Anion-exchange Fast Protein Liquid Research Articles

Bioengineered miR-34a modulates mitochondrial inner membrane protein 17 like 2 (MPV17L2) expression toward the control of cancer cell mitochondrial functions

ABSTRACT Genome-derived microRNAs (miRNAs or miRs) control post-transcriptional gene expression critical for various cellular processes. Recently, we have invented a novel platform technology to achieve high-yield production of fully humanized, bioengineered miRNA agents (hBERAs) for research and development. This study is aimed to produce and utilize a new biologic miR-34a-5p (or miR-34a) molecule, namely, hBERA/miR-34a, to delineate the role of miR-34a-5p in the regulation of mitochondrial functions in human carcinoma cells. Bioengineered hBERA/miR-34a was produced through in vivo fermentation production and purified by anion exchange fast protein liquid chromatography. hEBRA/miR-34a was processed to target miR-34a-5p in human osteosarcoma and lung cancer cells, as determined by selective stem-loop reverse transcription quantitative polymerase chain reaction analysis. The mitochondrial inner membrane protein MPV17 like 2 (MPV17L2) was validated as a direct target for miR-34a-5p by dual luciferase reporter assay. Western blot analysis revealed that bioengineered miR-34a-5p effectively reduced MPV17L2 protein outcomes, leading to much lower levels of respiratory chain Complex I activities and intracellular ATP that were determined with specific assay kits. Moreover, Seahorse Mito Stress Test assay was conducted, and the results showed that biologic miR-34a-5p sharply reduced cancer cell mitochondrial respiration capacity, accompanied by a remarkable increase of oxidative stress and elevated apoptotic cell death, which are manifested by greater levels of reactive oxygen species and selective apoptosis biomarkers, respectively. These results demonstrate the presence and involvement of the miR-34a-5p-MPV17L2 pathway in the control of mitochondrial functions in human carcinoma cells and support the utility of novel bioengineered miRNA molecules for functional studies.

Characterization of human plasma lipoproteins using anion exchange fast protein liquid chromatography and targeted mass spectrometry assay.

We described a targeted mass spectrometry assay based on selected reaction monitoring (SRM) for five apolipoproteins (apoA1, apoB, apoJ, apoD, and apoE) in plasma lipoproteins isolated by anion exchange fast protein liquid chromatography using only 100 μL of plasma. We performed analytical characterization of the SRM assay and evaluated reproducibility of the entire workflow. The limit of detections for apoA1, apoB, apoD, apoJ, and apoE were 0.6, 4.6, 0.8, 1.2, and 0.7 nM, respectively; the limit of quantifications was 8.3 nM for all peptides except apoD (4.2 nM). The SRM assay was linear from 0.4 to 1667 nM. The intra-day and inter-day and total repeatability (CV%) of the assay ranged from 2.2% to 21.7% for all five peptides. The intra-day and inter-day and total reproducibility of the entire workflow ranged from 12.2% to 43.9% for all five peptides in fractionated high-density lipoprotein, low-density lipoprotein, and IDL. In the future, we will apply this workflow to investigate the association of fractionated plasma lipoproteins with amyloid deposition and cognitive changes in the context of Alzheimer's disease.

Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells

The nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor in the regulation of many oxidative enzymes and efflux transporters critical for oxidative stress and cellular defense against xenobiotics. NRF2 is dysregulated in patient osteosarcoma (OS) tissues and correlates with therapeutic outcomes. Nevertheless, research on the NRF2 regulatory pathways and its potential as a therapeutic target is limited to the use of synthetic small interfering RNA (siRNA) carrying extensive artificial modifications. Herein, we report successful high-level expression of recombinant siRNA against NRF2 in Escherichia coli using our newly established noncoding RNA bioengineering technology, which was purified to >99% homogeneity using an anion-exchange fast protein liquid chromatography method. Bioengineered NRF2-siRNA was able to significantly knock down NRF2 mRNA and protein levels in human OS 143B and MG63 cells, and subsequently suppressed the expression of NRF2-regulated oxidative enzymes [heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1] and elevated intracellular levels of reactive oxygen species. In addition, recombinant NRF2-siRNA was effective to sensitize both 143B and MG63 cells to doxorubicin, cisplatin, and sorafenib, which was associated with significant downregulation of NRF2-targeted ATP-binding cassette (ABC) efflux transporters (ABCC3, ABCC4, and ABCG2). These findings support that targeting NRF2 signaling pathways may improve the sensitivity of cancer cells to chemotherapy, and bioengineered siRNA molecules should be added to current tools for related research.

