Regulatory Peptides最新文献

Dipeptidyl peptidases (DPPs) are proteolytic enzymes that regulate many physiological systems by degrading signaling peptides. DPP8 and DPP9 are distinct from DPP4 in sequence, cellular localization and expression levels, thus implying distinct functions. However, DPP8 and DPP9 expression needs further delineation. We evaluated DPP4, DPP8 and DPP9 expression using three independent methods at the mRNA, protein, and functional levels to better understand the local physiological contribution of each enzyme. Sprague Dawley rats and cynomolgus monkeys were selected for DPP4, DPP8 and DPP9 expression profiling to represent animal species commonly utilized for drug preclinical safety evaluation. A novel Xhibit assay of DPP protease activity was applied in addition to newly available antibodies for immunohistochemical localization. This combined approach can facilitate a functional evaluation of protease expression, which is important for understanding physiological relevance. Few inter-species differences were observed. Tissue mRNA and protein levels generally correlated to functional DPP4 and DPP8/9 enzymatic activity. All three proteins were seen in epithelial cells, lymphoid cells and some endothelial and vascular smooth muscle cells. Combined DPP8/DPP9 enzymatic activity was uniformly intracellular across tissues at approximately 10-fold lower levels than non-renal DPP4. Consistent levels of each DPP were detected among most non-renal tissues in rats and monkeys. DPP4 was ubiquitous, principally detected on cell membranes of epithelial and endothelial cells and was greatest in the kidney. The expression patterns suggest that DPP8 and DPP9 may act similarly across tissues, and that their actions might in part overlap with DPP4.

Ghrelin is an important gastrointestinal hormone involved in the regulation of feeding in both mammals and fish. In this study, the preproghrelin cDNA sequence was cloning in the gut of Schizothorax prenanti (S. prenanti). The preproghrelin gene, encoding 103-amino acids, was strongly expressed in the gut and brain using real-time quantitative RT-PCR (qPCR). The S. prenanti preproghrelin was detected in embryonic developmental stages. Further, it was detectable in unfertilized eggs, suggesting that ghrelin could be classified as maternal mRNA. An experiment was conducted to determine the expression profile of ghrelin during post-feeding and fasting status of the brain and gut. The results revealed a significant postprandial decrease in ghrelin mRNA expression in the gut 6 h post-feeding (hpf) and brain (1.5 and 9 hpf) compared to an unfed control group, indicating that food intake and processing affect the regulation of expression of ghrelin in S. prenanti. The constructed recombinant plasmid pMD-19 T-ghrelin was transformed to Escherichia coli BL21 and induced with IPTG, and the expressed product was identified by SDS-PAGE. The prokaryotic expression vector for ghrelin was constructed successfully, and fusion protein was expressed in E. coli BL21, which laid the foundation for the further study on the function of this protein and its mechanism. Overall, our results provide evidence for a highly conserved structure and biological actions of ghrelin in S. prenanti. Further studies are required to identify the tissue specific functions of ghrelin in S. prenanti.

The bursa of Fabricius (BF) is the acknowledged central humoral immune organ unique to birds which plays important roles in B cell development and antibody production. Little information on immunomodulatory functions of BF is reported, except for several reported active bursal-derived peptides. Three peptides were identified and characterized from BF through RP-HPLC and MADIL-TOF methods. They are named as bursal peptide (BP)-I, BP-II, BP-III. These peptides promoted CFU pre-B formation and decreased PU.1 expression. The different immunomodulatory activities of these three bursal peptides on antibody and cytokine productions were verified by the immunization comparative experiment. The results showed the three bursal peptides enhanced AIV-specific antibody and cytokine production, T-cell immunophenotyping at reachable concentrations. These results indicate the important orientations for the comprehensive understanding of the humoral central immune system, and provide a novel insight on new experimental reagents for immuno-adjuvant or immunopharmacological.

Neuropeptide Y (NPY) is a peptide found in the brain and autonomic nervous system, which is associated with anxiety, depression, epilepsy, learning and memory, sleep, obesity and circadian rhythms. NPY has recently gained much attention as an endogenous antiepileptic and antidepressant agent, as drugs with antiepileptic and/or mood-stabilizing properties may exert their action by increasing NPY concentrations, which in turn can reduce anxiety and depression levels, dampen seizures or increase seizure threshold.

