AR mRNAs Research Articles

Cross-talk between alpha1D-adrenoceptors and transient receptor potential vanilloid type 1 triggers prostate cancer cell proliferation.

BackgroundThere is evidence that calcium (Ca2+) increases the proliferation of human advanced prostate cancer (PCa) cells but the ion channels involved are not fully understood. Here, we investigated the correlation between alpha1D-adrenergic receptor (alpha1D-AR) and the transient receptor potential vanilloid type 1 (TRPV1) expression levels in human PCa tissues and evaluated the ability of alpha1D-AR to cross-talk with TRPV1 in PCa cell lines.MethodsThe expression of alpha1D-AR and TRPV1 was examined in human PCa tissues by quantitative RT-PCR and in PCa cell lines (DU145, PC3 and LNCaP) by cytofluorimetry. Moreover, alpha1D-AR and TRPV1 colocalization was investigated by confocal microscopy in PCa cell lines and by fluorescence microscopy in benign prostate hyperplasia (BPH) and PCa tissues. Cell proliferation was assessed by BrdU incorporation. Alpha1D-AR/TRPV1 knockdown was obtained using siRNA transfection. Signalling pathways were evaluated by measurement of extracellular acidification rate, Ca2+ flux, IP3 production, western blot and MTT assay.ResultsThe levels of the alpha1D-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha1D-AR and TRPV1 expression levels was found. Moreover, alpha1D-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens. Noradrenaline (NA) induced an alpha1D-AR- and TRPV1-dependent protons release and Ca2+ flux in PC3 cell lines; NA by triggering the activation of phospholipase C (PLC), protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways stimulated PC3 cell proliferation, that was completely inhibited by clopenphendioxan (WS433) and capsazepine (CPZ) combination or by alpha1D-AR/TRPV1 double knockdown.ConclusionsWe demonstrate a cross-talk between alpha1D-AR and TRPV1, that is involved in the control of PC3 cell proliferation. These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha1D-AR and TRPV1 channels.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-921) contains supplementary material, which is available to authorized users.

Testicular toxicity of methyl thiophanate in the Italian wall lizard (Podarcis sicula): morphological and molecular evaluation

The effects of the fungicide methyl thiophanate (MT) on testis were determined in the Italian wall lizard (Podarcis sicula) using morphological and molecular analyzes. Three experimental trials were performed: an acute test using six doses, a two-week chronic test, and "ecotoxicological" exposure (3 weeks). The minimal lethal dose (LD(50)) of pure MT, reached by the acute test, was 100 mg/kg body weight. Testicular histopathology of surviving animals showed a reduced lumen and several multinucleated giant cells 24 h after injection followed by large decreases in spermatogonia (72%) and secondary spermatocytes (58%) and a loss of spermatids and sperms 7 days after. In the chronic test, a dose equivalent to 1/100 of LD(50) was injected on alternate days. Complete shutting of the lumen and a great decrease in spermatogonia (82%) were observed. In "ecotoxicological" exposure, achieved with a commercial MT compound, testis showed a decrease in primary spermatocytes (20%) and several vacuoles. An increase in germ cell apoptosis was observed in all experimental groups using TUNEL assay. A decrease in expression of androgen and estrogen receptor (AR and ER) mRNAs was seen in all experimental groups. The reduction in AR and ER mRNAs was correlated to exposure time. Indeed, in the "ecotoxicological" treatment (30 days), the decrease reached 82 and 90% for AR and ER mRNAs, respectively. These data strongly indicate that treatment with MT, damaging the seminiferous epithelium and decreasing steroid receptor expression, might render exposed lizards infertile.

