Enzymatic Kit Research Articles

Increased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistance in the Metastatic 4T1 Breast Cancer Model

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.

Inhibitory effect of schisandrin B on free fatty acid-induced steatosis in L-02 cells

To investigate the effects of schisandrin B (Sch B) on free fatty acid (FFA)-induced steatosis in L-02 cells. Cellular steatosis was induced by incubating L-02 cells with a FFA mixture (oleate and palmitate at the ratio of 2:1) for 24 h. Cytotoxicity and apoptosis were evaluated by 3-(4, 5-dmethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay and Annexin V/propidium iodide staining, respectively. Cellular total lipid was determined using a photocolorimetric method after Nile red staining, and triglyceride content was measured using an enzymatic kit. To study the effects of Sch B on steatosis, L-02 cells were treated with Sch B (1-100 μmol/L) in the absence or presence of 1 mmol/L FFA for 24 h, and cellular total lipid and triglyceride levels were measured. To explore the mechanisms of action of Sch B in the steatotic L-02 cells, mRNA levels of several regulators of hepatic lipid metabolism including adipose differentiation related protein (ADRP), sterol regulatory element binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ were measured by quantitative real-time polymerase chain reaction (PCR), and protein levels of ADRP and SREBP-1 were measured by immunoblotting. Treatment with 1 mmol/L FFA for 24 h induced intracellular lipid accumulation in L-02 cells comparable to that in human steatotic livers without causing apparent apoptosis and cytotoxicity. Sch B mitigated cellular total lipid and triglyceride accumulations in the steatotic L-02 cells in a dose-dependent manner. Quantitative real-time PCR and Western blot analyses revealed that treatment of L-02 cells with 100 μmol/L Sch B reverted the FFA-stimulated up-regulation of ADRP and SREBP-1. Sch B inhibits FFA-induced steatosis in L-02 cells by, at least in part, reversing the up-regulation of ADRP and SREBP-1.

Smoking is associated with reduced serum paraoxonase, antioxidants and increased oxidative stress in normolipidaemic acute myocardial infarct patients

BackgroundParaoxonase is a high-density lipoprotein (HDL)-associated enzyme that protects lipoproteins from oxidative modifications and from becoming atherogenic in nature. Smoking is a well-known major cardiovascular risk factor that promotes lipid...

Effects of Honey Against the Accumulation of Adipose Tissue and the Increased Blood Pressure on Carbohydrate-Induced Obesity in Rat

This study was designed to assess the effect of honey supplementation and sugar-based hypercaloric regimen on weight gain and blood pressure (BP) in Wistar rats. Animals were fed for 8 weeks with standard diet (S-free) or a hypercaloric diet (standard chow and 30% sugar in drinking water), (SF), or standard chow supplemented with fat and honey and 10% sugar in drinking water (HF). Overall weight gain and body fat levels were significantly higher in SF and HF than in S-free. Fat cells were significantly larger in SF compared with HF. Compared with SF and S-free, HF had higher glucose, but triglycerides, and LDLc levels were not different. BP was significantly higher in SF but not in HF compared to S-free. These observations indicate that honey may afford a protection against increase in BP and in fat cell size resulting from a hypercaloric diet. Keywords: Carbohydrate, Honey, Hypertension, Obesity, Rats, Fat cell size, weight gain, blood pressure, SF, HF, overweight, glucose, sucrose, fructose, metabolic syndrome, adipose tissue, dyslipidemia, cytokine, Diet, LDLc, enzymatic colorimetric kit, Histology, Tuckey test, Body Weight, adipocyte size, Plasma Glucose, Triglycerides, hypercaloric diet, metabolism, hypertrophy, hyperplasia

Amperometric carbon paste enzyme electrodes for uric acid determination with different mediators

