Enzyme Assay Techniques Research Articles

Exploration of a novel protease from Phytolacca dodecandra (Endod) berries for potential application in detergent formulation

AbstractConventional detergent formulations contain either petroleum‐derived or synthetic chemicals. Hence, the replacement of these chemicals with natural products such as enzymes is considered a green technology. Among these enzymes, protease is renowned in detergent formulations. Therefore, the objective of this study was to explore and characterize a new protease from Phytolacca dodecandra, because the plant is traditionally known for detergency purposes. Enzyme extraction was performed with Glycine/NaOH buffer, pH = 10.0. Gelatin hydrolysis and agar well diffusion tests were implemented as qualitative assays, whereas the enzyme assay technique was applied for the quantitative investigation of the new protease from plant extracts. The crude enzyme was partially characterized concerning temperature and pH tolerance, washing performance, detergent compatibility, and stability. The finding of this study revealed that Phytolacca dodecandra contains protease and is characterized by 1418.84 U/mL of enzyme activity, where 1 unit (U) of enzyme activity refers to the amount of enzyme required to liberate 1 μg of tyrosine/mL/min under the defined assay conditions. The enzyme showed maximum activity at pH 10.5 and 45°C. The enzyme was stable and maintained 99.52% activity at 40°C and 91.73% at pH 10.0 after 2 h pre‐incubation. The enzyme showed excellent wash performance and could potentially remove blood stains completely within 3 min. After 2 h of pre‐incubation the enzyme maintained 69.06% and 54.88% of its activity in the presence of Azzy and Sunlight, respectively. The enzyme maintained 71.99% of its residual activity after 60 days of storage. If further studies are conducted, all these properties of the newly investigated protease can make the enzyme applicable in detergent formulations.

Characterization of alcohol acetyltransferases in the ripe flesh of ‘Monthong’ and ‘Chanthaburi 1′ durians

Durian is economically significant in Southeast Asia due to its distinctive aroma and palatability. During fruit ripening, the flesh generates a substantial quantity of esters and some sulfur-containing compounds. This study aimed to analyze the ester profiles and characteristics of alcohol acetyltransferase (AAT; EC. 2.3.1.84) in the ripe flesh of two Thai durian (Durio zibethinus Merr.) cultivars, ‘Chanthaburi 1' (a hybrid cultivar with a soft aroma) and ‘Monthong’ (a renowned cultivar with a medium scent). The primary esters responsible for the aromatic compounds found in durian are ethyl-2-methyl butanoate, ethyl hexanoate, and ethyl octanoate. The AAT's efficacy was assessed in its ability to catalyze the synthesis of acetate esters through the reaction between acetyl CoA and different alcohols. The AAT enzymes extracted from ‘Chanthaburi 1′ and ‘Monthong’ cultivars exhibited a notable affinity towards 3-methyl-1-butanol and hexanol as alcohol substrates. Propanol and butanol exhibited moderate activity as AAT substrates, whereas methanol and ethanol demonstrated the lowest. Both durians exhibited favorable enzyme activity at a temperature of 30 °C. However, ‘Monthong’ AAT demonstrated superior performance across a broader pH range compared to ‘Chanthaburi 1′ AAT. The partially purified proteins precipitated with ammonium sulfate and subsequently gel-filtered through a DEAE-Sephadex® column enhanced the potency of ‘Chanthaburi 1′ AAT, resulting in increased purity (1.20-fold) and specificity (1.08-fold) compared to ‘Monthong’. The AAT of ‘Chanthaburi 1′ and ‘Monthong’ exhibited molecular weights of 39.52 and 41.51 kDa, respectively. This study presents the initial documentation of AAT in durians through an enzyme assay and activity staining technique.

Effect of three typical sulfide mineral flotation collectors on soil microbial activity.

