Cathodal Bands Research Articles

Microheterogeneity of Serum β‐Hexosaminidase in Chronic Alcohol Abusers in a Driver's License Regranting Program

Carbohydrate-deficient transferrin (CDT) is one of the best indicators for chronic alcohol abuse and detection of relapse. In this study, we explore the microheterogeneity of β-hexosaminidase (β-HEX) in chronic alcohol abusers in the framework of a driver's license regranting program. Studies have shown that increased serum activity of β-HEX B (isoforms P, I, and B) may be a sensitive marker for chronic alcohol abuse. Here, we describe methodology, limitations, and correlation of β-HEX isoforms with CDT. CDT was assayed at the central laboratory of the Ghent University Hospital by capillary zone electrophoresis, measured on the Capillarys 2™ system and was expressed as a percentage of total serum transferrin (%CDT). Serum of chronic alcohol abusers was compared to nonheavy drinkers using agarose gel isoelectric focusing (IEF). Total β-HEX activity was assayed fluorimetrically following preparative IEF in 81 subjects. β-HEX isoforms were investigated and compared between nonheavy drinkers and heavy drinkers. Agarose gel IEF shows additional cathodal bands in serum of chronic alcohol abusers. Mean total β-HEX activity between pH 6.8 and 7.7, designated as HEX-7, showed the highest correlation with %CDT (r = 0.70, p < 0.0001, n = 68). In a selected subgroup, where CDT could not be quantified (n = 13) because of an atypical electropherogram, HEX-7 was in concordance with either estimated %CDT value or liver enzyme activities. In this proof-of-concept study, we introduce a novel approach to quantify β-HEX isoforms using preparative IEF and fluorimetry. A highly significant correlation of HEX-7 and %CDT has been found. Because of exclusion of the P isoform, HEX-7 could be a useful supplementary marker for detecting chronic alcohol abuse.

Increased expression of β-hexosaminidase α chain in cultured skin fibroblasts from patients with carbohydrate-deficient glycoprotein syndrome type I

Carbohydrate-deficient glycoprotein (CDG) syndrome type I is an autosomal recessive multisystem disorder characterized by multiple serum glycoproteins with deficient oligosaccharide chains. This characteristic under-glycosylation is found in several serum glycoproteins. We studied secreted forms of lysosomal enzymes, β-hexosaminidase and α-fucosidase, in serum from the patients and media of cultured fibroblasts. Both β-hexosaminidase and α-fucosidase activities were increased in sera from three CDG patients. The enzyme activity staining using the fluorogenic substrate-4-methylumbelliferyl- α- l-fucopyranoside after polyacrylamide gel isoelectric focusing revealed abnormal cathodal bands in sera from CDG patients. On the other hand, no abnormal secreted forms of β-hexosaminidase and α-fucosidase were detected in media from cultured CDG fibroblasts by isoelectric focusing and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis. However, SDS-polyacrylamide gel electrophoresis and Western blotting analysis of β-hexosaminidase using anti- β-hexosaminidase A (anti- α+ β chains) antibody, showed an increase of a 55-kDa mature form of the α chain. Northern blotting analysis identified an increase in mRNA levels of β-hexosaminidase α chain in CDG fibroblasts. Although under-glycosylated fractions of these lysosomal enzymes were not detected in cultured fibroblasts, it was suggested that intracellular processing of these lysosomal enzymes in CDG patients might be altered.

Caractérisation des variants génétiques de l'albumine humaine par focalisation isoélectrique

Normal human serum albumin and bisalbuminic fractions from genetic variants of european origin have been studied by ultrathin-layer isoelectric focusing performed on whole sera or after purification of albumin fractions by affinity chromatography on Blue-Trisacryl. Narrow range ampholytes giving pH gradient between 5 and 8, together with the use of 8 M urea, provided suitable patterns allowing to discriminate the various allotypes. In these conditions, both normal albumin and heterozygous variants were microheterogeneous with several main bands; in the case of normal albumin, four major bands were found, while variants exhibited additional bands differing in number and pI. The position of additional bands comparatively to that of normal albumin was consistent with the electrophoretic behavior of variants. Fast moving allotypes exhibited additional bands with more anodal pIs, whereas slow moving variants were characterized by cathodal additional bands. The number and position of these bands allowed to characterize some variants, when they failed to distinguish between some others, identical patterns being correlated to identical mutations arising at different locations on the albumin molecule. These observations indicate that isoelectric focusing could allow a clear distinction of albumin variants, provided that their mutation were different, or could confirm the occurrence of identical mutation when IEF patterns are indistinguishable. In addition to electrophoretic mobilities at various pH, analytical isoelectric focusing could be a useful technique employed as a second step in the identification of allotypes prior to the determination of the structural change characterizing the variant.

