Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Lipidomic profile as a noninvasive tool to predict endometrial receptivity

For the present study we asked whether the endometrial fluid lipidomic may be a useful approach to predict endometrial receptivity in freeze‐all cycles. For this case‐control study, endometrial fluid samples were collected from 41 patients undergoing freeze‐all cycles. Samples were split depending on the pregnancy outcome: positive group (n = 24) and negative group (n = 17). Data were acquired by the matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS‐DA) were applied. A list of potential biomarker ion ratios was obtained and the values were used to build a receiver operating characteristic (ROC) curve to predict pregnancy success. The lipid categories were attributed by LIPID MAPS database. Ion ratios were established according to their correlations and used for the analysis. The PCA showed a tendency of separation between the studied groups, whereas the PLS‐DA was able to clearly distinguish them. Fifteen ratios (13 hyper‐represented in the negative and two hyper‐represented in the positive group) were selected according to their importance for model prediction. These ratios were used to build the ROC curve, which presented an area under curve of 84.0% (95%CI: 69.2–97.4%; p = 0.009). These findings suggest that lipidomic profiling of endometrial fluid may be a valuable tool for identifying the time interval comprising the window of implantation.     

Adaptations against heme toxicity in blood-feeding arthropods

A blood-sucking habit appeared independently several times in the course of arthropod evolution. However, from more than a million species of insects and arachnids presently living on earth, only about 14,000 species developed the capacity to feed on vertebrate blood. This figure suggests the existence of severe physiological constraints for the evolution of hematophagy, implying the selective advantage of special adaptations related to the use of blood as a food source. Digestion of vertebrate hemoglobin in the midgut of blood-feeding arthropods results in the production of large amounts of heme, a potentially cytotoxic molecule. Here we will review mechanisms by which heme can exert biological damage, together with a wide spectrum of adaptations developed by blood-feeding insects and ticks to counteract its deleterious effects. In spite of the existence of a great molecular diversity of protective mechanisms, different hematophagous organisms developed convergent solutions that may be physiologically equivalent.     

Analysis of the subproteomes of proteinases and heparin‐binding toxins of eight Bothrops venoms

Viperid snakes show the most complex snake‐venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2‐DE, and their subproteomes of proteinases were explored by 2‐D immunostaining and 2‐D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti‐coagulant and anti‐inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high‐affinity for heparin as composed of mainly serine proteinases and C‐type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin‐binding toxins. Only the non‐bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin‐bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.     

Astaxanthin ameliorates the redox imbalance in lymphocytes of experimental diabetic rats

Diabetes mellitus is a syndrome of impaired insulin secretion/sensitivity and frequently diagnosed by hyperglycemia, lipid abnormalities, and vascular complications. The diabetic ‘glucolipotoxicity’ also induces immunodepression in patients by redox impairment of immune cells. Astaxanthin (ASTA) is a pinkish-orange carotenoid found in many marine foods (e.g. shrimp, crabs, salmon), which has powerful antioxidant, photoprotective, antitumor, and cardioprotective properties. Aiming for an antioxidant therapy against diabetic immunodepression, we here tested the ability of prophylactic ASTA supplementation (30 days, 20 mg ASTA/kg BW) to oppose the redox impairment observed in isolated lymphocytes from alloxan-induced diabetic Wistar rats. The redox status of lymphocytes were thoroughly screened by measuring: (i) production of superoxide (O₂^?), nitric oxide (NO), and hydrogen peroxide (H₂O₂); (ii) cytosolic Ca²⁺; (iii) indexes of oxidative injury; and (iv) activities of major antioxidant enzymes. Hypolipidemic and antioxidant effects of ASTA in plasma of ASTA-fed/diabetic rats were apparently reflected in the circulating lymphocytes, since lower activities of catalase, restored ratio between glutathione peroxidase and glutathione reductase activities and lower scores of lipid oxidation were concomitantly measured in those immune cells. Noteworthy, lower production of NO and O₂^? (precursors of peroxynitrite), and lower cytosolic Ca²⁺ indicate a hypothetical antiapoptotic effect of ASTA in diabetic lymphocytes. However, questions are still open regarding the proper ASTA supplementation dose needed to balance efficient antioxidant protection and essential NO/H₂O₂-mediated proliferative capacities of diabetic lymphocytes.     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

Trimetazidine improves left ventricular function in diabetic patients with coronary artery disease: a double-blind placebo-controlled study

Background

Patients with diabetic cardiomyopathy have an impaired myocardial glucose handling and distal distribution of coronary atherosclerosis. Trimetazidine, an anti-ischemic metabolic agent, improves myocardial glucose utilization though inhibition of fatty acid oxidation. Aim of the present study was to evaluate whether the metabolic effect of trimetazidine on left ventricular function in patients with diabetic cardiomyopathy.

Methods

32 patients (24 males and 8 females, mean (SE) age = 67 ± 6 years) with type 2 diabetes and ischemic cardiomyopathy were randomized to receive either trimetazidine (20 mg, t.d.s.) or placebo (t.d.s.) for six months in a randomized parallel study. Patients performed an echocardiogram at baseline and after 6 months.

Results

Demographic data were comparable between the two groups. After six month baseline left ventricular end-diastolic diameters increased from 62.4 ± 1.7 to 63 ± 2.1 mm in the placebo group, while decreased from 63.2 ± 2.1 to 58 ± 1.6 mm (p < 0.01 compared to baseline) in the trimetazidine group. Compared to baseline, left ventricular ejection fraction increased by 5.4 ± 0.5% (p < 0.05) in the trimetazidine group while remained unchanged in the placebo group -2.4 ± 1.1% (NS), p < 0.01 between groups. A significant improvement in wall motion score index and in the E/A wave ratio was detected in patients treated with trimetazidine, but not in those receiving placebo.

Conclusion

in diabetic patients with ischemic heart disease trimetazidine added to standard medical therapy has beneficial effect on left ventricular volumes and on left ventricular ejection fraction compared to placebo. This effect may be related to the effect of trimetazidine upon cardiac glucose utilization.     

Cell cycle -dependent proteolysis in plants. Identification Of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor mg132

It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable.     

Role of calcium in signal transduction during the hypersensitive response caused by basidiospore-derived infection of the cowpea rust fungus

The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea-cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection.