Isoelectric Focusing Patterns Research Articles

Implications of monoclonal gammopathy and isoelectric focusing pattern 5 on the free light chain kappa diagnostics in cerebrospinal fluid.

Oligoclonal bands (OCB) analysis is the reference standard for detecting an intrathecal IgG synthesis. Alongside OCB, free light chains kappa (FLCκ) are considered an additional sensitive biomarker for determining patterns 2 or 3, indicating intrathecal Ig synthesis. However, kFLC IF is not suitable for detecting a monoclonal pattern 5. The primary aim of this study was to evaluate the impact of incorporating FLCκ analysis into routine cerebrospinal fluid (CSF) diagnostics instead of OCB testing on the rate of missed monoclonal IgG detection. A two-center retrospective biomarker study was conducted. OCB were identified using isoelectric focusing in polyacrylamide gels followed by silver staining or in agarose gels followed by immunofixation. FLCκ were quantified using nephelometry and FLCκ assay (Siemens). Out of a combined total of 17,755 OCB analyses conducted between 2011 and 2021, a subset of 269 cases (1.5 %) exhibited pattern 5. 98 samples (36 %), which included 18 samples with intrathecal inflammation as determined by additional OCB pattern 2 were included in the FLCκ analysis. Of those, 16 (89 %) had intrathecal FLCκ synthesis. While FLCκ offers a promising avenue for detecting an intrathecal inflammation, the pattern 5, though rare, remains a valuable additional finding of OCB analysis. A combined approach of FLCκ and OCB analysis is recommended for a comprehensive assessment of the humoral intrathecal immune response.

Marker-Free Isoelectric Focusing Patterns for Identification of Meat Samples via Deep Learning.

Isoelectric focusing (IEF) is a powerful tool for resolving complex protein samples, which generates IEF patterns consisting of multiplex analyte bands. However, the interpretation of IEF patterns requires the careful selection of isoelectric point (pI) markers for profiling the pH gradient and a trivial process of pI labeling, resulting in low IEF efficiency. Here, we for the first time proposed a marker-free IEF method for the efficient and accurate classification of IEF patterns by using a convolutional neural network (CNN) model. To verify our method, we identified 21 meat samples whose IEF patterns comprised different bands of meat hemoglobin, myoglobin, and their oxygen-binding variants but no pI marker. Thanks to the high throughput and short assay time of the microstrip IEF, we efficiently collected 1449 IEF patterns to construct the data set for model training. Despite the absence of pI markers, we experimentally introduced the severe pH gradient drift into 189 IEF patterns in the data set, thereby omitting the need for profiling the pH gradient. To enhance the model robustness, we further employed data augmentation during the model training to mimic pH gradient drift. With the advantages of simple preprocessing, a rapid inference of 50 ms, and a high accuracy of 97.1%, the CNN model outperformed the traditional algorithm for simultaneously identifying meat species and cuts of meat of 105 IEF patterns, suggesting its great potential of being combined with microstrip IEF for large-scale IEF analyses of complicated protein samples.

A Novel System for Newborn Screening of Thalassemia and Hemoglobinopathies Using Capillary Electrophoresis Is Superior Than Isoelectric Focusing