Abstract 541: Application of novel RNA aptamer-based RNase I activity assay for pancreatic cancer biomarker development

Abstract Background: Ribonuclease I (RNase I) is highly expressed in pancreas and also seen in serum. Previous reports have noted its differential activity and association to a number of cancers including pancreatic adenocarcinoma (PDAC). In search of clinically relevant biomarkers for PDAC detection, in this study, we have pursued classification characterization by a novel plasma RNase I activity assay for discriminating PDAC cases from benign conditions and other malignancies. Methods: We have developed a new assay for accurate and precise quantification of RNase activities based on a specific and strong fluorescence intensity of malachite green (MG) bound to an RNA aptamer (MGA). The MGA chimeras were produced in E. coli using a novel optional noncoding RNA scaffold (OnRS) based recombinant RNA technology established in our lab. MGA chimeras were thus purified to a high degree of homogeneity (>98% pure) with an anion-exchange fast protein liquid chromatograph (FPLC) method and utilized in our assays. The cleavage of MGA by RNases (dominantly by RNase I) results in a dose- and time-dependent decrease in the fluorescence intensity at 630/650 nm (excitation/emission) for MGA-bound MG, which could be abolished by RNase inhibitor and utilized to determine RNase activities in any given sample. We applied this assay to quantitate the serum RNase activity levels in a set of prospectively collected serum from our IRB-approved Pancreas Registry including PDAC, benign cases (including chronic pancreatitis) and other (non-PDAC) malignancies, and normalized to that of a pooled serum sample from healthy subjects. Data analysis was performed for group comparison of the RNase activity levels and significance evaluated by Mann-Whitney U test (P<0.05 significant). Results: A total of 140 patients’ serum specimen was analyzed. 60 PDAC cases (stage I (n = 8), stage II (n = 25), stage III/IV (n = 27)), 60 benign conditions (chronic pancreatitis (n = 15), acute pancreatitis (n = 3), benign cysts (n = 17), normal pancreas (n = 25)), and 20 non-PDAC malignancies (Pancreatic neuroendocrine tumor (n = 11), CholangioCA(n = 2), Ampullary CA(n = 3), Hepatoma (n = 2), GIST (n = 1), lymphoma (n = 1)) Group comparison of RNase activity: PDAC vs benign: Median PDAC = 1.454; median benign group = 1.181 (P-value, <0.0001) Resectable-stage PDAC (stages I & II) vs benign: Median PDAC = 1.616; median benign group = 1.181 (P-value, <0.0001) PDAC vs other Malignancies: Median PDAC = 1.454; median other Malignancies = 1.242 (P-value = 0.0748) Conclusion: RNase activity level successfully discriminates PDAC cases from the benign conditions including chronic pancreatitis. Specifically, early-stage PDAC group is significantly separated from the benign group. Further testing is being performed for evaluating the test as a potential early detection diagnostic modality. Citation Format: Shiro Urayama, Aiming M. Yu. Application of novel RNA aptamer-based RNase I activity assay for pancreatic cancer biomarker development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 541. doi:10.1158/1538-7445.AM2015-541

