Metabolic stability, receptor binding, cAMP generation, insulin secretion and antihyperglycaemic activity of novel N-terminal Glu(9)-substituted analogues of glucagon-like peptide-1

Glucagonlike peptide-1(7 36)amide (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. Rapid removal of the Nterminal dipeptide, His7-Ala8, by the ubiquitous enzyme dipeptidyl peptidase IV (DPP IV) curtails the biological activity of GLP-1. Chemical modifications or substitutions of GLP-1 at His7 or Ala8 improve resistance to DPPIV action, but this often reduces potency. Little attention has focused on the metabolic stability and functional activity of GLP-1 analogues with amino acid substitution at Glu9, adjacent to the DPP IV cleavage site. We generated three novel Glu9-substituted GLP-1 analogues, (Pro9)GLP-1, (Phe9)GLP-1 and (Tyr9)GLP-1 and show for the first time that Glu9 of GLP-1 is important in DPP IV degradation, since replacing this amino acid, particularly with proline, substantially reduced susceptibility to degradation. All three novel GLP-1 analogues showed similar or slightly enhanced insulinotropic activity compared with native GLP-1 despite a moderate 4 10-fold reduction in receptor binding and cAMP generation. In addition, (Pro9)GLP 1 showed significant ability to moderate the plasma glucose excursion and increase circulating insulin concentrations in severely insulin resistant obese diabetic (ob/ob) mice. These observations indicate the importance of Glu9 for the biological activity of GLP-1 and susceptibility to DPP IVmediated degradation.

Enhanced cAMP generation and insulin-releasing potency of two novel Tyr(1)-modified enzyme-resistant forms of glucose-dependent insulinotropic polypeptide is associated with significant antihyperglycaemic activity in spontaneous obesity-diabetes

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin hormone, which potentiates glucose-induced insulin secretion. Antihyperglycaemic actions of GIP provide significant potential in Type 11 diabetes therapy. However, inactivation of GIP by the enzyme dipeptidyl peptidase IV (DPP IV) and its consequent short circulating half-life limit its therapeutic use. Therefore two novel Tyr(1)-Modified analogues of GIP, N-Fmoc-GIP (where Fmoc is 9-fluorenylmethoxycarbonyl) and N-palmitate-GIP, were synthesized and tested for metabolic stability and biological activity. Both GIP analogues were resistant to degradation by DPP IV and human plasma. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, both analogues exhibited a 2-fold increase in cAMP-generating potency compared with native GIP (EC50 values of 9.4, 10.0 and 18.2 nM respectively). Using clonal BRIN-BD11 cells, both analogues demonstrated strong insulinotropic activity compared with native GIP (P <0.01 to P <0.001). In obese diabetic (ob/ob) mice, administration of N-Fmoc-GIP or N-palmitate-GIP (25 nmol/kg) together with glucose (18 mmol/kg) significantly reduced the peak 15 min glucose excursion (1.4- and 1.5-fold respectively; P <0.05 to P <0.01) compared with glucose alone. The area under the curve (AUC) for glucose was significantly lower after administration of either analogue compared with glucose administered alone or in combination with native GIP (1.5-fold; P <0.05). This was associated with a significantly greater AUC for insulin (2.1-fold; P <0.001) for both analogues compared with native GIP. A similar pattern of in vivo responsiveness was evident in lean control mice. These data indicate that novel N-terminal Tyr(1) modification of GIP with an Fmoc or palmitate group confers resistance to degradation by DPP IV in plasma, which is reflected by increased in vitro potency and greater insulinotropic and antihyperglycaemic activities in an animal model of Type 11 diabetes mellitus.

