Growth Hormone mRNA Research Articles

Enhancement of transfection efficiency with NLS and SPB-NLS

Low positive cell screening efficiency severely hinders the development of transgenic animals. The major rate-limiting step of positive cell screening is DNA entering the nucleus, particularly for large DNA molecules. To enhance the transport of large DNA molecules into the nucleus, particularly for the production of transgenic animals, nuclear localization sequence (NLS) peptides and the peptide derivative succinimidyl-[4-(psoralen-8-yloxy)]-butyrate (SPB)-NLS were synthesized to mediate transfection in vitro. To investigate the function of NLS and SPB-NLS in vitro, the expression levels of growth hormone (GH) mRNA and green fluorescent protein (GFP) protein were analyzed following transfection mediated by NLS and SPB-NLS. The results demonstrated that the expression of GH mRNA was significantly higher in the NLS (increased by 69%) and SPB-NLS (330%) groups than that in the liposome/pGN group. Similarly, GFP expression was found to be higher in the SPB-NLS group than that in the liposome group, while the expression in the NLS group was lower than that in the liposome group. Further analysis demonstrated that SPB-NLS enhanced the expression of insulin-like growth factor 1 in hard-to-transfect goat mammary epithelia cells. The results of the microscopy analysis revealed that transfected DNA entered the nucleus via the nuclear pores, facilitated by NLS. Analysis of the cell cycle demonstrated that the cytotoxic effects of NLS and SPB-NLS were low. In conclusion, the results of the present study demonstrate that SPB-NLS acts as a transfection-enhancing agent and may be used both to enhance nuclear delivery and for the development of genetically modified animals.

DNA methylation of pituitary growth hormone is involved in male growth superiority of Nile tilapia ( Oreochromis niloticus )

Growth hormone (GH) and its receptors are critical regulators of somatic growth and metabolism. It has been shown in mammals that the methylation of cytosines within the GH promoter plays a key role in regulating transcripts expression. In the present study, the GH, GHR1 and GHR2 proximal promoters were identified and the methylation levels of these genes in corresponding tissues were assayed. The results suggested that significant arising of GH putative promoter methylation levels in pituitary was observed in females compared with males. However, no such sex-specific changes were found in GHR1 and GHR2 promoters. The GH mRNA expression also was influenced by GH promoter methylation levels in pituitary, which resulted in the higher growth rate of Nile tilapia males. Meanwhile, the methylation levels of GH putative promoter were negatively correlated with growth rate as well as mRNA expression of GH. Furthermore, the methylation of specific E-Box CpG site is also negatively related to the mRNA expression of GH in pituitary. Taken together, our data provide an epigenetic mechanism of explicating the sex duality in phenotypic plasticity of growth rate in male and female of Nile tilapia.

Construction and functional study of pGN, a mammary gland-specific expression plasmid.

The aim of this study was to construct a mammary gland-specific expression vector, pGN, and to validate its function in expressing growth hormone (GH) both in vitro and in vivo. First, the GH gene was amplified and inserted downstream of the b-casein 5'-arm. Next, the neo gene was cloned downstream of the b-casein 3'-arm as a selection marker. To analyze the bioactivity of the pGN plasmid, we expressed pGN in a Bcap-37 cell line and in the goat mammary gland. Quantitative PCR analysis revealed that the expression of GH mRNA in the pGN-transfected group was higher than that of the control group in Bcap-37 cells. Results of a radioimmunoassay and an enzyme-linked immunosorbent assay demonstrated that the pGN-transfected group expressed much more GH protein than the non-transfected group (P < 0.05). Upon injection of the pGN plasmid into the goat mammary gland, GH mRNA and growth hormone receptor mRNA expressions increased 2-fold. In vivo analyses revealed that GH protein expression was higher in the injected group than in the control group. Together, these results strongly demonstrated that the pGN plasmid was constructed correctly and exhibited favorable bioactivity in efficiently expressing GH both in vitro and in vivo.

