Polyacrylamide Gel Isoelectric Focusing Research Articles

Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis

NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [12C6]/[13C6] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS). A superior amount of low sialylated glycans and less branched structures were detected in NeuroEPO plus compare to other commercial rhEPOs. At the intact glycoprotein level, NeuroEPO plus glycoforms were separated by capillary zone electrophoresis with ultraviolet detection (CE-UV), optimizing the composition and pH of the separation electrolyte. Moreover, an isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) method was also optimized for the simultaneous analysis of this basic rhEPO and conventional acidic rhEPO products. The proposed glycomic and intact glycoprotein methods provide a robust and reliable analytical platform for NeuroEPO plus characterization and for its future implementation as biopharmaceutical in neurodegenerative diseases.

Transthyretin chemical chaperoning by flavonoids: Structure–activity insights towards the design of potent amyloidosis inhibitors

BackgroundMany polyphenols have been proposed as broad-spectrum inhibitors of amyloid formation. To investigate structure–activity relationships relevant for the interaction of flavonoids with transthyretin (TTR), the protein associated with familial amyloid polyneuropathy (FAP), we compared the effects of major tea catechins and their larger polymers theaflavins, side-by-side, on TTR amyloid formation process. MethodsInteraction of flavonoids with TTR and effect on TTR stability were assessed through binding assays and isoelectric focusing in polyacrylamide gel. TTR aggregation was studied, in vitro, by dynamic light scattering (DLS), transmission electron microscopy (TEM) and in cell culture, through cytotoxicity assays. ResultsTested flavonoids bound to TTR and stabilized the TTR tetramer, with different potencies. The flavonoids also inhibited in vitro formation of TTR small oligomeric species and in cell culture inhibited pathways involving caspase-3 activation and ER stress that are induced by TTR oligomers. In all assays performed the galloyl esters presented higher potency to inhibit aggregation than the non-gallated flavonoids tested. ConclusionsOur results highlight the presence of gallate ester moiety as key structural feature of flavonoids in chemical chaperoning of TTR aggregation. Upon binding to the native tetramer, gallated flavonoids redirect the TTR amyloidogenic pathway into unstructured nontoxic aggregation assemblies more efficiently than their non-gallated forms. General significanceOur findings suggest that galloyl moieties greatly enhance flavonoid anti-amyloid chaperone activity and this should be taken into consideration in therapeutic candidate drug discovery.

AB0052 Human Complement Components C4B and C4A Subisotypes Patterns: Resistant Visible Biomarkers of the System Infectious and Rheumatologic Diseases

BackgroundIsotypes C4A and C4B of human complement component C4 are innate immunity factors. We found that gel electrophoresis of patient sera in the new diagnostic area of C4 complexes/aggregates (pI...

ISOLATION OF A TRYPSIN–CHYMOTRYPSIN INHIBITOR AND ITS FUNCTIONAL PROPERTIES

A novel trypsin inhibitor with thermal and pH stability, designated Merrtine, was isolated from Glycine max L. merr. The procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, and affinity chromatography on Affi-gel blue gel. The 20 N-terminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high homology with the sequence of Bowman–Birk type trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing to be 20.0 kD and 5.8, respectively. Trypsin could be completely inhibited by Merrtine when the molar ratio was 8.1. The inhibitory activity of Merrtine was unaffected after exposure to temperatures up to 85°C, as well as within the pH range 2–12. Besides inhibiting trypsin–chymotrypsin, the inhibitor demonstrated additional antifungal activity against the species of Alternaria alternate, Fusarium oxysporum, Pythium aphanidermatum, Physalospora piricola, Botrytis cinerea, and Fusarium solani. We herein report not only the trypsin inhibitor's extraction and isolation for the first time, but also its physiochemical and antifungal properties.

