Cloning and Sequence Analysis on cDNA of Growth Hormone Releasing Hormone Receptor Gene from Wuzhishan Miniature Pig

[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.

Gastric motor effects of ghrelin and growth hormone releasing peptide 6 in diabetic mice with gastroparesis

AIM:To investigate the potential therapeutic significance of ghrelin and growth hormone releasing peptide 6 (GHRP-6) in diabetic mice with gastric motility disorders. METHODS: A diabetic mouse model was established by intraperitoneal (ip) injection of alloxan. Diabetic mice were injected ip with ghrelin or GHRP-6 (20-200 μg/kg), and the effects on gastric emptying were measured after intragastric application of phenol red. The effect of atropine, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) or D-Lys3-GHRP-6 (a growth hormone secretagogue receptor (GHS-R) antagonist) on the gastroprokinetic effect of ghrelin or GHRP-6 (100 μg/kg) was also investigated. The effects of ghrelin or GHRP-6 (0.01-10 μmol/L) on spontaneous or carbachol-induced contractile amplitude were also investigated in vitro, in gastric fundic circular strips taken from diabetic mice. The presence of growth hormone secretagogue receptor 1a transcripts in the fundic strips of diabetic mice was detected by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: We established a diabetic mouse model with delayed gastric emptying. Ghrelin and GHRP-6 accelerated gastric emptying in diabetic mice with gastroparesis. In the presence of atropine or L-NAME, which delayed gastric emptying, ghrelin and GHRP-6 (100 μg/kg) failed to accelerate gastric emptying. D-Lys3-GHRP-6 also delayed gastric emptying induced by the GHS-R agonist. Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in gastric fundicstrips taken from diabetic mice. RT-PCR confirmed the presence of GHS-R mRNA in the strip preparations. CONCLUSION: Ghrelin and GHRP-6 increase gastric emptying in diabetic mice with gastroparesis, perhaps by activating peripheral cholinergic pathways in the enteric nervous system.

Synthesis of large FeSe superconductor crystals via ion release/introduction and property characterization

Large superconducting Fe Se crystals of（001） orientation have been prepared via a hydrothermal ion release/introduction route for the first time. The hydrothermally derived Fe Se crystals are up to 10 mm×5 mm×0.3 mm in dimension. The pure tetragonal FeSe phase has been confirmed by x-ray diffraction（XRD） and the composition determined by both inductively coupled plasma atomic emission spectroscopy（ICP-AES） and energy dispersive x-ray spectroscopy（EDX）. The superconducting transition of the Fe Se samples has been characterized by magnetic and transport measurements. The zero-temperature upper critical field H（c2） is calculated to be 13.2–16.7 T from a two-band model. The normal-state cooperative paramagnetism is found to be predominated by strong spin frustrations below the characteristic temperature T（sn）, where the Ising spin nematicity has been discerned in the FeSe superconductor crystals as reported elsewhere.

Effect of growth hormone-releasing peptide on cardiac cholinergic nerve fiber density distribution in a rat model of heart failure

BACKGROUND： Changes in the cardiac autonomic nerve are considered to be important factors in the mechanisms of heart failure. It is possible to reduce or slow down nerve degeneration and necrosis, provided that patients take effective neuroprotectants during the early stages of heart failure. Moreover, it is possible to relieve the pathological process and reduce the risk of death. OBJECTIVE： To study the effect of growth hormone releasing peptide （GHRP） on cardiac cholinergic nerve fiber density distribution in a rat model of heart failure, and verify whether GHRP can ameliorate denervation. DESIGN, TIME AND SETTING： A randomized controlled study was performed at the Key Laboratory of Anatomy, Harbin Medical University, between June and October 2009. MATERIALS： Fifty adult, healthy, female, Wistar rats, weighing （200± 20） g, were randomly divided into GHRP （n = 30）, model （n = 10）, and sham operation （n = 10） groups. GHRP-2 was made in Shanghai, China （batch No. z071212-03）. METHODS： Acute myocardial infarction was established by ligating the left anterior descending coronary artery in the GHRP and model groups. Five weeks later, myocardial function was detected using color ultrasound electrocardiograph a successful marker of chronic heart failure models Ejection fraction 〈 60% was considered to be However, the left anterior descending coronary artery was not ligated in the sham operation group. The GHRP group was injected with 100 μ g/kg GHRP-2, and the other two groups were injected with the same volume of physiological saline, once per day. MAIN OUTCOME MEASURES： After 4 weeks, pathological changes in cardiac cholinergic nerve fibers were detected under optic microscopy following hematoxylin/eosin staining. In addition, density distribution was measured using a multi-function color pathological image system. RESULTS： In the sham operation group, myocardial cells were regular, uniformly stained, and no inflammatory cells were present. In the model group, myocardial cells were unevenly stained, exhibited nuclear atrophy, degeneration, dissolution, or disappearance. In the GHRP group, myocardial damage was less than in the model group; cardiac muscle fibers exhibited slight degeneration. The myocardium in the sham operation group was serried, spreading the cholinergic innervations along the cardiac fiber. In the model group, there was a decreased number of cholinergic nerve fibers decreased, which also became shorter and smaller, compared with the sham operation group （P 〈 0.01）. In the GHRP group, cholinergic positive nerve fibers were significantly increased compared with the model group （P 〈 0.01）, but still less than the sham surgery group （P 〈 0.05）. CONCLUSION： GHRP delayed denervation and reduced nerve reconstitution following heart failure in rats.

