Evaluation of 16K Prolactin to prevent metastatic spread through inhibition of angiogenesis

Poster (2006)

Adenovirus-mediated gene transfer of 16K prolactin inhibits B16-F10 tumor growth and metastasis

Poster (2006)

Molecular targeting of antiangiogenic factor 16K hPRL inhibits oxygen-induced retinopathy in mice

in Investigative Ophthalmology & Visual Science (2004), 45(7), 2413-2419

PURPOSE. To examine the ability and mechanism of the 16 kDa N-terminal fragment of human prolactin (16K hPRL) in the inhibition of abnormal retinal neovascularization. METHODS. The 16K hPRL-encoding ... [more ▼]

PURPOSE. To examine the ability and mechanism of the 16 kDa N-terminal fragment of human prolactin (16K hPRL) in the inhibition of abnormal retinal neovascularization. METHODS. The 16K hPRL-encoding sequence was inserted into an adenoviral vector (16K-Ad). Western blot analysis verified the expression of 16K hPRL and inhibition of proliferation, confirming functional activity of the 16K hPRL in virus-infected adult bovine aortic endothelial (ABAE) cells. 16K hPRL inhibited retinal neovascularization in a mouse model of oxygen-induced retinopathy. The ability of recombinant 16K hPRL expressed in E. coli (r16K hPRL) was compared to that of endostatin in inducing apoptosis of cultured human retinal endothelial cells (HREC). RESULTS. 16K was expressed in virus-infected ABAE cells and resulted in a dose-dependent inhibition of cell proliferation. Eyes injected with 16K-Ad showed a reduction in preretinal neovascularization of 82.3 +/- 9.3% (P < 0.00001) when compared to uninjected controls. r16K hPRL was 100 times more potent than endostatin in inducing apoptosis in HRECs. CONCLUSIONS. Intravitreal administration of 16K hPRL inhibited neovascularization in the mouse model of oxygen-induced retinopathy. 16K hPRL stimulated apoptosis in HRECs and inhibited cell proliferation in ABAE cells. These results suggested a potential therapeutic role for 16K hPRL in the treatment of proliferative retinopathies. [less ▲]

The 16 kDa N-terminal fragment of the human prolactin. Design and production of minimal peptides retaining antiangiogenic and antitumoral properties

Poster (2004)

16K hPRL prevents angiogenesis by inducing both apoptosis and cell cycle arrest of endothelial cells

Poster (2003)

Detection of illegal growth promoters in biological samples using receptor binding assays

in Analytica Chimica Acta (2002), 473(1-2), 135-141

In the European Union (EU), the use of growth-promoting substances in meat production is banned. The control of growth promoters, especially steroid hormones, is presently based on expensive and time ... [more ▼]

In the European Union (EU), the use of growth-promoting substances in meat production is banned. The control of growth promoters, especially steroid hormones, is presently based on expensive and time-consuming chromatographic methods of analysis or, sometimes, for screening purposes, on radio- or enzyme-immunoassays, all of which are often too specific to allow effective multi-analyte control. In order to develop rapid and inexpensive multi-analyte detection tests, we proposed the use of hormonal receptors as detection tools. The system described here (radio-receptor assays) is based on a direct bindin g assay of steroid hormones to their respective receptors. Human receptors to estrogens (hERalpha), androgens (hAR), progestagens (hPR) and glucocorticoids (hGR) have been produced by genetic engineering in bacteria or in eucaryotic cells. Binding analyses revealed that the obtained receptor proteins retained a high affinity for their corresponding native ligand. In addition, competition studies continued that each of the four receptors displays a specificity profile for a series of analogs in agreement with the literature. Finally, the stability of these recombinant receptors is sufficient to allow their use in test kits. (C) 2002 Elsevier Science B.V. All rights reserved. [less ▲]

Evidence of rainbow trout prolactin interaction with its receptor through unstable homodimerisation.

in Molecular & Cellular Endocrinology (2001), 172(1-2), 105-13

This study aims to characterise Prolactin receptor (PRLR) in rainbow trout for which no information is available despite the availability of Salmonid PRL preparations. By screening a freshwater rainbow ... [more ▼]