Introduction:Thalassemia (thal) and hemoglobinopathies (Hb variants) is highly prevalent in Thailand and nearly all Southeast Asian countries. In developed countries such as US and European countries, a newborn screening program for thal and hemoglobinopathies (NBS-Hb) has been incorporated in a routine healthcare to detect those with abnormal Hb in particular Hb S (Sickle cell disease) to provide early intervention for affected patients. Application of detecting patients and carriers could be useful for many developing countries such as Thailand as a part of our prevention and control program at a national level. At presence, isoelectric focusing (IEF) is a widely used platform for NBS-Hb, however a new capillary electrophoresis (CE) has recently been developed. It is of interest to evaluate whether which system provides a better efficiency under a circumstance with various types of thal and Hb variants.Objectives:To evaluate whether IEF or CE, is more effective for Hb screening at neonatal period and to develop new diagnostic criterion for different types of Hb variants in ThailandMethods:After informed consent by parent, the dried blood spot (DBS) samples were collected by heel prick puncture from 2-day old babies and processed in two Hb screening methods; IEF & ISOSCAN RESOLVE® SYSTEM Neonatal Hemoglobin Kits (Perkin Elmer, Turku, Finland) and Capillarys 2 NEONAT FAST® (SEBIA, Évry, France). These DBS were also extracted gDNA used for gold standard molecular method covering >98% of common globin mutations found in Thailand; 7-deletional α-thal GAP-PCR, 6-non-deletional α-thal ARMS-PCR, 16 mutations of β-thal ARMS-PCR, PCR-RFLP by Mnl I restriction cleavage for Hb E disorder and/or DNA sequencing. The statistics analysis was performed by PASW 18 Software (SPSS Inc, 2015) to analyze the detection efficiency of IEF vs. CE. Cut-off levels of Hb variants were also determined by using receiver operating characteristic (ROC) curve to set up diagnostic criterion.Results: The study recruited 1,213 healthy newborns (48.4% male and 51.6% female) with average 38 weeks of gestational age (GA). The profiles of IEF pattern and CE histogram were analyzed. Hb E was the most common Hb variants of β-globin gene found and Hb Bart's (γ4) was indicative of α-thal, both were confirmed by molecular methods. Comparison between the efficiency of IEF vs. CE on detecting Hb variants showed a higher efficiency of CE on Hb Bart's detection for most α-thal carriers and on Hb E detection in Hb E trait than those of IEF for sensitivity, specificity, and accuracy as shown in Table 1. There was no difference between two platforms on Hb H disease, homozygous Hb E and β-thal/Hb E. In order to set up cut-off levels of Hb Bart's, Hb E and Hb A for each globin genotypes by ROC curve analysis, the cut-off levels provided from CE seemed to be more clear and discriminative than those of IEF as shown in Table 2. The cut-off levels of Hb Bart's of ≥7.40%, ≥0.75%, ≥0.45%, and ≥0.10% were suggested different α-globin abnormalities; Hb H, αº-, non-deletional and deletional α+-thal carrier, respectively. Detection of Hb E of ≥0.25%, ≥4.85%, and ≥0.95% were suggestive of Hb E carrier, Hb E homozygote, and β-thal/Hb E, respectively. Interestingly, we can screen for β-thal carriers using a lower Hb A level (≤10.35%) and Hb A/Hb F ratio (≤0.115). This data was analyzed from 11 β-thal carrier newborns confirmed by DNA testing compared to 148 GA-sex matched newborn controls with normal β-globin genotype. These cut-off levels based on CE were powerful on detecting thal diseases (Hb H and β-thal/Hb E) and carriers of Hb E, αº- and non-deletional α+-thal. However, our screening criterions still have a limitation on fully detecting deletional α+-thal and β-thal traits.Conclusions:Our data suggested that neonatal CE platform is more effective system for Hb screening with excellent efficiency and precision to detect Hb variants compared to conventional IEF. Using NBS-Hb by CE could successfully screen for all important thal carriers in one simple test. Implementing our NBS-Hb to the national level would be simplest way to generate a national registry that can be linked to our national health database of severe diseases and carriers. By applying this approach into a national scale might be the only measure to effectively control the health burden of thal and hemoglobinopathies in Thailand where provides high prevalence of defective globin genes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cambios estructurales en las proteínas de dos especies del género vigna por efecto de diferentes tratamientos

A study of the thermal treatment effect on the protein structure of the rice bean and cowpea bean was conduced. The protein was isolated by isoelectric focusing and electrophoretic pattern, FT-IR and FT-Raman spectroscopy were used for their characterization. The results showed that in both species the thermal treatment originate modifications on the conformational structure modifying the secondary structure, disorder level and agregation fractions. The FT-IR and FT-Raman spectros indicate that the main secondary structure is the protein of both species, was the β-sheet with a smaller contribution of the α-helix structure. The structure 310 helix and the sulfhydril groups were detected in the protein of both species, in addition the presence of some aminoacids also were observed (Cys, Lys, Trp, Phe, Tyr and Met). The electrophoretic pattern showed a significantl reduction in the number of high molecular weight subunits by effect of thermal treatment and isolation process, two hight molecular weight bands are mantained before and after treatments (23 kDa and 50 kDa), these fractions could be a stable subunits and common ancestor in both species.

