Protein Content Of Extracts Research Articles

Electrophoretic pattern of proteins in carcinoma of the gallbladder

Carcinoma of the gallbladder is a common health problem in the northern region of India and in spite of improved diagnostic techniques it is generally diagnosed at an advanced stage. This study was carried out in 60 patients, 30 of whom were diagnosed with gallbladder carcinoma and 30 with cholelithiasis. The protein content in tissue extracts and serum was determined using the Folin method of Lowry. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed for the identification of proteins according to Laemmli. The results showed the mean concentration of total protein in gallbladder tissue of patients with carcinoma of the gallbladder was 51.83 +/- 3.36 mg/g tissue (45.33-57.80) and in patients with cholelithiasis it was 38.82 +/- 9.11 mg/g tissue (29.55-50.99) (P<0.001). Protein electrophoresis of gallbladder tissue from the patients with carcinoma of the gallbladder showed three additional bands of protein (two protein bands were present in the region of 50-55 kDa and the third band was present in the region of 35 kDa), which were absent in the gallbladder tissue of cholelithiasis patients. Mean total protein content in serum was 72.2 +/- 1.73 g/l (67.98-74.99) in patients of carcinoma of the gallbladder, whereas it was 71.01 +/- 3.4 g/l (60.00-78.99) (P>0.05) in the patients with cholelithiasis. Electrophoretic analysis of serum protein revealed at least two additional protein bands in patients with carcinoma of the gallbladder as compared with electrophoretic pattern in cholelithiasis. These two bands were in the range of 80 and 25 kDa proteins. In conclusion, the presence of these new bands of protein in the gallbladder tissue and serum of the patients with carcinoma of the gallbladder indicate their role in the pathogenesis of the carcinoma of the gallbladder.

Limb regeneration and molting processes under chronic methoprene exposure in the mud fiddler crab, Uca pugnax

Insect growth regulator application for wetland mosquito control remains controversial due to the potential for disruption of normal development and growth processes in non-target crustaceans and beneficial arthropods, e.g. Apis mellifera. Concerns include slow-release methoprene formulations and its environmental breakdown products which mimic an endogenous crustacean hormone and retinoids, respectively. Our primary objective was to evaluate the effect that a chronic methoprene exposure would have on male and female Uca pugnax limb regeneration and molting. After single limb autonomy, limb growth and molt stage were monitored every two days while eyestalk ablation was used to induce proecdysis. Dorsal carapace was collected 6 days post-molt to determine protein and chitin content. In post-molt crabs, methoprene-exposed individuals displayed lower percent gain in body weight. Male crabs lost more weight per body volume than females, took significantly longer to proceed through proecdysis than females exposed to 0.1 µg/L methoprene and exhibited significantly elevated frequency for abnormal limb formation at 1.0 µg/L while females displayed no such trend. Methoprene did not significantly alter extractable exoskeleton protein or chitin content. However, variable water-soluble protein expression increased with exposure at 1.0 µg/L (1 ppb) which contributed to overall variability in total protein content. Our findings suggest that adult male U. pugnax possess greater sensitivity to chronic methoprene exposure during limb regeneration and molting, potentially affecting their post-molt fitness. Furthermore, methoprene has the potential to impact post-molt biomass and exocuticle quality.

Signal Peptide Peptidase and γ-Secretase Share Equivalent Inhibitor Binding Pharmacology

The enzyme gamma-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of gamma-secretase) and signal peptide peptidase (SPP) are related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and gamma-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid beta-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, gamma-secretase inhibitor radioligands were used to evaluate SPP and gamma-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for gamma-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of gamma-secretase binding sites, making it essential to use selective radioligands for evaluation of gamma-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family.