Purification and characterization of a novel antifungal protein with antiproliferation and anti-HIV-1 reverse transcriptase activities from <italic>Peganum harmala</italic> seeds

A novel antifungal protein, designated as PHP, was isolated from the seeds of Peganum harmala, by cationic exchange chromatography on Resource S column and gel filtration on Sephadex 75 10/300 GL column. PHP was found to form a homodimer of about 16 kDa. Isoelectric focusing polyacrylamide gel electrophoresis analysis showed that the isoelectric point of PHP was ∼8.4. The N-terminal 20-amino acid sequence of PHP, ITCPQVTQSLAPCVPYLISG, resembles the non-specific lipid transfer proteins in certain plants. PHP exhibited lipid-binding activity. Furthermore, PHP exerted antifungal activity against Alternaria alternate, Penicillium degitatum, Rhizopus stuolonifer, and Magnaporthe grisea, and its antifungal activity was stable in the temperature range 4-60°C, and in the pH range 4-10. It inhibited the mycelial growth in A. alternate, P. degitatum, R. stuolonifer, and M. grisea with 50% inhibitory concentration (IC(50)) of 1.5, 37.5, 8.44, and 12.19 μM, respectively. PHP was also able to inhibit the proliferation of esophagus carcinoma (Eca-109), cervical carcinoma (HeLa), gastric carcinoma (MGC-7), and melanoma (B16) cells with IC(50) of 0.7, 2.74, 3.13, and 1.47 μM, respectively. Moreover, PHP significantly inhibited HIV-1 reverse transcriptase (RT) with an IC(50) of 1.26 μM. It did not have hemagglutinating and antibacterial activities. In conclusion, a novel antifungal protein with antiproliferation and anti-HIV-1 RT activities was obtained from P. harmala seeds.

Doping control of biosimilar epoetin kappa and other recombinant erythropoietins after intravenous application

Since the expiration of patent protection, a number of new recombinant erythropoietin (rEPO) biosimilars have appeared on the worldwide market. In 2010, epoetin kappa, which is biosimilar to epoetin alfa, was clinically approved in Japan. Currently, both isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) are approved by the World Anti-Doping Agency (WADA) for detection of rEPO doping. Because it was unclear whether epoetin kappa could be detected by WADA-accredited detection methods, intravenous administration studies of epoetin kappa, epoetin alfa, and epoetin beta were performed to test the applicability of these methods. The isoform bands of epoetin kappa expanded more widely towards the basic area and the profile appeared to be composed of at least eight bands, which were clearly different from those of other epoetins. The results showed that epoetin kappa also contains isoforms of higher molecular masses than those of originator epoetins on SDS-PAGE; the mass distribution was confirmed by electrospray ionization time-of-flight mass spectrometry. We clearly detected epoetin kappa after its administration up to 10 h by IEF-PAGE and 24 h by SDS-PAGE; the detection window of the SDS-PAGE is longer than that of the IEF-PAGE. SDS-PAGE compensates for the disadvantages of IEF-PAGE in detecting urinary epoetin kappa. We also concluded that athletes abusing rEPO might move to intravenous injections for shorter clearance times instead of subcutaneous injections. In conclusion, out-of-competition tests need to be applied more frequently to improve the effectiveness of the rEPO detection.

Practicing IEF‐PAGE of EPO: The impact of detergents and sample application methods on analytical performance in doping control

Electrophoretic techniques, namely isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are key techniques used for confirming the doping-related abuse of recombinant erythropoietins and analogs. IEF-PAGE is performed on horizontal slab-gels with samples applied to the surface of the gel. Different sample application techniques can be employed, but application pieces and applicator strips are most frequently used. However, defective application pieces cause lane streaking during IEF of erythropoietin (EPO), which is especially pronounced in the acidic region of the gel. The effect is due to an incompatibility of the substance used for enhancing the wettability of the cellulose-based commercial product and is batch-dependent. A detailed mass spectrometric study was performed, which revealed that defective sample application pieces (bought between 2007 and 2010) contained a complex mixture of alcohol ethoxylates, alcohol ethoxysulfates, and alkyl sulfates (e.g. SDS). Anionic detergents, like the sulfates contained in these application pieces, are in general incompatible with IEF. Alternative application techniques proved partly useful. While homemade pieces made of blotting paper are a good alternative, the usage of applicator strips or shims is hampered by the risk of leaking wells, which lead to laterally diffused samples. Casting IEF-gels with wells appears to be the best solution, since sustained release of retained proteins from the application pieces can be avoided. Edge effects do not occur if wells are correctly filled with the samples. The evaluation of EPO-profiles with defects is prohibited by the technical document on EPO-analytics (TD2009EPO) of the World Anti-Doping Agency (WADA).