The sulfide mineral flotation collectors are wildly used in China, whereas their toxic effect on soil microbial activity remains largely unexplored. In this study, isothermal microcalorimetric technique and soil enzyme assay techniques were employed to investigate the toxic effect of typical sulfide mineral flotation collectors on soil microbial activity. Soil samples were treated with different concentrations (0-100μg•g - 1 soil) of butyl xanthate, butyl dithiophosphate, and sodium diethyldithiocarbamate. Results showed a significant adverse effect of butyl xanthate (p < 0.05), butyl dithiophosphate, and sodium diethyldithiocarbamate (p < 0.01) on soil microbial activity. The growth rate constants k decreased along with the increase of flotation collectors concentration from 20.0 to 100.0μg•g(-1). However, the adverse effects of these three floatation collectors showed significant difference. The IC 20 of the investigated flotation reagents followed such an order: IC 20 (butyl xanthate) > IC 20 (sodium diethyldithiocarbamate) > IC 20 (butyl dithiophosphate) with their respective inhibitory concentration as 47.03, 38.36, and 33.34μg•g(-1). Besides, soil enzyme activities revealed that these three flotation collectors had an obvious effect on fluorescein diacetate hydrolysis (FDA) enzyme and catalase (CAT) enzyme. The proposed methods can provide meaningful toxicological information of flotation reagents to soil microbes in the view of metabolism and biochemistry, which are consistent and correlated to each other.

Lack of association between glutathione peroxidase1 (GPx1) activity, Pro198Leu polymorphism and stenosis of coronary arteries: A population-based prediction

BackgroundWe studied the association between erythrocyte glutathione peroxidase1 (GPx1) activity and rs1050450 (Pro198Leu) site with the stenosis of coronary arteries and, evaluated the Pro/Leu position within the predicted tertiary structure. MethodsSubjects were recruited from who underwent coronary angiography (controls; n=55, Stenosis <5% and Patients; n=95, Stenosis ≥50%). The GPx1 activity and rs1050450 C/T variants were determined using enzyme assay and RFLP-PCR techniques, respectively. The conserved regions and GPx1 tertiary structure were predicted using bioinformatics tools. ResultsWe did not find significant association between GPx1 activity (P=0.96), rs1050450 genotype distribution and coronary artery disease (adjusted OR=0.79; 95%CI 0.28–2.2, P=0.6). The polymorphic variants were not located at the predicted structural and functional domains so that it had not the significant role on the GPx1stability and function. ConclusionsIn agreement with the results predicted from bioinformatics tools, we suggested that the GPx1 activity and rs1050450 (Pro198Leu) site are not involved in the development of stenosis of coronary arteries in the study population.

P33. NK-92 cellular immunotherapy as an alternative to donor derived peripheral blood NK cells

<h3>INTRODUCTION</h3> Hypermethylation of the promoter region of the <i>hMLH1</i> gene is associated with absent expression of MLH1 protein in sporadic colorectal cancers with microsatellite instability (MSI+), and it has been proposed that methylation may be a mechanism of inactivation in Knudson9s hypothesis. The incidence of hypermethylation of the<i>hMLH1</i> promoter in hereditary non-polyposis colorectal cancer (HNPCC) versus MSI+ sporadic colorectal cancer was investigated and compared. <h3>METHODS</h3> DNA was available from 10 HNPCC colorectal cancers (median age 58 years, range 39-67) with germline mutations in <i>hMLH1 </i>and 10 MSI+ sporadic colorectal cancers (mean age 79 years, range 41-85). MSI was determined by amplification of BAT26 and TGF-β RII. The methylation status of the <i>hMLH1</i> promoter was studied by the polymerase chain reaction (PCR) based<i>Hpa</i>II restriction enzyme assay technique. Evidence of allelic loss at <i>hMLH1 </i>was searched for in the HNPCC colorectal cancers. <h3>RESULTS</h3> All cases were confirmed to be MSI+. The promoter region of <i>hMLH1 </i>was hypermethylated in seven of 10 MSI+ sporadic cancers versus 0 of 10 HNPCC cancers (p&lt;0.002). Evidence of loss of heterozygosity at<i>hMLH1 </i>was observed in eight of the 10 HNPCC colorectal cancers. <h3>CONCLUSION</h3> While mutations and allelic loss are responsible for the MSI+ phenotype in HNPCC cancers, the majority of MSI+ sporadic cancers are hypermethylated in the promoter region of <i>hMLH1.</i> These data further support our argument that tumours from HNPCC patients, which almost always acquire a raised mutation rate, mostly follow a different pathway from MSI+ sporadic tumours.

