Effect of interferon-TAU protein secretion in bovine endometrial cells and its modulation by steroid hormones

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Identification and bioactivity evaluation of two novel temporins from the skin secretion of the European edible frog, Pelophylax kl. Esculentus

The amphibian temporins, amongst the smallest antimicrobial peptides (AMPs), are α-helical, amphipathic, hydrophobic and cationic and are active mainly against Gram-positive bacteria but inactive or weakly active against Gram-negative bacteria. Here, we report two novel members of the temporin family, named temporin-1Ee (FLPVIAGVLSKLFamide) and temporin-1Re (FLPGLLAGLLamide), whose biosynthetic precursor structures were deduced from clones obtained from skin secretion-derived cDNA libraries of the European edible frog, Pelophylax kl. esculentus, by ‘shotgun’ cloning. Deduction of the molecular masses of each mature processed peptide from respective cloned cDNAs was used to locate respective molecules in reverse-phase HPLC fractions of secretion. Temporin-1Ee (MIC = 10 μM) and temporin-1Re (MIC = 60 μM) were both found to be active against Gram-positive Staphylococcus aureus, but retaining a weak haemolytic activity. To our knowledge, Single-site substitutions can dramatically change the spectrum of activity of a given temporin. Compared with temporine-1Ec, just one chemically-conservative substitution (Val8 instead of Leu8), temporin-1Ee bearing a net charge of +2 displays broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC = 40 μM). These factors bode well for translating temporins to be potential drug candidates for the design of new and valuable anti-infective agents.

Baltikinin: A New Myotropic Tryptophyllin-3 Peptide Isolated from the Skin Secretion of the Purple-Sided Leaf Frog, Phyllomedusa baltea

Here we report the identification of a novel tryptophyllin-3 peptide with arterial smooth muscle relaxation activity from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea. This new peptide was named baltikinin and had the following primary structure, pGluDKPFGPPPIYPV, as determined by tandem mass spectrometry (MS/MS) fragmentation sequencing and from cloned skin precursor-encoding cDNA. A synthetic replicate of baltikinin was found to have a similar potency to bradykinin in relaxing arterial smooth muscle (half maximal effective concentration (EC50) is 7.2 nM). These data illustrate how amphibian skin secretions can continue to provide novel potent peptides that act through functional targets in mammalian tissues.

Identification of Miscellaneous Peptides from the Skin Secretion of the European Edible Frog, Pelophylax kl. Esculentus

The chemical compounds synthesised and secreted from the dermal glands of amphibian have diverse bioactivities that play key roles in the hosts' innate immune system and in causing diverse pharmacological effects in predators that may ingest the defensive skin secretions. As new biotechnological methods have developed, increasing numbers of novel peptides with novel activities have been discovered from this source of natural compounds. In this study, a number of defensive skin secretion peptide sequences were obtained from the European edible frog, P. kl. esculentus, using a 'shotgun' cloning technique developed previously within our laboratory. Some of these sequences have been previously reported but had either obtained from other species or were isolated using different methods. Two new skin peptides are described here for the first time. Esculentin-2c and Brevinin-2Tbe belong to the Esculentin-2 and Brevinin-2 families, respectively, and both are very similar to their respective analogues but with a few amino acid differences. Further, [Asn-3, Lys-6, Phe-13] 3-14-bombesin isolated previously from the skin of the marsh frog, Rana ridibunda, was identified here in the skin of P. kl. esculentus. Studies such as this can provide a rapid elucidation of peptide and corresponding DNA sequences from unstudied species of frogs and can rapidly provide a basis for related scientific studies such as those involved in systematic or the evolution of a large diverse gene family and usage by biomedical researchers as a source of potential novel drug leads or pharmacological agents.

EspZ of enteropathogenic and enterohemorrhagic Escherichia coli regulates type III secretion system protein translocation

UNLABELLED: Translocation of effector proteins via a type III secretion system (T3SS) is a widespread infection strategy among Gram-negative bacterial pathogens. Each pathogen translocates a particular set of effectors that subvert cell signaling in a way that suits its particular infection cycle. However, as effector unbalance might lead to cytotoxicity, the pathogens must employ mechanisms that regulate the intracellular effector concentration. We present evidence that the effector EspZ controls T3SS effector translocation from enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Consistently, an EPEC espZ mutant is highly cytotoxic. Following ectopic expression, we found that EspZ inhibited the formation of actin pedestals as it blocked the translocation of Tir, as well as other effectors, including Map and EspF. Moreover, during infection EspZ inhibited effector translocation following superinfection. Importantly, while EspZ of EHEC O157:H7 had a universal "translocation stop" activity, EspZ of EPEC inhibited effector translocation from typical EPEC strains but not from EHEC O157:H7 or its progenitor, atypical EPEC O55:H7. We found that the N and C termini of EspZ, which contains two transmembrane domains, face the cytosolic leaflet of the plasma membrane at the site of bacterial attachment, while the extracellular loop of EspZ is responsible for its strain-specific activity. These results show that EPEC and EHEC acquired a sophisticated mechanism to regulate the effector translocation.