Changes in the proteomic and metabolic profiles of Beta vulgaris root tips in response to iron deficiency and resupply.

BackgroundPlants grown under iron deficiency show different morphological, biochemical and physiological changes. These changes include, among others, the elicitation of different strategies to improve the acquisition of Fe from the rhizosphere, the adjustment of Fe homeostasis processes and a reorganization of carbohydrate metabolism. The application of modern techniques that allow the simultaneous and untargeted analysis of multiple proteins and metabolites can provide insight into multiple processes taking place in plants under Fe deficiency. The objective of this study was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply.ResultsRoot tip extract proteome maps were obtained by 2-D isoelectric focusing polyacrylamide gel electrophoresis, and approximately 140 spots were detected. Iron deficiency resulted in changes in the relative amounts of 61 polypeptides, and 22 of them were identified by mass spectrometry (MS). Metabolites in root tip extracts were analyzed by gas chromatography-MS, and more than 300 metabolites were resolved. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. Iron deficiency induced increases in the relative amounts of proteins and metabolites associated to glycolysis, tri-carboxylic acid cycle and anaerobic respiration, confirming previous studies. Furthermore, a protein not present in Fe-sufficient roots, dimethyl-8-ribityllumazine (DMRL) synthase, was present in high amounts in root tips from Fe-deficient sugar beet plants and gene transcript levels were higher in Fe-deficient root tips. Also, a marked increase in the relative amounts of the raffinose family of oligosaccharides (RFOs) was observed in Fe-deficient plants, and a further increase in these compounds occurred upon short term Fe resupply.ConclusionsThe increases in DMRL synthase and in RFO sugars were the major changes induced by Fe deficiency and resupply in root tips of sugar beet plants. Flavin synthesis could be involved in Fe uptake, whereas RFO sugars could be involved in the alleviation of oxidative stress, C trafficking or cell signalling. Our data also confirm the increase in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways.

Expression, purification and characterization of the recombinant chimeric IgE Fc-fragment opossum–human–opossum (OSO), an active immunotherapeutic vaccine component

The active vaccine component recombinant chimeric IgE Fc-fragment opossum–human–opossum (OSO) has been expressed in CHO-K1 cells. It contains two identical polypeptide chains with 338 amino acid residues in each chain connected by two disulfide bridges. The cell lines were adapted to suspension culture in a serum-free medium. An expression level of 60 mg/L was obtained after 8 days in a shaking flask at a temperature of 31.5 °C. The OSO protein has been purified to homogeneity by a combination of three chromatographic steps. Virus inactivation and reduction by solvent detergent treatment and nano-filtration were included in the process. The residual host cell protein content was less than 50 ng/mg OSO as analyzed by ELISA. Purity was analyzed by SDS–PAGE under reducing and non-reducing conditions and was estimated by densitometry to be above 99.0%. The dimer content was less than 0.1% as estimated by analytical size exclusion chromatography. The molecular mass, as estimated by SDS–PAGE, is 90 kDa. A value of around 74 kDa was calculated from its amino acid composition. This indicates that the protein is heavily glycosylated containing around 18% carbohydrate. Isoelectric focusing in polyacrylamide gel disclosed a ladder type band pattern with p I values in the pH-range 7.0–8.3, indicating a variation in the sialic acid content. The OSO protein is not stable at temperatures above 40 °C and at pH values below 4 indicating that virus inactivation by incubating the protein solution at higher temperature or at lower pH is not possible.

Biochemical differentiation between the Wild rabbit (Oryctolagus cuniculus L.), the Domestic rabbit and the Brown hare (Lepus europaeus Pallas)

Twenty five specimens of the brown hare and fourteen specimens of the wild rabbit were examined for genetic differentiation and polymorphism in 28 enzyme systems by means of horizontal starch gel electrophoresis and isoelectric focusing in polyacrylamide gels. Indices of genetic variation show values similar to those averaged over numerous mammalian species. A D-value of 0, 0288 between wild and domestic rabbits suggests a high amount of gene frequency changes at enzyme loci during domestication. Evolutionary time of divergence between the rabbit and the hare (about 2430000 years) calculated from electrophoretic data is in good agreement with palaeontological findings.

Antibodies in oligoclonal immunoglobulins in CSF from patients with acute cerebrovascular disease.