Hemoglobin variants identified in the Uganda Sickle Surveillance Study

AbstractThe Uganda Sickle Surveillance Study analyzed dried blood spots that were collected from almost 100 000 infants and young children from all 10 regions and 112 districts in the Republic of Uganda, with the primary objective of determining the prevalence of sickle cell trait and disease. An overall prevalence of 13.3% sickle cell trait and 0.7% sickle cell disease was recently reported. The isoelectric focusing electrophoresis technique coincidentally revealed numerous hemoglobin (Hb) variants (defined as an electrophoresis band that was not Hb A, Hb F, Hb S, or Hb C) with an overall country-wide prevalence of 0.5%, but with considerable geographic variability, being highest in the northwest regions and districts. To elucidate these Hb variants, the original isoelectric focusing (IEF) gels were reviewed to identify and locate the variant samples; corresponding dried blood spots were retrieved for further testing. Subsequent DNA-based investigation of 5 predominant isoelectric focusing patterns identified 2 α-globin variants (Hb Stanleyville II, Asn78Lys; Hb G-Pest, Asp74Asn), 1 β-globin variant (Hb O-Arab, Glu121Lys), and 2 fusion globin variants (Hb P-Nilotic, β31-δ50; Hb Kenya, Aγ81Leu-β86Ala). Compound heterozygotes containing an Hb variant plus Hb S were also identified, including both Hb S/O-Arab and HbS/Kenya. Regional differences in the types and prevalence of these hemoglobin variants likely reflect tribal ancestries and migration patterns. Algorithms are proposed to characterize these Hb variants, which will be helpful for emerging neonatal hemoglobinopathy screening programs that are under way in sub-Saharan Africa.

High Frequency of Hb E-Saskatoon (HBB: c.67G > A) in Brazilians: A New Genetic Origin?

Hb E-Saskatoon [β22(B4)Glu→Lys, HBB: c.67G > A] is a rare, nonpathological β-globin variant that was first described in a Canadian woman of Scottish and Dutch ancestry and has since then been detected in several populations. The aim of the present study was to identify the origin of Hb E-Saskatoon in Brazil using β-globin haplotypes and genetic ancestry in carriers of this hemoglobin (Hb) variant. Blood samples were investigated by isoelectric focusing (IEF) and high performance liquid chromatography (HPLC) using commercial kits. Hb E-Saskatoon was confirmed by amplification of the HBB gene, followed by sequence analysis. Haplotypes of the β-globin gene were determined by polymerase chain reaction (PCR), followed by digestion with specific restriction enzymes. Individual ancestry was estimated with 48 biallelic insertion/deletions using three 16-plex PCR amplifications. The IEF pattern was similar to Hbs C (HBB: c.19G > A) and Hb E (HBB: c.79G > A) [isoelectric point (pI): 7.59-7.65], and HPLC results showed an elution in the Hb S (HBB: c.20A > T) window [retention time (RT): 4.26-4.38]. DNA sequencing of the amplified β-globin gene showed a mutation at codon 22 (GAA>AAA) corresponding to Hb E-Saskatoon. A total of 11 cases of this variant were identified. In nine unrelated individuals, Hb E-Saskatoon was in linkage disequilibrium with haplotype 2 [+ – – – –]. All subjects showed a high degree of European contribution (mean = 0.85). Hb E-Saskatoon occurred on the β-globin gene of haplotype 2 in all Brazilian carriers. These findings suggest a different genetic origin for this Hb variant from that previously described.

Complex changes in von Willebrand factor-associated parameters are acquired during uncomplicated pregnancy.