Gene expression of endothelin-1 and its receptors in the heart of broiler chickens with T 3-induced pulmonary hypertension

To investigate the effect of T 3-induced pulmonary hypertension on endothelin (ET) production and genes expression of ET-1, ET A and ET B receptors (ET AR and ET BR) during rearing, semiquantitative RT-PCR and enzyme immunometric assay were performed in the heart ventricles and serum, respectively. The ET-1 and its receptor genes were expressed in the right and left ventricles of control and T 3-treated broilers at 12, 28 and 49 days of age. There were significant ( P < 0.05) reductions of the relative amounts of ET-1 (in both ventricles) and ET AR (in the right ventricle) mRNAs at 28 and 49 days of age, in T 3-treated broilers compared to controls. The relative amounts of ET BR mRNA in the right and left ventricles did not significantly differ between control and T 3-treated broilers at any age. The serum level of ET was significantly ( P < 0.05) increased in T 3-treated chickens at 28 and 49 days of age when compared with that of the control. It is concluded that ET-1, ET AR and ET BR genes are normally expressed in the heart ventricles of broilers. It is likely that increased serum level of ET and decreased ET-1/ET AR genes expression in the ventricles are involved in the heart dysfunction of broiler chickens with developmental pulmonary hypertension.

Expression of androgen receptor mRNA in the ovary of Japanese eel, Anguilla japonica, during artificially induced ovarian development

In order to elucidate how androgens may mediate their effects on ovarian growth, we investigated the mRNA levels of two subtypes of androgen receptor ( ara and arb) in the ovary of feminized Japanese eel ( Anguilla japonica) during artificially induced ovarian development by quantitative real-time reverse transcriptase polymerase chain reaction and in situ hybridization. Ara mRNA levels were high from the late oil droplet stage to the late vitellogenic stage, whereas arb mRNA levels were high from the late oil droplet stage to the midvitellogenic stage. Both ar mRNAs were predominantly observed in the follicle cells and the epithelial cells of the ovigerous lamellae in all stages. In the oil droplet stage, oogonia exhibited intense signals for ar mRNAs. There was no obvious difference in localization pattern between ara and arb in all ovaries examined, irrespective of maturational stage. It was difficult to identify the follicle cell types that were positive for ar mRNA during ovarian development. Only in post-ovulatory follicles could theca and granulosa cells be clearly identified, and ar signals were observed in both layers. The predominant localization of ar mRNA in the follicle cells suggests that androgens play important roles in oocyte growth by acting on these cells in this species. We have shown the expression profile and localization of ar mRNA during ovarian development for the first time in an oviparous vertebrate.

Up-regulation of Endothelin-1 and Endothelin type A receptor genes expression in the heart of broiler chickens versus layer chickens

To compare Endothelin (ET) production and genes expression of ET-1 and ET A receptor (ET AR) between broiler and layer chickens during rearing, semi-quantitative RT-PCR and enzyme immunometric assay were performed in the heart ventricles and serum. There were gradual elevations of ET-1 and ET AR mRNAs in the left ventricle of broiler and layer chicken groups that were mainly significant ( P < 0.05) at 28, 35 and 42 days of age with compared to previous days whereas were not significant between two groups. These gradual elevations of ET-1 and ET AR mRNAs were also observed in the right ventricle that were significant ( P < 0.05) at 28, 35 and 42 days of age in broilers and 42 days of age in layers with compared to previous days. Increasing of these mRNAs in the right ventricle of broiler chickens were significantly ( P < 0.05) more than layer chickens at 28, 35 and 42 days. Serum ET in broilers was significantly ( P < 0.05) higher than layer chickens at 28 and 42 days of age. It is concluded that circulating ET and cardiac ET-1, ET AR genes expression is higher in broiler chickens than in layer chickens particularly after 21 days of age. It is probably that these breed differences make broiler chickens to be more susceptible to Endothelin related-cardiomyopathies such as congestive heart failure and ascites.

Abstract 3163: Posttranscriptional regulation of androgen receptor mRNA by an ErbB3 binding protein 1 (EBP1) in prostate cancer