English

The aim of this study was to compare the physicochemical parameters of milk samples of five different species: cow, goat, donkey, camel and human. Also the analysis of whey protein profile in different milk samples was performed by anion-exchange fast protein liquid chromatography (FPLC) while polyacrylamide gel electrophoresis was used to identify a single fraction. Camel milk was the most acid (pH 6.460±0.005) and the richest in total proteins (3.41±0.31 %) and ash (0.750±0.102 %), whereas donkey milk had a neutral pH (7.03±0.02) and characterised by low proteins (1.12±0.40 %) and fat (0.97±0.03 %) content, being very close to human milk. Proteomic analysis of cow, goat, donkey, camel and human milk highlighted significant interspecies differences. Camel milk was similar to human milk in lacking of β-lactoglobulin and richness of α-lactalbumin. The knowledge gained from the proteomic comparison of the milk samples analysed within this study might be of relevance, both, in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products.

Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization.

Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre-miR-34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (>98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non-small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNA-based therapies.

Rapid production of novel pre‐microRNA agent hsa‐mir‐27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells

MicroRNAs (miRNAs or miRs) are a large family of noncoding RNAs that have been identified as critical epigenetic factors in the regulation of various cellular processes including drug metabolism and disposition. However, the study on miRNA functions is limited to the use of synthetic RNA and recombinant DNA agents. Herein, we show that novel pre‐miRNA‐27b (mir‐27b) agents could be biosynthesized in Escherichia coli using tRNA‐based recombinant RNA technology, and recombinant tRNA/mir‐27b chimera was readily purified to a high degree of homogeneity (> 95%) using anion‐exchange fast protein liquid chromatography. The tRNA‐fusion mir‐27b was revealed to be processed to mature miRNA miR‐27b in human carcinoma LS‐180 cells in a dose and time dependent manner. Moreover, recombinant tRNA/mir‐27b agents were biologically active in reducing the mRNA and protein expression levels of cytochrome P450 3A4 (CYP3A4) which consequently led to a lower midazolam 1'‐hydroxylase activity. These findings demonstrate that human pre‐miRNAs may be produced in Escherichia coli by recombinant RNA technology as novel ncRNA agents for functional studies in drug metabolism.

Rapid production of novel pre-microRNA agent hsa-mir-27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells.

Noncoding microRNAs (miRNAs or miRs) have been revealed as critical epigenetic factors in the regulation of various cellular processes, including drug metabolism and disposition. However, research on miRNA functions is limited to the use of synthetic RNA and recombinant DNA agents. Herein, we show that novel pre-miRNA-27b (miR-27b) agents can be biosynthesized in Escherichia coli using recombinant RNA technology, and recombinant transfer RNA (tRNA)/mir-27b chimera was readily purified to a high degree of homogeneity (>95%) using anion-exchange fast protein liquid chromatography. The tRNA-fusion miR-27b was revealed to be processed to mature miRNA miR-27b in human carcinoma LS-180 cells in a dose- and time-dependent manner. Moreover, recombinant tRNA/miR-27b agents were biologically active in reducing the mRNA and protein expression levels of cytochrome P450 3A4 (CYP3A4), which consequently led to lower midazolam 1'-hydroxylase activity. These findings demonstrate that pre-miRNA agents can be produced by recombinant RNA technology for functional studies.

Purification and characterization of tinamou egg white ovotransferrin as an antimicrobial agent against foodborne pathogenic bacteria

