Anion-exchange Fast Protein Liquid Chromatography Research Articles

Physico-chemical characterization of a milk-clotting fraction extracted from turkey (Meleagris gallopavo) proventriculus

Abstract Valorization of avian by-products such as turkey (Meleagris gallopavo) proventriculus in the preparation of milk clotting enzymes constitutes an alternative to commercial rennet. The clotting enzyme of Meleagris gallopavo has been prepared by ammonium sulfate at 50% saturation followed by purification by cation-exchange fast protein liquid chromatography (FPLC), and determination of the clotting protease activity performed on milk incubated at 35 °C. The clotting fractions are concentrated and dialyzed against Tris/HCl pH 7.0 through a membrane with cutoff 8000 Da, and separated by anion-exchange FPLC. The coagulating fractions obtained by cation-exchange chromatography are also separated by reversed-phase high-performance liquid chromatography, which revealed only one fraction able to coagulate milk. Electrophoresis analysis revealed a band with apparent molecular mass of 36.5 kDa and seemed to correspond to pepsin-like enzyme according to primary sequence alignment analyses. Maximal clotting activity was obtained for optimal conditions of temperature of 55 °C and pH 5.4.

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.

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.

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.

Biosynthesis of UDP-N-acetyl-l-fucosamine, a Precursor to the Biosynthesis of Lipopolysaccharide in Pseudomonas aeruginosa Serotype O11

UDP-N-acetyl-L-fucosamine is a precursor to l-fucosamine in the lipopolysaccharide of Pseudomonas aeruginosa serotype O11 and the capsule of Staphylococcus aureus type 5. We have demonstrated previously the involvement of three enzymes, WbjB, WbjC, and WbjD, in the biosynthesis of UDP-2-acetamido-2,6-dideoxy-L-galactose or UDP-N-acetyl-L-fucosamine (UDP-l-FucNAc). An intermediate compound from the coupled-reaction of WbjB-WbjC with the initial substrate UDP-2-acetamido-2-deoxy-alpha-D-glucose or UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) was purified, and the structure was determined by NMR spectroscopy to be UDP-2-acetamido-2,6-dideoxy-L-talose (UDP-L-PneNAc). WbjD could then convert this intermediate into a new product with the same mass, consistent with a C-2 epimerization reaction. Those results led us to propose a pathway for the biosynthesis of UDP-L-FucNAc; however, the exact enzymatic activity of each of these proteins has not been defined. Here, we describe a fast protein liquid chromatography (FPLC)-based anion-exchange procedure, which allowed the separation and purification of the products of C-2 epimerization due to WbjD. Also, the application of a cryogenically cooled probe in NMR spectrometry offers the greatest sensitivity for determining the structures of minute quantities of materials, allowing the identification of the final product of the pathway. Our results showed that WbjB is bifunctional, catalyzing firstly C-4, C-6 dehydration and secondly C-5 epimerization in the reaction with the substrate UDP-D-GlcNAc, producing two intermediates. WbjC is also bifunctional, catalyzing C-3 epimerization of the second intermediate followed by reduction at C-4. The FPLC-based procedure provided good resolution of the final product of WbjD reaction from its epimer/substrate UDP-l-PneNAc, and the use of the cryogenically cooled probe in NMR revealed unequivocally that the final product is UDP-L-FucNAc.

Determination of high molecular mass Al species in serum and spent CAPD fluids of dialysis patients combining SEC and anion-exchange FPLC with ETAAS detection

Speciation of high molecular mass Al compounds (HMM-Al) in human serum is commonly performed by size-exclusion or ion-exchange chromatography. In general, size-exclusion chromatographic (SEC) columns are not able to completely separate transferrin from albumin. Fast protein liquid chromatography (FPLC), using an anion-exchange column, enables separation of transferrin from albumin. However, the possibility exists of coelution of LMM–Al species (e.g., citrate, phosphate) with HMM–Al species. In this case the concentration of HMM–Al species could be overestimated. It is therefore important to remove LMM–Al species before anion-exchange FPLC separation. For this purpose a novel analytical approach was developed for speciation of HMM–Al species in human serum by combination of SEC (Superdex 75 HR 10/30) and anion-exchange FPLC (Mono Q HR 5/5 anion-exchange FPLC) with UV detection at 278 nm. 1 cm3 of serum was injected onto the SEC column. Isocratic elution using 0.05 mol dm−3 TRIS-HCl + 0.03 mol dm−3 NaHCO3 was applied. It was experimentally proved that proteins were eluted in a 5 cm3 peak that was collected into a polyethylene cup. A 0.5 cm3 aliquot of the sample was then injected onto the anion-exchange FPLC column. The separation of serum proteins was obtained by applying linear gradient elution from 100% buffer A (0.05 mol dm−3 TRIS-HCl + 0.03 mol dm−3 NaHCO3) to 100% buffer B (A + 0.25 mol dm−3 NaCl). Well-resolved protein peaks were obtained. 0.25 cm3 fractions were collected during the chromatographic run and Al determined by ETAAS. It was experimentally proved that 90 ± 5% of Al in spiked serum from a dialysis patient was eluted under the transferrin peak which was identified, not only on the basis of the retention volume, but also by the SDS-PAGE electrophoresis. The same procedure may be applied for speciation of Al in spent CAPD fluids of dialysis patients if the concentration of Al is higher than10 ng cm−3. The proposed speciation procedure removes LMM–Al species and enables reliable determination of the concentration and composition of Al bound to proteins by anion-exchange FPLC-ETAAS.