Short-term effect of aniline on soil microbial activity: a combined study by isothermal microcalorimetry, glucose analysis, and enzyme assay techniques

The accidents of aniline spill and explosion happened almost every year in China, whereas the toxic effect of aniline on soil microbial activity remained largely unexplored. In this study, isothermal microcalorimetric technique, glucose analysis, and soil enzyme assay techniques were employed to investigate the toxic effect of aniline on microbial activity in Chinese soil for the first time. Soil samples were treated with aniline from 0 to 2.5mg/g soil to tie in with the fact of aniline spill. Results from microcalorimetric analysis showed that the introduction of aniline had a significant adverse effect on soil microbial activity at the exposure concentrations ≥0.4mg/g soil (p < 0.05) and ≥0.8mg/g soil (p < 0.01), and the activity was totally inhibited when the concentration increased to 2.5mg/g soil. The glucose analysis indicated that aniline significantly decreased the soil microbial respiratory activity at the concentrations ≥0.8mg/g soil (p < 0.05) and ≥1.5mg/g soil (p < 0.01). Soil enzyme activities for β-glucosidase, urease, acid-phosphatase, and dehydrogenase revealed that aniline had a significant effect (p < 0.05) on the nutrient cycling of C, N, and P as well as the oxidative capacity of soil microorganisms, respectively. All of these results showed an intensively toxic effect of aniline on soil microbial activity. The proposed methods can provide toxicological information of aniline to soil microbes from the metabolic and biochemical point of views which are consistent with and correlated to each other.

The development and use of an ELISA-based method to follow the distribution of cellulase monocomponents during the hydrolysis of pretreated corn stover

BackgroundIt is widely recognised that fast, effective hydrolysis of pretreated lignocellulosic substrates requires the synergistic action of multiple types of hydrolytic and some non-hydrolytic proteins. However, due to the complexity of the enzyme mixture, the enzymes interaction with and interference from the substrate and a lack of specific methods to follow the distribution of individual enzymes during hydrolysis, most of enzyme-substrate interaction studies have used purified enzymes and pure cellulose model substrates. As the enzymes present in a typical “cellulase mixture” need to work cooperatively to achieve effective hydrolysis, the action of one enzyme is likely to influence the behaviour of others. The action of the enzymes will be further influenced by the nature of the lignocellulosic substrate. Therefore, it would be beneficial if a method could be developed that allowed us to follow some of the individual enzymes present in a cellulase mixture during hydrolysis of more commercially realistic biomass substrates.ResultsA high throughput immunoassay that could quantitatively and specifically follow individual cellulase enzymes during hydrolysis was developed. Using monoclonal and polyclonal antibodies (MAb and PAb, respectively), a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to specifically quantify cellulase enzymes from Trichoderma reesei: cellobiohydrolase I (Cel7A), cellobiohydrolase II (Cel6A), and endoglucanase I (Cel7B). The interference from substrate materials present in lignocellulosic supernatants could be minimized by dilution.ConclusionA double-antibody sandwich ELISA was able to detect and quantify individual enzymes when present in cellulase mixtures. The assay was sensitive over a range of relatively low enzyme concentration (0 – 1 μg/ml), provided the enzymes were first pH adjusted and heat treated to increase their antigenicity. The immunoassay was employed to quantitatively monitor the adsorption of cellulase monocomponents, Cel7A, Cel6A, and Cel7B, that were present in both Celluclast and Accellerase 1000, during the hydrolysis of steam-pretreated corn stover (SPCS). All three enzymes exhibited different individual adsorption profiles. The specific and quantitative adsorption profiles observed with the ELISA method were in agreement with earlier work where more labour intensive enzyme assay techniques were used.

Multi-component, Self-assembled, Functional Soft Materials

Supramolecular chemistry is an emerging tool for devising materials that can perform specified functions. The self-assembly of facially amphiphilic bile acid molecules has been extensively utilized for the development of functional soft materials. Supramolecular hydrogels derived from the bile acid backbone act as useful templates for the intercalation of multiple components. Based on this, synthesis of gel-nanoparticle hybrid materials, photoluminescent coating materials, development of a new enzyme assay technique, etc. were achieved in the author's laboratory. The present account highlights some of these achievements.

