Changes In Electrophoretic Pattern Research Articles

The biochemical changes of ultrasonic waves on protein metabolism

The biochemical changes in liver marker enzymes and protein metabolism after repeated exposure todiagnostic ultrasonic waves in rabbits. Thirty-five male rabbits were used for the study; animals wereclassified to five equal groups. Group I (control group) not exposed to ultrasound (control group) whileother groups (II, III, IV and V) were exposed to diagnostic ultrasound (5.5 MHz) 2 times per week fordifferent time (15, 30, 45 and 60min) for 5 weeks, respectively. Our results revealed that a highlysignificant increase of serum liver marker enzymes (ALT, AST, GGT, Alp activities, total protein),protein concentration in liver tissue, and changes in protein electrophoresis were observed after ten timesof ultrasound exposure. Also a significant increase of serum urea and uric acid concentrations wereobserved in groups III, IV and V. with changes in electrophoretic pattern of liver protein electrophoresisafter five and ten times of exposure to ultrasound. Our results showed that, ultrasound exposure hadpositive anabolic effect on protein metabolism when used for short period as 15 min. However, it had anegative effect on protein metabolism when used for long durations more than 15 min of exposure

Changes in the electrophoretic pattern of glucosidases during apple seeds stratification

The technique of localization of glucosidases on disc electropherograms, based on reduction of 2,3,5-triphenyltetrazolium chloride by enzymatically liberated sugar is described. The changes in electrophoretic patterns of amygdaline, phloridzin and p-nitrophenyl-β-glucoside hydrolysing glucosidases during apple seed stratification were studied. The changes were correlated with earlier described changes in endogenous apple seed amygdaline and phloridzin contents.

Changes in electrophoretic patterns of protein in several tissues of the silkworm, Bombyx mori L., during metamorphosis

Changes in electrophoretic patterns of protein in several tissues of the silkworm, Bombyx mori L., during metamorphosis

Developmental changes in electrophoretic patterns of sericin in the silkgland of Bombyx mori

Developmental changes in electrophoretic patterns of sericin in the silkgland of Bombyx mori

Changes in cathepsin gene expression and relative enzymatic activity during gilthead sea bream oogenesis

The aim of this study was to provide evidence on the modulation of lysosomal enzymes in terms of both gene expression and enzymatic activity during follicle maturation. For this purpose three lysosomal enzymes, cathepsins B, D, and L, were studied in relation to yolk formation and degradation, during the main phases of ovarian follicle growth in the pelagophil species, the sea bream Sparus aurata. Specific attention was focused on the gene expression quantification method, on the assay of enzymatic activities, and on the relationship between the proteolytic cleavage of yolk proteins (YPs), cathepsin gene expression and cathepsin activities. For the gene expression study, the cathepsins B-like and L-like mRNAs were isolated and partially or fully characterized, respectively; the sequences were used as design specific primers for the quantification of cathepsin gene expression by real-time PCR, in follicles at different stages of maturation. The enzymatic assays for cathepsins B, D, and L were optimized in terms of specificity, sensitivity and reliability, using specific substrates and inhibitors. In ovulated eggs, the lipovitellin I (LV I) was degraded and the changes in electrophoretic pattern were preceded by an increase in the activity of a cysteine proteinase, cathepsin L, and its mRNA. Cathepsin B did not appear to be involved in YP changes during the final maturation stage.

INFLUENCE OF PARAQUAT, GLYPHOSATE, AND CADMIUM ON THE ACTIVITY OF SOME SERUM ENZYMES AND PROTEIN ELECTROPHORETIC BEHAVIOR (IN VITRO)

