Enzymatic Reagent Research Articles

An integrated device for the rapid and sensitive detection of the influenza hemagglutinin.

Influenza is a viral respiratory tract infection responsible for up to 5 million cases of severe infection and nearly 600 000 deaths worldwide each year. While treatments for influenza exist, diagnostics for the virus at the point of care are limited in their sensitivity and ability to differentiate between subtypes. We have developed an integrated two-dimensional paper network (2DPN) for the detection of the influenza virus by the surface glycoprotein, hemagglutinin. The hemagglutinin assay was developed using proteins computationally designed to bind with high affinity to the highly-conserved sialic acid binding site. The integrated 2DPN uses a novel geometry that allows automated introduction of an enzymatic amplification reagent directly to the detection zone. This assay was integrated into a prototype device and demonstrated successful detection of clinically relevant virus concentrations spiked into 70 μL of virus-free pediatric nasal swab samples. Using this novel geometry, we found improved assay performance on the device (compared to a manually-operated dipstick method), with a sensitivity of 4.45 × 102 TCID50 per mL on device.

Gene to diagnostic: Self immobilizing protein for silica microparticle biosensor, modelled with sarcosine oxidase

A rational design approach is proposed for a multifunctional enzyme reagent for point-of-care diagnostics. The biomaterial reduces downstream isolation steps and eliminates immobilization coupling chemicals for integration in a diagnostic platform. Fusion constructs combined the central functional assay protein (e.g. monomeric sarcosine oxidase, mSOx, horseradish peroxidase, HRP), a visualizing protein (e.g. mCherry) and an in-built immobilization peptide (e.g. R5). Monitoring protein expression in E.coli was facilitated by following the increase in mCherry fluorescence, which could be matched to a color card, indicating when good protein expression has occurred. The R5 peptide (SSKKSGSYSGSKGSKRRIL) provided inbuilt affinity for silica and an immobilization capability for a silica based diagnostic, without requiring additional chemical coupling reagents. Silica particles extracted from beach sand were used to collect protein from crude protein extract with 85–95% selective uptake. The silica immobilized R5 proteins were stable for more than 2 months at room temperature. The Km for the silica-R52-mCh-mSOx-R5-6H was 16.5 ± 0.9 mM (compared with 16.5 ± 0.4 mM, 16.3 ± 0.3 mM, and 16.1 ± 0.4 mM for R52-mCh-mSOx-R5-6H, mSOx-R5-6H and mSOx-6H respectively in solution). The use of the “silica-enzymes” in sarcosine and peroxide assays was shown, and a design using particle sedimentation through the sample was examined. Using shadowgraphy and particle image velocimetry the particle trajectory through the sample was mapped and an hourglass design with a narrow waist shown to give good control of particle position. The hourglass biosensor was demonstrated for sarcosine assay in the clinically useful range of 2.5–10 μM in both a dynamic and end point measurement regime.

Hydrogen Production from Energy Poplar Preceded by MEA Pre-Treatment and Enzymatic Hydrolysis.

The need to pre-treat lignocellulosic biomass prior to dark fermentation results primarily from the composition of lignocellulose because lignin hinders the processing of hard wood towards useful products. Hence, in this work a two-step approach for the pre-treatment of energy poplar, including alkaline pre-treatment and enzymatic saccharification followed by fermentation has been studied. Monoethanolamine (MEA) was used as the alkaline catalyst and diatomite immobilized bed enzymes were used during saccharification. The response surface methodology (RSM) method was used to determine the optimal alkaline pre-treatment conditions resulting in the highest values of both total released sugars (TRS) yield and degree of lignin removal. Three variable parameters (temperature, MEA concentration, time) were selected to optimize the alkaline pre-treatment conditions. The research was carried out using the Box-Behnken design. Additionally, the possibility of the re-use of both alkaline as well as enzymatic reagents was investigated. Obtained hydrolysates were subjected to dark fermentation in batch reactors performed by Enterobacter aerogenes ATCC 13048 with a final result of 22.99 mL H2/g energy poplar (0.6 mol H2/mol TRS).

