Electrophoretic Variants Research Articles

Evaluation of the biological efficiency of Terminalia chebula fruit extract against neurochemical changes induced in brain of diabetic rats: an epigenetic study

Diabetes mellitus (DM) is a chronic and progressive metabolic disorder that can stimulate neuroinflammation and increase oxidative stress in the brain. Therefore, the present study was aimed to assess the efficacy of ethanolic Terminalia chebula extract against the neurochemical and histopathological changes induced in the brains of diabetic rats. The study clarified the reduction in oxidative stress induced in the brains of diabetic rats by the significant (P ≤ 0.05) increase in levels of the antioxidants with decreasing the peroxidation products via ethanolic T. chebula extract at both doses (400 and 600 mg/kg). Moreover, T. chebula extract improved the brain integrity by lowering levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), β-amyloid (Aβ) content, monocyte chemoattractant protein-1 (MCP-1) and acetylcholine esterase (ACHE) significantly (P ≤ 0.05) in a dose dependent manner compared to brain of diabetic rats. Severe nuclear pyknosis and degeneration were noticed in neurons of the cerebral cortex, hippocampus and striatum in brains of diabetic rats. The severity of these alterations decreased with T. chebula extract at a dose of 600 mg/kg compared to the other treated groups. The different electrophoretic protein and isoenzyme assays revealed that the lowest similarity index (SI%) values exist in the brains of diabetic rats compared to the control group. The quantity of the most native proteins and isoenzyme types increased significantly (P ≤ 0.05) in the brains of diabetic rats, and these electrophoretic variations were completely diminished by T. chebula extract. The study concluded that T. chebula extract ameliorated the biochemical, histopathological and electrophoretic abnormalities induced in the brains of diabetic rats when administered at a dose of 600 mg/kg.

Identification of the gene coding for seed cotyledon albumin SCA in the pea (Pisum L.) genome.

Albumins SCA and SAA are short, highly hydrophilic proteins accumulated in large quantities in the cotyledons and seed axes, respectively, of a dry pea (Pisum sativum L.) seed. SCA was earlier shown to have two allelic variants differing in mobility in polyacrylamide gel electrophoresis in acid medium. Using them, the corresponding gene SCA was mapped on Linkage Group V. This protein was used as a useful genetic and phylogeographical marker, which still required electrophoretic analysis of the protein while the DNA sequence of the corresponding SCA gene remained unknown. Based on the length, the positive charge under acidic conditions and the number of lysine residues of SCA and SAA albumins, estimated earlier electrophoretically, the data available in public databases were searched for candidates for the SCA gene among coding sequences residing in the region of the pea genome which, taking into account the synteny of the pea and Medicago truncatula genomes, corresponds to the map position of SCA. Then we sequenced them in a number of pea accessions. Concordance of the earlier electrophoretic data and sequence variation indicated the sequence Psat0s797g0160 of the reference pea genome to be the SCA gene. The sequence Psat0s797g0240 could encode a minor related albumin SA-a2, while a candidate gene for albumin SAA is still missing (as well as electrophoretic variation of both latter albumins). DNA amplif ication using original primers SCA1_3f and SCA1_3r from genomic DNA and restriction by endonuclease HindII made it possible to distinguish the SCA alleles coding for protein products with different charges without sequencing the gene. Thus, the gene encoding the highly hydrophilic albumin SCA accumulated in pea seeds, the alleles of which are useful for classif ication of pea wild relatives, has now been identif ied in the pea genome and a convenient CAPS marker has been developed on its basis.

МОЛЕКУЛЯРНО-ГЕНЕТИЧЕСКИЙ АНАЛИЗ РАЗМНОЖЕННЫХ IN VITRO КЛОНОВ POPULUS ALBA L. И POPULUS TREMULA L. С ИСПОЛЬЗОВАНИЕМ МИКРОСАТЕЛЛИТНЫХ МАРКЕРОВ

