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Identifying the type(s) of body fluid present in forensic casework exhibits can assist with scene reconstruction and indicate potential activity types. Confirmatory body fluid identification can be achieved using mRNA-based profiling assays. This commonly involves endpoint reverse-transcription PCR (RT-PCR) with capillary electrophoresis (CE) detection. In comparison, real-time quantitative RT-qPCR is more sensitive, quantitative, and does not require post-PCR processing. We have developed real-time RT-qPCR assays for forensic body fluid identification. This study compared the performance (sensitivity, PCR efficiency, precision) of five real-time RT-qPCR kits across circulatory blood, buccal, semen, and vaginal fluid samples with normal and extended (3-step) PCR cycling. An objective scoring system for the experimental performance parameters was considered along with other features of the commercial kits. Statistical analysis by ANOVA and post-hoc Tukey of the slope estimates, which relate to PCR efficiency, revealed that many observed differences were insignificant (p > 0.05). Sensitivity and precision were also similar across most kits and PCR cycling protocols. Using the scoring system, the five highest performing kit and cycling combinations were: TaqMan-extended, followed by the TaqPath-normal, QuantiNova-extended, QuantiNova-extended, then UltraPlex-normal. Based upon high performance, room temperature set-up, and multiplexing capability, the UltraPlex kit was selected for our body fluid identification assays. [ABSTRACT FROM AUTHOR]

Background: G protein‐coupled receptors (GPCRs) play crucial roles in regulating islet function, with Gαs‐ and Gαq‐coupled receptors being linked to the stimulation of insulin secretion. We have quantified the mRNA expression of 384 non‐olfactory GPCRs in islets isolated from lean and obese organ donors to determine alterations in islet GPCR mRNA expression in obesity. Methods: RT‐qPCR was used to quantify GPCR mRNAs relative to five reference genes (ACTB, GAPDH, PPIA, TBP, and TFRC) in human islets isolated from lean (BMI = 22.6 ± 0.5) and obese (BMI = 32.0 ± 0.8) donors. Results: Overall, 197 and 256 GPCR mRNAs were detected above trace level in islets from lean and obese donors, respectively, with 191 GPCR mRNAs being common to the lean and obese groups. 40.9% (n = 157) and 27.1% (n = 104) of the mRNAs were expressed at trace level whilst 7.8% and 6.3% were absent in islets from lean and obese donors, respectively. Hundred and seventeen GPCR mRNAs were upregulated at least twofold in islets from obese donors, and there was >twofold downregulation of 21 GPCR mRNAs. Of particular interest, several receptors signalling via Gαs or Gαq showed significant mRNA upregulation in islets from obese donors (fold increase: PTH2R: 54.0 ± 14.6; MC2R: 34.3 ± 11.5; RXFP1: 8.5 ± 2.1; HTR2B: 6.0 ± 2.0; GPR110: 3.9 ± 1.2; PROKR2: 3.9 ± 0.7). Conclusions: Under conditions of obesity, human islets showed significant alterations in mRNAs encoding numerous GPCRs. The increased expression of Gαs‐ and Gαq‐coupled receptors that have not previously been investigated in β‐cells opens up possibilities of novel therapeutic candidates that may lead to the potentiation of insulin secretion and/or β‐cell mass to regulate glucose homeostasis. [ABSTRACT FROM AUTHOR]

Objective To optimize the extraction method of total RNA from pancreatic cancer and adjacent tissues, and provide high-quality experimental samples for the pathogenesis of pancreatic-related diseases. Methods The pretreatment of pancreatic cancer and paracancerous tissues was accomplished through shredding, RNase-free washing and RNase inhibition. RNA was isolated by liquid nitrogen grinding and trizol-chloroform extraction. The integrity of RNA was identified by agarose gel electrophoresis and bioanalyzer. With the same amount of RNA as the reverse transcription template, oligo dT primers were used for reverse transcription of messenger RNA (mRNA); stem-loop structure primers were used to reverse transcription of microRNA (miRNA). The expression levels of mRNA and miRNA were detected by quantitative RT-PCR. Compared with conventional methods, RNA samples extracted by the optimized method had a lower degradation degree and higher integrity. Results Compared with pancreatic cancer tissue, RNA in adjacent tissue is more easily degraded, and the expression level of mRNA tends to decrease. However, there is no significant difference in miRNA expression between pancreatic cancer and adjacent tissue. Conclusions Pretreatment of pancreatic cancer and adjacent tissues could reduce the RNA degradation, and provide high-quality experimental samples for RNA investigation and high experimental accuracy. [ABSTRACT FROM AUTHOR]

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for molecular diagnostics and evaluating prognosis in cancer. The utility of mRNA expression biomarkers relies heavily on the accuracy and precision of quantification, which is still challenging for low abundance transcripts. The critical step for quantification is accurate estimation of efficiency needed for computing a relative qRT-PCR expression. We propose a new approach to estimating qRT-PCR efficiency based on modeling dynamics of polymerase chain reaction amplification. In contrast, only models for fluorescence intensity as a function of polymerase chain reaction cycle have been used so far for quantification. The dynamics of qRT-PCR efficiency is modeled using an ordinary differential equation model, and the fitted ordinary differential equation model is used to obtain effective polymerase chain reaction efficiency estimates needed for efficiency-adjusted quantification. The proposed new qRT-PCR efficiency estimates were used to quantify GUCY2C (Guanylate Cyclase 2C) mRNA expression in the blood of colorectal cancer patients. Time to recurrence and GUCY2C expression ratios were analyzed in a joint model for survival and longitudinal outcomes. The joint model with GUCY2C quantified using the proposed polymerase chain reaction efficiency estimates provided clinically meaningful results for association between time to recurrence and longitudinal trends in GUCY2C expression. [ABSTRACT FROM AUTHOR]

Highlights • The mRNA expression of PTTG1 was studied in canine pituitary adenomas. • PTTG1 was overexpressed in adenoma tissue approximately 3-fold. • Dogs with high PTTG1 expression had significantly shorter disease-free intervals. • PTTG1 can be used as a clinical prognosticator in canine Cushing's disease. Abstract Pituitary-dependent hypercortisolism (PDH) is a common endocrinopathy in dogs, but the promotors and initiators of the tumourigenesis of corticotroph pituitary adenomas remain unknown. Based on human data, we investigated mRNA expression of pituitary tumour transforming gene 1 (PTTG1) with quantitative RT-PCR in canine corticotroph pituitary adenomas. PTTG1 was overexpressed in adenomas approximately 3-fold. A strong association was observed between PTTG1 expression and disease-free interval; dogs with high PTTG1 expression had a significantly (4 times; P = 0.02) shorter disease-free interval than dogs with low PTTG1 expression. This paper shows that PTTG1 expression is a negative prognosticator in relation to disease-free interval and recurrence in dogs undergoing transsphenoidal hypophysectomy as treatment for PDH. [ABSTRACT FROM AUTHOR]