PERIODS OF MOUTH OPENING AND YOLK ABSORPTION RELATED TO THE ACTIVITY OF DIGESTIVE ENZYMES IN THE EARLY STADIA FROM THE SECOND GENERATION OF HUMPBACK GROUPER BROODSTOCK

The experiment was carried out to determine the exposure time of the first mouth opening on humpback grouper larval from F2 broodstock on hatchery until 10 DAH (days after hatching) because this phase is a critical phase of larval adaptation from food endogenous (from yolk sac) to the ability of absorbing nutrients from external (exogenous). The eggs used were collected from natural spawning of second generation of humpback grouper reared in the tank size of 150 litre. The mouth opening was observed at 90 and 45 degrees under computerize integrated microscope and processed by a specific software. The rate of yolk absorption was observed in larvae from the beginning to the end of breeding, while the activities of digestive enzymes (trypsin and chymotripsin) were analyzed by enzyme assay techniques. The results showed that the period of mouth opening of larvae of humpback grouper occurred at 3 DAH with mouth openings of 45° as large as 0.103 mm and of 90° as large as 0.156 mm. In the early stadia, the diameter of yolk was 0.1875 mm, while the oil globule was 0.0537 mm. At 4 days after hatching, the yolk and oil globule had been absorbed, while the enzymes trypsin and chymotripsin were detected in the early stadia. The results also showed that the larvae was started to use foods from outside after the age of 4 DAH. Up to the age of 10 days, the mouth opening increased and the activity of trypsin and chymotripsin were still detected.Keywords: progeny larvae of humpback grouper F2, mouth opening, yolk sac, enzyme

PERIODS OF MOUTH OPENING AND YOLK ABSORPTION RELATED TO THE ACTIVITY OF DIGESTIVE ENZYMES IN THE EARLY STADIA FROM THE SECOND GENERATION OF HUMPBACK GROUPER BROODSTOCK

&lt;p&gt;The experiment was carried out to determine the exposure time of the first mouth opening on humpback grouper larval from F2 broodstock on hatchery until 10 DAH (days after hatching) because this phase is a critical phase of larval adaptation from food endogenous (from yolk sac) to the ability of absorbing nutrients from external (exogenous). The eggs used were collected from natural spawning of second generation of humpback grouper reared in the tank size of 150 litre. The mouth opening was observed at 90 and 45 degrees under computerize integrated microscope and processed by a specific software. The rate of yolk absorption was observed in larvae from the beginning to the end of breeding, while the activities of digestive enzymes (trypsin and chymotripsin) were analyzed by enzyme assay techniques. The results showed that the period of mouth opening of larvae of humpback grouper occurred at 3 DAH with mouth openings of 45° as large as 0.103 mm and of 90° as large as 0.156 mm. In the early stadia, the diameter of yolk was 0.1875 mm, while the oil globule was 0.0537 mm. At 4 days after hatching, the yolk and oil globule had been absorbed, while the enzymes trypsin and chymotripsin were detected in the early stadia. The results also showed that the larvae was started to use foods from outside after the age of 4 DAH. Up to the age of 10 days, the mouth opening increased and the activity of trypsin and chymotripsin were still detected.&lt;/p&gt;&lt;p&gt;Keywords: progeny larvae of humpback grouper F2, mouth opening, yolk sac, enzyme&lt;/p&gt;

Impact of beta-cypermethrin on soil microbial community associated with its bioavailability: A combined study by isothermal microcalorimetry and enzyme assay techniques

In this study, an isothermal microcalorimetric technique has been used to show that beta-cypermethrin (CYP) had no significant effect ( p > 0.05) on soil microbial activity at 80 μg g −1 soil. Our soil enzyme data indicated that beta-CYP ranging 10–80 μg g −1 soil had no significant effect ( p > 0.05) on soil enzyme activities such as β-glucosidase, urease, acid-phosphatase, and dehydrogenase. Therefore, our results infer that beta-CYP would not pose severe toxicity to soil microbial community, but its toxic level may vary greatly with environment that associates with its increase in bioavailability: the level in soil (at μg g −1) < the level in sediment (varying from μg g −1 to μg L −1) < the level in water (at μg L −1). The comparison of the results of solvent volatilization on soil microbial activity has shown that the acetone-treated sample had no significant difference with the control ( p > 0.05). These results suggest that the heavy application of beta-CYP may not cause damage to soil microbial community which is very different from its high toxicity to the aquatic organism.

