Anion-exchange HPLC Research Articles

Quantitative Determination of A Sirna (Ad00370) in Rat Plasma Using Peptide Nucleic Acid Probe and HPLC with Fluorescence Detection

Toxicokinetic and pharmacokinetic studies of therapeutic oligonucleotides require validated bioanalytical methods for sensitive and specific quantification of oligonucleotide drug candidates in biological samples. A peptide nucleic acid (PNA) hybridization-based HPLC-fluorescence assay was developed and validated for quantification of Arrowhead Pharmaceuticals' proprietary siRNA in rat plasma samples via hybridization and anion-exchange-HPLC (AEX-HPLC) with fluorescence detection. The validated method provided a sensitive and selective approach for quantification of siRNA in biological samples at a linear quantitation range of 1-1000 ng/ml. The assay requires only 25 μl of plasma sample and shows excellent accuracy and precision even without using an internal standard, providing a useful quantification method for siRNA determination in biological matrix with limited sample volume.

Understanding of decreased sialylation of Fc‐fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N‐glycosylation gene expression and N‐linked glycan antennary profile

To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (qFc ) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After 3 days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. Biotechnol. Bioeng. 2017;114: 1721-1732. © 2017 Wiley Periodicals, Inc.

Reprint of Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT)

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.

One single, fast and robust capillary electrophoresis method for the direct quantification of intact adenovirus particles in upstream and downstream processing samples

During development of adenovirus-based vaccines, samples have to be analyzed in order to either monitor the production process or control the quality and safety of the product. An important quality attribute is the total concentration of intact adenoviruses, which currently is determined by quantitative polymerase chain reaction (qPCR) or anion exchange-HPLC. Capillary Electrophoresis (CE) was evaluated as alternative to the current methods with the aim to have one single method that allows reliable and fast quantification of adenovirus particles throughout the full process. Intact adenoviruses samples from downstream processing and upstream processing were analyzed directly by CE with UV-detection at 214nm. Only the samples with high amounts of DNA required a simple sample pretreatment by benzonase. Adenovirus particles were separated from matrix components such as cell debris, residual cell DNA, and/or proteins on a PVA-coated capillary using a BGE consisting of 125mM Tris, 338mM tricine and 0.2% v/v polysorbate-20 at pH 7.7. Full factorial design of experiments was used for method optimization as part of the analytical quality by design (AQbD) method development approach. The method was validated for the quantification of adenoviruses on five representative samples from the manufacturing process in the range of 0.5×1011–1.5×1011 adenovirus particles per ml (~80 to 250pmol/l). The CE method showed intermediate precision of 7.8% RSD on concentration and an accuracy (spiked recovery) of 95–110%. CE proved highly useful for process development support and is being implemented for in-process control testing for adenovirus vaccine manufacturing.

Evaluation of human nonmercaptalbumin as a marker for oxidative stress and its association with various parameters in blood.

Oxidative status of albumin was not a useful biomarker for oxidative stress in practical use due to time-consuming measuring method. We evaluated oxidized, human nonmercaptalbumin measured more quickly than ever by a novel method using anion-exchange HPLC. In 60 subjects taking a general health examination, mean serum human nonmercaptalbumin level was 25.1 ± 3.0% with no gender difference but positive correlation with age. There were no links between human nonmercaptalbumin and C-reactive protein, γ-glutamyltransferase or iron, reportedly associated with oxidative stress. Human nonmercaptalbumin correlated with systolic blood pressure, pulse pressure and body mass index among physical findings. Positive correlations were observed between human nonmercaptalbumin and AST, LDH, BUN, or creatinine, suggesting that oxidative stress may link with liver injury and renal function. Human nonmercaptalbumin correlated with uric acid in female but not in male, suggesting that higher uric acid levels may be associated with increased oxidative stress only in female. As another gender difference, white blood cell counts correlated with human nonmercaptalbumin in female, while the parameters for red blood cells correlated with human nonmercaptalbumin in male. In conclusion, serum human nonmercaptalbumin level in healthy subjects was approximately 25% as previously reported. Oxidative stress may be closely associated with hypertension, obesity, liver injury, renal function, and anemia.

Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation.

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298nm and λ em 550nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.

Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT)

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.