Like Xenopus laevis, some species of the Rana genus are also used to study endocrine disrupting chemicals (EDCs). Although ribosomal protein L8 (rpl8) is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rana, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rpl8 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rpl8 cDNA consisted of 919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rpl8 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rpl8 gene was found in all tissues examined (brain, liver, intestine, tail, testis and ovary) during R. nigromaculata development. Finally, we investigated rpl8 expression under exposure to hormones. No change in rpl8 mRNA expression was found under exposure to thyroid hormone (T4) and estrogen (estradiol), whereas expression of the corresponding biomarker genes was induced. Our results show that rpl8 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rpl8 as a reference gene in other Rana species due to the high conservation of rpl8 among the Rana genus. [ABSTRACT FROM AUTHOR]

Oocytes and eggs of the African clawed frog, Xenopus laevis, are commonly used in gene expression studies. However, monitoring transcript levels in the individual living oocytes remains challenging. To address this challenge, we used a technique based on multiple repeated collections of nanoliter volumes of cytoplasmic material from a single oocyte. Transcript quantification was performed by quantitative RT-PCR. The technique allowed monitoring of heterologous gene expression in a single oocyte without affecting its viability. We also used this approach to profile the expression of endogenous genes in living Xenopus oocytes. Although frog oocytes are traditionally viewed as a homogenous cell population, a significant degree of gene expression variation was observed among the individual oocytes. A lognormal distribution of transcript levels was revealed in the oocyte population. Finally, using this technique, we observed a dramatic decrease in the content of various cytoplasmic mRNAs in aging unfertilized eggs but not in oocytes, suggesting a link between mRNA degradation and egg apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Bm86 midgut protein has been used in order to control ticks of the Hyalomma genus. Previous studies demonstrated the inefficacity of this antigen in the control of Hyalomma scupense, whereas recombinant Hd86 antigen, the Bm86 ortholog in H. scupense produced in Pichia pastoris, was protective against larval H. scupense tick stage infestations but ineffective in the control of the adult stage. One possible explanation for this result is the variation in Hd86 expression levels between these two developmental stages. To test this hypothesis, Hd86 mRNA levels were characterized in H. scupense developmental stages. The expression profile of Hd86 demonstrated a significant variation between tick life stages and showed a significant reduction in the number of transcripts during feeding and, particularly after molting to adults. The most interesting result was noted after molting of engorged nymphs in unfed adults where the expression levels decreased significantly by 12.78 (10.77–17.39) (p <0.001) and 9.25 (5.77–15.72)-fold (p <0.001) in unfed males and unfed females, respectively. Comparing unfed nymphs to unfed adult ticks, the Hd86 expression levels decreased by 13.82 (5.39–24.45) (p =0.035) and 9.93 (2.87–22.08)-fold (p =0.038) in males and females respectively. Lower Hd86 mRNA levels in adult ticks should result in lower protein levels and thus less antibody–antigen interactions necessary for vaccine efficacy in ticks fed on vaccinated animals. Thus, the observed differences in Hd86 expression profile between immature and adult stages might explain, in part, the discrepancy of the Hd86 vaccine efficacy against these two life stages of H. scupense. [Copyright &y& Elsevier]

Abstract: We report two novel hypofibrinogenemias, Shizuoka III and Kanazawa II, which are caused by heterozygous mutations in FGG. Shizuoka III showed c.147delT and 147_149insACA in FGG exon 3 and a subsequent frameshift mutation, resulting in mature protein γ23X (native protein: γ49X), and Kanazawa II showed c.1205G>A in FGG exon 9, resulting in γ376X (native protein: γ402X). To determine whether the truncated γ-chains, γ23X and γ376X, were synthesized and participated in the assembly of fibrinogen, mutant-type cDNA vectors were transfected into Chinese hamster ovary (CHO) cells. Significant levels of mutant fibrinogen were not detected by ELISA in the culture media and cell lysates. Immunoblot analysis of cell lysates revealed that the mutant γ-chain of γ376X was observed but intact fibrinogen was not. On the other hand, mutant γ-chain was not observed in γ23X-expressing cells. To demonstrate the involvement of the mechanisms of nonsense-mediated mRNA decay (NMD), we cloned wild- and mutant-type mini-genes containing γ23 or γ376 codon and transfected these into CHO cell lines in the absence or presence of cycloheximide as an NMD inhibitor. mRNA levels were determined using real-time quantitative RT-PCR in CHO cells. In the absence of cycloheximide, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of cycloheximide, levels of mRNAs transcribed from the mutant gene increased dose-dependently. Finally, these results demonstrated that mRNAs containing γ23X or γ376X are degraded by the NMD system and translation of the truncated γ-chain polypeptide decrease in patients’ hepatocytes, resulting in hypofibrinogenemias. [Copyright &y& Elsevier]

Abstract: We investigated whether correlations between mRNA levels of cytokines versus other proteins from patchy lesion could estimate cytokine paracrine signaling in vivo. Experiments with rat experimental autoimmune myocarditis (EAM), a patchy myocarditis model, indicated IL-1 and other protein levels were correlated, indicating paracrine signaling pathways in vivo. [Copyright &y& Elsevier]

Abstract: The YAP1 gene encodes a potent new oncogene and stem cell factor. However, in some cancers, the YAP1 gene plays a role of tumor suppressor. At present, the gene and its products are intensely studied and its cDNAs are used as transgenes in cellular and animal models. Here, we report 4 new potential mRNA splicing isoforms of the YAP1 gene, bringing the total number of isoforms to 8. We detected all 8 YAP1 isoforms in a panel of human tissues and evaluated the expression of the longest isoform of YAP1 (YAP1-2δ) using Real Time PCR. All YAP1 isoforms are barely detectable in human leukocytes compared to fair levels of expression found in other human tissues. We analyzed the structure of the genomic region that gave rise to alternatively spliced YAP1 transcripts in different metazoans. We found that YAP1 isoforms, which utilize exon 6 emerged in evolution with the appearance of amniotes. Interestingly, 6 YAP1 isoforms, which contain the exon 5 extension, exon 6 or both would have their leucine zipper region disrupted in the predicted protein product, compared to the intact leucine zipper found in two YAP1 (α) isoforms. This observation has direct functional ramifications for YAP1 signaling. We also propose a normalized nomenclature for the mRNA splice variants of the YAP1 gene, which should aid in the characterization of signaling differences among the potential protein products of the YAP1 gene. [Copyright &y& Elsevier]

Abstract: Background: In breast cancer, little is known about the consequences of co-expression of ERα with the second estrogen receptor, ERβ, and its isoforms in light of their joint prognostic value. Previously reported correlations have been based mostly on independent ERα and ERβ expression levels in breast tumors. Purpose: To address whether the expression ratio of ERα and ERβ and its isoforms may be a more important parameter than their absolute levels, we analyzed relative mRNA expression ratios of ERβ1 to ERβ2 and ERα in 74 clinical samples of invasive breast cancer including 39 early-onset and 35 late-onset breast cancers. Expression levels were correlated with clinical and histopathological parameters and disease-free interval. Results: A specific correlation of ERβ1 expression levels with tumor size was detected in early-onset breast cancer patients and of ERβ2 levels with tumor size in late-onset patients. Expression of both ERβ isoforms inversely correlated with expression of the two estrogen regulated genes, progesterone receptor and pS2 in both groups. Higher levels of ERβ2 than ERβ1 isoform were associated with a better outcome in late-onset patients. Conclusions: Our results suggest that different isoforms of ERβ may be involved in suppression of tumor growth in young and elder patients and may have different prognostic values. [Copyright &y& Elsevier]