GLP-1 and related peptides cause concentration-dependent relaxation of rat aorta through a pathway involving K_ATP and cAMP

increasing evidence from both clinical and experimental studies indicates that the insulin-releasing hormone, glucagon-like peptide-1 (GLP-1) may exert additional protective/reparative effects on the cardiovascular system. The aim of this study was to examine vasorelaxant effects of GLP-1(7-36)amide, three structurally-related peptides and a non-peptide GLP-1 agonist in rat aorta. Interestingly, all GLP-1 compounds, including the established GLP-1 receptor antagonist, exendin (9-39) caused concentration-dependent relaxation. Mechanistic studies employing hyperpolarising concentrations of potassium or glybenclamide revealed that these relaxant effects are mediated via specific activation of ATP-sensitive potassium channels. Further experiments using a specific membrane-permeable cyclic AMP (cAMP) antagonist, and demonstration of increased cAMP production in response to GLP-1 illustrated the critical importance of this pathway. These data significantly extend previous observations suggesting that GLP-1 may modulate vascular function, and indicate that this effect may be mediated by the GLP-1 receptor. However, further studies are required in order to establish whether GLP-1 related agents may confer additional cardiovascular benefits to diabetic patients. (c) 2008 Elsevier Inc. All rights reserved.

cAMP/PKA-dependent increases in Ca Sparks, oscillations and SR Ca stores in retinal arteriolar myocytes after exposure to vasopressin.

PURPOSE: To investigate the effects of arginine vasopressin (AVP) on Ca(2+) sparks and oscillations and on sarcoplasmic reticulum (SR) Ca(2+) content in retinal arteriolar myocytes. METHODS: Fluo-4-loaded smooth muscle in intact segments of freshly isolated porcine retinal arteriole was imaged by confocal laser microscopy. SR Ca(2+) store content was assessed by recording caffeine-induced Ca(2+) transients with microfluorimetry and fura-2. RESULTS: The frequencies of Ca(2+) sparks and oscillations were increased both during exposure to, and 10 minutes after washout of AVP (10 nM). Caffeine transients were increased in amplitude 10 and 90 minutes after a 3-minute application of AVP. Both AVP-induced Ca(2+) transients and the enhancement of caffeine responses after AVP washout were inhibited by SR 49059, a V(1a) receptor blocker. Forskolin, an activator of adenylyl cyclase, also persistently enhanced caffeine transients. Rp-8-HA-cAMPS, a membrane-permeant PKA inhibitor, prevented enhancement of caffeine transients by both AVP and forskolin. Forskolin, but not AVP, produced a reversible, Rp-8-HA-cAMPS insensitive reduction in basal [Ca(2+)](i). CONCLUSIONS: AVP activates a cAMP/PKA-dependent pathway via V(1a) receptors in retinal arteriolar smooth muscle. This effect persistently increases SR Ca(2+) loading, upregulating Ca(2+) sparks and oscillations, and may favor prolonged agonist activity despite receptor desensitization.

Exchange protein directly activated by cAMP 1 (Epac1) is expressed in human neutrophils and mediates cAMP-dependent activation of the monomeric GTPase Rap1

Epac1 and Epac2 bind cAMP and mediate cAMP-dependent activation of Rap1. cAMP is produced in neutrophils in response to many chemoattractants. This second messenger plays a key role in the regulation of the functions of neutrophils. However, it is still not known whether Epacs are expressed in human neutrophils. We found that stimulation of PLB-985 cells differentiated into neutrophil-like cells, human neutrophils with 8CPT-2Me-cAMP (a selective activator of Epacs), or FK (a diterpene that augments the intracellular level of cAMP) led to GTP-loading of Rap1. Epac1 mRNA was expressed in UND and DF PLB-985 cells, but Epac1 protein was only detected in DF PLB-985 cells. In human neutrophils, the Epac1 transcript was present, and Epac1 protein could be detected by Western blot analysis if the cells had been treated with the serine protease inhibitor PMSF. FK induced adhesion of PLB-985 cells and human neutrophils on fibrinogen, a ligand for beta 2 integrins. Interestingly, in DF PLB-985 cells, but not in human neutrophils, 8CPT-2Me-cAMP induced beta 2 integrin-dependent adhesion. The failure of 8CPT-2Me-cAMP to induce beta 2 integrin-dependent human neutrophil adhesion could be explained by the fact that this compound did not induce a switch of the beta 2 integrins from a low-affinity to a high-affinity ligand-binding conformation. We concluded that Epac1 is expressed in human neutrophils and is involved in cAMP-dependent regulation of Rap1. However, the loading of GTP on Rap1 per se is not sufficient to promote activation of beta 2 integrins. J. Leukoc. Biol. 90: 741-749; 2011.