Effects of Solid-State Yeast Treatment on the Antioxidant Properties and Protein and Fiber Compositions of Common Hard Wheat Bran

The bran fraction of wheat grain is known to contain significant quantities of bioactive components. This study evaluated the potential of solid-state yeast fermentation to improve the health beneficial properties of wheat bran, including extractable antioxidant properties, protein contents, and soluble and insoluble fiber compositions. Three commercial food grade yeast preparations were evaluated in the study along with the effects of yeast dose, treatment time, and their interaction with the beneficial components. Solid-state yeast treatments were able to significantly increase releasable antioxidant properties ranging from 28 to 65, from 0 to 20, from 13 to 19, from 0 to 25, from 50 to 100, and from 3 to 333% for scavenging capacities against peroxyl (ORAC), ABTS cation, DPPH and hydroxyl radicals, total phenolic contents (TPC), and phenolic acids, respectively. Yeast treatment increased protein content 11-12% but did not significantly alter the fiber composition of wheat bran. Effects of solid-state yeast treatment on both ORAC and TPC of wheat bran were altered by yeast dose, treatment time, and their interaction. Results suggest that solid-state yeast treatment may be a commercially viable postharvest procedure for improving the health beneficial properties of wheat bran and other wheat-based food ingredients.

Relationship between tissue retention efficiency and production traits in a slow-growing broiler population

An experiment was conducted using 96 individually caged male broilers between 49 and 77 d of age. One objective was to establish phenotypic relationships between some production traits [feed conversion ratio (FCR), feed consumption, residual feed consumption (RFC), relative weight gain (RWG), weight gain (WG) and live weight (LW)] and “tissue retention efficiency” (TRE) traits in a slow-growing broiler population. The other objective was the characterization of Campero-INTA broilers for TRE traits. Weight and feed consumption were recorded weekly. Forty four broilers were slaughtered at 44 d of age to estimate initial body composition while the remaining birds were slaughtered at 79 d of age. Ether extract and crude protein content of the carcasses were used to estimate TRE traits: Energy retained as protein (ERP), energy retained as fat (ERF), ERP/(ERP+ERF), ERF/(ERP+ERF), protein retention efficiency and lipid-protein ratio. Correlation coefficients between traits were obtained and regression analyses were done for the evaluation of the influence of production traits on TRE traits. The independent variable that best explained ERF was WG (R2 = 0.49). Inclusion of final LW and RWG raised the R2 to 0.58 and decreased the error term. The ERP was best explained by RWG (R2 = 0.37); lipid-protein ratio by final LW (R2 = 0.49); protein retention efficiency by FCR (R2 = 0.34) and fraction of retained energy (ERF-ERP/ERP+ERF) by WG (R2 = 0.29). The TRE traits were not well predicted by the measured production traits. The high phenotypic variability observed in some of the TRE traits suggested a need for further studies on these characteristics. Keywords : Broiler; tissue retention; efficiency; slow-growing stock South African Journal of Animal Science Vol. 37 (3) 2007: pp.164-169

A comparison of intradermal testing and detection of allergen-specific immunoglobulin E in serum by enzyme-linked immunosorbent assay in horses affected with skin hypersensitivity

Skin hypersensitivities (allergies) in horses are often diagnosed using clinical signs only. Intradermal testing or serological assays are diagnostic options to confirm the allergic nature of the disease and to identify the allergen(s). Our objective was to develop an allergen-specific enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specific for horse IgE and to examine its potential for allergen detection in serum in comparison to intradermal testing. Intradermal testing with 61 allergen extracts was performed on 10 horses affected with skin hypersensitivity. Their sera were analyzed by ELISA for IgE antibodies to the same allergens. The kappa test of concordance was used for comparison of the results of both tests. Out of 61 allergen extracts, only two (Timothy and Quack) had kappa values greater than 0.60, suggesting a substantial agreement between skin testing and IgE ELISA. The statistical comparison of the remaining 59 allergens showed little or no concordance between the tests beyond chance. To identify parameters that may influence the sensitivity of the ELISA, the assay was modified to detect allergen-specific IgGb and IgG(T) in serum, and the protein content in all allergen extracts was determined by SDS-PAGE. The commercial allergen extracts revealed a high variation in detectable protein. High concentrations of allergen-specific IgG in horse serum were found to compete with IgE for binding to the plates. In conclusion, an ELISA using whole serum and crude allergen preparations provides limited diagnostic information in horses. The reliable diagnosis of allergens in equine skin hypersensitivity is essential to improve allergen-specific treatments, such as hyposensitization, or the development of allergy vaccines.