Thin-layer polyacrylamide gel isoelectric focusing (PAG IEF) of CSF and serum, and subsequent immunofixation with viral and structural brain components followed by autoradiography revealed in eight out of nine selected patients with oligoclonal CSF IgG and cerebrovascular disease local synthesis within the CNS of antibodies against one or more of the viruses tested: six patients against measles, five against herpes simplex virus type 1, and two against varicella virus. This finding may reflect a polyclonal B cell activation secondary to brain damage and elaboration of certain structural brain components. None of the patients had local synthesis of antibodies against the other viruses tested (mumps, rubella and cytomegalovirus), or against structural brain components (crude saline, lipid-proteolipid, myelin basic protein extracts from human brain and purified bovine myelin basic protein).

Production, Purification, and Characterization of a Potential Thermostable Galactosidase for Milk Lactose Hydrolysis from Bacillus stearothermophilus

β-Galactosidase, commonly named lactase, is one of the most important enzymes used in dairy processing; it catalyzes the hydrolysis of lactose to its constituent monosaccharides glucose and galactose. Here, a thermostable β-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. sub-tilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified β-galactosidase appeared as a single protein band in sodium dodecyl sulfate-PAGE gel with a molecular mass of approximately 70 kDa. Its isoelectric point, determined by polyacryl-amide gel isoelectric focusing, was close to 5.1. The optimum temperature and pH for this β-galactosidase activity were 70°C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70°C were 50 and 9h, respectively, and the Km and Vmax values were 2.96mM and 6.62μmol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe2+, Zn2+, Cu2+, Pb2+, and Sn2+, inhibited its activity. Thiol reagents had no effect on the enzyme activity, and sulfhydryl group blocking reagents inactivated the enzyme. This enzyme possessed a high level of transgalactosylation activity in hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galactooligosaccharides in milk processing.

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.

Lactobacilli and bifidobacteria lectins as possible signal molecules regulating intra- and inter-population bacteria

Lectins (natural or artificial nonimmunoglobulin origin proteins/glycoproteins) are able to bind noncovalently with carbohydrate targets. Lectins and lectin-like substances of lactobacilli (L. acidophilus K 3 III 24 ; 100ash; NK1; L. plantarum 8RA3) and bifidobacteria ( B . adolescentis MC42; B gallinarum, B. bifidum 1) have been investigated. Bacteria were grown in semi-anaerobic conditions in a casein-yeast broth. Medium was microfiltrated and ultrafiltrated for isolation of 30 kDa compounds which were then separated by isoelectric focusing in polyacrylamide gel in the presence of urea and sucrose. Components isolated were electrotransferred to immobillon P. Blots obtained were then treated with a set of biotinylated artificial linear homopolysaccharides revealed by streptavidin-peroxidase conjugate. Chemiluminescence registration of peroxidase bound to blots was performed using the ‘BioChemi System’ camera. A spectrum of different polysaccharidebinding individual components reacting with such homopolymers as mannan-alpha (phosphorylated or not), GalNAcalpha- polysaccharide of mucin-like-type or galactan-beta (sulfated or not)-binding was found in lactobacilli and bifidobacteria. Lactobacilli produced a more limited spectrum of lectins or lectin-like compounds with higher affinities to polysaccharides tested than bifidobacteria having more extended pI polysaccharide-binding activity spectrum. Interaction of isolated lectins with T and B lymphocytes, macrophages, and thrombocytes was demonstrated. The importance of lactobacilli and bifidobacteria lectins/lectin-like components and their effector complexes in cell mitoses, blood coagulation, aerobic metabolic reactions, and other biological activities is discussed. Key words: bacterial lectin, lactobacilli, bifidobacteria, bacterial communication, bacteriahost cross-talk, lectin isolation, lectin identification, lectin biological activity

Screening for hetero-transglycosylating activities in extracts from nasturtium ( Tropaeolum majus)

