[Identification of endogenous control genes for gene expression studies in peripheral blood of patients with coronary artery disease].

The selection of stable endogenous control genes is critical for normalization of quantitative real-time PCR (qPCR) data. In this study, we aimed to identify a suitable set of control genes to be used as endogenous references for gene expression evaluation in human peripheral blood samples among coronary artery disease patients. The expression levels of 12 endogenous control genes procured from TATAA Biocenter (Goteborg, Sweden) were measured in five acute coronary syndrome patients and five chronic stable angina patients. Gene expression stability was analyzed using two different software applications i.e geNorm and NormFinder. Results suggested that beta-glucuronidase is the most stable endogenous control, followed by hypoxanthine-guanine phosphoribosyltransferase. The NormFinder analysis further confirmed that beta-glucuronidase and hypoxanthine-guanine phosphoribosyltransferase were on the first rank order with the most stable expression among endogenous control genes analyzed and 60S acidic ribosomal protein P0. Besides this, the expression levels of 18S rRNA were revealed to be highly variable between coronary heart disease patients. We thus recommend the use of beta-glucuronidase and hypoxanthine-guanine phosphoribosyltransferase as reference genes for accurate normalization of relative quantities of gene expression levels in coronary artery disease patients using qPCR. Also the use of 18S rRNA as a control gene should be avoided.

Development of triplex real-time PCR and detection of Toxoplasma gondii DNA in infected mice tissues and spiked human samples.

Toxoplasma gondii is an important pathogen in veterinary and human medicine. In this study, a new multiplex TaqMan real-time PCR for detection of T. gondii DNA was developed. This assay consisted of new sets of primers and probes which targeted B1 gene and ITS-1 region of T. gondii, with Vibrio cholera gene as internal control. The B1 gene primers were designed to detect T. gondii RH strain, while the ITS-1 region primers detected most T. gondii strains. Specificity test using common protozoal and bacterial DNA revealed that the assay was very specific to T. gondii. Standard curves constructed using human body fluids spiked with T. gondii (RH and ME49 strains) showed that the sensitivity of the assay was one parasite, with R² value of 0.975 to 0.999 and efficiency of 97% to 99% for all types of samples. The assay performed on DNA extracted from tissues of mice infected with T. gondii showed that liver contained the highest parasite load for both strains of T. gondii. The multiplex real-time PCR developed in this study would be potentially useful for detection of T. gondii in human and animal samples.

Inhibition of Staphylococcus aureus by crude and fractionated extract from lactic acid bacteria.

Increasing levels of antibiotic resistance by Staphylococcus aureus have posed a need to search for non-antibiotic alternatives. This study aimed to assess the inhibitory effects of crude and fractionated cell-free supernatants (CFS) of locally isolated lactic acid bacteria (LAB) against a clinical strain of S. aureus. A total of 42 LAB strains were isolated and identified from fresh vegetables, fresh fruits and fermented products prior to evaluation of inhibitory activities. CFS of LAB strains exhibiting a stronger inhibitive effect against S. aureus were fractionated into crude protein, polysaccharide and lipid fractions. Crude protein fractions showed greater inhibition against S. aureus compared to polysaccharide and lipid fractions, with a more prevalent effect from Lactobacillus plantarum 8513 and L. plantarum BT8513. Crude protein, polysaccharide and lipid fractions were also characterised with glycine, mannose and oleic acid being detected as the major component of each fraction, respectively. Scanning electron microscopy revealed roughed and wrinkled membrane morphology of S. aureus upon treatment with crude protein fractions of LAB, suggesting an inhibitory effect via the destruction of cellular membrane. This research illustrated the potential application of fractionated extracts from LAB to inhibit S. aureus for use in the food and health industry.

Sulphate conjugation in human liver. Direct measurement of N-acetyldopamine-sulphate.

The overall three-step sulphate conjugating activity of the human liver was determined by the direct measurement of N-acetyldopamine (NADA)-sulphate from ATP and inorganic sulphate. NADA-sulphate was separated from NADA and quantified by HPLC-ECD (high-pressure liquid chromatography-electrochemical detection). The overall sulphate conjugation of NADA showed at pH optimum of 8.0 with apparent Km values for sodium sulphate and NADA of 103 microM and 4.8 microM. respectively. The optimum concentration of ATP was 5 mM and that for Mg2+ ions was 7 mM. The specific activities of 17 samples of human liver, measured by this HPLC-ECD procedure ranged from 940 to 23,128 pmol NADA-sulphate/hr/mg protein. A comparison of these values with those determined by the radiometric method developed previously showed a direct correlation coefficient, r = 0.89. The rates of overall sulphate conjugation of NADA and dopamine measured in these samples by the radiometric assay procedure were also significantly correlated with r = 0.82.