In vitro study for the determination of the toxicity of some pesticides (gly-phospate and paraquat) and cadmium chloride (CdCl2) on the activities of serum acetylcholinesterase (AChE), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AlP), and acid phosphatase (AcP) is described. Changes in electrophoretic patterns of serum proteins were also tested. Results revealed that glyphosate was effective on all enzymes except AcP. Its IC50 values (the concentration of compound that inhibits 50% of the enzyme activity in 1 h at 37°C) were 714.3, 750, 54.2, 270.8, and 71.4 mM for AChE, LDH, AST, ALT, and AlP, respectively. The inhibitory effect of paraquat varied markedly among all enzymes. The IC50 values of paraquat were 321.4 and 750 mM for AST and ALT, respectively. It had mild effect on AChE and LDH; and no effect on the activities of AlP and AcP. The effect of CdCl2 was pronounced with AChE, ALT, AlP, and AcP, and no effect on LDH and AST was found. The corresponding IC50 values were 77.7, 22.2, 33.3, and 83.3 mM for AChE, ALT, AlP, and AcP, respectively. Polyacrylamide gel electrophoretic patterns of serum proteins showed marked differences with glyphosate and CdCl2 but not with paraquat. The results suggest that the in vitro enzyme-activity test seems to have a potential for the assessment of pesticide and heavy metal toxicity.

Simultaneous analysis of serum immunoglobulins in patients with M protein using cellulose acetate membrane isoelectric focusing.

We developed a method for the simultaneous analysis of microheterogeneity of human serum IgG, IgA, IgM, IgD, and IgE, and serum protein pattern using cellulose acetate membrane isoelectric focusing, and analyzed in 11 healthy subjects and 67 patients with M protein (17 cases of multiple myeloma [MM] and 50 cases of monoclonal gammopathy of undetermined significance [MGUS]). Using this method, bands indicating the microheterogeneity of each immunoglobulin could clearly be detected.Among healthy subjects, the detected IgG, IgA, and IgM bands did not vary, but the detected IgE and IgD bands did vary. Therefore, IgA, IgM, and IgG were selected for comparison of serum immunoglobulins in MM and in MGUS. In the IgA-type M protein group, normal IgM and IgG bands were decreased in MM patients compared to MGUS patients, while the M band and other bands were increased in MM patients compared to MGUS patients, but the differences between the two groups were not significant. In the IgG-type M protein group, normal IgM, IgA, and IgG were significantly decreased in MM patients compared to MGUS patients. We examined the changes in electrophoretic pattern in six MM patients and eight MGUS patients with IgA-type M protein after neuraminidase treatment. The width of the M band in MM patients with IgA-type M protein decreased with neuraminidase treatment. On the other hand, the width of the M band in MGUS patients with IgA-type M protein increased with neuraminidase treatment. We concluded that the decrease of the normal immunoglobulins in MM patients with IgG type M protein could be detected by this method, and IgA type of M protein binding sugar chain were different between MM and MGUS patients.

Lipid apheresis in an animal model causes in vivo changes in lipoprotein electrophoretic patterns.

Lipid apheresis, a new extracorporeal procedure based on plasma delipidation and showing promise as a possible treatment for atherosclerosis, was recently reported for the first time from this laboratory [Cham et al., J Clin Apheresis 10:61-69, 1995]. In the present study lipid apheresis was applied to hypercholesterolemic and normocholesterolemic roosters to examine its effect on plasma lipoprotein particles. This procedure resulted in conspicuous changes in electrophoretic patterns of plasma lipoproteins. The electrophoretic mobilities of all the lipoprotein fractions had changed considerably. Lipid stainable material was present in at least three bands in the alpha-globulin area. In particular, changes in the electrophoretic region of high-density lipoproteins were observed. Lipid apheresis markedly induced the anti-atherogenic pre- beta-high-density lipoproteins. The observed changes induced by lipid apheresis were more pronounced in the hyperlipidemic animals compared with the normocholesterolemic controls. A novel pre-alpha-lipoprotein band was observed soon after lipid apheresis. This lipoprotein band had a density larger than 1.21. At approximately 150 minutes after lipid apheresis, the electrophoretic pattern had almost returned to its original base pattern. Lipid apheresis results in plasma lipoprotein changes which may induce reverse cholesterol transport and shows promise as a possible treatment of atherosclerosis.

