteins were separated on a 2D gel, trypsinized in situ and identified by MALDI-TOF/TOF mass spectrometry. MASCOT search engine was used to analyze Mass Data. Results: Part of proteome that physically interacts with crocin was found to consist of beta-actin-like protein 2, cytochrome b-c1 complex subunit 1, ATP synthase subunit beta, tubulin beta-3 chain, tubulin beta-6 chain, 14-3-3 protein beta/alpha, V-type proton ATPase catalytic subunitA, 60 kDa heat shock protein, creatine kinase b-type, peroxiredoxin-2, cytochrome b-c1 complex subunit 2, acetyl-coA acetyltransferase, cytochrome c1, proteasome subunit alpha type-6 and proteasome subunit alpha type-4. Conclusion: The present findings revealed that crocin physically binds to a wide range of cellular proteins such as structural proteins, membrane transporters, and enzymes involved in ATP and redox homeostasis and signal transduction. [ABSTRACT FROM AUTHOR]

Galkeun-tang (GKT; Galgen-tang in Chinese and Kakkon-to in Japanese), a traditional herbal formula, has been used for treatment of the common cold. Here, we report in vitro antioxidant and antiadipogenic effects of GKT. GKT increased the activities of scavenging 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. GKT also significantly reduced the malondialdehyde (MDA) generation during low-density lipoprotein (LDL) oxidation and the electrophoretic mobility of oxidized LDL, indicating inhibitory effects of GKT on Cu2+-mediated oxidation of LDL. Regarding antiadipogenic activity, GKT treatment significantly suppressed lipid accumulation, triglyceride production, and glycerol-3- phosphate dehydrogenase (GPDH) activity in differentiated 3T3-L1 adipocytes. Consistent with this, GKT significantly reduced the secretion of leptin, a major adipokine, in differentiated 3T3-L1 adipocytes. Overall, our findings suggest that GKT has the potential for antioxidative and antiadipogenic properties. [ABSTRACT FROM AUTHOR]

Context.-It is recommended that hemoglobin (Hb) A1c (Hb A1c) not be used to assess average glycemia in patients who have altered red blood cell life span. Objective.-To investigate the frequency of reporting an Hb A1c value for Hb variant samples that do not include Hb A. Design.-Hb A1c samples (n ¼ 500) were procured and screened for Hb variants that may affect Hb A1c interpretation (Hb SS, Hb SC, and Hb S-b-thalassemia). Five of each of these samples were tested by ion-exchange high-performance liquid chromatography, immunoturbidimetric assay, second-generation immunoturbidimetric assay, and affinity chromatography. Results.-Eleven (2.2%) homozygous Hb SS, 6 (1.2%) Hb SC, and 5 (1.0%) Hb S-b-thalassemia samples were identified out of the 500 samples tested. Three of 4 instruments investigated in this study are known to not be plagued by analytic interference from these Hb variants but disturbingly reported Hb A1c values in the absence of Hb A. Conclusions.-The improved analytic specificity of Hb A1c platforms has by and large eliminated interferences from the most common heterozygous Hb variants. A consequence, however, is the potential for unintended reporting of Hb A1c results in the presence of homozygous and compound heterozygous Hb variants that lack Hb A and the inability to distinguish those samples not recommended to be used for patient care. The ability to identify samples harboring Hb variants that preclude the utility of Hb A1c may be beneficial in high prevalence populations. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was production of a recombinant antigen from envelope protein of dengue virus, in order to achieve high level expression in soluble form. Therefore, the possibility of envelope protein domain III expression in bacterial host was studied. Methods: Multiple sequence alignment for domain III sequences was carried out using Megalign software. The Modeller and Optimizer software were used for protein structure prediction and sequence optimization. After gene synthesis and subcloning in the pET21a expression vector, the optimization of recombinant protein expression was carried out in Escherichia coli. Ni-NTA chromatography columns were used for protein purification. The efficiency of protein expression was analyzed using electrophoresis and western blotting methods. Findings: A consensus sequence for domain III of dengue virus type 2 envelope protein was provided. The structural properties of target protein were predicted using bioinformatics methods. For high expression level, the sequence of EDIII2 gene was optimized for appropriate codon usage and GC content. The coding gene sequence was approved by sequencing and enzymatic digestion. For disulfide bound formation in protein structure, the Origami (DE3) expression host was used. At the final step, expression of target protein was optimized, and a high concentration of recombinant protein was achieved in soluble form. Conclusion: The results of this study showed that the optimization of domain III sequence led to obtain high concentration (20 mg/l) of produced protein. This expression system can be used for production of dengue virus envelope protein domain III. [ABSTRACT FROM AUTHOR]

