Hybrid Isoelectric Focusing Research Articles

Hydrolysis Mechanism of New Anticancer Drug Transplatin Analogues Containing One Nonplanar Heterocyclic Amine Ligand

Hydrolysis processes of novel anticancer transplatin analogues,trans-[PtCl2(NH3)(Am)](Am:nonplanar heterocycle piperidine or piperazine),were explored using the B3LYP hybrid functional and isoelectric focusing polarized continuum model(IEF-PCM).Stationary points on the potential energy surfaces for the first and second hydrolysis steps that proceed via a general SN2 pathway were fully optimized and characterized.The most remarkable structural variations in the hydrolysis process were found to occur in the equatorial plane of five-coordinate trigonal-bipyramidal(TBP) like structures of the intermediates and transition states.We obtained lower activation energies for trans-[PtCl2(NH3)(piperazine)] and a slightly higher activation energy for the first step and a slightly lower activation energy for the second step during the hydrolysis of trans-[PtCl2(NH3)(piperidine)] by comparison to previous work on the hydrolysis reactions of cisplatin.Our calculations suggest that this class of non-classical transplatin analogues with one nonplanar heterocyclic amine decreases the equatorial steric effect and the hydrolysis reaction barriers.

Glycolytic enzyme expression in human granulosa cells

To examine the differences in specific protein expression between mural and cumulus granulosa cells following 24-hour in vitro culture. Laboratory study. University Hospital. Human granulosa cells were collected at the time of egg collection during assisted reproduction. Cumulus cells associated with the oocyte were separated from mural cells from the periphery of the follicle before in vitro culture for 24 hours. Cells were then lysed and subjected to two-dimensional gel electrophoresis. Given that cumulus (cGC) and mural granulosa cells (mGC) differentiate from a single layer, it is likely that phenotypic differences between them may reflect specific molecular processes and structural adaptations. Computer-assisted analysis using dedicated software enabled the presence, absence, or relative volume of each individual protein spot to be estimated. Differentially expressed spots were identified using tandem mass spectrometry. The mean number of separate protein spots detected in mGC gels was 1,105 +/- 146, and in cGC it was 887 +/- 236, although there was no statistically significant difference between the two. Five enzymes of the glycolytic pathway were never expressed in cGC after 24 hours in vitro; these were triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase 1, and two isoforms of alpha enolase. These are the first data collected in humans consistent with a recent demonstration that isolated murine cGC cultured in vitro exhibit decreased expression of mRNA encoding glycolytic enzymes, and support the suggestion that some factor or factors secreted by the oocyte may be responsible for the maintenance of glycolysis in the adjacent cGC.

Convenient high-resolution isoelectric focusing (IEF) method for the separation of alpha1-proteinase inhibitor (A1PI) isoforms in A1PI concentrates

Currently, high-resolution separation of A1PI is done in highly specialized laboratories using gels made in-house. This paper presents a high-resolution method for the separation of A1PI concentrates and human plasma using commercially available gels. Hybrid IEF was performed with carrier ampholytes and the gels were stained with Coomassie Brilliant Blue G-250. In addition, a sensitive immunoblotting procedure is described. The IEF method allowed the reproducible and convenient determination of the IEF pattern of A1PI in concentrates including resolution of glycan-dependent isoforms and isoproteins with secondary modifications such a C-terminal Lys-truncation. Furthermore, a shift in the IEF pattern of A1PI occurring upon reduction could be detected. Finally, in combination with a sample pretreatment step, the method proved able to monitor complex A1PI isoform patterns in samples with low A1PI concentrations as present for example in bronchoalveolar lavage solutions.

Biochemical and population genetics of the rabbit, Oryctolagus cuniculus, carbonic anhydrases I and II, from the Iberian Peninsula and France.

Available studies on the biochemical and electrophoretic characterization of European rabbit (Oryctolagus cuniculus) carbonic anhydrases I and II (CAI, CAII) show contradictory results about their relative electrophoretic mobility and substrate specificity. After positive identification of carbonic anhydrase activity by CO2 hydration, the differential esterase activity of CAI and CAII toward beta-napththyl acetate and flourescein diacetate,respectively, were used to identify the banding patterns corresponding to each locus. Electrophoretic and hybrid isoelectric focusing analyses of the CAI and CAII loci in 1 domestic and 19 wild rabbit populations led to the recognition of genetic polymorphism at the CAI locus and of extensive variability at the CAII locus. Four and nine alleles at the CAI and CAII loci, respectively, are described. The geographic distribution of genetic variability is consistent with the existence of two evolutionary groups within O. cuniculus.