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma.

Establishment and characterization of dairy cow growth hormone secreting anterior pituitary cell model

A dairy cow anterior pituitary cell (DCAPC) model was established in vitro for the study of growth hormone (GH) synthesis and secretion in the anterior pituitary gland of the dairy cow. Pituitary glands were obtained from Holstein dairy cows' heads cut by electric saw, and the posterior pituitary glands were removed to obtain integrated anterior pituitary glands. Immunohistochemistry assay of GH in the anterior pituitary glands showed that most somatotrophs were located within the lateral wings of the anterior pituitary. Tissues of the lateral wings of the anterior pituitary were dispersed and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The DCAPCs displayed a monolayer, cobblestone, epithelial-like morphology which are the typical characteristics of the anterior pituitary cells. The DCAPCs were subcultured continuously over ten passages. GH immunoreactivity was present in DCAPCs at passage 10. The transcription of the bovine GH mRNA in DCAPCs at passage 10 was decreased to below 50% compared with the lateral wings of the anterior pituitary tissues. Thus, our DCAPCs model is effective for the in vitro examination of GH synthesis and secretion in the dairy cow anterior pituitary gland. The effects of transforming growth factor beta 1 (TGF-β1) and interferon-γ (IFN-γ) on the expression of GH mRNA in DCAPCs at passage 3 were also investigated. There were no obvious changes in transcription of the GH gene after treatment with TGF-β1 for 24 h, while IFN-γ increased transcription of the GH gene in a dose-dependent manner.

Expression and function of chicken bursal growth hormone (GH)

Growth hormone (GH) has several effects on the immune system. Our group has shown that GH is produced in the chicken bursa of Fabricius (BF) where it may act as an autocrine/paracrine modulator that participates in B-cell differentiation and maturation. The time course of GH mRNA and protein expression in the BF suggests that GH may be involved in development and involution of the BF, since GH is known to be present mainly in B lymphocytes and epithelial cells. In addition, as GH is anti-apoptotic in other tissues, we assessed the possibility that GH promotes cell survival in the BF. This work focused on determining the mechanism by which GH can inhibit apoptosis of B cells and if the PI3K/Akt pathway is activated. Bursal cell cultures were treated with a range of GH concentrations (0.1–100nM). The addition of 10nM GH significantly increased viability (16.7±0.6%) compared with the control and decreased caspase-3 activity to 40.6±6.5% of the control. Together, these data indicate that GH is produced locally in the BF and that the presence of exogenous GH in B cell cultures has antiapoptotic effects and increases B cell survival, probably through the PI3k/Akt pathway.

Glucocorticoid-induced changes in gene expression in embryonic anterior pituitary cells

Within the anterior pituitary gland, glucocorticoids such as corticosterone (CORT) provide negative feedback to inhibit adrenocorticotropic hormone secretion and act to regulate production of other hormones including growth hormone (GH). The ontogeny of GH production during chicken embryonic and rat fetal development is controlled by glucocorticoids. The present study was conducted to characterize effects of glucocorticoids on gene expression within embryonic pituitary cells and to identify genes that are rapidly and directly regulated by glucocorticoids. Chicken embryonic pituitary cells were cultured with CORT for 1.5, 3, 6, 12, and 24 h in the absence and presence of cycloheximide (CHX) to inhibit protein synthesis. RNA was analyzed with custom microarrays containing 14,053 chicken cDNAs, and results for selected genes were confirmed by quantitative reverse transcription real-time PCR (qRT-PCR). Levels of GH mRNA were maximally induced by 6 h of CORT treatment, and this response was blocked by CHX. Expression of 396 genes was affected by CORT, and of these, mRNA levels for 46 genes were induced or repressed within 6 h. Pathway analysis of genes regulated by CORT in the absence of CHX revealed networks of genes associated with endocrine system development and cellular development. Eleven genes that were induced within 6 h in the absence and presence of CHX were identified, and eight were confirmed by qRT-PCR. The expression profiles and canonical pathways defined in this study will be useful for future analyses of glucocorticoid action and regulation of pituitary function.

