Monitoring solid-phase glycoside synthesis with (19)F NMR spectroscopy

[structure: see text] A simple and efficient method for monitoring and optimizing carbohydrate synthesis on polymeric support by using (19)F NMR spectroscopy is described. The method relies on the use of fluorinated variants of protective groups that are in common use in oligosaccharide synthesis.     

Rare variants analysis by risk-based variable-threshold method

Genome-wide association studies, as a powerful approach for detecting common variants associated with diseases, have revealed many disease-associated loci. However, the traditional association analysis methods do not have enough power for detecting the effects of rare variants with limited sample size. As a solution to this problem, pooling rare variants by their functions into a composite variant provides an alternative way for identifying susceptible genes. In this paper, we propose a new pooling method to test the variant–disease association and to identify the functional rare variants related with the disease. Variants with smaller and larger risk measures defined as the ratio of allele frequencies between cases and controls are pooled and a chi-square test of the resultant pooled table is calculated. We vary the threshold of pooling over all possible values and use the maximal chi-square as test statistic. The maximal chi-square is in fact the global maximum over all possible poolings. Our approach is similar to the existing variable-threshold method, but we threshold on the risk measure instead of allele frequencies of controls. Simulation results show that our method performs better in both association testing and variant selection.     

Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants

A new RP-HPLC method for the separation and quantification of the most common genetic variants of bovine milk proteins is described. A reversed-phase analytical column C8 (Zorbax 300SB-C8 RP, 3.5 microm, 300A, 150 x 4.6 I.D.) was used. All the most common casein (CN) and whey protein genetic variants, including beta-CN(I) were detected and separated simultaneously in less then 40 min, with the exception of alpha(S1)-CN(B) and CN(C) variants. Purified protein genetic variants were employed in calibration and showed different absorbances at 214 nm. The procedure was developed using 40 raw individual milk samples of cows belonging to four different breeds and certified skim milk powder BCR-063R. Method validation consisted in testing linearity, repeatability, reproducibility and accuracy. A linear relationship (R(2)>0.99) between the concentrations of proteins and peak areas was observed over the concentration range, with low detection limits. Repeatability and reproducibility were satisfactory for both retention times and peak areas. The RSD of peak areas ranged from 0.92 to 4.32% within analytical day and from 0.85 to 9.52% across analytical days. The recoveries, calculated using mixtures of samples previously quantified, ranged from 98.1 to 103.7%.     

Genetic microheterogeneity of human transthyretin detected by IEF

Mutations of the human transthyretin (TTR) gene have attracted medical interest as a cause of amyloidosis. Recently, we have described in detail an electrophoretic procedure with PAGE followed by IEF in urea gradients for the study of the microheterogeneity of TTR monomers (Altland, K., Winter, P., Sauerborn, M. K., Electrophoresis 1999, 20, 1349-1364). In this paper, we present a study on 49 different mutations of TTR including 33 that result in electrically neutral amino acid substitutions. The aims of the investigation were to test the sensitivity of the procedure to detect TTR variants in patients with TTR amyloidosis and their relatives and to identify some common characteristics that could explain the amyloidogenicity of these variants. We found that all tested amyloidogenic mutations could be detected by our method with the exception of those for which the corresponding variant was absent in plasma samples. Most of the electrically neutral amyloidogenic TTR variants had in common a reduced conformational stability of monomers by the activity of protons and urea. For three variants, e.g. TTR-F64L, TTR-I107V and TTR-V122I, the monomers had a conformational stability close to that of normal monomers but we found experimental and structural arguments for a weakening of the monomer-monomer contact. All types of amyloidogenic mutations affected the stability of TTR tetramers.     

Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activity-structure relationships to design new bioactive agents

A prominent group of bioactive lipopeptides produced by Bacillus species is constituted by iturins, surfactins and lichenysins. Interest in such substances results in their exceptional surfactant power, and their valuable antifungal, antibacterial, antitumoral and anti-Mycoplasma properties. As is typical for peptidic secondary-metabolites synthesized by the polyenzymic pathway, they are produced as mixtures of components varying in the peptidic and/or in the lipidic structure. In the context of structure-activity relationships, it is possible to take advantage of the adaptability of the biosynthesis system by systematically adding selected amino acids in the culture medium of the producing bacterium. When an amino acid is used as the sole nitrogen source, it is inserted directly into selected positions of the peptide sequence, thus amplifying the original structural microheterogeneity via a production of variants. This method revealed very efficient for increasing the amounts of preexisting variants and for building new variants of surfactins and lichenysins but totally inefficient with iturins. In this group, the peptidic diversity strictly depends on the selected strain. So far the screening remained the only method to discover new iturins. Another interesting peculiarity is the common occurrence in a single strain of two lipopeptides with different core structures such as surfactins and iturins. Taken together, these features led to an extensive metabolite pattern. Besides, engineered variants and chemical derivatives enlarged the array of available molecules. Despite the high degree of chemical similarity, the separation of variants and/or homologues was successfully achieved by reversed-phase HPLC leading to well-separated compounds ideally suited to investigation of structure-activity relationships. Improved physical techniques such as 2D-NMR and mass spectrometry allowed to describe efficiently and rapidly the composition of cyclic lipopeptides even in mixtures containing several variants. From NMR, the 3D structure and dynamics gave crucial data for fine structure-activity relationships as well as for understanding of the properties at the membrane and/or at the air/water interface. Here the role of residues was identified in the context of hydrophobic and electrostatic interactions that play a leader role. Such a comprehensive approach, based on both structural and biosynthesis knowledge, opened the way to rational design for enhanced properties and its validity was confirmed with 10 fold higher surfactant efficacy.     