Chilean tinamou (Nothoprocta perdicaria) egg white ovotransferrin was purified with anion-exchange Fast Protein Liquid Chromatography and identified by peptide mass fingerprinting. Tinamou egg white ovotransferrin was compared to chicken (Gallus gallus) and emu (Dromaius novaehollandiae) ovotransferrin by SDS-PAGE analysis. Though all three species contained similar molecular weight ovotransferrins, tinamou egg white contained 20% more ovotransferrin than chicken, but 30% less than emu.The effectiveness of tinamou ovotransferrin as a natural antimicrobial agent was compared to chicken ovotransferrin against two food related pathogens, Escherichia coli O157:H7 and Staphylococcus aureus. The bacteriostatic and bactericidal activities of the native, apo and holo forms of the proteins were dependent on the presence of 50mM bicarbonate and protein iron saturation. Native ovotransferrins were the most effective at a concentration of 10mg/ml with 50mM bicarbonate, exhibiting bacteriostatic and bactericidal activities against both pathogens. Holo ovotransferrins exhibited moderate antimicrobial activity in the presence of bicarbonate. Different antimicrobial activities were observed between chicken and tinamou ovotransferrins, suggesting tinamou ovotransferrin may possess unique antimicrobial motifs that warrant further investigation of its amino acids composition. Further, our results suggest that tinamou ovotransferrin may represent a natural antimicrobial agent suitable for use in food matrices or on food preparation surfaces.

CaCO3 crystallization and morphology control by using purified soluble protein related to shell regeneration

The soluble protein related to shell regeneration (SPSR) plays an important role to repair the damaged shell. In this study, the SPSR was used as a biocatalyst to control the crystal growth and morphology during in vitro CaCO3 crystallization. Anion exchange fast protein liquid chromatography (FPLC) was applied to separate the SPSR constituents. Each fraction from purification of the SPSR was subjected to CaCO3 crystallization to identify the fraction's effect on controlling the CaCO3 crystal morphology. CaCO3 crystallization experiments were performed by mixing solutions of CaCl2 and purified SPSR in the presence of vaporized (NH4)2CO3. The morphology of the CaCO3 crystals was characterized by field emission scanning electron microscopy (FE-SEM). The synthesized CaCO3 was characterized by Fourier transform infrared spectroscopy (FT-IR) to reveal the type of crystals formed. In vitro CaCO3 crystallization process showed the effect of SPSR on the morphology change of CaCO3 crystals. We identified a condition for rapid crystal growth and specific morphology of CaCO3 in the presence of SPSR. Thus, our study confirms that SPSR governs CaCO3 crystallization and influences the observed crystal morphology.

A Simulated MS/MS Library for Spectrum-to-spectrum Searching in Large Scale Identification of Proteins

Identifying peptides from mass spectrometric fragmentation data (MS/MS spectra) using search strategies that map protein sequences to spectra is computationally expensive. An alternative strategy uses direct spectrum-to-spectrum matching against a reference library of previously observed MS/MS that has the advantage of evaluating matches using fragment ion intensities and other ion types than the simple set normally used. However, this approach is limited by the small sizes of the available peptide MS/MS libraries and the inability to evaluate the rate of false assignments. In this study, we observed good performance of simulated spectra generated by the kinetic model implemented in MassAnalyzer (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922; Zhang, Z. (2005) Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges. Anal. Chem. 77, 6364-6373) as a substitute for the reference libraries used by the spectrum-to-spectrum search programs X!Hunter and BiblioSpec and similar results in comparison with the spectrum-to-sequence program Mascot. We also demonstrate the use of simulated spectra for searching against decoy sequences to estimate false discovery rates. Although we found lower score discrimination with spectrum-to-spectrum searches than with Mascot, particularly for higher charge forms, comparable peptide assignments with low false discovery rate were achieved by examining consensus between X!Hunter and Mascot, filtering results by mass accuracy, and ignoring score thresholds. Protein identification results are comparable to those achieved when evaluating consensus between Sequest and Mascot. Run times with large scale data sets using X!Hunter with the simulated spectral library are 7 times faster than Mascot and 80 times faster than Sequest with the human International Protein Index (IPI) database. We conclude that simulated spectral libraries greatly expand the search space available for spectrum-to-spectrum searching while enabling principled analyses and that the approach can be used in consensus strategies for large scale studies while reducing search times.