This study aims to characterise Prolactin receptor (PRLR) in rainbow trout for which no information is available despite the availability of Salmonid PRL preparations. By screening a freshwater rainbow trout intestine cDNA library with a probe corresponding to the extracellular domain (ECD) of tilapia PRLR, we have cloned a 2.5 kb insert coding for the PRLR. The mature protein of 614 amino acid residues is similar to PRLR isolated in tilapia and also the long form of mammalian PRLR. Analysis of PRLR gene expression in osmoregulatory organs revealed the presence of a unique transcript, thus confirming the involvement of this hormone in the control of osmoregulation in this fish species. By using surface plasmon resonance (SPR) technology, kinetic measurement of interaction between trout PRL and its receptor ECD was studied. This approach allowed us to demonstrate the formation of a transient, unstable homodimeric complex. This unstability could explain the inability to perform binding experiments using homologous PRL. In contrast, heterologous lactogenic ligands were able to interact through a more stable complex. Whether these characteristics of PRL-receptor interaction in rainbow trout are different to what occurs in tilapia where a homologous radioreceptor assay was developed would require further studies. [less ▲]

Recombinant prolactin Receptor Extracellular Domain of Rainbouw trout (Oncorhynchus mykiss): subcloning, preparation and characterization

in General and Comparative Endocrinology (2000), 118

The cDNA of the extracellular domain of rainbow trout (Oncorhynchus mykiss) prolactin receptor (trPRLR-ECD) was cloned in the prokaryotic expression vector pMON to enable its expression in Escherichia ... [more ▼]

The cDNA of the extracellular domain of rainbow trout (Oncorhynchus mykiss) prolactin receptor (trPRLR-ECD) was cloned in the prokaryotic expression vector pMON to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed trPRLR-ECD protein, contained within the refractile body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 26-kDa fraction was eluted in 0.2 M NaCl, yielding 20 mg/2.5 L of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent and by chromatography on a Superdex column. Binding experiments using [125I]ovine placental lactogen (oPL) as a ligand revealed that human growth hormone (hGH), oPL, and ovine prolactin (oPRL) were the most effective competitors, with respective IC50 values of 1.32, 2.27, and 2.70 nM. Chicken (ch) PRL did not compete at all, and homologous trPRL was much less effective, with a corresponding IC50 value of 1826 nM. Gel-filtration was used to determine the stoichiometry of trPRLR-ECD's interaction with oPL, hGH, and oPRL. Only oPL yielded a 2:1 complex, whereas hGH and oPRL formed only 1:1 complexes, with excess trPRLR-ECD being seen at the initial 2:1 trPRLR-ECD:hGH or trPRLR-ECD:oPRL ratios. No studies were performed with chPRL because of its inability to compete with [125I]oPL or with trPRL because of its low affinity toward trPRLR-ECD. The present results agree with previous findings indicating, as in mammals, that homologous PRL interacts transiently with its receptor and suggest that transient homologous PRL-induced homodimerization of the receptor is sufficient to initiate a biological signal, despite the fact that, in classical binding experiments, only low specific binding can be detected. [less ▲]

Lys13 plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures

in Journal of Biological Chemistry (1999), 274(27), 19181-7

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair ... [more ▼]

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-A resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption. [less ▲]

APH-1, a POU homeobox gene expressed in the salt gland of the crustacean Artemia franciscana

in Mechanisms of Development (1999), 87(1-2), 207-12

We characterized the first POU-homeoprotein in a crustacean (designated APH-1 for Artemia POU-Homeoprotein, EMBL Y15070). The amino acid sequence of the APH-1 POU-domain is identical, except for two ... [more ▼]

We characterized the first POU-homeoprotein in a crustacean (designated APH-1 for Artemia POU-Homeoprotein, EMBL Y15070). The amino acid sequence of the APH-1 POU-domain is identical, except for two residues, to that of the two class III POU proteins Cf1-a (Drosophila) and POU-M1 (Bombyx mori). Southern blot analysis suggests that crustaceans have only one class III POU gene. RT-PCR and whole-mount in situ hybridization show that APH-1 mRNA is present in larvae specifically in the salt gland, an organ which is involved in osmoregulation, and disappears in the adult. [less ▲]