Oxidative impairment of hippocampal long‐term potentiation involves activation of protein phosphatase 2A and is prevented by ketone bodies

Previous studies have shown that ketone bodies (KB) exert antioxidant effects in experimental models of neurological disease. In the present study, we explored the effects of the KB acetoacetate (ACA) and beta-hydroxybutyrate (BHB) on impairment of hippocampal long-term potentiation (LTP) in rats by hydrogen peroxide (H(2)O(2)) using electrophysiological, fluorescence imaging, and enzyme assay techniques. We found that: 1) a combination of ACA and BHB (1 mM each) prevented impairment of LTP by H(2)O(2) (200 microM); 2) KB significantly lowered intracellular levels of reactive oxygen species (ROS)--measured with the fluorescent indicator carboxy-H(2)DCFDA (carboxy-2',7'-dichlorodihydrofluorescein diacetate)--in CA1 pyramidal neurons exposed to H(2)O(2); 3) the effect of KB on LTP was replicated by the protein phosphatase 2A (PP2A) inhibitor fostriecin; 4) KB prevented impairment of LTP by the PP2A activator C(6) ceramide; 5) fostriecin did not prevent the increase in ROS levels in CA1 pyramidal neurons exposed to H(2)O(2), and C(6) ceramide did not increase ROS levels; 6) PP2A activity was enhanced by both H(2)O(2) and rotenone (a mitochondrial complex I inhibitor that increases endogenous superoxide production); and 7) KB inhibited PP2A activity in protein extracts from brain tissue treated with either H(2)O(2) or ceramide. We propose that oxidative impairment of hippocampal LTP is associated with PP2A activation, and that KB prevent this impairment in part by inducing PP2A inhibition through an antioxidant mechanism.

Primary sludge floc degradation is accelerated under biosulphidogenic conditions: Enzymological aspects

The mechanism of enhanced sludge floc fracture due to the action of enzymes hydrolysing important structural components, remains a key element in our understanding of how the floc integrity in systems utilising a sulphate-reducing system is compromised. Commercial β-glucosidase and cellulase as well as proteases were added to methanogenic bioreactor and sulphidogenic bioreactor sludge and the effect of these enzymes on sludge floc size (diameter) distribution was investigated using bright field and differential interference contrast light microscopy. Cellulolytic activity of the enzymes was monitored using standard enzyme assay techniques and Bradford, Somogyi–Nelson, and total carbohydrate assays were performed. Sludge flocs present in the sulphide-rich environment of the sulphidogenic bioreactor were found to be of smaller average diameter (23 μm) than those flocs present in the methanogenic bioreactor (average diameter of 40 μm). The addition of commercially available enzymes resulted in an increased rate of matrix hydrolysis, leading to increased rates of floc fracture and smaller flocs. The presence of β-glucosidase activity and protease, naturally residing within the sludge flocs, was confirmed. The addition of commercial enzymes may be prohibitively costly on a large scale, and ways of increasing the activities of these enzymes naturally residing within the floc matrix were investigated. As the bulk of the extracellular polymeric substances (EPS) was shown to be composed of mainly polysaccharides and protein, it was proposed that, by increasing the activities of the native β-glucosidases and proteases naturally residing within the floc matrix, the process of floc hydrolysis could be enhanced. Sulphide was shown to increase the activity of these key enzymes, and we propose that this is one of the factors contributing to the accelerated rate of sludge solubilisation under sulphate-reducing conditions. It was also confirmed that this effect of sulphide occurred at the enzymatic level.