Advancing the analysis of terbutaline in urine samples using novel enzyme hydrolysis.

Aim: To investigate the efficiency of two new fast-acting enzymes, recombinant arylsulfatase (IMCS-PSF) and mutant β-glucuronidase from Escherichia coli (IMCSzyme), in hydrolyzing specific terbutaline metabolites. Materials & methods: Two purified novel enzymes are used to precisely determine the amount of each metabolite in urine at different time points after oral administration. After systematically evaluating the hydrolysis efficiency of the novel enzymes compared with commercially available enzymes, these recently developed enzymes were applied to establish the separate urine concentration profiles of terbutaline and each metabolite. Results & discussion: The results highlight the highly efficient arylsulfatase enzyme expressed from E. coli for urine analysis of terbutaline while suggesting sulfoconjugates as the main terbutaline metabolites. Conclusion: This study demonstrated the high efficiency of the IMCS-PSF enzyme in hydrolyzing terbutaline conjugates in comparison with other enzyme reagents typically used for the analysis of terbutaline and sulfoconjugates are the main terbutaline metabolites in urine.

A simplified and optimized protocol for total DNA extraction from insect species: applicable for studying genetic diversity and PCR-based specimen identification via partial amplification of cytochrome oxidase I (COI) gene

The efficient DNA extraction from insects has been suggested as a critical and main step affecting molecular entomology for taxonomic identification, the establishment of DNA barcoding library and analysis of genetic diversity relationship between insect populations. For successfully apply these molecular techniques, high-quantity and high-quality of the extracted DNA are required. Several protocols for efficient genomic DNA extraction from insects have been developed. In this research, we represent a rapid, reliable and cost-effective method that it is not reliant on poisonous and enzymatic reagents for DNA extraction from insect tissues. Results showed that high quantity and high-quality of the isolated DNA by this method is suitable and can be used directly for PCR, also is enough to do hundreds of molecular reactions. In conclusion, we described a fast, cost-effective, non-toxic and enzyme-free protocol for high yield genomic DNA extraction from green Lacewings (Chrysoperla carnea) tissues in basic equipment laboratories.

A novel combination of quick response code and microfluidic paper-based analytical devices for rapid and quantitative detection.

Nowadays, there is great interest in the use of microfluidic paper-based analytical devices (μPADs) for low-cost diagnostics. In most cases, the test equipment is needed. Here we report a new type of device-independent detection method based on timer-paper-based analytical devices, which can be tested by smartphone, and its application for cholesterol detection. The Quick Response code was designed as the timer component of this device. Wax printing method was employed to print the pattern on filter paper. The color and enzyme reagents have been immobilized in the hydrophilic channel to complete the colorimetric detection for cholesterol. Under laminar flow conditions of the cellulose network, the liquid volume of detection zone has been quantified by monitoring the fluid residence time on different area of the timer-paper-based devices. The precise monitoring of detection time can promote the accuracy of colorimetric detection. This is very important for the quantitative detection of paper-based analytical devices. One significant outcome of this report is that simple and accurate timer can be used for detection process self-clocking. The factors of total detection time have been investigated. The linear range of the calibration curve was 3.0 ~ 6.0mmolL-1, with correlation coefficient of 0.9956. With the characteristic of easy to use, low cost and accurate monitoring of detection time, this kind of timer-paper-based devices can be applied to cholesterol or other substances rapid detection.

