Agar Gel Electrophoresis Research Articles

Biochemical and Immunogenetical Variability of Proteins in Cestodes Echinococcus granulosus

The paper presents data on electrophoretic variability of proteins in larval stage of Echinococcus granulosus helminths, which indicate that under electrophoresis of proteins of the studied helminth in Polyacrylamide, they fractionate into the following number of fractions: echinococcus fluid −18, extracts of scolices −20 and extracts of helminthic membranes −16 fractions. The comparison of electrophoregrammes of proteins in echinococci of sheep and pigs revealed that in the latter the number of fractions was higher and their electrophoretic mobility was lower. Under electrophoresis in agar gel, the obtained echinococcus antigens fractionated into two main albumin and betaglobulin fractions. The immunization of rabbits by echinococcus antigens induced synthesis of antibodies against both protein fractions. However, merely 70% of sera of immunoreactive rabbits formed precipitation link against the betaglobulin fraction of echinococcus antigen. All rabbit antisera showed obvious precipitation reaction (a si...

Early features of apoptosis detected by four different flow cytometry assays.

The objective of this study was to investigate the sensitivity, specificity and reproducibility of some frequently used apoptosis assays. The degree of apoptosis was tested in two T-lymphoblastoid cell lines, HSB and Jurkat, in which apoptosis was induced by ionizing radiation. HSB and Jurkat samples were taken before, and 0, 2, 4, 6, 8 and 24 h after irradiation with 6 and 10 Gray, or with 10 and 14 Gray, respectively. Four frequently used flow cytometric techniques were evaluated: (i) Annexin V/Propidium Iodide assay, detecting the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, simultaneously with preservation of the membrane integrity; (ii) Terminal deoxynucleotidyl Transferase (TdT) Uridine triphosphate (UTP) nick end labelling (TUNEL), revealing the presence of DNA strand breaks; (iii) DNA-flow cytometry, measuring DNA-stainability (DNA-fragmentation assay) and (iv) Phycoerythrin-labelled (PE) Apo2.7-assay, a monoclonal antibody against 7A6 antigen, a protein, which becomes exposed upon the mitochondrial membrane during apoptosis. As a general standard for identifying that apoptosis had occurred, the cells were assessed for the presence of DNA-laddering on agar gel electrophoresis and by demonstration of characteristic cell morphology. Results were as follows: Fluorescein Isothiocyanate (FITC)-labelled Annexin V/Propidium iodide flow cytometry appeared to be the most sensitive, the most specific and the most user-friendly test for measurement of apoptosis of cells in culture conditions in suspension. The expression of 7A6 antigen on the mitochondrial membrane appeared to be not specific for apoptotic cell death.

Ethanol-induced cell death in cultured rat astroglia.

Because of the important role of glial cells in brain maturation and reports on their delayed proliferation following ethanol exposure, it was considered of interest to investigate the mechanism of ethanol action on these cells. Biochemical parameters related to the apoptotic and necrotic processes in astroglial cells exposed for 1 week to 50 and 100 mM ethanol were examined. Ethanol increased intracellular calcium levels without changing transglutaminase activity and nitrite levels. Moreover, DNA fragmentation was noted with flow cytometry and with the random oligonucleotide primed synthesis assay but neither following agar gel electrophoresis nor in UV microscopy of cell nuclei. The DNA patterns obtained were different from these seen in programmed cell death. Additionally, immunocytochemical analysis showed greater fragility of astrocytes than oligodendrocytes to ethanol. These results support the hypothesis that astroglial cells in vitro exposed to ethanol die due to necrotic but not apoptotic mechanisms.

Hemoglobin Genotypes in Turbot (Scophthalmus maximus Rafinesque), Their Oxygen Affinity Properties and Relation With Growth

A growth trial lasting 15 months was conducted with 80 individually tagged turbot at four temperatures (10, 13, 16, and 19°C). Haemoglobins of the individually tagged fish were analysed by agar gel electrophoresis and the fish segregated into three genotypes named; Hb-I(1/1), Hb-I(1/2) and Hb-I(2/2). The genotype specific growth rates of the fish were analysed. In addition, the oxygen affinity properties of the hemoglobin genotypes were studied at 10 and 19°C. The genotype Hb-I(2/2) displayed the overall best growth at 10, 13 and 16°C, but no differences in growth among genotypes were seen at 19°C. Further, Hb-I(2/2) had the highest oxygen affinity at 10 and 19°C, whereas Hb-I(1/1) had the lowest oxygen affinity at both temperatures. In conclusion, the hemoglobin polymorphism in turbot seems to be correlated with physiological performance, and thus useful in future selection programs for turbot as a correlated trait with production characteristics.