Inhibitory potential of biosurfactants from Bacillus amyloliquefaciens derived from mangrove soil against Vibrio parahaemolyticus

Vibrio parahaemolyticus is a halophilic bacterium that causes seafood-borne gastroenteritis which can occur through direct or cross-contamination. In this study, biosurfactant-producing marine bacteria were isolated from 28 soil samples collected from mangrove and coastal regions. Using the cross streak technique, 26 isolates were found to inhibit the growth of V. parahaemolyticus. Biosurfactant lipopeptides were obtained by acid precipitation and their antimicrobial potentials were assessed by the agar well diffusion technique. The extract of a bacterial isolate SM11 derived from mangrove soil showed the strongest inhibitory activity against V. parahaemolyticus. The inhibition zones against V. parahaemolyticus of the extract obtained by chloroform and methanol at concentrations of 900, 1800 and 2600 μg/mL were 16.9 ± 0.2, 18.4 ± 0.5 and 25.0 ± 0.1 mm, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract were 256 to 512 and 512 to 1024 μg/mL, respectively. The extract of biosurfactants showed more than 60 % reduction in V. parahaemolyticus adhesion. 16S rRNA gene sequencing of SM11 revealed that this isolate is Bacillus amyloliquefaciens. The active fractions obtained from anion exchange chromatography and HPLC as analysed by ESI-Q-TOF mass spectrometry indicated that those biosurfactants were mycosubtilin, surfactin and iturin A. Besides highlighting the merits of biosurfactants as antagonistic agents, this study suggests the possibility of using them to decrease cross-contamination of V. parahaemolyticus on cooking or food processing surfaces.

Chemical synthesis of RNA with site-specific methylphosphonate modifications

Methylphosphonate(mP)-modified RNA serves as valuable probe to evaluate biomolecular interactions between the nucleic acid backbone and binding partners, such as proteins or small molecules. Here, we describe an efficient workflow for the synthesis of RNA with a single mP modification in diastereomerically pure form. While the automated assembly of mP-modified RNA is straightforward, its deprotection under basic conditions is challenging; a carefully optimized step-by-step procedure is provided. In addition, we demonstrate purification and separation strategies for the RP and SP-configurated RNA diastereomers using a combination of anion-exchange and reversed-phase HPLC, and comment on troubleshooting if their separation appears difficult. Furthermore, we demonstrate the stereochemical assignment of short RP and SP mP-modified RNA diastereomers based on 2D ROESY NMR spectroscopy and we report on the impact of the mP modification on thermal and thermodynamic stabilities of RNA-DNA hybrid and RNA-RNA duplexes.

Ion chromatography for the separation of heparin and structurally related glycoaminoglycans: A review.

The global crisis resulting from adulterated heparin in late 2007 and early 2008 revived the importance of analytical techniques for the purity analysis of heparin products. The utilization of ion chromatography techniques for the separation, detection, and structural determination of heparin and structurally related glycoaminoglycans, including their corresponding oligosaccharides, has become increasingly important. This review summarizes the primary HPLC approaches, particularly strong anion exchange, weak ion exchange, and reversed-phase ion-pair, used for heparin purity analysis as well as structural characterization. Strong anion exchange HPLC has been studied most extensively and currently offers the best separation of crude heparin and heparin-like compounds. Weak anion exchange HPLC has been shown to provide shorter analysis times with lower salt concentrations in the mobile phase but is not as widely developed for the separation of all glycoaminoglycans of interest. Reversed-phase ion-pair HPLC offers fast and effective separations of oligosaccharides derived from glycoaminoglycans that can be coupled to mass spectrometry for structural analysis. However, this method generally does not provide sufficient retention of intact glycoaminoglycans.

Metatartaric acid: physicochemical characterization and analytical detection in wines and grape juices

Seven commercial metatartaric acid (MTA) products used for tartaric stabilization of wines were investigated regarding molecular masses and composition. Combined size-exclusion chromatography with MALS detection showed that the products had medium molecular masses in a range of 2–9 kDa. All samples were highly polydisperse products with a broad molecular weight distribution. Beside oligomeric or polymeric contents, the products contained on average 13 g/100 g monomeric tartaric acid, thus reducing the effectiveness of MTA in respect of achieving tartaric stability. An analytical method to determine MTA in wines and grape juices based on an anion-exchange cleanup step, HPLC separation, and MS detection was developed. The method shows a sufficient robustness to evaluate important enological or food legal aspects in connection with MTA.

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice.