A rapid enzyme assay for � -galactosidase using optically gated sample introduction on a microfabricated chip

The ability to perform enzyme assays on microchips is demonstrated using optically gated sample introduction. The hydrolysis of fluorescein mono- beta- d-galactopyranoside (FMG) by beta- d-galactosidase ( beta-Gal) is continuously monitored using a microchip for 5 to 10 min. The outcome of the reaction was analyzed by performing serial on-chip separations of fluorescent substrate, FMG, and product, fluorescein. Kinetic information about beta-Gal has been successfully obtained by varying the concentration of FMG. beta-Gal enzymes from two different sources including bovine liver and E. coli., have been examined and compared to each other and to results obtained using traditional assay methods. In addition, the competitive inhibition of beta-Gal by phenylethyl beta- d-thiogalactoside (PETG) and beta-lactose has been studied using this technique. PETG is found to have higher inhibition than lactose in the hydrolysis. This separation-based enzyme assay technique avoids the possible fluorescence interference between FMG and fluorescein, which is a problem with the traditional plate assay method. Additionally, the amount of the enzyme and substrate required with this technique is at least four orders of magnitude lower than the traditional plate assay method. By using optically gated sample introduction, microchips allow continuous serial injections and separations without any potential switch, thus making this technique ideal as a sensor for enzyme assays. This technique should therefore be valuable for high-throughput screening in the drug discovery industry.

Is there a place for preconditioning during cardiac operations in humans?

Background. Activation of the kinase cascade (protein kinase C (PKC), tyrosine kinase (TK), and mitogen-activated protein kinase (MAPK) is a key feature of the transduction pathway, elicited by preconditioning signals and mediating their cardioprotective effects. We assessed whether such an activation occurred during cardiac operations and could thus represent a target for cardioprotective strategies. Methods. A total of 20 patients undergoing coronary artery bypass grafting surgery were studied. During the first 10 minutes of cardiopulmonary bypass (CPB), 10 were treated with sevoflurane (2.5 minimum alveolar concentration), an inhalational anesthetic that mimics preconditioning through a similar activation of the kinase cascade. Ten case-matched patients undergoing 10 minutes of sevoflurane-free CPB served as controls. Right atrial biopsies were taken before and 10 minutes after CPB and were then processed for the measurement of PKC, TK, and p38 MAPK activities by enzyme assay techniques. Troponin I was also monitored over the first 2 postoperative days. Results. Compared with pre-CPB values, PKC and p38 MAPK activities (in nanomoles per milligram of protein per minute and arbitrary units, respectively) increased significantly and to the same extent in both groups: PKC, from 20.7 ± 0.7 to 29.9 ± 3.9 in controls ( p = 0.037) and from 18.4 ± 1.1 to 23.9 ± 1.8 in sevoflurane ( p = 0.016); p38 MAPK, from 88.6 ± 8.5 to 312.9 ± 66.2 in controls ( p = 0.005) and from 114.6 ± 14.7 to 213.4 ± 51.8 in sevoflurane ( p = 0.045). Conversely, sevoflurane triggered a significant increase in TK activity (from 68.5 ± 1.4 to 83.7 ± 2.9 picomoles per milligram of protein per minute p = 0.0015) which did not occur in controls (from 67.5 ± 1.9 to 76.8 ± 4.2 picomoles per milligram of protein per minute, p = 0.09). Likewise, the peak postoperative value of troponin I was not different between controls and sevoflurane-treated patients (3.4 ± 0.6 vs 2.4 ± 0.4, p = 0.21). Conclusions. Cardiopulmonary bypass triggers an activation of the kinase cascade that is mechanistically linked to opening of potassium channels. The direct opening of these channels by the anesthetic sevoflurane does not increase kinase activation further, nor does it improve markers of cell necrosis, thus suggesting that pharmacologically targeting potassium channels may overlap the preconditioning-like effects of CPB alone.