A simple quantitative assay for the activity of thymidine kinase 1 in solid tumors

The activity of the pyrimidine salvage pathway enzyme thymidine kinase 1 (TK1) is tightly cell cycle regulated and has been investigated as a prognostic indicator of cancer in a variety of tissues. However, using the in vitro assay of TK1 to rank order a series of unique tumor samples by their TK1 activity can be problematic due to the complex nature of TK1 enzyme substrate kinetics. We present a refined TK1 in vitro assay and method of analysis which address these problems. Extracts were prepared of the resected lung lesions from eight patients and assayed for TK1 activity using an in vitro assay modified to account for nonlinearities in extract protein concentration. A separate extract of exponentially growing A549 human lung carcinoma cells was used as a cross-assay control. In extracts prepared from eight frozen samples of resected human lung lesions, TK1 activity (mean=0.0070+/-0.0077 pmol [(3)H]-TMP/microg protein/minute) was 2 orders of magnitude below that of exponentially growing A549 human lung carcinoma cells (mean=0.1572+/-0.0218 pmol [(3)H]-TMP/microg protein/minute; n=9). TK1 activity was nonlinear with respect to extract protein concentration in both groups, with A549 cell extracts exhibiting evidence of positive cooperativity which could not be explained by the presence of detergents in the cell lysis buffer. Lung tumor extracts demonstrated evidence of negative cooperativity. The modified TK1 assay takes into account these nonlinearities by averaging the results of several complete time-course curves measured over a range of extract protein concentrations. An extract prepared from exponentially growing A549 cells is included in each assay for use as a cross-assay control. We demonstrate that these modifications allow for the accurate rank ordering of TK1 activity in solid tumors.

Fasting-induced changes in ECL cell gene expression

Gastric enterochromaffin-like (ECL) cells release histamine in response to food because of elevation of gastrin and neural release of pituitary adenylate cyclase-activating peptide (PACAP). Acid secretion is at a basal level in the absence of food but is rapidly stimulated with feeding. Rats fasted for 24 h showed a significant decrease of mucosal histamine despite steady-state expression of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Comparative transcriptomal analysis using gene expression oligonucleotide microarrays of 95% pure ECL cells from fed and 24-h fasted rats, thereby eliminating mRNA contamination from other gastric mucosal cell types, identified significantly increased gene expression of the enzymes histidase and urocanase catabolizing the HDC substrate L-histidine but significantly decreased expression of the cellular L-histidine uptake transporter SN2 and of the vesicular monoamine transporter 2 (VMAT-2) responsible for histamine uptake into secretory vesicles. This was confirmed by reverse transcriptase-quantitative polymerase chain reaction of gastric fundic mucosal samples from fed and 24-h fasted rats. The decrease of VMAT-2 gene expression was also shown by a decrease in VMAT-2 protein content in protein extracts from fed and 24-h fasted rats compared with equal amounts of HDC protein and Na-K-ATPase alpha(1)-subunit protein content. These results indicate that rat gastric ECL cells regulate their histamine content during 24-h fasting not by a change in HDC gene or protein expression but by regulation of substrate concentration for HDC and a decreased histamine secretory pool.

Direct Interactions of Runx2 and Canonical Wnt Signaling Induce FGF18

Canonical Wnt signaling is clearly required for skeletal development and bone formation. However, the targets of Wnt signaling that convert this signal into bone are unclear. Identification of these targets will yield insight into normal bone physiology and suggest new therapeutics for treatment of bone disease. Here we show that an essential regulator of bone development, FGF18, is a direct target of canonical Wnt signaling. A single DNA binding site for the Wnt-dependent transcription factors TCF/Lef accounted for the stimulation of the fgf18 promoter in response to Wnt signaling. Additionally, targeted disruption of betacat blocked fgf18 expression in vivo. Partially overlapping the TCF/Lef binding site is a Runx2 binding site and experiments showed that Runx2 and TCF/Lef work cooperatively to induce fgf18 expression. RNA interference knockdown of Runx2 inhibited and Runx2 forced expression augmented the induction of fgf18 by canonical Wnt signaling. Significantly, Runx2 formed a complex with Lef1 or TCF4 and this complex bound the composite binding site in the fgf18 promoter. These results demonstrate that two transcription pathways that are essential for bone, physically and functionally converge at the fgf18 promoter.

