Joseph A. Martial

Antagonistic Properties of Human Prolactin Analogs That Show Paradoxical Agonistic Activity in the Nb2 Bioassay

Based on the assumption that the prolactin receptor (PRLR) is activated by PRL-induced sequential dimerization, potential human PRL (hPRL) antagonists were designed that sterically interfere with binding site 2. We previously reported the unexpected agonistic properties of these hPRL analogs in the rat Nb2 bioassay (Goffin, V., Struman, I., Mainfroid, V., Kinet, S., and Martial, J. A. (1994) J. Biol. Chem. 269, 32598-32606). In order to investigate whether such paradoxical agonistic behavior might result from characteristic features of the Nb2 assay (e.g. species specificity), we transfected in the same cell system the cDNA encoding the PRLR from rat or human species along with reporter genes containing PRL-responsive DNA sequences. We characterized the agonistic, self-antagonistic and/or antagonistic effects of wild type rat PRL, wild type hPRL, and three hPRL analogs, mutated either at binding site 1 or at binding site 2. Our results clearly show that the agonistic/antagonistic properties of PRLs are species-specific. We thus propose different models of receptor activation, depending on the relative affinities of each hormonal binding site, which is directed by species specificity. Finally, this is the first report of hPRL binding site 2 analogs showing antagonistic properties on human and, to a lesser extent, rat receptors.

Plasminogen activator inhibitor-1 protects endothelial cells from FasL-mediated apoptosis.

Plasminogen activator inhibitor-1 (PAI-1) paradoxically enhances tumor progression and angiogenesis; however, the mechanism supporting this role is not known. Here we provide evidence that PAI-1 is essential to protect endothelial cells (ECs) from FasL-mediated apoptosis. In the absence of host-derived PAI-1, human neuroblastoma cells implanted in PAI-1-deficient mice form smaller and poorly vascularized tumors containing an increased number of apoptotic ECs. We observed that knockdown of PAI-1 in ECs enhances cell-associated plasmin activity and increases spontaneous apoptosis in vitro. We further demonstrate that plasmin cleaves FasL at Arg144-Lys145, releasing a soluble proapoptotic FasL fragment from the surface of ECs. The data provide a mechanism explaining the proangiogenic activity of PAI-1.

Cloning and expression analysis of an inducible HSP70 gene from tilapia fish

We isolated and characterized the tilapia (Oreochromis mossambicus) HSP70 gene, highly homologous to other HSP70 genes. A dramatic increase of tilapia HSP70 mRNA levels was observed after heat shock of whole animals in all organs tested. Reporter constructs were tested for transient expression in carp cells and in microinjected zebrafish embryos. The entire isolated regulatory region (−851/+157) was able to mediate heat shock inducible expression of the reporter gene, with no preference for a particular tissue. Our studies represent the first transcriptional analysis of a HSP70 promoter from fish, revealing a powerful tool to direct controlled, tissue‐independent gene expression in fish.

Nucleotide sequence of part of the gene for human chorionic somatomammotropin: Purification of DNA complementary to predominant mRNA species

Abstract A novel purification procedure for DNA complementary to individual mRNA species has been developed by using restriction endonuclease cleavage of cDNA transcribed from a complex mixture of mRNAs. This procedure has allowed us to isolate and analyze DNA fragments complementary to the mRNA coding for the human peptide hormone chorionic somatomammotropin (HCS). The mRNA for this hormone is a major constituent of placental polyadenylated RNA as shown by in vitro translation of placental RNA and by nucleic acid hybridization using HCS cDNA as a specific probe. The purification of HCS cDNA was achieved by Hae III and Hha I restriction endonuclease cleavage of single-stranded cDNA synthesized in vitro from total polyadenylated placental RNA. Polyacrylamide gel electrophoresis of the products allowed detection and purification of discrete DNA fragments. A comparison of the nucleotide sequence of these fragments with that predicted from the amino acid sequence of HCS demonstrated that the fragments are transcripts of HCS mRNA, containing most of the translated and 3′ untranslated regions. The latter region is characterized by a UAG termination codon immediately adjacent to the translated region (a second in phase UAG occurs 9 nucleotides away) and a palindromic sequence (GUGACCCCUCCCCAGUG) centered 27 nucleotides from the termination codon. The purification scheme outlined for HCS cCNA should be applicable to DNA sequences complementary to mRNA species which represent at least 2% of any polyadenylated RNA preparation. This was demonstrated by restriction endonuclease cleavage of cDNA synthesized from a mixture of purified rabbit globin and total polyadenylated human placental RNAs.