Abstract Introduction: Regulation of AR mRNA stability and translation, two central processes that control AR expression, is little known. ErbB3 binding protein 1 (EBP1) overexpression decreases AR-target genes including AR itself at the mRNA and protein levels. Interestingly, AR protein levels were down regulated to a greater extent than mRNA levels. EBP1 has recently been demonstrated to be an RNA binding protein. We therefore examined the ability of EBP1 to regulate AR post-transcriptionally. Experimental procedure: The stability of AR mRNA was analyzed in LNCaP cells engineered with EBP1 cDNA or shRNA at different times after treatment with actinomycin D by measuring the remaining levels of AR and GAPDH mRNAs by RT-qPCR. The steady-state levels of AR mRNA and protein were also detected by quantitative RT-PCR and immunoblotting analysis. To assess the association of endogenous EBP1 and AR mRNA, LNCaP cell lysates were immunoprecipitated with antibodies recognizing EBP1 or with control IgG, followed by PCR amplification using primers specific for AR. The interaction between the biotinylated transcripts spanning a 397-nt region of the 3’UTR containing the AR UC-rich element, or CAG polyglutamine repeat, which is predicted to form a stable stem-loop structure, at the 5’-end of the coding sequence in the AR mRNA, and recombinant His-EBP1 fusion proteins was assessed by biotin pull-down assays followed by Western blot analysis. In similar way, the domain of EBP1 responsible for the binding to CAG and UC-rich sequences was mapped. To determine if EBP1 affects the ability of the UC-rich region to accelerate AR mRNA decay, a pGL3-Luc-UC plasmid was constructed by fusing the UC-rich region in the 3’UTR of AR mRNA to the 3’ end of the Firefly-luciferase coding sequence and luciferase reporter assays performed in LNCaP cells engineered with EBP1 cDNA or shRNA. To test if EBP1 may modulate not only AR stability, but also its translation, linear sucrose gradient fractionation was performed to investigate the distribution of AR mRNA associated with polysomes in LNCaP cells engineered with EBP1 cDNA or shRNA. A summary of the data: EBP1 promoted AR mRNA decay through physical interaction with a conserved UC-rich motif within the 3’-UTR of AR. The ability of EBP1 to accelerate AR mRNA decay was further enhanced by HRG treatment. EBP1 also bound to a CAG-formed stem-loop in the 5’ coding region of AR mRNA and reduced percentage of AR mRNA in a translationally active polysome pool. Conclusion: Our current work indentified EBP1 as a novel AR mRNA-binding protein regulating AR mRNA stability and translation, and suggests that targeting EBP1 has potential mechanistic and functional significance in the therapeutic management of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3163.

Expression analysis of steroid hormone receptor mRNAs during zebrafish embryogenesis

We have analyzed by qRT-PCR and/or RT-PCR the abundance and degradation rate of maternal mRNAs for nine steroid hormone receptors and their possible replacement by corresponding embryonic transcripts in both ovulated oocytes and embryos of zebrafish collected at 0, 1, 2, 4, 8, 12, 24 and 48 h post-fertilization (hpf). The mRNAs encoded the nuclear receptors for progesterone ( pr), androgen ( ar), estrogen ( erα, erβ1 and erβ2), glucocorticoids ( gr), mineralocorticoids ( mr) and the membrane progestin receptor-α and β ( mprα and β). gr mRNA was the most abundant maternal transcript in oocytes and early embryos followed by erβ2 and ar mRNAs. They declined during the first 8 hpf, being replaced, thereafter, by the embryonic messengers. erβ1 and mr transcript levels were low until 8 hpf, but increased steadily during embryonic transcription from 24 to 48 hpf. pr transcripts were detectable only in ovulated oocytes and at 24 and 48 hpf. At these stages, there was a slight increase of erα mRNA that initially was very low. mPrα and β mRNAs were expressed in ovulated oocytes and faintly persisted during the first 4 hpf. There was no subsequent embryonic expression of these transcripts. The possible involvement of maternal mRNAs for glucocorticoid and sex hormone receptors in the programming of early zebrafish development is intriguing, since they mainly occur at stages in which gene replication predominates over transcription.

Expression of α-AR Subtypes in T Lymphocytes and Role of the α-ARs in Mediating Modulation of T Cell Function