Relationship of oligosaccharide modification to the cause of serum thyroxine-binding globulin excess.

T4-binding globulin (TBG), a glycoprotein with four N-glycosyl complex oligosaccharide chains, exhibits sialic acid-dependent microheterogeneity on isoelectric focusing (IEF). Increasing the sialic acid content of TBG increases its anodal IEF mobility and decreases its rate of in vivo degradation, a mechanism for the elevation of serum TBG levels in pregnancy. In this study, the structure of oligosaccharides in TBG from subjects with various conditions associated with TBG excess was determined by measuring the proportion that bound to Concanavalin-A (Con-A). Since oligosaccharides with three or more branches (antennae) attached to the trimannosyl core are excluded from binding to Con-A, the percentage of serum TBG not bound to Con-A (% peak A) represented the portion of TBG molecules with three or more antennae in all oligosaccharide chains interacting with Con-A. Peak A contained the most anodal IEF bands, while the Con-A-bound TBG (peak B) contained the cathodal bands. Serum samples from 10 normal men and 10 premenopausal women did not significantly differ in terms of TBG levels, % peak A, or IEF mobility and were combined as a single group (normal). Eight subjects with elevated serum TBG levels due to inherited TBG excess [62.0 +/- 10.1 (+/- SD) mg/L] or 2 receiving 5-fluorouracil treatment (26.2 and 31.3 mg/L) compared to 20 normal (14.7 +/- 3.3 mg/L) had % peak A values and IEF mobility similar to those in normal subjects. On the other hand, high serum TBG levels in 8 women during the third trimester of pregnancy (39.2 +/- 5.3 mg/L), 2 women taking oral contraceptives (25.7 and 27.0 mg/L), and 3 women with acute hepatitis (34.8 +/- 4.8 mg/L) were associated with significant elevations of % peak A values (5.68 +/- 1.73%, 3.31% and 2.41%, and 3.25 +/- 0.78%, respectively) compared to those in normal subjects (1.33 +/- 0.40%), as well as increased anodal mobility on IEF. Treatment of a man for 3 days with ethinyl estradiol produced similar changes. Using data from densitometry measurements of IEF bands of TBG, the degree of anodal shift was quantitated (anodal index). This index correlated with the % peak A (r = 0.92) in all study subjects. We conclude that increased sites for sialylation, resulting from the increased proportions of triantennary oligosaccharide chains, account for the increased anodal mobility of TBG in hyperestrogenemia and hepatitis. Thus, in these two conditions, a reduced TBG degradation rate resulting from oligosaccharide modification is the likely mechanism of increased serum TBG levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Preliminary evidence for a cholinergic-like system in lichen morphogenesis.

Membrane acetylcholinesterase activity is considered to be a marker for a cholinergic system. When temporarily expressed in differentiating cells other than the nervous or muscular ones, it may play a role in morphogenesis. In the lichen Parmelia caperata (L.) Ach., acetylcholinesterase is histochemically localized mainly in the cell walls and/or membranes of both symbionts just where they proliferate and form well-organized propagation structures, the soredia. The enzyme activity is first detected in a few algae undergoing aplanosporogenesis and later in medullary hyphae that reach the dividing algae by elongating perpendicularly to the thallus surface. This histochemical pattern that is associated with algal proliferation and oriented hyphal growth is characteristic of early morphogenesis of the soredia; when fully differentiated, they consist of an inner dividing alga and an outer hyphal envelope, both showing cholinesterase activity. Substrate specificity and inhibitor sensitivity of the histochemical staining indicate an acetylcholinesterase-like activity. However, extracts of the thallus areas where soredia develop give four bands of cholinesterase activity on disc electrophoresis: the two cathodal bands have the characteristics of acetylcholinesterase, the others of pseudocholinesterase. One of the latter hydrolyses propionylthiocholine very rapidly. The findings suggest that in lichen symbiosis, a cholinergic-like system participates in regulating morphogenetic processes such as cell division, oriented tip growth and alga-fungus membrane interactions. Environmental stimuli, particularly light, might trigger the development of soredia by modulating the activity of the cholinergic mechanism.