Abstract: Background: The actions of endogenous opioid peptides are mediated by 3 main classes of opioid receptors; mu (MOR), kappa (KOR), and delta (DOR). Methods: We developed an absolute quantitative real-time reverse transcriptase PCR (AQ-rt-RT-PCR) assay to quantify MOR, DOR, and KOR mRNA in 22 human tissues. Results: MOR mRNA was greatly enriched (12–20×106 copies/μg) in the cerebellum, nucleus accumbens, and caudate nucleus; moderate (6×106 copies/μg) in the dorsal root ganglion, spinal cord, and adrenal gland; low (2×104 copies/μg) in the pancreas and small intestine; and absent in the lung, spleen, kidney, heart, skeletal muscle, liver, and thymus. High levels (>8.8×106 copies/μg) of DOR mRNA were expressed in the brain and dorsal root ganglion; moderate (1.5×106 copies/μg) in the adrenal gland and pancreas; low (2×104–6.5×105 copies/μg in the cerebellum, spinal cord, small intestine, skeletal muscle, thymus, lung, and kidney); and very low (3.8×103 copies/μg) in the heart. DOR mRNA was not detected in the spleen or liver. KOR mRNA was moderate (1×106 copies/μg) in brain regions and dorsal root ganglion, but low (1.6–7×105 copies/μg) in the cerebellum, temporal lobe and all other peripheral tissues. Conclusions: Our data demonstrate that the AQ-rt-RT-PCR is a highly reproducible and precise method to study the expression of opioid receptors in various tissues and under different disease conditions. [Copyright &y& Elsevier]

Background: In previous studies we found that MHC2TA +1614 genotype frequency was very different when MS patients with and without human herpesvirus 6 (HHV-6) in serum samples were compared; a different clinical behavior was also described. The purpose of the study was: 1. To evaluate if MHC2TA expression in MS patients was influenced by interferon beta (IFN-beta) treatment. 2. To study MHC2TA expression in MS patients with and without minor allele C. 3. To analyze the relation between MHC2TA mRNA levels and HHV-6 active infection in MS patients. Methods: Blood and serum samples of 154 MS patients were collected in five programmed visits: basal (prior to beginning IFN-beta treatment), six, twelve, eighteen and twenty-four months later. HHV-6 in serum and MHC2TA mRNA levels were evaluated by PCR and RT-PCR, respectively. Neutralizing antibodies (NAbs) against IFN-beta were analyzed by the cytopathic effect assay. Results: We found that MHC2TA mRNA levels were significantly lower among MS patients with HHV-6 active infection at the basal visit (without treatment) than in those MS patients without HHV-6 active infection at the basal visit (p = 0.012); in all the positive samples we only found variant A. Furthermore, 58/99 (58.6%) MS patients without HHV-6 along the five programmed visits and an increase of MHC2TA expression after two-years of IFN-beta treatment were clinical responders vs. 5/21 (23.8%) among those MS patients with HHV-6 and a decrease of MHC2TA mRNA levels along the two-years with IFN-beta treatment (p = 0.004); no differences were found between patients with and without NAbs. Conclusions: MHC2TA mRNA levels could be decreased by the active replication of HHV-6; the absence of HHV-6 in serum and the increase of MHC2TA expression could be further studied as markers of good clinical response to IFN-beta treatment. [ABSTRACT FROM AUTHOR]

iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3-18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT-PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies. [ABSTRACT FROM AUTHOR]

Abstract: TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4+ T-helper cell population in the resting (=naïve) and activated (=effector) state, and 3) two commonly used CD4+ Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4+ T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca2+-dependent proliferation of primary CD4+ T-cells indicating that TRPC3 may be involved in Ca2+ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca2+ entry. [Copyright &y& Elsevier]

Aim To investigate the characteristics of side population (SP) cells derived from the dental pulp of young and aged rats. Methodology Maxillary and mandibular incisors were extracted from 5-week-old (young) rats and 60- to 80-week-old (aged) rats. Coronal pulp tissue was removed mechanically, and single-cell suspensions were prepared using collagenase and dispase. Cells were stained with Hoechst 33342 and sorted with an fluorescence-activated cell sorter (FACS). Isolated SP and main population (MP) cells were analysed by realtime reverse transcription polymerase chain reaction, immunohistochemical localization and cell cycle determination. Two-way analysis of variance and the multiple comparison Scheffe` test were used for statistical analysis (P < 0.05). Results Approximately 0.40% of pulp cells in young rats and 0.11% in aged rats comprised SP cells. SP cells expressed a higher mRNA level of ATP-binding cassette transporter G2 (ABCG2), but lower mRNA levels of nestin, alkaline phosphatase, p16 and p57 than MP cells in both age groups. Immunohistochemical observation revealed ABCG2-positive cells localized in the cell-rich zone and nestin in the odontoblastic layer in both groups. Furthermore, the majority of both young and aged SP and MP cells were in growth arrest of the G0/G1 phase. Conclusion The FACS analysis revealed a decrease in the proportion of SP cells with age, whilst p16 mRNA expression indicated an increase in cell senescence. The cell cycles of SP and MP cells from both young and aged dental pulp were generally in the G0/ G1 phase. [ABSTRACT FROM AUTHOR]

Abstract: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a highly specific and sensitive technique for the quantification of gene expression on the mRNA levels. But use of unconfirmed housekeeping genes (HKGs) could lead to misinterpretation of the expression of genes of interest (GOI). In this study, the stability and suitability of 11 frequently used housekeeping genes, namely 18S rRNA, ACTB, B2M, CYPA, GADPH, GUSB, HMBS, HPRT1, RPL13A, SDHA and TBP in 36 lung tissues isolated from either wild-type (WT) mice or p50 knock out (p50−/−) mice or p105 knock-out (p105−/−) mice which were treated with either carbon nanoparticle (CNP) or H2O or non-treated, have been validated by geNorm, NormFinder and BestKeeper programs. The expression levels of ACTB, GUSB and RPL13A were the most constant in lung tissues across three genotypes and three kinds of treatments. A set of three most stable genes is found sufficient to be used as housekeeping genes for lung tissues in studies of similar design. [Copyright &y& Elsevier]

Abstract: Background: Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. Design and methods: Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. Results: Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. Conclusions: mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type. [Copyright &y& Elsevier]

