Two monoclonal antibodies against prolactin-receptor are internalized in epithelial mammary cells without mimetic prolactin effect on casein secretion*

Summary— Prolactin exerts an early stimulatory effect on casein secretion which was qualified as a secretagogue effect. After binding to its receptor, the hormone transits intracellularly through the mammary epithelial cell. When this transit is slowed down the secretagogue effect does not occur. Different monoclonal antibodies which bind to the rabbit prolactin receptor have been previously developed. One of them (A917) mimics prolactin effect on casein gene expression. Another (M110) blocks this prolactin effect. In order to study the respective role of the hormone and its receptor, we have examined the binding of the two monoclonal antibodies (M110 and A917), labeled with biotin or colloidal gold, to the receptor of lactating rabbit mammary epithelial cells in incubation. Subsequently, the intracellular movement of these antibodies and the secretory response have been measured. Irrespective of the labeling (biotin or colloidal gold) or the preparation of tissues (fragments or enzymatically dissociated cells), Ml 10 and A917 bound to the basal membrane of mammary epithelial cells. However, only M110 bound to apical membrane of dissociated cell when this membrane was in direct contact with the incubation medium, showing that the two antibodies discriminate the receptor located on the apical membrane. Following internalization, each antibody was carried via a peculiar pathway. M110 remained associated with the cells during a 1-h incubation, mainly in endosomes, multivesicular bodies and lysosomes like vesicles. In contrast, A917 was very quickly detectable in endosomes, multivesicular bodies and vesicles of the Golgi region and was carried throughout the cell to the lumen of the acini. M110 and A917 were extremely rare in secretory vesicles containing casein micelles. When mammary fragments pulse labeled for 3 mm with ³H-leucine were chased for 60 mm in the presence of prolactin, M110 or A917, only prolactin was able to increase casein secretion. These results show that two monoclonal antibodies against prolactin receptor are internalized after binding to the surface of the mammary cell. They are carried intracellularly by different routes. Internalization of these antibodies is not sufficient to mimic the secretagogue effect of prolactin.     

Prolactin and its cleaved 16 kDa fragment

Prolactin, owing to its origins, actions and molecular forms, is an ubiquitous and pleiotropic hormone. Indeed prolactin, initially thought to be essentially synthesized in the hypophysis, is also produced by several tissues in mammals. It is involved in more than 300 different biological activities, such as reproduction, developmental immunity and behaviour. It is also described under several molecular forms resulting from co- or post-translational modifications and enzymatic cleavage. Among these, the 16 kDa form, derived from native prolactin, has received particular attention because of its inhibitory effect on angiogenesis. Recent results have suggested an important role of tissue enzymes in the production of this form in several tissues (retina, myocardium and mammary gland). The cleavage leading to the production of 16 kDa prolactin may occur outside the cells, in the interstitial medium and therefore in the vicinity of blood capillaries. This process implies tissue-specific mechanisms of regulation. A better knowledge of the location of the cleavage and of the regulation of these activities of the cleaving enzymes is now essential for controlling the processes. This knowledge will allow a better understanding of the relationships between some pathologies (cardiomyopathy, pre-eclampsia, retinopathy) and modification of the production of the anti-angiogenic form of prolactin.     

Natural hybridization between 2 sympatric species of mice, Mus musculus domesticus L. and Mus spretus Lataste

Using protein loci and DNA markers, we show by a multilocus genetic analysis that certain populations of the two sympatric mouse species Mus musculus domesticus and Mus spretus show clear signs of partial introgression. Given the sterility of F1 males and the known partial genetic incompatibilities between the genomes of the two species, our finding does not invalidate the biological species complex, but allows to think that very limited genetic exchanges remain possible even long after the divergence of taxa. This may have some consequences on the dynamics of certain kinds of invasive or advantageous DNAs like transposable elements or pathogen resistance genes.     

