Effects of pituitary adenylate cyclase-activating polypeptide on the cyclooxygenase pathway of rat platelets and on platelet aggregation

Several data suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of local circulation. One possible role of PACAP in the regulation of circulation is that, it may modify the cyclooxygenase pathway of the arachidonate cascade in platelets. Our study was designed to study the effect of PACAP on the cyclooxygenase pathway of rat platelets and on platelet aggregation. PACAP (10(-7) and 10(-6) M) significantly inhibited the cyclooxygenase pathway of platelets, mostly the thromboxane synthesis. Pretreatment with a PACAP receptor antagonist, PACAP(6-38), or with an inhibitor of protein kinase A, H-89, shows that the effects of PACAP on the cyclooxygenase pathway were diminished. In the aggregation studies, PACAP inhibited both the arachidonic acid-induced and the thrombin-induced platelet aggregation. It can be concluded that PACAP inhibits the cyclooxygenase pathway of rat platelets via a specific PACAP receptor-activated, cAMP-dependent pathway, and these effects of PACAP are involved in the inhibition of platelet aggregation.     

Ecophysiological responses of desiccation-tolerant cryptobiotic crusts

In our present studies, the recovery of photosynthetic activity after rehydration was demonstrated. We measured chlorophyll fluorescence, CO₂ gas exchange and the pigment composition in the previously long-term air-dried cryptogamic inselberg crusts collected from two tropical areas. The cryptobiotic crusts were collected from different localities on similar ecological and climatic conditions from extreme habitats of inselbergs (outcrops). These inselbergs are characterized by a dry microclimate and are covered by scarce soil. We found that the ecophysiological responses of both cryptogamic inselberg crusts showed an extremely high degree of desiccation-tolerance due to the fast and full recovery during rehydration. The photosynthetic activity of the cryptobiotic crusts were restored and regained within 15 and 40 min, respectively, after rehydration. Photosynthetic activity of the crusts was retained at all applied light intensities when enough water was available, however the degree of the recovery was different between the crusts. Photosynthetic pigment contents were strongly and positively correlated with water content. Our results indicated that tropical desiccation-tolerant cryptogamic crusts found on inselberg rock surfaces have CO₂ fixation ability in the range of cyanobacteria and lichens, suggesting that at a global scale they can assimilate CO₂ in a significant amount.     

Occurrence of laccases in lichenized ascomycetes of the Peltigerineae

Following our previous findings of high extracellular redox activity in lichens, the results of the work presented here identify the enzymes involved as laccases. Despite numerous data on laccases in fungi and flowering plants, this is the first report of the occurrence of laccases in lichenized ascomycetes. Extracellular laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from suborder Peltigerineae, 18 displayed laccase activity, while activity was absent in species tested from other lichen groups. Identification of the enzymes as laccases was confirmed by the ability of lichen leachates to readily metabolize substrates such as 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in the absence of hydrogen peroxide, sensitivity of the enzymes to cyanide and azide, the enzymes having typical laccase pH and temperature optima, and an absorption spectrum with a peak at 614 nm. Desiccation and wounding stimulated laccase activity. Laccase activity was not increased after treatment with normal inducers of laccase synthesis, suggesting that they are constitutively expressed. Electrophoresis showed that the active form of laccase from Peltigera malacea was a tetramer with an unusually high molecular mass of 340 kDa and an isoelectric point (pI) of 4.7. The finding of abundant extracellular redox enzymes known to actively produce reactive oxygen species suggest that their roles may include increasing nutrient supply to lichens by delignification, and deterring pathogens by contributing to the oxidative burst. Furthermore, once released into the environment, they may participate in the carbon cycle by facilitating the breakdown or formation of humic substances.     

Optimalized transient transfection of chondrogenic primary cell cultures

We aimed to find a transfection method which provides high efficiency with minimal cytotoxic and/or apoptotic effects for gene transfer into multilayer primary chondrogenic cell cultures. The pEGFP-C1 plasmid was introduced into the cell culture and the efficiency of transformation quantified by GFP fluorescence; the resulting nucleofection was effective but resulted in severe apoptosis. Two liposomal reagents designed to allow transfection into adherent cells did not deliver the plasmids sufficiently and cartilage formation did not occur. In addition, a third liposomal compound, recommended for transfection into either adherent or suspension cell cultures, lead to acceptable transfection efficiency but no cartilage formation. When an amphiphilic reagent was used however, there was acceptable transfection efficiency as well as cartilage formation. The viability of the cells which were transfected using the amphiphilic reagent remained unaffected but proliferation was severely diminished, particularly in the presence of GFP. In addition, the amount of cartilage decreased when GFP was expressed, despite unchanged levels of mRNAs of sox9 and aggrecan core protein, factors reflecting on the efficiency of chondrogenesis. Overexpression of both the constitutively active delta and gamma isoforms of catalytic subunit of calcineurin, a protein phosphatase described as a positive regulator of chondrogenesis, decreased protein level of Sox9 and subsequent cartilage formation. In conclusion, we found that amphiphilic reagent applied prior to the adhesion of cells provides a useful means to transfer plasmids to primary differentiating chondrogenic cells.     