The presence of regional lymph node metastases is one of the most important prognostic factors in colon cancer. Nevertheless, up to 30% of the lymph node negative patients experience disease recurrence. Possibly, this patient group may be identified by more sensitive techniques than routine histopathological examination of the lymph nodes. In the present study, we have evaluated the detection of colon cancer lymph node metastases by real-time RT-PCR quantitation of the epithelial-specific cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNAs. Both assays were able to detect dilutions of tumor cells down to one tumor cell in 106 normal lymphocytes. CK20 and MUC2 mRNA were quantitated in 52 normal lymph nodes from 12 patients undergoing surgery for benign bowel diseases and in 144 primary colon tumors. The median tumor level of both markers were more than 104-fold higher than the highest level in normal lymph nodes, indicating that the markers had a potential for metastasis detection in a clinical context. We applied the assays to 61 lymph nodes with known metastases detected by routine staining. Elevated CK20 or MUC2 mRNA levels were detected in 57 (95%) of the 61 LNs. Thus, CK20 and MUC2 quantitation by real-time RT-PCR seems to be a promising, sensitive tool to detect metastases in regional lymph nodes from colon cancer patients. [ABSTRACT FROM AUTHOR]

Purpose: Deviant expression of platelet-derived growth factor receptor-β (PDGFRβ) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFRβ and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Methods and Materials: Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFRβ and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG, 0-1). Results: The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFRβ ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFRβ ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFRβ and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFRβ ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFRβ and c-kit mRNA expression and the pathologic regression rate. Conclusion: Cetuximab-based chemoradiotherapy increased PDGFRβ levels even further compared with the pretreatment samples and deserves further investigation. [Copyright &y& Elsevier]

Objectives: Several clinical studies have indicated that the rates of invasive growth and metastatic disease in cancer depend on the degree of hypoxia, which is mediated by hypoxia-inducible factor 1 α (HIF-1α). To determine its potential role as a marker for prostate cancer (CaP) diagnosis, HIF-1α mRNA levels were measured in blood samples of patients diagnosed with different stages of prostatic disease. Methods: HIF-1α mRNA levels were measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and correlated with accurate clinicopathological data. Quantitative data were compared with serum prostate-specific antigen (PSA) measurements to determine variations in the accuracy of the CaP diagnosis. Results: HIF-1α mRNA levels were significantly upregulated in patients with localized CaP (LocCaP; n=63; p<0.0001), compared with patients with no evidence of malignancy (NEOM) and benign prostatic hyperplasia (BPH) (n=35 for both patient groups combined). Receiver operator characteristic (ROC) curve analysis demonstrated that HIF-1α specificity for the NEOM/BPH diagnosis was 88.6%. Sensitivity for LocCaP was 74.6% with an overall diagnostic efficiency of 79.6%. Specificity of the NEOM diagnosis at PSA levels of 4.0 ng ml-1 was 28.6% and sensitivity of the LocCap diagnosis was 65.1%, demonstrating a reduced overall diagnostic efficiency, compared with that given by HIF-1α measurements, of 52.0%. Levels of HIF-1α in patients with metastatic CaP (MetCaP; n=27)) were similar to those in the NEOM/BPH group. Conclusions: HIF-1α is upregulated early in CaP development with subsequent downregulation at later metastatic stages. This study demonstrates increased accuracy of early-stage disease diagnosis using HIF-1α qRT-PCR compared with serum PSA measurements. HIF-1α may therefore be a useful adjunct, together with other diagnostic markers used in relative qRT-PCR and current diagnostic techniques (including serum PSA and PSA velocity) to minimize unnecessary biopsies indicated by elevated serum PSA levels alone. [ABSTRACT FROM AUTHOR]

The aim of this study is to compare the expression pattern of matrix metalloproteinases (MMP) in primary CNS lymphoma (PCNSL) with that of nodal lymphoma (NL). PCNSL possesses a characteristic pattern of brain infiltration, specifically, perivascular aggregation and destruction of the surrounding connective tissue. Hypotheses have been proposed that this infiltration pattern indicates the infiltration of lympoma cells from outside the CNS. However, the mechanism of this infiltration pattern has not been clearly explained. Here we performed quantitative analysis of mRNA expression of MMP1, 2, 3, 7 and 9 in lymphoma cells from 10 cases of PCNSL, all of which were diagnosed as diffuse large B-cell lymphoma (DLBCL), and 14 cases of nodal DLBCL. Immunohistochemistry was also performed for phenotyping of lymphoma cells and for examining the localization of MMPs. The expression level of MMP7 in PCNSL (median ratio to normal lymophocytes, 45.6) was significantly higher than the level in NL (median, 7.55). By contrast, the expression levels of MMP2 (median, 31.7) and MMP9 (median, 1.30) in PCNSL were significantly lower than those in NL (median, 120 and 16.5, respectively). The expression levels of MMP1 and MMP3 were very low and not different between PCNSL and NL (almost comparable to those in normal lymphocytes). Immunohistochemical phenotyping revealed that the frequency of non-germinal center type DLBCL was significantly higher in PCNSL than in NL. These results, notably the significantly increased expression of MMP7 in PCNSL, suggest the involvement of this MMP in the characteristic infiltration pattern of PCNSL. They also support the existing hypothesis that PCNSL and NL are of different origins. [ABSTRACT FROM AUTHOR]

Abstract: Bursicon is the ultimate hormone in insect ecdysis, which is involved in cuticle hardening. Here we show that mRNAs encoding the heterodimeric cystine knot protein bursicon (Burs α, β), are present in crustaceans, suggesting ubiquity of this hormone in arthropods. We firstly report the cloning, sequencing of mRNAs encoding subunits from the water flea, Daphnia arenata and the CNS of the crab, Carcinus maenas, in comparison with insect bursicon subunits. Expression patterns of α and β burs mRNAs were examined by in-situ hybridisation (ISH) and quantitative RT-PCR. In the thoracic ganglion, burs α and β mRNAs were completely colocalised in neurones expressing crustacean cardioactive peptide (CCAP). However, in the brain and eyestalk, bursicon transcripts were never observed, despite a complex expression pattern of CCAP interneurones. Patterns of expression of burs α and β mRNAs were constitutive during the moult cycle of adult crabs, in stark contrast to the situation in insects. Whilst copy numbers of burs β transcripts closely matched those of CCAP, those of burs α mRNA were around 3-fold higher than burs β. This pattern was apparent during embryogenesis, where bursicon transcripts were first observed at around 50% development—the same time as first expression of CCAP mRNA. Transcript ratios (burs α: β) increased during development. Our studies have shown, for the first time, that bursicon mRNAs are expressed in identified neurones in the nervous system of crustaceans. These findings will now promote further investigation into the functions of bursicon during the moult cycle and development of crustaceans. [Copyright &y& Elsevier]

Abstract: Events occurring during early filarial nematode migrations are central to parasite establishment but rarely studied. Brugia pahangi larvae injected intradermal (ID) into the hind limb of the gerbil (Meriones unguiculatus) can be recovered from the popliteal lymph node (POP) at 3 days post-infection (DPI). They have been designated migrating larvae (IDL3). Alternatively, L3 recovered at 3DPI from the peritoneal cavity (IPL3) do not migrate. Subtracted cDNA libraries using IDL3 and IPL3 revealed distinct gene profiles between IDL3 and IPL3. Troponin-c was significantly upregulated in IDL3, while Cathepsin L was significantly increased in IPL3. Differences in mRNA levels were also observed with these and other genes between IDL3, IPL3 and L3 isolated from mosquitoes (VL3). These data suggest that migratory activity, exposure to potentially different host environments and/or host location may be important external factors in influencing larval gene expression. [Copyright &y& Elsevier]