Comparative breast cancer research, lessons from companion animals.

Comparative breast cancer research, lessons from companion animals.

Neuronostatin alters gene expression in rat anterior pituitary cells: a possible role in energy balance and glucose homeostasis

Neuronostatin is a recently discovered peptide hormone derived from the somatostain preprohormone (JBC 287:4572, 2008). We now have identified that the orphan G protein‐coupled receptor, GPR107, is a potential cognate receptor for neuronostatin (AJP 303:R941, 2012). Because of the abundant expression of GPR107 in anterior pituitary cells, and because neuronostatin is present in somatostatin‐producing neurons of the hypothalamus that project to median eminence, we hypothesized that neuronostatin would alter hormone production in anterior pituitary. The ability of neuronostatin to activate early gene expression in pituitary gland further suggested trophic effects; however, we were initially unable to detect acute effects of the peptide on basal, stimulated or inhibited hormone secretion in cells in culture. Therefore, anterior pituitary cells were isolated from male rats, treated for longer time periods with neuronostatin, and changes in hormone gene expression were examined using real‐time polymerase chain reaction. We found that neuronostatin activated early gene expression and induced an increase in growth hormone mRNA levels, suggesting that neuronostatin may regulate growth hormone production, which could have important implications for energy homeostasis and glucose metabolism in addition to possible direct effects in the pancreas where the peptide also activates early gene expression. (NIH HL066023)

In vitro Suppression of Prolactin During Later Stages of Egg Lay in Domestic Hen (Gallus gallus Domesticus) Anterior Pituitcytes by RNA Interference

2 Abstract: Prolactin (PRL) is a peptide hormone secreted by anterior pituitary gland. PRL increases as per the age and reproductive cycle of birds from normal physiological levels to extremely higher level there by affecting the ovulation, egg formation, oviposition, egg production and hyperprolactinemia. This is mor e pronounced and persistent after 72 weeks of age in birds. Hence a study was conducted in anterior pituitary primary cultured cells obtained from 72 and 82 weeks old white leg horn (WLH) hens to knock down the PRL gene expression by siRNA and observing its effects on PRL, PRL mRNA, protein content of PRL, prolactin receptor (PRLR) and Growth Hormone (GH) mRNA to unravel the functional role of PRL at 72 and 82 week age by siRNA for PRL. Three siRNAs were designed as per standard siRNA protocols and studied the suppression of PRL gene expression in primary cultured cells procured from adult chicken anterior pituitary glands in in vitro conditions. Average percentage reduction of PRL in anterior pituitary primary cell culture following siRNA transfection was 82 and 60% at 72 and 82 weeks respectively. Protein content of PRL was significantly (p<0.01) decreased in siRNA transfected cells compared to controls. Growth Hormone (GH) mRNA and PRL receptor (PRLR) mRNA levels did not change significantly (p<0.01) between control and treated cells. Results clearly suggested that the siRNA designed for PRL specifically decreased PRL gene expression in in vitro conditions. Level of PRLR mRNA and GH mRNA levels expression did not follow the similar pattern of PRL gene expression in anterior pituitary cells. It is concluded that, construction of short specific siRNA for PRL significantly decreased PRL, PRL mRNA and protein content of PRL without showing any effect on PRLR and GH between the two age groups of birds. These results may lead to construction of short specific siRNA for stable and chronic suppression of PRL gene expression during embryogenesis before an increase of PRL occurs for long term knock down of PRL in in vivo conditions. In conclusion , understanding of hyperprolactinemia and the involvement of PRL may provide the basis for the development of therapeutic drugs or methods against hyperprolactinemia by RNA interference.

