Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar. The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples. Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive. When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×105 copies per reaction (equals to 1.58×103 to 1.58×107 cfu/ml L. monocytogenes) were detectable. As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels.

Nucleolin is regulated both at the level of transcription and translation

Nucleolin is a multi-functional protein that is located to the nucleolus. In tissue Culture cells, the stability of nucleolin is related to the proliferation status of the cell. During development, rat cardiomyocytes proliferate actively with increases in the mass of the heart being due to both hyperplasia and hypertrophy. The timing of this shift in the phenotype of the myocyte from one capable of undergoing hyperplasia to one that can grow only by hypertrophy occurs within 4 days of post-natal development. Thus, cardiomyocytes are an ideal model system in which to study the regulation of nucleolin during growth in vivo. Using Western blot and quantitative RT-PCR (TaqMan) we found that the amount of nucleolin is regulated both at the level of transcription and translation during the development of the cardiomyocyte. However, in cells which had exited the cell cycle and were subsequently given a hypertrophic stimulus, nucleolin was regulated post-transcriptionally. (c) 2005 Elsevier Inc. All rights reserved.

Identification of novel expressed sequences, up-regulated in the leucocytes of chronic fatigue syndrome patients

BACKGROUND: Chronic fatigue syndrome (CFS) is an increasing medical phenomenon of unknown aetiology leading to high levels of chronic morbidity. Of the many hypotheses that purport to explain this disease, immune system activation, as a central feature, has remained prominent but unsubstantiated. Supporting this, a number of important cytokines have previously been shown to be over-expressed in disease subjects. The diagnosis of CFS is highly problematic since no biological markers specific to this disease have been identified. The discovery of genes relating to this condition is an important goal in seeking to correctly categorize and understand this complex syndrome. OBJECTIVE: The aim of this study was to screen for changes in gene expression in the lymphocytes of CFS patients. METHODS: 'Differential Display' is a method for comparing mRNA populations for the induction or suppression of genes. In this technique, mRNA populations from control and test subjects can be 'displayed' by gel electrophoresis and screened for differing banding patterns. These differences are indicative of altered gene expression between samples, and the genes that correspond to these bands can be cloned and identified. Differential display has been used to compare expression levels between four control subjects and seven CFS patients. RESULTS: Twelve short expressed sequence tags have been identified that were over-expressed in lymphocytes from CFS patients. Two of these correspond to cathepsin C and MAIL1 - genes known to be upregulated in activated lymphocytes. The expression level of seven of the differentially displayed sequences have been verified by quantifying relative level of these transcripts using TAQman quantitative PCR. CONCLUSION: Taken as a whole, the identification of novel gene tags up-regulated in CFS patients adds weight to the idea that CFS is a disease characterized by subtle changes in the immune system.

Anti-proliferative effects of physiological concentrations of enterolactone in models of prostate tumourigenesis

SCOPE: There is evidence that a mammalian lignan, enterolactone (ENL), decreases the proliferation rate of prostate cancer cells, although previous studies have used concentrations difficult to achieve through dietary modification. We have therefore investigated the anti-proliferative effects of ENL in an in vitro model of prostate tumourigenesis at concentrations reported to occur in a range of male populations. METHODS AND RESULTS: The effects of 0.1 and 1 μM ENL on three markers of viability and proliferation (metabolic activity, growth kinetics, and cell cycle progression) were assessed in the RWPE-1, WPE1-NA22, WPE1-NB14, WPE1-NB11, WPE1-NB26, LNCaP, and PC-3 cell lines over 72 h. Based on these data, we quantified the expression levels of 12 genes involved in the control of DNA replication initiation using TaqMan real-time PCR in the WPE1-NA22, WPE1-NB14, WPE1-NB11, and WPE1-NB26 cell lines. ENL significantly inhibited the abnormal proliferation of the WPE1-NB14 and WPE1-NB11 cell lines and appears to be a consequence of decreased expression of abnormal chromatin licensing and DNA replication factor 1. CONCLUSION: In contrast to previous studies, concentrations of ENL that are reported after dietary intervention restrict the proliferation of early-stage tumourigenic prostate cell lines by inhibiting the abnormal formation of complexes that initiate DNA replication.

Long-term depression activates transcription of immediate early transcription factor genes: involvement of serum response factor/Elk-1

Long-term depression (LTD) is one of the paradigms used in vivo or ex vivo for studying memory formation. In order to identify genes with potential relevance for memory formation we used mouse organotypic hippocampal slice cultures in which chemical LTD was induced by applications of 3,5-dihydroxyphenylglycine (DHPG). The induction of chemical LTD was robust, as monitored electrophysiologically. Gene expression analysis after chemical LTD induction was performed using cDNA microarrays containing >7,000 probes. The DHPG-induced expression of immediate early genes (c-fos, junB, egr1 and nr4a1) was subsequently verified by TaqMan polymerase chain reaction. Bioinformatic analysis suggested a common regulator element [serum response factor (SRF)/Elk-1 binding sites] within the promoter region of these genes. Indeed, here we could show a DHPG-dependent binding of SRF at the SRF response element (SRE) site within the promoter region of c-fos and junB. However, SRF binding to egr1 promoter sites was constitutive. The phosphorylation of the ternary complex factor Elk-1 and its localization in the nucleus of hippocampal neurones after DHPG treatment was shown by immunofluorescence using a phosphospecific antibody. We suggest that LTD leads to SRF/Elk-1-regulated gene expression of immediate early transcription factors, which could in turn promote a second broader wave of gene expression.