Reference intervals for serum 1,5-anhydroglucitol in children, adolescents, adults, and pregnant women

Background1,5-anhydroglucitol (1,5-AG) is a validated marker of short-term glycemic control. We determined the reference intervals of 1,5-AG in different age groups and during pregnancy. MethodsBlood samples were collected from 2303 Euro-Brazilian healthy subjects: 580 children, 496 adolescents, 922 adults matched by age and sex, and 305 pregnant women in four gestational periods. Serum 1,5-AG was measured using an enzymatic reagent in an automated system. ResultsThe calculated reference intervals (nonparametric, 2.5th–97.5th) for males and females were, respectively: children, 96–302 and 89–277 μmol/l; adolescents, 84–311 and 79–277 μmol/l; and adults, 80–260 and 62–241 μmol/l. Males consistently showed significantly higher concentrations than females. 1,5-AG reference intervals in pregnant women were 56–298 μmol/l at <23 weeks gestation (n = 110), 37–166 μmol/l at 24–28 weeks gestation (n = 106), 34–155 μmol/l at 29–32 weeks gestation (n = 52), and 33–246 μmol/l at >32 weeks gestation (n = 37). No significant differences in 1,5-AG concentration were observed between non-pregnant and pregnant women at <23 weeks of gestation. A negative correlation (r = −0.287; p < .001) between 1,5-AG concentration and age was observed. ConclusionsThe reference intervals for 1,5-AG were affected by sex and age.

Bioethanol production from rice straw by simultaneous saccharification and fermentation with statistical optimized cellulase cocktail and fermenting fungus

BackgroundRice straw is one of the abundant lignocellulosic biomass with potential as a feedstock for bioethanol production. To produce ethanol from the biomass biologically, enzymatic hydrolysis is necessary that can effectively degrade rice straw into fermentable sugars such as glucose and xylose. Many researches utilized many kinds of commercial cellulase reagents on the hydrolysis of cellulose. Since these have different enzyme activities, enzyme reagents suitable for each biomass must be selected. In this study, three appropriate cellulase reagents were selected by multivariate analysis technique and then optimized by design of experiments method for efficient hydrolysis of alkali-pretreated rice straw. Moreover, an ethanol production from the treated straw was performed by simultaneous saccharification and fermentation (SSF) with the optimized enzyme cocktail and xylose-fermenting fungus of Mucor circinelloides.ResultsPretreatment by alkali solution of rice straw resulted in the increase of fermentable sugar content from 56.3 to 80.0%. The desirable commercial enzyme reagents for saccharification of the straw were determined as a combination of “Cellulase Onozuka 3S”, “Cellulase T Amano 4”, and “Pectinase G Amano” by a multivariate analysis based on various cellulolytic activities of each reagent. The optimum mixing ratio was calculated by response surface method based on design of experiment method. The optimized cocktail successfully achieved 75.3 g/L in production of the total fermentable sugar by hydrolysis of alkali-treated rice straw that agreed with the hydrolysis efficiency of 94.1%. SSF of 100 g/L treated rice straw with the optimal cocktail and M. circinelloides under aerobic condition resulted in 30.5 g/L ethanol concentration for 36 h.ConclusionThe construction of cellulase cocktail by the proposed statistical method enabled efficient hydrolysis of alkali-treated rice straw. SSF process combining the optimized cocktail and a xylose-fermenting fungus could be expected as a promising system for ethanol production from various lignocellulosic biomasses.

Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis.

Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.

Diagnosis of spontaneous bacterial peritonitis in children using leukocyte esterase reagent strips and granulocyte elastase immunoassay.