The primary structure and enzymic properties of porcine prochymosin and chymosin

Preliminary investigations by N-terminal sequence analysis showed that pig and calf chymosin possessed 80% amino acid sequence identity but showed considerable differences in their enzymatic properties. A comparison of their structures may therefore contribute to an understanding of the significance of the amino acid residues responsible for the differences in these properties. Pig chymosis was extracted from the stomachs of pigs of less than 3 weeks of age, and was purified by ion exchange chromatography. Half of the primary structure was determined by amino acid sequencing and the complete structure was deduced from a cloned chymosin cDNA. Results showed that the zymogen showed 81% sequence identity with calf prochymosin and 57% identity with pig pepsinogen A. The size of the propart and location of the residue which becomes the N-terminus in the active molecule were the same in the prochymosins. The maximum general proteolytic activity at pH 3.5 of pig chymosin was 2–3% of that of the activity of pig pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of pig chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.3 of stomach extracts of individual pigs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or N-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and pig chymosin differ in the following positions (pig pepsin numbering, subsites in parentheses): Ser 12 Thr (S 4), Leu 30 Val (S 1/S 3), His 74 Gln (S 2′), Val 111 Ile (S 1/S 3), Lys 220 Met (S 4). With regard to the low general proteolytic activity of pig chymosin, the substitution Asp 303 Val relative to calf chymosin may contribute to an explanation of this.

Long-term beta-blocker treatment prevents chronic creatine kinase and lactate dehydrogenase system changes in rat hearts after myocardial infarction

Objectives. We tested the hypothesis that long-term beta-blocker treatment with bisoprolol prevents creatine kinase (CK) and lactate dehydrogenase system changes that occur after chronic myocardial infarction.Background. The mechanism of the beneficial effect of beta-blocker therapy is still unclear.Methods. Six groups of rats were studied. Sham operated (sham) and hearts with ligated left anterior descending coronary artery (myocardial infarction) were untreated, treated early (beginning 30 min after infarction) or treated late (beginning 14 days after infarction). After 8 weeks, hearts were isolated and buffer perfused isovolumetrically. With a left ventricular balloon, mechanical function was recorded at all end-diastolic pressure of 10 mm Hg. Biopsy samples of noninfarcted left ventricular tissue were taken. Enzyme activities were measured spectrophotometrically; isoenzymes were separated by agar gel electrophoresis; and total creatine levels were measured with high performance liquid chromatography.Results. The decrease in left ventricular developed pressure in untreated hearts (120 ± 9 vs. 104 ± 5 mm Hg [mean ± SE], p < 0.05, sham vs. myocardial infarction) after myocardial infarction was prevented by early treatment (118 ± 9 vs. 113 ± 4 mm Hg). Late treatment failed to improve mechanical function. Reduction of CK activity occurring in untreated infarcted hearts (6.4 ± 0.3 vs. 5.1 ± 0.3 IU/mg protein, p < 0.05, sham vs. myocardial infarction) was prevented by early beta-blocker therapy. The increase in CK isoenzyme BB and MB levels, decrease in mitochondrial CK isoenzyme levels and increase in anaerobic lactate dehydrogenase isoenzyme levels in untreated infarcted hearts did not occur during bisoprolol treatment. The decrease in total creatine levels after myocardial infarction (74.2 ± 4.9 vs. 54.9 ± 3.3 nmol/mg protein, p < 0.05, sham vs. myocardial infarction) was prevented by bisoprolol treatment. Early treatment was more effective than late therapy in preventing CK and lactate dehydrogenase system changes. In addition, in sham hearts, a 40% increase of creatine levels above normal levels was detected.Conclusions. Bisoprolol prevented changes in CK and lactate dehydrogenase systems that occur after myocardial infarction. These observations may be related to the beneficial effects of long-term beta-blocker treatment in patients with chronic myocardial infarction.