TPA Enhances Growth Horm one (GH) Secretion Effect ofGH-Releasing Horm one (GHRH) by Hum an gsp-PositivePi-tuitary Som atotrophinom as

In recent years, one of the most exciting advances in the researches of pituitary adenomas is the discovery that 30 %-40 % of human pituitary somatotrophinomas carry somatic mutations of the gene for the α subunit of the stimulatory GTP binding protein, G s (G sα). These mutations, termed gsp oncogenes, may play an important role in the tumorigenesis of pituitary adenomas. Of 10 somatotrophinomas examined, 3 (30 %) were proved to be gsp positive, as determined by sequence analysis of DNA generated by the polymerase chain reaction (PCR). GHRH exerted a significant stimulatory effect on GH secretion in 2 of 3 gsp positive and 4 of 7 gsp negative tumors. Moreover, phorbol ester, 1, 2 tetradecanoylphorbol 13 acetate (TPA), enhanced stimulation of lated the GH secretion effect exerted by GHRH in gsp positive somatotrophinomas, whereas this effect was not observed in gsp negative tumors. This result suggests that the protein kinase C signal system as well as adenylyl cyclase cAMP protein kinase A intracellular signal transduction system plays a pivotal role in GH secretory control of GHRH, which may work together via a cross talk mechanism.

Targeted therapy in advanced metastatic colorectal cancer: Current concepts and perspectives

The introduction of new cytotoxic substances as well as agents that target vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) signaling has improved clinical outcome of patients with metastatic colorectal cancer (mCRC). In this review we summarize the most relevant clinical data on VEGF and EGFR targeting regimens in mCRC. The effects of available treatment strategies for mCRC are often temporary, with resistance and disease progression developing in most patients. Thus, new treatment strategies are urgently needed. Some GI peptides including gastrin and gastrin releasing peptide, certain growth factors such as insulin-like growth factor-I and II and neuropeptides such as growth hormone releasing hormone (GHRH) are implicated in the growth of CRC. Experimental investigations in CRC with antagonistic analogs of bombesin/gastrin-releasing peptide, GHRH, and with cytotoxic peptides that can be targeted to peptide receptors on tumors, are summarized in the second part of the review.

Bioactive hydrogel microcapsules for guiding stem cell fate decisions by release and reloading of growth factors

Human pluripotent stem cells(hPSC)hold considerable promise as a source of adult cells for treatment of diseases ranging from diabetes to liver failure.Some of the challenges that limit the clinical/translational impact of hPSCs are high cost and difficulty in scaling-up of existing differentiation protocols.In this paper,we sought to address these challenges through the development of bioactive microcapsules.A co-axial flow focusing microfluidic device was used to encapsulate hPSCs in microcapsules comprised of an aqueous core and a hydrogel shell.Importantly,the shell contained heparin moieties for growth factor(GF)binding and release.The aqueous core enabled rapid aggregation of hPSCs into 3D spheroids while the bioactive hydrogel shell was used to load inductive cues driving pluripotency maintenance and endodermal differentiation.Specifically,we demonstrated that one-time,1 h long loading of pluripotency signals,fibroblast growth factor(FGF)-2 and transforming growth factor(TGF)-β1,into bioactive microcapsules was sufficient to induce and maintain pluripotency of hPSCs over the course of 5 days at levels similar to or better than a standard protocol with soluble GFs.Furthermore,stem cell-carrying microcapsules that previously contained pluripotency signals could be reloaded with an endodermal cue,Nodal,resulting in higher levels of endodermal markers compared to stem cells differentiated in a standard protocol.Overall,bioactive heparin-containing core-shell microcapsules decreased GF usage five-fold while improving stem cell phenotype and are well suited for 3D cultivation of hPSCs.

The Role of Protein Kinase C and Its Effect on GHRH in the Regulation of Hormone Secretion by Somatotrophinomas

Summary: Phorbol ester-induced release of growth hormone (GH) and prolactin (PRL) from hu- man somatotrophic tumors was examined in vitro. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) strongly stimulated GH and PRL secretion and showed an additive effect on GH secretion if used in combination with GH releasing hormone (GHRH). In contrast, staurosporine exerted a variable inhibitory effect on GH release. There was no correlation between such effects and gsp mutations. The findings suggested that TPA doesn't act directly through cAMP signal transduction system.

