Perturbation in liver mitochondrial Ca2+ homeostasis in experimental iron overload: a possible factor in cell injury

The functional state of isolated mitochondria and specifically the integrity of the inner membrane, were investigated in the liver of rats made siderotic by dietary supplementation with carbonyl iron. The concentration of iron in the hepatic tissue increased progressively up to nearly 40 days and reached a steady-state level. When the iron content reached a threshold value (higher than 90 nmol/mg protein) the occurrence of in vivo lipid peroxidation in the mitochondrial membrane was detected. This process did not result in gross alterations in the mitochondrial membrane, as indicated by electron microscopy, phosphorylative capability and membrane potential measurements. On the contrary, the induction of lipoperoxidative reaction appeared to be associated with the activation of Ca2+ release from mitochondria. This was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine nucleotides. The induction of this Ca2+ release from iron-treated mitochondria resulted in enhancement of Ca2+ cycling, a process which dissipates energy to reaccumulate into mitochondria the released Ca2+. The perturbation in mitochondrial Ca2+ homeostasis reported here may be a factor in the onset of cell damage in this experimental model of hepatic iron overload.     

Zinc content of normal human serum and its correlation with some hematic parameters

Numerous studies have demonstrated that several diseases and stress conditions are associated with changes in the levels of zinc in the blood plasma and cellular elements. In this research the association between serum zinc concentrations and other hematic parameters of diagnostic interest has been evaluated. Quantitative determinations of zinc, total plasmatic proteins, albumin, hemoglobin and calculation of mean corpuscular volume were performed on blood samples from 58 males aged 20–61 years. Concentrations measured in our sample are comparable with reference values. Statistically significant correlation coefficients were found between age and albumin (r = - 0.562, P < 0.001), serum zinc and albumin (r = 0.328, P < 0.05), serum zinc and hemoglobin (r = 0.291, P < 0.05), and total plasmatic proteins and albumin (r = 0.463, P < 0.001). These correlation coefficients were significant even after adjustment for age effect. The determination of serum zinc concentration may be useful in the assessment of clinical scenarios. Particularly, it may provide additional information for the diagnosis of specific pathologies, such as hepatic malfunctions. It could also be useful in the identification of different stages of anemia.     

Peroxidation and phospholipase A2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine.

The relationship between lipid peroxidation and phospholipase A2 (PLA2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37 degrees C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA2.     

Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion

BACKGROUND: The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts. METHODS: By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy. RESULTS: Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer. CONCLUSIONS: These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.     

Population size and identity influence the reaction norm of the rare,endemic plant Cochlearia bavarica across a gradient of environmental stress

Habitat degradation and loss can result in population decline and genetic erosion, limiting the ability of organisms to cope with environmental change, whether this is through evolutionary genetic response (requiring genetic variation) or through phenotypic plasticity (i.e., the ability of a given genotype to express a variable phenotype across environments). Here we address the question whether plants from small populations are less plastic or more susceptible to environmental stress than plants from large populations. We collected seed families from small (<100) versus large natural populations (>1,000 flowering plants) of the rare, endemic plant Cochlearia bavarica (Brassicaceae). We exposed the seedlings to a range of environments, created by manipulating water supply and light intensity in a 2 x 2 factorial design in the greenhouse. We monitored plant growth and survival for 300 days. Significant effects of offspring environment on offspring characters demonstrated that there is phenotypic plasticity in the responses to environmental stress in this species. Significant effects of population size group, but mainly of population identity within the population size groups, and of maternal plant identity within populations indicated variation due to genetic (plus potentially maternal) variation for offspring traits. The environment x maternal plant identity interaction was rarely significant, providing little evidence for genetically- (plus potentially maternally-) based variation in plasticity within populations. However, significant environment x population-size-group and environment x population-identity interactions suggested that populations differed in the amount of plasticity, the mean amount being smaller in small populations than in large populations. Whereas on day 210 the differences between small and large populations were largest in the environment in which plants grew biggest (i.e., under benign conditions), on day 270 the difference was largest in stressful environments. These results show that population size and population identity can affect growth and survival differently across environmental stress gradients. Moreover, these effects can themselves be modified by time-dependent variation in the interaction between plants and their environment.     