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.

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit.

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.

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.

Speciation of low molecular weight Al complexes in serum of CAPD patients.

Speciation of LMW--Al complexes was performed in human serum of six continuous ambulatory peritoneal dialysis (CAPD) patients in order to investigate the individual variability in the percentage and the composition of LMW--Al species. The total concentration of Al in serum ranged from 10 to 120 ng ml(-1). The samples with high total concentration of Al were analysed directly, while those of low total Al concentration were spiked with Al(3+). Spiked and non-spiked samples (100--120 ng ml(-1) of total Al) were microultrafiltered through a membrane filter (cut-off 30,000 Da) to separate Al-transferrin from LMW-Al complexes. On an anion-exchange fast protein liquid chromatography (FPLC) column, 0.2 ml of filtrate was injected. An aqueous -- 4 mol l(-1) NH(4)NO(3) linear gradient elution was applied for 10 min to separate LMW--Al complexes. Fractions of 0.2 ml collected throughout the chromatographic run were diluted 1:1 with water and Al determined 'off line' by electrothermal atomic absorption spectrometry (ETAAS). The characterisation of LMW-Al species eluted under the chromatographic peaks was performed also by electrospray tandem mass spectrometric (ES-MS-MS) analysis. It was found experimentally that the percentage of LMW--Al species in spiked and non-spiked serum ranged from 25 to 50% (in one non-spiked sample 100%). The following LMW--Al species were separated and identified during the chromatographic run: Al-phosphate and a mixture of Al-citrate and ternary Al-citrate--phosphate complexes. It was found experimentally that the distribution of these species varied among particular patients. Similar distribution of LMW--Al species was found in spiked serum of healthy volunteers.

Determination of hexavalent chromium in lime-treated sewage sludge by anion-exchange fast protein liquid chromatography with electrothermal atomic absorption spectrometry detection

The applicability of an anion-exchange fast protein liquid chromatographic–electrothermal atomic absorption spectrometric procedure (FPLC-ETAAS) was investigated for the determination of trace amounts of Cr(VI) in lime-treated sewage sludge. Samples of sewage sludge and sludge mixed with sawdust (volume ratio 1 + 1) with a pH of 8.0 and 7.8, respectively, as well as their mixtures with 5% of quicklime with a pH of 12 were analysed. Liming was applied for sludge stabilisation and disinfection. 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. It was experimentally proven that Cr(VI) was reproducibly and quantitatively eluted from 12.0 to 13.0 min with a maximum peak at 12.5 min. The repeatability of measurement for a standard solution of 25.0 ng cm−3 of Cr(VI) was found to be 4.0% and the LOD 1.5 ng cm−3 of Cr(VI). The technique was successfully applied for the determination of Cr(VI) in lime-treated sewage sludge. In aqueous extracts of untreated sewage sludge Cr(VI) was not detected. In aqueous extracts of lime-treated sewage sludge Cr(VI) was determined 48 h after liming [the concentration was about 200 ng g−1 Cr(VI)] and was monitored over a time span of six months. The validation of the procedure was performed by analysis of a certified reference material, spiking of samples (recoveries from 93 to 101%) and comparative analysis of Cr(VI) in the fraction eluted at 12.5 min by the ion chromatography thermal lens spectrometry technique. The applied FPLC-ETAAS procedure was found to be a reliable and sensitive technique for the determination of traces of Cr(VI) in lime-treated sewage sludge and investigation of oxidation–reduction processes of Cr. The LOD for sludge samples analysed was 20 ng g−1 of Cr(VI) and the repeatability of measurement 6.5%.

N-Glycosylated Variants of Growth Hormone in Human Pituitary Extracts

This study was designed to investigate the existence, in human pituitary extracts, of growth hormone (GH) variants not encoded by the hGH-N gene. Using anion exchange-fast protein liquid chromatography followed by SDS-PAGE, we isolated several basic forms of pituitary GH. Incubation of these basic forms with endoglycosidase F/N-glycosidase F revealed that two of them (about 34 and 12 kD) were N-glycosylated. In contrast, no changes were found when samples were incubated with the O-linked glycosylation-specific O-glycosidase. Since the GH-N molecule lacks consensus sequences for N-linked glycosylation, our findings suggest that GH genes other than hGH-N are expressed in the human pituitary gland.