A high-resolution capillary isoelectric focusing method for the determination of therapeutic recombinant monoclonal antibody

Capillary isoelectric focusing (CIEF) is a common choice for separation and analysis of the charge variants and impurities of therapeutic proteins. In this study, we developed a sensitive CIEF analysis method for determining the charge heterogeneity of therapeutic monoclonal antibody (mAb) using Beckman PA800 plus platform. The mixture of 5% Pharmalyte 8-10.5 and 1% Pharmalyte 3-10 was used to overcome the limitation of using single Pharmalyte 3-10 in detecting charge heterogeneity of basic mAb. This approach largely improved the resolution of the heterogeneous peaks. In addition, 3 M urea and 50 mM arginine (Arg) were used to improve the separation as solubilizer and cathodic stabilizer, respectively. Under optimized condition, both acidic and basic peaks of the mAb were separated well. Method qualification results showed good specificity, precision, and linearity within the concentration range of 0.03-0.20 mg/mL for mAb R1. The method was then used for C-terminal lysine (Lys) variants characterization and glycosylation profiles analysis. Furthermore, it also had a wide application in the clone screening process. The highly sensitive and repeatable results highlighted the wide application prospects of this method in biopharmaceutical industry.     

Forensic aspects of haptoglobin: electrophoretic patterns of haptoglobin allotype products and an evaluation of typing procedure

A procedure used for haptoglobin (Hp) typing in paternity cases has been evaluated. All serum samples have been subtyped with a one-dimensional isoelectric focusing/immunoblotting method, and samples with rare or questionable patterns have been further examined by two-dimensional electrophoresis with isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The electrophoretic Hp-patterns of common and rare alpha- and beta-chain variants are shown, including allotype patterns of two new beta-chain variants and three new alpha-chain variants. Retyping of nearly 2000 individuals at intervals between 1 to 12 months revealed a typing error frequency of about 0.3%, which is considered acceptable, provided new blood samples are required in every case of paternity exclusion. Comparison of typing results obtained with the present procedure and with routine starch gel electrophoresis in more than 5000 serum samples gave conflicting results in 6 samples. The sensitivity of the described one-dimensional subtyping method was slightly better than that of starch gel electrophoresis. In 4110 unrelated individuals, involved in cases of disputed paternity the Hp 2SS 0.038, Hp 2FF 0.004, and Hp 3 (Johnson) 0.0005. These allele frequencies give a theoretical paternity exclusion rate of 32.5%, which is in accordance with the observed rate in 2200 paternity cases with more than 600 non-fathers. It is concluded that the present procedure represents a definite improvement for Hp subtyping in practical paternity diagnostics. Preliminary results with retyping of weak Hp patterns using a staining technique involving the biotin/avidin complex indicate that the sensitivity of the one-dimensional subtyping method may be substantially increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simplified hemoglobin chain detection by capillary electrophoresis

Hemoglobin (Hb) chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb chains also after sample extraction. We describe a CE method for analysis of Hb chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the chains were denatured and separated from the heme during electrophoresis. The common variants of the beta-chains, such as beta(S), beta(C), and beta(E), are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the hemoglobinopathies.     

Enantioselective synthesis of bridged bicyclic ring systems

The type 2 intramolecular Diels-Alder (IMDA) reaction is a valuable method for synthesis of both carbocyclic and heterocyclic bridged bicyclo[5.3.1]undecane and bicyclo[4.3.1]decane ring systems. These structures are common to a number of biologically important natural products. Asymmetric variants of the type 2 IMDA reaction incorporating oxazolidinone chiral auxiliaries have been evaluated. This study has resulted in systems that deliver bridged bicyclic [5.3.1] and [4.3.1] ring systems in high diastereomeric (97-99% de) and enantiomeric purity.     

