Travel-associated and autochthonous Zika virus infection in mainland France, 1 January to 15 July 2016.

During summer 2016, all the conditions for local mosquito-borne transmission of Zika virus (ZIKV) are met in mainland France: a competent vector, Aedes albopictus, a large number of travellers returning from ZIKV-affected areas, and an immunologically naive population. From 1 January to 15 July 2016, 625 persons with evidence of recent ZIKV infection were reported in mainland France. We describe the surveillance system in place and control measures implemented to reduce the risk of infection.

Characterization of the epidermal growth factor receptor in human meningioma.

We have characterized the epidermal growth factor (EGF) receptor in human meningioma (biopsy) microsomes, cellularly derived microsomes, and intact meningioma cells in culture. Scatchard analysis of competition studies reveals both high and low affinity EGF binding sites in the meningiomas tested [dissociation constant (Kd) = 0.9 nM, maximum number of binding sites (Bmax) = 280 fmol/mg protein; Kd = 5.0 nM, Bmax = 660 fmol/mg protein, respectively]. The binding of 125I-EGF is specific since it is abolished by excess unlabeled EGF but not by excess unlabeled platelet-derived growth factor or insulin. Meningioma cultures preincubated with platelet-derived growth factor (10 ng/ml) at 37 degrees C shifted the 125I-EGF competition curve to the right but did not affect receptor number (100,000 sites/cell) when compared to cultures preincubated at 4 degrees C. Cross-linking studies performed with ethyleneglycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal a major band of specifically bound EGF (Mr approximately 150,000), although the normal (Mr approximately 170,000) and another putative proteolytic form (Mr approximately 125,000) can also be seen. These results indicate that human meningiomas contain a mixed population of EGF binding sites and exhibit properties of previously described EGF receptors.

Multiple regulation of prolactin receptor gene expression in rat liver.

Sex steroids are major regulators of PRL receptor expression in rat liver. Using a probe encoding the rat PRL receptor we have studied receptor mRNA levels in female rat liver during ontogeny and in response to estrogen treatment. Steady state mRNA levels were determined by Northern blot and densitometric analysis. Messenger RNA levels have been compared to the number of binding sites, which was assessed by Scatchard analysis of [125I]ovine PRL binding in membrane preparations. Our results show that steady state mRNA and binding levels of PRL receptors are both regulated by development and estrogens, but that binding does not exactly parallel mRNA levels. From the developmental stages of prepuberty to adult, receptor numbers increase 8-fold, whereas mRNA levels increase 3-fold. Estrogen treatment stimulates receptor levels 6-fold, but mRNA levels are only increased 3-fold. These results suggest that PRL receptor gene expression in rat liver is regulated at the transcriptional or posttranscriptional level as well as at the translational level.

Hepatic prolactin receptors in the rat: characterization using monoclonal antireceptor antibodies.

Monoclonal antibodies (mAbs) to the rat hepatic PRL receptor were produced and used for characterization of the receptor. A microsomal fraction from female rat liver was solubilized, purified 300- to 500-fold by affinity chromatography, and injected into mice. Two hybridoma clones (E21 and E29) were established, and immunoglobulin G fraction was obtained. Both E21 and E29 at 200 micrograms/ml could inhibit [125I]ovine PRL (oPRL) binding to microsomes from rat liver by 40% and 95%, respectively. E29 also inhibited PRL binding to solubilized receptors, whereas E21 stimulated PRL binding by about 50%. The action of E21 was markedly attenuated when [125I]human GH (hGH) was used as tracer in both microsomal (inhibition) and solubilized (stimulation) receptors. Specificity studies using microsomes from other tissues showed that both mAbs were specific to rat tissues (mammary gland, ovary, prostate, testis, and adrenal) and did not cross-react with tissues from other species (rabbit, mouse, human, pig, and cat) examined. Immunoprecipitation of PRL receptors with mAbs were assessed using 125I-labeled or [125I]oPRL-labeled PRL receptors. Both E21 and E29 were capable of immunoprecipitating a 44,000 mol wt band, the migration of which on a sodium dodecyl sulfate-electrophoresis gel was not affected by the absence or presence of a reducing agent. Only E21 was able to precipitate [125I]oPRL-receptor complexes. Binding studies of 125I-labeled mAb to microsomal receptors showed that oPRL could inhibit 90% of specific E29 binding, whereas inhibition of E21 binding was only 30%. Immunoblotting of PRL receptors confirmed the finding of immunoprecipitation; a band with a similar mol wt was identified with E21, although two closely located bands could be distinguished. There was no reaction in the presence of a reducing agent. These studies demonstrate that E21 recognizes a region distinct from the lactogen-binding site, while E29 binds to a closely related but not completely coincident domain; those regions recognized by both antibodies are specific to PRL receptors in the rat but not to those in other species; from immunoprecipitation and immunoblotting studies, the mol wt of the PRL receptor (or its subunit) is estimated to be 42-46K, similar to that reported for the rabbit mammary gland; this receptor molecule does not appear to bind to other receptor molecules through disulfide linkages; and hGH appears to recognize the PRL receptor-binding site in a somewhat different manner from that of oPRL.

