Performance of RT-PCR-ELISA for the detection of peste des petits ruminants virus

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.

Abnormal levels of heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) in tumour tissue and blood samples from patients diagnosed with lung cancer

Lung cancer is the second most common type of cancer in the world and is the most common cause of cancer-related death in both men and women. Research into causes, prevention and treatment of lung cancer is ongoing and much progress has been made recently in these areas, however survival rates have not significantly improved. Therefore, it is essential to develop biomarkers for early diagnosis of lung cancer, prediction of metastasis and evaluation of treatment efficiency, as well as using these molecules to provide some understanding about tumour biology and translate highly promising findings in basic science research to clinical application. In this investigation, two-dimensional difference gel electrophoresis and mass spectrometry were initially used to analyse conditioned media from a panel of lung cancer and normal bronchial epithelial cell lines. Significant proteins were identified with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), pyruvate kinase M2 isoform (PKM2), Hsc-70 interacting protein and lactate dehydrogenase A (LDHA) selected for analysis in serum from healthy individuals and lung cancer patients. hnRNPA2B1, PKM2 and LDHA were found to be statistically significant in all comparisons. Tissue analysis and knockdown of hnRNPA2B1 using siRNA subsequently demonstrated both the overexpression and potential role for this molecule in lung tumorigenesis. The data presented highlights a number of in vitro derived candidate biomarkers subsequently verified in patient samples and also provides some insight into their roles in the complex intracellular mechanisms associated with tumour progression.

Consent Practices and Biomedical Knowledge Production in Tissue Economies

The use of human tissue sample collections has become an important tool in biomedical research. The collection, use and distribution of human tissue samples, which include blood and diagnostic tissue samples, from which DNA can be extracted and analyzed has also become a major bio-political preoccupation, not only in national contexts, but also at the transnational level. The foundation of medical research rests on the relationship between the doctor and the research subject. This relationship is a social one, in that it is based on informed consent, privacy and autonomy, where research subjects are made aware of what they are getting involved in and are then able to make an informed decision as to whether or not to participate. Within the post-genomic era, however, our understanding of what constitutes informed consent, privacy and autonomy is changing in relation to the needs of researchers, but also as a reflection of policy aspirations. This reflects a change in the power relations between the rights of the individual in relation to the interests of science and society. Using the notions of tissue economies and biovalue (Waldby, 2002) this research explores the changing relationship between sources and users of samples in biomedical research by examining the contexts under which human tissue samples and the information that is extracted from them are acquired, circulated and exchanged in Finland. The research examines how individual rights, particularly informed consent, are being configured in relation to the production of scientific knowledge in tissue economies in Finland from the 1990s to the present. The research examines the production of biovalue through the organization of scientific knowledge production by examining the policy context of knowledge production as well as three case studies (Tampere Research Tissue Bank, Hereditary Non-polyposis Colorectal Cancer and the Finnish Genome Information Center) in which tissues are acquired, circulated and exchanged in Finland. The research shows how interpretations of informed consent have become divergent and the elements and processes that have contributed to these differences. This inquiry shows how the relationship between the interests of individuals is re-configured in relation to the interests of science and society. It indicates how the boundary between interpretations of informed consent, on the one hand, and social and scientific interests, on the other, are being re-drawn and that this process is underscored, in part, by the economic, commercial and preventive potential that research using tissue samples are believed to produce. This can be said to fundamentally challenge the western notion that the rights of the individual are absolute and inalienable within biomedical legislation.

Array-based gene expression, CGH and tissue data defines a 12q24 gain in neuroblastic tumors with prognostic implication

Neuroblastoma has successfully served as a model system for the identification of neuroectoderm-derived oncogenes. However, in spite of various efforts, only a few clinically useful prognostic markers have been found. Here, we present a framework, which integrates DNA, RNA and tissue data to identify and prioritize genetic events that represent clinically relevant new therapeutic targets and prognostic biomarkers for neuroblastoma.