Effect of glycerol on GHR and IGF-1 gene expression in breast muscle and on growth of Japanese meat quails

We evaluated messenger RNA (mRNA) expression of the growth-hormone (GHR) and insulin-like growth factor (IGF-1) genes in 28-day-old Japanese meat quails fed diets containing 0, 8, or 12% dietary glycerol in substitution of corn. Total RNA was extracted from the breast muscle and the DNA was amplified with specific primers using real-time PCR. Feed conversion ratio and feed intake were evaluated. The birds fed 8 and 12% glycerol presented higher IGF-1 mRNA expression [0.059 and 0.049 arbitrary units (AU), respectively] relative to those not fed with glycerol (0.029 AU), while 12% glycerol reduced GHR mRNA expression (0.022 AU). Dietary inclusion of 8% glycerol promoted similar performance results (feed conversion) as the diet with no glycerol. We conclude that inclusion of glycerol in the diet affects GHR and IGF-1 gene expression in Japanese meat quails. However, considering the performance results and the expression of the GHR and IGF-1 genes, 8% glycerol may be safely included in the diet of meat quails.

Progesterone Receptor Isoforms in the Mammary Gland of Cats and Dogs

Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed.

Insulin-Like Growth Factor 1 Mediates Negative Feedback to Somatotroph GH Expression via POU1F1/CREB Binding Protein Interactions

Circulating insulin-like growth factor 1 (IGF-1) has been shown to act as a negative feedback regulator of growth hormone (GH) gene expression; however, the mechanism of this negative feedback is poorly understood. Activation and regulation of GH gene expression require the binding of the transcription factor POU1F1 to the GH promoter along with cyclic AMP (cAMP) response element binding protein (CREB) binding protein (CBP). We investigate the role of CBP as a target of IGF-1 somatotroph regulation using the MtT/S somatotroph cell line. IGF-1 significantly inhibits basal GH mRNA levels but not POU1F1 levels. Chromatin immunoprecipitation assays demonstrate inhibition of CBP binding to the GH promoter after IGF-1 treatment. We hypothesized that IGF-1 receptor (IGF-1R) signaling disrupts the POU1F1/CBP complex to inhibit gene expression. In support, the use of a mutant CBP (S436A) construct, which lacks a critical phosphorylation site, leads to the loss of IGF-1 inhibition. The studies of CBP (S436A) knock-in mice show elevated serum GH levels, a greater response to GH releasing hormone (GHRH) stimulation along with lower weight gain, and decreased body fat. Our data confirm the inhibitory effects of IGF-1 on GH expression at the level of the promoter and provide evidence of CBP's role as a target of IGF-1R signaling.

Effects of cysteamine on mRNA levels of growth hormone and its receptors and growth in orange-spotted grouper (Epinephelus coioides)

Effects of cysteamine (CS) on growth hormone (GH) mRNA, two types of growth hormone receptor (GHR) mRNAs and growth rate in orange-spotted grouper (Epinephelus coioides) were investigated. CS could cause a modification in the structure of somatostatin, which is the most important neuroendocrine inhibitor of basal and stimulated growth hormone synthesis and release, and renders it nonimmunoreactive probably through interaction with the disulfide bonds. In the present study, cysteamine hydrochloride (CSH) enhanced the level of pituitary GH mRNA in a dose-dependent manner through attenuating or deleting the inhibiting action of somatostatin on GH mRNA expression. CSH at relatively low doses (from 1 to 3mg/g diet) enhanced the levels of two types of GHR mRNAs in dose-dependent manner, whereas the stimulation induced by CSH declined from the peak at higher dose of CSH (4mg/g diet). It might be attributed to the variation in GH-induced up-regulation of GHRs at different doses of GH. Feeding of CSH could induce remarkable enhancement of growth rate in orange-spotted grouper. In addition, the stimulatory effect of CSH could be potentiated by the additive effect of luteinizing hormone-releasing hormone analog (LHRH-A). Compared with individual treatments, combined feeding of CSH and LHRH-A caused more efficient elevation of growth rate after 8weeks of feeding. CSH and LHRH-A individually and in combination remarkably increased the levels of GH and GHR mRNAs compared with the control. The combined administration of CSH and LHRH-A in diet was most effective to enhance the level of GH and GHR1 mRNA. The morphological characteristics of the experimental fish were evaluated. Compared with control, the ratios of muscle RNA/DNA, condition factors (CF) and feed conversion efficiency (FCE) were significantly enhanced in the treated groups, while the highest values were observed in the combined treatment. All the results suggested that CSH (1-3mg/g diet) is an effective, economical and feasible feed additive in orange-spotted grouper culture.