High resolution isoelectric focusing in a narrow pH interval for the phenotyping of vitamin D-binding protein (Gc-globulin).

A routine method for the analysis of Vitamin D-binding protein (Gc-globulin) phenotypes has been developed. The assay is based on isoelectric focusing in a narrow pH interval (4.5-5.4) using Agarose IEF (1%) or polyacrylamide (T5% C3%) gels, followed by immunofixation. When focused in this pH gradient the anodal and cathodal bands of the phenotype Gc 1-2 are separately by 16-18 mm. The "fast" and "slow" bands of the phenotype Gc 1-1 can also be easily distinguished from each other. The isoelectric focusing technique which has been developed resolves the Vitamin D-binding protein phenotypes in a fast, convenient and reproducible manner. Since agarose gels are so easy to cast and process, Agarose IEF is recommended as the matrix of choice.

Cathodal bands in electrophoretograms of creatine kinase isoenzymes in serum collected after cardiac surgery: a poor prognostic sign.

Of 708 patients who had undergone cardiac surgery, the serum of 23 showed one or two enzyme bands cathodal to CK-MM in creatine kinase (CK) isoenzyme electrophoretograms. Postoperative mortality rate during hospitalization was: no bands, 8.8%; one band, 13%, two bands, 63% (p less than 0.001). Patients whose sera showed cathodal bands were slightly older than those without, and their postoperative serum lactate dehydrogenase (LD) activity was greater, with very high proportions of LD 5. The two cathodal bands in patients who died differed in cathodal electrophoretic mobility from the two bands in survivors, implying that different enzyme forms were involved. All cathodal bands were inhibited by reagent containing diadenosine pentaphosphate, as was adenylate kinase from erythrocytes and liver cytoplasm. Mitochondrial CK from liver and presumed mitochondrial CK in serum from a patient with malignancy were not inhibited. We conclude that the appearance of two enzyme bands cathodal to CK-MM, probably representing adenylate kinase and possibly originating from various tissues, is associated with a poor prognosis in patients after cardiac surgery.

Lactate dehydrogenase isozymes in the genus Xenopus: Analyses of complex isozyme patterns

1. 1. Previous work showed that seven out of 13 Xenopus species and subspecies possess a simple pattern of 5 LDH isozymes as it is common to most vertebrates. 2. 2. The remaining six species with more complicated patterns are further analyzed in the present paper. 3. 3. X. l. laevis, X. vestitus and X. wittei are characterized by the appearance of an anodal “sixth band”. Two explanations are given. 4. 4. A comparison of organs in X. borealis reveals the bands additional to the five banded pattern as secondary isozymes. 5. 5. The five bands in the A 4 region in X. ruwenzoriensis, combinations of A subunits in nature as demonstrated by treatment with antibodies against the purified A 4 homopolymer, must be considered as being the result of a duplicated A. locus. 6. 6. The same is possibly true for the closely spaced cathodal bands in X. fraseri. 7. 7. The occurrence of only few expressed duplicated loci among the genus Xenopus indicates progressed diploidization within this genus.

Apparent mitochondrial creatine kinase in the serum of a patient with metastatic cancer to the liver.

Creatine kinase (CK, EC 2.7.3.2) activity in the serum of a patient with metastatic carcinoma migrated as two distinct bands cathodal to the origin and to CK-3 on agarose gel electrophoresis. The more cathodal isoenzyme (CKm-2) is of high molecular mass, is precipitated by ammonium sulfate at 30% of saturation, and is not retarded by Sephadex G-100. Treatment with urea at a concentration of 6 mol/L caused CKm-2 to elute with proteins of lower molecular mass on a G-100 column and shifted the electrophoretic migration to a position just cathodal to the origin (CKm-1). Antibody to CK-1 and CK-2 did not affect the activity of either CKm-1 or CKm-2. Similarities between these cathodal bands of CK activity and mitochondrial CK suggest the mitochondrial origin of these isoenzymes. These cathodal CK isoenzymes reacted unpredictably with different commercial reagent systems for determination of CK activity in serum or in agarose gel.