Development of Analytical Procedure for the Determination of Exchangeable Cr(VI) in Soils by Anion-exchange Fast Protein Liquid Chromatography with Electrothermal Atomic Absorption Spectrometry Detection

Analytical procedure for the determination of exchangeable Cr(VI) was developed. In order to optimise the extraction procedure, the efficiency of extraction of exchangeable Cr(VI) in soil samples was investigated in KH2PO4–K2HPO4 buffer solutions (0.015 up to 0.2 mol l−1), adjusted to the pH of the soil. Phosphate buffer was used to efficiently desorb Cr(VI) from soil particles. The extraction time (mechanical shaking) ranged from 1 up to 72 h. Cr(VI) in soil extracts was determined by anion-exchange fast protein liquid chromatography with electrothermal atomic absorption detection (FPLC-ETAAS). The study was performed on soil samples from the field treated with the tannery waste for seventeen years. Samples were analysed in the 16 year after the last waste application. It was experimentally proven that the optimal phosphate buffer concentration was 0.1 mol l−1 and extraction time 16 h. An additional experiment was done to confirm that during the extraction, soluble Cr(III) was not oxidised to Cr(VI) by Mn(IV) oxides present in soil samples. For this purpose soil with the same characteristics, but not treated with tannery waste, was spiked with Cr(III) and the analytical procedure performed. No measurable Cr(VI) concentrations were detected. The repeatability of measurement was 2.5%, while the reproducibility of measurement was 6.9%. The accuracy of the analytical procedure was tested by spiking of soil samples with Cr(VI). The recoveries were better than 95%. The analytical procedure with limit of detection (LOD) 15 ng g−1 of Cr(VI) was sensitive enough for the determination of exchangeable Cr(VI) in soils. In field soil samples analysed the concentrations of exchangeable Cr(VI) were found to be about 200 ng g−1.

Speciation analysis of platinum antitumoral drugs in impacted tissues

Chemical compounds containing platinum have been employed since 1978 as drugs to beat certain type of tumours. Nevertheless, besides of their exceptional antitumoral properties, these drugs also have important deleterious side effects, such as, nephrotoxicity and ototoxicity. A study of Pt accumulation and a speciation analysis has been performed by ICP-MS in samples from kidney and inner ear in a controlled population of Wistar rats treated with, either, cisplatin, carboplatin or oxaliplatin. The results on Pt accumulation point out to drug structure and not only to Pt content as the responsible for the alteration of organ functionality. Speciation studies in the samples from kidney and inner ear were performed coupling two-dimensional liquid chromatography (2D-LC) to ICP-MS. Size exclusion (SEC) and anion exchange fast protein liquid chromatography (FPLC) was employed for 2D orthogonal separation. After these separations, free drug peaks were not observed in any of the samples. The binding of Pt to biomolecules was demonstrated by SEC and, independently of the drug used, Pt eluted as two main bands with molecular weights of 12 kDa and 25–65 kDa for inner ear samples, and as two different bands with 20 kDa and 50–60 kDa in the samples from kidney. However, the relative band intensity presented important differences for the three drugs. Using the same chromatographic conditions, it was shown that a metallothionein (MT) standard eluted in the same position as some of the cytosolic Pt-biomolecules. High Pt-containing fractions eluting from the SEC column were analysed by anion exchange FPLC after a preconcentration step. Among the different preconcentration methods tested, sample focusing on the head of the FPLC column shows main advantages. In this way, the separation by 2D chromatography of the high molecular Pt-species has been considerably improved.