Screening and biochemical characterization of transthyretin variants in the Portuguese population.

The study of pathogenic and nonpathogenic transthyretin (TTR) variants is very important for the understanding of such TTR-related diseases as hereditary amyloidosis and also to establish a relationship between the structure and function of the molecule. Variants with clinical manifestations can be easily detected, but clinically silent variants can be detected only by population screening programs using specialized techniques. Hybrid isoelectric focusing (HIEF) in extremely flattened immobilized pH gradients (IPG) allows the detection of even neutral amino acid substitutions and has been used to analyze approximately 5,000 samples from the Portuguese population. Comparison with samples from carriers of three known TTR mutations (Met 30 associated with hereditary amyloidosis, Met 119, and Asn 90) was also made. In this study we detected: (1) 8 individuals carriers of TTR Met 30, (2) 35 carriers of TTR Met 119, (3) 12 carriers of TTR Asn 90, (4) 1 compound heterozygote for TTR Met 30/Met 119, and (5) 5 variants that presented a different pattern from the controls used. We also performed DNA sequencing analyses of two of the variants with the different band pattern in HIEF. The individuals were found to be carriers of TTR Ile 122 and TTR Thr 190, respectively. All the mutations detected, except for Asn 90, result from substitutions in CpG hot spots and thus can be rather frequent in the populations. Studies on the clinical evolution of the compound heterozygotes and on the physical-chemical properties of these hybrid TTRs will help to understand the pathogenicity associated with TTR.

Screening and biochemical characterization of transthyretin variants in the Portuguese population

The study of pathogenic and nonpathogenic transthyretin (TTR) variants is very important for the understanding of such TTR-related diseases as hereditary amyloidosis and also to establish a relationship between the structure and function of the molecule. Variants with clinical manifestations can be easily detected, but clinically silent variants can be detected only by population screening programs using specialized techniques. Hybrid isoelectric focusing (HIEF) in extremely flattened immobilized pH gradients (IPG) allows the detection of even neutral aminoacid substitutions and has been used to analyze ∼5,000 samples from the Portuguese population. Comparison with samples from carriers of three known TTR mutations (Met 30 associated with hereditary amyloidosis, Met 119, and Asn 90) was also made. In this study we detected: (1) 8 individuals carriers of TTR Met 30, (2) 35 carriers of TTR Met 119, (3) 12 carriers of TTR Asn 90, (4) 1 compound heterozygote for TTR Met 30/Met 119, and (5) 5 variants that presented a different pattern from the controls used. We also performed DNA sequencing analyses of two of the variants with the different band pattern in HIEF. The individuals were found to be carriers of TTR Ile 122 and TTR Thr 109, respectively. All the mutations detected, except for Asn 90, result from substitutions in CpG hot spots and thus can be rather frequent in the populations. Studies on the clinical evolution of the compound heterozygotes and on the physical-chemical properties of these hybrid TTRs will help to understand the pathogenicity associated with TTR. Hum Mutat 9:226–233, 1997 © 1997 Wiley-Liss, Inc.

Mannitol prevents methionine sulphoxidation mediated electrophoretic heterogeneity of apolipoprotein A-I.

Hybrid isoelectric focusing of apolipoprotein A-I in polyacrylamide gels with immobilized pH-gradients under non-denaturing conditions resulted in the occurrence of additional bands which could prevent the specific and sensitive detection of genetic variants. Hybrid isoelectric focusing of two chromatographically distinguishable apolipoprotein A-I isoforms that differ by sulphoxidation of methionine residues, apo A-I(Met) and apo A-I(MetSO), revealed that the additional bands were caused by this post-translational modification. Several antioxidative additives and conditions were compared for their ability to prevent methionine sulphoxidation in apolipoprotein A-I. In the presence of 200 g/L mannitol in the gel, apolipoprotein A/I focused as a single band. Since methionine sulphoxidation in proteins is a general phenomenon either taking place in vivo or in vitro by isoelectric focusing, we conclude that isoelectric focusing in the presence of mannitol will improve the quality of resolution of many proteins in gels with immobilized pH-gradients.