Using combinations of different polysaccharides as glycosyl donors and of oligosaccharides fluorescently labeled by sulforhodamine (SR) as glycosyl acceptors, we screened for the presence of transglycosylating activities in extracts from nasturtium ( Tropaeolum majus). Besides xyloglucan endotransglycosylase/hydrolase (XTH/XET, EC 2.4.1.207) activity, which transfers xyloglucanosyl residues from xyloglucan (XG) to XG-derived oligosaccharides (XGOs), a glycosyl transfer from XG to SR-labeled cellooligosaccharides and laminarioligosaccharides has been detected. The XGOs also served as acceptors for the glycosyl transfer from soluble cellulose derivatives carboxymethyl cellulose and hydroxyethylcellulose. The effectivity of these polysaccharides as glycosyl donors for transfer to XG-derived octasaccharide [1- 3H]XXLGol decreased in the order XG > HEC > CMC. Isoelectric focusing in polyacrylamide gels showed that bands corresponding to hetero-transglycosylase activities coincided with zones corresponding to XTH/XET. These results can be explained as due either to substrate non-specificity of certain isoenzymes of XTH/XET or to existence of enzymes catalyzing a hetero-transfer, that is the formation of covalent linkages between different types of carbohydrate polymers.

Lactobacilli and bifidobacteria lectins as possible signal molecules regulating intra- and inter-population bacteria–bacteria and host–bacteria relationships. Part I. Methods of bacterial lectin isolation, physico-chemical characterization and some biological activity investigation

Lectins (natural or artificial nonimmunoglobulin origin proteins/glycoproteins) are able to bind noncovalently with carbohydrate targets. Lectins and lectin-like substances of lactobacilli (L. acidophilus K3III24; 100ash; NK1; L. plantarum 8RA3) and bifidobacteria (B. adolescentis MC42; B gallinarum, B. bifidum 1) have been investigated. Bacteria were grown in semi-anaerobic conditions in a casein-yeast broth. Medium was microfiltrated and ultrafiltrated for isolation of 30 kDa compounds which were then separated by isoelectric focusing in polyacrylamide gel in the presence of urea and sucrose. Components isolated were electrotransferred to immobillon P. Blots obtained were then treated with a set of biotinylated artificial linear homopolysaccharides revealed by streptavidin-peroxidase conjugate. Chemiluminescence registration of peroxidase bound to blots was performed using the ‘BioChemi System’ camera. A spectrum of different polysaccharide-binding individual components reacting with such homopolymers a...

Sensitive detection of transglycosylating activity of xyloglucan endotransglycosylase/hydrolase (XTH) after isoelectric focusing in polyacrylamide gels

The paper describes a sensitive and rapid zymogram technique for detection of transglycosylating activity (XET) of xyloglucan endotransglycosylase/hydrolase (XTH; EC 2.4.1.207) in polyacrylamide isoelectric focusing gels. After the electrophoresis, the separating gel was overlaid and incubated with an agarose detection gel containing XET substrates: tamarind-seed xyloglucan as the glycosyl donor and sulphorhodamine-labeled xyloglucan-derived oligosaccharides (XGO-SRs) as the glycosyl acceptors. The transglycosylation catalyzed by XTH caused incorporation of the fluorescent label into the high-M r polysaccharide. Selective removal of unreacted XGO-SRs from the agarose replicas by washing with organic solvents revealed the zones corresponding to XET activity as bright pink fluorescent spots under UV-light. The method appears suitable for a number of purposes such as analysis of the isoenzyme composition of XTHs with XET activity in crude extracts from various plants and plant organs, monitoring the enzyme expression at various stages of plant development and/or for checking enzyme purity in the course of its isolation procedure.

Red cell enzyme and serum protein polymorphisms (ACP1, PGM1, GLO1, ESD, HP, PI) in Turkish population