Speciation of aluminium in tea infusions by use of SEC and FPLC with ICP–OES and ES–MS–MS detection

Speciation of Al in tea infusions was studied by size exclusion chromatography (SEC) and anion-exchange fast protein liquid chromatography (FPLC). Fractions were collected throughout the chromatographic separations and Al was determined "off line" by inductively coupled plasma optical emission spectroscopy (ICP-OES). Black, green, and red tea samples were investigated. The total concentration of Al in tea infusions was determined by ICP-OES and ranged between 0.5 and 4 mg dm(-3). The pH of tea infusions ranged between 5.3 and 5.5. Data from SEC-ICP-OES analysis indicated that 10-35% of total Al in tea infusions was eluted at a retention volume corresponding to a molecular mass of approximately 3800 Da. The remaining Al was adsorbed on the column resin. The same tea infusions were also analysed by anion-exchange FPLC-ICP-OES. It was found experimentally that the same percentage of total Al as from the SEC column was eluted at a retention volume that corresponded to negatively charged Al-citrate. The remaining Al was adsorbed on the column resin. Identification of Al-binding ligands eluting under the chromatographic peak was performed by electrospray ionisation tandem mass spectrometry (ES-MS-MS) analysis. It was proven that ionic Al species in tea infusions (10-35% of the total Al) corresponded to negatively charged Al-citrate. The remaining species that was adsorbed on the SEC or FPLC columns was most probably bound to phenolic compounds. Speciation of Al in tea with milk or lemon was also studied. Results for tea with milk indicated that Al-citrate was not transformed and that approximately 60% of total Al was transformed into high-molecular-mass Al species. This fraction was subjected to sodium dodecyl sulfonate polyacryl gel electrophoresis (SDS-PAGE). The results indicated that Al was occluded by milk proteins (mostly caseins). When citric acid was added to tea infusions the percentage of negatively charged Al-citrate remained either the same or increased to 40% of total Al.

Physicochemical Characterization of Casein Phosphopeptide-Amorphous Calcium Phosphate Nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.

Extraction, Purification, and Inhibitory Effect of Alpha - Amylase Inhibitor from Wheat (Triticum aestivum Var. Zarrin)

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. They are also drug-design targets for treatment of diabetes and digestion disorderes. These inhibitors also known as sensitizing agents in human. The numerous form of alpha-amylase inhibitors was reported. In this study alpha-amylase inhibitor was extracted from Iranian wheat cultivar (Triticum aestivum v zarrin), precipitated and purified by anion exchange fast protein liquid chromatography. Electrophoresis of purified protein showed 0.66 relative mobility. Total hydrolytic activity of human salivary and bacillus subtilis alpha-amylase were inhibited 97.07% and 89.97% respectively by collected purified alpha-amylase inhibitor.

Multiple forms of equine α-lactalbumin: evidence for N-glycosylated and deamidated forms

Equine raw milk whey proteins were separated by anion-exchange fast protein liquid chromatography and characterised by alkaline polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulphate PAGE, and bi-dimensional PAGE. Approximately, 1% of α-lactalbumin (α-LA) was N-glycosylated. A minor N-glycosylated form of lysozyme was also found in equine milk. On the other hand, two non-glycosylated α-LA isoforms with similar molecular masses (14,215±4 Da) were shown to be present. Their respective apparent isoelectric points were 5.25 and 4.94. These isoforms did not correspond to different genetic variants and were not the result of α-LA modulation by calcium ions. They corresponded rather to a non-enzymatic deamidation process of a single asparagine side-chain, the most acidic isoform being spontaneously generated from the less acidic isoform by simple incubation of α-LA at 37°C. The initial rate of this chemical degradation was 4.5 μm ammonia liberated per hour, in 150 mm sodium phosphate buffer pH 7.4 at 37°C. Deamidation induced a slight variation in secondary structure content, but no significant change in the tertiary structure of the equine α-LA was studied by circular dichroism in the near- and far-UV regions.

Determination of Cr(VI) in welding fumes by anion-exchange fast protein liquid chromatography with electrothermal atomic absorption spectrometric detection.