In this study, two new amperometric carbon paste enzyme electrodes for determination of uric acid were developed. The carbon paste was prepared by mixing uricase enzyme, 1,4-benzoquinone or poly(vinylferrocene) (PVF) as a mediator, graphite powder, paraffin oil and then the paste was placed into cavity of a teflon electrode body. Determination of uric acid was performed by oxidation of enzymatically generated H2O2. The effects of enzyme loading, mediator amount, buffer type, pH, buffer concentration, working potential and temperature were investigated for both electrodes. The working range of the 1,4-benzoquinone modified enzyme electrode was 1.9 × 10–8–2.7 × 10–3 M, detection limit 1.9 × 10–8 M and response time 150 s. Optimum buffer type, pH, buffer concentration, working potential, temperature and amounts of enzyme and mediator for 1,4-benzoquinone modified enzyme electrode were found to be Tris, 8.0, 0.20 M, +0.25 V, 30 °C, 2.0 Unit and 13%, respectively. The working range of the PVF modified enzyme electrode was 7.4 × 10–8–7.0 × 10–3 M, detection limit 7.4 × 10–8 M and response time 120 s. Optimum buffer type, pH, buffer concentration, working potential, temperature and amounts of enzyme and mediator for PVF modified enzyme electrode were found to be phosphate, 8.0, 0.05 M, +0.70 and +0.30 V, 40 °C, 2.0 Unit and 10.9%, respectively. The repeatability, storage stability of the enzyme electrodes and interference effects were also investigated. Enzyme electrodes were used for determination of uric acid in serum samples and the results were in a good agreement with those obtained by commercial enzymatic kits.

Influence of the soluble fibres inulin and oat β-glucan on quality of dough and bread

Bread represents a suitable food product for the addition of functional ingredients, such as the cholesterol-lowering dietary fibre oat β-glucan and the prebiotic inulin. Therefore, these soluble fibres were incorporated into wheat as well as gluten-free bread, and their effects on rheological properties of the dough, on bread quality and on crumb microstructure were compared. The level of remaining β-glucan as well as its molecular weight was determined using an enzyme kit and size-exclusion chromatography. The addition of oat β-glucan resulted in a higher water addition level, whereas incorporation of inulin had the opposite effect. Rheological testing showed that the incorporation of oat β-glucan results in a more elastic dough. The baking characteristics mainly affected by fibre addition were volume and crust colour, with inulin increasing and oat β-glucan decreasing loaf-specific volume in the gluten-free breads. Inulin led to a darkening of the crust of both bread types, whereas addition of oat β-glucan resulted in a lighter crust of gluten-free bread. Oat β-glucan softened the crumb of gluten-free bread, but had the opposite effect on wheat bread. Inulin resulted in an increased crumb hardness as well as the rate of staling. Beta-glucan breakdown was more pronounced in wheat bread than in gluten-free bread. The results show that the use of β-glucan to increase the nutritional value of wheat bread is limited due to negative influences on technological properties. However, this soluble fibre is highly suitable for incorporation into gluten-free bread.

ChIP-on-Chip Analysis Identified HLXB9 as a Transcription Factor In Hematopoietic Cells and Alters the Expression of Genes Involved In Cell-Adhesion