Modified enzyme activity assay to determine biofilm biomass

An assay of potential exoproteolytic enzyme activity was modified to quantitatively measure the biomass of attached biofilm. The assay utilized the nonfluorescent compound l-leucine-β-naphthylamide (LLβN) that becomes fluorescent when bacterial exoenzymes break the peptide bond, releasing the fluorochrome β-naphthylamine. Fluorescence development was measured by pumping the liquid phase of a biofilm sample through a fluorescence detector and recording the detector output using a personal computer. A significant linear relationship was shown to exist between the rate of fluorescence development and the biofilm's biomass as carbon, determined using total direct cell counts, measured cell volumes and an existing relationship between cell volume and cell carbon. The technique was used to measure biofilm biomass for experiments where iron oxides were the substratum. Biofilm biomass measurements made using heterotrophic plate counts (HPCs) on R2A medium were shown to correlate well to biomass measurements made using the modified enzyme assay. The technique was shown to be sufficiently sensitive to measure biomass on samples containing little biofilm biomass, such as those exposed to free chlorine. While granular and porous media were used for the experiments presented, small biofilm coupons could easily be used to measure biofilm biomass, expanding the number of possible applications for the enzyme assay technique.

The role of hypermethylation of thehMLH1 promoter region in HNPCC versus MSI+ sporadic colorectal cancers

INTRODUCTIONHypermethylation of the promoter region of the hMLH1 gene is associated with absent expression of MLH1 protein in sporadic colorectal cancers with microsatellite instability (MSI+), and it has been proposed...

Effect of electron acceptor conditions on hydrolytic enzyme synthesis in bacterial cultures

An experimental study was undertaken to observe the effect of electron acceptor conditions on the enzyme synthesis potential in pure cultures of Bacillus amyloliquefaciens (gram positive) and Pseudomonas saccharophilia (gram negative). An appropriate experimental methodology was developed to observe enzymes synthesis over a time period by using enzyme assay technique. It was found that the development of the starch hydrolytic enzyme system in both the cultures was strongly affected by the type of electron acceptor conditions. The relative change in the enzyme synthesis due to change in electron acceptor conditions was more pronounced in B. amyloliquefaciens as compared to P. saccharophilia. In case of B. amyloliquefaciens the calculated reduction factors η no3 and η fe ranged from 0.13 to 0.21 and 0.04 to 0.06, respectively, whereas for P. saccharophila they were found to range from 0.40 to 0.54 and 0.20 to 0.27, respectively. An apparent relationship between the growth rates of the cultures during active growth phase in minimal starch medium and their specific enzyme synthesis potential was observed. Further, the hydrolytic enzymes were found to be released in the bulk solution and the activity of the synthesized enzymes was found to be independent of electron acceptor conditions.

Facile enzyme immobilization and assay technique using polyethyleneimine-cellulose thin-layer chromatography as a solid support

In the search for a plate-shaped solid support for enzyme immobilization, we found commercially obtainable polyethyleneimine (PEI)-cellulose thin-layer chromatography (TLC) plates to have adequate physical and chemical properties for enzyme immobilization. Through Benedict's, Tollen's and ninhydrin tests, the PEI-cellulose coating proved to have enough physical strength and amine concentration, and its properties could be maintained nearly constantly during the processes of enzyme immobilization. To confirm that the PEI-cellulose TLC plate was a suitable support for enzyme immobilization, alkaline phosphatase was immobilized using glutaraldehyde as a crosslinking reagent. The activity of the immobilized alkaline phosphatase was assayed with a reiterative method using p-nitrophenyl phosphate as a substrate. By analyzing Lineweaver-Burk plots from the results of successive assays, we proved the immobilized alkaline phosphatase to be reusable many times.

Secretory leukocyte protease inhibitor in the cervical mucus and in the fetal membranes

The purpose was to determine the concentration of leukocyte elastase and SLPI in the cervical mucus plug, cervical tissue, fetal membranes, amniotic fluid and serum from pregnant women. Samples were analysed using radioimmunoassay, immunodiffusion, immunohistological and enzyme assay techniques. Statistical comparisons were performed by the Mann-Whitney two-sample rank sum test. Concentrations of SLPI and elastase in cervical tissue are significantly increased during pregnancy and after delivery. Very high concentrations of SLPI and elastase were found in the cervical mucus plug. The concentrations of SLPI and elastase in human fetal membranes are highest at the rupture site. The concentration of elastase in amnion from preterm delivery was significantly higher than in amnion from term deliveries. The high molar ratio of SLPI/elastase in the cervical mucus plug suggests that inhibition of elastase is important during normal pregnancy. The role of SLPI may be protection of the fetal membranes and cervical tissue.