Biosynthesis of PAPS in vitro by human liver. Measurement by two independent assay procedures.

The biosynthesis of 3'-phosphoadenosine-5'-phosphosulphate (PAPS) by extracts of human liver from inorganic sulphate and APS was assayed by transferring the "active sulphate" to two different acceptors, namely N-acetyldopamine (NADA) and 4-methylumbelliferone (4-MU). NADA-sulphate was quantified by an HPLC-ECD method while the decrease in 4-MU was monitored continuously by a fluorimetric procedure. The optimum pH was 8.0 for both the PAPS generation and APS-kinase reaction. The apparent Km value for APS determined by the fluorimetric and HPLC-ECD procedures was 17.6 and 16.8 microM, respectively. PAPS-generation measured in 13 samples of human liver showed excellent correlation between the two assay procedures, with correlation coefficients (r) of 0.96 and 0.95 for PAPS generation from inorganic sulphate and APS, respectively. The fluorimetric assay was preferred as it is simple, equally sensitive and more reproducible. There was also a good correlation between the APS-kinase reaction and the two-step PAPS-generation from inorganic sulphate, with r = 0.97 and 0.91, as determined by the fluorimetric and HPLC-ECD procedures. The rate of PAPS generation from inorganic sulphate was only marginally less than that from APS in each of the 13 human liver samples, suggesting that the coupling of ATP-sulphurylase to APS-kinase facilitates "sulphate activation" and releases it from the constraints imposed by the unfavourable ATP sulphurylase reaction.

An HPLC-ECD procedure for measuring total phenolsulfotransferase (PST) activity in human liver, platelets and blood.

The phenolsulfotransferase (PST) activity in human liver, platelets and blood was measured under saturating concentrations of the conjugating agent, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Conventional PST assays employ PAP35S at suboptimal concentrations. In addition, the sulfate conjugate formed, namely N-acetyldopamine-sulfate (NADA-sulfate) was quantified directly by high-pressure liquid chromatography cum electrochemical detection (HPLC-ECD). NADA, the biogenic amine acceptor used in this study appeared from kinetic data to be a substrate of both the P and M forms of PST when used in micromolar concentration. Two apparent Km values of 4.2 mumol/l and 22.6 mumol/l were observed. In contrast, only one apparent Km value was evident when the assay was carried out in the presence of 2,6-dichloro-4-nitrophenol (DCNP), a selective inhibitor of the P form of PST or after heat treatment under specified conditions which inactivates the M form of PST. Thus measurement of PST activity with NADA as the acceptor substrate permits the determination of total PST activity and a parallel assay with the inclusion of DCNP would distinguish the two variants of PST, both of which appear to be present in all human tissues.

Phenolsulfotransferase (PST) and PAPS in catecholamine metabolism in human tissues: an overview.

Sulfate conjugation in vitro was studied in a number of human tissues, such as the platelets, liver as well as in human foetal liver cells in culture. Essentially, the two enzymes involved in PAPS generation and PST were measured either singly or in combination, representing PAPS generation, PST and the overall three-step sulfate conjugation. These activities in the liver and platelets were comparable in magnitude. In the platelets, PAPS generation and PST activities showed a good correlation to the overall sulfate conjugation. With the expression of these activities in human foetal liver cells in culture, it is anticipated that such cells can be employed as model systems for the study of sulfate conjugation in man.

The human platelet as an independent unit for sulfate conjugation.

The human platelets possess a full complement of enzymes capable of synthesizing N-acetyldopamine (NADA) 35sulfate from ATP, Mg++ and sodium 35sulfate. The pH optimum for this three-step overall sulfate conjugation (comprising of the ATP sulfurylase, APS kinase and phenolsulfotransferase reactions) is 8.6 and the reactions proceeded progressively for several hours. Both ATP and Mg++ ions, above their respective optimal concentrations of 5 and 7 mM, inhibited the sulfate conjugation of NADA. The apparent Km values for NADA as determined by the phenolsulfotransferase (PST) and overall reactions were similar in magnitude being 2.6 and 4.8 microM, respectively, while that for sodium 35sulfate was 202 microM. A comparison of these two activities in 62 platelet preparations of normal subjects showed that the rate of the PST reaction was generally higher than the overall reaction even though the PST assay was carried out at suboptimal concentration of PAPS. There was a positive correlation (r = 0.82) between the two sets of data, suggesting that the PST reaction probably has some control over the rate of overall sulfate conjugation.

Does PAPS generation determine the overall sulfate conjugation in human platelets?