Publications on the diagnosis of various diseases and a search for their biomarkers based on the characteristic chromatographic or electrophoretic profiles of biologically active compounds followed by chemometric treatment are discussed. [ABSTRACT FROM AUTHOR]

Single-dimension separations are routinely coupled in series to achieve two-dimensional separations, yet little has been done to simultaneously exploit multiple dimensions during separation. In this work, simultaneous chromatography and electrophoresis is introduced and evaluated for its potential to achieve two-dimensional separations. In simultaneous chromatography and electrophoresis, chromatography occurs via capillary action while an orthogonal electric field concurrently promotes electrophoresis in a second dimension. A novel apparatus with a dual solvent reservoir was designed to apply the concurrent electric field. Various compounds were used to characterize the apparatus and technique, i.e., vitamins, amino acids, and dyes. Improved separation is reported with equivalent analysis times in comparison to planar chromatography alone. The feasibility of simultaneously employing chromatography and electrophoresis in two dimensions is discussed. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Nowadays antidepressant drugs like selective serotonin reuptake inhibitors (SSRIs) and selective norepinephrine reuptake inhibitors (SNRIs) represent the first choice in the treatment of moderate to severe depressive illness, various phobias, and personality disorders. In spite of the therapeutic aspects, they often produce very severe and toxic effects in deliberate and accidental cases of poisoning. These are also considered as date-rape drugs used for drugged victims for raping or robbing. Therefore, in recent years, their analyses in different biological matrices for clinical and toxicological analysis purposes has been a target worthy of interest. Thus, the review focuses on recent advancements of various separation techniques like chromatography and electrophoresis that are concernd with the determination of selective serotonin reuptake inhibitor and selective norepinephrine reuptake inhibitor drugs and their metabolites in various biological matrices. In addition to this, a critical discussion on analytical approaches has also been incorporated, suggesting their applicability and limitations for further implementations. Thus, this paper will definitely help in the selection and development of proper analytical methodologies to achieve satisfactory results, better scientific understanding, and test interpretation. [ABSTRACT FROM AUTHOR]

AbstractBanana oligosaccharides (BOS) were extracted with water, and then separated and purified using column chromatography. Gel penetration chromatography was used to determine the molecular weights. Thin layer chromatogram and capillary electrophoresis were employed to analyze the monosaccharide composition. The indican bond and structure of the BOS molecule were determined using Fourier transform infrared spectroscopy and nuclear magnetic resonance. Results showed that BOS were probably composed of eight β-D-pyran glucose units linked with 1→6 indican bonds. The laxative effects of BOS were investigated in mice using the method described in “Handbook of Technical Standards for Testing and Assessment of Health Food in China.” The length of the small intestine over which a carbon suspension solution advanced in mice treated with low-, middle-, and high-dose BOS was significantly greater than that in the model group, suggesting that BOS are effective in accelerating the movement of the small intestine. [ABSTRACT FROM AUTHOR]

Bovine seminal (BS) RNase, the unique natively dimeric member of the RNase super-family, represents a special case not only for its additional biological actions but also for the singular features of 3D domain swapping. The native enzyme is indeed a mixture of two isoforms: M=M, a dimer held together by two inter-subunit disulfide bonds, and MxM, 70% of the total, which, besides the two mentioned disulfides, is additionally stabilized by the swapping of its N-termini. When lyophilized from 40% acetic acid, BS-RNase oligomerizes as the super-family proto-type RNase A does. In this paper, we induced BS-RNase self-association and analyzed the multimers by size-exclusion chromatography, cross-linking, electrophoresis, mutagenesis, dynamic light scattering, molecular modelling. Finally, we evaluated their enzymatic and cytotoxic activities. Several BS-RNase domain-swapped oligomers were detected, including two tetramers, one exchanging only the N-termini, the other being either N- or C-swapped. The C-swapping event, confirmed by results on a BS-K113N mutant, has been firstly seen in BS-RNase here, and probably stabilizes also multimers larger than tetramers. Interestingly, all BS-RNase oligomers are more enzymatically active than the native dimer and, above all, they display a cytotoxic activity that definitely increases with the molecular weight of the multimers. This latter feature, to date unknown for BS-RNase, suggests again that the self-association of RNases strongly modulates their biological and potentially therapeutic properties. [ABSTRACT FROM AUTHOR]