Objectives: Previous work in our laboratory has shown that α-adrenoreceptors (α-ARs) and β-ARs exist on lymphocytes from functional profile, and that the receptors mediate the regulation of lymphocyte function by catecholamines. In the present study, we directly examined the expression of α-AR subtypes, α₁-AR and α₂-AR mRNAs, in T lymphocytes and explored the roles of the α-AR subtypes and intracellular signal transduction mechanisms linked to the receptors in mediating the modulation of T lymphocyte function. Methods: T lymphocytes from mesenteric lymph nodes of rats were purified by using a nylon wool column. Reverse transcription polymerase chain reaction was used to detect the expression of α₁-AR and α₂-AR mRNAs in the freshly isolated T cells and the mitogen concanavalin A (Con A)-activated lymphocytes. Colorimetric methylthiazoletetrazolium assay was employed to measure lymphocyte proliferation induced by Con A. Interferon-γ (IFN-γ) and interleukin-4 (IL-4) levels in the Con A-stimulated lymphocyte culture supernatants were examined by enzyme-linked immunosorbent assay. Results: T cells expressed both α₁-AR and α₂-AR mRNAs. The expression of both α₁-AR and α₂-AR mRNAs was significantly higher in the Con A-activated lymphocytes than in the resting lymphocytes. Phenylephrine, a selective α₁-AR agonist, had no evident effect on lymphocyte proliferation nor on IFN-γ and IL-4 production induced by Con A. However, the selective α₂-AR agonist clonidine attenuated Con A-induced lymphocyte proliferation as well as IFN-γ and IL-4 production. The inhibited lymphocyte proliferation and IFN-γ and IL-4 production by clonidine were blocked by yohimbine, an α₂-AR antagonist. Either phospholipase C inhibitor U-73122 or protein kinase C inhibitor chelerythrine partially prevented the suppressive effect of clonidine on Con A-stimulated lymphocyte proliferation and IL-4 production. Conclusions: T lymphocytes express both α₁-ARs and α₂-ARs, but only the α₂-ARs participate in the suppressive modulation of lymphocyte proliferation and cytokine production in vitro. The inhibitory effect of α₂-AR stimulation on lymphocyte function is partially mediated via the phospholipase C-protein kinase C pathway.

Prostaglandin E2 stimulates expression of osmoprotective genes in MDCK cells and promotes survival under hypertonic conditions

The cells of the renal medulla produce large amounts of prostaglandin E2 (PGE2) via cyclooxygenases (COX)-1 and -2. PGE2 is well known to play a critical role in salt and water balance and maintenance of medullary blood flow. Since renal medullary PGE2 production increases in antidiuresis, and since COX inhibition is associated with damage to the renal medulla during water deprivation, PGE2 may promote the adaptation of renal papillary cells to high interstitial solute concentrations. To address this question, MDCK cells were exposed to a gradual tonicity increase in the presence or absence of 20 microM PGE2 prior to analysis of (i) cell survival, (ii) expression of osmoprotective genes (AR, BGT1, SMIT, HSP70 and COX-2), (iii) subcellular TonEBP/NFAT5 abundance, (iv) TonEBP/NFAT5 transcriptional activity and (v) aldose reductase promoter activity. Cell survival and apoptotic indices after raising the medium tonicity improved markedly in the presence of PGE2. PGE2 significantly increased tonicity-mediated up-regulation of AR, SMIT and HSP70 mRNAs. However, neither nuclear abundance nor TonEBP/NFAT5-driven reporter activity were elevated by PGE2, but aldose reductase promoter activity was significantly increased by PGE2. Interestingly, tonicity-induced COX-2 expression and activity was also stimulated by PGE2, suggesting the existence of a positive feedback loop. These results demonstrate that the major medullary prostanoid, PGE2, stimulates the expression of osmoprotective genes and favours the adaptation of medullary cells to increasing interstitial tonicities, an effect that is not explained directly by the presence of TonEs in the promoter region of the respective target genes. These findings may be relevant in the pathophysiology of medullary damage associated with analgesic drugs.