The formation of 3'-phospho-adenosine-5'phosphosulfate (PAPS) depends on essentially two enzymic reactions catalysed by ATP-sulfurylase and APS-kinase (adenosine 5'-phosphosulfate-kinase). In this paper, PAPS generation by human platelets was determined by the transfer of 35sulfate from PAP35S formed in vitro to N-acetyldopamine (NADA), using the phenolsulfotransferase extracted from rat liver. A pre-requisite of this quantitative procedure was the prior inhibition of the sulfate-activating system in the latter enzyme preparation. This was accomplished by the addition of 10 mM EDTA and 14 mM pyrophosphate. The PAPS-generating system of human platelets exhibited two pH peaks with higher activity at pH 8 than pH 6. Optimal concentrations of ATP and Mg++ at 7 mM were required for the two reactions. PAPS generation so measured showed a highly significant correlation with the overall sulfate conjugation of NADA: a correlation coefficient of 0.96 was established from data obtained from 60 platelet preparations of normal subjects.

Molecular cloning and characterisation of peroxisome proliferator activated receptor gamma1 (PPARgamma1) cDNA gene from guinea pig (Cavia porcellus): tissue distribution.

The coding region of guinea pig peroxisome proliferator activated receptor gamma1 (gpPPARgamma1) cDNA was successfully cloned from adipose tissue by reverse transcription polymerase chain reaction (RT-PCR) using the designated primers based on the conserved regions of the other mammalian PPARgamma1 sequence. From RT-PCR, a combination of three cDNA fragments that comprised of the full length coding region PPARgamma1 cDNA gene were amplified, with the size of 498, 550 and 557 bp, respectively. All three fragments were then successfully assembled by utilising the internal restriction sites present at the overlapping regions to give rise to the full-length coding region of gpPPARgamma1 with the size of 1428 bp and consisting of 475 amino acids. Guinea pig PPARgamma1 is highly conserved with those of other species at protein and nucleotide levels. Gene expression studies showed that gpPPARgamma mRNA was predominantly expressed in adipose tissue followed by lung and spleen. However, at the protein level, PPARgamma was also found to be expressed in skeletal muscle.

Ultrasound enhanced growth and cholesterol removal of Lactobacillus fermentum FTDC 1311 in the parent cells but not the subsequent passages.

The aim of this study was to evaluate the effect of ultrasound on the intestinal adherence ability, cell growth, and cholesterol removal ability of parent cells and subsequent passages of Lactobacillus fermentum FTDC 1311. Ultrasound significantly decreased the intestinal adherence ability of treated parent cells compared to that of the control by 11.32% (P<0.05), which may be due to the protein denaturation upon local heating. Growth of treated parent cells also decreased by 4.45% (P<0.05) immediately upon ultrasound (0-4h) and showed an increase (P<0.05) in the viability by 2.18-2.34% during the later stage of fermentation (12-20 h) compared to that of the control. In addition, an increase (P<0.05) in assimilation of cholesterol (>9.74%) was also observed for treated parent cells compared to that of the control, accompanied by increased (P<0.05) incorporation of cholesterol into the cellular membrane. This was supported by the increased ratio of membrane cholesterol:phospholipids (C:P), saturation of cholesterol in the apolar regions, upper phospholipids regions, and polar regions of membrane phospholipids of parent cells compared to that of the control (P<0.05). However, such traits were not inherited by the subsequent passages of treated cells (first, second, and third passages). Our data suggested that ultrasound treatment could be used to improve cholesterol removal ability of parent cells without inducing permanent damage/defects on treated cells of subsequent passages.

Optimization of Toxoplasma gondii cultivation in VERO cell line.

In vitro culture of Toxoplasma gondii can provide tachyzoites which are active, viable and with desirable purity. Thus the aim of this study was to optimize the cell culture method for T. gondii propagation to obtain a consistent source of parasites with maximum yield and viability, but minimum host cell contamination for use in production of excretory-secretory antigen. Tachyzoites with seed counts of 1x10(6), 1x10(7) and 1x10(8) harvested from infected mice were added to VERO cells of different degrees of confluence, namely 50%, 85% and 100%, and examined periodically using an inverted microscope. When the maximum release of the tachyzoites was observed from the host cells, the culture supernatant was removed and the tachyzoites harvested. Using a Neubauer chamber, the percentages of viable tachyzoites and host cell contamination were determined using trypan blue stain. Parameters that gave the best yield and purity of viable tachyzoites were found to be as follows: VERO cells at 85% confluence in DMEM medium and inoculum comprising 1x10(7) tachyzoites. After about 3 days post infection, the tachyzoites multiplied 78x, with a yield of ~7.8x10(8) per flask, 99% viability and 3% host cell contamination. This study has successfully optimized the method of propagation of T. gondii tachyzoites in VERO cells which produce parasites with high yield, purity and viability.