Use of planting material of forest trees with improved hereditary characteristics is one of the ways to increase the productivity and biological stability of forest stands. It requires taking measures to develop and improve selection base using modern approaches and methods of genetics and biotechnology. A molecular genetics assessment of clone plants of aspen (Populus tremula L.) and white poplar (Populus alba L.) from a long-term in vitro collection (up to 24 years), planted in a greenhouse and field conditions (nursery), was carried out. SSR loci of the PTR series (PTR5, PTR7, PTR8, PTR12, PTR14) were used as DNA markers. Evaluation of clones' ploidy was carried out on the basis of the diagnosis of "loss of heterozygosity" (LOH) effect. Analysis of 5 microsatellite loci of the specimens showed their high intraclonal genotypic stability and homogeneity in vitro and ex vitro. For the first time, data on the results of a comparative determination of ploidy using karyological and microsatellite analysis were presented. Based on the results of the SSR analysis, it can be concluded that the structure of molecular markers is stable among the samples of one clone that are in long-term cultivation. The ratio of the representation (dose) of electrophoretic variants of PCR products serves as an indirect sign of determining ploidy, but for its reliable assessment it is necessary to study the number of loci that are three times larger than the main set of chromosomes. The specimen also requires information on the amplification coefficient of the markers under study. Thus, it is necessary to use both chromosomal and microsatellite analyzes for reliable assessment of intraclonal homogeneity of various specimens, the development of understanding of clone genotypes formation and determination of their ploidy

Comparative studies on seed protein characteristics in eight lines of two Gossypium species

BackgroundIn order to achieve the targets aiming at the improvement of protein quality, knowledge regarding seed protein fractions and polypeptides constituting them in different crops is essential. Besides having high nutritional value as animal feed and human food, the protein isolates from cottonseed meal have also been proven promising as industrial raw materials for a number of applications. As far as Indian work on the characterization of cotton seed proteins is concerned, relatively meagre reports are available. Keeping in mind the importance of cotton seed proteins, lines belonging to Gossypium arboreum L. (Indian cotton) and G. hirsutum L. (American cotton) which are grown in all the major cotton growing states in India were selected for analysing their seed protein characteristics.ResultsWhereas G. arboreum (A-genome) lines revealed a lower range of seed protein content i.e. 19.5~24.3%, an upper range (21.8~29.5%) could be observed in lines of G. hirsutum (AD-genome). Globulins represented dominating fraction in both species followed by albumins, glutelins and prolamins. A significant positive correlation between albumins/globulins and seed protein content in G. arboreum /G. hirsutum, respectively, was observed. Intraspecific electrophoretic variation in seed protein extracts was observed in the region of molecular weight 22 kDa - 27 kDa in lines of both the species; however some lines with A-genome showed similarity in banding pattern with AD-genome. Four polypeptides with disulphide-linkages were also reported for the first time. Albumins were observed to reveal more variations in their electrophoretic pattern between the lines of two species followed by globulins.ConclusionOn the basis of present and previous studies, screening the lines with low or high protein content will lead the selection of lines with superior polypeptide fraction important for nutritional and industrial purposes. On comparing the composition and behaviour of four 2-S linked polypeptides with other plant groups, these were suggested to be legumin-like in nature. The similarity in banding patterns between the lines of A-genome and AD-genome species marked towards the close evolutionary relationship between these two. Albumin fractions on the basis of our results could be taken for cultivar differentiation in cotton crop.

Изменчивость и специфичность сибирских видов Elymus transbaicalensis, E. komarovii, E. sajanensis, E. kronokensis (Poaceae) и некоторых морфологически отклоняющихся форм по гистону H1

As the result of introgression and genetic recombination between the species a number of morphologically deviating forms arises in many mixed populations of the genus Elymus L. The study of electrophoretic variability in histone H1 among Siberian species Elymuskomarovii, E. transbaicalensis, E. kronokensisand E. sajanensis  to reveal microevolutionary levels of differentiation was carried out. It has been shown that the variation in E. komarovii was higher than in E. transbaicalensis in the Republic of Altai, and some biotypes E. komarovii were identical on this characteristic to the majority of biotypes of E. transbaicalensis. Morphologically deviating forms of E. transbaicalensiswith atypical for this species glabrate lemmas and approximating on this character to E. kronokensis are just one of the morphotypes of E. transbaicalensis. The biotypes of E. komarovii from the classic habitat in the Eastern Sayan were identical by histone H1 to some biotypes from the Altai Mountains, where the center of diversity for E. komarovii is situated presumably. E. sajanensis is the most variable in a composition of histone H1 subtypes in Siberia. This may be due to the fact that the species is formed by a number of isolated Alpine populations, some of which are derived from local biotypes of E. transbaicalensis. Some Siberian biotypes corresponding to E. sajanensis and E. kronokensis from close habitats had identical profiles of histone H1. This indirectly suggests that Siberian biotypes which are similar morphologically to Far Eastern biotypes of E. kronokensis are distant phylogenetically from the last, and probably need to be relegated to independent taxonomic rank.