Interference of phenolic compounds in Brassica napus, Brassica rapa and Sinapis alba seed extracts with the Lowry protein assay

The protein content of aqueous extracts of Brassica napus, Brassica rapa and Sinapis alba meal was determined by the Lowry and Kjeldahl nitrogen assays. Phenolic compounds interfered with the Lowry method to different extents based on the lines studied as well as the extraction procedure used. Three ways to correct for this interference were studied; acid precipitation of the protein before analysis, analyzing in the presence and absence of copper and the binding of free phenolics using non-ionic, porous polystyrene (Amberlite XAD-4). Analysis in presence and absence of copper, and using the difference in absorption at 660 nm between these analyses, proved to be the best way to correct for phenolic interference in the Lowry assay. Extractability of Cruciferae seed phenolics may be pH dependant thus the contribution of phenolics to the Lowry protein assay varies with the pH used for extraction.

Degradation of malathion in aqueous extracts obtained from different developmental stages of asparagus (Asparagus officinalis)

AbstractWhen malathion was incubated in aqueous extracts obtained from 17 vegetables and fruits at pH 7.4 and 37 °C for 4 h, asparagus extract exhibited the highest malathion degradation. The degradation effects of the extracts ranged from 100% (asparagus) to 0.5% (papaya). The highest malathion‐degradation effects obtained from extracts of asparagus at different growing stages incubated at pH 7.4 and 37 °C for 1 h was 94.4 ± 0.45% (stem length, 5–10 cm), followed by 88.0 ± 0.31% (10–15 cm), 85.1 ± 0.94 (15–20 cm), 82.2 ± 0.52 (20–25 cm), 81.2 ± 1.4% (pre‐emergent), 67.3 ± 0.67% (&gt;25 cm), and 42.7 ± 2.1% (fern). The protein content in an asparagus extract from different growing stages was not consistent with the degradation activity of malathion. Results of time course studies on malathion degradation in asparagus extracts from different growing stages yielded half‐life times of malathion degradation ranging from 51.7 min (fern stage) to 17.3 min (stem length, 5–10 cm). Formation of α‐ and β‐malathion mono acids from malathion was detected in an incubated sample by gas chromatography/mass spectrometry. The results support the presence of carboxylesterases in asparagus extracts. Copyright © 2006 Society of Chemical Industry

Large Hepatitis Delta Antigen Modulates Transforming Growth Factor-β Signaling Cascades: Implication of Hepatitis Delta Virus–Induced Liver Fibrosis

Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of liver disease. TGF-beta is involved in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV-encoded-only protein, the small hepatitis delta antigen (SHDAg), and the large hepatitis delta antigen (LHDAg), on TGF-beta- and c-Jun-induced signaling cascades. The effects of either SHDAg or LHDAg on TGF-beta- and c-Jun-induced signaling cascades in Huh7 and Cos7 cells were investigated by luciferase reporter gene assay, immunoprecipitation assay, electrophoretic mobility shift assay, Western blot analysis, and confocal microscopy analysis. The LHDAg, but not the SHDAg, potentiated TGF-beta- and c-Jun-induced signal activation, and the isoprenylation of LHDAg played a major role in signaling cascades. LHDAg synergistically activated hepatitis B virus X protein-mediated TGF-beta and AP-1 signaling cascades. In addition, LHDAg enhanced the protein expression level of TGF-beta-induced plasminogen activator inhibitor-1. LHDAg may induce liver fibrosis through the regulation of TGF-beta-induced signal transductions. This regulation of TGF-beta-mediated signaling is accomplished by the isoprenylation of LHDAg, which is a novel mechanism involved in HDV pathogenesis.