THE ROLE OF CALCIUM ION IN THE PATHOGENESIS OF HUMAN PITUITARY GH－SECRETING ADENOMAS

To study the role of Ca2+ in the pathogenesis of pituitary growth hormone secreting adenomas, the function of Ca2+ in 23 cases of human Prturtary GH-secreting adenoma was investigated in monolayer cell culture. It was found that Ca2+ channel blockers nicardipin and nifedipin inhibrted basal and growth hormone releasing hormone (GRH)stimulated GH secretion in 87. 5 % and 100. 0 % of the GH adenomas . respectively, demonstrating that in most human pituitary GH adenomas, the basal and GRH regulated GH secretion is Ca2+ dependent. The GRH and sometostatin (SRIF) agonist octreotide regulated the processes of GH secretion via Ca2+ had defects in different steps including receptor ,postreceptor Ca2+ channel and Ca2+GH secreting coupling in 6 (66. 6%) and 5 (55. 5 % ) cases of 9 GH adenomas respectively. Among them,the defects in GRH receptor and SRIF regulated Ca2+ channel are the main causes of the dysfunction of GH adenomas. These defects may be related to GH hypersecretion in GH adenomas. Our data provides advance evidences for intrinsic defects of GH adenomas.

Therapeutic effects of ghrelin and growth hormone releasing peptide 6 on gastroparesis in streptozotocin-induced diabetic guinea pigs in vivo and in vitro

Background Diabetic gastroparesis is a disabling condition with no consistently effective treatment. In normal animals, both ghrelin and its synthetic peptide, growth hormone releasing peptide 6 （GHRP-6）, increase gastric emptying. Thus, we investigated the potential therapeutic significance of ghrelin and GHRP-6 in diabetic guinea pigs with gastric motility disorders. Methods A diabetic guinea pig model was produced by intraperitoneal （i.p.） injection of streptozotocin （STZ, 280 mg/kg）. Diabetic guinea pigs were injected i.p. with ghrelin or GHRP-6 （10-100 μg/kg）, and the effects on gastric emptying were measured after intragastric application of phenol red. The effect of atropine or a growth hormone secretagogue receptor （GHS-R） antagonist, D-Lys^3-GHRP-6, on the gastroprokinetic effects of ghrelin or GHRP-6 （100 μg/kg） was also investigated. Further, the in vitro effects of ghrelin or GHRP-6 （0.01-10 μmol/L） on spontaneous or carbachol-induced contractile amplitude in gastric fundic circular strips taken from diabetic guinea pigs were examined. Growth hormone secretagogue receptor transcripts in the fundic strips of diabetic guinea pigs were detected by reverse transcriptase polymerase chain reaction （RT-PCR）. Results We established a guinea pig model of delayed gastric emptying. Ghrelin （20, 50, or 100 μg/kg） and GHRP-6 （20, 50, or 100 μg/kg） accelerated gastric emptying in diabetic guinea pigs with gastroparesis （n=6, P 〈0.05）. In the presence of atropine, which delayed gastric emptying, ghrelin and GHRP-6 （100 μg/kg） failed to accelerate gastric emptying （n=6, P 〈0.05）. D-Lys^3-GHRP-6 also delayed gastric emptying induced by the GHS-R agonist （n=6, P 〈0.05）. Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in gastric fundic strips taken from diabetic guinea pigs （n=6, P〈0.05）. RT-PCR confirmed the presence of GHS-R mRNA in the strip preparations. Conclusions Ghrelin and GHRP-6 increased gastric emptying in diabetic guinea pigs with gastroparesis, potentially, by activating the peripheral cholinergic pathways in the enteric nervous system.

Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-β_1 delivery for cartilage tissue engineering

Background Natural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-β1 （TGF-β1） to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-β1 delivery and the effect of released TGF-β1 on the chondrogenic potential of chondrocytes. Methods Chitosan scaffolds and chitosan microspheres loaded with TGF-β1 were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-β1 microspheres and incubated in vitro for 3 weeks. Histological examination and type Ⅱ collagen immunohistochemical staining was performed to evaluate the effects of released TGF-β1 on cell adhesivity, proliferation and synthesis of the extracellular matrix. Results TGF-β1 was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-β1 was high （90.1%）. During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-β1 could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-β1 was 37.4%, 50.7%, 61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type II collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type II in control group （32.7%± 10.4%） was significantly lower than that in the controlled TGF-β1 release group （92.4%±4.8%, P〈0.05）. Both the proliferation rate and production of collagen type Ⅱ in the transforming growth factor-β1 microsphere incorporated scaffolds were significantly higher than those in the scaffolds without microspheres, indicating that the activity of TGF-β1 was retained during microsphere fabrication and after growth factor release. Conclusion Chitosan microspheres can serve as delivery vehicles for controlled release of TGF-β1, and the released growth factor can augment chondrocytes proliferation and synthesis of extracellular matrix. Chitosan scaffolds incorporated with chitosan microspheres loaded with TGF-β1 possess a promising potential to be applied for controlled cytokine delivery and cartilage tissue engineering.