Abstract: Aspergillus niger is a widely used expression host for homologous and heterologous protein production in biotechnological processes. In order to increase product yields, a thorough optimisation of these cultivation processes is necessary. Considering mRNA as the key molecule, which transports the genetic information between DNA and protein production side, the quantification of product specific gene expression provides useful information about product formation already on the level of transcription. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a powerful tool to obtain data about gene transcription. However, using this technique the choice of an appropriate reference system is a crucial aspect to provide optimal data normalisation. A prominent approach is the use of so called housekeeping genes as internal references. However, validation of the usability of these reference genes is the fundamental step before starting with qRT-PCR experiments. Adequate reference genes for A. niger have not been published so far. Therefore, 10 possible candidate genes from different functional classes were selected and their applicability as internal references validated. Transcript levels of these genes were compared in sets of 9, 41 and 19 samples from diverse cultivations of A. niger. Under the chosen experimental conditions, the genes act, sarA and cox5 have been identified as genes with the most stable gene expression. The three reference genes were used to normalise qRT-PCR data for glaA gene expression which showed a high correlation with glucoamylase production in continuous cultivations. [Copyright &y& Elsevier]

In addition to Estrogen Receptor α (ERα) and Progesterone Receptor (PR), the Second Estrogen Receptor (ERβ) appears to play an important role not only in estrogen signaling, but also in the pathogenesis of cancer in estrogen dependent tissues. The existence of various isoforms and splice variants of both ERs additionally complicates elucidation of their physiological role and involvement in the process of carcinogenesis. In this study, the expression of ERβ1 mRNA (wild type of β receptor) and splice variant ERβΔ5 mRNA (which codes for truncated protein) was measured by the quantitative RT-PCR (q RT-PCR) in the 60 samples of Breast Cancer (BC) and correlated with ERα and PR protein levels and with clinical and histopathological parameters. We found the inverse correlation of ERβΔ5 mRNA expression with the levels of PR and ERα proteins in the group of postmenopausal patients; we also report the lower expression of ERβ1 and ERβΔ5 mRNA in the larger tumors (>20 mm, T2, and T3) than in smaller ones (≤20 mm, T1). The decrease of ERβΔ5 mRNA expression in larger tumors is found to arise from ER-positive breast carcinomas. In addition, the portion of tumors with concomitant high expression of both transcripts matches up the known percentage of tumors resistant to endocrine therapy in patients with different ER/PR status. As far as we know, this is the first study in which ERβΔ5 mRNA splice variant was quantified by real-time RT-PCR in the clinical samples of breast cancer tissue. Until now, the focus of clinical reports was the level of ERβ1, ERβ2, and ERβ5 isoforms. The higher expression of ERβΔ5 mRNA is associated with the indicators of low biological aggressiveness of tumor (low tumor size within ER-positive status in our study) suggesting that the uncontrolled local tumor growth may occur as the expression of ERβΔ5 mRNA decreases in estrogen-dependent breast cancer. [ABSTRACT FROM AUTHOR]

Micro (mi)RNAs are small, noncoding RNAs that regulate gene expression through binding to the 3'-untranslated region of mRNAs by complementary base pairing and mainly act through cleavage or translational inhibition of mRNAs. Recent studies have shown the roles of miRNAs in development and cancer, revealing the physiological and pathological importance of these tiny molecules. Therefore, as with mRNAs, researchers have focused on the global analyses of miRNAs to seek their potential use as biomarkers for physiological and pathological states of a cell. Methods developed for miRNA profiling are briefly discussed in this review. Recent evidences supporting the use of miRNAs as biomarkers in both differentiation and cancer are presented. The profiling studies may highlight the clinical relevance of miRNAs and will enable the researchers to uncover the enormous potential of these tiny molecules. In the near future, selected miRNA genes based on expression abnormalities will be tested as candidates for miRNA-based cancer gene therapy. [ABSTRACT FROM AUTHOR]

Abstract: A fluorescence resonance energy transfer (FRET) based quantitative RT-PCR method (qRT PCR) was developed in this study for measuring the mRNA expression of trypsins Y and I in the Atlantic cod. Atlantic cod β-actin was used as the reference gene and standard curves were created for quantification of the mRNA expression levels. For yet unknown reasons, the Atlantic cod (Gadus morhua) produces several trypsins with different characteristics. Trypsin I is the most common and best characterized of these but trypsin Y is a recently discovered enzyme. The recombinant form of trypsin Y was found to have unique characteristics relative to trypsin I. The native form of trypsin Y has proven difficult to isolate from the cod and activity assays do not distinguish between the activities of trypsin I and trypsin Y. The results show that trypsin Y mRNA is expressed in a very low copy number relative to that of trypsin I (ratio of 1:1340), which may explain the difficulty of isolating the native form of trypsin Y. [Copyright &y& Elsevier]

The current investigation evaluates the expression of phosphatidylinositol kinase (PIK) genes in the parasitic protozoan, Giardia lamblia. The G. lamblia Genome Database revealed the presence of two putative phosphatidylinositol-3-kinase (gPI3K) and one phosphatidylinositol-4-kinase (gPI4K) genes resembling the catalytic subunit of eukaryotic PIKs. Primers, designed to amplify mRNA of these three genes, were used to measure transcription by quantitative reverse-transcriptase polymerase chain reactions. Results suggest that all three PIK genes are expressed in non-encysting and encysting trophozoites. The relative levels of the mRNA were highest in parasites cultured in pre-encysting medium that contained no bile. Two inhibitors of PI3K, LY 294002 and wortmannin were found to inhibit the growth of the trophozoite in culture. However, wortmannin was more effective than LY294002. Altogether, the present study indicates that Giardia is capable of expressing PIKs that are necessary for the growth and differentiation of this pathogen. [ABSTRACT FROM AUTHOR]

Abstract: Members of the Caenorhabditis elegans REF-1 family of bHLH proteins are atypical in that each protein contains two bHLH domains. In this study we describe a functional and molecular characterization of the REF-1 family members, hlh-29/hlh-28. 5′-RACE results confirm the presence of two bHLH domain coding regions in a single transcript and quantitative PCR (qPCR) shows that hlh-29/hlh-28 mRNA is detected in wild-type animals throughout development. A promoter fusion of hlh-29 to the green fluorescent protein shows post-embryonic reporter activity in cells of the vulva, the somatic gonad, the intestine and in neuronal cells of the head and tail. Loss of hlh-29/hlh-28 function via RNA interference (RNAi) results in multiple phenotypes including late embryonic lethality, yolk protein accumulation, everted vulva, bordering behavior, and alter chemosensory responses. [Copyright &y& Elsevier]