Effect of auxin on cell wall glycanhydrolytic enzymes in epicotyls of Cicer arietinum

Cell wall glycanhydrolytic enzymes have been related to cell wall loosening and cell growth, although the mechanism of this relationship has not been clarified. Since auxins are plant hormones that stimulate growth in elongating organs, in the present work we studied the effect of auxin on cell wall glycanhydrolytic enzymes, which were extracted with LiCl. Our results show that incubation of sections of Cicer arietinum epicotyls with indoleacetic acid elicit some minor changes in electrophoretic patterns of cell wall proteins when compared with control sections. This indicates that there is no appearance of a specific polypeptide synthesized de novo in response to the hormone, although there are increases in the intensity of some of the polypeptides, which could indicate an enhancement of wall protein biosynthesis. Brief incubation with IAA led to a general increase in the specific activities of these different cell wall enzyme fractions separated by chromatography, with the exception of the α‐fraction, with α‐galactosidase activity. Longer incubation resulted in an increase in the amount of protein associated with some of the enzyme fractions. In particular, it induced a large increase in the amount of protein associated with the β111‐galactosidase fraction that is involved in the autolytic process of cell walls of chick‐pea epicotyls. Our results indicate that auxin‐enhanced growth could be the result of the action of the hormone al the level of the cell wall glycanhydrolytic proteins that have been related to the wall‐loosening process.

Capillary electrophoresis of rat liver microsomes in polymer solutions.

Rat liver microsome components were separated by capillary zone electrophoresis in buffers containing substituted agarose, agarose crosslinked polyacrylamide, polyacrylamide, polyethylene glycol and dextran of various molecular weights. The best resolution of the components was obtained with polymers of 10-21 nm geometric mean radius. Both the crude and the purified preparations of microsomes exhibit a single major peak. In a Tris-borate-EDTA (TBE) buffer, containing polyacrylamide of 5 x 10(6) molecular weight, it has a retardation coefficient, KR, of 0.77 +/- 0.02. Translation of the KR value to geometric mean radii, R, on the basis of the standard curve applicable to polymer solutions, KR vs. R, with polystyrene carboxylate size standards in both dextran and polyacrylamide solution allows one to estimate a value of R as 13-16 nm for the major microsome component. The value is smaller than expected from electron microscopic measurements (100-250 nm), possibly due to the chemical and geometric differences between microsomes and the polystyrene particles used as size standards. The crude preparation also contains a minor component which is smaller and less charged than the major component. Another component, apparently very much larger than the major one, is seen in TBE buffer but not in a potassium-N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) buffer and is therefore thought to be an artifact of interaction with borate. After a short incubation under conditions promoting delayed microsome fusion, i.e. in presence of GTP and Mg++ and in the absence of polyethylene glycol, the electrophoretic pattern changes dramatically: it now exhibits five unretarded, highly mobile and, therefore, presumably large components in addition to the two original retarded components of the microsome and a less highly charged species similar in KR to the smaller of the original two components.

Early phases in in vitro culture of tomato cotyledons: Starch accumulation and protein pattern in relation to the hormonal treatment

Tomato cotyledon explants, cultured in vitro in the presence of sucrose, were subjected to different hormonal treatments to establish whether the induction of different organogenic programmes could be correlated with differences in starch accumulation and protein electrophoretic pattern. The cytohistological changes in explants over the first 15 days of culture were studied by light and electron microscopy. It was found that starch accumulation occurs under all conditions, though varying in duration and amount. Over the first 2 days of culture the protein electrophoretic pattern changes in a similar way under all conditions, while after 7 days changes take place which are probably related to the different developmental programmes induced by the treatments.

Interactions of isothiocyanates with egg white proteins

AbstractGlucosinolates and their very highly reactive breakdown products (mainly isothiocyanates = ITC) belong to one of the most important natural toxicants. This paper deals with the interactions of allyl‐, butyl‐, phenyl‐ and benzyl ITC with egg white proteins resulting in the formation of ITC‐protein‐conjugates. The ITC's react with amino groups by forming thiourea derivatives. Such reactions are well described by changes in the amounts of free amino groups and available lysine. Further reaction sites are sulphhydryl side chains of proteins leading to dithiocarbamate esters. The investigations further show that such reactions are accompanied with a decrease in the solubility of the prepared conjugates. Simultaneously a shift of the isoelectric range to a lower value of pH can also be observed. Changes in electrophoretic patterns and mobility can also be seen due to the strong electrophilic attack of ITC to proteins. Comparison of the 4 investigated preparations shows that benzyl‐ITC is the most reactive partner for egg white proteins.