Phylogeography and demographic history of Shaw's Jird (Meriones shawii complex) in North Africa

Palaeoenvironmental data and climatic reconstructions show that the Mediterranean ecoregion of North Africa underwent drastic ecological changes during the Pleistocene. Given its rich palaeontological record, North Africa is a pertinent region for documenting the role of climate change and human mediated‐habitat changes on the demography and genetic structure of faunal species. In the present study, we collected data from this species in Morocco, Algeria, and Tunisia, and we combined molecular (mitochondrial and nuclear DNA sequences, microsatellites), fossil, palaeoenvironmental, and human context data to propose an explanation for the fluctuations of populations belonging to the Meriones shawii complex in the past. Genetic and fossil data both indicate a strong bottleneck in Moroccan populations at the Middle Holocene (last interglacial optimum) compared to the Late Pleistocene. Our mitochondrial DNA data suggest a diversification event within Morocco corresponding to the 130–125 kya interglacial optimum. Given that (1) major demographic changes in the M. shawii complex coincide with the interglacial optimums, and (2) the impact of human activities on the landscape and faunal communities was moderate during the Middle Holocene (beginnings of the Neolithic culture), our results demonstrate that climate, rather than anthropogenic influences, likely explains the M. shawii complex population decline in the Holocene.     

Real-Time PCR for Measles Virus Detection on Clinical Specimens with Negative IgM Result in Morocco

Since the confirmation of measles cases represents an important indicator regarding the performance of the measles-elimination program, the aim of this study was to evaluate the effectiveness of the routine procedures followed in Morocco for the laboratory confirmation of measles cases. Suspected cases reported between January 2010 and December 2012 were assessed for the timeliness of the sample collection, occurrence of measles clinical symptoms, and the results of the laboratory diagnoses. For 88% of the 2,708 suspected cases, a clinical specimen was collected within 7d of rash onset, of which 50% were IgM-positive and 2.6% were equivocal. The measles symptoms were reported in 91.4% of the cases; the occurrence of symptoms showed a positive association with the serological results (odds ratio [OR] = 2.9883, 95% confidence interval [CI] 2.2238–4.0157). Of the negative samples, 52% (n = 116) tested positive by real-time polymerase chain reaction (PCR). These results are in favor of using molecular detection to complement serological diagnosis in the context of measles surveillance approach in Morocco. In addition, the introduction of additional laboratory methods for differential diagnosis is required for the final classification of suspected cases with maculopapular rash and fever in the context of the measles elimination program.     

Genetic differentiation of European anchovy (Engraulis encrasicolus) along the Moroccan coast reveals a phylogeographic break around the 25th parallel North

Seven microsatellite markers were used to investigate the population structure of the offshore ecotype of the European anchovy (Engraulis encrasicolus) by comparing 12 marine samples collected off the Moroccan coast with an inshore sample taken as a reference for the lagoonal ecotype. F-statistics, correspondence analysis and Bayesian assignment all concurred to cluster the European anchovy in this region into three groups: (i) one reference lagoonal sample, (ii) samples north of the 25°N latitude and (iii) samples south of it. Moreover, the Bayesian cluster analysis pointed toward the existence of an admixture between the group north of 25°N and the lagoonal ecotype, while this was not detectable with the group south of 25°N. Differential introgression between the two ecotypes could be one of the plausible explanations for the observed genetic structure and reveals the possible existence of a phylogeographic break around the 25th parallel North. Our study illustrates the fact that, for those species that encompass several incompletely isolated ecotypes, the level of gene flow among them may vary in space and serve as a tool for stock identification. This information may be useful to improve fishery management of this important harvested species along the Moroccan coast.     

