The Significance of PCR Primer Design in Genetic Diversity Studies: Exemplified by Recent Research into the Genetic Structure of Marine Species.

Genetic markers are widely applied in the study of genetic diversity for many species. The approach incorporates a Polymerase Chain Reaction (PCR) amplification of targeted sequences in the genome. Crucial for the overall success of a PCR experiment is the careful design of synthetic oligonucleotide primers. Ideally designed primer pairs will ensure the efficiency and specificity of the amplification reaction, resulting in a high yield of the desired amplicon. Important criteria such as primer-sequence, -length, and -melting temperature (Tm) are fundamental for the selection of primers and amplification of targeted nucleotide sequences from a DNA template. There are many computational tools available to assist with critical bioinformatics issues related to primer design. These resources allow the user to define parameters and criteria that need to be taken into account when designing primers. Following the initial in silico selection, a primer pair should be further tested in vivo for their amplification efficiency and robustness.Using examples taken from genetic diversity studies in a marine crustacean, this chapter provides outlines for the application of PCR technology and discusses details for the design of primers for the development and characterization of microsatellite and SNP-markers.

Vitamin d status and anthropometric indices of an omani study population.

OBJECTIVES: Serum 25-hydroxyvitamin D [25(OH)D] concentrations reflect vitamin D status, with deficiency implicated as causative of many diseases. This study assessed vitamin D status and anthropometric indices in a sample of healthy Omanis. METHODS: Serum 25(OH)D concentrations were measured by high performance liquid chromatography in 206 healthy Omanis, aged 18-55 years (mean age: men 31.1, women 26.8) in Muscat, Oman. Of this number, 95% indicated that they had never taken vitamin D supplements. Findings were compared with published values for populations domiciled in more northerly latitudes. Classical procedures were used to determine global obesity (body mass index [BMI]), and central obesity determined by waist circumference, waist-to-hip ratio (WHR), and waist-to-height ratio. RESULTS: Women, as compared to men, had markedly lower concentrations of 25(OH)D. Applying the cut-off point of serum 25(OH)D levels at 50 nmol/L, the prevalence of vitamin D deficiency in the study population was 87.5%; this was higher than the rates reported for the British, and European-, Hispanic-, and African-Americans. At a BMI cut-point of ≥30 kg/m(2), the prevalence of obesity was 14.6%; this was lower than the rates reported for European-, Hispanic-, and African-Americans. Levels of 25(OH) D increased relative to age and obesity. WHR was the main predictor of 25(OH)D levels. CONCLUSION: The striking vitamin D deficiency seen in the study population, relative to more northerly populations, may be linked to sun avoidance, inadequate dietary vitamin D, and virtual non-intake of supplemental vitamin D. Age and male-gender determined the status of vitamin D and of obesity.

VEGF-A and VEGFR-3 correlate with nodal status in operable non-small cell lung cancer: inverse correlation between expression in tumor and stromal cells.

BACKGROUND: Lymph node metastasis is an essential determinant for stage and clinical management of non-small cell lung cancer (NSCLC). The vascular endothelial growth factors (VEGFs) and receptors (VEGFRs) are fundamental molecules in angiogenesis and lymphangiogenesis. We aimed to explore the correlations between nodal metastasis and the expression of VEGFs and VEGFRs in tumor cells and in tumor-related stroma. PATIENTS AND METHODS: Tumor tissue samples from 335 resected patients with stage I-IIIA NSCLC were obtained and tissue microarrays were constructed from duplicate cores of tumor cells and surrounding stromal tissue from each resected specimen. Immunohistochemistry was used to evaluate the expression of VEGF-A, VEGF-C, and VEGF-D and VEGFR-1, VEGFR-2 and VEGFR-3. RESULTS: There were 232 N0 and 103 N+ patients (76 N1, 27 N2). In multivariate analyses, low stromal VEGF-A expression (P=0.018) is associated with N+ status. In tumor cells, strong correlations exist between high VEGF-A expression (P=0.032) and N+ status, and high VEGFR-3 expression (P<0.001) and N2-status. CONCLUSION: The converse impact by stromal VEGF-A versus tumor cell VEGF-A expression on nodal metastasis may allude the importance of the tumor-stroma interaction when trying to understand lymphatic metastasis in NSCLC.

Inverse prognostic impact of angiogenic marker expression in tumor cells versus stromal cells in non small cell lung cancer.

PURPOSE: The vascular endothelial growth factors (VEGF-A, -C, -D) and the VEGF receptors (VEGFR-1, -2, and -3) are important molecular markers in angiogenesis and lymphangiogenesis. This study elucidates the prognostic significance of these molecular markers in tumor cells as well as in the tumor stroma of resected non-small cell lung cancer tumors. EXPERIMENTAL DESIGN: Tumor tissue samples from 335 resected patients with stage I to IIIA disease were obtained and tissue microarrays were constructed from duplicate cores of tumor cells and surrounding stromal tissue from each resected specimen. Immunohistochemistry was used to evaluate the expression of each molecular marker. Microvessel density was assessed by CD34 immunohistochemical staining. RESULTS: In univariate analyses, high tumor cell expression of VEGF-A (P = 0.0005), VEGFR-1 (P = 0.013), VEGFR-2 (P = 0.006), and VEGFR-3 (P = 0.0003) were negative prognostic indicators for disease-specific survival (DSS). In tumor stroma, however, high expression of VEGF-A (P = 0.017), VEGF-C (P = 0.003), VEGF-D (P = 0.009), VEGFR-1 (P = 0.01), and VEGFR-2 (P = 0.019) correlated with good prognosis. There was no significant correlation between microvessel density and DSS. In multivariate analyses, high expression in tumor cells of VEGFR-3 (P = 0.007) was an independent negative prognostic factor for DSS, whereas in stromal cells, high VEGF-C (P = 0.004) expression had an independent positive survival impact. CONCLUSION: These are the first tissue microarray data in non-small cell lung cancers showing a positive prognostic impact by highly expressed angiogenic markers in tumor stroma, with VEGF-C as a major independent prognostic indicator.