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasmagondii (T.gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T.gondii which are able to mature DCs and consequently instruct immune responses in tumor-bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion-exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL-12p70 and IL-10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor-bearing mice. We found that ammonium sulfate fraction one (A1) matured-DCs produced higher IL-12 level and IL-12/IL-10 ratio; therefore, this fraction was selected for further fractionation by anion-exchange HPLC. The results showed that anion-exchange HPLC fraction 14 (C14) matured-DCs secrete higher levels of IL-12p70 and IL-12p70/IL-10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS-PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T.gondii are able to mature DCs more efficient.

Feasibility of asymmetric flow field-flow fractionation coupled to ICP-MS for the characterization of wear metal particles and metalloproteins in biofluids from hip replacement patients.

Hip replacements are used to improve the quality of life of people with orthopaedic conditions, but the use of metal-on-metal (MoM) arthroplasty has led to poor outcomes for some patients. These problems are related to the generation of micro- to nanosized metal wear particles containing Cr, Co or other elements, but the current analytical methods used to investigate the processes involved do not provide sufficient information to understand the size or composition of the wear particles generated in vivo. In this qualitative feasibility study, asymmetric flow field-flow fractionation (AF(4)) coupled with inductively coupled plasma mass spectrometry (ICP-MS) was used to investigate metal protein binding and the size and composition of wear metal particles present in serum and hip aspirates from MoM hip replacement patients. A well-established HPLC anion exchange chromatography (AEC) separation system coupled to ICP-MS was used to confirm the metal-protein associations in the serum samples. Off-line single particle ICP-MS (spICP-MS) analysis was used to confirm the approximate size distribution indicated by AF(4) of the wear particles in hip aspirates. In the serum samples, AF(4) -ICP-MS suggested that Cr was associated with transferrin (Tf) and Co with albumin (Alb) and an unidentified species; AEC-ICP-MS confirmed these associations and also indicated an association of Cr with Alb. In the hip aspirate sample, AF(4)-ICP-MS suggested that Cr was associated with Alb and Tf and that Co was associated with Alb and two unidentified compounds; AEC analysis confirmed the Cr results and the association of Co with Alb and a second compound. Enzymatic digestion of the hip aspirate sample, followed by separation using AF(4) with detection by UV absorption (280nm), multi-angle light scattering and ICP-MS, suggested that the sizes of the Cr-, Co- and Mo-containing wear particles in a hip aspirate sample were in the range 40-150nm. Off-line spICP-MS was used to confirm these findings for the Co- and Cr-containing nanoparticles. Whilst limited in scope, the results are sufficient to show the interaction of ions with transport proteins and give an indication of particle size, providing useful pathological indices. As such, the methods indicate a new way forward for in vivo investigation of the processes which lead to tissue necrosis and hip loosening in patients with MoM hip replacements.

Arsenic speciation and Fucoxanthin analysis from seaweed dietary supplements using LC-ICP-MS

The study involves the analysis of total arsenic in metallic form, organic and inorganic arsenic species from seaweeds and dietary supplements. The analysis provides data for dietary exposure estimates of inorganic species which are considered more toxic to humans than organic arsenic and total arsenic. Total arsenic was determined by acid digestion followed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The optimum arsenic speciation method was determined to be water extraction followed by anion exchange HPLC coupled with ICP-MS. Optimization of chromatographic conditions led to baseline separation for six arsenic species, including arsenic acid, arsenous acid, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, and arsenocholine, in approximately 8 min using gradient elution. Detection limits for all 6 compounds were in the range from 10 – 15 ng/mL. The data presented here will be valuable for the quality assurance of analytical method development and surveys of total arsenic and arsenic species in dietary supplements. The most abundant arsenic species found were arsenite (As III) and arsenate (As V). In addition, the dietary supplements purported to contain fucoxanthin, a carotenoid having pharmacological activities were analyzed using UHPLC-UV-MS.

Arsenic speciation and fucoxanthin analysis from seaweed dietary supplements using LC-MS.