Low molecular weight DNA in the heteromeric macronuclei of two cyrtophorid ciliates

Summary— The size range of the native DNA molecules in the heteromeric macronuclei of two cyrtophorid ciliates (Trithigmostoma cucullulus, Chilodonella uncinata) was mainly investigated by using agarose gel electrophoresis. Numerous bands superimposed on a continuous spectrum of molecular sizes between about 0.35 kb and 30 kb were resolved by conventional electrophoresis. Species-specific banding patterns indicate a variation between species in the copy number of individual DNA fragments. A slight intra-specific variability of banding patterns can exist. Electrophoretic distributions for two strains of T cucullulus were indeed found to differ by at least one more intense band (‘overamplified’ sequences?). Fractionation by contour-clamped homogeneous electric field (CHEF) gel electrophoresis revealed that the size continum of macronuclear DNA molecules does not extend beyond 60–70 kb. The average size was estimated to be around 4 kb. Unresolved DNA fraction (> 1000 kb) accounted for less than 10% of the mass of cellular DNA entering CHEF gels. Macronuclear ribosomal DNA of each cyrtophorid species was identified by Southern hybridization with a Tetrahymena rDNA probe. The hybridization signal was observed on a single band of low molecular weight DNA. The corresponding size was close to 14.5 kb in Trithigmostoma and 15.5 kb in Chilodonella, which is about twice the size of monomeric rDNA in hypotrichous ciliates. We showed that S1 nuclease resistant duplexes wit half the length of the native rDNA can be formed by rapid renaturation of heat-denatured molecules and hybridized with native rDNA. This strongly suggests that the nucleotide sequence of this rDNA is a large palindrome. Unlike the hypotrichs, macronuclear rDNA in cyrtophorids should be organized into palindromic dimers as in Tetrahymena species.     

Hydrogenated and fluorinated surfactants derived from Tris(hydroxymethyl)-acrylamidomethane allow the purification of a highly active yeast F1-F0 ATP-synthase with an enhanced stability

Loss of stability and integrity of large membrane protein complexes as well as their aggregation in a non-lipidic environment are the major bottlenecks to their structural studies. We have tested C₁₂H₂₅-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H₁₂-TAC) among many other detergents for extracting the yeast F₁F₀ ATP-synthase. H₁₂-TAC was found to be a very efficient detergent for removing the enzyme from mitochondrial membranes without altering its sensitivity towards specific ATP-synthase inhibitors. This extracted enzyme was then solubilized by either dodecyl maltoside (DDM), H₁₂-TAC or fluorinated surfactants such as C₂H₅-C₆F₁₂-C₂H₄-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H₂F₆-TAC) or C₆F₁₃-C₂H₄-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F₆-TAC), two surfactants exhibiting a comparable polar head to H₁₂-TAC but bearing a fluorinated hydrophobic tail. Preparations from enzymes purified in the presence of H₁₂-TAC were found to be more adapted for AFM imaging than ATP-synthase purified with DDM. Keeping H₁₂-TAC during the Ni-NTA IMAC purification step or replacing it by DDM at low concentrations did not however allow preserving enzyme activity, while fluorinated surfactants H₂F₆-TAC and F₆-TAC were found to enhance enzyme stability and integrity as indicated by sensitivity towards inhibitors. ATPase specific activity was higher with F₆-TAC than with H₂F₆-TAC. When enzymes were mixed with egg phosphatidylcholine, ATP-synthases purified in the presence of H₂F₆-TAC or F₆-TAC were more stable upon time than the DDM purified enzyme. Furthermore, in the presence of lipids, an activation of ATP-synthases was observed that was transitory for enzymes purified with DDM, but lasted for weeks for ATP-synthases isolated in the presence of molecules with Tris polyalcoholic moieties. Relipidated enzymes prepared with fluorinated surfactants remained highly sensitive towards inhibitors, even after 6 weeks.     

Application of airborne imaging spectrometry system data to intertidal seaweed classification and mapping

Hydrobiologia - The aim of this paper is to test the ability of imaging radiometers to describe the principal seaweed and seagrass beds along the coast of Brittany (France). In this work we used...