Interaction of the vitamin D-binding protein (group-specific component) and its ligand 25-hydroxy-vitamin D3: binding differences of the various genetic types disclosed by isoelectric focusing

The three common variants of the vitamin D binding protein, also known as group specific component (Gc), namely types 1S, 1F and 2, as well as some rare variants were studied by thin-layer polyacrylamide gel isoelectric focusing in a pH 4.5-5.4 carrier ampholyte generated pH gradient, additionally containing N-(2-acetamido)-2-aminoethanesulfonic acid (ACES). Prior to isoelectric focusing, whole serum or purified preparations of the vitamin D binding protein were incubated with 25-hydroxycholecalciferol at various ligand/protein ratios. Binding differences were found for the anodal and cathodal isoforms of Gc 1 variants and also for various allelic types. Isoforms with higher isoelectric points generally had a lower affinity for the ligand than the variants with lower isoelectric points.     

Optimal drug cocktail design: methods for targeting molecular ensembles and insights from theoretical model systems

Drug resistance is a significant obstacle in the effective treatment of diseases with rapidly mutating targets, such as AIDS, malaria, and certain forms of cancer. Such targets are remarkably efficient at exploring the space of functional mutants and at evolving to evade drug binding while still maintaining their biological role. To overcome this challenge, drug regimens must be active against potential target variants. Such a goal may be accomplished by one drug molecule that recognizes multiple variants or by a drug "cocktail"--a small collection of drug molecules that collectively binds all desired variants. Ideally, one wants the smallest cocktail possible due to the potential for increased toxicity with each additional drug. Therefore, the task of designing a regimen for multiple target variants can be framed as an optimization problem--find the smallest collection of molecules that together "covers" the relevant target variants. In this work, we formulate and apply this optimization framework to theoretical model target ensembles. These results are analyzed to develop an understanding of how the physical properties of a target ensemble relate to the properties of the optimal cocktail. We focus on electrostatic variation within target ensembles, as it is one important mechanism by which drug resistance is achieved. Using integer programming, we systematically designed optimal cocktails to cover model target ensembles. We found that certain drug molecules covered much larger regions of target space than others, a phenomenon explained by theory grounded in continuum electrostatics. Molecules within optimal cocktails were often dissimilar, such that each drug was responsible for binding variants with a certain electrostatic property in common. On average, the number of molecules in the optimal cocktails correlated with the number of variants, the differences in the variants' electrostatic properties at the binding interface, and the level of binding affinity required. We also treated cases in which a subset of target variants was to be avoided, modeling the common challenge of closely related host molecules that may be implicated in drug toxicity. Such decoys generally increased the size of the required cocktail and more often resulted in infeasible optimizations. Taken together, this work provides practical optimization methods for the design of drug cocktails and a theoretical, physics-based framework through which useful insights can be achieved.     

Application of capillary isoelectric focusing and peptide mass fingerprinting in carbohydrate-deficient transferrin detection

Carbohydrate-deficient transferrin (CDT) is a specific biomarker of alcohol abuse and is widely used in clinical diagnosis to detect and follow up excessive alcohol consumption. However, false %CDT results still exist in CDT detection, because of interference from genetic variants and the lack of standardization in CDT analysis. Therefore, it is still very important to find a method with high sensitivity and high accuracy for CDT detection. Here, we compared the detection sensitivity and accuracy of pI values based methods [isoelectric focusing on polyacrylamide gel electrophoresis (IEF-PAGE) and capillary isoelectric focusing (CIEF)] with hydrophobic characteristic based methods [reversed-phase high-performance liquid chromatography (RP-HPLC)] on CDT detection. Moreover, we investigated the potential of peptide mass fingerprinting (PMF), a method based on the mass spectrometry to identify human transferrin (HTf) variants including CDT isoforms and genetic variants, based on their specific peptide masses. Results indicated that PMF can identify HTf variants including CDT isoforms and genetic variants based on their specific peptides, and CIEF showed higher sensitivity detection of HTf variants than RP-HPLC and IEF-PAGE did. Accordingly, we suggest that PMF is suitable for identifying CDT with high accuracy, and CIEF has potential for detection of CDT and genetic variants with high sensitivity; moreover, they are both worth further investigation in clinical diagnosis.     

Separation of hemoglobin variants with similar charge by capillary isoelectric focusing: value of isoelectric point for identification of common and uncommon hemoglobin variants

Clinical assays for the primary evaluation of congenital hemoglobin (Hb) disorders must detect and identify a variety of Hb variants. We analyzed hemolysates containing Hb variants with similar charge to evaluate the diagnostic sensitivity and specificity of automated capillary isoelectric focusing (CIEF). Peak separation was observed for each variant in samples containing Hb S, D, and G. The calculated isoelectric points (pI) of these variants were significantly different such that each could be identified in a single run with pI as the sole criterion of identification. The pI of Hb C was significantly different from that of Hb E, C-Harlem, and O-Arab. Hb E, C-Harlem, and O-Arab had similar pI and were not readily differentiated. Hb Koln, M-Saskatoon, Aida, and S/Aida hybrid were readily separated from common Hb variants and detected by CIEF. We conclude that CIEF exhibits both diagnostic sensitivity and specificity, and that pI is an objective and specific criterion of Hb variant identification.     