Genetic analysis of population structure of tusk in the North Atlantic

To study the population structure of tusk Brosme brosme in the North Atlantic, samples from six fishing grounds were collected: St Kilda; Storegga; Faroe Islands; Rockall bank; Davis Strait and East Greenland. In addition two samples from the Norwegian coast; Kragerø and Øygarden, were included. Nine enzymes and haemoglobin were analysed by starch gel and agar gel electrophoresis. In addition isoelectric focusing (IEF) was carried out. At least 22 loci were represented. Polymorphisms were shown in eight loci and average heterozygosity was found to be about 0.05. Highest heterozygosity was found in haemoglobin analysed by Smithies buffer pH 8.6. Malate dehydrogenase (MDH‐2) analysed in IEF (pH 3.5‐9.5), and haemoglobin were found as valuable tools for studies of stock structure in tusk. In the Greenland samples only one of the MDH‐2 heterozygotes was found, while in the East Atlantic both the observed heterozygotes were more frequent. The frequencies of the the Hb*b allele were about 0.40 and 0.70 in the samples from east and west Atlantic respectively.

Ontogeny of haemoglobin polymorphism in cod

Haemoglobins of 0‐group cod from 30 to 150 mm length were analysed by agar gel electrophoresis and isoelectric focusing to identify the lower size limit for determination of the haemoglobin polymorphisms in cod; both the main system which has been known for more than 30 years and the recently described subgroups. The juvenile fish studied had higher concentration of the HbII‐component (split into three bands at these fish sizes) compared to older fish. The main haemoglobin types could be distinguished clearly and reliably at a fish size of about 35 mm and above. The subgroups of the HbI‐components, however, could not be identified reliably until the cod were about 80 mm.

The SCID mouse as a model for multiple myeloma.

The SCID mouse was investigated as a potential animal model for human multiple myeloma (MM). Duplicate samples of bone marrow mononuclear cells (BMMC) and/or peripheral blood mononuclear cells (PBMC) of six MM patients in different clinical phases and one patient with monoclonal gammapathy of undetermined significance (MGUS) were injected intraperitoneally into SCID mice. Human immunoglobulins (Ig) in the SCID sera were quantified with a light-chain isotype-specific ELISA, and their monoclonality biochemically characterized, using a sensitive immunoblotting technique after agar gel electrophoresis. Successful transplantation of bone marrow derived-tumour cells in SCID mice was obtained with BMCC of two MM patients with progressive disease. Human plasma cells were detected in the mesenteric fat tissue around the pancreas and the spleen. This model in SCID mice may facilitate studies on processes involved in tumour progression and provides a new tool for therapeutic approaches in MM.

Correlation of Est erase Isozymes to Malathion Resistance in the Small Brown Planthopper (Homoptera: Delphacidae)

Variation of esterase and its importance to malathion resistance was investigated in the small brown planthopper, Laodelphax striatellus (Fallen). It was confirmed that increased activity of malathion carboxylesterase was one of the most important correlative factors for malathion resistance and that the insensitivity of acetylcholinesterase to malaoxon was a rather minor factor in the small brown planthopper. Increased carhoxylesterase activity was detected by aliesterase activity. Several aliesterase isozymes, which were separated by isoelectrofocusing, showed a high activity in the resistant strain. Among them, activities of two isozymes (pI = 5.3 and 5.0) were correlated with malathion resistance, and they were found to be able to hydrolyze both aliphatic esters and 14C-malathion. These two isozymes were found to be components of an E7 band separated by agar gel electrophoresis, a band already known to be controlled by a single, codominant, autosomal factor.

Establishment of a Mucin‐Degrading Intestinal Microflora During the First Two Years of Human Life

The establishment of a mucin‐degrading intestinal microflora was followed in 30 healthy Swedish children by agar gel electrophoresis of fecal samples taken at 0, 1, 3, 6, 9, 12, 15, 18, 21, and 24 months of age. At 3 months, 18 children showed a limited degree of mucin degradation, while 12 children showed no degradation. In children exclusively breast‐fed for at least 4 months, mucin degradation was initiated significantly later than in children who received formula, with or without breast milk, by 1 month of age. Complete degradation of mucin was seen in 21 children after 1 year of age and in all children at 2 years. A positive correlation between increasing age and increased degradation of mucin was found between birth and 1 month and between 6 and 9 months, after standardization for the period of exposure to foods other than breast milk.