β3‐Adrenoceptors in urinary bladder

The beta-adrenoceptor (AR) is currently classified into beta(1), beta(2), and beta(3) subtypes. A third subtype, beta(3)-AR, was first identified in adipose tissue, but has also been identified in smooth muscle tissue, particularly in the gastrointestinal tract and urinary bladder smooth muscle. There is a predominant expression of beta(3)-AR messenger RNA (mRNA) in human bladder, with 97% of total beta-AR mRNA being represented by the beta(3)-AR subtype and only 1.5 and 1.4% by the beta(1)-AR and beta (2)-AR subtypes, respectively. Moreover, the presence of beta(1)-, beta(2)-, and beta(3)-AR mRNAs in the urothelium of human bladder has been identified. The distribution of beta-AR subtypes mediating detrusor muscle relaxation is species dependent, the predominant subtype being the beta(3)-AR in humans. Recent studies have suggested that cAMP-dependent routes are not exclusive mechanisms triggering the beta-AR-mediated relaxation of smooth muscle. It has been demonstrated in rats detrusor muscle that cAMP plays a greater role in beta-adrenergic relaxation against basal tone than against KCl-induced tone and that conversely calcium-activated K(+) channels (BKca channels) play a greater role under the latter circumstances. In rat models, beta(3)-AR agonists increase bladder capacity without influencing bladder contraction and have only weak cardiovascular side effects. Although this evidence points toward the clinical utility of beta(3)-AR agonists as therapy for overactive bladder (OAB), pharmacological differences exist between rat and human beta(3)-ARs. Development of compounds with high selectivity for the human beta(3)-AR, identified by screening techniques using cell lines transfected with the human beta(1)-, beta(2)-, and beta(3)-AR genes, may mitigate against such problems. The association between the tryptophan 64 arginine polymorphism in the beta(3)-AR gene and idiopathic OAB is discussed.

Expression of Estrogen Receptor ESR1 and Its 46-kDa Variant in the Gubernaculum Testis1

Testicular descent corresponds to migration of the testis from the abdominal cavity to the scrotum and is essential for proper functioning of the testis. Recent advances in the characterization of estrogen receptor (ESR) subtypes and isoforms in various tissues prompted us to study ESRs within the gubernaculum testis, a structure involved in testicular descent. In the rat gubernaculum, we searched for ESR alpha (Esr1) and beta (Esr2) and for the androgen receptor (Ar), androgens being known to regulate testicular descent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that Esr1, Esr2, and Ar mRNAs were all expressed in the gubernaculum. Using PEETA (Primer extension, Electrophoresis, Elution, Tailing, and Amplification), we established that all Esr1 leader exons, previously identified in other organs, such as the uterus and pituitary, were transcribed in the gubernaculum, with the major form being O/B. The RNA protection assays, RT-PCR, and Western blot experiments revealed that isoform-specific mRNA transcripts generated by alternative splicing of the C-leader sequence on coding exons 1 and 2 of the Esr1 gene gave the 46- and 66-kDa ESR1 proteins. The ESR1 and AR proteins were found to colocalize in the parenchymal cells of the gubernaculum early in development, whereas AR also was strongly expressed in the muscular cells, both during fetal and postnatal life. The ESR2 protein was weakly expressed, principally in the muscular cells, but only once testicular descent had occurred. The levels of the 46-kDa ESR1 variant (ER46) exceeded those of the 66-kDa ESR1 form (ER66) at periods when the gubernaculum developed. Conversely, the 66-kDa form appears to predominate clearly when the gubernaculum growth was low or completed. The possible role of estrogens on the modulation of the androgen-dependent growth of the gubernaculum and, more widely, on testicular descent is discussed.

Effects of perinatal exposure to flutamide on sex hormone responsiveness in F1 male rats.

Pregnant rats were administered flutamide (0 and 10 mg/kg, p.o.) from gestation Day 14 to post-parturition Day 3 and effects on responsiveness to androgens (testosterone propionate, TP; dihydrotestosterone, DHT) in male offspring were examined with a Hershberger assay. Male pups of each group were assigned to 6 subgroups as follows: Group 1, castration and euthanized at postnatal Day 46 (PND 46); Group 2, castration + vehicle; Group 3, castration + TP; Group 4, castration + DHT; Group 5, vehicle; Group 6, DHT. After castrations were conducted at PND 36, animals were treated with TP (2 mg/kg in corn oil, s.c.) or DHT (1.25 mg/kg in corn oil, s.c.) once a day for 10 days, beginning at PND 46. At PND 56, the following organs/tissues were removed and weighed: ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, levator ani muscle plus bulbocavernosus muscle, Cowper's gland, and glands penis. Analysis of serum testosterone, LH and FSH in Groups 2, 3, 4, 5 and 6, and RT-PCR using prostate tissue from Groups 2, 3 and 4 were carried out. Perinatal exposure to flutamide caused decreased weights of androgen-dependent organs. Responses to androgens were recognized in organs of all castrated groups, with increased organ weights, especially in animals administered TP where values were essentially equal to or greater than those of intact animals in both the control and the 10 mg/kg group. On the other hand, the degree of weight increase of the ventral prostate and seminal vesicles with TP or DHT treatment in castrated animals was smaller in the flutamide administration group than in the controls. In hormone assays, castrated + vehicle animals showed higher serum LH than the other groups. Serum FSH was high in the castrated groups (Group 2>Group 4>Group 3), while in the noncastrated group a constant level was noted, with or without flutamide. No effect of flutamide administration was observed regarding sex hormone. RT-PCR using ventral prostate tissue revealed no significant differences in expression of AR, C3, VEGF, TGF-beta1, beta2, KGF and CK8 mRNA after androgen treatment between the control and flutamide treatment groups. C3 mRNA was increased in androgen-treated animals, whereas AR, TGF-beta and KGF mRNAs were decreased. Perinatal exposure to anti-androgen causes irreversible abnormalities in male pups. Concerning the responsiveness to TP and DHT, the degrees of weight changes in ventral prostate and seminal vesicles in castrated animals were decreased. However, the other organ weights, the sex hormone levels and androgen-reactive gene expression in the ventral prostate were not influenced by perinatal flutamide treatment in the present study.