Arylesterase isoenzymes and activity in normal healthy adults and in patients with cancer and with other diseases

Serum arylesterase isozyme patterns were studied in 184 normal healthy individuals, 290 cancer patients and 466 patients with various diseases. No abnormal patterns were seen in the normal healthy subjects. Several abnormal patterns found in the group of cancer patients and patients with various diseases are described. In the majority of patients with cancer of the liver there is an abnormal additional cathodal band. The most cathodal band in normals or the two most cathodal bands in the patients with hepatoma with double cathodal bands stained for cholinesterase as well as for arylesterase. We also studied serum arylesterase activity on the basis of the kinetic release of beta-naphthol in these groups. The mean activity in normal healthy individuals agrees with that reported earlier. In patients with cancer and with miscellaneous other diseases, the mean activity is lower but the range of values in the two groups is very wide.

Two HLA-linked loci controlling the fourth component of human complement.

An electrophoretic polymorphism of the fourth component of human complement (C4) is described. Three patterns of bands of C4 were observed in EDTA plasma from a panel of unrelated blood donors and family members by using the technique of immunofixation electrophoresis. These patterns consisted of four fast-moving anodal bands (F), four slow-moving cathodal bands (S), or a combination of both the F and S bands (FS). The C4 patterns of bands were observed in EDTA plasma and not in serum. Family studies showed that this polymorphism of C4 did not segregate with HLA histocompatibility genes in a fashion governed by two codominant alleles at a single genetic locus. The family data are in agreement with the hypothesis that two different genetic loci control the electrophoretic patterns of C4. One locus controls the presence (F) or absence (f0) of the four anodal (F) bands and the other controls the presence (S) or absence (s0) of the four cathodal (S) bands. The C4F and C4S loci are both closely linked to HLA-B.

Chido and Rodgers blood groups are distinct antigenic components of human complement C4.

THE HLA complex consists of many closely linked genes which code for a variety of characters, particularly the expression of cell surface antigens found on lymphocytes (HLA-A, B, C, DR antigens) or erythrocytes (Chido and Rodgers blood groups) and polymorphic variation of certain complement components (Bf, C2, C4). We1 have recently suggested that the polymorphism of the fourth component of human complement (C4), detected by immunofixation electrophoresis, is controlled by two genes linked to HLA and not by codominant alleles at a single locus as previously described2,3. One C4 variant, designated F, is controlled by one locus with alleles determining the presence (F+) or absence (f0) of four fast-moving anodal bands of C4 while the second variant, S, is controlled by a second locus with alleles determining the presence (S+) or absence (s0) of four slow-moving cathodal bands. We noted that C4 F individuals homozygous for the s0 allele and lacking the four cathodal bands were always Chido (Cha) negative, whereas individuals homozygous for the f0 allele and lacking the four anodal bands of C4 (C4 S) were always Rodgers (Rga) negative. This intriguing observation led us to study the relationship of Chido and Rodgers red cell antigens, which are also found in serum and C4. We show here that Chido and Rodgers are distinct antigenic components of human C4, a finding which provides a biological explanation as to why these unique red cell antigens are controlled by genes of the HLA complex.

Biochemical basis of hybrid vigour. The genetics of grain weight of Oryza sativa

Four cathodal bands (C1, C2, C3 and C4) of esterase (E1, C1. 3.1) were correlated with the grain weight of rice (Oryza sativa L.). Zymogram patterns indicated intensity differences among these bands infinegrain and coarse-grain varieties. Bands C1. and C2 were dark in fine grain varieties whereas C3 and C4 were dark in coarse grain varieties. These bands were specific to endosperm. Observations on fine-grain (Kalanamak), coarse grain (SR(26)B) varieties and their reciprocal hybrids indicated the presence of 4 esterase loci G1, G2, G3 and G4, corresponding to bands C1, C2, C3 and C4, respectively. A possible model for heterosis in grain weight of rice was proposed which supports the dominance theory of heterosis. In hybrid vigour the 4 esterase loci appear to be associated with grain weight and they complemented each other in an additive manner.

Further evidence of molecular alteration and aberration of erythrocyte pyruvate kinase

Human erythrocyte pyruvate kinase (PK) was separated by electrophoresis into two bands with different migration from L-type PK of the liver. During erythrocyte aging, alterations of these electrophoretic patterns were observed in which younger cells had denser cathodal bands than older cells. Erythrocyte PK seems to be closely related to L-type PK since erythrocyte PK could be converted to L-type PK and has the same antigenicity. Aberrant PK molecules were shown to exist by electrophoretic and immunologie studies as well as the usual kinetic studies.