Establishment of a mucin-degrading intestinal microflora during the first two years of human life.

The establishment of a mucin-degrading intestinal microflora was followed in 30 healthy Swedish children by agar gel electrophoresis of fecal samples taken at 0, 1, 3, 6, 9, 12, 15, 18, 21, and 24 months of age. At 3 months, 18 children showed a limited degree of mucin degradation, while 12 children showed no degradation. In children exclusively breast-fed for at least 4 months, mucin degradation was initiated significantly later than in children who received formula, with or without breast milk, by 1 month of age. Complete degradation of mucin was seen in 21 children after 1 year of age and in all children at 2 years. A positive correlation between increasing age and increased degradation of mucin was found between birth and 1 month and between 6 and 9 months, after standardization for the period of exposure to foods other than breast milk.

Study of biochemical gene markers in the Mongolian gerbil (Meriones unguiculatus)

The present study was conducted to analyze biochemical marker genes and to clarify genetic variation in 4 Mongolian gerbil (Meriones unguiculatus) strains (MGS/Sea, MON/Jms Gbs, Kwl: Mongolian Gerbil and Hos: Mongolian gerbil) maintained in Japan. Cellulose acetate, starch gel or agar gel electrophoresis was used. Of the 23 kinds of proteins detected, variation was found only in liver acid phosphatase (Acp2); no variation in other proteins was found within or among strains. The results suggest that these 4 Mongolian gerbil strains are genetically similar. A strain difference was found in the Acp2 locus of liver acid phosphatase. The Kwl: Mongolian Gerbil strain had a band of Acp2 locus, whereas MGS/Sea, MON/Jms and Hos: Mongolian gerbil strains did not.

Activation of porcine pepsinogen A

Reaction products formed during activation of porcine pepsinogen A at pH 2 were characterized by native agar-gel electrophoresis and by denaturing SDS/PAGE. The results revealed the presence of non-covalent intermediates between prosegment peptides and pepsin. The complexes Leu1p-Leu44p/pepsin and Leu1p-Leu16p/pepsin were isolated (the prosegment residues are characterized by the suffix p; numbering of residues starts again from the N-terminus of pepsin). Relative to mature pepsin, the inherent milk-clotting activities of the intermediates were 3% and 18%, respectively. The intermediates were incubated at pH 8.5 for 20 min at 28 degrees C and the residual proteolytic activities were tested at pH 2. The stabilities at pH 8.5 were between those of pepsinogen and pepsin, Leu1p-Leu44p/pepsin being most stable. The implications of these findings for the conformational changes that occur during the activation of pepsinogen are discussed.

Beta-thalassemia mutations in the Portuguese; high frequencies of two alleles in restricted populations.

We report the characterization of seven different beta-thalassemia mutations in 131 newly diagnosed Portuguese beta-thalassemia heterozygotes. Methodology included the detection of abnormal fragments by agar gel electrophoresis of PCR-amplified DNA fragments after digestion with specific restriction endonucleases, as well as hybridization with synthetic nucleotide probes and sequencing of amplified DNA. Four mutations, including the newly discovered TGG-->TGA change at codon 15, occurred in excess of 10% and accounted for some 90% of the beta-thalassemia alleles in this population. The geographical distribution is uneven; the TGG-->TGA mutation at codon 15 was primarily observed in the coastal region north of Lisbon, while the IVS-I-6 (T-->C) mutation was confined to the central part of the country.

Inhibition of interleukin 1-mediated proteoglycan degradation in bovine articular cartilage explants by addition of sodium hyaluronate

Summary The effect of exogenous hyaluronate on normal cartilage metabolism and interleukin-1 (il-1)-induced cartilage matrix degradation was investigated in a bovine cartilage explant culture system. Addition of hyaluronate at a concentration of 1.5 mg/ml to cartilage culture explants consistently decreased normal proteoglycan release from the matrix to a value less than that found in control cultures. Addition of 1.5 mg of hyaluronate/ml to il-1 stimulated cartilage culture systems reduced proteoglycan release from the matrix by 83 to 113%. The reduction in control and il-1-stimulated proteoglycan degradation by hyaluronate had a concentration-dependent trend. Evaluation of alterations in protein (enzyme) release by il-1-stimulated chondrocytes after introduction of hyaluronate was evaluated by use of sodium dodecyl sulfate agar gel electrophoresis of cartilage-conditioned media. The quantity or the molecular weight profile of il-1-induced proteins did not differ after introduction of hyaluronate into the culture system. Results indicate that introduction of high molecular weight hyaluronate into cartilage culture systems results in a decrease in proteoglycan release from the matrix in control systems, as well as in cultures incubated with il-1. Because il-1-stimulated protein synthesis by chondrocytes remains unchanged after addition of exogenous hyaluronate, the mechanism of inhibition of matrix degradation does not appear to be interference with binding of il-1 to chondrocytes or to be inhibition of the production of neutral metalloproteases, including stromelysin.