BackgroundThe coagulation protein von Willebrand Factor (VWF) is known to be elevated in pregnancy. However, the timing and nature of changes in VWF and associated parameters throughout pregnancy are not well understood.ObjectivesTo better understand the changes in VWF provoked by pregnancy, we studied VWF-associated parameters in samples collected over the course of healthy pregnancies.MethodsWe measured VWF antigen (VWF:Ag), VWF propeptide (VWFpp), Factor VIII (FVIII), and ADAMTS13 activity in samples collected from 46 women during pregnancy and at non-pregnant baseline. We also characterized pregnant vs. non-pregnant VWF multimer structure in 21 pregnancies, and performed isoelectric focusing (IEF) of VWF in two pregnancies which had samples from multiple trimesters.ResultsVWF:Ag and FVIII levels were significantly increased during pregnancy. ADAMTS13 activity was unchanged. VWFpp levels increased much later in pregnancy than VWF:Ag, resulting in a progressive decrease in VWFpp:Ag ratios. FVIII:VWF ratios also decreased in pregnancy. Most pregnancies exhibited a clear loss of larger VWF multimers and altered VWF triplet structure. Further evidence of acquired VWF qualitative changes in pregnancy was found in progressive, reversible shifts in VWF IEF patterns over gestation.ConclusionsThese data support a new view of pregnancy in which VWF can acquire qualitative changes associated with advancing gestational age. Modeling supports a scenario in which both increased VWF production and doubling of the VWF half-life would account for the data observed. We propose that gestation induces a prolongation in VWF survival, which likely contributes to increased total VWF levels and altered VWF structure.

Adult phenotype and further phenotypic variability in SRD5A3-CDG

BackgroundSRD5A3 is responsible for SRD5A3-CDG, a type of congenital disorder of glycosylation, and mutations have been reported in 15 children. All the mutations are recessive and truncating.Case presentationWe present 2 brothers at the age of 38 and 40 years with an initial diagnosis of cerebellar ataxia. We found the candidate disease loci via linkage analysis using data from single nucleotide polymorphism genome scans and homozygous truncating mutation SRD5A3 p.W19X, which was previously reported in 3 unrelated children, by exome sequencing.Clinical investigations included physical and ocular examinations and blood tests. Severe ocular involvement with retinal bone spicule pigmentation and optic atrophy are the most prominent disabling clinical features of the disease. The serum transferrin isoelectric focusing (TIEF) pattern is abnormal in the patient investigated.ConclusionOur patients are older, with later onset and milder clinical phenotypes than all patients with SRD5A3-CDG reported so far. They also have atypical ocular findings and variable phenotypes. Our findings widen the spectrum of phenotypes resulting from SRD5A3 mutations and the clinical variability of SRD5A3-CDG, and suggest screening for SRD5A3 mutations in new patients with at least a few of the clinical symptoms of SRD5A3-CDG.

A New Method for the Rapid Diagnosis of Protein N-linked Congenital Disorders of Glycosylation

The Congenital Disorders of Glycosylation (CDG) are a devastating group of genetic disorders that encompass a spectrum of glycosylation defects and are characterized by the underglycosylation of or the presence of abnormal glycans on glycoproteins. The N-linked CDG disorders (Type I and II) are usually diagnosed in chemical pathology laboratories by an abnormal serum transferrin isoelectric focusing (IEF) pattern. Transferrin has been the protein of choice for CDG analysis because it is well characterized, highly abundant, and easily detected in plasma. However, IEF provides limited information on the glycosylation defect and requires a separate and extensive glycan analysis to diagnose CDG Type II. We have therefore developed a simple bead-based immunoaffinity and mass spectrometry-based assay to address these issues. Our method uses immuno-purified transferrin and proteolytic digestion followed by a rapid 30 min mass spectral analysis and allows us to identify both micro- and macroheterogeneity of transferrin by sequencing of peptides and glycopeptides. In summary, we have developed a simple, rapid test for N-linked glycosylation disorders that is a significant improvement on existing laboratory tests currently used for investigating defective N-linked glycosylation.