Aim of the studyWe aimed to assess the utility and rapidity of granulocyte elastase (GE) latex immunoassay and leukocyte esterase (LE) reagent strips for the diagnosis of spontaneous bacterial peritonitis (SBP) in hepatic children with ascites.Material and methodsThis study included 80 ascitic fluid (AF) samples from 45 patients with chronic liver diseases. They were divided into 2 groups (SBP and non-SBP groups). White blood cells > 500 cell/mm3 or polymorphonuclear leukocytes > 250 cell/mm3 were considered as a positive result for the diagnosis of SBP. The AF obtained at bedside was immediately tested with an LE enzyme reagent strip, and another sample was aliquoted for measurement of serum GE.ResultsOn doing LE dipstick strips, all the non-SBP group gave no coloration with LE strips while 62% of the SBP group gave coloration. LE strips had accuracy of 86.25% in differentiating SBP and non-SBP at a cut-off value of 1 (color grade 1). The diagnostic performance of GE in differentiating SBP and non-SBP was assessed and showed accuracy of 70% for a cutoff value of 123.5 ng/ml.ConclusionsApplication of LE reagent strips is a bedside, rapid, easy-to-use, and inexpensive method for diagnosis of SBP. It has an accuracy of 86.25% in differentiating SBP and non-SBP, which is higher than more complicated and delayed methods such as GE latex immunoassay, which has an accuracy of 70%.

Sol-Gel Derived Adsorbents with Enzymatic and Complexonate Functions for Complex Water Remediation.

Sol-gel technology is a versatile tool for preparation of complex silica-based materials with targeting functions for use as adsorbents in water purification. Most efficient removal of organic pollutants is achieved by using enzymatic reagents grafted on nano-carriers. However, enzymes are easily deactivated in the presence of heavy metal cations. In this work, we avoided inactivation of immobilized urease by Cu (II) and Cd (II) ions using magnetic nanoparticles provided with additional complexonate (diethylene triamine pentaacetic acid or DTPA) functions. Obtained nanomaterials were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM). According to TGA, the obtained Fe3O4/SiO2-NH2-DTPA nanoadsorbents contained up to 0.401 mmol/g of DTPA groups. In the concentration range Ceq = 0–50 mmol/L, maximum adsorption capacities towards Cu (II) and Cd (II) ions were 1.1 mmol/g and 1.7 mmol/g, respectively. Langmuir adsorption model fits experimental data in concentration range Ceq = 0–10 mmol/L. The adsorption mechanisms have been evaluated for both of cations. Crosslinking of 5 wt % of immobilized urease with glutaraldehyde prevented the loss of the enzyme in repeated use of the adsorbent and improved the stability of the enzymatic function leading to unchanged activity in at least 18 cycles. Crosslinking of 10 wt % urease on the surface of the particles allowed a decrease in urea concentration in 20 mmol/L model solutions to 2 mmol/L in up to 10 consequent decomposition cycles. Due to the presence of DTPA groups, Cu2+ ions in concentration 1 µmol/L did not significantly affect the urease activity. Obtained magnetic Fe3O4/SiO2-NH2-DTPA-Urease nanocomposite sorbents revealed a high potential for urease decomposition, even in presence of heavy metal ions.

Performance verification of six enzymatic glycated albumin reagents

Objective To validate the performance of six enzymatic glycated albumin reagents and evaluate their clinical application. Methods The performance of six enzymatic glycated albumin reagents(labled as A, B, C, D, E, F) from Beijing Jiuqiang Co, Beijing Lideman Co, Ningbomeikang Co, Beijing Haomai Co, Sichuan Maike Co and Asahi Kasei Co were assessed on Olympus AU5800 automatic biochemistry analyzer. According to the standard of CLSI, the precision, interference and linear correlation of these reagents were assessed.To assess the accuracy of GA% , we used GA standard material whose value had been assigned using ID-LC/MS method provided by ReCCS.To do the method comparison and determine the consistency of assay, 50 fresh serum samples of T2DM outpatient and 80 fresh serum samples of apparently healthy people in Jan 2016 were tested using six kits.According to the EP28-A3C protocol, the reference range for GA%was validated in 122 apparently healthy individuals undertaking medical examination from January to February 2016 in PUMC. Results The precision, and the ability of anti-interference of the six reagents were good.The accuracy percentage deviation of six reagents was -19.3%-9.2%.The correlation coefficient of domestic reagents A to E and imported reagents F in the determination of GA% was 0.966-0.999, the average absolute bias was 7.0%-10.4%.The coincidence rate of A-E and F in determining abnormal GA% was between 88.5% and 96.9%.The coincidence rate was increased after switching to the reference range for preliminary clinical evaluation. Conclusion Six GA enzymatic kits used in automatic biochemical analyzer have high precision and strong anti-interference ability.Accuracy still needs to be improved.((Chin J Lab Med, 2017, 40: 346-442) Key words: Serum albumin; Enzymic assays; Reference value

Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a non enzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

THE NEW METALL-BETA-LACTAMASE’S INHIBITOR EFFICACY IN A MODEL SYSTEM IN VITRO

The Enterobacteriaceae antibiotics resistance depends on a combination of several mechanisms, such as the beta-lactamases overproduction, the microbial cell reduction outer membrane permeability (usually associated with loss of protein porin), the presence of efflux systems. Particularly noteworthy are the metallo-beta-lactamases (MBL) whose presence causes resistance of gram-negative microorganisms to all beta-lactam antibiotics (in some cases except aztreonam). Currently there are no MBL inhibitors permitted for use in the clinic. The effective inhibitors search for carbapenem-resistant bacteria MBL authorized for use in the clinic and reinforcing effects of carbapenems, served as the basis for the present study. The work was carried out in three stages: 1) creating a model system using a standard enzyme reagent metallo-beta-lactamase P. aeruginosa recombinant expressed in E. coli, to evaluate the increasing of minimal inhibitory concentrations (MIC) of carbapenems against previously sensitive Gram-negative microorganisms strains in vitro;&#x0D; 2) evaluation of MBL promising inhibitors in the presence of the same standard enzyme reagent; 3) evaluation of the ability of the identified inhibitors increase the carbapenems effects against clinical isolates of Gram-negative microorganisms producing MBL, in terms of the their MIC and fractional inhibitory concentration index (FIC index). The checkerboard array was modified to evaluate the combined use of carbapenems and potential MBL inhibitor a drug from the group of bisphosphonates etidronic acid. Using a standard enzyme reagent metallo-beta-lactamase P. aeruginosa recombinant expressed in E. coli, we created a model system that allows to assess the prospects of new inhibitors MBL gram-negative microorganisms. A dose-dependent effect of increasing the meropenem level MIC from reagent MBL quantity in a model system against previously antibiotic sensitive reference strains of microorganisms was revealed. MBL enzyme inactivation was noted in the presence of even small doses of bisphosphonate, in the tests the appearance of logarithmic phase of P. aeru ginosa ATCC 27853 growth was shown delayed up to 12 hours compared to the control. In this case the maximum dose of etidronic acid 50 000100 000 g/ml completely inhibited the MBL, there was no a log phase microbes growth due to the effect of meropenem on the reference level of sensitivity (2 g/ml). The synergistic effect (FIC index 0.5) of combined meropenem with etidronic acid use was identified against clinical isolates Gram-negative microorganisms resistant to carbapenems and producing MBL, wherein the enhancing action of the antibiotic was more 8512 times compared with the initial MIC levels.

An enhanced chemiluminescence bioplatform by confining glucose oxidase in hollow calcium carbonate particles.

A chemiluminescence (CL) amplification platform based on HCC/Lucigenin&GOx (HLG) film was developed. Hollow structural calcium carbonate (HCC) particles were used as alternative materials for carrying both enzyme and CL reagent. The model enzyme (GOx), immobilized in confined space of HCC particles, exhibited an improved biocatalysis. The Michaelis constant (Km) and the enzymatic rate constant (kcat) were determined to be 0.209 μM and 2.21 s−1, respectively, which are much better than those of either free GOx in aqueous solution or the GOx immobilized on common nanomaterials. Based on the HLG platform, CL signal was effectively amplified and visualized after adding trace glucose, which could be attributed to the HCC particles’ high biocompatibility, large specific surface area, attractive interfacial properties and efficient interaction with analyses. The visual CL bioplatform showed an excellent performance with high selectivity, wide linear range and low detection limit for sensing trace glucose. Because it eliminates the need of complicated assembly procedure and enables visualization by the naked eye, the sensitive and selective CL bioplatform would provide wide potential applications in disease diagnosis and food safety.

