Female reproductive behaviour,ovarian hormones and vaginal cytology of the induced ovulator, <Emphasis Type="Italic">Ctenomys talarum</Emphasis>

In the present study, we evaluated whether reproductive condition affects female reproductive behaviour in the induced ovulator Ctenomys talarum. We also explored the effect of the interaction with a male on the reproductive condition of females. To evaluate this, we arranged mating trials and evaluated female reproductive behaviour. Reproductive status of females was evaluated using a combined approach of vaginal smears, urinary progesterone and oestradiol, and ovarian histology. Behaviours denoting attraction (‘male sniff’ and ‘mount attempts’) and mutual courtship behaviours (‘spin’ and copula) were correlated with vaginal cytology before and oestradiol and progesterone levels in urine 12 h after male–female encounter. After 24 h of the interaction, oestradiol levels and vaginal epithelization increased while progesterone levels decreased in soliciting females. C. talarum females’ reproductive behaviour was related to its physiological reproductive state and vaginal cytology. The kind of male interaction, whether couples copulated or remained indifferent affected the later status of females. Females are induced ovulators by mating but male presence and interaction also affected other components of their reproductive physiology such as ovarian hormones and vaginal cytology.     

Electrical impedance spectroscopy measurements using a four-electrode configuration improve on-line monitoring of cell concentration in adherent animal cell cultures

This paper describes the improvement in the use of electrical impedance spectroscopy (EIS) for animal cell concentration monitoring of adherent cultures by using a four-electrode configuration instead of the commonly used two-electrode configuration. This four-electrode configuration prevents cell concentration measurements from external masking effects such as the electrode covering ratio, the degree of cellular adherence to the electrodes and the impedance of the measuring electrodes. Cell concentration was monitored using both four-electrode and two-electrode configurations in vero cell and human mesenchymal stem cell cultures in order to analyze the attained improvement in two cell lines with opposite growth characteristics. The experiments performed with vero cell cultures evidenced that the four-electrode configuration enables cell concentration measurements along all culture phases, even once the culture reached cell confluence (over 2×10(5) cells/cm(2)), confirming that this configuration is less effected by all the external influences. The experiments performed with human mesenchymal stem cells demonstrated good sensitivity of the measurement at very low cell concentrations, as well as a very good robustness all over the 12-days experiment. Finally, off-line cell measurements during cell cultures proved good accuracy of impedance measurements carried out with a four-electrode configuration along all cell growth phases, enabling determination of relevant cell growth parameters.     

Essential oils from aromatic herbs as antimicrobial agents

Bacterial resistance to multiple antibiotics is a health problem. Essential oils (EOs) possess antibacterial properties and have been screened as potential sources of novel antimicrobial compounds. Terpenes and terpenoids are components derived from EOs. Some of these EOs show inhibitory activity against Staphylococcus aureus. Carvacrol has specific effects on S. aureus and Staphylococcus epidermidis. Perilla oil suppresses expression of α-toxin, Staphylococcus enterotoxin A and B and toxic shock syndrome toxin. Geraniol shows good activity in modulating drug resistance in several gram-negative species. EOs could act as biopreservatives, reducing or eliminating pathogenic bacteria and increasing the overall quality of animal and vegetable food products. Although clinical studies are scarce, the uses of EOs for topical administration and as penetration enhancers for antiseptics are promising. Little information exists for oral administration.     

PpiA, a surface PPIase of the cyclophilin family in Lactococcus lactis

Background

Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes.

Results

In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H₂O₂) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H₂O₂. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities.

Conclusions

Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.     

Dual role of respiratory syncytial virus glycoprotein fragment as a mucosal immunogen and chemotactic adjuvant

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.     

Relationships of hepatic and pancreatic biomarkers with the cholestatic syndrome and tumor stage in pancreatic cancer

We analyzed relationships of hepatic and pancreatic biomarkers with the cholestatic syndrome and tumor stage in exocrine pancreatic cancer (N = 183). Information on laboratory tests and on signs and symptoms was obtained from medical records and patient interviews. Bilirubin, aspartate aminotransferase (AST), γ-glutamyltransferase (GGT) and alkaline phosphatase were lower in tumor stage IV. The association was due to the relationship between cholestatic syndrome and earlier presentation of patients. There was no association between hepatic biomarkers and stage when adjusting by cholestatic syndrome. Relationships of hepatic and pancreatic biomarkers with pancreatic symptoms and tumor stage must be controlled in "-omics" and other studies using biomarkers.     

Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.     

LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

Actin exists as a monomer (G-actin) which can be polymerized to filaments) F-actin) that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl₂ or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.