The Other Side of the Fence:Reconceptualizing the ���Camp��� and Migration Zones at the Borders of Spain

This article explores the dynamics of the space of exception at the borders of Europe in the Spanish enclave of Melilla, and the neighboring Moroccan city of Oujda. Building upon field research conducted in the spring of 2008, I ask how we can understand the political space of migration not simply as exceptional, but as shaped by the mobility of the irregular migrants moving outside of the frameworks, policies, and practices of the state. By privileging the migrant narrative and making use of Ranci��re's conception of politics as shaped by the demands of those who ���have no part,��� I suggest an alternative way of understanding the politics of exception and agency of non-citizens���that is, one of disruption and demands to open up powerful potentials for change in an otherwise rigid regime.

The Opportunistic Pathogen Propionibacterium acnes: Insights into Typing, Human Disease, Clonal Diversification and CAMP Factor Evolution

We previously described a Multilocus Sequence Typing (MLST) scheme based on eight genes that facilitates population genetic and evolutionary analysis of P. acnes. While MLST is a portable method for unambiguous typing of bacteria, it is expensive and labour intensive. Against this background, we now describe a refined version of this scheme based on two housekeeping (aroE; guaA) and two putative virulence (tly; camp2) genes (MLST) that correctly predicted the phylogroup (IA, IA, IB, IC, II, III), clonal complex (CC) and sequence type (ST) (novel or described) status for 91% isolates (n = 372) via cross-referencing of the four gene allelic profiles to the full eight gene versions available in the MLST database (http://pubmlst.org/pacnes/). Even in the small number of cases where specific STs were not completely resolved, the MLST method still correctly determined phylogroup and CC membership. Examination of nucleotide changes within all the MLST loci provides evidence that point mutations generate new alleles approximately 1.5 times as frequently as recombination; although the latter still plays an important role in the bacterium's evolution. The secreted/cell-associated 'virulence' factors tly and camp2 show no clear evidence of episodic or pervasive positive selection and have diversified at a rate similar to housekeeping loci. The co-evolution of these genes with the core genome might also indicate a role in commensal/normal existence constraining their diversity and preventing their loss from the P. acnes population. The possibility that members of the expanded CAMP factor protein family, including camp2, may have been lost from other propionibacteria, but not P. acnes, would further argue for a possible role in niche/host adaption leading to their retention within the genome. These evolutionary insights may prove important for discussions surrounding camp2 as an immunotherapy target for acne, and the effect such treatments may have on commensal lineages. �� 2013 McDowell et al.

Increased expression of the RI�� subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer

The primary element in the cAMP signal transduction pathway is the cAMP-dependent protein kinase (PKA). Expression of the RI�� subunit of type I PKA is elevated in a variety of human tumours and cancer cell lines. The purpose of this study was to assess the prognostic importance of RI�� expression in patients with ovarian cancer. We have evaluated the expression of RI�� in a panel of human ovarian tumours (n = 40) and five human ovarian cancer cell lines using quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The human ovarian cell lines OAW42 and OTN14 express high endogenous levels of RI�� mRNA and protein (at significantly higher mRNA levels than high tissue expressors, P < 0.05). The ovarian cell line A2780 expresses low endogenous levels of RI�� mRNA and protein (also at higher mRNA levels than low tissue expressors, P < 0.05). Quantitative RT-PCR revealed no significant difference in RI�� mRNA expression between different ovarian histological subtypes in this study. No associations were found between RI�� mRNA expression and differentiation state. RI�� mRNA expression was significantly associated with tumour stage (P = 0.0036), and this remained significant in univariate analysis (P = 0.0002). A trend emerged between RI�� mRNA expression levels and overall survival in univariate analysis (P = 0.051), however, by multivariate analysis, stage remained the major determinant of overall survival (P = 0.0001). This study indicates that in ovarian epithelial tumours high RI�� mRNA expression is associated with advanced stage disease. RI�� expression may be of predictive value in ovarian cancer and may be associated with dysfunctional signalling pathways in this cancer type.