Effect of dietary cholesterol and ubiquinone on isoprene synthesis in rat liver

The effect of dietary cholesterol and ubiquinone on the synthesis of isoprene compounds in the liver, as tested by the incorporation of acetate-1-14C and mevalonate-2-14C, was studied in rats. In cholesterol feeding, there appears to be a second site of inhibition after squalene in addition to the previously known primary site of inhibition at the β-hydroxy-β-methyl glutaryl-CoA reductase. Feeding ubiquinone inhibited at some common step between acetate and mevalonate in the synthesis of both cholesterol and ubiquinone, without affecting the acetate activation or fatty acid synthesis, and also at a step in the synthesis of ubiquinone not common with the synthesis of cholesterol. These results are suggestive of a role for ubiquinone in the regulation of isoprene synthesis.

The conversion of 3-hydroxyanthranilic acid to cinnabarinic acid by the nuclear fraction of rat liver

Although several authors have implicated 3-hydroxyanthranilic acid (3-OHA) as an intermediate in tryptophaniacin pathway in animals (Kaplan, 1961), alternative pathways of metabolism of this compound have not been fully explored. Madhusudanan Nair obtained an enzyme from spinach leaves which could convert 3-OHA to cinnabarinic acid (private communication). Viollier and Süllmann (1950) reported the conversion of 3-OHA to an unidentified red compound by rat liver homogenates. The present investigation describes the identification of this product as cinnabarinic acid (2-amino-3-H-isophenoxazine-3-one-1,9-dicarboxylic acid). Cinnabarinic acid is known to occur in nature along with cinnabarin is olated from the fungus Polystictus sanguineus (Gripenberg et al., 1957; Gripenberg, 1958).

Intracellular distribution of coenzyme Q in rat liver

Coenzyme Q was found to be distributed in rat liver cell fractions. Mitochondria accounted for only 40–60% of the total. The presence of coenzyme Q in nuclei, isolated by several methods, could always be correlated with the presence of oxidative enzymes. It has been established that coenzyme Q is a constituent of microsomes. Administered coenzyme Q10-C14 was preferentially taken up by mitochondrial and microsomal fractions. Exogenous coenzyme Q appears to be rapidly metabolized.

Distribution of coenzyme Q in rat liver cell fractions

COENZYME Q (CoQ), which is widely distributed in animal, plant and microbial sources, has been implicated in electron transport1 and generally assumed to be associated with mitochondria. However, it has also been found in non-mitochondrial fractions of green leaves, although it appears to be concentrated in mitochondria2. A similar distribution has now been demonstrated in rat liver cell fractions.

Fatty acid component of vitamin A ester in sheep liver

Vitamin A, when extracted along with other lipids from sheep liver, had an E1cm.1% value of 14.4, which was raised to 45.57 on removal of the phospholipids by cold acetone. Selective hydrolysis of triglycerides by an extract of acetone-dried sheep pancreas in the presence of HgCl2 as inhibitor of vitamin A esterase, followed by chromatography through alumina gave a product with E1cm.1% value of 276. This on chromatography through magnesium oxide raised the E1cm.1, value to 601.5, representing 64% pure vitamin A ester calculated as palmitate, and the total recovery was 23% of the starting oil. The purified ester preparation, when subjected to reverse-phase chromatography on silicone-impregnated paper, gave a single ultraviolet fluorescent band. The fluorescent band on hydrolysis gave only one fatty acid. This was conclusively identified to be palmitic acid.

Studies on nucleotide pyrophosphatase I. Partial purification and properties of a sheep liver enzyme that catalyzes the hydrolysis of dinucleotides

