Isoelectric Focusing Sodium Dodecyl Sulfate Research Articles

Protective ability of phenolics from white grape vinification by-products against structural damage of bovine serum albumin induced by glycation

Grape skins recovered from white grape vinification processes were studied as possible anti-glycation agents. Total phenolics were characterised by the Folin Ciocalteu assay, proanthocyanidins by depolymerisation with n-butanol/HCl, flavonols by HPLC-DAD, reducing capacity by ferric ion reducing antioxidant power assay (FRAP) and anti-glycation activity by a bovine serum albumin (BSA)/fructose model system. Structural modifications of BSA were investigated by 2D isoelectric focusing sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF/SDS–PAGE) and fluorescence measurements. Both pI and Mr. of BSA were modified upon glycation reaction. These changes attributable to the involvement of free amino groups in Maillard-type reactions were inhibited by the white grape skin extracts. The anti-glycation activity ranged between 250 and 711mmol aminoguanidine Eq/kg. These results raise the interest in the potential health benefits of by-products of white grape vinification that could have a secondary use as an ingredient for new functional foods targeting wellbeing of diabetic and elderly people.

Pancreatic duct mucosa following bile salt injury in cats

We studied pancreatic duct mucosal morphology and barrier function to both activated and nonactivated pancreatic exocrine proteins following bile salt injury in cats. Prograde pancreatic duct perfusion with 15 mM glycodeoxycholate caused epithelial disruption with focal epithelial loss in the majority of animals. In these cats, flow of preanalyzed nonactivated rat pancreatic juice along the duct resulted in a selective loss of zymogens from the duct lumen as determined by two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis and reversed-phase high-performance liquid chromatography. Proteins lost had a low isoelectric point (< or = 5.7) and a molecular weight as large as 47,000 Da and included chymotrypsinogen 1, trypsinogens 1 and 2, and procarboxypeptidases B. Proteins with a high isoelectric point (> 5.7), a high molecular weight (> 47,000 Da), or both, were completely recovered from the duct lumen. Flow of activated rat pancreatic juice along a pancreatic duct with bile salt-induced epithelial disruptions caused additional morphologic alterations including an increase in epithelial destruction and occasional necrosis of the duct interstitial and vascular tissue. In some cats, in which the integrity of the ductal epithelium remained preserved following exposure to the bile salt, neither loss of rat pancreatic exocrine zymogens from the duct lumen nor degradation of the duct mucosa by activated rat pancreatic juice was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid high voltage isoelectric focusing of proteins in rod gels.

A rapid procedure of isoelectric focusing (IEF) of proteins in polyacrylamide rod gels (i.d., 1.1 mm; length, 7.5 cm) is described. The time required for IEF can be reduced to 0.5 h by using high voltages up to 3000 V in the presence or absence of urea in the gels. When used as the first dimension of a two-dimensional technique for IEF sodium dodecyl sulphate electrophoresis, high voltage IEF gives smaller protein spots on the second dimension gel, associated with an increase in resolution. The method has been tested by a two-dimensional separation of an eye sample of the goodeid fish Xenotoca eiseni.

顎，口腔へん平上皮癌細胞蛋白質の２次元電気泳動法による解析

The cellular proteins of 12 specimens of maxilla and oral squamous cell carcinoma were analyzed by two dimensional isoelectric focusing-sodium dodecyl sulfate gel electro-phoresis. The number of polypeptide spots detectable in individual gels was about 110 per 200μg of proteins with pIs from 4.5 to 8.5 and MWs from 22, 000 to 72, 000. Many of the polypeptides were common to these lesions. However, there were nine main polypeptides which showed differences between normal tongue mucosa and squamous cell carcinoma. In the carcinoma, two polypeptides (7.0/45 K, 7.2/47 K, pI/MW) were decreased and four polypeptides (4.8/33 K, 6.9/49 K, 7.1/38 K, 7.4/59 K) were increased in an amount but only polypeptide, 7.9/38 K was varied in an amount by differentiation of carcinoma. This polypeptide was increased in moderately and lower differentiation and decreased in well differentiation. The remaining two polypeptides (4.8/38 K, 4.9/37 K) that were not normally present appeared in carcinoma, but these two new polypeptides were not specific to carcinoma because they were also present in dysplastic lesion. These results suggest that two dimensional electrophoresis may be useful in biochemical diagnosis of maxilla and oral squamous cell carcinoma.