A highly efficient electrophoretic method for discrimination between two Neoscytalidium species using a specific fungal internal transcribed spacer (ITS) fragment.

Neoscytalidium (or N.) dimidiatum and N. novaehollandiae are two aggressive plant pathogenic species that affect several agricultural crops. Early detection and identification of these fungi are of critical importance to bring about the effective minimization to the threat they pose to the infected plants. Herein, two species of Neoscytalidium were rapidly discriminated by utilizing the rRNA internal transcribed (ITS4-5.8S-ITS5) PCR primers. A total of 100 isolates of Neoscytalidium species, which were isolated from Iraqi canker-infected fig trees, were included in this study. Two discrete electrophoretic PCR bands were observed in Neoscytalidium isolates-A-variants were about 546bp, while B-variants were about 993bp in length. The comprehensive phylogenetic analysis of both DNA variants revealed that A-variants resided between N. novaehollandiae and N. hyalinum, while B-variants were closely related to N. dimidiatum. Furthermore, the highly specific re-constructed tree of both electrophoretic variants demonstrated that B-variants share a high similarity with N. novaehollandiae. Additionally, the secondary structures for both variants were predicted computationally to reveal the structural patterns that each variant follows. In conclusion, a small rRNA locus comprising 22 nucleotides that differs in the two variants is potentially responsible for this species-specific classification. The main divergence in the amplified loci led to the classification of these fungal variants into two main species, namely N. dimidiatum and N. novaehollandiae, demonstrating that the amplification by ITS4-ITS5 rRNA fragment is a beneficial strategy that can be employed for the assessment of Neoscytalidium diversity in the natural ecosystems.

Electrophoretic variation in seed proteins and interrelationships of species in the genus Oryza

Comparative studies of two cultivated and sixteen wild species of the genus Oryza were carried out using one- and two-dimensional gel electrophoresis for variation in their seed proteins for interrelationships of these species. A number of polypeptides in the range of molecular weight 13–110 kDa were seen to vary. Under reducing conditions, polypeptides spread over the regions of mol. wt. 33–40.5, 25–27 and 19–21.5 kDa exhibited maximum variation in their patterns. Two-dimensional gel electrophoresis revealed the occurrence of disulphide-linked glutelin polypeptide pairs of mol. wt. 60, 58, 52 and 25 kDa breaking into a large and a small subunit each in the range of mol. wt. 18–40.5 and 16–25 kDa respectively in Oryza sativa. The number of such polypeptide pairs varied from 2 to 6 in different species and also in O. sativa showed variation in mol. wts. of their constituent subunits. The UPGMA dendrogram revealed that most of the Oryza species occurred in different clusters and subclusters and thus did not share very close relationships. The undisputed and closest relationship observed was that of cultivated rice O. sativa with the O. rufipogon followed by that with O. nivara. The African cultivated O. glaberrima occurring on the nearest branch of the same subcluster, thereby, supporting the phylogenetic of these species suggested in earlier studies. Eight diploid species and seven tetraploid species were included in one part of the dendrogram while the remaining two species with AA genome i.e. O. glumaepatula and O. meridionalis and one with FF i.e. O. brachyantha stood separately from these as scattered in the group of seven tetraploid species with BBCC, CCDD and HHJJ genomes. The tetraploids O. alta, O. latifolia and O. grandiglumis with CCDD genomes which occurred on the farthest part were distantly related with O. sativa. The cyanogen bromide peptide maps and two dimensional gel electrophoresis also supported the closest relationship between O. sativa and O. rufipogon.