Regulation of single exocytotic events by the cAMP signaling pathway

Permanece por esclarecer como a via de sinaliza����o do cAMP modula a exocitose regulada. Os principais objetivos deste trabalho foram: i) avaliar o efeito do cAMP nos eventos exocit��ticos, nas propriedades dos poros de fus��o e na secre����o hormonal; ii) perceber o impacto da sinaliza����o por cAMP-HCN na exocitose e nas propriedades do poro de fus��o; e iii) estudar as propriedades do poro de fus��o na presen��a de um agente neurot��xico comum, como o alum��nio. Lactotrofos, isolados a partir da hip��fise anterior de ratos Wistar machos, foram usados como modelo celular. Os eventos unit��rios de fus��o exocit��tica e a prolactina (PRL) libertada foram avaliados, respetivamente, em ensaios eletrofisiol��gicos efectuados segundo a t��cnica de contacto herm��tico no modo sobre a c��lula aderida �� pipeta porta-el��trodo e com recurso a m��todos imunol��gicos de dete����o. Os n��veis intracelulares de cAMP foram aumentados por 3-isobutil-1-metilxantina (IMBX), forscolina e N6,2'-O-dibutiril adenosina- 3',5'-monofosfato c��clico (dbcAMP). A express��o dos canais HCN foi determinada por Western-blot, qRT-PCR e imunocitoqu��mica em combina����o com microscopia confocal. Culturas prim��rias de lactotrofos foram tamb��m transfetadas com DNA plasm��dico que codifica HCN2 juntamente com a prote��na-verde-fluorescente e um agente farmacol��gico foi usado para avaliar o efeito de cAMP-HCN na exocitose. Observou-se que os lactotrofos responderam �� forscolina e ao dbcAMP libertando PRL de um modo bif��sico e dependente da concentra����o, uma vez que a secre����o aumentou e diminuiu, respectivamente, na gama de baixas e altas concentra����es. Os compostos que elevaram os n��veis de cAMP aumentaram os eventos transientes e impediram a fus��o completa. Al��m disso, o dbcAMP promoveu o aparecimento de eventos exocit��ticos transientes de elevada periodicidade, cujos poros de fus��o, de maior di��metro, se mativeram abertos durante mais tempo. A express��o das quatro isoformas de HCN foi confirmada nos lactotrofos ao n��vel do mRNA e, tal como no cora����o, rim e hip��fise, o mais abundante codifica a isoforma HCN2. Nos lactotrofos com sobre-express��o desta isoforma, o dbcAMP n��o s�� aumentou a frequ��ncia dos eventos transientes e a condut��ncia dos poros, mas tamb��m a frequ��ncia dos eventos de fus��o completa. Enquanto o bloqueador dos canais HCN, ZD7288, reduziu a frequ��ncia dos eventos transientes e de fus��o completa desencadeados por dbcAMP e diminuiu o di��metro dos poros de fus��o. A simult��nea diminui����o da liberta����o de PRL, da frequ��ncia dos eventos transientes e do di��metro dos poros de fus��o representaram as principais altera����es observados ap��s pr��-tratamento dos lactotrofos com concentra����o micromolar de alum��nio. Em conclus��o, os resultados demonstram que elevados n��veis de cAMP reduzem a secre����o de PRL devido �� estabiliza����o dos poros de fus��o no estado de maior abertura. Al��m disso, a via de sinaliza����o cAMP-HCN afecta a actividade exocit��tica e modifica as propriedades dos poros de fus��o, que parecem ser igualmente importantes na citotoxicidade induzida por alum��nio.

La pesca de la anchoveta ��� Estadística de pesca y esfuerzo en octubre, noviembre y diciembre de 1961

Estadísticas de la pesca de anchoveta por dos fuentes: del desembarque por viaje de las embarcaciones pesqueras y de los datos del consumo de materia prima por d��a que proporcionan todas las f��bricas de elaboraci��n de harina de pescado.