Characterization and applications of monoclonal antibodies to the prolactin receptor.

Monoclonal antibodies (mAbs) were produced in BALB/c mice immunized with partially purified PRL receptors from rat liver. Two mAbs (T1 and T6) were able to completely inhibit [125I]ovine PRL ([125I]oPRL) binding to solubilized rat liver PRL receptors, while two other mAbs (U5 and U6) showed only a small effect on PRL binding, but were able to precipitate hormone-receptor complexes. Scatchard analysis of [125I]oPRL binding to rat liver microsomes in the presence of mAbs resulted in a decrease in the number of sites without changing the affinity of PRL binding by T1 and T6, whereas U5 and U6 altered neither parameter. [125I]mAb binding to rat liver microsomes was performed in the presence of various concentrations of unlabeled mAbs or oPRL to examine the interaction between mAbs. Competition of binding to the receptor was observed, respectively, between T1 and T6, U5 and U6, and U5 and E21 (a mAb to the rat liver PRL receptor previously produced). Both [125I]T1 and [125I]T6 binding were inhibited by oPRL, although not completely (80% inhibition at the higher concentrations). When [125I]T1 binding was analyzed by Scatchard analysis, two classes of binding sites to rat liver microsomes were found, of which only the number of higher affinity sites was affected by the presence of oPRL in incubation. Similar results were observed for [125I]T6 binding. [125I]mAb binding to microsomes from other tissues and species was examined. All five mAbs were able to bind to microsomes from rat tissues (liver, ovary, adrenal, prostate, and Nb2 lymphoma cells), similar to the level of [125I]oPRL binding in these tissues. The binding characteristics of [125I]T6 or [125I]U5 were essentially identical in all rat tissues examined. Although T1, U6, and E21 showed strong species specificity, there was significant binding of T6 to rabbit liver and mammary gland and of U5 to rabbit and pig mammary gland and mouse liver Competition curves of [125I]U5 binding were parallel for rat, rabbit, and mouse tissues, while [125I]T6 binding was able to distinguish PRL receptors in rabbit mammary gland from those in rat tissues. The use of 125I-labeled mAb in immunoblot analysis of the PRL receptor resulted in a marked increase in sensitivity. All mAbs detected microsomal PRL receptors in rat liver with mol wt of 84,000, 42,000 and 40,000. As little as 4 fmol receptors can be identified using this approach. Microsomal PRL receptors from rat ovary, prostate, and Nb2 cells and purified receptors from pig and rabbit mammary gland were subjected to immunoblot analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the structure and glycosylation properties of intracellular and cell surface rat hepatic prolactin receptors.

In this study we examined the structure of the PRL receptor of rat liver. We used immunoblotting with monoclonal antibodies to binding and nonbinding site epitopes of the PRL receptor to assess receptor subtypes in different hepatic subcellular fractions. Analysis of frozen-thawed cell fractions revealed 40- and 42-kilodalton (kDa) species. Digestion with neuraminidase indicated that both species were terminally sialylated. Freshly isolated membranes exhibited a single 42-kDa species in all subcellular fractions, whereas freeze-thawing generated the 40-kDa species. The carbohydrate linkages present in the PRL receptor were examined using enzymes to deglycosylate iodinated purified receptors. These studies indicated that oligosaccharides comprise about 7 kDa of receptor mass and that they are exclusively N-linked, consisting of tri- and/or tetra-antennary complex glycans. Monoclonal antibodies to the receptor recognized the deglycosylated receptor. Maximally deglycosylated receptors retained about 85% of their binding capacity. After in vivo tunicamycin treatment of rats, total PRL receptors (as determined by immunoblot analysis and binding activity) disappeared with a half-time of about 25 min. In this circumstance, no aglycosylated receptor species were recognized by monoclonal antibodies. Since deglycosylation of mature receptors did not markedly reduce binding capacity, we infer that mature receptors do not accumulate during the blockade of glycosylation by tunicamycin. Thus, glycosylation appears to be required for the acquisition of a mature receptor status, but is not necessary for the maintenance of that status.

Purification and protein sequence analysis of rat liver prolactin receptor.

Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)