Proteomic characterization by 2-DE in bovine serum and whey from healthy and mastitis affected farm animals

Acute phase proteins (APP) have been identified in whey and sera from healthy and mastitis cows through the proteomic analysis using two-dimensional electrophoresis (2-DE) coupled with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Although normal and mastitis serum samples show relatively similar protein composition, marked differences in expression levels and patterns can be observed. Conversely, normal and mastitis whey showed a very different composition, likely due to extravasation of blood proteins to the mammary gland. Different isoforms from the most abundant protein in milk, casein, were detected in both normal and mastitis whey. Other proteins, such as lactotransferrin, were only detected in the inflamed animal samples. Immunoglobulins showed different patterns but not increased levels in the inflamed whey. Also, many cellular proteins in mastitis cow's whey, that were absent from healthy cow's milk. They are responsible for the great change in composition between normal and mastitis whey, especially those which exert a biological function related to immune defense. Data collected in this work are of interest for gaining information about physiological changes in protein patterns in different fluids and, the correspondent modifications as result of an acute phase process in farm. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Different expression of adrenoceptors and GRKs in the human myocardium depends on heart failure ethiology and correlates to clinical variables

Downregulation of β(1)- adrenergic receptors (β(1)-ARs) and increased expression/function of G-protein-coupled receptor kinase 2 (GRK2) have been observed in human heart failure, but changes in expression of other ARs and GRKs have not been established. Another unresolved question is the incidence of these compensatory mechanisms depending on heart failure etiology and treatment. To analyze these questions, we quantified the mRNA/protein expressions of six ARs (α(1A), α(1B), α(1D), β(1), β(2), and β(3)) and three GRKs (GRK2, GRK3, and GRK5) in left (LV) and right ventricle (RV) from four donors, 10 patients with ischemic cardiomyopathy (IC), 14 patients with dilated cardiomyopathy (DC), and 10 patients with nonischemic, nondilated cardiopathies (NINDC). We correlated the changes in the expressions of ARs and GRKs with clinical variables such as left ventricular ejection fraction (LVEF) and left ventricular end-systolic and left ventricular end-diastolic diameter (LVESD and LVEDD, respectively). The main findings were 1) the expression of the α(1A)-AR in the LV positively correlates with LVEF; 2) the expression of GRK3 and GRK5 inversely correlates with LVESD and LVEDD, supporting previous observations about a protective role for both kinases in failing hearts; and 3) β(1)-AR expression is downregulated in the LV and RV of IC, in the LV of DC, and in the RV of NINDC. This difference, better than an increased expression of GRK2 (not observed in IC), determines the lower LVEF in IC and DC vs. NINDC.     

Mycobacterium bovis in wildlife in France

In early 2001, tuberculosis-like lesions were detected in three hunter-killed red deer (Cervus elaphus) in the Brotonne Forest (Normandy, France), and Mycobacterium bovis was isolated. In subsequent hunting seasons, two surveys were conducted in the area. In the first survey (2001-02 hunting season), nine (13%) of 72 red deer sampled were positive for M. bovis. In the 2005-06 hunting season, the prevalence of M. bovis infection increased to 24% (chi2=3.85, df=1, P=0.05; 33 positive among 138 sampled). The prevalence remained stable in juveniles, but it increased significantly in adults: from 13% in 2001-02 to 32% in 2005-06 (chi2=5.13, df=1, P=0.02). Wild boar (Sus scrofa) were heavily infected in both surveys. One roe deer (Capreolus capreolus) and one red fox (Vulpes vulpes) also tested positive in the second survey. Mycobacterium bovis was not isolated from Eurasian badgers (Meles meles). Spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat analysis demonstrated that all M. bovis strains isolated from wildlife were of the same genotype. Thus, the wildlife outbreak involved only a single strain, and this strain was the same as that circulating in nearby cattle herds since 1995. Sensitivity, specificity, and predictive values of the presence of macroscopic lesions as a diagnostic criterion were evaluated from the data obtained from red deer. Necropsy seems to be satisfactory as a routine tool to monitor the disease in wild red deer populations in which bovine tuberculosis has become established.     

Derivatization of biomolecules for chemiluminescent detection in capillary electrophoresis

An overview is presented on the power and drawbacks of the relatively unfamiliar chemiluminescence-based detection technique applied in analysis by capillary electrophoresis, for determining chemically derivatized biomolecules. Examples of the most common systems are given for many series of biologically active compounds as well as for some pharmaceuticals. The most common chemiluminescent systems include the application of peroxyoxalate ester chemiluminescence, acridinium esters, luminol and derivatives, detection based on the tris(2,2'-bipyridine)ruthenium(III) system, the huge potentials offered by direct oxidations-though often with still unelucidated reaction mechanisms-and the powerful area of bioluminescence techniques, revealing as well the fast developing area of microchip-based analysis employing this specific luminescence principle.     