Human Erythrocyte Insulin Receptor Processing Is Affected by the Oxidizing Agent Menadione

Insulin-induced down-regulation of erythrocyte insulin receptors is a simplified model that can provide useful information on the cell surface regulative phenomena and on role of the plasma membrane and cytoskeleton in such physiological processes. Oxidative imbalance was examined since it was shown to play an important role in numerous cellular pathologies as well as in cell aging. Specifically, the free radical inducer menadione was used in order to evaluate if this compound is able to modify (and in which manner) the down-regulation process. Biochemical, biophysical, and ultrastructural approaches were used. The results obtained seem to indicate that menadione-induced oxidative damage was able to decrease the insulin-induced down-regulation process, as measured by binding assays. This effect was accompanied by slight alterations in plasma membrane ultrastructure and insignificant variations in plasma membrane lipid composition. In addition, the decrease in membrane order, measured by electron paramagnetic resonance, which was shown to usually occur during the process of down-regulation, was not observed. In contrast, cytoskeletal protein assembly, as previously shown in other in vitro systems, appeared to be remarkably altered. Such changes in specific cytoskeletal elements could lead to the decrease of down-regulation phenomenon induced by menadione. Changes in electrophoretic pattern of some cytoskeletal proteins (e.g., spectrin) reenforce this hypothesis. Considering the importance of free radicals in cell injury, data reported here could represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be modified by changes in some intracellular molecule redox status and cell ionic homeostasis.

Changes in Electrophoretic Patterns of Oocyte Proteins during Oocyte Maturation in Oryzias latipes.

Changes in the two-dimensional SDS-electrophoretic patterns of extracts of maturing denuded oocytes of the medaka (Oryzias latipes) were surveyed. In oocytes without follicular constituents several proteins became detectable in the area between the acidic and slightly basic proteins on the two-dimensional electrophoretograms, while a few of the protein spots disappeared during the process of oocyte maturation. The former proteins were detected also in oocytes that were induced to mature in vivo without breakdown of the germinal vesicle. Several proteins newly observed in extracts of post-vitellogenic oocytes during maturation after breakdown of the germinal vesicle were also identified by two-dimensional electrophoresis. Of several proteins that exhibited noticeable changes in maturing oocytes, only one spot incorporated 14 C-labeled amino acid during maturation, suggesting that post-translational modification of many proteins occurred during oocyte maturation.

Isoelectric Focusing of Human Hair Keratins: Correlation with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Patterns and Effect of Cosmetic Treatments

A new isoelectric focusing (IEF) technique in polyacrylamide gels with 6M urea and 1.5% Nonidet P40 has been developed to characterize human hair samples. The phenotypes demonstrated with this procedure has been correlated with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns described by other authors. The method described can be applied in the forensic science analysis of a single human hair. Using the same IEF technique we have studied the changes in electrophoretic patterns of cosmetically treated hair. The characteristics of the modifications observed and its utility in forensic science work are also discussed in this paper.

Changes in electrophoretic patterns of acetic acid-insoluble wheat flour proteins during dough mixing

Acetic acid-insoluble (residue) protein fractions of wheat flours and respective doughs were studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS—PAGE). Gel patterns of unreduced SDS buffer extracts from residue fractions showed that dough mixing caused systematic changes in the staining intensity of a group of relatively high molecular weight proteins comprising triplet band proteins and neighboring bands. Together, these proteins were named in this study the triplet zone proteins. Continued mixing of control doughs resulted in a progressive decrease in the intensity of the triplet zone bands, whereas mixing in the presence of potassium iodate (KIO 3 ) and N -ethylmaleimide (NEMI) caused an increase in staining intensity of these bands. These results appear to be related to sulfhydryl-disulfide interchange reactions that have been proposed to occur during dough mixing. Results for doughs from water-extracted flour suggested water soluble components of the flour interacted during mixing with the triplet zone proteins. SDS—PAGE patterns of reduced proteins that could not enter the separating gel under non-reducing conditions (slot protein) resembled the patterns of purified glutenin. Electrophoretic patterns of slot protein of SDS buffer extracts from acetic acid-insoluble residues showed the presence of subunits that corresponded in molecular weight to subunits of triplet band proteins. This suggested the possibility of strong interaction between triplet band proteins and SDS-buffer extractable glutenin in flour and dough residues.