Fibronectin Increases Matrix Metalloproteinase 9 Expression through Activation of c-Fos via Extracellular-regulated Kinase and Phosphatidylinositol 3-Kinase Pathways in Human Lung Carcinoma Cells

Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human lung tumor invasion and/or metastasis. We have demonstrated that fibronectin (FN), a matrix glycoprotein, stimulates human non-small cell lung carcinoma (NSCLC) cell proliferation. The current study examines the effect of FN on MMP-9 expression in NSCLC cells. We show that FN increases MMP-9 protein, mRNA expression, and gelatinolytic activity in NSCLC cells. The integrin alpha5beta1 mediated the effects of FN because alpha5 small interfering RNA blocked FN-stimulated MMP-9 protein expression, and also abrogated FN-induced phosphorylation of ERK and phosphatidylinositol 3-kinase (PI3K) signals. The inhibitor of ERK, PD98095, and of PI3K, wortmannin, but not that of protein kinase A, H89, of Rho kinase, Y-27632, of mTOR, rapamycin, or of JNK, SP600125, prevented FN-induced MMP-9 gelatinolytic activity and gene expression. FN enhanced MMP-9 gene promoter activity; however, there was no response to FN in DNA constructs with an AP-1 site mutation. FN increased AP-1 DNA binding activity, and this was abrogated by cyclic AMP response element decoy oligonucleotides, which also diminished FN-induced MMP-9 promoter activity. FN increased the expression of the AP-1 subunit c-Fos protein, but not in the presence of PD98095 and wortmannin. The AP-1 inhibitor, nordihydroguaiaretic acid, and a c-Fos small interfering RNA eliminated the effect of FN on MMP-9 expression. This study indicates that FN, by binding to the integrin alpha5beta1 receptor, stimulates the expression of MMP-9 through increased AP-1/DNA binding and c-Fos protein expression via ERK and PI3K signaling pathways. The data unveils a novel mechanism by which FN could promote NSCLC cell invasion and metastasis.

Endoplasmic Reticulum Stress Induction of Insulin-like Growth Factor-binding Protein-1 Involves ATF4

Endoplasmic reticulum (ER) stress is sensed by cells in different physiopathological conditions in which there is an accumulation of unfolded proteins in the ER. A coordinated adaptive program called the unfolded protein response is triggered and includes translation inhibition, transcriptional activation of a set of genes encoding mostly intracellular proteins, and ultimately apoptosis. Here we show that insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), a secreted protein that modulates IGF bioavailability and has other IGF-independent effects, is potently induced during ER stress in human hepatocytes. Various ER stress-inducing agents were able to increase IGFBP-1 mRNA levels, as well as cellular and secreted IGFBP-1 protein up to 20-fold. A distal regulatory region of the human IGFBP-1 gene (-6682/-6384) containing an activating transcription factor 4 (ATF4) composite site was required for promoter activation upon ER stress. Mutation of the ATF4 composite site led to the loss of IGFBP-1 regulation. Electrophoretic mobility shift assay revealed an ER stress-inducible complex that was displaced by an ATF4 antibody. Knockdown of ATF4 expression using two specific small interfering RNAs impaired up-regulation of IGFBP-1 mRNA, which highlights the relevance of ATF4 in endogenous IGFBP-1 gene induction. In addition to intracellular proteins involved in secretory and metabolic pathways, we conclude that ER stress induces the synthesis of secreted proteins. Increased secretion of IGFBP-1 during hepatic ER stress may thus constitute a signal to modulate cell growth and metabolism and induce a systemic adaptive response.

The inhibitory effects of freshwater clam ( Corbicula fluminea, Muller) muscle protein hydrolysates on angiotensin I converting enzyme

