Specific labelling by [125I]helodermin of high-affinity VIP receptors in rat liver membranes

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.     

Differences in primary structure among five phospholipases A2 from Heloderma suspectum

Five increasingly anionic phospholipases A2 (Pa1-Pa5) exist in the venom of the lizard Heloderma suspectum. We recently elucidated the sequence of Pa5, the most abundant and most active variant, towards emulsified phosphatidylcholines. Here we present the primary structures of Pa2, Pa3 (subvariants a and b) and Pa4, based on Edman degradation of tryptic, endoproteinase Arg-C and chymotryptic fragments of the reduced and S-carboxymethylated proteins. Pa1-Pa5, considered collectively, belong to an original class of secretory phospholipases A2 with 141-143 residues, a short hydrophobic N-terminus, 10 half-cystine residues and an extended C-terminus. The only known phospholipase A2 with characteristics close enough to be a member of the same class is that present in the venom from the insect Apis mellifera. More specifically, the sequences of Pa3 and Pa5 are almost identical, and those of Pa2 and Pa4 are also quite similar. Both groups diverge enough to indicate the translation of two mRNA species in the venom gland. The primary structure of Pa3 reveals the existence of subvariants a and b, the sequence of which is identical to that previously defined for Pa5, except that the C-terminal tripeptide GEG in Pa5 is replaced by the dipeptide GE in Pa3a and the tetrapeptide GEGR in Pa3b, Pa4, when compared to Pa5, shows 21 substitutions with a cluster of five modified amino acids in positions 40-44, immediately after the catalytic segment amino acids 30-39, and added changes scattered before the C-terminus. Pa2 differs from Pa4 only by the absence of the Gly142 C-terminal residue. The 15% difference in primary structure observed between the Pa3-Pa5 and Pa2-Pa4 subgroups might be largely responsible for their distinct biological properties.     

Antagonistic properties are shifted back to agonistic properties by further N-terminal shortening of pituitary adenylate-cyclase-activating peptides in human neuroblastoma NB-OK-1 cell membranes.

N-terminally shortened analogs of the 27-amino-acid and 38-amino-acid forms of the pituitary-adenylate-cyclase-activating neuropeptide, PACAP(1-27) and PACAP(1-38), were synthesized by a solid-phase method. Systematic deletion of the first 13 amino acids of both PACAP was tested by evaluating their ability to occupy the specific and selective PACAP receptor of human neuroblastoma NB-OK-1 cell membranes and to stimulate adenylate cyclase or, when inactive per se, to inhibit PACAP-stimulated adenylate cyclase activity. For each peptide, the Kact (concentration required for half-maximal adenylate cyclase activation) or Ki [concentration required to shift the dose/response curve of PACAP(1-27) twofold to the right] was in good agreement with the corresponding IC50 [concentration inhibiting 50% of 125I-[AcHis1]PACAP(1-27) binding to membranes], suggesting interaction with the same homogeneous class of adenylate cyclase-coupled receptors. The deletion of the two first amino acids (His1 and Ser2) sufficed to decrease the affinity for receptors and to suppress the capacity to activate adenylate cyclase. The shorter fragments 3-27 and 3-38, 4-27 and 4-38, 5-27 and 5-38, 6-27 and 6-38, 7-27 and 7-38, 8-27 and 8-38, and 9-27 and 9-38 were all competitive antagonists of PACAP(1-27)-stimulated activity with the N-terminally shortened PACAP(1-38) derivatives being 4-30-fold more potent than the equivalent PACAP(1-27) derivatives. In this group PACAP(6-38) was the most potent antagonist (Ki 1.5 nM). Surprisingly, the N-terminally shorter fragments 10-27 and 10-38, 11-27 and 11-38, 12-27 and 12-38, 13-27 and 13-38, and 14-27 and 14-38 were again able to stimulate adenylate cyclase, the smallest fragments, PACAP(14-27) and PACAP(14-38), being the most potent and efficient (Kact 2 microM and 0.1 microM, respectively). In this group of agonists, PACAP(1-38) derivatives deleted at the N-terminus were also more potent than the equivalent PACAP(1-27) derivatives.     