The study involves the analysis of total arsenic (As) in metallic form, and organic and inorganic As species from seaweeds and dietary supplements. The analysis provides data for dietary exposure estimates of inorganic species that are considered more toxic to humans than organic and total As. Total As was determined by acid digestion followed by inductively coupled plasma (ICP)-MS. To characterize the As species, solvent extraction with sonication and microwave extraction using various aqueous and aqueous/organic solvent mixtures were initially evaluated. The optimum As speciation method was determined to be water extraction followed by anion exchange HPLC coupled with ICP-MS. Optimization of chromatographic conditions led to baseline separation for six As species, including As acid, arsenous acid, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, and arsenocholine, in approximately 8 min using gradient elution. Detection limits for all six compounds were in the range of 10-15 ng/mL. The data presented here will be valuable for the QA of analytical method development and surveys of total As and As species in dietary supplements. The most abundant As species found were arsenite [As(III)] and arsenate [As(V)]. The sum of inorganic As species present in the dietary supplements ranged from 1.2 to 31 μg/day. In addition, the dietary supplements purported to contain fucoxanthin, a carotenoid having pharmacological activities, were analyzed using ultra-performance LC-UV/MS.

Heparosan-glucuronate 5-epimerase: Molecular cloning and characterization of a novel enzyme

Iduronic acid (IdoA) is a critical component of heparan sulfate in its interaction with functional proteins. Heparosan-N-sulfate-glucuronate 5-epimerase (HNSG-5epi) converts d-glucuronic acid (GlcA) residues in N-sulfated heparosan (NS-heparosan), as an intermediate in heparan sulfate biosynthesis, to IdoA. In the present study, the authors discovered a different 5-epimerase, designated HG-5epi (heparosan-glucuronate 5-epimerase), that is involved in acharan sulfate biosynthesis and possesses novel substrate specificity. A candidate cDNA of HG-5epi was cloned from the cDNA library of Achatina fulica. The cloned cDNA contained a whole coding region that predicts a type II transmembrane protein composed of 601 amino acid residues. The amino acid sequence of HG-5epi is homologous to that of HNSG-5epi. Recombinant HG-5epi was expressed in insect cells and its enzymatic properties characterized. As expected, HG-5epi epimerizes GlcA residues in heparosan, but not in NS-heparosan. Conversion of IdoA to GlcA was also catalyzed by HG-5epi when completely desulfated N-acetylated heparin was used as the substrate, indicating a reversible reaction mechanism. At equilibrium of the epimerization, the proportion of IdoA in the reaction product reached up to 30% of total hexuronic acid. To our knowledge, this is the first report to describe an enzyme that catalyzes the epimerization of non-sulfated heparosan. This new enzyme may be applied to the study of synthetic heparan sulfate-related polysaccharides having certain biological and pharmacological activities. In addition, a new method using anion-exchange HPLC connected to a post-column fluorescent labeling system was developed for analyzing hexuronic acid isomers.

Structural and Functional Study of d-Glucuronyl C5-epimerase

Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.

16 BIOCHEMICAL ANALYSIS OF COMPONENT IN SEMINAL GEL SECRETED WITH BOAR SEMEN

Seminal gel (SG), a part of semen, of the boar originates from secretions from the Cowper's gland and has a high viscosity and water-holding capacity, preventing backflow of semen at natural mating. However, there are is little information available about biochemical and functional characteristics of boar SG. In this study, as a first step to elucidate the chemical features of the SG, we examined the structure of O-glycans and the primary structure of protein from the boar SG. Seminal gel was collected from ejaculated semen of a Berkshire boar with high fertility and freeze-dried. Samples were preserved in a refrigerator until experiments were conducted. For Exp. 1 the presence of O-glycans in SG was confirmed by detection of the amino sugar, galactosamine (GalNH2), from acid hydrolysis of GalNAc. The freeze-dried SG (1 mg) was hydrolyzed with 4N trifluoroacetic acid at 110°C for 2 h. The resulting amino sugar was labelled with phenyl isothiocyanate (PITC) and then analysed by RP-HPLC. The GalNAc was detected as a main amino sugar, suggesting that the SG contains O-glycosylated glycoprotein. For Exp. 2 the O-glycans were prepared from the freeze-dried SG (5 mg) by hydrazinolysis at 100°C for 2 h. After N-acetylation, the O-glycans were pyridylaminated. The structures were identified by anion-exchange HPLC, size-fractionation HPLC, glycosidase digestion, and ESI-MS and MS/MS analysis. Almost all glycans were digested by α2–3,6-sialidasae, indicating that these O-glycans are sialylated and give the glycoproteins viscosity. Furthermore, the MS analysis showed that the de-sialylated O-glycans consist of HexNAc-PA (m/z 300.0) and Hex-HexNAc-PA (m/z 462.0) and major glycans are di- or tri-saccharides. For Exp. 3 proteins in the SG were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition with 5% 2-mercaptoethanol. Proteins were stained with Coomassie Brilliant Blue R-250. Three bands (~160, 140, and 70 kDa) were found on 7.5% polyacrylamide gel, but two bands (160, 140 kDa) were converted to ~130 kDa after the sialidase digestion, indicating that native two proteins (160 and 140 kDa) may be highly sialylated. For Exp. 4 internal amino acid sequence was analysed using one of the peptic peptides. The freeze-dried SG (5 mg) was digested with porcine pepsin in 5% formic acid at 37°C for 3 h. The resulting peptides were separated by RP-HPLC. N-terminal sequence of one of the peptic peptides was WSEKYGIPGGKAH. The amino acid sequence showed a high homology with tyrosine-protein kinase ZAP-70. These results suggest that boar SG contains mucin-like glycoproteins carrying heavily sialylated O-glycans. Additionally, the current study suggests a possibility that some protein components of the boar SG derive from high concentration of the kinase in (dead) sperms.

