Naloxone and TRH affect regional blood flows in the anesthetized rabbit

The cardiovascular effects of IV naloxone and a subsequent administration of TRH IV were studied in the rabbit. Naloxone caused a vasodilation in the myocardium and adrenal glands. Naloxone elicited an increment in cerebral blood flow in several regions which attenuated the cerebrovasodilating effect of TRH in a few regions. The blockade of endogenous opioids with naloxone did not modify the peripheral vasoconstricting effect of TRH or affect the vascular effects of TRH mediated by the peripheral sympathetic nerves. The results indicate that naloxone has a vasodilating effect in the myocardium and CNS in anesthetized rabbits. The major part of the cardiovascular effect of TRH is not dependent on mechanisms sensitive to naloxone.     

Aging bone marrow mesenchymal stromal cells have altered membrane glycerophospholipid composition and functionality

Human mesenchymal stem/stromal cells (hMSC) are increasingly used in advanced cellular therapies. The clinical use of hMSCs demands sequential cell expansions. As it is well established that membrane glycerophospholipids (GPL) provide precursors for signaling lipids that modulate cellular functions, we studied the effect of the donor''s age and cell doublings on the GPL profile of human bone marrow MSC (hBMSC). The hBMSCs, which were harvested from five young and five old adults, showed clear compositional changes during expansion seen at the level of lipid classes, lipid species, and acyl chains. The ratio of phosphatidylinositol to phosphatidylserine increased toward the late-passage samples. Furthermore, 20:4n-6-containing species of phosphatidylcholine and phosphatidylethanolamine accumulated while the species containing monounsaturated fatty acids (FA) decreased during passaging. Additionally, in the total FA pool of the cells, 20:4n-6 increased, which happened at the expense of n-3 polyunsaturated FAs, especially 22:6n-3. The GPL and FA correlated with the decreased immunosuppressive capacity of hBMSCs during expansion. Our observations were further supported by alterations in the gene expression levels of several enzymes involved in lipid metabolism and immunomodulation. The results show that extensive expansion of hBMSCs harmfully modulates membrane GPLs, affecting lipid signaling and eventually impairing functionality.     

Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells)

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.     

A novel automated fluorometric assay to evaluate sperm viability and fertility in dairy bulls

The artificial insemination (AI) industry is in need of an objective and rapid, but inexpensive method to evaluate frozen thawed bull semen ejaculates. This study presents a new fluorescence method that uses an automatized fluorometer and fluorophore stain propidium iodide that stains only those cells with damaged membranes. The fluorescence of the semen sample and the totally killed subsample were measured simultaneously, and viability was calculated. Every semen batch was analyzed before use in AI. For fertility evaluation, the nonreturn rates (NR%) obtained from 92,120 inseminations with the analyzed batches were recorded from 166 bulls (436 batches). This study confirms a 3.9% better NR% for the Finnish Holstein-Friesian breed than for Finnish Ayrshire. There was a clear seasonality in NR%: it differed (5.3%) significantly, being best in summer to autumn (June to October) and lowest in winter (January to March). The fluorometer method was fast and easy. The correlation between the total number of viable spermatozoa in an insemination dose and field fertility was low but significant (r = 0.051, P = 0.016), suggesting that the plasma membrane integrity evaluation can serve as a cost-beneficial quality control method of frozen-thawed semen at bull stations.     

Spectrum of styrene-induced DNA adducts: the relationship to other biomarkers and prospects in human biomonitoring

Styrene is an important industrial chemical that has shown genotoxicity in many toxicology assays. This is believed to be related to the DNA-binding properties of styrene-7,8-oxide (SO), a major metabolite of styrene. In this review, we have summarized knowledge on various aspects of styrene genotoxicity, especially in order to understand the formation and removal of primary DNA lesions, and the usefulness of biomarkers for risk assessment. Biological significances of specific DNA adducts and their role in the cascade of genotoxic events are discussed. Links between markers of external and internal exposure are evaluated, as well as metabolic aspects leading to the formation of DNA adducts and influencing biomarkers of biological effect. Finally, we suggest a design of a population study, which may contribute to our understanding genotoxic events in the exposure either to single xenobiotic or complex mixture.     

Microsatellite data resolve phylogeographic patterns in European grayling, Thymallus thymallus, Salmonidae

The phylogeography of an endangered salmonid, European grayling (Thymallus thymallus), was studied based on analysis of 17 nuclear microsatellite DNA loci. In agreement with earlier mitochondrial DNA (mtDNA) studies, phylogenetic relationships of the populations suggested that northern Europe was colonized from two distinct Pleistocene refugia. Furthermore, microsatellites revealed highly supported grouping of mainland Swedish, Norwegian, Danish, German and Slovenian populations, suggesting that grayling from the northwestern and central Europe have descended from their southern conspecifics. The level of divergence between populations was substantial, even across short geographical distances. Although this was in part due to postglacial colonization patterns and contemporary barriers for gene flow, the high divergence estimates between hydrologically connected sampling locations implied efficient interpopulation reproductive isolation. Microsatellites revealed that the populations exhibited, on average, only 3.5 (+/-2.2) alleles per locus, indicating that T. thymallus has strikingly low levels of intrapopulation genetic diversity as compared with other freshwater fish species. Accordingly, as indicated by analysis of molecular variance (AMOVA), only 49.1-58.0% of the total grayling microsatellite diversity resided within populations. A latitudinal genetic diversity gradient, potentially resulting from glaciation-mediated founder events, was not evident. Alternatively, it is possible that grayling display limited dispersal behaviour/capability, leading to low long-term effective population sizes and, consequently, depauperate intrapopulation polymorphism. These findings have implications for conservation of T. thymallus. Importantly, they exemplify that microsatellites can be highly informative for intraspecific phylogeography studies dealing with substantial divergence scales.