Although many proteins have been described involved in Escherichia coli colonization and infection, only few reports have shown lectins as important components in these processes. Because the mechanisms underlying E. coli colonization process involving lectins are not fully understood, we sought to identify the presence of other non-described lectins in E. coli. Here, we isolated a 75-kDa protein from E. coli on Sepharose column and identified it as ferric aerobactin receptor (IutA). Since IutA is controversially associated with virulence of some E. coli strains, mainly in uropathogenic E. coli (UPEC), we evaluated the presence of iutA gene in UPEC isolated from patients with urinary infection. This gene was present in only 38% of the isolates, suggesting a weak association with virulence. Because there is a redundancy in the siderophore-mediated uptake systems, we suggest that IutA can be advantageous but not essential for UPEC. [ABSTRACT FROM AUTHOR]

The use of a new class of high-molecular-weight substances, dendritic polymers (dendrimers and hyperbranched polymers), as components of chromatographic and electrophoretic systems is discussed; the effect of their introduction into a background electrolyte or an eluent on the separation efficiency and selectivity of organic compounds of different classes is considered. [ABSTRACT FROM AUTHOR]

A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession), and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6-8 and in the temperature range 35-60?C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 μM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities. [ABSTRACT FROM AUTHOR]

Purpose. Pigment epithelium-derived factor (PEDF) is a multifunctional serpin. The purpose of this study is to identify PEDF protein forms and investigate their biological activities on tumor cell lines. Methods. Recombinant human PEDF proteins were purified by cation- and anion-exchange column chromatography. They were subjected to SDS-PAGE, IEF, deglycosylation, heparin affinity chromatography, and limited proteolysis. Cell viability, real-time electrical impedance of cells, and wound healing assays were performed using bladder and breast cancer cell lines, rat retinal R28, and human ARPE-19 cells. Results. Two PEDF protein peaks were identified after anion-exchange column chromatography: PEDF-1 eluting with lower ionic strength than PEDF-2. PEDF-1 had higher pI value and lower apparent molecular weight than PEDF-2. Both PEDF forms were glycosylated, bound to heparin, and had identical patterns by limited proteolysis. However, PEDF-2 emerged as being highly potent in lowering cell viability in all tumor cell lines tested, and in inhibiting tumor and ARPE-19 cell migration. In contrast, PEDF-1 minimally affected tumor cell viability and cell migration but protected R28 cells against death caused by serum starvation. Conclusion. Two distinct biochemical forms of PEDF varying in overall charge have distinct biological effects on tumor cell viability and migration. The existence of PEDF forms may explain the multifunctional modality of PEDF. [ABSTRACT FROM AUTHOR]

A lectin detected in haemolymph from the Australian spiny lobster Panulirus cygnus agglutinated human ABO Group A cells to a higher titre than Group O or B. The lectin also agglutinated rat and sheep erythrocytes, with reactivity with rat erythrocytes strongly enhanced by treatment with the proteolytic enzyme papain, an observation consistent with reactivity via a glycolipid. The lectin, purified by affinity chromatography on fixed rat-erythrocyte stroma, was inhibited equally by N-acetylglucosamine and N-acetylgalactosamine. Comparison of data from gel filtration of haemolymph (behaving as a 1,800,000 Da macromolecule), and polyacrylamide gel electrophoresis of purified lectin (a single 67,000 Da band), suggested that in haemolymph the lecin was amultimer. The purified anti-A lectin autoprecipitated unless the storage solution contained chaotropic inhibitors (125 mmol/L sucrose: 500 mmol/L urea). The properties of this anti-A lectin and other similar lectins are consistent with a role in innate immunity in these invertebrates. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The analytical potencies of ligand-exchange capillary electrophoresis ( LECE) in regard to the determination of bioactive substances are discussed. As it is shown, complexation with copper(II) in the electrophoretic determination with UV detection makes possible the determination of amino acids, amines, and sugars, which do not absorb in the UV region, with the limits of detection 5-10 mg/L, and reduce the detection limits of the absorbing analytes, such as tryptophane, tyrosine, and histamine by 2-3 times. Prospects for using online preconcentration for the reduction of c are shown on an example of aliphatic amino acids, i.e., thirtyfold preconcentration is attained. LECE is shown to be applicable to the simultaneous determination of glucose, Na(), and K() in human blood serum. Also versions of ligand-exchange capillary electrophoresis with direct UV detection and capillary zone electrophoresis with indirect one are compared in terms of efficiency, time of analysis, and limit of detection. The potencies of the LECE method are compared with those of ligand-exchange chromatography. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscle fibres represent one of the most abundant cell types in mammals. Their highly specialised contractile and metabolic functions depend on a large number of membrane-associated proteins with very high molecular masses, proteins with extensive posttranslational modifications and components that exist in highly complex supramolecular structures. This makes it extremely difficult to perform conventional biochemical studies of potential changes in protein clusters during physiological adaptations or pathological processes. Results: Skeletal muscle proteomics attempts to establish the global identification and biochemical characterisation of all members of the muscle-associated protein complement. A considerable number of proteomic studies have employed large-scale separation techniques, such as high-resolution two-dimensional gel electrophoresis or liquid chromatography, and combined them with mass spectrometry as the method of choice for high-throughput protein identification. Muscle proteomics has been applied to the comprehensive biochemical profiling of developing, maturing and aging muscle, as well as the analysis of contractile tissues undergoing physiological adaptations seen in disuse atrophy, physical exercise and chronic muscle transformation. Biomedical investigations into proteome-wide alterations in skeletal muscle tissues were also used to establish novel biomarker signatures of neuromuscular disorders. Importantly, mass spectrometric studies have confirmed the enormous complexity of posttranslational modifications in skeletal muscle proteins. Conclusions: This review critically examines the scientific impact of modern muscle proteomics and discusses its successful application for a better understanding of muscle biology, but also outlines its technical limitations and emerging techniques to establish new biomarker candidates. [ABSTRACT FROM AUTHOR]