Growth rate related to genotype of 0-group cod at three environmental temperatures

Abstract Growth rates of cod of an initial weight of about one gram were studied at three environmental temperatures (6, 10, and 14° C) for about 70 days. Samples of all three groups of fish were genotyped for haemoglobin and several tissue enzymes by agar or starch gel electrophoresis. Size at termination of the experiment, regarded to represent individual growth rate, was studied separately for frequent genotypes within temperature groups. No clear associations between mean growth rate and genotypes of the enzymes LDH or PGI were found at any temperature. However, fish of the genotype Hb-I(2/2) showed the highest mean growth rate regardless of environmental temperature, and fish of genotype Hb-1(1/1) grew on average slowest at the two higher temperatures. This observation was partly in contrast to earlier findings indicating that the performance of the different genotypes is temperature dependent, with Hb-1(2/2) as the more efficient at lowest temperatures.

Separation of porcine pepsinogen A and progastricsin. Sequencing of the first 73 amino acid residues in progastricsin

Porcine pepsinogen A (EC 3.4.23.1) and progastricsin (EC 3.4.23.3) have been separated by chromatography on DEAE-cellulose followed by chromatography on DEAE-Sepharose. Agar gel electrophoresis at pH 6.0 showed the presence of three components of pepsinogen A and two of progastricsin. During activation at pH 2 a segment of 43 amino acid residues (the prosegment peptide) is cleaved from the N-terminus of progastricsin. The sequence of this was determined; in addition, the first 30 residues of gastricsin were sequenced. The sequence of the first 73 amino acid residues of progastricsin shows an overall identity with progastricsins from man, monkey and rat of 67%. The overall identity with other zymogens for gastric proteinases is 27%. The highly conserved Lys36p (pig pepsinogen A numbering) is changed to Arg in porcine progastricsin.

Macroamylasemia in a 5‐Year‐Old Girl

SummaryIn macroamylasemia, a macromolecular complex consisting of amylase linked to immunoglobulins circulates in the plasma and usually causes benign hyperamylasemia with low or normal amylasuria. Macroamylasemia is extremely rare in pediatric patients as it has been described in only four patients. We report herein the case of a 5‐year‐old girl with abdominal pain and macroamylasemia. To recognize macroamylase, we used agar gel electrophoresis, PEG precipitation, and fast protein liquid chromatography (FPLC). In our case, FPLC was found to be the most reliable method for the identification of the macromolecular complex. Macroamylasemia is merely a biochemical abnormality that is not associated with any kind of pathology. Its identification is therefore essential in order to avoid a wrong diagnosis, i.e., pancreatitis, with consequent inappropriate therapies.

Analysis of the Survival of Mature Human Eosinophils: Interleukin-5 Prevents Apoptosis in Mature Human Eosinophils

AbstractWe and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in an in vitro liquid culture system. Mature eosinophils did not proliferate but their survival was maintained in the presence of IL-5. Using this culture system, we investigated the mechanism of IL-5–mediated survival. In the absence of human IL-5 (hlL-5) mature eosinophils succumbed after 4 days, while in the presence of hlL-5 they survived up to 10 days. When DNA extracts of cultured eosinophils were analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hlL-5, while no significant DNA fragmentation was observed in the culture with hlL-5 for 48 hours. The DNA fragmentation appeared as early as 6 to 12 hours after hlL-5 deprivation. Concomitantly, IL-5 stimulated total RNA and protein synthesis, but did not induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protection of apoptosis by hlL-5, some new RNA and protein synthesis appeared to be required in this phenomena. These findings indicate that IL-5 maintains survival of mature eosinophils with induction of new RNA and protein synthesis, thus leading to the inhibition of apoptosis.