Identification and characterization of a prevacuolar compartment in stigmas of nicotiana alata

The stigmas of the ornamental tobacco plant Nicotiana alata accumulate large quantities of a series of 6-kD proteinase inhibitors (PIs) in the central vacuole that are derived from a 40-kD precursor protein, Na-PI. The sorting information that directs Na-PI to the vacuole is likely to reside in a C-terminal propeptide domain of 25 amino acids that forms an amphipathic alpha helix. Using cell fractionation techniques, we have examined transit of Na-PI through the endomembrane system and have identified a prevacuolar compartment that contains Na-PI with an intact targeting signal. In contrast, the targeting signal is not present on the predominant form of Na-PI in the vacuole. The prevacuolar compartment is marked by the presence of homologs of both the t-SNARE, PEP12p, and the putative vacuolar sorting receptor BP-80. Cross-linking and affinity precipitation studies revealed that Na-PI associates with BP-80 within this compartment, providing in vivo evidence for the function of BP-80 as a sorting receptor for a protein with a C-terminal vacuolar targeting signal.     

Tumor‐associated macrophages (TAMs) depend on ZEB1 for their cancer‐promoting roles

Accumulation of tumor‐associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro‐tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial‐to‐mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor‐promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80^low pro‐tumor phenotype, including direct activation of Ccr2. In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem‐like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs’ tumor‐promoting functions.     

Mangrove reforestation: greening or grabbing coastal zones and deltas? Case studies in Senegal§

Besides their important contribution to global biodiversity, mangroves provide many services. Nevertheless, due to an increase of human activities and to climate change, in less than 20 years these ecosystems have lost one fifth of their global surface area. In response to this decrease, mangrove reforestation incentives have spread throughout the world. The scientific and societal legitimacy of reforestation actions still remain in question. Focusing on two case studies, the Saloum Delta and Lower Casamance, Senegal, our methodology was mainly based on the analysis of environmental narratives and discourses between 2009 and 2013, and on reforestation campaigns conducted by NGOs. We highlight the complexity of the system of values associated with the mangroves, as well as the positive and negative interactions between the services. Even although the reforestation campaigns were generally successful in terms of reforested surfaces and international visibility, they were poor in terms of biological and cultural diversities and led to spatial injustice. Moreover, the extensive reforestation with a unique mangrove species, Rhizophora mangle, was perceived as means of ‘green grabbing’, and the simultaneous buying of carbon tax by industrial conglomerates induced disempowerment of the local communities. More integrated research programmes must be developed towards the extensive knowledge of the mangroves.     

Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection

Background  

Methodologies like phage display selection, in vitro mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised. In the current study we show that high-resolution melt curve analysis (HRMA) is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge.     

Caffeic Acid Phenylethyl Ester and MG-132 Have Apoptotic and Antiproliferative Effects on Leukemic Cells But Not on Normal Mononuclear Cells

Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor κB (NF-κB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-κB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-κB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias.     

Thyroid hormone plasmatic levels in rats treated with serotonin in acute and chronic way

Many experiments show that serotonin (5-HT) controls thyroidal function at hypothalamic level, inhibiting the TRH secretion. The majority of experiments are done in an acute way, consisting of a single serotonin dose injected intraperitoneally (ip) or intracerebroventricularly (ic) with the effect registered after a short time (usually 1 h) as in normal environmental conditions similar to the TSH stimulation test, that consists of transfer of the experimental animals from 30°C to 4°C for 30 min, thus inducing stimulation of the hypothalamus-hypophysis-thyroid axis. The aim of the present research was to study the correlation between 5-HT and the thyroidal function, measuring plasmatic thyroid hormone levels in rats ip treated in chronic (injected daily for 10 days with different doses of 5-HT), and in acute way (after 1 h from a single 2.0 mg/kg bw 5-HT dose) in normal environmental conditions to evidence the serotonin site action activity outside the blood-brain barrier. The results of the chronic experiment show an inhibitory effect of 5-HT, on T3 and T4 plasmatic level, only when it is injected at medium doses (0.2 and 0.4 mg/kg bw for T3, and 0.2 for T4), while the results of the acute experiment do not evidence any modification. These results show that in normal environmental conditions the outside 5-HT site action is active only when the 5-HT is injected chronically at defined doses, probably for a down-regulation phenomenon.