Thyroxine binding in a TTR Met 119 kindred.

Recently, a transthyretin variant, TTR Met 119, in which methionine substitutes for threonine 119, a component of the protein's iodothyronine binding site, was identified in individuals with transient euthyroid hyperthyroxinemia. Healthy carriers of Met 119 have normal serum thyroid hormone concentrations, but two studies of Met 119 carriers have differed as to whether T4 binding to TTR is increased. An additional kindred has been identified by hybrid isoelectric focusing in an ongoing screening program for TTR variants in the Portuguese population with TTR Met 30 associated familial amyloidotic polyneuropathy. Cyanogen bromide peptide mapping and DNA restriction length polymorphism analyses showed that the propositus was a compound heterozygote for two TTR variants: Asn 90 and Met 119. Family analysis revealed that he inherited the TTR Met 119 variant from the mother and the TTR Asn 90 variant from the father. Neither the compound heterozygote nor his parents had symptoms of familial amyloidotic polyneuropathy. Serum dialysis with stepwise saturation of iodothyronine binding sites confirmed that TTR binding of T4 is increased in TTR Met 119. The increased binding is due to a higher TTR concentration rather than an increased association constant for T4. Because of the small proportion of serum T4 bound by TTR, increased T4 binding by TTR did not affect the ratio of free to bound T4 or T4 concentrations. In contrast, plasma retinol binding protein, almost all of which is bound by TTR, was elevated. The Asn 90 mutation does not affect either the concentration or the hormone binding characteristics of the protein. Possible long-term effects of these mutations and the combined heterozygotic state remain to be determined.

Cardiac amyloidosis: a review and report of a new transthyretin (prealbumin) variant.

Cardiac amyloidosis is caused by amyloid deposits derived from different human plasma proteins. It can lead to cardiac conduction disturbances, restrictive cardiomyopathy, and low output heart failure. The heart is variably involved during the development of systemic amyloidosis and seems to be more frequently affected in immunoglobulin (primary) than in reactive (secondary) amyloidosis. Amyloid is common in the elderly. Isolated atrial amyloid, for which a major subunit is the atrial natriuretic peptide, seems to be three times more frequent than senile cardiac amyloid, which is derived from normal prealbumin (transthyretin). Like polyneuropathy, cardiac amyloidosis is a prominent clinical feature of hereditary amyloidosis, namely of the autosomal dominant transthyretin (TTR) type. All 28 cases of TTR amyloidoses reported so far were heterozygotes for a single nucleotide change in the gene for TTR that resulted in amino acid substitutions in the mature protein. A new TTR genetic variant is reported in a German family where the index patient presented at the age of 63 with anginal pain and arrhythmia. Electrocardiography was suggestive of a pseudoinfarction pattern, and echocardiography and cardiac catheterisation showed signs of hypertrophic nonobstructive cardiomyopathy with increased ventricular filling pressures and a prominent "a" wave. Amyloid of the TTR type was identified by immunohistochemistry in the endomyocardial biopsy specimen. Hybrid isoelectric focusing established heterozygosity by showing normal TTR protein and an electrically neutral TTR variant differing from all known TTR variants so far. The patient died in an accident before investigations were complete. Electrophoretic analysis of the plasma from his first degree relatives (son, daughter, brother, and mother) identified the asymptomatic 22 year old son as an apparently heterozygous carrier of the mutant TTR protein. Comparative tryptic peptide mapping and sequencing showed that isoleucine at position 68 of the amino acid sequence was replaced by leucine.

Reliable phenotyping of alpha‐1‐antitrypsin by hybrid isoelectric focusing in an ultranarrow immobilized pH gradient

Genetically determined phenotypes of the highly polymorphic human alpha 1-antitrypsin were examined by hybrid isoelectric focusing in a narrow immobilized pH gradient. The chosen pH range from 4.45 to 4.75 was useful for identification and classification of the common PI M subtypes and a number of PI variants in the microheterogeneous regions of m6, m7, and m8. A high degree of resolution and an improved sharpness of PI bands was achieved with this excellent technique. It allowed the distinction of a new PI M variant, which has been designated M8, or Mingolstadt, according to the PI nomenclature. The pI difference of this mutant to the slightly cathodically located subtype M3 is approximately 0.001 pH unit. In addition, some common as well as rare phenotypes are presented.