Profiles of mRNA expression for prolactin, growth hormone, and somatolactin in Japanese eels, Anguilla japonica: The effect of salinity, silvering and seasonal change

For understanding the functions of the growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family of hormones, we examined pituitary mRNA expression of these hormones in anguillid eels in relation to salinity difference, silvering, and seasonal change. Female Japanese eels (Anguilla japonica) were collected in the brackish Hamana Lake and its freshwater rivers from July to December. To clarify the effect of salinity, the habitat use history of the eels were determined using otolith microchemistry. Expression levels of mRNA of each hormone were determined using real time PCR. Although GH and PRL have been known to be osmoregulatory hormones, there were no consistent differences in expression levels of these hormones between different salinity habitats. In contrast, SL mRNA expression was higher in eels from freshwater rivers than from the brackish lake. GH mRNA expression clearly decreased during silvering, whereas PRL and SL mRNA expression did not change. We also showed that PRL mRNA and SL mRNA decreased in the brackish lake and PRL mRNA increased in freshwater rivers from autumn to early winter. These findings provide basic knowledge for a further understanding of the role of these hormones.

OR07-2 Simultaneous stimulation of human 20 kDa growth hormone and 22 kDa growth hormone secretion by ghrelin

The World Anti Doping Agency has banned the use of recombinant human growth hormone (rhGH). Regardless, rogue athletes may use rhGH in pursuit of improved performance. Current immunoassaybased detection of illicit use of rhGH is complicated by the short half-life of injected rhGH, by the chemical identity of rhGH and a native growth hormone (GH), and by the physiologically high peaks of GH that occur with the pulsatile secretion of GH by a normal pituitary. We hypothesized that injection of rhGH changes the expression patterns of GHmessenger RNA (mRNA) in circulating white blood cells and of microRNA (miRNA) in plasma. Objective: To measure circulating GH mRNA and miRNAs in humans. Methods: We used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) with nested primers to detect and quantify GHmRNA.We usedmiRNAmicroarrays to identify rhGH-dependent miRNA expression and then measured the concentrations of selected miRNAs by RT-qPCR. Following Institutional Review Board approval, we obtained whole-blood or plasma, from 9 healthy volunteers and from 36 patient volunteers. All patients had known or suspected disorders of growth hormone production including panhypopituitarism treated with physiologic doses of rhGH (0.2 to 0.8mg/day) and acromegaly. Results: GH mRNA was quantified in blood from all healthy volunteers and patients with disorders of GH at concentrations ranging from 36 to 4815 copies/100 mL blood. The intra-individual GH mRNA concentration varied less than 3-fold in 17 samples collected over the period of one year in a healthy, athletic volunteer; however, variability increased markedly in the group with pituitary disorders. Initially, 9 specimens from: 3 patients receiving therapeutic growth hormone replacement, 4 patients with acromegaly, and 2 normal controls, were subject to plasma-derived miRNA microarrays. Of 1105 miRNA’s measured in each microarray, 3 were present in plasma of all rhGH replacement users and undetectable in all patients with acromegaly and normal controls. Conclusions: The narrow intra-individual range of its concentration suggests that GH mRNA in blood is not present as a result of pulsatile secretion and thus is worthy of study as a marker of doping with supraphysiologic amounts of rhGH. Further investigation of miRNA in plasma should be performed on samples obtained before and after administration of supraphysiologic amounts of rhGH to confirm the preliminary results in patients receiving therapeutic replacement doses of rhGH.