Abstract: Botulinum toxin is produced by Clostridium botulinum as a large toxin complex (L-TC) non-covalently assembled with a neurotoxin (NT), a non-toxic non-hemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33, and HA-17). In this study, the gene expressions of five individual L-TC components were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in C. botulinum serotype D strain 4947 (D-4947) during cell growth. Transcripts for the five component genes were successfully detected in the mid-exponential growth phase (6.5 h), reaching a maximum at the early stationary growth phase (12 h). The ratio of the mRNA transcripts of nt and ntnha was approximately 1:1, suggesting that nt and ntnha are bicistronically transcribed. On the other hand, the transcript levels of the ha genes were several-fold higher than those of nt and ntnha, although the mRNA transcript level of ha-33 was less than the other two ha subcomponent genes. The results based on qRT-PCR indicate that a shortage of HA-33 among the proteins associated with botulinum TC could explain the production by D-4947 of other smaller-sized L-TCs (610, 540 and 410 kDa) with fewer HA-33 molecules than the mature 650 kDa L-TC. Western blot analysis demonstrated that TC species in cell lysate were initially observed in the mid-exponential phase, while extracellular TCs were detected subsequently in the early stationary phase. [Copyright &y& Elsevier]

Background: We and others have recently explored the use of quantitative real-time RT-PCR analysis for the detection of circulating tumour cells in blood of patients with breast cancer (BC). One major problem in these experiments is the in vitro instability of the cellular RNA. The copy number of mRNA can change during storage and transport at room temperature and this may hamper accurate quantitative measurements of specific transcripts, especially when working with small numbers of target mRNAs. Methods: Peripheral blood samples were obtained from two healthy volunteers and 13 patients with BC. Blood was stored at room temperature for 0, 1, 2, 4, 6, 24, 48 and 72 h. The potential alteration of gene expression for six target genes was investigated by quantitative real-time RT-PCR. Results: For β-actin, GAPDH, cytokeratin-19 ( CK-19) and HER2, a significant decrease in expression level occurs after 4 h ( CK-19 and HER2), 6 h (β-actin) or 24 h ( GAPDH). Mammaglobin expression was only measurable in two samples and seems to be stable for at least 6 h. For vascular endothelial growth factor ( VEGF), a statistically significant increase in expression level is observed in samples processed 24 h after collection. Conclusions: Most transcripts were reduced in samples that were stored overnight at room temperature compared with fresh samples, but upregulation of transcripts, probably as an active response to cellular stress, also occurs when blood is removed from its in vivo environment and stored ex vivo. Optimally, blood samples and RNA should be processed or stabilised within 3 h after collection to avoid interference of the in vivo gene expression signature by ex vivo stress responses. Clin Chem Lab Med 2006;44:1082–7. [ABSTRACT FROM AUTHOR]

Mutations in two kinases, PTEN induced kinase 1 (PINK1) and leucine-rich repeat kinase 2 (LRRK2), have been shown to segregate with familial forms of Parkinson's disease. Although these two genes are expected to be involved in molecular mechanisms relevant to Parkinson's disease, their precise anatomical localization in mammalian brain is unknown. We have mapped the expression of PINK1 and LRRK2 mRNA in the rat and mouse brain via in situ hybridization histochemistry using riboprobes. We found that both genes are broadly expressed throughout the brain with similar neuroanatomical distribution in mouse compared to rat. PINK1 mRNA abundance was rather uniform throughout the different brain regions with expression in cortex, striatum, thalamus, brainstem and cerebellum. LRRK2, on the other hand, showed strong regional differences in expression levels with highest levels seen in the striatum, cortex and hippocampus. Weak LRRK2 expression was seen in the hypothalamus, olfactory bulb and substantia nigra. We confirmed these distributions for both genes using quantitative RT-PCR and for LRRK2 by western immunoblot. As their broad expression patterns contrast with localized neuropathology in Parkinson's disease, the pathogenicity of clinical mutant forms of PINK1 and LRRK2 may be mediated by nigrostriatal-specific mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: Epidemiological studies show that the obstructive sleep apnea syndrome (OSAS) is strongly associated with obesity, hypertension and diabetes, the three conditions characteristic of the metabolic syndrome. Since metabolic disorders usually involve altered homeostatic mechanisms both centrally and peripherally, it is likely that so it is in OSAS, but the underlying mechanisms remain largely unknown. We used an established rodent model to test whether chronic intermittent hypoxia (CIH) similar to that experienced by OSAS patients leads to distinct and relevant for metabolic regulation transcriptional changes in the posterior hypothalamus. Using quantitative reverse transcription–polymerase chain reaction, we found that rats exposed to CIH for 35 days (n =9) had twice higher levels of the adrenergic α2A receptor mRNA than the rats simultaneously submitted to a matching sham treatment (n =9). The mRNA levels of three members of the family of signal transducers and activators of transcription, STAT1, STAT3 and STAT5b, were also increased 2–4 times. The increases occurred only in the perifornical region, whereas no changes were detected in the ventromedial region comprising the ventromedial and arcuate nuclei or the dorsomedial region comprising the dorsomedial and paraventricular nuclei. These results show that, at least at the transcriptional level, CIH exerts a distinct and regionally selective central effect on the expression of selected mRNAs involved in metabolic regulation through adrenergic, leptinergic and inflammatory pathways. [Copyright &y& Elsevier]

Prostaglandin E1 leads to penile erection, mainly via prostaglandin E2 (EP) receptors. This study aimed to identify the expression profile of EP receptor genes in human corpus cavernosum. Using the quantitative real-time reverse transcription polymerase chain reaction, the mRNA levels of EP receptor subtypes were measured. In addition, expressions of EP receptor subtype proteins were determined by immunohistochemical method. Among the four subtypes, EP4 receptor mRNA expression was the highest, and EP2 receptor mRNA followed, whereas EP1 and EP3 receptor mRNAs were hardly observed. Expression level of EP4 receptor mRNA was significantly higher than that of EP2 receptor mRNA. Expression of both EP2 and EP4 receptor proteins were clearly detected in the cavernous smooth muscle. These results may suggest that EP4 receptor plays an important role among four EP receptor subtypes for relaxation of smooth muscle in the human corpus cavernosum.International Journal of Impotence Research (2006) 18, 275–281. doi:10.1038/sj.ijir.3901408; published online 20 October 2005 [ABSTRACT FROM AUTHOR]

Abstract: Objectives: : Our aim was to verify whether the quantitative determination of PSA mRNA in circulating cells is helpful in diagnosing and scoring localized prostate cancer (PC). Design and methods: : The study included 145 patients with benign prostatic hyperplasia (BPH), 138 with localized PC and 28 healthy controls (CS). PSA cDNA was amplified by real-time PCR from circulating mononuclear cells. Serum total and free PSA were determined. Prostate cancers were histologically scored according to the Gleason criteria. Results: : The most sensitive index of PC was tPSA (70%), and the most specific was f/t PSA (80%). High PSA mRNA was found more frequently in PC patients with poorly differentiated (23.1%) than in those with well (4.5%) or moderately (4.3%) differentiated tumors. Conclusions: : tPSA and f/t PSA are the best available tools for discriminating between localized PC and BPH. The quantitative assessment of PSA mRNA in blood might be helpful in the biochemical grading of prostate cancer. [Copyright &y& Elsevier]