Familial amyloid polyneuropathy: Alanine‐for‐threonine substitution in the transthyretin (prealbumin) molecule

A previously reported family with amyloid polyneuropathy (FAP) was reinvestigated to determine the type of mutation in the transthyretin (prealbumin) molecule. Transthyretin was isolated from amyloid-laden myocardium and serum, and tryptic peptides were resolved by high-performance liquid chromatography. Amino acid sequencing of an anomalous peptide revealed an alanine-for-threonine substitution corresponding to position No. 60 of the transthyretin monomer. Detection of the FAP gene in asymptomatic carriers was accomplished by hybrid isoelectric focusing of transthyretin in the presence of dithiothreitol and high concentrations of urea, and by Southern blotting of Pvull-digested leukocyte deoxyribonucleic acid. This type of FAP was found to be identical to the previously described Appalachian amyloid. Patients with FAP and their asymptomatic gene-carrying offspring had significantly reduced levels of serum transthyretin and retinol-binding protein.

Orosomucoid (ORM1 and ORM2) types in the Spanish Basque Country, Galicia and Northern Portugal

The genetic variation of orosomucoid (ORM1 and ORM2) in three south-western European populations (Galicia, Spanish Basque Country and northern Portugal) was investigated using hybrid isoelectric focusing. Three common ORM1 alleles were observed in these populations, the frequencies of ORM1 *S observed in Galicia and northern Portugal being the highest found among populations of European origin. Rare variants were observed for both the ORM1 and ORM2 loci.

The use of hybrid isoelectric focusing for the detection of polymorphic proteins in bloodstains

α 1-Antitrypsin (Pi), transferrin (Tf) and orosomucoid (ORM) were determined in bloodstain extracts by isolelectric focusing (IEF) with carrier ampholytes (CA) and also with a mixture of immobilines (HIEF). HIEF yields superior results from proteins typing in bloodstain extracts, since phenotypes are better distinguished and the bands are straighter and sharper. Also the sensitivity of HIEF is similar to IEF with CA.

Determination of phosphoglucomutase (PGM1), acid phosphatase (ACP), and esterase D (ESD) in human bloodstains by hybrid isoelectric focusing (HIEF)

PGM1, ESD, and ACP were determined in bloodstain extracts by isoelectric focusing (IEF) with carrier ampholytes (CA) and HIEF. HIEF yields superior results in PGM typing from bloodstain extracts, whereas for ESD and ACP typing isoelectric focusing with carrier ampholytes seems to be the method of choice.

Human alpha-1-antitrypsin subtyping by hybrid isoelectric focusing in miniaturized polyacrylamide gel.

The polymorphism of alpha-1-antitrypsin (PI) has been studied by hybrid isoelectric focusing in miniaturized immobilized pH gradient gels, with an interelectrode distance of 55 mm, in two narrow ranges of pH 4.35-4.75 and 4.35-4.55), following rehydration with pH 4.2-4.9 carrier ampholytes. The use of the separator N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) in combination with carrier ampholytes for gel rehydration has been shown to enhance PI band sharpness. The influence of different additives (sucrose, sorbitol and glycerol) on the PI band pattern has also been evaluated. Glycerol has been shown to be responsible for the change in the relative mobility of the M3 band. The analysis of the minor M-7 isoprotein zone by hybrid isoelectric focusing followed by silver staining has permitted a more reliable classification of PIM subtypes. A population study carried out with 164 unrelated individuals living in Spain is also presented.

Determination of Orosomucoid (ORM) Phenotypes by Hybrid Isoelectric Focusing

ORM phenotypes were studied by hybrid isoelectric focusing (HIEF) followed by immunofixation and silver staining. The method was applied to the study of ORM in human serum and minute bloodstains. Given the promising results observed, the use of HIEF for forensic purposes is recommended. © 1988 Taylor & Francis Group, LLC.

PI Mtoyoura: a new PI M subtype found by separator and hybrid isoelectric focusing.