&lt;i&gt;Ixeris dentata&lt;/i&gt; (Thunb) Nakai Ethylacetate Extract Attenuates Sterol Regulatory Element-Binding Proteins-1c via AMP-Activated Protein Kinase Activation

Purpose: To investigate the molecular mechanisms underlying the role of Ixeris dentataa extract (IDE) in the prevention of high glucose induced lipid accumulation in human HepG-2 hepatocytes.Methods: IDE extract was prepared by maceration in ethyl acetate. Its fractionation was carried out by column chromatography. HepG-2 cells were pretreated with various concentration of IDE (0, 10, 20, 40 and 80 μg/mL) and then treated with serum-free medium with normal glucose (5 mM) for 1 h, followed by exposure to high glucose (30 mM D-glucose) for 24 h. Cell viability and cytotoxicity parameters were measured using lactate dehydrogenase (LDH) and MTT assay while triglyceride and total cholesterol levels were evaluated using respective enzymatic reagent kits. Protein expressional levels were analyzed by Western blotting.Results: IDE did not influence the cell viability (up to 200 μg/mL) and did not show any signs of cytotoxicity (up to 80 μg/mlL. IDE significantly attenuated lipid accumulation in human HepG2 hepatocytes when exposed to high glucose (30 mM D-glucose) in a dose-dependent manner (p &lt; 0.05, 0.01 and 0.001 at 20, 40 and 80 μg/mL concentrations, respectively). Nile red staining showed that 10, 20, 40 and 80 μg/mL concentrations of IDE reduced lipid accumulation by 23.4, 34.8 (p &lt; 0.05), 46.5 (p &lt; 0.01) and 53.2 % (p &lt; 0.001), respectively. The increased levels of triglycerides and total cholesterol were also attenuated by IDE (p &lt; 0.001 at 80 μg/mL). Further, IDE attenuated the expression of fatty acid synthase and sterol regulatory element-binding protein-1. Adenosine monophosphate-activated protein kinase was also activated by IDE treatment when exposed to high glucose level (30 mM Dglucose) in human HepG2 hepatocytes.Conclusion: The findings indicate that IDE exerts hypolipidemic effect by inhibiting lipid biosynthesis, mediated via AMPK signaling. This probably explains the extract’s beneficial effect in various inflammation disorders.Keywords: Ixeris dentata extract, Lipogenesis, AMP-activated protein kinase, Sterol regulatory element-binding protein 1 -1, Anti-obesity, HepG-2 cell

Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin

The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (μ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research.

N-Linked Glycosylation Regulates CALHM1 Channel Function and Subcellular Localization

Calcium homeostasis modulator 1 (CALHM1) was discovered as a susceptibility gene for late-onset Alzheimer's disease. CALHM1 has been established as a pore-forming subunit of a homohexameric voltage-gated, non-selective ion channel and its important physiological roles including neuronal excitability and taste perception have also been revealed. However, other modes of CALHM1 channel regulation including ones mediated by post-translational modifications remain to be elucidated. On a western blot, heterologously expressed mouse CALHM1 (mCALHM1) immunosignal appears as three bands with one band at the size of a mCALHM1 monomer, approximately 37 kDa, and the other two bands at higher molecular weights, suggesting post-translational modifications on CALHM1. The two higher molecular weight bands disppeared after peptide-N-glycosidase F treatment, demonstrating that mCALHM1 is glycosylated at asparagine (N) residue(s). Point mutation studies determined that a conserved N139 is the only N-glycosylation site on mCALHM1. The two glycosylated forms of mCALHM1 showed different sinsitivities to endoglycosidase H, demonstrating that conversion of the acquired N-linked glycan chain from high-mannose type to complex type takes place. Furthermore, by using chemical and enzymatic reagents, point mutation studies, and glycosylation mutant cell lines, we examined roles of acquisition and conversion of the N-linked glycan on function and subcellular localization of mCALHM1 channel. Our data provide insights into a novel regulation of CALHM1 channel by N-linked glycosylation.