We have used human neuroblastoma SH-SY5Y cells to investigate the effect of valproate (VPA) and amitriptyline (AMI) on NPY expression at therapeutic plasma concentrations of 0.6 mM and 630 nM, respectively. In addition, 12-O-tetradecanoylphorbol-13-acetate (TPA) known to differentiate SH-SY5Y cells into a neuronal phenotype and to increase NPY expression through activation of protein kinase C (PKC) was applied as a positive control (16 nM). Cell viability after drug treatment was tested with a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. NPY expression was measured using immunofluorescence and quantitative RT-PCR (qRT-PCR). Results from immunocytochemistry have shown NPY levels to be significantly increased following a 72 h but not 24 h VPA treatment. A further increase in expression was observed with simultaneous VPA and TPA treatment, suggesting that the two agents may increase NPY expression through different mechanisms. The increase in NPY mRNA by VPA and TPA was confirmed with qRT-PCR after 72 h. In contrast, AMI had no effect on NPY expression in SH-SY5Y cells.

Together, the data point to an elevation of human NPY mRNA and peptide levels by therapeutic concentrations of VPA following chronic treatment. Thus, upregulation of NPY may have an impact in anti-cancer treatment of neuroblastomas with VPA, and antagonizing hypothalamic NPY effects may help to ameliorate VPA-induced weight gain and obesity without interfering with the desired central effects of VPA.

Objective

To determine the effect of gestational hypertension on the developmental origins of blood pressure (BP), altered kidney gene expression, salt-sensitivity and cardiac hypertrophy (CH) in adult offspring.

Methods

Female mice lacking atrial natriuretic peptide (ANP −/−) were used as a model of gestational hypertension. Heterozygous ANP +/− offspring was bred from crossing either ANP +/+ females with ANP −/− males yielding ANP +/−^WT offspring, or from ANP −/− females with ANP +/+ males yielding ANP +/−^KO offspring. Maternal BP during pregnancy was measured using radiotelemetry. At 14 weeks of age, offspring BP, gene and protein expression were measured in the kidney with real-time quantitative PCR, receptor binding assay and ELISA.

Results

ANP +/−^KO offspring exhibited normal BP at 14 weeks of age, but displayed significant CH (P < 0.001) as compared to ANP +/−^WT offspring. ANP +/−^KO offspring exhibited significantly increased gene expression of natriuretic peptide receptor A (NPR-A) (P < 0.001) and radioligand binding studies demonstrated significantly reduced NPR-C binding (P = 0.01) in the kidney. Treatment with high salt diet increased BP (P < 0.01) and caused LV hypertrophy (P < 0.001) and interstitial myocardial fibrosis only in ANP +/−^WT and not ANP +/−^KO offspring, suggesting gestational hypertension programs the offspring to show resistance to salt-induced hypertension and LV remodeling. Our data demonstrate that altered maternal environments can determine the salt-sensitive phenotype of offspring.

Endogenous daily rhythms are generated by the hierarchically organized circadian system predominantly synchronized by the external light (L): dark (D) cycle. During recent years several humoral signals have been found to influence the generation and manifestation of daily rhythm. Since most studies have been performed under in vitro conditions, the mechanisms employed under in vivo conditions need to be investigated. Our study focused on angiotensin II (angII)-mediated regulation of Per2 expression in the suprachiasmatic nuclei (SCN) and heart and spontaneous locomotor activity in Wistar rats under synchronized conditions. Angiotensin II was infused (100 ng/kg/min) via subcutaneously implanted osmotic minipumps for 7 or 28 days. Samples were taken in 4-h intervals during a 24 h cycle and after a light pulse applied in the first and second part of the dark phase. Gene expression was measured using real time PCR. Locomotor activity was monitored using an infrared camera with a remote control installed in the animal facility. Seven days of angII infusion caused an increase in blood pressure and heart/body weight index and 28 days of angII infusion also increased water intake in comparison with controls. We observed a distinct daily rhythm in Per2 expression in the SCN and heart of control rats and infused rats. Seven days of angII infusion did not influence Per2 expression in the heart. 28 days of angII treatment caused significant phase advance and a decrease in nighttime expression of Per2 and influenced expression of clock controlled genes Rev-erb alpha and Dbp in the heart compared to the control. Four weeks of angII infusion decreased the responsiveness of Per2 expression in the SCN to a light pulse at the end of the dark phase of the 24 h cycle. Expression of mRNA coding angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) showed a daily rhythm in the heart of control rats. Four weeks of angII infusion caused a decrease in amplitude of rhythmic expression of Ace, the disappearance of rhythm and an increase in Ace2 expression. The Ace/Ace2 ratio showed a rhythmic pattern in the heart of control rats with peak levels during the dark phase. Angiotensin II infusion decreased the mean Ace/Ace2 mRNA ratio in the heart. We observed a significant daily rhythm in expression of brain natriuretic peptide (BNP) in the heart of control rats. In hypertensive rats mean value of Bnp expression increased. Locomotor activity showed a distinct daily rhythm in both groups. Angiotensin II time dependently decreased ratio of locomotor activity in active versus passive phase of 24 h cycle. To conclude, 28 days of subcutaneous