Nanoparticle-based material is a revolutionary scientific and engineering venture that will invariably impact the existing analytical separation and preconcentration for a variety of analytes. Nanoparticles can be regarded as a hybrid between small molecule and bulk material. A material on the nanoscale produces considerable changes on various properties, making them size- and shape-dependent. Gold nanoparticles (Au NPs), one of the wide variety of core materials available, coupled with tunable surface properties in the form of inorganic or inorganic-organic hybrid have been reported as an excellent platform for a broad range of analytical methods. This review aims to introduce the basic principles, examples, and descriptions of methods for the characterization of Au NPs by using chromatography, electrophoresis, and self-assembly strategies for separation science. Some of the latest important applications of using Au NPs as stationary phases toward open-tubular capillary electrochromatography, gas chromatography, and liquid chromatography as well as roles of run buffer additive to enhance separation and preconcentration in the field of chromatographic, electrophoretic and in chip-based systems are reviewed. Additionally, we review Au NPs-assisted state-of-the-art techniques involving the use of micellar electrokinetic chromatography, an online diode array detector, solid-phase extraction, and mass spectrometry for the preconcentration of some chemical compounds and biomolecules. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The article offers information on the study conducted by the authors related to the functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi. It states that lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. It mentions that the strategy of the study enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis.

Surface charge, molecular weight, and folding state are known to influence protein chromatographic behavior onto ion-exchangers. Experimentally, information related to such factors can be gathered via two-dimensional electrophoretic (2-DE) methods. The separation behavior depicted by the insect cultured-cells proteome, which is an important host for recombinant protein production, was explored in this study. Experimental evidence showed a correlation between apparent isoelectric point distributions and the mobile phase conductivity. It was observed that the information contained in the isoelectric point (pI) value(s) obtained with a 2-DE routine showed a good correlation with the IEX chromatographic behavior, for a number of commercial adsorbents. This correlation was observed irrespective of the pH of the feedstock within the range 6 to 8. An initial prediction of protein ion-exchange chromatographic behavior could be possible utilizing an experimental approach based on the mentioned orthogonal methods. This technique is providing information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock. [ABSTRACT FROM AUTHOR]

Structural control of single-walled carbon nanotubes (SWNTs) is attracting enormous interest in view of their applications to nanoelectronics and nanooptics. Actually, more than 200 papers regarding separation of SWNTs have been published since 1998. In this review, they are classified into the following five sections according to the separation methods; electrophoresis, centrifugation, chromatography, selective solubilization and selective reaction. In each method, all literature is summarized in tables showing the separated objects (metallic/semiconducting (M/S), length, diameter, (n, m) structure and/or handedness), the production process of the used SWNTs (CoMoCAT, HiPco, arc discharge and/or laser vaporization) and the employed chemicals, such as detergents and polymers. Changes in annual number of publications related to this subject are also discussed. [ABSTRACT FROM AUTHOR]