Use of dried blood spot as an alternative method to estimate serum triglyceride in field studies

Introduction: Cardiovascular diseases are showing an increasing trend in both developing as well as developed countries. Raised triglycerides is considered as an important risk factor of cardio vascular diseases. Collection, storage and transportation of the blood samples from the field becomes a major limiting factor in areas lacking modern laboratory facilities. In such situations, dry blood or serum spots can become an alternative for the estimation of triglyceride. The objectives of our study was to evaluate the use of dry blood or dry serum spot to estimate triglyceride levels as an alternative method to estimate serum triglyceride and also to study the effect of storage time and storage temperature on the triglyceride content in the dry blood and serum spots. Methodology: Blood samples were collected from 100 study participants. Seven spots each of blood and serum from these samples were made on Whatman paper 3. After drying, one dried spot each from blood and serum was analysed on same day. Three of blood and three of serum dried spots were kept at room temperature and remaining at a temperature of 2-8° in separate re-sealable bags which were analysed on days 7, 15 and 30. Triglyceride was measured by Glycerol-3-phosphate oxidase-Peroxidase method. For estimating triglycerides in the eluate, 100 microlitre of the extract from the test-tube was added to 1 ml of the commercially available enzymatic reagent kit and incubated at 37°C for 15 min, and the absorbance was measured at 500 nm using a spectrophotometer. The data collected was analysed by Pearson’s correlation using SPSSv15. Results: Study showed a good correlation between the serum triglyceride values and the dry serum or blood spot triglyceride values. Dried serum spot triglyceride was found to be stable for about 15 days at room temperature and 7 days at 2-8°C with good correlation with serum value. The triglyceride concentration in the dry blood spots remained stable for 7 days at both room temperature and at 2-8°C. Conclusion: Dry blood/serum spots can be used as an alternative to serum for the estimation of triglyceride level which can be used to estimate various biochemical parameters at community level in remote areas which lack modern laboratory facilities. Key words: Serum triglycerides, Dry blood spot, Dry serum spot, cardiovascular diseases, surveillance

Salivary alkaline phosphatase levels speak about association of smoking, diabetes and potentially malignant diseases???

Background:Alkaline phosphatase (ALP) is a hydrolase intracellular enzyme participating in the metabolic processes of cells. Rise in salivary ALP (S-ALP) levels reflects inflammation and destruction of healthy tissues suggesting it as a clinical biomarker. S-ALP is used in analyzing the severity of the disease occurrence in smokers and nonsmokers who are diabetic and nondiabetic. S-ALP levels are analyzed using autoanalyzer in 40 patients who visited our department.Aims and Objectives:To determine the levels of S-ALP in diagnosing potentially malignant conditions and debilitating diseases in early stages of inflammation and altered cellular metabolism.Materials and Methods:The study groups include: (1) Group A - 10 smokers who are diabetic. (2) Group B - 10 smokers who are nondiabetic. (3) Group C - 10 nonsmokers who are diabetic. (4) Group D - 10 nonsmokers and nondiabetic as control. Unstimulated saliva samples are collected and run in auto-analyzer with ALP enzyme reagent to analyze ALP levels. Comparison is made between all the four groups.Results:Results were statistically significant with increased activity of ALP levels in saliva from Group A when compared to Group D. The results are Group A > Group B > Group C > Group D. The results also revealed significant raise in levels of ALP levels in saliva from smokers when compared to diabetes. Thus explaining adverse effects of smoking.Conclusion:S-ALP can be considered to be the biomarker for evaluating adverse effects of smoking, diabetes and other debilitating diseases in early stages.