９，１０‐ｄｉｍｅｔｈｙｌ‐１，２‐ｂｅｎｚａｎｔｈｒａｃｅｎｅ誘発ハムスター舌癌および上皮異形成細胞蛋白質の２次元電気泳動法による解析

The cellular proteins of hamster tongue squamous cell carcinoma and epithelial dysplastic lesions induced by 9,10-dimethyl-1,2-benzanthracend (DMBA) were analyzed by two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis. Many of the polypeptides were common to these lesions. However, there were thirteen main polypeptides which differed between normal tongue mucosa and tongue carcinoma. In the carcinoma, eight polypeptides (5.2/53 K, 5.8/47 K, 6.9/46 K, 6.9/50 K, 7.1/55 K, 7.2/46 K, 7.3/75 K, 7.4/48 K, pI/MW) were decreased and three polypeptides (4.9/32 K, 6.3/50 K, 7.2/60 K) were increased in an amount. The remaining two polypeptides (4.8/37 K, 4.9/36 K) that were not normally present were appeared in carcinoma, but these two new polypeptides were not specific to carcinoma because they were also present in dysplastic lesions. There were marked differences in the polypeptide between various degree of dysplastic lesions and carcinoma on the one hand and normal mucosa on the others. These results suggests that two-dimensional electrophoresis may have usefulness in further research on transformations from normal mucosa to malignant cancer cells.

Metabolic behavior of cell surface biotinylated proteins.

The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with sodium dodecyl sulfate and mercaptoethanol, and separated on polyacrylamide gels. Biotin-epsilon-aminocaproic acid N-hydroxysuccinimide ester (BNHS) or N-biotinoyl-N'-(maleimidohexanoyl)hydrazine (BM) were the derivatizing agents. Only 10-12 bands were adsorbed onto streptavidin-agarose from undervatized cells or from derivatized cells treated with free avidin at 4 degrees C. Two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis resolved greater than 100 BNHS-derivatized proteins and greater than 40 BM-derivatized proteins. There appeared to be little overlap between the two groups of derivatized proteins. Short-term pulse-chase studies showed an accumulation of label into both groups of biotinylated proteins up until 1-2 h of chase and a rapid decrease over the next 1-5 h. Delayed appearance of labeled protein at the cell surface was attributed to transit time from site of synthesis. The unexpected and unexplained rapid disappearance of pulse-labeled proteins from the cell surface was invariant for all two-dimensionally resolved proteins and was sensitive to temperature reduction to 18 degrees C. Long-term pulse-chase experiments beginning 4-8 h after the initiation of chase showed the disappearance of derivatized proteins to be a simple first-order process having a half-life of 115 h in the case of BNHS-derivatized proteins and 30 h in the case of BM-derivatized proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of Turgor Induces Rapid Changes in Leaf Translatable RNA

The turgor of pea (Pisum sativum) leaves was reduced by exposing excised pea shoots to a stream of 23 degrees C air for 20 min. Poly(A)(+) RNA was isolated from control and wilted shoots, translated in vitro and radiolabeled translation products separated by electrophoresis on two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gels. This analysis showed that the levels of several poly(A)(+) RNAs increased in wilted plants. Most of the poly(A)(+) RNAs induced in wilted plants did not accumulate in response to heat shock or exogenously applied ABA even though endogenous ABA levels were found to increase in shoots 30 min after wilting and by 4 h had increased 50-fold (1 versus 0.02 microgram per gram fresh weight). A lambdagt10 cDNA library was constructed using poly(A)(+) RNA from wilted shoots which had been incubated for 4 hours. Differential screening of the library identified four clones corresponding to poly(A)(+) RNAs which are induced in wilted shoots.

Water Deficit-Induced Changes in Abscisic Acid, Growth, Polysomes, and Translatable RNA in Soybean Hypocotyls

Soybean seedlings (Glycine max L.) were germinated and dark-grown in water-saturated vermiculite (water potential = -0.01 megapascal) for 48 hours, then transferred either to water-saturated vermiculite or to low water potential vermiculite (water potential = -0.30 megapascal). A decrease in growth rate was detectable within 0.8 hour post-transfer to low water potential vermiculite. A fourfold increase in the abscisic acid content of the elongating region was observed within 0.5 hour. At 24 hours post-transfer, hypocotyl elongation was severely arrested and abscisic acid reached its highest measured level: 3.7 nanograms per milligram dry weight (74-fold increase). A comparison of the polyA(+) RNA populations isolated at 24 hours post-transfer from the elongating region of water-saturated and low water potential vermiculite-grown seedlings was made by two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gel analysis of in vitro translation products. It revealed both increases and decreases in the relative amounts of a number of translation products. Rewatering seedlings grown in low water potential vermiculite at 24 hours post-transfer led to a total recovery in growth rate within 0.5 hour, while abscisic acid in the elongating hypocotyl region required 1 to 2 hours to return to uninduced levels. Application of 1.0 millimolar (+/-) abscisic acid to well-watered seedlings resulted in a 48% reduction in hypocotyl growth rate during the first 2 hours after treatment. Plants treated with abscisic acid for 24 hours had a lower polysome content than control plants. However, hypocotyl growth inhibition in abscisic acid-treated seedlings preceded the decline in polysome content.