AbstractAbstract 2486Infants with t (7;12)/HLXB9-TEL positive Acute Myeloid Leukemia (AML) have an Event-Free Survival (EFS) of 0 % and are characterized by concomitant HLXB9 (MNX1) expression. However, the role of the homeobox protein HLXB9 on hematopoietic cell development remains unknown. Expression profiling of t (7;12) and t (11;X) positive leukemias revealed up-regulation of cell-cell interacting genes in t (7;12) positive leukemia (Wildenhain et al., 2010). Furthermore, no increased expression of HOX-Genes, like HOXA9 and MEIS1, could be observed in t (7;12) positive leukemia compared to t (11;X) positive leukemia. Based on the altered gene expression profile in t (7;12) positive leukemia we investigated the role of HLXB9 as a transcription factor in hematopoietic cells using ChIP-on-chip analysis and its impact on the cellular gene expression pattern using Affymetrix expression arrays. The myeloid cell line HL60 was stable transfected with a CMV-HLXB9 (HL60/HLXB9) expression vector or an empty vector control (HL60/control). Microarray analysis was performed using “Human Gene 1.0 ST Arrays” (Affymetrix) and data from the HL60/HLXB9 cells were normalized to HL60/control cells. ChIP-on-chip analysis was performed using the “SimpleChIP Enzymatic Chromatin IP Kit” (Cell Signaling Technologies). Hybridisation on “385K RefSeq Promoter arrays” and analysis of raw data were performed by NimbleGen using the NimbleScan software. Data were visualized with the SignalMap software. Altered expression analyses as well as enrichment of promoter regions were validated by quantitative RT-PCR. Expression analysis revealed 81 differentially expressed genes, whereof 63 were down-regulated indicating that HLXB9 acts as a transcriptional repressor, as characteristic for homeobox proteins. CLEC5A, normally expressed in mature myeloid cells, is the highest differentially repressed gene. Further, we identified several differentially expressed genes which interfere in cell-adhesion and/or angiogenesis (e.g. IL8, ZYX, SELL, SPP1, EMILIN2). Western blot analysis of nuclear extracts confirmed the translocation of HLXB9 into the nucleus. ChIP-on-chip analysis revealed binding of HLXB9 to several promoter regions, among them the promoters of ZYX and IL8. Binding of HLXB9 to those promoters results in a decreased gene expression.These data strengthens the hypothesis, that HLXB9 plays a major role in cell adhesion and/or cell interactions. Further we observed increased expression of the adhesion molecule CD11b, when culturing HL60/HLXB9 cells in All-Trans Retinoic Acid (ATRA) containing medium in contrast to HL60/control cells. In summary, this study shows that HLXB9 acts as a transcription factor in hematopoietic cells and has a repressive function on gene expression. HLXB9 target genes regulate cell-adhesion and angiogenesis. This study provides the first molecular results of HLXB9 function in hematopoietic cells and supports the previously published data showing the importance on altered gene expression of cell-cell interacting genes in the pathogenesis of t (7;12) positive leukemia. Disclosures:No relevant conflicts of interest to declare.

Impact of serum amyloid A on high density lipoprotein composition and levels

Serum amyloid A (SAA) is an acute-phase protein mainly associated with HDL. To study the role of SAA in mediating changes in HDL composition and metabolism during inflammation, we generated mice in which the two major acute-phase SAA isoforms, SAA1.1 and SAA2.1, were deleted [SAA knockout (SAAKO) mice], and induced an acute phase to compare lipid and apolipoprotein parameters between wild-type (WT) and SAAKO mice. Our data indicate that SAA does not affect apolipoprotein A-I (apoA-I) levels or clearance under steady-state conditions. HDL and plasma triglyceride levels following lipopolysaccharide administration, as well as the decline in liver expression of apoA-I and apoA-II, did not differ between both groups of mice. The expected size increase of WT acute-phase HDL was surprisingly also seen in SAAKO acute-phase HDL despite the absence of SAA. HDLs from both mice showed increased phospholipid and unesterified cholesterol content during the acute phase. We therefore conclude that in the mouse, SAA does not impact HDL levels, apoA-I clearance, or HDL size during the acute phase and that the increased size of acute-phase HDL in mice is associated with an increased content of surface lipids, particularly phospholipids, and not surface proteins. These data need to be transferred to humans with caution due to differences in apoA-I structure and remodeling functions.

Pax6 Controls the Expression of Critical Genes Involved in Pancreatic α Cell Differentiation and Function*

The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased β and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.