OR07-1 Circulating RNAs as markers of growth hormone doping

The World Anti Doping Agency has banned the use of recombinant human growth hormone (rhGH). Regardless, rogue athletes may use rhGH in pursuit of improved performance. Current immunoassaybased detection of illicit use of rhGH is complicated by the short half-life of injected rhGH, by the chemical identity of rhGH and a native growth hormone (GH), and by the physiologically high peaks of GH that occur with the pulsatile secretion of GH by a normal pituitary. We hypothesized that injection of rhGH changes the expression patterns of GHmessenger RNA (mRNA) in circulating white blood cells and of microRNA (miRNA) in plasma. Objective: To measure circulating GH mRNA and miRNAs in humans. Methods: We used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) with nested primers to detect and quantify GHmRNA.We usedmiRNAmicroarrays to identify rhGH-dependent miRNA expression and then measured the concentrations of selected miRNAs by RT-qPCR. Following Institutional Review Board approval, we obtained whole-blood or plasma, from 9 healthy volunteers and from 36 patient volunteers. All patients had known or suspected disorders of growth hormone production including panhypopituitarism treated with physiologic doses of rhGH (0.2 to 0.8mg/day) and acromegaly. Results: GH mRNA was quantified in blood from all healthy volunteers and patients with disorders of GH at concentrations ranging from 36 to 4815 copies/100 mL blood. The intra-individual GH mRNA concentration varied less than 3-fold in 17 samples collected over the period of one year in a healthy, athletic volunteer; however, variability increased markedly in the group with pituitary disorders. Initially, 9 specimens from: 3 patients receiving therapeutic growth hormone replacement, 4 patients with acromegaly, and 2 normal controls, were subject to plasma-derived miRNA microarrays. Of 1105 miRNA’s measured in each microarray, 3 were present in plasma of all rhGH replacement users and undetectable in all patients with acromegaly and normal controls. Conclusions: The narrow intra-individual range of its concentration suggests that GH mRNA in blood is not present as a result of pulsatile secretion and thus is worthy of study as a marker of doping with supraphysiologic amounts of rhGH. Further investigation of miRNA in plasma should be performed on samples obtained before and after administration of supraphysiologic amounts of rhGH to confirm the preliminary results in patients receiving therapeutic replacement doses of rhGH.

Structural and functional characterization of pituitary adenylyl cyclase-activating polypeptide (PACAP)/PACAP-related peptide (PRP) and its receptor in olive flounder (Paralichthys olivaceus)

We identified full-length cDNAs encoding pituitary adenylyl cyclase-activating polypeptide (PACAP), PACAP-related peptide (PRP), and PACAP-specific receptor (PAC1R) from olive flounder, Paralichthys olivaceus. Two variant mRNA forms were created by alternative splicing. Comparison of genomic and cDNA sequences of the PRP-PACAP precursor revealed that skipping of exon 4 within PRP resulted in two variant transcripts: a long form encoding both PRP and PACAP and a short form encoding PACAP only. Both transcripts were constitutively observed only in the brain, whereas the short form appeared in gut tissues, such as the intestine and pyloric cecum in fish challenged with a pathogen, but not in healthy fish. Furthermore, expression of the long PRP/PACAP transcript gradually increased in the intestine of flounder challenged with bacteria, suggesting that PRP and/or PACAP may serve as a regulator(s) of the immune system, especially in the gastrointestinal tract of olive flounder. The biological functions of PACAP and PRP were investigated by exogenous treatment of flounder embryogenic cells (hirame natural embryonic cells, HINAE cells) with synthetic peptides of fPACAP-38 and/or fPRP-45. Intracellular cyclic adenosine monophosphate (cAMP) production in PAC1R-overexpressing HINAE cells was regulated by fPACAP-38 in a concentration-dependent manner, but was not regulated by fPRP-45. Results from real-time quantitative polymerase chain reaction revealed that PAC1R mRNA was specifically induced by fPACAP-38 but not by fPRP-45; PACAP significantly increased TNF-α mRNA but not growth hormone (GH) mRNA in HINAE cells; however, PRP affected GH but not TNF-α mRNA expression. These results suggest that the expression ratio of PRP and PACAP is regulated at the transcriptional level depending on the tissues and conditions, and that the unique biological roles of PRP and PACAP differ from that of mammalian PRP.