Relationship between intracellular ionic strength and expression of tonicity-responsive genes in rat papillary collecting duct cells.

Intracellular ionic strength may play an important role in regulating the expression of genes encoding osmolyte-accumulating molecules. To establish whether a strict relation exists between these variables, intracellular ionic strength (sum of Na+, Cl- and K+ concentrations) and the relative abundance of mRNA derived from various tonicity-sensitive genes was examined using electron microprobe analysis and Northern blots on primary cultures of rat papillary collecting duct (PCD) cells following acute or long-term alterations in medium tonicity. Hypertonic medium (450 mosmol kg(-1)) evoked an initial rise in intracellular ionic strength (269 +/- 5 vs. 194 +/- 7 mmol (kg wet weight (wt))(-1) in isotonic controls; means +/- S.E.M.), which subsequently declined gradually, and a significantly higher abundance of bgt1 (Na+- and Cl- -dependent betaine transporter), smit (Na+/myo-inositol cotransporter), ar (aldose reductase) and osp94 (osmotic stress protein 94) mRNAs. Conversely, exposure to hypotonic medium (200 mosmol kg(-1)) for 12 h was associated with significantly reduced intracellular ionic strength (153 +/- 4 mmol (kg wet wt)(-1)) and significantly reduced the abundance of smit and ar mRNAs. PCD cells preconditioned in hypotonic medium and re-exposed to isotonic medium showed significantly higher abundance of these mRNAs than isotonic controls, although the intracellular ionic strength did not differ. Two further tonicity-sensitive genes responded differently to medium tonicity: while the abundance of hsp70 (heat shock protein 70) mRNA increased significantly following both hypo- and hypertonic stress, inos (inducible nitric oxide synthase) mRNA abundance correlated inversely with medium tonicity. These findings support the view that the effect of intracellular ionic strength on the expression of bgt1, smit, ar and osp94 is modulated by additional factors such as cell volume, and that its effect on the pathways regulating hsp70 and inos is even more complex.

Real-time RT-PCR for the Detection of Beta-adrenoceptor Messenger RNAs in Small Human Endomyocardial Biopsies