Effects of vitamin C and melatonin on cysteamine-induced duodenal ulcer in a cholestatic rat model: A controlled experimental study

Background: Superoxide dismutase (SOD) is one of the defense mechanisms against free radicals. Cysteamine is a cytotoxic agent, acting through generation of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, and superoxide, and may decrease defense activity of SOD against ROS and induce duodenal ulcer. Melatonin is a suicidal antioxidant that has a protective effect against ROS and cytoprotective effect through inhibition of the decrease in SOD activity. Objectives: The primary aim of this study was to assess the effects of pretreatment with vitamin C and melatonin on cysteamine-induced duodenal ulcer. Secondary aims were to compare the ulcerogenic effect of cysteamine and the antiulcer effects of vitamin C and melatonin. Methods: This study was performed in male Wistar rats (200–250 g) in 3 groups of equal size (n = 24): bile duct ligation-induced cholestasis (test), sham, and control groups. In the test and sham groups, laparotomy was performed under general anesthesia and the common bile duct was identified; in sham rats, the common bile duct was left in situ, but in test rats, the common bile duct was isolated and doubly ligated to induce cholestasis. Animals in each group were also divided into 4 equal subgroups (n = 6). These subgroups were treated with vitamin C plus cysteamine, melatonin plus cysteamine, cysteamine alone, and saline, respectively. All animals were euthanized via overdose of ether anesthesia 24 hours after the last injection of cysteamine or saline, and 0.5 mL of blood was collected from the heart ventricle. The duodenum was cut open, washed with saline, fixed, and prepared for calculation of ulcer index (Szabo method) and histopathologic assessment. SOD activity was measured using a branded enzyme kit. Results: In all 3 groups, animals treated with cysteamine had significantly increased mean (SE) ulcer index (test, 4.00 [0.10] vs 1.17 [0.30]; sham, 3.83 [0.16] vs 0.50 [0.22]; control, 3.67 [0.21] vs 0 [0]) and decreased SOD activity (test, 146.41 [2.16] vs 299.83 [1.94] U/mL; sham, 154.75 [2.02] vs 303.08 [0.35] U/mL; control, 157.08 [1.67] vs 314.50 [1.14] U/mL) compared with saline-treated rats (all, P < 0.001). In the test rats, ulcer index was significantly increased and SOD activity was significantly decreased compared with the sham and control groups (both, P < 0.001). Pretreatment with vitamin C and melatonin was associated with attenuation of ulcer index and increased SOD activity compared with rats treated with cysteamine alone ( P < 0.001). There were no significant differences in ulcer index or SOD activity between groups administered vitamin C or melatonin. Conclusions: In this experimental study, pretreatment with melatonin or vitamin C in all rats produced significant attenuation of the ulcer index and enhanced SOD activity. Cysteamine-induced duodenal mucosal damage was greater in cholestatic rats compared with sham and control rats.

Formulation and stability of a novel artificial human sweat under conditions of storage and use

A limitation of most artificial sweat formulations used for in vitro assessment of chemical release from materials in contact with skin have little biological relevance to human sweat. The purposes of this paper are to provide guidance for preparation of a novel artificial sweat with chemical constituents at concentrations that match human sweat and to characterize chemical stability. The artificial sweat was characterized under conditions of use (with and without sebum at 36 °C) and storage (without sebum at −4, 4, and 23 °C) over 28 days by gas chromatography–mass spectroscopy, high-performance liquid chromatography, enzymatic assay kits, and ion-selective electrodes. Seven indicator constituents were tracked: sodium, chloride, glucose, lactic acid, urea, pantothenic acid, and alanine. With or without sebum at 36 °C, the sweat solvent was chemically stable for 14 days. Storage by refrigeration at 4 °C retained the chemical integrity of the solvent longest. Based on these results, the solvent should be used within 14 days of preparation. The artificial sweat model presented herein is most similar to human sweat and has applications as a dissolution solvent, donor solution in diffusion cells, or vehicle for patch testing. This sweat model may aid researchers in understanding potential release and percutaneous absorption of chemicals in contact with human skin surface liquids.

Momordica charantia (bitter melon) inhibits primary human adipocyte differentiation by modulating adipogenic genes.