IMPORTANCE: Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important diarrheal pathogens responsible for significant morbidity and mortality in developing countries and the developed world, respectively. The virulence strategy of EPEC and EHEC revolves around a conserved type III secretion system (T3SS), which translocates bacterial proteins known as effectors directly into host cells. Previous studies have shown that when cells are infected in two waves with EPEC, the first wave inhibits effector translocation by the second wave in a T3SS-dependent manner, although the factor involved was not known. Importantly, we identified EspZ as the effector responsible for blocking protein translocation following a secondary EPEC infection. Interestingly, we found that while EspZ of EHEC can block protein translocation from both EPEC and EHEC strains, EPEC EspZ cannot block translocation from EHEC. These studies show that EPEC and EHEC employ a novel infection strategy to regulate T3SS translocation.

A Combined Molecular Cloning and Mass Spectrometric Method to Identify, Characterize, and Design Frenatin Peptides from the Skin Secretion of Litoria infrafrenata

Amphibian skin secretions are unique sources of bioactive molecules, particularly bioactive peptides. In this study, the skin secretion of the white-lipped tree frog (Litoria infrafrenata) was obtained to identify peptides with putative therapeutic potential. By utilizing skin secretion-derived mRNA, a cDNA library was constructed, a frenatin gene was cloned and its encoded peptides were deduced and confirmed using RP-HPLC, MALDI-TOF and MS/MS. The deduced peptides were identified as frenatin 4.1 (GFLEKLKTGAKDFASAFVNSIKGT) and a post-translationally modified peptide, frenatin 4.2 (GFLEKLKTGAKDFASAFVNSIK.NH2). Antimicrobial activity of the peptides was assessed by determining their minimal inhibitory concentrations (MICs) using standard model microorganisms. Through studying structure–activity relationships, analogues of the two peptides were designed, resulting in synthesis of frenatin 4.1a (GFLEKLKKGAKDFASALVNSIKGT) and frenatin 4.2a (GFLLKLKLGAKLFASAFVNSIK.NH2). Both analogues exhibited improved antimicrobial activities, especially frenatin 4.2a, which displayed significant enhancement of broad spectrum antimicrobial efficiency. The peptide modifications applied in this study, may provide new ideas for the generation of leads for the design of antimicrobial peptides with therapeutic applications.

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P=0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P=0.001), sustained into adolescence (P=0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P=0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P=0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P=0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion

Collagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.

Tear Secretion Induced by Selective Stimulation of Corneal and Conjunctival Sensory Nerve Fibers

Purpose. To measure the increase in tear secretion evoked by selective stimulation of the different populations of sensory receptors of the cornea and conjunctiva by using moderate and intense mechanical, chemical, and cold stimuli. Methods. Six healthy subjects participated in the study. Tear secretion was measured in both eyes by the Schirmer’s test conducted under control conditions and after stimulation of the center of the cornea and the temporal conjunctiva with a gas esthesiometer. Mechanical stimulation consisted in three pulses of 3 seconds’ duration of warmed air (at 34°C on the eye surface) applied at moderate (170 mL/min) and high (260 mL/min) flow rates. Cold thermal stimulation was made with cooled air that produced a corneal temperature drop of −1°C or −4.5°C. Chemical (acidic) stimulation was performed with a jet of gas containing a mixture of 80% CO2 in air. Results. The basal volume of tear secretion increased significantly (P < 0.05, paired t-test) after stimulation of the cornea with high-flow mechanical stimuli (260 mL/min), intense cooling pulses (−4.5°C), and chemical stimulation (80% CO2). The same stimuli were ineffective when applied to the conjunctiva. Moderate mechanical (170 mL/min) and cold (−1°C) stimulation of the cornea or the conjunctiva did not change significantly the volume of tear secretion. Conclusions. Reflex tear secretion caused by corneal stimulation seems to be chiefly due to activation of corneal polymodal nociceptors, whereas selective excitation of corneal mechanonociceptors or cold receptors appears to be less effective in evoking an augmented lacrimal secretion. Conjunctival receptors stimulated at equivalent levels do not evoke an increased tear secretion.

Role of phospholipase A(2) and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Clostridium difficile-associated disease causes diarrhea to fulminant colitis and death. We investigated the role of phospholipase A(2) (PLA(2)) inhibitors, aristolochic acid (AA), bromophenacyl bromide BPB and quinacrine (QUIN) on the C. difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion. Toxin A caused severe hemorrhagic and inflammatory fluid secretion at 6-8 h in rabbit ileal segments, an effect that was significantly inhibited by QUIN (71%, P < 0.01), AA (87%, P < 0.0001) or by BPB (51%, P < 0.01). The secretory effect of toxin A was also inhibited in segments adjacent to those with AA (89%, P < 0.01). Furthermore, QUIN or AA substantially reduced the histologic damage seen after 6-8 h in rabbit ileal segments. The cyclooxygenase inhibitor, indomethacin, also significantly inhibited (96%; n = 6) the secretory effects of toxin A in ligated rabbit intestinal segments. The destruction by toxin A of F-actin at the light junctions of T-84 cell monolayers was not inhibited by AA or BPB. AA or QUIN had no effect on the T-84 cell tissue resistance reduction over 8-24 h after toxin A exposure. All the inhibitors were shown to be effective in the doses administered direct in ileal loops to inhibit PLA(2) activity. The data suggest that PLA(2) is involved in the major pathway of toxin A-induced histologic inflammatory damage and hemorrhagic fluid secretion. Cop. right (C) 2008 John Wiley & Sons, Ltd.