Abstract: Idiopathic canine eosinophilic bronchopneumopathy (EBP) is a disease characterized by eosinophilic infiltration of the pulmonary interstitium and bronchial mucosa, a cause for which has not yet been discovered. A recent study, examining the relative proportion of various lymphocyte cell subsets within bronchoalveolar lavage fluid from dogs with EBP, has shown a selective increase in CD4+ T-cells and a selective decrease in CD8+ T-cells, suggesting that a similar Th2 immune response might occur in EBP. The aim of the present study was to determine the profile of cytokine, chemokine and CC chemokine receptor 3 (CCR3) messenger RNA (mRNA) expression in bronchial tissue from dogs with EBP. Real-time RT-PCR assays were used for the quantification of mRNA encoding for a panel of cytokines, CC chemokines and CCR3 in perendoscopic bronchial biopsies from eight dogs with EBP and seven age-matched control dogs. Messenger RNA transcribed from the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used for normalisation of the threshold cycle in order to determine the relative copy numbers of the transcripts. No significant difference in the expression of any cytokine, MCP-1, -2, -4 and CCR3 was found between control and EBP dogs. The expression of transcript for MCP-3, eotaxin-2 and -3 was significantly greater in bronchial biopsies from dogs with EBP than in samples from control dogs while there was significantly less mRNA encoding RANTES in the mucosa of dogs with EBP. In conclusion, the cytokine mRNA expression profile in perendoscopic bronchial biopsies is similar in dogs with EBP and dogs without respiratory disease. Further studies on the quantification of mRNA encoding cytokines in isolated T lymphocytes from bronchoalveolar lavage fluid or bronchial biopsies are needed before any conclusion on the cytokine profile in canine EBP can be drawn. Eotaxin-2, -3 and MCP-3 appear to be implicated in the pathogenesis of the disease. [Copyright &y& Elsevier]

Abstract: This study was performed to prove our hypothesis that the metabolite(s) of polycyclic aromatic hydrocarbons (PAHs) caused the activation or phosphorylation of p53 via DNA damage to suppress the liver X receptor (LXR)-mediated signal transductions as a probably more direct mechanism. We found that LXR-mediated trans-activation was inhibited by 3-methylchoranthrene (MC) and doxorubicin (Dox) in HepG2 cells carrying wild-type p53, but not in Hep3B cells possessing mutant p53. The exogenous expression of wild-type p53 suppressed the LXR-mediated trans-activation in Hep3B cells. The expression of mRNA for ATP binding cassette A1 was suppressed by MC and Dox in HepG2 cells. The protein expression of retinoid X receptor (RXR), a partner of LXR to form a heterodimer, was suppressed by MC and Dox in HepG2 cells. [Copyright &y& Elsevier]

Background. The aim of this study was to confirm the vulnerability of fatty liver to heat stress using fatty liver rats from the viewpoint of the induction of apoptosis. Methods. We exposed rats with and without a fatty liver to heat stress and then looked for apoptotic cells within the liver tissue using two apoptosis detection kits. We also determined the mRNA expression of heat shock protein (HSP) 70, caspase-3, bcl-2, and bax using a quantitative reverse transcription-polymerase chain reaction method. Results. Following heat stress, apoptosis was strongly visible in the fatty liver comparing with that noted in the normal liver. The expression of HSP70 was increased following heat stress in both livers, but the volume of its expression was significantly less in the fatty liver than in the normal liver. The ratio of bcl-2/ bax expression tended to increase in the normal liver but decrease in the fatty liver following heat stress. Caspase-3 demonstrated no significant change following heat stress in both livers. Conclusions. The detection of apoptosis, together with changes in the mRNA expression of HSP70 and the expression ratio bcl-2/bax mRNA may indicate vulnerability of a fatty liver to heat stress and may support the hypothesis that morphologic change is induced in a fatty liver by exposure to heat stress. These results suggest that fatty liver may be more vulnerable to heat stress than normal liver. [ABSTRACT FROM AUTHOR]

Abstract: Background: A new basis for diagnostic tests is being provided by the vast amount of data on gene expression that are now becoming available through large-scale measurement of mRNA abundance. The insights gained from these resources are most likely going to provide both a better basic understanding of disease mechanisms, and to identify molecular markers for more precise diagnoses and for prediction of prognosis and treatment response. Methods: Some quantitative RT–PCR assays are utilized today for diagnosis of both malignant and non-malignant disease, but the use of gene expression measurements in clinical medicine can be expected to increase dramatically. Conclusions: There are important technical issues that must be adequately solved in order to obtain robust assays, such as standardized protocols with appropriate quality controls that ensure reliable data for the specific samples being analysed and good inter-laboratory reproducibility. [Copyright &y& Elsevier]

Aim To investigate the expression of osteocalcin mRNA in young and in aged human dental pulp tissue to determine the characteristics of osteocalcin expression. Methodology Human dental pulp tissues of the third molars were obtained from healthy young (17–23 years) and aged (>50 years) subjects, and total RNA was extracted. Osteocalcin mRNA expression was determined by RT-PCR and by quantitative real-time RT-PCR (QRT-PCR). The threshold cycle (Ct) value, which reflects the amount of PCR, was calculated and the difference between the value in young and aged pulp was statistically analysed. Results Osteocalcin mRNA was detected in all samples of human dental pulp tissue homogenates by RT-PCR analysis. Osteocalcin mRNA was expressed in young adult dental pulp but was decreased in aged human dental pulp. QRT-PCR analysis also showed a reduced expression of osteocalcin mRNA in aged human pulp. Expression of osteocalcin in young human pulp was significantly higher (about sixfold) than in aged pulp ( P < 0.01, Mann–Whitney U-test). Conclusion Reduction of osteocalcin expression may be associated with the loss of viability in human dental pulp tissue, and may be a characteristic of aged human dental pulps. [ABSTRACT FROM AUTHOR]

Abstract: Real-time RT-PCR has been used widely, both in fundamental research and in clinical diagnostics, for instance for quantification of RNA levels in human tissues and tissue biopsies. In the present study we provide a strategy to validate primers/probes for real-time RT-PCR quantification of baboon samples. The method is based on the TaqMan system and uses primers/probes that have been designed and validated for human real-time RT-PCR. A prerequisite for the accuracy of this strategy is a similar amplification efficiency between human and baboon PCR reactions. We propose two different methods, i.e. by calculating PCR efficiencies from the slope of a dilution curve or by using the linear regression method, to compare the amplification efficiency between human and baboon samples. In conclusion, by performing a simple validation experiment, real-time PCR assays based on human sequences, which are easily available, can be applied for analysis of baboon samples. [Copyright &y& Elsevier]