Starch gel electrophoresis of Naja nigricollis venom. Enzymatic and toxicity studies.

Abstract Starch gel electrophoresis of N. nigricollis venom revealed eleven bands, nine were cathodal, one at site of application and one anodal. Phospholipase A activity and indirect hemolysis were demonstrated in the least mobile cathodal bands, C7–9, the eluates of which were not lethal. ATPase activity was present in C7, and also in C4. Proteinase activity was detected in C3. No cholinesterase activity could be detected in the venom or any of its bands. Eluates of the two fast moving cathodal bands, C1 and C2, were lethal to mice and showed none of the tested enzyme activities. Acid boiled venom could be fractionated into a hemolytic fraction with phospholipase A activity, consisting of three bands, C′2–4, and a lethal fraction consisting of one band, C′1.

Studies on the characterization and localization of rat placental esterases.

The nonspecific carboxylic esterases of the rat placenta were studied by electrophoretic, biochemical and histochemical methods, with naphthyl and naphthol AS esters as substrates. The enzymes were characterized by the effects upon their activity of a group of organophosphate inhibitors and of eserine, p-chloromercuribenzoate and urea solution. After electrophoresis at pH 7.5, zymograms show an extensive pattern of anodal and cathodal bands similar for both maternal and foetal portions of the placenta. The B (carboxyl) esterases are sensitive to inhibition with E600 and diisopropyl phosphorofluoridate at very low concentrations. The A (aryl) and C (acetyl) esterases undergo progressive inhibition at concentrations greater than 0.01 mM, and only one A esterase resists a 1.0 mM concentration. Mipafox resembles the other organophosphates in its effect on esterases only when it is used fresh. Comparison of the zymograms with the histochemical preparations permits the localization of some of the individual enzyme species separated by electrophoresis.

Comparative studies of the isozymes of lactic dehydrogenase in rabbit and man. Observations during development and in tissue culture.

During development of rabbit tissues, characteristic sequential alterations in the LDH isozyme pattern occur, and consist for liver and muscle in loss of the most rapidly migrating anodal bands, and increased activity in the cathodal bands and slower migrating anodal bands. In heart the reverse changes were observed. Comparison of the isozyme patterns observed in various fetal and adult human tissues suggests that these same sequential alterations probably occur. A species-specific isozyme pattern is obtained in long term culture of rabbit, chick, and human cells. The alterations in tissue culture are characterized by a gradual redistribution of total LDH activity in which there is decreased intensity of rapidly migrating anodal bands. These sequential alterations are independent of the organ of origin. The number of bands observed in the starch gel is partly dependent upon the total activities applied. Isozymes may provide a convenient method for determining the species of origin of cell lines in common use and for investigating the effects of various alterations in the in vitro environment on cells grown in tissue culture.

Dialyzable cofactor in nerve growth promoting protein from mouse salivary glands.

Cohen's method for preparing the nerve growth factor from mouse submaxillary glands was followed to the last ammonium sulfate fraction. Further purification was accomplished with carboxymethyl- and diethylaminoethyl- column chromatography. Three peaks were obtained for each column; the third peak obtained with diethylaminoethyl cellulose was the most active. Electrophoresis of this active fraction produced one anodal and two cathodal bands. Each band was inactive for nerve outgrowth. However combinations of the cathodal bands produced 3+ growth. One cathodal band was not dialyzable; the other was dialyzable and negative for ultraviolet absorption at 280 millimicrons.

Sequential alterations of lactic dehydrogenase isozymes during embryonic development and in tissue culture.

Summary1. During development fetal chick liver and muscle show sequential alterations in distribution of LDH isozymes. These changes are characterized by a progressive increase in concentration of cathodal bands relative to anodal bands. In chick heart the LDH isozyme pattern remains relatively constant during development. The number of bands depends on the activity applied to the gel, and with high LDH activity chick heart, muscle and liver 10 days after fertilization show the full tissue complement of LDH isozymes. 2. The chick embryo tissues studied in tissue culture reveal under the conditions employed in this study a common pattern characterized by 1 or 2 heavily stained cathodal bands and 1 or 2 lightly stained anodal bands. During prolonged culture the anodal bands decrease in concentration. Isozymes may prove to be a sensitive index of the maintenance of physiological activities of cells grown in tissue culture.