Interactions between Intralipid dispersion and local anesthetics (bupivacaine, prilocaine, and lidocaine) were investigated. The amount of bupivacaine (the most cardiotoxic analyte of the local anesthetics studied) entrapped in Intralipid in the presence of plasma was studied using an off-line filtration and solid phase extraction method combined with capillary zone electrophoresis for quantification of free unbound bupivacaine. To confirm interactions between the analytes and Intralipid at lower concentrations, direct injection mass spectrometry was used. The use of immobilized Intralipid chromatography-atmospheric pressure ionization–ion trap mass spectrometry in the study of interactions between drugs and Intralipid dispersion is demonstrated. Finally, interactions between Intralipid dispersion and local anesthetics were investigated by electrokinetic capillary chromatography. The electrophoretic mobility of the Intralipid dispersed phase was calculated using the iterative procedure and a homologous series of alkyl phenyl benzoates (C1–C6), and the retention factors for the analytes were determined. [ABSTRACT FROM AUTHOR]

Background: Brain capillary endothelial cells (BCECs) form the physiological basis of the blood-brain barrier (BBB). The barrier function is (at least in part) due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein). Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the wellestablished in vitro BBB model developed in our laboratory. Results: A total of 215 protein spots (corresponding to 130 distinct proteins) were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin) and constitutes valuable evidence for predictions based on genome annotation. Conclusions: Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets. [ABSTRACT FROM AUTHOR]

Abstract: This review focuses on analytical techniques used for separation and characterization of dendrimers and their derivatives. These macromolecules have been attractive material for a development of new drug carriers and imaging agents. They are also interesting for many biological and industrial applications. The review mentions a few of them. [Copyright &y& Elsevier]

Pentocin 31-1, an anti-Listeria bacteriocin produced by Lactobacillus pentosus 31-1 from the traditional Chinese fermented Xuan-Wei ham, was successfully purified by the pH-mediated cell adsorption-desorption method and then purified by gel chromatography with Sephadex G-10. The purification resulted in a 1,381.9-fold increase in specific activity with a yield of 76.8% of the original activity. Using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular mass of the purified peptide was found to be between 3,500 and 6,400 Da, and bacteriocin activity was confirmed by overlayer techniques. When subjected to mass spectrometry analysis, the protein was highly pure and its molecular mass was 5,592.225 Da. The partial N-terminal sequence of pentocin 31-1 was the following: NH2-VIADYGNGVRXATLL. Compared with the sequence of other bacteriocins, pentocin 31-1 has the consensus sequence YGNGV in its N-terminal region, and therefore it belongs to the class Ha of bacteriocins. [ABSTRACT FROM AUTHOR]

An extension of the unified equation of chromatography to directly access reaction rate constants k1 of first-order reaction in on-column chromatography is presented. This extended equation reflects different response factors in the detection of the reaction educt and product which arise from structural changes by elimination or addition, e.g., under pseudo-first-order reaction conditions. The reaction rate constants k1 and Gibbs activation energies $$ \Delta G^{ \ne } $$ of first-order reactions taking place in a chromatographic system can be directly calculated from the chromatographic parameters, i.e., retention times of the educt E and product P ( $$ t_{\text{R}}^{\text{A}} $$ and $$ t_{\text{R}}^{\text{B}} $$), peak widths at half height ( wA and wB), the relative plateau height ( hp) of the conversion profile, and the individual response factors fA and fB. The evaluation of on-column reaction gas chromatographic experiments is exemplified by the evaluation of elution profiles obtained by ring-closing metathesis reaction of N, N-diallytrifluoroacetamide in presence of Grubbs second-generation catalyst, dissolved in polydimethylsiloxane (GE SE 30). [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: The removal efficiency of algae and microcystins (MCs) of a pilot setup based on biological degradation of enrichment microbes by artificial media was studied. The results showed that when chlorophyll-a in Lake Taihu was 3.76–266.1μgL−1, 62.8% of chlorophyll-a could be removed within a hydraulic retention time of 6–7 days. High-performance liquid chromatography (HPLC) analysis for the detection of MCs was applied. When total microcystins RR and LR (TMC-RR and TMC-LR) and extracellular microcystins RR and LR (EMC-RR and EMC-LR) were 0.23–8.93, 0.14–4.73, 0.12–1.15, 0.02–0.17μgL−1, respectively, and in source water, the average removal efficiencies of TMC-RR, TMC-LR, EMC-RR, EMC-LR were 67.0%, 40.5%, 40.0% and 66.0%, respectively. The polymerase chain reaction (PCR) electrophoresis chart revealed that there was a large amount of algae-lysing bacteria such as Pseudomonas spp. and Bacillus spp. on the artificial medium. The protozoa number in the assembled medium was higher than in lake water. Enrichment microbes on the artificial medium could effectively degrade algae and microcystins in Lake Taihu. [Copyright &y& Elsevier]