Antivirally Active Ribavirin Analogues – 4,5-disubstituted 1,2,3-triazole Nucleosides: Biological Evaluation against Certain Respiratory Viruses and Computational Modelling

Ribavirin is a broad-spectrum antiviral agent that derives some of its activity from inhibition of cellular inosine monophosphate dehydrogenase (IMPDH), resulting in lower guanosine triphosphate (GTP) levels. Here we report the biological activities of three ribavirin analogues. Antiviral activities of test compounds were performed by in vitro cytopathic effect inhibition assays against influenza A (H1N1, H3N2 and H5N1), influenza B, measles, parainfluenza type 3 (PIV-3) and respiratory syncytial viruses. Compounds were modelled into the ribavirin 5'-monophosphate binding site of the crystallographic structure of the human type II IMPDH (hIMPDH2) ternary complex. Effects of compounds on intracellular GTP levels were performed by strong anion exchange HPLC analysis. Of the three compounds evaluated, the 5-ethynyl nucleoside (ETCAR) exhibited virus-inhibitory activities (at 1.2-20 μM, depending upon the virus) against most of the viruses, except for weak activity against PIV-3 (62 μM). Antiviral activity of ETCAR was similar to ribavirin; however, cytotoxicity of ETCAR was greater than ribavirin. Replacing the 5-ethynyl group with a 5-propynyl or bromo substituent (BrCAR) considerably reduced antiviral activity. Computational studies of ternary complexes of hIMPDH2 enzyme with 5'-monophosphates of the compounds helped rationalize the observed differences in biological activity. All compounds suppressed GTP levels in cells; additionally, BrCAR suppressed adenosine triphosphate and elevated uridine triphosphate levels. Three compounds related to ribavirin inhibited IMPDH and had weak to moderate antiviral activity. Cytotoxicity adversely affected the antiviral selectivity of ETCAR. As with ribavirin, reduction in intracellular GTP may play a role in virus inhibition.

Anion Exchange HPLC Isolation of High-Density Lipoprotein (HDL) and On-Line Estimation of Proinflammatory HDL

Proinflammatory high-density lipoprotein (p-HDL) is a biomarker of cardiovascular disease. Sickle cell disease (SCD) is characterized by chronic states of oxidative stress that many consider to play a role in forming p-HDL. To measure p-HDL, apolipoprotein (apo) B containing lipoproteins are precipitated. Supernatant HDL is incubated with an oxidant/LDL or an oxidant alone and rates of HDL oxidation monitored with dichlorofluorescein (DCFH). Although apoB precipitation is convenient for isolating HDL, the resulting supernatant matrix likely influences HDL oxidation. To determine effects of supernatants on p-HDL measurements we purified HDL from plasma from SCD subjects by anion exchange (AE) chromatography, determined its rate of oxidation relative to supernatant HDL. SCD decreased total cholesterol but not triglycerides or HDL and increased cell-free (cf) hemoglobin (Hb) and xanthine oxidase (XO). HDL isolated by AE-HPLC had lower p-HDL levels than HDL in supernatants after apoB precipitation. XO+xanthine (X) and cf Hb accelerated purified HDL oxidation. Although the plate and AE-HPLC assays both showed p-HDL directly correlated with cf-Hb in SCD plasma, the plate assay yielded p-HDL data that was influenced more by cf-Hb than AE-HPLC generated p-HDL data. The AE-HPLC p-HDL assay reduces the influence of the supernatants and shows that SCD increases p-HDL.