BackgroundEscalating trends of obesity and associated type 2 diabetes (T2D) has prompted an increase in the use of alternative and complementary functional foods. Momordica charantia or bitter melon (BM) that is traditionally used to treat diabetes and complications has been demonstrated to alleviate hyperglycemia as well as reduce adiposity in rodents. However, its effects on human adipocytes remain unknown. The objective of our study was to investigate the effects of BM juice (BMJ) on lipid accumulation and adipocyte differentiation transcription factors in primary human differentiating preadipocytes and adipocytes.MethodsCommercially available cryopreserved primary human preadipocytes were treated with and without BMJ during and after differentiation. Cytotoxicity, lipid accumulation, and adipogenic genes mRNA expression was measured by commercial enzymatic assay kits and semi-quantitative RT-PCR (RT-PCR).ResultsPreadipocytes treated with varying concentrations of BMJ during differentiation demonstrated significant reduction in lipid content with a concomitant reduction in mRNA expression of adipocyte transcription factors such as, peroxisome proliferator-associated receptor γ (PPARγ) and sterol regulatory element-binding protein 1c (SREBP-1c) and adipocytokine, resistin. Similarly, adipocytes treated with BMJ for 48 h demonstrated reduced lipid content, perilipin mRNA expression, and increased lipolysis as measured by the release of glycerol.ConclusionOur data suggests that BMJ is a potent inhibitor of lipogenesis and stimulator of lipolysis activity in human adipocytes. BMJ may therefore prove to be an effective complementary or alternative therapy to reduce adipogenesis in humans.

Microarray-based Transcriptional and Epigenetic Profiling of Matrix Metalloproteinases, Collagens, and Related Genes in Cancer

Epigenetic parameters (DNA methylation, histone modifications, and miRNAs) play a significant role in cancer. To identify the common epigenetic signatures of both the individual matrix metalloproteinases (MMPs) and the additional genes, the function of which is also linked to proteolysis, migration, and tumorigenesis, we performed epigenetic profiling of 486 selected genes in unrelated non-migratory MCF-7 breast carcinoma and highly migratory U251 glioma cells. Genome-wide transcriptional profiling, quantitative reverse transcription-PCR, and microRNA analyses were used to support the results of our epigenetic studies. Transcriptional silencing in both glioma and breast carcinoma cells predominantly involved the repressive histone H3 Lys-27 trimethylation (H3K27me3) mark. In turn, epigenetic stimulation was primarily performed through a gain in the histone H3 Lys-4 dimethylation (H3K4me2) and H3 hyperacetylation and by a global reduction of H3K27me3. Inactive pro-invasive genes in MCF-7 cells but not in U251 cells frequently exhibited a stem cell-like bivalent mark (enrichment in both H3K27me3 and H3K4me2), a characteristic of developmental genes. In contrast with other MMPs, MMP-8 was epigenetically silenced in both cell types, thus providing evidence for the strict epigenetic control of this anti-tumorigenic proteinase in cancer. Epigenetic stimulation of multiple collagen genes observed in cultured glioma cells was then directly confirmed using orthotopic xenografts and tumor specimens. We suggest that the epigenetic mechanisms allow gliomas to deposit an invasion-promoting collagen-enriched matrix and then to use this matrix to accomplish their rapid migration through the brain tissue.

Reduced Creatine Kinase B Activity in Multiple Sclerosis Normal Appearing White Matter