Identification of Post-translational Modifications of Plasmodium yoeliiGlyceraldehyde-3-phosphate dehydrogenase by Mass Spectrometry

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme from Plasmodium yoelii exhibits diverse sub-cellular distribution with a multitude of electrophoretic variants. Recent studies have implicated this protein in multiple non-glycolytic functions such as vesicular transport and facilitating host cell invasion by merozoites and sporozoites . In the absence of any organelle specific signal sequence in GAPDH, PTMs that could enlarge molecular species of a protein with distinct functions are likely to form the structural basis for its diverse localization and functions. Such considerations have enthused our interest in chemically characterizing all species of this protein in the parasite. Here, an attempt was made for a comprehensive determination of the PTMs in PyGAPDH in blood stage parasites using LC-ESI-MS/MS of peptides obtained from in-gel digestion of appropriate protein bands. Twelve residues were identified that underwent modifications. These changes consisted of four phosphorylations ( pS 144 , pT 146 , pS 204 and pS 213 ), five ubiquitinations ( u K73 , uK 218 , uK 222 , uK 230 and uK 336 ) , three acetylations (ac K 163 , ac K 230 , acK 301 ), two methylations (m K 218 and mK 230 ), one dimethylation (m2K 230 ) and one nitrosylation (n C 157 ). It is hoped that such comprehensive analysis of PTMs in a single protein will pave the way to correlate structure with specific functions and provide the molecular basis for diverse intracellular distribution. Keywords : Glyceraldehyde-3-phosphatedehydrogenase; Mass Spectrometry; Moonlighting Functions; Plasmodium; Post-Translational Modifications; Protein Species

Hubby and Lewontin on Protein Variation in Natural Populations: When Molecular Genetics Came to the Rescue of Population Genetics.

The 1966 GENETICS papers by John Hubby and Richard Lewontin were a landmark in the study of genome-wide levels of variability. They used the technique of gel electrophoresis of enzymes and proteins to study variation in natural populations of Drosophila pseudoobscura, at a set of loci that had been chosen purely for technical convenience, without prior knowledge of their levels of variability. Together with the independent study of human populations by Harry Harris, this seminal study provided the first relatively unbiased picture of the extent of genetic variability in protein sequences within populations, revealing that many genes had surprisingly high levels of diversity. These papers stimulated a large research program that found similarly high electrophoretic variability in many different species and led to statistical tools for interpreting the data in terms of population genetics processes such as genetic drift, balancing and purifying selection, and the effects of selection on linked variants. The current use of whole-genome sequences in studies of variation is the direct descendant of this pioneering work.

Chicken Ovotransferrin Variants OTFB and OTFC Harboring Substitution of GAT (Asp) to AAT (Asn) in the Codon 500 and their Antimicrobial Activity.

Chicken egg white ovotransferrin (OTF) has been reported to exist in three electrophoretic variants (OTFA, OTFB and OTFC). In this report, we identified a causal polymorphism between the OTFB and OTFC variants in Japanese and Taiwanese native chickens and compared the antibacterial activity between these two variants. The cDNA sequence analyses from Satsumadori oviducts revealed that three non-synonymous SNPs T1809G (Ser52Ala), A2258G (Ile96Val) and G7823A (Asp500Asn) corresponded to the OTF electrophoretic phenotypes. Of the three SNPs, the G7823A mutation perfectly corresponded to the electrophoretic phenotypes OTFB (G/G, Asp500Asp), OTFB/C (G/A, Asp500Asn) and OTFC (A/A, Asn500Asn) in three chicken populations. The variants OTFB and OTFC exhibited similar antibacterial potency against Gram-positive and Gram-negative bacteria. This study provides, for the first time, molecular information on polymorphism of OTFB and OTFC variants of chicken ovotransferrin and its effect on the antimicrobial activity of the respective variants.

Typing Candida Species Using Microsatellite Length Polymorphism and Multilocus Sequence Typing.

To gain more insight into the epidemiological relationships between isolates of Candida spp. obtained from various origins, several molecular typing techniques have been developed. Two methods have emerged in the 2000s as soon as enough knowledge of the Candida spp. genomes was available to choose adequate loci and primers, namely microsatellite length polymorphism (MLP) and multilocus sequence typing (MLST). To contrast with previous PCR-based methods, specific amplifications with stringent conditions easily reproducible are the basis of MLP and MLST. MLST relies on Sanger sequencing to detect single-nucleotide polymorphisms within housekeeping genes. MLP needs a first in silico step to select tandemly repeated stretches of two to five nucleotides. One of the two primers used to amplify a microsatellite locus is labeled and fragment sizing is automatically performed using high-resolution electrophoresis platforms. MLST provides results easily comparable between laboratories and active MLST schemes are publicly available for the main Candida species. For comparative studies, MLP needs standards to compensate for the electrophoretic variations depending on the platforms used. Both methods can help us gain insight into the genetic relatedness of fungal isolates, both with advantages and drawbacks, and the choice of one method rather than the other depends on the task in question.