Quantification of mRNAs from extremely small human samples remains a challenge. Requiring minimal amounts of tissue and no post-reaction manipulation, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is an attractive method to quantitatively assess the expression of rare mRNAs. We evaluated the applicability of the technique on RNA extracted from human endomyocardial biopsies and isolated cardiomyocytes, and compared the technique to the RT-competitive PCR approach. Primers and probes were designed to amplify the three subtypes of human β -adrenoceptors (β 1-,β 2- and β 3 AR), as well as reference genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hypoxanthine-guanine phosphoribosyltransferase (HPRT), and the oncogene ABL by real-time RT-PCR. Specific primers and a deleted competitor were synthetized to compare the quantitation of the β 3 AR mRNA expression by RT-competitive PCR. We validated the technique on human cardiomyocytes either freshly isolated or selectively excised from fixed sections of human myocardium by Laser Capture Microdissection. The standard curves obtained for the cDNA's analysed showed mean slopes comprised between −3.3 and −3.7. Inter- and intra-assay variablility of gene quantitation was reflected by mean values of the variance coefficients of Ct of 4.84±1.13% and 2.73±0.39% or 3.32±1.03% and 2.21±0.24% (corresponding to percent variances of copy numbers of 83.07±12.72% and 34.45±9.03% or 47.40±8.59% and 23.83±3.16%) for human β 3 AR and GAPDH genes, respectively. The expression of GAPDH, HPRT and ABL mRNA was characterized by a very low dispersion of individual values across cardiac pathologies, suggesting that these genes may be used as reference genes in quantitative PCR studies. Finally, we applied the technique to detect rare mRNAs, such as β -AR mRNAs, from small human endomyocardial biopsies and even isolated cardiomyocytes. Real-time RT-PCR is appropriate to quantitate rare messenger RNAs, including in extremely small human tissue samples. This method appears very promising for futures studies of gene expression in several pathophysiological conditions, including heart failure.

Occurrence of androgen and estrogen receptor mRNAs in the Harderian gland: A comparative survey

In Rana esculenta the presence of an androgen receptor in both the male and female Harderian gland (HG) has been demonstrated. Hybridization analysis has evidenced a high degree of homology between the rat androgen receptor cDNA and the frog androgen receptor mRNA (fARmRNA). Correspondingly the molecular size of fARmRNA is similar to those described in mammals (9.4 kb). In in vivo experiments testosterone (T) increases the levels of fARmRNA. The use of the antiandrogen alone or in combination with T prevents the increase of fARmRNA. In the control animals a loss of fARmRNA has been observed. In primary cultures of HG cells, the steady-state levels of fARmRNA increase in the cells exposed to T. These results suggest that T exerts an autoinduction on its own receptor, increasing the levels of fARmRNA. In Xenopus laevis the HG shows a sexual dimorphism of the protein pattern. The female shows two major proteins (210 and 180 kDa). Administration of estradiol to the male shifts the protein pattern into the female one. In this respect an estrogen receptor mRNA (ERmRNA) has been found in the female gland and can be induced in the male one. No ARmRNA has been detected in either sexes. A similar sex dimorphism has been found in Gallus domesticus. The female pattern is characterised by a protein fraction of about 210 kDa, the male one by a protein fraction of about 180 kDa. In 4-day-old chicks no sex differences have been found. An ERmRNA is expressed in the female, while no ARmRNA has been detected in both sexes. Neither AR nor ER mRNAs have been detected in the chick HG. Among mammals the HG of the hamster (Mesocricetus auratus) shows an androgen-dependent sex dimorphism. In in vitro experiments T 10−12 M induces a onefold increase of ARm-RNA with respect to unexposed cells. This effect reaches its maximum (4.4-fold) when cells are exposed to T 10−8 M. The size of the hamster ARmRNA is similar to that observed in other mammals (9.5 kb). The above results suggest that in the HG the phenomenon of autoinduction occurs and that there is a relationship between the androgen or estrogen dependence of the HG and the digamety of the species. © 1996 Wiley-Liss, Inc.

Molecular analysis of the armadillo locus: uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene.

During Drosophila embryogenesis, the segment polarity genes are required for the formation of specific pattern domains within each segment. Mutations in the armadillo (arm) gene primarily affect the posterior part of the segment and lead to the production of anterior structures within this region. To examine the molecular basis for these effects, we have cloned the arm region and identified the gene by germ-line transformation. The arm gene produces two types of very abundant 3.2-kb transcripts that differ only in their first exons. These RNAs appear to be formed by independent transcriptional initiation but have similar patterns of expression throughout development. Both arm transcripts are present in virtually all of the cell types contained in embryos, third-instar larvae, and adult ovaries, suggesting that arm may be required in all cells. In addition, the arm transcripts are uniformly distributed in embryonic segments, so the regional pattern defects associated with its embryonic phenotype may result from interactions between arm and other localized factors. Both arm RNAs encode the same 91-kD polypeptide. This protein has no probable secretory or membrane-spanning regions and contains a series of novel internal repeats that are conserved in sequence, length, and spacing. Considering these results and previous genetic observations, we discuss potential roles for the arm gene in pattern formation processes.