The muscle of golden freshwater clam was extracted using hot water. The residual meat was freeze-dried then hydrolyzed at 50 °C first by Protamex (PX) as primary hydrolysis followed by a secondary hydrolysis (Flavourzyme, F). The inhibitory effects of hot water extracts and hydrolysates against angiotensin I converting enzyme (ACE) were investigated. The soluble protein content and peptide content of the hot water extracts was 85 and 84 mg/g, respectively. The IC 50 on ACE was 1.95 mg/ml. The highest values of soluble protein (571 mg/g) and peptide content (272 mg/g) of hydrolysate prepared by PX hydrolysis for 5 h (PX5) or PX hydrolysis for 5 h followed by F hydrolysis for 0.5 h (PX5F0.5). PX5 showed the lowest IC 50 on ACE (0.043 mg/ml), and a mixed-type inhibition kinetics while Captopril showed competitive inhibition. Their K i values were 0.032 mg/ml and 0.0067 μg/ml, respectively. The PX5 hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25. A fraction of the PX5 had molecular weight ranged 420–380 Da showed the highest inhibitory efficiency ratio (IER) being 1314.7%/mg/ml. The amino acid sequences of the two peaks of this fraction PX-D1 and PX-D2 were Val-Lys-Pro and Val-Lys-Lys, of which IC 50 value were 3.7 and 1045 μM, respectively. This freshwater clam hydrolysate (PX5) (peptide concentration 5 mg/ml) was used as drinks administered to spontaneously hypertensive rats (SHR) for 8 weeks. The systolic blood pressure and diastolic blood pressure of the SHR were significantly reduced by 22.0 and 13.2 mmHg, respectively, indicating a hypotensive effect by oral administration.

Latent Pathway Activation and Increased Pathway Capacity Enable Escherichia coli Adaptation to Loss of Key Metabolic Enzymes

The ability of biological systems to adapt to genetic and environmental perturbations is a fundamental but poorly understood process at the molecular level. By quantifying metabolic fluxes and global mRNA abundance, we investigated the genetic and metabolic mechanisms that underlie adaptive evolution of four metabolic gene deletion mutants of Escherichia coli (delta pgi, delta ppc, delta pta, and delta tpi) in parallel evolution experiments of each mutant. The initial response to the gene deletions was flux rerouting through local bypass reactions or normally latent pathways. The principal effect of evolution was improved capacity of already active pathways, whereas new flux distributions were not observed. Combinatorial changes in capacity and pathway activation, however, led to different intracellular flux states that enabled evolution in three of the four parallel cases tested. The molecular bases of the evolved phenotypes were then elucidated by global mRNA transcript analyses. Activation of latent pathways and flux changes in the tricarboxylic acid cycle were found to correlate well with molecular changes at the transcriptional level. Flux alterations in other central metabolic pathways, in contrast, were apparently not connected to changes in the transcriptional network. These results give new insight into the dynamics of the evolutionary process by demonstrating the flexibility of the metabolic network of E. coli to compensate for genetic perturbations and the utility of combining multiple high throughput data sets to differentiate between causal and noncausal mechanistic changes.

The use of five techniques to extract protein, oil and gossypol content of Okra seeds (&lt;i&gt;Abelmosches esculentus (L) moench&lt;/I&gt;

The use of five techniques to extract protein, oil and gossypol content of Okra seeds (&lt;i&gt;Abelmosches esculentus (L) moench&lt;/I&gt;

The influence of γ-irradiation on some biological activities and electrophoresis patterns of wheat grain albumin fraction

Water-soluble proteins were extracted from irradiated wheat grain for the purpose of assaying biological activities, reducing sugars content and SDS–PAGE electrophoresis. The differences between samples after γ-irradiation were tested by analysis of variance at the level of significance ( P ⩽ 0.05). Ionising radiation (0.05–10 kGy) caused an increase in the activity of endogenous amylases, statistically significant at doses of 5 and 10 kGy ( P ⩽ 0.05), which was well correlated with the highest extractable protein content. γ-Irradiation of wheat grain at a dose of 0.05 kGy caused an increase of inhibition activity against Sitophilus granarius L. α-Amylase, whereas there was a decrease at 10 kGy ( P ⩽ 0.05). On the other hand, grain irradiated by 0.5 and 1 kGy doses showed a significant increase in inhibition activity against α-amylase of Tribolium confusum Duv. ( P ⩽ 0.05) while at the remaining doses the inhibition activity was on the same level as the control grain. Decrease of Ephesitia kuehniella Zell. α-amylase was observed only at 5 kGy radiation dose ( P ⩽ 0.05). At the remaining doses this activity was comparable to non-irradiated grain. The highest increase of antiamylolytic activity against human saliva α-amylase was noted in albumin proteins of wheat grain treated by γ-radiation doses >0.5 kGy while there were decreases at radiation doses <0.5 kGy ( P ⩽ 0.05). The increase of inhibition activity against hog pancreas α-amylase was noted at radiation doses 0.1 kGy ( P ⩽ 0.05). γ-Radiation doses of 0.05, 0.5 and 1 kGy caused an increase of the antitryptic activity while 10 kGy decreased this activity ( P ⩽ 0.05). SDS–PAGE electrophoresis patterns of albumin sample at 0.1 kGy showed 15 proteins while in the control sample only 11 bands were found. Among studied wheat albumins, the lower protein bands 5 and 8 were detected in wheat grain samples irradiated by 10 and 0.5 kGy, respectively. The differences in inhibitor activities and electrophoretic protein bands of wheat albumin from γ-irradiated wheat grain give additional evidence that these proteinaceous inhibitors against enzymes studied are different in nature.