Isozyme analysis based genetic fidelity assessment of micropropagated banana plants

Isozyme studies of micropropagated and mother plants of banana cvs. Matti, Ney Poovan, Kechulepa, Dwarf Cavendish, Malbhog, Champa, B.B. Battisa and FHIA-1 were done to test their genetic fidelity. The banding patterns as revealed by electrophoretic variations were evaluated with respect to isozymes of acid phosphatase, catalase, esterase and peroxidase as markers. The genetic fidelity of micropropagated plants and the relationship of the different cultivars were determined by dendrogram using numerical taxonomy and multivariate analysis system(NTSYS). A clustered dendrogram was prepared by unweighted pair group method using averages (UPGAMA) method. At 87% similarity, the micropropagated and mother plants were clustered in four groups reflecting their genomic constitution. Cvs. Matti (AA) and Dwarf Cavendish (AAA) with similar ‘A’ genome were categorized in Cluster I. Cluster II comprised of cvs. Ney Poovan (AB), B.B. Battisa (ABB) and FHIA-1(AAAB) with genomic constitution of both ‘A’ and ‘B’ type. Cvs. Champa (AAB) and Malbhog (AAB) with similar genome were grouped inCluster III. Cluster IV contained the cv. Kechulepa (BB) having only ‘B’ genome. However, there was no somaclonal variation among the micropropagated plants and they showed 100% genetic similarity. Thus, the isozyme studies could be a reliable marker for testing the genetic fidelity of micropropagated plants and for evaluating the diversity among the banana germplasm.

Genetic variation and racial admixture in the Miskito of the southern Mosquito Shore, Nicaragua

Se estudió la variación electroforétíca de II loei que codifican para enzimas eritrocítícas, globinas y proteínas séricas en una muestra de 59 indígenas misquitos originarios de la región sur de la Mosquitia de Nicaragua. Siete loci, ALB, 03-, f-y o-globinas, LDHA, LDHB y TPI fueron monomórficos, mientras que API, CP, HP y TF fueron polimórficos. Esto se traduce en una proporción de,..loci polimórficos (P) de 0.364 y una heterocigosis media (H) de 0.077. Ambos valores se encuentran dentro del ámbito de variación mostrado por diez grupos indígenas de estirpe chibchense de Costa Rica y Panamá evaluados para los mismos loei [(P) = 0.364-182, (H) = 0.104-0.052). El análisis permitió una estimación de los porcentages mínimo (m) = 0.0), medio (mm = 7.34) y máximo (m, = 21.9) de mezcla racial con negros. A manera de comparación, esta mezcla se calculó también a partir de los resultados de A. Matson y su grupo, quienes estudiaron, en 1960, la variación genética, usando primordialmente grupos sanguíneos, en una muestra de misquitos de la región fronteriza entre Nicaragua y Honduras. Los resultados (m) = 6.05), (mm = I 1.0) Y (m, = 18.1) no fueron diferentes estadístícrunente, para los valores medios, bajo la suposición de que los alelos no-indígenas siguen la distribución de Poisson (P=0.42). Además, la disponibilidad de una fecha en la que se supone se inició la mezcla de los mísquitos con negros, en 1641, permitió el cálculo de la tasa de mezcla por generación. Dada una duración generacional de 27 años, su valor máximo se encuentra entre el 1.68 y el 1.91 %. Estos resultados indican que el acervo genético de los mísquitos aun conserva características propias de los grupos amerindios.

Alterations in Protein and Isozymes Profiles during Accelerated Ageing in Soybean (Glycine Max(L.) Merrill)

This study was done to observe alterations in protein and isozyme profiles for soybean (Glycine max (L.) Merrill), during accelerated aeging. During the aeging period some physiological (percent germination, seedling vigour index and germination index) and biochemical (electrical conductivity, malonaldehyde content, total soluble sugars, total soluble proteins, peroxidase and dehydrogenase activity) changes were also observed in relation to seed vigour and viability in fresh and aged soybean seeds. Results showed an overall decrease in germination, vigour, soluble sugars and soluble protein content with increase in ageing duration. Increased activity of lipid peroxidation and electrical conductivity coupled with significant reduction in activities of peroxidase and dehydrogenase were noticed in accelerated aged seeds. Alterations in protein and isozymes profiles in aged seeds compared to fresh seeds indicated that the loss in seeds quality might be due to the adverse effect of ageing on proteins. The electrophoretic variations in protein and isozymes noticed in this study could be used as a criterion in assessing the quality of soybean.