A partially purified sheep liver enzyme that hydrolyzed dinucleotides at the pyrophosphate bond was obtained by solubilizing the 18,000g sediment with n-butanol and fractionating the solubilized enzyme with acetone. The enzyme activity when measured using FAD as substrate, (FAD → FMN + AMP), was optimal at pH 9.7 and temperatures between 30 °–36 ° and at 60 °. The rate of release of FMN with time occurred with an initial lag of 30 sec, a linear increase for 1 min, and a subsequent irregular rate. In the presence of orthophosphate (Pi; 10 μImage ), FMN was released at an uniformly continuous and enhanced rate. 32Pi was not incorporated into the substrate or products. Sodium arsenate counteracted the effects of Pi. The apparent Km and Vmax were 0.133 mImage and 100 units; and 0.133 mImage and 200 units, in the absence and presence of Pi, respectively. The temperature optimum was 42 ° in the presence of Pi.Negative cooperative interactions observed at low concentrations of FAD were abolished by the addition of Pi. The inhibition by AMP was sigmoid and Pi abolished this sigmoidal response. The enzyme hydrolyzed in addition to FAD, NAD+ and NADP+. Nucleoside triphosphates were potent inhibitors of the enzyme activity. The partial inhibition of the enzyme by o-phenanthroline and by p-hydroxymercuribenzoate could be reversed by Fe2+ ions and by reduced glutathione, respectively.

Model For Regulation Of Delta-Aminolevulinate Synthetase Induction In Rat-Liver

A reciprocal relationship exists between the cytochrome P-450 content and d-aminolaevulinate synthetase activity in adult rats. In young rats the basal d-aminolaevulinate synthetase activity is higher and the cytochrome P-450 content is lower compared with the adult rat liver. Administration of allylisopropylacetamide neither induces the enzyme nor causes degradation of cytochrome P-450 in the young rat liver, unlike adult rat liver. Allylisopropylacetamide fails to induce d-aminolaevulinate synthetase in adrenalectomized–ovariectomized animals or intact animals pretreated with successive doses of the drug, in the absence of cortisol. The cortisol-mediated induction of the enzyme is sensitive to actinomycin D. Allylisopropylacetamide administration degrades microsomal haem but not nuclear haem. Haem does not counteract the decrease in cytochrome P-450 content caused by allylisopropylacetamide administration, but there is evidence for the formation of drug-resistant protein-bound haem in liver microsomal material under these conditions. Phenobarbital induces d-aminolaevulinate synthetase under conditions when there is no breakdown of cytochrome P-450. On the basis of these results and those already published, a model is proposed for the regulation of d-aminolaevulinate synthetase induction in rat liver.

Studies on induction of delta-aminolevulinic acid synthetase in mouse liver

Administration of 3,5-diethoxy carbonyl-1,4-dihydrocollidine (DDC) to mice resulted in a striking increase in the level of δ-aminolevulinic acid (ALA) synthetase in liver. Although the enzyme activity was primarily localized in mitochondria and postmicrosomal supernatant fluid, a significant level of activity was also detected in purified nuclei. The time course of induction showed a close parallelism between the bound and free enzyme activities with the former always accounting for a higher percentage of the total activity as compared to the latter. Studies with cycloheximide indicated a half-life of around 3 hr for both the bound and free ALA synthetase. Actinomycin D and hemin prevented enzyme induction when administered along with DDC, but when administered 12 hr after DDC treatment Actinomycin D did not lead to a decay of either the bound or free enzyme activity and hemin inhibited the bound enzyme activity but not the free enzyme level. The molecular sizes of the mitochondrial and cytosolic ALA synthetase(s) were found to be similar on sephadex columns.

A fast time-domain algorithm for the assessment of tissue blood flow in laser-Doppler flowmetry

In this study, we derive a fast, novel time-domain algorithm to compute the nth-order moment of the power spectral density of the photoelectric current as measured in laser-Doppler flowmetry (LDF). It is well established that in the LDF literature these moments are closely related to fundamental physiological parameters, i.e. concentration of moving erythrocytes and blood flow. In particular, we take advantage of the link between moments in the Fourier domain and fractional derivatives in the temporal domain. Using Parseval's theorem, we establish an exact analytical equivalence between the time-domain expression and the conventional frequency-domain counterpart. Moreover, we demonstrate the appropriateness of estimating the zeroth-, first- and second-order moments using Monte Carlo simulations. Finally, we briefly discuss the feasibility of implementing the proposed algorithm in hardware.