Abstract: The basic helix–loop–helix (bHLH) transcription factor family regulates numerous developmental events in eukaryotic cells. In the model system, C. elegans, thirty-seven bHLH proteins have been identified via genome-wide sequence analysis and fourteen have been genetically characterized to date. These proteins influence cell fate specification of neural lineages and differentiation of myogenic lineages and have distinct roles in somatic gonadogenesis. We report here on the molecular characterization of HLH-17, a protein whose putative bHLH domain is homologous to the mammalian bHLH proteins BETA3 and bHLHB5. The gene hlh-17 is transcriptionally active at all developmental stages, with the highest steady state accumulation of hlh-17 mRNA during embryogenesis. An upstream hlh-17 sequence drives expression of GFP in the sheath cells of the cephalic sensilla. Finally, animals that are defective in HLH-17 via RNAi display egg-laying defects, while those carrying null mutations in hlh-17 do not develop beyond the L2 stage and are less attracted to potassium and sodium ions. We propose that hlh-17 affects the ability of C. elegans to respond to food cues, with possible downstream effects on insulin-signaling genes involved in the normal development and reproductive viability of the worm. [Copyright &y& Elsevier]

Abstract: The differential expression of AMPA receptor subunit mRNA was analyzed in the adult rat brain and spinal cord using quantitative RT-PCR with laser capture microdissection. The expression of all four AMPA receptor subunits was demonstrable, with the mRNA expression level for GluR2 being the highest in all the brain areas and neuronal subsets examined. Both the absolute expression level of GluR2 mRNA and its expression relative to the other subunits were the lowest in motoneurons. The unique AMPA receptor expression profile of motoneurons may render them selectively vulnerable to AMPA receptor-mediated excitotoxicity. [Copyright &y& Elsevier]

In breast cancer, vascular endothelial growth factor (VEGF) is a prognostic factor, but the relationship of VEGF mRNA levels with various parameters or tumor progression is unclear. VEGF mRNA levels were measured in 48 cases of invasive ductal carcinoma by using laser capture microdissection and quantitative reverse transcription–polymerase chain reaction (RT-PCR). The mean VEGF mRNA levels were compared among different histological types and grades in 41 and 29 samples of invasive and intraductal components, respectively. VEGF mRNA levels were always higher in cancerous cells than in non-cancerous cells, but mean VEGF mRNA levels were not significantly different between invasive component (3.24 ± 3.18-fold the value of non-cancerous tissue) and intraductal component (4.14 ± 4.43-fold). They were higher in papillotubular type than in other types, and higher in grade 2 carcinomas than in grade 3 carcinomas of invasive component, and higher in comedo type than in other types of intraductal component. Mean VEGF mRNA levels were higher in the VEGF-immunopositive group than in the VEGF-immunonegative group. There was no correlation between VEGF mRNA levels and tumor size, nodal status, or hormone receptor status. VEGF expression may play an important role in the development of both invasive and intraductal carcinoma components, especially those carcinoma components of less aggressive histological features. [ABSTRACT FROM AUTHOR]

Abstract: We quantitated the copy number of mRNAs (NTG1, NTG2, OGG1, APN1, APN2, MSH2, MSH6, REV3, RAD30) encoding different DNA repair enzymes and translesion-synthesis polymerases in yeast. Quantitations reported examine how the steady-state number of each transcript is modulated in association with the growth in glucose-fermentative medium, and evaluate the respective contribution of the rate of mRNA degradation and transcription initiation to the specific mRNA level profile of each gene. Each transcript displayed a unique growth-related profile, therefore altering the relative abundance of mRNAs coding for proteins with similar functions, as cells proceed from exponential to stationary phase. Nonetheless, as general trend, they exhibited maximal levels when cells proliferate rapidly and minimal values when cells cease proliferation. We found that previous calculations on the stability of the investigated mRNAs might be biased, in particular regarding those that respond to heat shock stress. Overall, the mRNAs experienced drastic increments in their stabilities in response to gradual depletion of essential nutrients in the culture. However, differences among the mRNA stability profiles suggest a dynamic modulation rather than a passive process. As general rule, the investigated genes were much more frequently transcribed during the fermentative growth than later during the diauxic arrest and the stationary phase, this finding conciliating low steady-state levels with increased mRNA stabilities. Interestingly, while the rate at which each gene is transcribed appeared as the only determinant of the number of mRNA copies at the exponential growth, later, when cell growth is arrested, the rate of mRNA degradation becomes also a key factor for gene expression. In short, our results raise the question of how important the respective contribution of transcription and mRNA stability mechanisms is for the steady-state profile of a given transcript, and how this contribution may change in response to nutrient-availability. [Copyright &y& Elsevier]

Abstract: We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90βa, Hsp90βb, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout. [Copyright &y& Elsevier]

Abstract: The mechanisms of action of an estrogenic chemical have been examined in a viviparous fish the eelpout (Zoarces viviparus), by identification of an upregulated estrogenic pathway—the induction of hepatic estrogen receptor mRNA, hepatic estrogen binding activity and plasma vitellogenin. A relative quantitative RT-PCR assay has been established to measure hepatic estrogen receptor α (ER) mRNA levels in eelpout. Assay conditions were optimised using control and induced samples to ascertain its applicability in the actual working range of ER mRNA concentrations. β-Actin was co-amplified and used as an internal standard. Time-course effects of water exposure to 0.5 μg/L 17β-estradiol (E2) and 25 μg/L of the xeno-estrogen 4-tert-octylphenol (4-tert-OP) on ER mRNA levels in the male eelpout was examined. After 48 h of exposure, ER transcripts were induced 15-fold and 6-fold in the E2- and OP-treated fish, respectively. This difference, however, was not apparent after 1 week of exposure, when similar high levels of ER mRNA were present in both groups (20-fold induction). This indicates that the estrogenic capacity of 4-tert-OP increases with exposure time. The effect of treatment was also evaluated by examining the induction of specific E2 binding capacity in hepatic cytosolic extracts and by measuring vitellogenin in plasma. Both parameters were also induced by the treatments, but later in the time course. The measurement of ER mRNA by the RT-PCR assay showed to be the most sensitive method for the detection of estrogenic responses in eelpout. [Copyright &y& Elsevier]

Muramatsu T, Hamano H, Ogami K, Ohta K, Inoue T, Shimono M.Reduction of connexin 43 expression in aged human dental pulp.International Endodontic Journal,37, 814–818, 2004.To investigate the expression of connexin 43 (CX43) mRNA in young and old human dental pulp tissues to determine the characteristics of CX43 expression.Samples were obtained from human dental pulp of healthy young (17–23 years) and aged (>50 years) subjects. CX43 expression was determined by RT-PCR and by quantitative real-time RT-PCR (QRT-PCR). The threshold cycle (Ct) value, which reflects the amount of PCR, was calculated and the difference between value in the young pulp and that in the aged pulp was statistically analysed.RT-PCR analysis of human dental pulp tissue detected CX43 mRNA in all the samples. CX43 was abundantly expressed in young adult dental pulp, but expression of CX43 mRNA was dramatically decreased in aged human dental pulp. QRT-PCR analysis also showed the reduced expression of CX43 in aged pulp, and expression of CX43 in young pulp was significantly higher (about 10-fold,P < 0.01, Mann–WhitneyU-test).Reduction of CX43 expression may be associated with the loss of viability in human dental pulp, and is considered as one characteristic of aged pulp. [ABSTRACT FROM AUTHOR]