Converting a β-Glycosidase into a β-Transglycosidase by Directed Evolution

Directed evolution was applied to the beta-glycosidase of Thermus thermophilus in order to increase its ability to synthesize oligosaccharide by transglycosylation. Wild-type enzyme was able to transfer the glycosyl residue with a yield of 50% by self-condensation and of about 8% by transglycosylation on disaccharides without nitrophenyl at their reducing end. By using a simple screening procedure, we could produce mutant enzymes possessing a high transferase activity. In one step of random mutagenesis and in vitro recombination, the hydrolysis of substrates and of transglycosylation products was considerably reduced. For certain mutants, synthesis by self-condensation of nitrophenyl glycosides became nearly quantitative, whereas synthesis by transglycosylation on maltose and on cellobiose could reach 60 and 75%, respectively. Because the most efficient mutations, F401S and N282T, were located just in front of the subsite (-1), molecular modeling techniques were used to explain their effects on the synthesis reaction; we can suggest that repositioning of the glycone in the (-1) subsite together with a better fit of the acceptor in the (+1) subsite might favor the attack of a glycosyl acceptor in the mutant at the expense of water. Thus these new transglycosidases constitute an interesting alternative for the synthesis of oligosaccharides by using stable and accessible donor substrates.

Nucleic acids and protein content in relation to growth and regression of buffalo (Bubalus bubalis) corpora lutea

Information on nucleic acids and protein content of buffalo corpus luteum (CL) in relation to growth, development and regression is not available. An experiment was thus conducted to investigate the variation and relationship between nucleic acids and protein content in CL during different developmental stages and to determine the qualitative differences in protein constituents in any of these stages. Buffalo corpora lutea of different developmental stages viz., developing (day 5–10, n = 16), developed (day 11–17, n = 12) and regressed (day 18–21, n = 10) stages were collected from non-pregnant and -pathological genitalia ( n = 38). The DNA, RNA and protein content in tissue extracts were determined and the proteins in pooled samples were analyzed by polyacrylamide gel electrophoresis. Developing stage CL had more total and per gram tissue level of DNA and RNA with significant positive relationship with total and per gram RNA and protein contents. Although there was no significant difference in total weight, a significant decrease in total DNA as well as per gram level of DNA and RNA was observed in developed stage compared to developing stage CL. The total protein content in developed stage CL was compared to developing and regressed stage CL. Non-denaturing PAGE analysis of CL proteins of different stages showed five protein bands of 210, 190, 82, 68 and 66 kDa and one that migrated with the dye front in all the stages however, not shown any differences in banding pattern. Denaturing PAGE showed 15 bands viz., 205, 66, 53, 42, 35, 27, 24, 22, 20, 18, 17, 14, 9, 7.5 and 6.5 kDa. Out of these 66 and 53 kDa bands appeared with maximum intensity in all the three stages of CL. Comparison of bands between the three stages revealed five 57, 31, 27, 19 and 16 kDa stage-specific bands in regressed stage CL. The present study indicated that the DNA, RNA and protein content of buffalo CL varied with the stages of development and regressed stage CL contained some unique protein bands which were not observed either in developed or developing stage CL.