Expression of smooth muscle-specific alpha-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation.

The relationship between growth and cytodifferentiation was studied in cultured rat aortic smooth muscle cells (SMCs) using expression of the smooth muscle (SM)-specific isoactins (Vanderkerckhove, J., and K. Weber, 1979, Differentiation, 14:123-133) as a marker for differentiation in these cells. Isoactin expression was evaluated by: (a) measurements of fractional isoactin content and synthesis ([35S]methionine incorporation) by densitometric evaluation of two-dimensional isoelectric focusing sodium dodecyl sulfate gels, and (b) immunocytological examination using SM-specific isoactin antibodies. Results showed the following: (a) Loss of alpha-SM isoactin was not a prerequisite for initiation of cellular proliferation in primary cultures of rat aortic SMCs. (b) alpha-SM isoactin synthesis and content were low in subconfluent log phase growth cells but increased nearly threefold in density-arrested postconfluent cells. Conversely, beta-nonmuscle actin synthesis and content were higher in rapidly dividing subconfluent cultures than in quiescent postconfluent cultures. These changes were observed in primary and subpassaged cultures. (c) alpha-SM actin synthesis was increased by growth arrest of sparse cultures in serum-free medium (SFM; Libby, P., and K. V. O'Brien, 1983, J. Cell. Physiol., 115:217-223) but reached levels equivalent to density-arrested cells only after extended periods in SFM (i.e., greater than 5 d). (d) SFM did not further augment alpha-SM actin synthesis in postconfluent SMC cultures. (e) Serum stimulation of cells that had been growth-arrested in SFM resulted in a dramatic decrease in alpha-SM actin synthesis that preceded the onset of cellular proliferation. These findings demonstrate that cultured vascular SMCs undergo differential expression of isoactins in relation to their growth state and indicate that growth arrest promotes cytodifferentiation in these cells.

Bean lectins

Seeds of forty bean cultivars having different lectin types based on two-dimensional isoelectric focusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE) were analyzed for quantities of lectin, phaseolin and total protein. Significant differences were found among groups of cultivars with different lectin types for the quantity of lectin and phaseolin. Cultivars with more complex lectin types based on IEF-SDS/PAGE tended to have higher quantities of lectin and lower quantities of phaseolin than cultivars with simple lectin types. An association between lectin type and the quantity of lectin and phaseolin was found also in the seeds of F2 plants that segregated in a Mendelian fashion for two lectin types. Seeds from plants with the complex lectin type had more lectin and less phaseolin than seeds from plants with the simple lectin type. Therefore, the genes controlling qualitative lectin variation also may influence the quantitative variation of lectin and phaseolin. The results of this study are related to other studies on the quantitative variation for seed proteins and to the possible molecular basis for variation in the quantity of lectins in beans.

Bean lectins IV: genetic variation in the non-denatured structure of lectins from different Phaseolus vulgaris L. cultivars

Variation in the native conformation of bean lectins was examined using electrophoresis of non-denatured total protein extracts and purified albumin and globulin lectin. The observed variation was related to the genetic variation reported previously for lectin polypeptide composition as revealed by two-dimensional isoelectricfocusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE). When eleven cultivars with different IEF-SDS/PAGE lectin polypeptide compositions were compared, eight had unique non-denatured lectin patterns and three had identical patterns. For some cultivars differences in non-denatured lectin patterns were observed between the purified albumin and globulin lectin preparations.

Cytochrome P-450 induction in genetically obese rats

Using two dimension iso-electric focusing sodium dodecyl sulphate polyacrylamide gel electrophoresis, we have shown that the lack of response by genetically obese Zucker rats to phenobarbital treatment is due to a decreased induction of cytochrome's P-450 especially cytochrome P-450-PB 3. We do not know whether this represents a defect in transcription or translation.