Protein kinase D induces transcription through direct phosphorylation of the cAMP-response element-binding protein.

Protein kinase D (PKD), a family of serine/threonine kinases, can be activated by a multitude of stimuli in a protein kinase C-dependent or -independent manner. PKD is involved in signal transduction pathways controlling cell proliferation, apoptosis, motility, and protein trafficking. Despite its versatile functions, few genuine in vivo substrates for PKD have been identified. In this study we demonstrate that the transcription factor cAMP-response element-binding protein (CREB) is a direct substrate for PKD. PKD1 and CREB interact in cells, and activated PKD1 provokes CREB phosphorylation at Ser-133 both in vitro and in vivo. A constitutive active mutant of PKD1 stimulates GAL4-CREB-mediated transcription in a Ser-133-dependent manner, activates CRE-responsive promoters, and increases the expression of CREB target genes. PKD1 also enhances transcription mediated by two other members of the CREB family, ATF-1 and CREM. Our results describe a novel mechanism for PKD-induced signaling through activation of the transcription factor CREB and suggest that stimulus-induced phosphorylation of CREB, reported to be mediated by protein kinase C, may involve downstream activated PKD.

The cAMP signalling pathway activates CREB through PKA, p38 and MSK1 in NIH 3T3 cells.

Cyclic adenosine 3',5'-monophosphate (cAMP) was originally shown to induce gene transcription through activation of cAMP-dependent protein kinase (PKA), and subsequent phosphorylation of the transcription factor cAMP response element-binding protein, CREB, at serine-133. However, elevated cAMP levels may activate multiple signalling pathways with protein kinases that can phosphorylate CREB at serine-133. We analysed the pathways involved in CREB phosphorylation and activation in NIH 3T3 cells exposed to the cAMP elevating agent forskolin. PKA represented the predominant pathway during the burst phase, while the mitogen-activated protein kinase p38 pathway became activated in a PKA-dependent fashion in forskolin treated cells. The phosphorylation kinetics of p38 was delayed compared to PKA activation. Activated p38 stimulated CREB-mediated transcription and potentiated the transcriptional strength of CREB provoked by forskolin. The p38-mediated activation of CREB was inhibited by dominant negative mutants of MSK-1 and by the PKA/MSK-1 inhibitor H89, but not by dominant negative mutants of MSK-2/RSK-B and MAPKAPK2. Our results suggest that forskolin-induced CREB phosphorylation and activation in NIH 3T3 cells is mediated directly by PKA and by a time-delayed PKA-dependent p38/MSK-1 pathway. This bifurcation and time-dependent regulation of the cAMP-responsive signalling pathways may enable the cell to endure and/or enforce a cellular response provoked by a cAMP-elevating stimulus.

What turns CREB on?

The transactivation domain of the cAMP response element-binding protein (CREB) consists of two major domains. The glutamine-rich Q2 domain, which interacts with the general transcription factor TAFII130/135, is sufficient for the recruitment of a functional RNA polymerase II complex and allows basal transcriptional activity. The kinase-inducible domain, however, mediates signal-induced activation of CREB-mediated transcription. It is generally believed that recruitment of the coactivators CREB-binding protein (CBP) and p300 after signal-induced phosphorylation of this domain at serine-133 strongly enhances CREB-dependent transcription. Transcriptional activity of CREB can also be potentiated by phosphoserine-133-independent mechanisms, and not all stimuli that provoke phosphorylation of serine-133 stimulate CREB-dependent transcription. This review presents an overview of the diversity of stimuli that induce CREB phosphorylation at Ser-133, focuses on phosphoserine-133-dependent and -independent mechanisms that affect CREB-mediated transcription, and discusses different models that may explain the discrepancy between CREB Ser-133 phosphorylation and activation of CREB-mediated transcription.

Synergistic activation of CREB-mediated transcription by forskolin and phorbol ester requires PKC and depends on the glutamine-rich Q2 transactivation domain.

Recruitment of a RNA polymerase II complex by the glutamine-rich Q2 domain of cAMP response element-binding protein (CREB) allows basal transcriptional activity, while recruitment of CBP/p300 through signal-induced phosphorylation of the kinase-inducible domain at serine-133 enhances CREB-dependent transcription. Here we demonstrate that co-administration of forskolin and phorbol ester TPA to NIH3T3 cells provoked a dose-dependent increase in phosphoserine-133. CREB- and Q2-dependent transcription, as well as transcription by other glutamine-rich transcription factors, but not by transcription factors lacking glutamine-rich regions, augmented synergistically in the presence of both stimuli. Synergistic activation was abograted by specific inhibition of protein kinase C (PKC), but not of PKA. Co-stimulation increased the basal activity of a minimal, CREB-independent promoter. Therefore, Q2, which directly interacts with the RNA polymerase II initiation complex, may transmit the increased basal promoter activity provoked by these stimuli to CREB, thereby contributing to synergistic activation of CREB-mediated transcription. This synergism may have important implications on glutamine-rich transcription factor-target genes.