S179D-Human PRL, a Pseudophosphorylated Human PRL Analog, Is an Agonist and Not an Antagonist

For many years, our group has been involved in the development of human PRL antagonists. In two recent publications, S179D-human PRL, a human PRL analog designed to mimic a putative S179-phosphorylated human PRL, was reported to be a highly potent antagonist of human PRL-induced proliferation and signaling in rat Nb2 cells. We prepared this analog with the aim of testing it in various bioassays involving the homologous, human PRL receptor. In our hands, S179Dhuman PRL was able to stimulate 1) the proliferation of rat Nb2 cells and of human mammary tumor epithelial cells (T47D), 2) transcriptional activation of the lactogenic hormone response element-luciferase reporter gene, and 3) activation of the Janus kinase/signal transducer and activator of transcription and MAPK pathways. Using the previously characterized antagonist G129R-human PRL as a control, we failed to observe any evidence for antagonism of S179D-human PRL toward any of the human PRL-induced effects analyzed, including cell proliferation, transcriptional activation, and signaling. In conclusion, our data argue that S179D-human PRL is an agonist displaying slightly reduced affinity and activity due to local alteration of receptor binding site 1, and that the antagonistic properties previously attributed to S179Dhuman PRL cannot be confirmed in any of the assays analyzed in this study. (Endocrinology 142: 3950 –3963, 2001)

Biological Properties of Human Prolactin Analogs Depend Not Only on Global Hormone Affinity, but Also on the Relative Affinities of Both Receptor Binding Sites

Zinc increases the affinity of human growth hormone (hGH) for the human prolactin receptor (hPRLR) due to the coordination of one zinc ion involving Glu-174hGH and His-18hGH. In contrast, binding of hPRL to the hPRLR is zinc-independent. We engineered in binding site 1 of hPRL a hGH-like zinc coordination site, by mutating Asp-183hPRL (homologous to Glu-174hGH) into Glu (D183E mutation). This mutation was also introduced into G129R hPRL, a binding site 2 mutant (Goffin, V., Kinet, S., Ferrag, F., Binart, N., Martial, J. A., and Kelly, P. A. (1996) J. Biol. Chem. 271, 16573–16579). These analogs were characterized using a stable clone expressing both the hPRLR and a PRLR-responsive reporter gene. The D183E mutationper se decreases the binding affinity and transcriptional activity of hPRL. However, this loss is partially rescued by the addition of zinc and the effect is much more marked on bioactivity than on binding affinity. These data indicate that the D183E mutation confers zinc sensitivity to hPRL biological properties. Due to an impaired site 2, the agonistic activity of G129R analog is almost nil. Although the double mutant D183E/G129R displays lower affinity (∼1 log) compared with G129R hPRL, it unexpectedly recovers partial agonistic activity in the absence of zinc. Moreover, whereas zinc increases the affinity of D183E/G129R, it paradoxically abolishes its agonistic activity. Our results demonstrate that the biological properties of hPRL analogs do not necessarily parallel their overall affinity. Rather, the relative affinities of the individual binding sites 1 and 2 may play an even more important role.

A green and bio-inspired process to afford durable anti-biofilm properties to stainless steel

A bio-inspired durable anti-biofilm coating was developed for industrial stainless steel (SS) surfaces. Two polymers inspired from the adhesive and cross-linking properties of mussels were designed and assembled from aqueous solutions onto SS surfaces to afford durable coatings. Trypsin, a commercially available broad spectrum serine protease, was grafted as the final active layer of the coating. Its proteolytic activity after long immersion periods was demonstrated against several substrata, viz. a synthetic molecule, N-α-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA), a protein, FTC-casein, and Gram-positive biofilm forming bacterium Staphylococcus epidermidis.

Effect of seasonal acclimatization on the expression of the carp transcription factor Pit-1

We isolated a clone comprising four exons of the carp Pit‐1 gene. Using synthetic oligonucleotide probes derived from the carp Pit‐1 sequence Pit‐1 expression was assessed by in situ hybridization in pituitary sections from summer‐ and winter‐acclimatized carp. Semiquantitative analyses of the hybridization signals revealed a significant higher Pit‐1 expression in the proximal pars distalis (PPD) and pars intermedia (PI) of the pituitary glands from summer‐acclimatized carp, compared to the winter‐acclimatized fish. In both adaptive states, relative to the PPD and PI, only a basal Pit‐1 expression was detected in the rostral pars distalis. Thus, during seasonal acclimatization of an eurythermal fish, Pit‐1 seems to be involved in the mechanisms that underlie the compensatory response.

Regulation of calcium-binding protein messenger RNA by 1,25-dihydroxycholecalciferol

Summary1,25-Dihydroxycholecalciferol modulates the synthesis of the intestinal calcium-binding protein. To determine if this effect is due to an increase in calcium-binding protein mRNA activity, we measured total cytoplasmic protein-specific mRNA activity from chick intestine in an in vitro wheat germ translation system. 1,25-Dihydroxy-cholecalciferol enhances calcium-binding protein specific mRNA activity. The sterol does not induce a general increase in mRNA synthesis since the concentration of only a few proteins increased. Thus 1,25-dihydroxycholecalciferol regulates only a subset of genes and specifically affects a transcriptional process involving calcium-binding protein synthesis.