Defining the Phenotype in Congenital Disorder of Glycosylation Due to ALG1 Mutations

Deficiency of β-1,4 mannosyltransferase (MT-1) congenital disorder of glycosylation (CDG), due to ALG1 gene mutations. Features in 9 patients reported previously consisted of prenatal growth retardation, pregnancy-induced maternal hypertension and fetal hydrops. Four patients died before 5 years of age, and survivors showed a severe psychomotor retardation. We report on 7 patients with psychomotor delay, microcephaly, strabismus and coagulation abnormalities, seizures and abnormal fat distribution. Four children had a stable clinical course, two had visual impairment, and 1 had hearing loss. Thrombotic and vascular events led to deterioration of the clinical outcome in 2 patients. Four novel ALG1 mutations were identified. Pathogenicity was determined in alg1 yeast mutants transformed with hALG1. Functional analyses showed all novel mutations representing hypomorphs associated with residual enzyme activity. We extend the phenotypic spectrum including the first description of deafness in MT1 deficiency, and report on mildly affected patients, surviving to adulthood. The dysmorphic features, including abnormal fat distribution and strabismus highly resemble CDG due to phosphomannomutase-2 deficiency (PMM2-CDG), the most common type of CDG. We suggest testing for ALG1 mutations in unsolved CDG patients with a type 1 transferrin isoelectric focusing pattern, especially with epilepsy, severe visual loss and hemorrhagic/thrombotic events.

Cell wall-bound cationic and anionic class III isoperoxidases of pea root: biochemical characterization and function in root growth.

Cell wall isolated from pea roots was used to separate and characterize two fractions possessing class III peroxidase activity: (i) ionically bound proteins and (ii) covalently bound proteins. Modified SDS–PAGE separated peroxidase isoforms by their apparent molecular weights: four bands of 56, 46, 44, and 41kDa were found in the ionically bound fraction (iPOD) and one band (70kDa) was resolved after treatment of the cell wall with cellulase and pectinase (cPOD). Isoelectric focusing (IEF) patterns for iPODs and cPODs were significantly different: five iPODs with highly cationic pI (9.5–9.2) were detected, whereas the nine cPODs were anionic with pI values between pH 3.7 and 5. iPODs and cPODs showed rather specific substrate affinity and different sensitivity to inhibitors, heat, and deglycosylation treatments. Peroxidase and oxidase activities and their IEF patterns for both fractions were determined in different zones along the root and in roots of different ages. New iPODs with pI 9.34 and 9.5 were induced with root growth, while the activity of cPODs was more related to the formation of the cell wall in non-elongating tissue. Treatment with auxin that inhibits root growth led to suppression of iPOD and induction of cPOD. A similar effect was obtained with the widely used elicitor, chitosan, which also induced cPODs with pI 5.3 and 5.7, which may be specifically related to pathogen defence. The differences reported here between biochemical properties of cPOD and iPOD and their differential induction during development and under specific treatments implicate that they are involved in specific and different physiological processes. Abbreviations:cPODcovalently bound peroxidaseDAB3,3'-diaminobenzidineDEPMPOspin-trap (5-diethoxy-phosphoryl-5-methyl-1-pyrroline-n-oxide)EPRelectron paramagnetic resonanceHRPhorseradish peroxidaseIAAindole-3-acetic acidHRPhorseradish peroxidaseIEFisoelectric focusingiPODionically bound peroxidaseNAAnaphthalene acetic acidPNGase Fpeptide N-glycosidase FPRpathogen-relatedSDS–PAGEsodium dodecyl sulphate–polyacrylamide gel electrophoresisSHAMsalicylhydroxamic acidTMBtetramethyl benzidineWGAwheat germ agglutinin

Isoelectric focusing pattern of plasminogen mutants of patients with hypoplasminogenemia