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans

The F₁F₀ complex of Paracoccus denitrificans (PdF₁F₀) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF₁F₀-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur. J. Biochem. 267, 993–1000) but its effect on the PdF₁F₀-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and ³²Pi ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K _0.5 for Mg²⁺-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC₅₀ of Mg²⁺-ADP for inhibition of the PdF₁F₀-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF₁F₀ complex. These effects are not due to membrane or PdF₁F₀ uncoupling. Kinetic and structural modifications that could account for these results are discussed.     

Stomatal response functions to environmental stress of dominant species in the tropical Andean páramo

ABSTRACT

Background: Stomatal response functions of dominant plant species can provide insights into the behaviour of ecosystems under environmental stress, and provide tools for modelling their response to climate change. However, they remain little studied in tropical Alpine ecosystems.

Aims: Our objective was to formulate and compare stomatal response functions for two dominant páramo species with different adaptive strategies to drought, the stress-tolerant shrub Hypericum laricifolium, and the stress avoiding giant rosette Espeletia schultzii and thus enable making projections as to their future fitness in a changing climate.

Methods: A reanalysis of data found in the literature and new ecophysiological and micrometeorological measurements were used to fit and test new stomatal response functions to environmental variables for these two species.

Results: The response functions of vapour pressure differences between leaf and air showed an exponential decrease for both species, while for photosynthetically active radiation (PAR), peak-form response functions provided the best fit. The response function for leaf water potential was linear for the drought-tolerant shrub and decreased exponentially for the stress avoiding giant rosette. Several thresholds prior to stomatal closure were also included in the functions.

Conclusions: Although stress-avoiding and stress-tolerant strategies are both successful in the Andean páramo, the response functions suggest that the tolerant shrub could be more resistant to more intense drought.     

Mitochondrial ATPase activity and membrane fluidity changes in rat liver in response to intoxication with Buckthorn (Karwinskia humboldtiana)

Background

Karwinskia humboldtiana (Kh) is a poisonous plant of the rhamnacea family. To elucidate some of the subcellular effects of Kh toxicity, membrane fluidity and ATPase activities as hydrolytic and as proton-pumping activity were assessed in rat liver submitochondrial particles. Rats were randomly assigned into control non-treated group and groups that received 1, 1.5 and 2 g/Kg body weight of dry powder of Kh fruit, respectively. Rats were euthanized at day 1 and 7 after treatment.

Results

Rats under Kh treatment at all dose levels tested, does not developed any neurologic symptoms. However, we detected alterations in membrane fluidity and ATPase activity. Lower dose of Kh on day 1 after treatment induced higher mitochondrial membrane fluidity than control group. This change was strongly correlated with increased ATPase activity and pH gradient driven by ATP hydrolysis. On the other hand, membrane fluidity was hardly affected on day 7 after treatment with Kh. Surprisingly, the pH gradient driven by ATPase activity was significantly higher than controls despite an diminution of the hydrolytic activity of ATPase.

Conclusions

The changes in ATPase activity and pH gradient driven by ATPase activity suggest an adaptive condition whereby the fluidity of the membrane is altered.     

Podocalyxin Is a Marker of Poor Prognosis in Pancreatic Ductal Adenocarcinoma

Aim of the Study

Podocalyxin-like 1 is a transmembrane glyco-protein whose overexpression associates in many cancers with poor prognosis and unfavorable clinicopathological characteristics. Until now, its prognostic value has never been studied in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to investigate podocalyxin expression in PDAC by a novel monoclonal antibody and a commercially available polyclonal antibody.

Patients and Materials

With tissue microarrays and immuno-histochemistry, podocalyxin expression evaluation involved 168 PDAC patients. The associa-tions of the podocalyxin tumor expression with clinicopathological variables were explored by Fisher’s exact test and the linear-by-linear test. Survival analyses were by Kaplan-Meier anal-ysis and the Cox proportional hazard model.

Results

The polyclonal antibody revealed membranous podocalyxin expression in 73 (44.0%) specimens and the monoclonal antibody was highly expressed in 36 (21.8%) cases. Membranous expression by the polyclonal antibody was associated with T classification (p=0.045) and perineural invasion (p=0.005), and high expression by the mono-clonal antibody with poor differentiation (p=0.033). High podocalyxin expression associated significantly with higher risk of death from PDAC by both the polyclonal antibody (hazard ratio (HR) = 1.62; 95% confidence interval (CI) 1.12-2.33; p=0.01) and the monoclonal antibody (HR = 2.10, 95% CI 1.38-3.20; p<0.001). The results remained significant in multivariate analysis, adjusted for age, gender, stage, lymph node ratio (≥/< 20%), and perivascular invasion (respectively as HR = 2.03; 95% CI 1.32-3.13, p=0.001; and as HR = 2.36; 95% CI 1.47-3.80, p<0.001).

Conclusion

We found podocalyxin to be an independent factor for poor prognosis in PDAC. To our knowledge, this is the first such report of its prognostic value.