In the course of a population study of alpha 1-antitrypsin polymorphism by separator isoelectric focusing, a variant phenotype having a somewhat narrower spacing than PI M1M3 was observed in a Japanese blood donor. Family studies by hybrid isoelectric focusing in a carrier ampholyte-supplemented immobilized pH gradient from 4.35-4.65 revealed that the products of the responsible gene, PI*Mtoyoura, were extremely close but slightly cathodal to those of PI*M1. The difference in isoelectric point between them corresponded to the resolving limit of isoelectric focusing. For this reason, although the propositus' father was deduced to have the genotype PI*M1/PI*Mtoyoura, the products of these two genes failed to form a double band pattern. Thus, the gene frequency for PI*Mtoyoura was unknown. These findings, however, indicate that a further microheterogeneity in the PI M subtype exists at least in the Japanese.

Analysis of the genetic polymorphism of coagulation factor XIIIB (FXIIIB) by isoelectric focusing.

The genetic variants of the coagulation factor XIIIB (FXIIIB) were analyzed by isoelectric focusing, carried out in agarose gels and followed by immunofixation. The FXIIIB phenotypes were visualized by a combined staining procedure with Coomassie Brilliant Blue R-250 and silver nitrate. Improved resolution was accomplished in polyacrylamide gels by hybrid isoelectric focusing in immobilized pH gradients supplemented with carrier ampholytes. We examined a total of 1,604 unrelated, healthy individuals from Southern Germany. The frequencies for the FXIIIB alleles were B*1 = 0.7581, B*2 = 0.0843, B*3 = 0.1568 and B*4 = 0.0019. The theoretical exclusion rate for disputed paternity is 22.35%.

Hybrid isoelectric focusing: Adsorption of proteins onto immobilized pH gradient matrices and desorption by carrier ampholytes

AbstractComparing the separation patterns achieved by hybrid isoelectric focusing in immobilized pH gradients of 1 and 3 meqv/pH/L buffering power, it is demonstrated that a significant proportion of sample protein is lost in gels with the higher buffering power. A buffering power of 1 meqv/pH/L was found to be the lowest acceptable limit for flat bed gel hybrid isoelectric focusing still ensuring adequate control of the immobilized pH gradient. Protein losses were manifested as diminished overall staining intensity, disappearance of individual zones in dilution series, tailing effects and residual material at the application site, all of the above effects being more pronounced for larger than smaller proteins. Adsorption of proteins to charged binding sites of the immobilized pH gradient offers a plausible explanation for the observed phenomena. The visible effects of adsorption could be reduced by the addition of carrier ampholytes, but not by urea or detergents like Triton X‐100, 3‐(3‐chol‐amidopropyl)‐dimethylammonio‐l‐propanesulfonate (CHAPS) or octyl β‐thio‐glucoside, indicating ionic interactions between the immobilized charged groups, carrier ampholytes and proteins. The adsorption phenomena may strongly affect the quantification of proteins in immobilized pH gradients.

Paraffin oil protected high resolution hybrid isoelectric focusing for the demonstration of substitutions of neutral amino acids in denatured proteins: The case of four human transthyretin (prealbumin) variants associated with familial amyloidotic polyneuropathy

AbstractAll four sequenced variants of human plasma transthyretin (TTR) with different substitutions of electrically neutral amino acids and associated with familial amyloidotic polyneuropathy (i. e. TTR(Met30), TTR(Ala60), TTR(Tyr77) and TTR(Ser84)) have been separated from the normal TTR monomer by hybrid isoelectric focusing (HIEF) under denaturing conditions. The pI differences from the normal monomer were between 0.0025 and 0.005 pH units corresponding to 0.7–1.4 % of a full charge unit difference. The detectability of these variants by HIEF under denaturing conditions (7 M urea, 50 mM dithiothreitol, 2 % Triton X–100) was explained assuming interaction of the amino acids at the substitution site with a charged amino acid in its close vicinity. It is proposed to distinguish a high from a low level of resolution for HIEF under denaturing conditions to underline the fact that the studied variants could only be detected by extreme flattening of the pH gradient. To provide protection against modification by the laboratory atmosphere the entire gel, including the electrode wicks and sample application site, was covered with a layer of paraffin oil.