Plasminogen (plg), the circulating proenzyme of plasmin in blood, is a polymorphic protein and most of these natural variants have been identified using isoelectric focusing (IEF) gel electrophoresis. Here, we show that a rare plg gene polymorphism 504R/W is associated with IEF phenotype A3 on the protein level. One healthy individual with homozygous plg gene polymorphism 504W studied so far exhibited low normal plg antigen and slightly decreased plg activity, suggesting that this polymorphism is associated with (mild) hypoplasminogenemia. In addition, we present the findings of IEF phenotyping of plg mutants of 26 patients with severe hypoplasminogenemia, showing one of the following four IEF patterns: A3-like, A3A-like, B-like and AB-like. In the plasma of most compound heterozygous patients, only one of the two plg mutants was detectable by IEF electrophoresis, probably due to undetectable plasma concentration of the 2nd plg mutant. In almost all cases, pI of plg mutants and variants predicted by computer modeling were in good agreement with the observed IEF band pattern. plg phenotyping by IEF in combination with molecular genetic analysis of the plg gene is a useful approach to characterize plg mutants and variants further.

A fast preparative method for detection of recombinant erythropoietin in blood samples

While firstand second-generation recombinant erythropoietins (EPOs) are detectable in urine, we showed that analysis in blood is more appropriate for the third-generation, continuous erythropoietin receptor activator (CERA), due to its poor excretion in urine. However, isoelectric focusing (IEF) of EPO in serum or plasma is not as simple as in urine since it requires an initial preparative step by immunoaffinity chromatography. That is why enzyme-linked immunosorbent assay (ELISA) and IEF are used as screening and confirmation tests for CERA detection, respectively. Due to the observation of false negative results of ELISA, we have developed a fast method for preparing plasma or serum samples, making IEF as easy to use in blood as in urine. While preparation by immunoaffinity is kept for confirmation analysis, this convenient method is proposed for screening analysis. In addition to CERA, Darbepoetin alfa is very easily identified in blood due to its typical IEF profile. Identification of first-generation EPO drugs will require criteria specific for blood. In comparison with urine, EPO analyses in blood appear beneficial in terms of sensitivity, stability during physical exercise, and prevention of sample adulteration at collection. Anti-doping control of erythropoietin (EPO) is based on the differentiation of natural endogenous from recombinant hormones by their isoelectric profiles.[1] SDS (sodium dodecyl sulfate) electrophoresis is a useful complementary method.[2] Both methods were developed for urine samples. While firstand second-generation recombinant EPOs are detectable in urine, we showed that analysis in blood is more appropriate for the thirdgeneration, continuous erythropoietin receptor activator (CERA), due to its poor excretion in urine.[3] However, isoelectric focusing (IEF) of EPO in serum or plasma is not as simple as in urine since it requires an initial preparative step by immunoaffinity chromatography.[4] We thus developed a convenient method (more than 20 samples can be treated in parallel) for preparing plasma or serum samples, making IEF as easy to use in blood as in urine.

Characterisation of phenol oxidase and peroxidase from maize silk

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non-browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o-diphenolic substrates, was abundant only in browning silk, and low or absent in non-browning silk. Pollination increased POD but not PPO activity. Isoelectric-focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN(3), EDTA, KCN, and L-cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.

A severe human metabolic disease caused by deficiency of the endoplasmatic mannosyltransferase hALG11 leads to congenital disorder of glycosylation-Ip

A new type of congenital disorders of glycosylation, designated CDG-Ip, is caused by the deficiency of GDP-Man:Man3GlcNAc2-PP-dolichol-alpha1,2-mannosyltransferase, encoded by the human ortholog of ALG11 from yeast. The patient presented with a multisystemic disorder characterized by muscular hypotonia, seizures, developmental retardation and death at the age of 2 years. The isoelectric focusing pattern of the patient's serum transferrin showed the partial loss of complete N-glycan side chains, which is a characteristic sign for CDG-I. Analysis of dolichol-linked oligosaccharides in patient-derived fibroblasts revealed an accumulation of Man3GlcNAc2-PP-dolichol and Man4GlcNAc2-PP-dolichol. Determination of mannosyltransferase activities of early steps of lipid-linked oligosaccharide biosynthesis in fibroblasts indicated that the patient was deficient in elongating Man3GlcNAc2-PP-dolichol. These findings gave rise to genetic analysis of the hALG11 cDNA, in which homozygosity for mutation c.T257C (p.L86S) was identified. Verification of the mutation as a primary cause for the genetic defect was proved by retroviral expression of human wild-type and mutated ALG11 cDNA in patient-derived fibroblasts as well as using a yeast alg11 deletion strain as a heterologous expression system for hALG11 variants. Immunofluorescence examinations combined with western blotting showed no differences of intracellular localization or expression of ALG11 between control and patient fibroblasts, respectively, indicating no mislocalization or degradation of the mutated transferase.