Two new esterase D (ESD) variants revealed by isoelectric focusing in agarose gel

Using isoelectric focusing in thin-layer agarose gel (AGIF) with the narrow pH range of 4.5-5.4, a high resolution of esterase D (ESD) isozyme banding patterns has been achieved. Some variant phenotypes which could not be distinguished from common ESD types by conventional electrophoresis have shown different patterns after AGIF. The IEF method permitted the distinction of two further variants in the ESD system, tentatively named ESD Rehren and ESD Ravensburg. We recommend, therefore, that for the classification of ESD phenotypes a high resolution IEF technique should be used.     

Application of plasminogen polymorphism to forensic hemogenetics

Plasminogen polymorphism (PLG) has attained considerable importance in forensic hemogenetics. PLG comprises two common, codominant autosomal alleles, PLG*A and PLG*B, more than 18 variants, and the silent allele PLG*Q0. Isoelectric focusing followed by functional or immunochemical detection seems to be the optimal method for the determination of phenotypes. PLG*A is the most common allele in all populations, having its highest frequency in Mongoloids, Amerindians and Eskimos, the lowest in Caucasoids. The functionally inactive plasminogen M5 so far has been seen exclusively in Japanese individuals. Silent PLG alleles were only observed in the heterozygous state. No clear differences in functional activity or plasma level could be ascertained for any of the other allotypes. PLG polymorphism is now widely used for many haemogenetic investigations. From the allele distribution in European Caucasoids a single exclusion chance of 17.2% for non-fathers in paternity testing may be calculated. The major prerequisites of a new genetic marker in the parentage expertise, established Mendelian inheritance, favorable distribution of common alleles, low frequency of silent alleles, and simple reproducible typing technology, are fulfilled.     

Separation of normal and abnormal hemoglobin chains by reversed-phase high-performance liquid chromatography

A review is presented of the elution patterns on reversed-phase columns of the normal and abnormal globin chains of different hemoglobin types, including 16 beta-chain variants, 7 alpha-chain variants, 9 gamma-chain variants, and 4 variants with fusion or hybrid chains. Separations appear to be based primarily on differences in hydrophobicity. The method is ideally suited for the detection of abnormal globin chains, their quantitation and their isolation. Semi-quantitative data based on the calculation of the delta/non-alpha ratios allow the detection of beta-thalassemic conditions in situations where the quantitation of hemoglobin A2 by other procedures is impossible or complicated.     

Ped_Outlier software for automatic identification of within-family outliers

A high-throughput resequencing technology has brought family based studies back into genetic research focus. Within-family outliers (the individuals whose phenotype is very much unlike the phenotype of relatives) may carry rare variants of large effects and thus resequencing of these provides a highly powered strategy for rare variants detection. On the other hand, such outliers may complicate search for common variants of smaller effects, because they may obscure a real linkage signal. We have developed a program Ped_Outlier allowing automatic detection of within-family outliers in a sample of pedigrees of arbitrary structure and size. We tested our program by identification of within-family outliers for adult height and intracranial volume in large pedigree. Results of linkage analysis of these traits demonstrated that identification of within-family outliers is one of the important steps of pedigree analysis. The program Ped_outlier is freely available at http://mga.bionet.nsc.ru/soft/index.html.     

Exon Array Analysis of Alternative Splicing of Genes in SOD1G93A Transgenic Mice

Alternative splicing is a common strategy for creating functional diversities of proteins. While conventional identification of splice variants generally targets individual genes in amyotrophic lateral sclerosis, we present a novel exon-centric array that allows genome-wide identification of splice variants and concurrently provides analysis of gene expression. Compare 1 was asymptomatic SOD1G93A transgenic mice with nontransgenic littermates; compare 2 was symptomatic with asymptomatic transgenic mice. RT-PCR was performed to validate. Pathway and GO analysis were performed on abnormal genes. These findings could guide us to demonstrated the potential influence of mutant human CuZn-SOD1 and of splicing regulation in pathological processes.     

A Comparison Between Full-COLD PCR/HRM and PCR Sequencing for Detection of Mutations in Exon 9 of PIK3CA in Breast Cancer Patients

Applied Biochemistry and Biotechnology - One of the most common somatic mutations in breast cancer is found in PIK3CA with a prevalence rate of 18–45%. Different variants of this gene are...