BACKGROUND: The allele frequencies in human populations are used in different areas such as population genetics, forensic genetics and anthropological studies. Various different populations have been investigated their allele frequency distributions of polymorphic traits. AIMS AND OBJECTIVES: The allele frequency distributions of four red cell enzyme (ACP1, PGM1, GLO1, ESD) and two serum protein systems (HP, PI subtypes) were analyzed in Turkish population from Adana area, Turkey, in order to enlarge our knowledge on the genetic composition of Turkish population. MATERIAL AND METHODS: Venous blood samples taken from 200 unrelated Turkish individuals were transported to the Forensic Serology Laboratory of Albert Szent-Gyorgyi Medical University (Szeged/Hungary) for phenotyping. The phenotypes of ACP1, PGM1, GLO1, ESD, and HP systems were determined by means of starch gel electrophoresis, while PI subtypes were obtained by polyacrylamide gel isoelectric focusing method. The allele frequencies were calculated by gene counting. RESULTS: The calculated frequencies of the alleles are as follows: ACP1*A = 0.250 ± 0.021, ACP1 *B = 0.693 ± 0.023, ACP1 *C = 0.057 ± 0.011; PGM1 *1 = 0.710 ± 0.022, PGM1*2 = 0.288 ± 0.022, PGM1*6 = 0.002 ± 0.002; GLO1*1 = 0.373 ± 0.024, GLO1*2 = 0.627 ± 0.024; ESD*1 = 0.805 ± 0.019, ESD*2 = 0.195 ± 0.019; HP*1 = 0.265 ± 0.022, HP*2 = 0.735 ± 0.022; PI*M1 = 0.887 ± 0.015, PI*M2 = 0.010 ± 0.004, PI*M3 = 0.095 ± 0.014, PI*S = 0.008 ± 0.004. CONCLUSIONS: The comparison of the data with those of Asian and European populations showed that the allele frequencies of ACP1, ESD and HP are similar or close to those of Asian populations, whereas those of PGM1 and PI come close to European populations. The allele frequency of GLO1 system is inbetween those of Asians and Europeans.

Isoelectrophoretic patterns of Der p 2 isoforms in house dust from different homes

Rationale Relative environmental exposures to different allergen isoforms are unknown. We aimed to determine the prevalence of different isoforms of the mite allergen Dermatophagoides pteronyssinus Der p 2 in house dust from different homes. Methods Mattress reservoir dust from 10 different homes was extracted. Eluates were concentrated by spin-filtration, separated by polyacrylamide gel (PAGE) isoelectric focusing and immunoblotted. Der p 2 isoforms were detected by the monoclonal antibody alphaDpX and a chemiluminescence-based reporter system. Radiographs were scanned, analyzed by standard densitometry software and the number and relative motilities of bands were compared between samples. Band molecular mass was estimated by PAGE. Recombinant isoforms rDer p 2.0101, rDer p 2.0102 and rDer p 2.0103 were used as standards, run on PAGE-isoelectric focusing and then analyzed by denaturing gel isoelectric focusing. Results Isoelectric focusing and immunoblotting: Multiple bands were detectable in house dust samples (range 3-8 bands). Relative band motility was similar; up to 5 different bands were common to samples from different homes. Native gel electrophoresis: Der p 2 had a tendency to form dimers and trimers. Denaturing gel electrophoresis of Der p 2 recombinant isoforms: Each isoform demonstrated a tendency to form 2 or 3 bands which could not easily be resolved under stringent conditions (6M urea). Conclusions Polyacrylamide gel isoelectric focusing indicates the presence of multiple Der p 2 isoforms in dust extracts. Some of the observed bands may represent multimeric complexes of Der p 2. Future studies to determine environmental exposure to different isoforms should take this into account.

Biochemical and molecular investigations on qualitative and quantitative Hb polymorphism in the river buffalo (Bubalus bubalis L.) population reared in Southern Italy

On 398 river buffalo samples, randomly collected in distinct breeding areas of the Campania region, high-resolution analytical systems were used to identify both qualitative and quantitative variations of the Hb phenotype. Polyacrylamide gel isoelectric focusing and HPLC were used to determine the ratio between HBA1 and HBA2 globin chains; restriction endonuclease analysis was performed to assess whether quantitative variations in Hb bands were related to an unusual number of a-globin genes. In the two buffalo subpopulations, allele frequencies of the alpha and beta globin systems were calculated, and F statistics (FIS, FIT and FST) were estimated as parameters of genetic diversity. The results suggest that: i) as shown by RFLP analysis, only a couple of associated a globin genes account for the quantitative variations recorded at the phenotypic level; ii) as expected, in the a globin gene system (HBA), the frequency of haplotype B (HBA-B) largely exceeded that of haplotype A (HBA-A) (95.1% vs 4.9%); iii) the frequency of the usual allele at the beta locus is 0.6, as opposed to 0.4 of the slow variant; iiii) the most significant component of variation of the genetic system of hemoglobin is between individuals within the same location.