Novel silent albumin variant (191Ala → Thr) detected by TOF MS of whole plasma

BackgroundSerum protein electrophoresis occasionally reveals multiple albumin bands referred to as bis- or alloalbuminaemia, and whilst the condition can be inherited it may also be acquired. MethodsWe present a new high resolution approach to the investigation of qualitative changes in albumin structure. The on-line reverse phase time-of-flight mass spectrometry procedure (TOF MS) elaborated here requires <0.2μl of plasma and takes ~10min to perform. Two plasma samples with classical bisalbuminaemia were used to verify the efficacy of the procedure for detecting genetic variants. When a novel mutation was detected, mass mapping of native unreduced albumin was used to pinpoint its location and inform targeted DNA sequencing of the albumin gene. ResultsNormal serum albumin showed its expected major isoform at 66,439Da and the electrophoretic variants showed co-equal expression of additional components at −14 and +744Da respectively. Surprisingly, one of the supposed controls showed paired albumin peaks at 66,439 and 66,469Da and this 30Da increase in mass was localised to between Arg114 and Arg197 and confirmed as being due to a novel 191Ala→Thr (+30Da) substitution through DNA sequencing. ConclusionsThe electrospray TOF MS approach developed here provides a rapid, sensitive and extremely precise method of revealing minute changes in albumin primary structure.

Reference intervals, longitudinal analyses, and index of individuality of commonly measured laboratory variables in captive bald eagles (Haliaeetus leucocephalus).

The objectives of this study were to determine reference intervals, perform longitudinal analyses, and determine the index of individuality (IoI) of 8 hematologic, and 13 biochemical and electrophoretic variables for a group of captive bald eagles (Haliaeetus leucocephalus). Reference intervals were determined from blood samples collected during annual wellness examinations for 41 eagles (23 male and 18 female) with ages ranging between 6 and 43 years (18.7 +/- 7.4, mean +/- SD) at the time of sample collection. Longitudinal analyses and IoI were determined for measured hematologic, biochemical, and protein electrophoretic variables, both individually and as a group, for a subset of 16 eagles (10 male and 6 female) during a 12-year period. This smaller group of eagles ranged in age between 2 and 20 years at the start of the study period, and between 14 and 32 years (21.9 +/- 5.0, mean +/- SD) at the end of the study period. Significant increases with age within the group of 16 eagles were observed only for red blood cells, percent heterophils, total protein, and beta-globulin protein fraction, while albumin:globulin decreased significantly with age. A low IoI (> or = 1.4) was determined for all hematologic and biochemical variables except gamma globulins, which had high IoI (< or = 0.6) for 3 individuals within the subset of 16.

Domestication and N.I. Vavilov’s law of homologous series in hereditary variability

Domestication traits in animal and plant species distinguishing them from closely related wild species are discussed. Data are given on what such traits are found not only at the phenotypic level but also in polymorphism of electrophoretic variants of protein groups with a different biochemical function, DNA fragments flanked by inverted short repeats. The assumption that retrovirus infections can participate in the formation of domestication traits is substantiated.

Genetic Polymorphism of the Complement Receptor-1 (CR1) Gene Correlates with the Clinical Response to Eculizumab of Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