Changes in Electrophoretic Patterns of Gadoid and Non‐gadoid Fish Muscle during Frozen Storage

ABSTRACTSodium dodecyl sulfate polyacrylamidc gel electrophoresis of fish muscle during frozen storage showed that a crosslinked protein of 280,000 daltons formed with gadoids, whereas this band did not occur with non‐gadoid fish species. For cod, 20 days at ‐7°C are needed for the crosslink to appear, whereas for whiting only 3 days arc needed. Formaldehyde (FA) may be responsible for the crosslinking. Adding ascorbic acid to cod mince prior to freezing accelerated dimcthylamine and FA formation during frozen storage and accelerated the formation of the crosslinked protein, while leaving cod fillets on ice for 10 days prior to freezing, reduced the Instron hardness and the crosslinked protein did not appear with 50 days of frozen storage at ‐7°C.

ニジュウヤホシテントウ成虫の蛋白質の電気泳動による分離と泳動パターンの前休眠期における変化

ニジュウヤホシテントウ成虫の体液,脂肪体の蛋白質をポリアクリルアミド・ゲル電気泳動によって分離し,それぞれの蛋白質成分の前休眠期および脂肪体発達に伴う変化を観察した。この昆虫では,雌特異的なvitellogenin様蛋白質は認められたが,それらは卵巣蛋白質の主要成分ではなかった。休眠に特異的な蛋白質は認められなかったが,ほとんどの体液,および,脂肪体蛋白質成分は前休眠期の進行につれて増加した。体液蛋白質の中に脂肪体が発達すると消失するものも認められ,一成分は光周期感受性消失後に消失した。体液中には色素蛋白質と呼ぶべきものが存在した。これらは羽化時には色素と結合しておらず,摂食が始まると色素と結合して電気泳動上の相対距離が小さいほうへ変化することが明らかになった。

Changes in flour thiols during storage, and improver action

Measurements of total and accessible thiols in three wheat flours, kept at room temperature for up to six months or so after milling, showed that there was a slow loss of total thiol. The rate of loss varied with the flour. The accessible thiol fell more rapidly than the total thiol but this was partly because some became inaccessible. When a flour was treated at room temperature or at 44°C with five common additives at 10 5 ppm, no significant differences could be found by amino acid analysis compared with controls. Electrophoresis and dough tests produced no evidence for peptide chain scission caused by the improvers ammonium persulphate (APS), azodicarbonamide, potassium bromate or the bleaching agent benzoyl peroxide (BP). APS and BP caused slight crosslinking, presumably by free radicals. l-Ascorbic acid produced unexplained minor changes in electrophoretic pattern, notably an extra doublet of high molecular weight. It is very unlikely that any of these changes would be significant at the levels used commercially and the results support the idea that loss of thiol groups is the reason for both the improving effect noticed during the first few months of storage and the action of improvers.

Occurrence of sugar non-specific nuclease in tobacco callus: Changes of nuclease activity during callus growth and plant regeneration

It was determined using electrophoresis in polyacrylamide gels containing native DNA or RNA that sugar non-specific nuclease active at pH 5.2 was expressed in tobacco callus. The nuclease had a relative molecular mass of about 34.6 kDaltons and degraded substrates in the following order of decreasing rate: denaturated DNA>poly dA>UV-irradiated native DNA>native DNA>alkylated native DNA>apurinated native DNA>poly dG≳poly dC. The nuclease activity changed during callus growth and plant regeneration, but no developmental changes in electrophoretic patterns were detected. The increase in specific DNAse activity of nuclease was maximal in the exponential phase of callus growth on both growth and regeneration media, except for activity in the cytokinin-independent cell strain grown on growth medium. The specific DNAse activity of nuclease decreased during the bud formation period, while total DNAse activity calculated per mg of dry weight was slightly higher in vegetative buds (9.1U) than in undifferentiated tissue of callus (8.5U). Specific DNAse activity was, on the average, several hundred-fold lower in the vegetative tissues of flowering tobacco plants than in calluses in the exponential phase of growth.