Enhancement of gene transfer efficiency in the Bcap-37 cell line by dimethyl sulphoxide and menthol

Simple and efficient gene transfer into the nucleus would facilitate non-viral gene delivery. One promising method of non-viral gene delivery is to apply penetration enhancers. Chemicals, such as dimethyl sulfoxide (DMSO) and menthol, may have promise as non-toxic vehicles in improving gene transfer efficiency. In this study, the cytotoxic effects of DMSO and menthol were evaluated using MTT assays. Gene delivery efficiency in a human breast cancer cell line (Bcap-37) was investigated by quantitative PCR, fluorescence microscopy and flow cytometry. Non-toxic concentrations of DMSO (2%) and menthol (12.5µM) enhanced the efficiency of liposome-mediated gene delivery in Bcap-37 cells. Quantitative PCR results showed that growth hormone (GH) mRNA expression in the post-menthol and pre-DMSO treatment groups was 10-fold higher compared to that in the liposome group, while in the pre-menthol and post-DMSO treatment groups, a 30-fold increase in GH mRNA expression was observed. Both DMSO and menthol treatments increased green fluorescent protein (GFP) expression efficiency as shown by fluorescence microscopy experiments. Compared to the liposome group, the number of positive cells in the pre-menthol and post-DMSO treatment groups was significantly increased by 15%. Furthermore, cell cycle analysis demonstrated that there were significant differences among the DMSO-treated group, the menthol-treated group and the normal group, which implied different effects of DMSO and menthol treatments. In conclusion, both non-toxic and harmless DMSO (2%) and menthol (12.5µM) treatments improve gene transfer efficiency, while post-DMSO treatment may be the most effective protocol in increasing transgene expression efficiency.

Quantification of growth hormone mRNA in blood

BackgroundGrowth hormone (GH) is present in human lymphocytes and the relative concentration of GH mRNA in circulating cells is reportedly increased in patients with acromegaly. No quantitative assay has been described to measure GH in circulating cells. MethodsTo measure GH mRNA, we used real-time reverse-transcriptase (RT) quantitative PCR (qPCR) with nested primers, a synthetic nucleotide standard and SYBR Green I detection. Blood samples from volunteers with and without pituitary disorders were collected in PAXgene Blood RNA tubes. Essential MIQE guidelines were followed during sample preparation and assay development. ResultsRT-qPCR assay was specific for GH mRNA and was linear from ≤90 to ≥90,000 copies of GH mRNA per reaction. Day-to-day imprecision (CV) was 31%–46% at mean concentrations of 3400 and 2630 copies/mL of blood, respectively (n=10). The variability (CV) of results in samples collected from a single individual over a year and assayed in a single run was 28% (n=17 samples). The mean concentrations of GH mRNA in blood were statistically indistinguishable in patients with acromegaly, control subjects, and patients receiving replacement doses of recombinant GH. Moreover, GH mRNA concentrations in blood were not correlated with concentrations of insulin-like growth factor-I in plasma. ConclusionsThe RT-PCR assay quantifies GH mRNA at concentrations present in circulating cells. The present study suggests that the absolute concentration of GH mRNA in circulating cells is not altered in patients with acromegaly.