Lithocholic acid,a bacterial metabolite reduces breast cancer cell proliferation and aggressiveness

Our study aimed at finding a mechanistic relationship between the gut microbiome and breast cancer. Breast cancer cells are not in direct contact with these microbes, but disease could be influenced by bacterial metabolites including secondary bile acids that are exclusively synthesized by the microbiome and known to enter the human circulation. In murine and bench experiments, a secondary bile acid, lithocholic acid (LCA) in concentrations corresponding to its tissue reference concentrations (< 1 μM), reduced cancer cell proliferation (by 10–20%) and VEGF production (by 37%), aggressiveness and metastatic potential of primary tumors through inducing mesenchymal-to-epithelial transition, increased antitumor immune response, OXPHOS and the TCA cycle. Part of these effects was due to activation of TGR5 by LCA. Early stage breast cancer patients, versus control women, had reduced serum LCA levels, reduced chenodeoxycholic acid to LCA ratio, and reduced abundance of the baiH (7α/β-hydroxysteroid dehydroxylase, the key enzyme in LCA generation) gene in fecal DNA, all suggesting reduced microbial generation of LCA in early breast cancer.     

Risk Factors and Relationship of Cutaneous and Uveal Melanocytic Lesions in Monozygotic and Dizygotic Twin Pairs

BackgroundThe similar genetic background of a pair of twins, and the similar environmental impacts to which they are exposed allow an exact and objective investigation of various constitutional and environmental factors in naevus development. As far as we are aware, this is the first published survey that simultaneously examines cutaneous and ocular pigmented lesions in an appreciable sample of identical and non-identical twins.Methods172 pairs of twins of Caucasian origin were included in this study. A whole-body skin examination and a detailed ophthalmological examination were performed to determine the density of melanocytic lesions. A standardized questionnaire was used to assess the data relating to constitutional, sun exposure and other variables.ResultsA notably high proportion of the subjects (36.78%) manifested one or more clinically atypical melanocytic naevi (CAMNs), and approximately one-third (31.4%) of them at least one benign uveal pigmented lesion (BUPL). The incidence of iris freckles (IFs), iris naevi (INs) and choroidal naevi (CHNs) proved to be 25.35%, 5.98% and 3.52%, respectively. The interclass correlation coefficients for common melanocytic naevi (CMNs), CAMNs, and INs were 0.77, 0.76 and 0.86 in monozygotic twins, as compared with 0.5, 0.27 and 0.25 in dizygotic twin pairs, respectively. A statistically significant correlation was found between the prevalence of CAMNs and that of INs.ConclusionsThis significant correlation suggests the existence of a subgroup of Caucasian people with an increased susceptibility to both cutaneous and ocular naevus formation. There is accumulating evidence that, besides the presence of cutaneous atypical naevi, INs can serve as a marker of a predisposed phenotype at risk of uveal melanoma. The correlation between cutaneous and ocular pigmented lesions underlines the need for the adequate ophthalmological screening of subjects with CAMNs and INs.     

Drosophila PATJ supports adherens junction stability by modulating Myosin light chain activity

The assembly and consolidation of the adherens junctions (AJs) are key events in the establishment of an intact epithelium. However, AJs are further modified to obtain flexibility for cell migration and morphogenetic movements. Intact AJs in turn are a prerequisite for the establishment and maintenance of apical–basal polarity in epithelial cells. In this study, we report that the conserved PDZ (PSD95, Discs large, ZO-1) domain–containing protein PATJ (Pals1-associated tight junction protein) was not per se crucial for the maintenance of apical–basal polarity in Drosophila melanogaster epithelial cells but rather regulated Myosin localization and phosphorylation. PATJ directly bound to the Myosin-binding subunit of Myosin phosphatase and decreased Myosin dephosphorylation, resulting in activated Myosin. Thereby, PATJ supports the stability of the Zonula Adherens. Notably, weakening of AJ in a PATJ mutant epithelium led first to a loss of Myosin from the AJ, subsequently to a disassembly of the AJ, and finally, to a loss of apical–basal polarity and disruption of the tissue.     

Effects of Kupffer cell blockade on the hepatic expression of metallothionein and heme oxygenase genes in endotoxemic rats with obstructive jaundice

AimsHeme oxygenase (HO) and metallothionein (MT) genes are rapidly upregulated in the liver by pro-inflammatory cytokines and/or endotoxin as protection against cellular stress and inflammation. Gadolinium chloride (GdCl₃)-induced Kupffer cell blockade has beneficial consequences in endotoxemia following bile duct ligation. Herein we further characterized the effects of Kupffer cell inhibition on the activation of the antioxidant defense system (HO and MT gene expressions, and antioxidant enzyme activities) in response to endotoxemia and obstructive jaundice.Main methodsThe isoform-specific expression of MT and HO genes was assessed (RT-PCR) in rat livers following 3-day bile duct ligation, 2-h lipopolysaccharide treatment (1 mg/kg) or their combination, with or without GdCl₃ pretreatment (10 mg/kg, 24 h before endotoxin). Lipid peroxidation, DNA damage and hepatic antioxidant enzyme activities were also assessed.Key findingsAll these challenges induced similar extents of DNA damage, whereas the lipid peroxidation increased only when endotoxemia was combined with biliary obstruction. The MT and HO mRNA levels displayed isoform-specific changes: those of MT-1 and HO-2 did not change appreciably, whereas those of MT-2 and HO-1 increased significantly in 2-h endotoxemia, with or without obstructive jaundice. Among the enzymes reflecting the endogenous protective mechanisms, the catalase and copper/zinc-superoxide dismutase levels decreased, while that of Mn-SOD slightly increased. Interestingly, GdCl₃ alone induced lipid peroxidation, DNA damage and MT-2 expression. In response to GdCl₃, HO-1 induction was significantly lower in each model.SignificanceDespite its moderate hepatocellular toxicity, the ameliorated stress-induced hepatic reactions provided by GdCl₃ may contribute to its protective effects.