BackgroundTwo studies using 31P-magnetic resonance spectroscopy (MRS) reported enhanced phosphocreatine (PCr) levels in normal appearing white matter (NAWM) of subjects with multiple sclerosis (MS), but this finding could not be properly explained.Methodology/Principal FindingsWe performed 31P-MRS and 1H-MRS in the NAWM in 36 subjects, including 17 with progressive MS, 9 with benign MS, and 10 healthy controls. Compared to controls, PCr/β-ATP and PCr/total 31P ratios were significantly increased in subjects with progressive MS, but not with benign MS. There was no correlation between PCr ratios and the N-acetylaspartate/creatine ratio, suggesting that elevated PCr levels in NAWM were not secondary to axonal loss. In the central nervous system, PCr is degraded by creatine kinase B (CK-B), which in the white matter is confined to astrocytes. In homogenates of NAWM from 10 subjects with progressive MS and 10 controls without central nervous system disease, we measured CK-B levels with an ELISA, and measured its activity with an enzymatic assay kit. Compared to controls, both CK-B levels and activity were decreased in subjects with MS (22.41 versus 46.28 µg/ml; p = 0.0007, and 2.89 versus 7.76 U/l; p<0.0001).Conclusions/SignificanceOur results suggest that PCr metabolism in the NAWM in MS is impaired due to decreased CK-B levels. Our findings raise the possibility that a defective PCr metabolism in astrocytes might contribute to the degeneration of oligodendrocytes and axons in MS.

Effect of simvastatin on paraoxonase 1 (PON1) activity and oxidative stress

Objective To investigate the effect of simvastatin treatment on lipid profile and oxidative stress in hypercholesterolaemic Indian population and determine the effect of simvastatin treatment on the activity of paraoxonase (PON). Methods Analyzed initially before medication administration and four months later after medication. Lipid and lipoprotein measurement were done by enzymatic kits, high density lipoprotein (HDL) was determined by phosphotungstic acid precipitation method and low density lipoprotein (LDL) was calculated by Friedewald's formula. Lipid peroxidation was measured by three markers namely, conjugated diene, total peroxide, and malondialdehyde. Conjugated diene was assayed by Buege and Aust method. Total peroxide was determined by FOX2 method. Malondialdehyde determination was carried out by Flemming method and total antioxidant status was determined by Ozacan. Paraoxonase activity was determined by measuring the absorbance inrease of p-nitrophenol at 405 nm. Arylesterase activity was calculated from the molar coefficient of 1 310 M −1 cm −1 . Results Simvastatin significantly reduced total cholesterol, triglycerides, LDL, conjugated diene, total peroxide and MDA levels, where as antioxidant status was significantly increased. Besides, simvastatin significantly increased PON1 activity towards paraoxon. Conclusions The results from the current study indicate simvastatin may have important antioxidant properties via increasing PON activity.

Genotyping of Polymorphisms in Alcohol and Aldehyde Dehydrogenase Genes by Direct Application of PCR-RFLP on Dried Blood without DNA Extraction

We have developed a simple, labor-saving, inexpensive, and rapid single nucleotide polymorphism (SNP) genotyping method that works directly on whole human blood. This single-tube genotyping method was used to successfully and reliably genotype ADH1B and ALDH2 polymorphisms without DNA isolation using a 1.2-mm disc of dried blood and the KOD FX PCR enzyme kit. SNP genotyping was performed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In addition to the labor and expense advantages, the possibility of sample contamination was considerably decreased, since the DNA extraction step was eliminated. In the post-genome era, a simple and inexpensive method for diagnostic analysis is in high demand, and this method will be very useful for genetic diagnoses in biological and medical laboratories.

Application of biosensors in early detection of contamination with lactic acid bacteria during apple juice and concentrate production

The aim of Collective Research Project QUALI-JUICE (COLL-CT-2005, Co Nr 012461) was to introduce the biosensors in control of microbiologically pure apple juice production. Three commercial lactate biosensors were used and compared with enzyme kits assays. Parallel the microbiological analysis of the production process at one juice producing enterprise was done. The results of lactic acid assay with biosensors were in good correlation with those obtained by enzyme kits. The main benefit of biosensor use was shortening of measurement time as compared with assay by enzyme kit and possibility to measure at line. The concentration of l-lactic acid in apple pulp could be correlated with the number of lactic acid bacteria. Pasteurization process was eliminating lactic acid bacteria and the concentration of lactic acid was at this stage not exceeding 0.1 g L −1. The final product (apple concentrate) contained in some cases very high amounts of lactic acid indicating secondary microbiological contamination after pasteurization step. Parallel microbiological analysis of production process and lactate assay indicated that the critical point during production was prolonged vacuum filtration after pasteurization.