Superoxide dismutase isoelectric focusing patterns as a tool to differentiate pathotypes of Globodera spp.

Abstract Isoelectric focusing was used to separate proteins from cyst extracts of potato cyst nematode (PCN) populations. In a first set of assays, cyst extracts from standard populations of Globodera rostochiensis pathotypes Ro1, Ro2, Ro3, Ro2/3, Ro4, and Ro5, and G. pallida pathotypes Pa2 and Pa3, were loaded on isoelectric focusing gels. Gels were stained for superoxide dismutase (SOD), esterase, and glucose-6-phosphate isomerase (GPI). Twelve bands of SOD activity were detected, six (B1-B6) migrating towards the basic zone and the other six (A1-A6) migrating towards the acidic zone, starting from the loading point. A cluster analysis was carried out based on a data matrix that reported the presence or absence of SOD bands on the isozyme electrophoresis patterns (IEPs). Globodera spp. were clearly distinguished and, within G. rostochiensis, Ro2 and Ro4 shared a high level of similarity, respectively, with Ro3 and Ro5; moreover, Ro1 could be clearly distinguished from Ro2/3 and Ro4/5. Globodera pallida Pa2 and Pa3 also shared a high level of similarity. In contrast, esterase and GPI IEPs did not discriminate among G. rostochiensis standard pathotypes. Subsequently, 14 field populations of G. rostochiensis, five from Italy and nine from Venezuela, and three field populations of G. pallida, two from Italy and one from Chile, were assayed to obtain SOD IEPs. Italian populations had previously been identified at pathotype level by bioassays according to the generally accepted international test using different resistant potato cultivars and clones. The cluster analysis carried out on the SOD IEPs of all the populations tested formed four distinct groups within G. rostochiensis and only one within G. pallida. Pathotype identification of Globodera populations by SOD IEPs was not able to discriminate between bioassay standard couples Ro2/Ro3, Ro4/Ro5 and Pa2/Pa3. Therefore, three groups were assigned to Ro1, Ro2/3 and Ro4/5, and a fourth group to Pa2/Pa3. Four Venezuelan populations, not identified at pathotype level by bioassays, formed a distinct fifth group. By means of the method described herein, four additional unknown Venezuelan populations could be assigned to Ro1 group and one to Ro2/Ro3 group; one G. pallida population from Chile was assigned to Pa2/Pa3 group.

Secondary disorders of glycosylation in inborn errors of fructose metabolism

Adamowicz and colleagues raised the alert in 2007 about patients with atypical hereditary fructose intolerance (HFI) primarily misdiagnosed as CDG Ix. We describe a girl with neonatal hypertonia, facial trismus, absent swallowing and coughing reflexes, gastro-oesophageal reflux and sporadically elevated Krebs cycle metabolites and lactate. At 14 months microcephaly and hepatomegaly were noted, with hypertransaminasaemia but normal blood coagulation, glucose, phosphate, and absent urinary reducing substances. Neurological impairment persisted. Because of hepatic and neurological abnormalities with developmental delay, Tf IEF was performed and showed a severe type 1 pattern, resulting in a wrong diagnosis of CDG. Subsequently, an aversion to fruits suggested HFI, confirmed by the finding of ALDOB mutations (p.A150P/p.N335K). The girl improved with fructose-free diet, but liver cirrhosis led to hepatic transplantation. She is now 7 years old with good evolution; facial trismus and hypertonia reversed, but microcephaly persists. Transferrin MALDI-TOF MS characterization revealed underoccupation of glycosylation sites and glycan abnormalities, which reversed with dietary treatment. High maternal fructose concentrations might have caused neonatal abnormalities. Although in our patient's mother there is no fructose accumulation at present, it is possible that increased ingestion of fruits and vegetables during pregnancy, together with her heterozygosity, caused an accumulation of fructose that finally affected the fetus. We also describe slightly abnormal transferrin isoelectric focusing and MALDI-TOF MS patterns of intact transferrin and N-glycans in a fructose-1,6-bisphosphatase (FBP1)-deficient patient. While HFI is a well-known cause of secondary CDG, we found no reports of abnormal transferrin isoelectric focusing patterns in FBP1 deficiency and we introduce this condition as a possible secondary cause for altered transferrin isoelectric focusing.