Comparative population genetic structure in a plant-pollinator/seed predator system

Comparative analyses of spatial genetic structure of populations of plants and the insects they interact with provide an indication of how gene flow, natural selection and genetic drift may jointly influence the distribution of genetic variation and potential for local co‐adaptation for interacting species. Here, we analysed the spatial scale of genetic structure within and among nine populations of an interacting species pair, the white campion Silene latifolia and the moth Hadena bicruris, along a latitudinal gradient across Northern/Central Europe. This dioecious, short‐lived perennial plant inhabits patchy, often disturbed environments. The moth H. bicruris acts both as its pollinator and specialist seed predator that reproduces by laying eggs in S. latifolia flowers. We used nine microsatellite markers for S. latifolia and eight newly developed markers for H. bicruris. We found high levels of inbreeding in most populations of both plant and pollinator/seed predator. Among populations, significant genetic structure was observed for S. latifolia but not for its pollinator/seed predator, suggesting that despite migration among populations of H. bicruris, pollen is not, or only rarely, carried over between populations, thus maintaining genetic structure among plant populations. There was a weak positive correlation between genetic distances of S. latifolia and H. bicruris. These results indicate that while significant structure of S. latifolia populations creates the potential for differentiation at traits relevant for the interaction with the pollinator/seed predator, substantial gene flow in H. bicruris may counteract this process in at least some populations.     

Functional study of CD4+CD25+ regulatory T cells in health and autoimmune hepatitis

Regulatory CD4(+)CD25(+) T cells (Tregs) are defective numerically and functionally in autoimmune hepatitis (AIH). We have investigated and compared the mechanism of action of Tregs in healthy subjects and in AIH patients using Transwell experiments, where Tregs are cultured either in direct contact with or separated from their targets by a semipermeable membrane. We also studied Treg FOXP3 expression and effect on apoptosis. Direct contact is necessary for Tregs to suppress proliferation and IFN-gamma production by CD4(+)CD25(-) and CD8(+) T cells in patients and controls. Moreover, in both, direct contact of Tregs with their targets leads to increased secretion of regulatory cytokines IL-4, IL-10, and TGF-beta, suggesting a mechanism of linked immunosuppression. Tregs/CD4(+)CD25(-) T cell cocultures lead to similar changes in IFN-gamma and IL-10 secretion in patients and controls, whereas increased TGF-beta secretion is significantly lower in patients. In contrast, in patients, Tregs/CD8(+) T cell cocultures lead to a higher increase of IL-4 secretion. In AIH, Treg FOXP3 expression is lower than in normal subjects. Both in patients and controls, FOXP3 expression is present also in CD4(+)CD25(-) T cells, although at a low level and not associated to suppressive function. Both in patients and controls, addition of Tregs does not influence target cell apoptosis, but in AIH, spontaneous apoptosis of CD4(+)CD25(-) T cells is reduced. In conclusion, Tregs act through a direct contact with their targets by modifying the cytokine profile and not inducing apoptosis. Deficient CD4(+)CD25(-) T cell spontaneous apoptosis may contribute to the development of autoimmunity.     

Sexual conflict over floral receptivity

In flowering plants, the onset and duration of female receptivity vary among species. In several species the receptive structures wilt upon pollination. Here we explore the hypothesis that postpollination wilting may be influenced by pollen and serve as a general means to secure paternity of the pollen donor at the expense of female fitness. Taking a game-theoretical approach, we examine the potential for the evolution of a pollen-borne wilting substance, and for the coevolution of a defense strategy by the recipient plant. The model without defense predicts an evolutionarily stable strategy (ESS) for the production of wilting substance. The ESS value is highest when pollinator visiting rates are intermediate and when the probability that pollen from several donors arrives at the same time is low. This finding has general implications in that it shows that male traits to secure paternity also can evolve in species, such as plants, where mating is not strictly sequential. We further model coevolution of the wilting substance with the timing of stigma receptivity. We assume that pollen-receiving plants can reduce the costs induced by toxic pollen by delaying the onset of stigmatic receptivity. The model predicts a joint ESS, but no female counter-adaptation when the wilting substance is highly toxic. This indicates that toxicity affects the probability that a male manipulative trait stays beneficial (i.e., not countered by female defense) over evolutionary time. We discuss parallels to male induced changes in female receptivity known to occur in animals and the role of harm for the evolution of male manipulative adaptations.     

The effects of inbreeding, genetic dissimilarity and phenotype on male reproductive success in a dioecious plant

Pollen fate can strongly affect the genetic structure of populations with restricted gene flow and significant inbreeding risk. We established an experimental population of inbred and outbred Silene latifolia plants to evaluate the effects of (i) inbreeding depression, (ii) phenotypic variation and (iii) relatedness between mates on male fitness under natural pollination. Paternity analysis revealed that outbred males sired significantly more offspring than inbred males. Independently of the effects of inbreeding, male fitness depended on several male traits, including a sexually dimorphic (flower number) and a gametophytic trait (in vitro pollen germination rate). In addition, full-sib matings were less frequent than randomly expected. Thus, inbreeding, phenotype and genetic dissimilarity simultaneously affect male fitness in this animal-pollinated plant. While inbreeding depression might threaten population persistence, the deficiency of effective matings between sibs and the higher fitness of outbred males will reduce its occurrence and counter genetic erosion.