Integrated multienzyme electrochemical biosensors for monitoring malolactic fermentation in wines

Integrated amperometric biosensors for the determination of L-malic and L-lactic acids were developed by coimmobilization of the enzymes L-malate dehydrogenase (MDH) and diaphorase (DP), or L-lactate oxidase (LOX) and horseradish peroxidase (HRP), respectively, together with the redox mediator tetrathiafulvalene (TTF), on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +100mV (vs. Ag/AgCl), and the reduction of TTF(+) at -50mV were used for the monitoring of the enzyme reactions involved in L-malic and L-lactic acid determinations, respectively. Experimental variables concerning the biosensors composition and the detection conditions were optimized for each biosensor. Good relative standard deviation values were obtained in both cases for the measurements carried out with the same biosensor, with no need of cleaning or pretreatment of the bioelectrodes surface, and with different biosensors constructed in the same manner. After 7 days of continuous use, the MDH/DP biosensor still exhibited 90% of the original sensitivity, while the LOX/HRP biosensor yielded a 91% of the original response after 5 days. Calibration graphs for L-malic and L-lactic were obtained with linear ranges of 5.2x10(-7) to 2.0x10(-5) and 4.2x10(-7) to 2.0x10(-5)M, respectively. The calculated detection limits were 5.2x10(-7) and 4.2x10(-7)M, respectively. The biosensors exhibited a high selectivity with no significant interferences. They were applied to monitor malolactic fermentation (MLF) induced by inoculation of Lactobacillus plantarum CECT 748(T) into a synthetic wine. Samples collected during MLF were assayed for L-malic and L-lactic acids, and the results obtained with the biosensors exhibited a very good correlation when plotted against those obtained by using commercial enzymatic kits.

Single-Tube Genotyping from a Human Hair Root by Direct PCR

We have developed a simple, labor-saving, inexpensive and rapid SNP genotyping method that directly uses a human hair root as the template. This single-tube genotyping method was used to successfully and reliably genotype the ADH1B and ALDH2 polymorphisms using a hair root (without DNA isolation) and the polymerase chain reaction (PCR) enzyme kit KOD FX. Since the DNA extraction step was eliminated, the possibility of sample contamination was considerably decreased. The single-tube SNP genotyping was performed by coupling the PCR enzyme kit with allele-specific primer (ASP)-PCR. In the post-genome era, a simple and inexpensive method for diagnostic analysis is in high demand, and this PCR method with a hair root as a template will be very useful for genetic diagnoses in biological and medical laboratories.

A flow-based procedure with solenoid micro-pumps for the spectrophotometric determination of uric acid in urine

Abstract The determination of uric acid in urine shows clinical importance, once it can be related to human organism dysfunctions, such as gout. An analytical procedure employing a multicommuted flow system was developed for the determination of uric acid in urine samples. Cu(II) ions are reduced by uric acid to Cu(I) that can be quantified by spectrophotometry in the presence of 2,2′-biquinoline 4,4′-dicarboxylic acid (BCA). The analytical response was linear between 10 and 100 μmol L − 1 uric acid with a detection limit of 3.0 μmol L − 1 (99.7% confidence level). Coefficient of variation of 1.2% and sampling rate of 150 determinations per hour were achieved. Per determination, 32 μg of CuSO 4 and 200 μg of BCA were consumed, generating 2.0 mL of waste. Recoveries from 91 to 112% were estimated and the results for 7 urine samples agreed with those obtained by the commercially available enzymatic kit for determination of uric acid. The procedure required 100-fold dilution of urine samples, minimizing sample consumption and interfering effects. In order to avoid the manual dilution step, on-line sample dilution was achieved by a simple system reconfiguration attaining a sampling rate of 95 h − 1 .