AbstractAbstract 983Eculizumab, a monoclonal antibody that binds to the complement component C5, controls intravascular hemolysis in PNH patients and causes significant clinical improvement. However, the extent to which patients respond is variable in terms of hemoglobin levels, and some patients remain red blood cell (RBC) transfusion-dependent in spite of regular eculizumab treatment. We have hypothesized that genetic polymorphisms of complement-related genes might contribute to determining this variability of response to eculizumab. In order to test this hypothesis, we have analyzed polymorphic alleles of the Complement Receptor 1 (CR1) gene and of the Complement Component 3 (C3) gene. We have genotyped by standard methods (1) the HindIII restriction length polymorphism (RFLP) in the CR1 intron 27 (rs11118133 A>T) whose co-dominant alleles, H (common) and L (rare), are associated with high and low levels of CR1 expression on RBC, respectively; (2) the single nucleotide polymorphism rs2230199 C>G of the C3 gene, responsible for the allelic electrophoretic variants, S (slow, common allele) and F (fast, rare allele), that influence the complement alternative pathway activity.We have studied 72 patients with hemolytic PNH treated with eculizumab for at least 6 months. Patients with clear evidences of bone marrow failure (platelets ≤ 50,000/μL, neutrophils ≤1000/μL) have not been included in this study because this condition may affect the clinical response independently of the control of hemolysis. In order for our criterion for response to be stringent and objective we used blood transfusion requirement: patients who received no RBC transfusion have been defined as Responders; patients who received any RBC transfusion have been defined as Poor Responders.In this series of 72 hemolytic PNH patients on eculizumab the allelic frequency of the CR1 rare allele L was 0.31, and that of the C3 rare allele F was 0.19. We found no correlation between response and the C3 genotype (Chi square for trend: p=0.939). On the other hand the proportion of Poor Responders is significantly higher in patients who were heterozygotes (H/L) for the CR1 polymorphism, and even higher in those who were homozygous for the rare allele (L/L) of the CR1 HindIII RFLP polymorphism (Chi square for trend: p=0.016): see figure 1. [Display omitted] We do not know yet the precise mechanism whereby the CR1 genotype affects the response to eculizumab. CR1 enhances the decay of C3 and C5 convertase by binding to C3b and C4b and it plays a role in the clearance of immune complex and in phagocytosis. Thus, the variable levels of CR1 expression on RBC membrane associated with the HindIII RFLP polymorphism may affect the regulation of complement alternative pathway. Moreover, it is well established that in almost all PNH patients on eculizumab a significant fraction of GPI-negative RBCs are opsonized by C3 and therefore may undergo extravascular hemolysis. Thus, a possible explanation of our findings is that the lower levels of CR1 expression on RBCs associated with H/L and L/L genotypes are responsible for a more intense extravascular hemolysis in PNH patients on eculizumab.In conclusion, our data show that the CR1 HindIII polymorphism may predict the poor response to eculizumab in PNH patients and provide additional insight in the mechanisms responsible for the variable clinical effectiveness of complement blockade. Disclosures:Risitano:Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding. Peffault de Latour:Alexion: Consultancy. Iori:Alexion: invited speaker Other. Socie:Alexion: Consultancy. Luzzatto:Alexion: Consultancy. Notaro:Alexion: Consultancy.

Determination of anionic impurities in hexafluorosilicic acid by capillary zone electrophoresis

This paper reports an alternative analytical methodology for the determination of chloride and sulphate impurities in hexafluorosilicic acid, FSA, using capillary zone electrophoresis. The influence of different electrophoretic and matrix variables on electrophoretic response was studied. Samples were injected hydrodynamically onto a fused silica capillary of 63.5 cm × 75 μm id, with an effective length of 55 cm. The capillary temperature was kept constant at 25 ± 1 °C throughout the experiments. A voltage of −25 kV was applied (in anionic mode). The electrophoretic zones were detected with a fixed-wavelength UV detector at 254 nm and a diode array detector at 254 and 370 nm. The final background electrolyte consisted of 20 mM sodium chromate at pH 8.0 containing 6% MeOH and 1.5 μM tetradecyltrimethylammonium hydroxide. In these electrophoretic conditions, the separation of chloride and sulphate anions is achieved in less than ten minutes. The introduction of a water gap before the sample injection increases considerably the capillary life. The electrophoretic method developed allows the determination of chloride and sulphate at mg L−1 concentration levels in industrial production FSA samples.

Chemometrics optimization of six antihistamines separations by capillary electrophoresis with electrochemiluminescence detection

This work expanded the knowledge of the use of chemometric experimental design in optimizing of six antihistamines separations by capillary electrophoresis with electrochemiluminescence detection. Specially, central composite design was employed for optimizing the three critical electrophoretic variables (Tris–H3PO4 buffer concentration, buffer pH value and separation voltage) using the chromatography resolution statistic function (CRS function) as the response variable. The optimum conditions were established from empirical model: 24.2mM Tris–H3PO4 buffer (pH 2.7) with separation voltage of 15.9kV. Applying theses conditions, the six antihistamines (carbinoxamine, chlorpheniramine, cyproheptadine, doxylamine, diphenhydramine and ephedrine) could be simultaneous separated in less than 22min. Our results indicate that the chemometrics optimization method can greatly simplify the optimization procedure for multi-component analysis. The proposed method was also validated for linearity, repeatability and sensitivity, and was successfully applied to determine these antihistamine drugs in urine.