Comparison between high performance liquid chromatography and capillary zone electrophoresis for the diagnosis of congenital disorders of glycosylation

Transferrin isoelectric focusing (IEF) is the most widely used method to screen for congenital disorders of glycosylation (CDG). Our aim was to compare high performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) procedures for serum sialotransferrin analysis. 58 serum samples were processed both by CZE and HPLC: 35 were from paediatric controls, 18 from patients with an altered sialotransferrin IEF pattern and 5 were transferrin variant samples. HPLC analysis was performed with an anion-exchange column with spectrophotometric detection at 470 nm. CZE analysis was done using the commercial CEofix-CDT kit with spectrophotometric detection at 200 nm. Passing-Bablok regression analysis showed good agreement for tri-, tetra- and penta-sialotransferrin by both procedures. But for disialotransferrin, higher values were observed by the HPLC procedure. The HPLC and CZE methods allowed reproducible separation and analysis of single transferrin glycoforms with similar peak patterns. All patients presented values outside the range established in our control population either by HPLC or by CZE, even in patients with moderate forms of CDG that had been difficult to detect by IEF. In conclusion, measurement of sialotransferrin isoforms and interpretation using method-specific reference values may offer some advantages for the diagnosis of CDG as compared with the standard IEF procedure.

Cerebrospinal fluid investigations in multi-infarct dementia and senile dementia.

Thirty-eight demented patients were clinically investigated and classified into a multi-infarct dementia (MID) and senile dementia (SD) group. Total protein, albumin, immunoglobulin G, agar gel electrophoretic protein and iso-electric focusing pattern were determined in CSF and serum as well as the lactate dehydrogenase, LD-iso-enzyme pattern and uric acid. No significant differences were found regarding total protein, albumin and immunoglobulin G. The agar gel electrophoresis showed a tau-globulin increase in 18% of the SD-group against 6% of the MID group. The patterns were, however, plasma-like in 56% of the MID group against 23% of the SD group. The iso-electric focusing pattern showed an abnormal band in some SD-patients which was not found in the MID group. The uric acid and LD-enzyme concentration was equal in both CSF and serum. An index [Formula: see text] was calculated to give an approximate correction for the influence of different permeability of the blood-brain and CSF barrier. A significantly higher LD-index was found in the SD-group, suggesting a continuous cell degeneration.

Comparison of IEF patterns of sarcoplasmic proteins of fish from North Atlantic and Aegean Sea

Identification of processed fish lacking morphological characteristics is of growing importance in the times of increasing global trade and illegal fishery. Protein electrophoresis techniques, like isoelectric focusing (IEF) of water-soluble proteins, are fast and inexpensive tools for identification of raw fishery products. Protein profiles of raw fish or fillet of the same species were found to be remarkably constant. However, it cannot be excluded that protein patterns of fish of the same species from distant stocks may show some variation. The protein profiles of commercially important fish from different fishing grounds were determined by collecting samples of 38 fish species from Aegean Sea and Turkish fish farms and of five species from North Atlantic waters. The raw fillet of the fish was analysed by IEF of water-soluble (sarcoplasmic) proteins. Species-specific protein profiles were obtained suited for identification of raw fish or fillet. When protein patterns and pI values were compared between Mediterranean and North Atlantic fish of the respective species, only minor differences were observed. These differences were related to the intensity of protein bands, but not to the position within the pH gradient of the IEF gel. The pI values (isoelectric points) of the prominent protein bands of the fish were determined to be used as a database.