Proteomic evaluation of sheep serum proteins

Background

Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation.

Results

A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours.

Conclusions

These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis.     

Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe-2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe-2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe-S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe-2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe-2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe-S clusters or in the regulation of the chloroplastic Fe-S cluster assembly machinery.     

Presynaptic functional trkB receptors mediate the release of excitatory neurotransmitters from primary afferent terminals in lamina II (substantia gelatinosa) of postnatal rat spinal cord

A subset of primary sensory neurons produces BDNF, which is implicated in control of nociceptive neurotransmission. We previously localized full-length trkB receptors on their terminals within lamina II. To functionally study these receptors, we here employed patch-clamp recordings, calcium imaging and immunocytochemistry on slices from 8-12 days post-natal rats. In this preparation, BDNF (100-500 ng/mL) enhances the release of sensory neurotransmitters (glutamate, substance P, CGRP) in lamina II by acting on trkB receptors expressed by primary afferent fibers of the peptidergic nociceptive type (PN-PAFs). Effect was blocked by trk antagonist K252a or anti-trkB antibody clone 47. A pre-synaptic mechanism was demonstrated after (i) patch-clamp recordings where the neurotrophin induced a significant increase in frequency, but not amplitude, of AMPA-mediated mEPSCs, (ii) real time calcium imaging, where sustained application of BDNF evoked an intense response in up to 57% lamina II neurons with a significant frequency rise. Antagonists of ionotropic glutamate receptors and NK(1) receptors completely inhibited the calcium response to BDNF. Reduction of CGRP (a specific marker of PN-PAFs) and substance P content in dorsal horn following BDNF preincubation, and analysis of the calcium response after depletion with capsaicin, confirmed that the neurotrophin presynaptically enhanced neurotransmitter release from PN-PAFs. This is the first demonstration that trkB receptors expressed by PN-PAF terminals in lamina II are functional during postnatal development. Implications of this finding are discussed considering that BDNF can be released by these same terminals and microglia, a fraction of which (as shown here) contains BDNF also in unactivated state.     

Conflicting human interests over the re-introduction of endangered wild dogs in South Africa

In South Africa, a plan was launched to manage separate sub-populations of endangered African wild dogs (Lycaon pictus) in several small, geographically isolated, conservation areas as a single meta-population. This intensive management approach involves the re-introduction of wild dogs into suitable conservation areas and periodic translocations among them. To assess the attitudes towards re-introduced wild dogs, we conducted a questionnaire survey of multiple stakeholders—local community members, private landowners and tourists—in and around Hluhluwe-iMfolozi Park (HiP), one of the meta-population conservation areas. Here, we document conflicting human interests over the re-introduced wild dogs. Tourists in HiP, on the one hand, expressed overwhelmingly positive opinions about wild dogs across personal details of the respondents, but especially after having seen free-ranging wild dogs. On the other hand, we found misconceptions and perceptions that were more negative among the rural population around HiP, again largely independent of personal details of the participants, although educated respondents voiced more favourable views of wild dogs. These negative attitudes were in particular due to perceived and real threats of livestock losses. In a follow-up questionnaire survey, we also discovered apparent shortcomings of a previous short-lived conservation education programme among the local communities adjacent to HiP. Consequently, the mitigation of the conflict between wild dogs and rural people requires an understanding of the conditions under which livestock predation occurs, the encouragement of practices that prevent such predation, and increasing local tolerance of co-existence with wild dogs through both economic and non-monetary incentive schemes as well as continued conservation education.     

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a β-adrenergic agonist with an affinity higher for receptor expressed in the heart (β₁) than for receptors expressed in the arteries (β₂). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dt_min), at baseline conditions and under increasing doses of dobutamine.As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dt_max and dP/dt_min. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.     

Sequential inoculum of <Emphasis Type="Italic">Hanseniaspora guilliermondii</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for winemaking Campanino on an industrial scale

In this study, the effect of sequential inoculation with non-Saccharomyces (Hanseniaspora guilliermondii) and Saccharomyces cerevisiae yeast on the distinctive characteristics of the Campanino white wine was investigated. For this purpose, three independent winemaking experiments were carried out on an industrial scale (batches A, B and C). In detail, the first one was carried out using the sequential inoculation technique while the other two, using a S. cerevisiae single-strain starter or no inoculation representing the control batches. Microbiological and chemical parameters and sensorial profiles of the wines were defined. Interestingly, the results showed that when sequential cultures (H. guilliermondii in a sequential mixture with S. cerevisiae) were used, a better wine aroma and quality was observed. More specifically, the wine obtained by sequential inoculation showed lower acetic acid values and enhanced volatile profiles than the wine from the control batches. Finally, sensorial analysis confirmed that the sequential cultures led to an improvement in wine flavour. Therefore, results suggest that the sequential inoculation using non-Saccharomyces and Saccharomyces yeast represents a biotechnological practice that can improve the quality features of traditional white wine. It has been shown for the first time that on an industrial scale H. guilliermondii could be used in sequential inoculum with S. cerevisiae in making white Campanino wine.

Graphical abstract

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Lysophosphatidic acid‐triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary‐like structures, migration, and proliferation. The HTR‐8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR‐8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein‐coupled receptors, LPA₁ and LPA₃, were expressed in HTR‐8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA₃. In addition, cyclooxygenase‐2 and inducible nitric oxide synthase pathways participated in LPA‐stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase‐2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration‐dependent manner. Finally, we observed that cyclooxygenase‐2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPA‐triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal‐fetal interface.     

Effects of 2.45 GHz microwave fields on liposomes entrapping glycoenzyme ascorbate oxidase: evidence for oligosaccharide side chain involvement.

Previous observations reported by our group indicate that 2.45 GHz microwave fields at specific absorption rate (SAR) of 5.6 W/kg reduce the enzyme activity rate of ascorbate oxidase (AO) trapped in liposomes. In this study, we report dose-response studies on these AO containing liposomes irradiated at different SAR values (1.4, 2.8, 4.2, and 5.6 W/kg). No response was observed for SAR below 5.6 W/kg. Liposomes entrapping functional AO in its deglycated form (AO-D) were also used. In this case, no MW related enzyme activity changes were observed, demonstrating a direct involvement of oligosaccharide chains of AO. Furthermore, the catalytic properties of both AO and AO-D were not impaired by MW irradiation, neither in homogeneous solution nor loaded in liposomes, excluding possible changes in the conformation of enzyme as a mechanism. Our results suggest that the oligosaccharide chains of AO are critical to elicit the microwave observed effects on lipid membrane.