The applicability of an anion-exchange fast protein liquid chromatographic-electrothermal atomic absorption spectrometric procedure (FPLC-ETAAS) was investigated for the determination of Cr(VI) in welding fumes after alkaline extraction of aerosols loaded on filters. Gas tungsten arc welding (GTAW) of stainless steel was applied. Samples of welding fumes were collected during regular welding on polycarbonate membrane filters of 8 microm and 0.4 microm pore size (inhalable and respirable aerosols). Alkaline extraction (2% NaOH-3% Na2CO3) of filters in a heated ultrasonic bath was applied to leach Cr from the airborne particulate matter. 0.5 cm3 of sample extract was then injected onto an anion-exchange FPLC column. Tris-HCl buffer (0.005 mol dm(-3), pH 8.0) and the same buffer with NaCl (0.5 mol dm(-3)) were employed in gradient elution (15 min, flow rate 1 cm3 min(-1)). The separated Cr species were determined "off line" by ETAAS in 0.5 cm3 fractions. Cr(VI) was reproducibly and quantitatively eluted from 12.0 to 13.0 min with a maximum peak at 12.5 min. Good repeatability of measurement (+/-3.0%) of alkaline extracts was obtained for Cr(VI). The LOD (3s) was found to be 0.035 microg m(-3) Cr(VI), when 2 m3 of aerosols were collected on the filter. Validation of the procedure was performed by spiking alkaline extracts and by the analysis of standard reference material CRM 545, Cr(VI) in welding dust loaded on a filter. The technique was successfully applied for the determination of Cr(VI) in welding fumes.

Identification of tropomyosin as a major allergen in the octopus Octopus vulgaris and elucidation of its IgE-binding epitopes

: The major allergen (named Oct v 1) in the muscle of the octopus Octopus vulgaris was purified by gel filtration on Sephacryl S-300, anion-exchange fast protein liquid chromatography on Mono Q and reverse-phase high-pressure liquid chromatography on TSKgel Octadecyl-4PW. In addition to the molecular mass, amino acid composition and cross-reactivity with Tur c 1 (turban shell Turbo cornutus allergen), the determined partial amino acid sequence clearly demonstrated that Oct v 1 is tropomyosin, similar to the known molluscan and crustacean allergens. Using peptide fragments isolated from the lysylendopeptidase digest of Oct v 1, competitive enzyme-linked immunosorbant assay inhibition experiments showed that IgE-binding epitopes of Oct v 1 are contained in two peptides (77–112 and 148–160) in the central region and one peptide (269–281) in the C-terminal region. In the peptide 77–112, the same sequence as the IgE-binding epitope proposed for Cra g 1 (oyster Crassostrea gigas allergen) is recognized at 92-105. Moreover, the peptide 148-160 partly overlaps with the IgE-binding epitopes suggested for Pen i 1 (shrimp Penaeus indicus allergen) and Pen a 1 (shrimp Penaeus aztecus allergen), and the peptide 269–281 with those for Tur c 1 and Pen a 1.

Effect of Osmolytes and Chaperone-like Action of P-protein on Folding of Nucleocapsid Protein of Chandipura Virus

Amino acid sequences of nucleocapsid proteins are mostly conserved among different rhabdoviruses. The protein plays a common functional role in different RNA viruses by enwrapping the viral genomic RNA in an RNase-resistant form. Upon expression of the nucleocapsid protein alone in COS cells and in bacteria, it forms large insoluble aggregates. In this work, we have reported for the first time the full-length cloning of the N gene of Chandipura virus and its expression in Escherichia coli in a soluble monomeric form and purification using nonionic detergents. The biological activity of the soluble recombinant protein has been tested, and it was found to possess efficient RNA-binding ability. The state of aggregation of the recombinant protein was monitored using light scattering. In the absence of nonionic detergents, it formed large aggregates. Aggregation was significantly reduced in the presence of osmolytes such as d-sorbitol. Aggregate formation was suppressed in the presence of another viral product, phosphoprotein P, in a chaperone-like manner. Both the osmolyte and phosphoprotein P also suppressed aggregation to a great extent during refolding from a guanidine hydrochloride-denatured form. The function of the phosphoprotein and osmolyte appears to be synergistic to keep the N-protein in a soluble biologically competent form in virus-infected cells.