Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes

Insulin in the presence of high concentrations of glucose has a beneficial trophic effect on the development of primary cultures of hepatocytes. Compared to the situation observed in hormone-free control cultures, the flattening of the reaggregated hepatocytes is enhanced, and the reconstituted cell trabeculae are enlarged and tend to form a confluent monolayer after 3 days; the survival time is prolonged from 3 to 5 or 6 days. Ultrastructural modifications are also initiated by insulin; numerous glycogen particles appear after 24 h, in between the cisternae of the proliferated smooth endoplasmic reticulum. After 48 h, large amounts of glycogen are stored, and numerous polysomes are present. A small number of cells showed an increased synthesis of lipid droplets in the lumen of the smooth endoplasmic reticulum and form liposomes at the same time. After 72 h, cytolysomes filled with glycogen develop, simulating glycogenosis type II. Simultaneously, microtubules and microfilaments, closely related to numerous polysomes, appear in cytoplasmic extensions constituting undulating membranes. The biochemical data demonstrate that, in the absence of insulin, a high concentration of glucose stimulates glycogenesis and hinders glycogenolysis. This effect of glucose on polysaccharide synthesis is progressively lost. The addition of insulin to the culture induces after 48 and 72 h, a three- to fivefold increase of the glucose incorporation into glycogen, as compared to the controls. The presence of insulin is required to maintain the hepatocyte's capacity to store glycogen. Glycogen synthetase is converted into its active form under the influence of glucose. Insulin increases the rate of activation.     

Estimation of heterozygosity for single-probe multilocus DNA fingerprints

In spite of the increasing application of DNA fingerprinting to natural populations and to the genetic identification of humans, explicit methods for estimation of basic population genetic parameters from DNA fingerprinting data have not been developed. Contributing to this omission is the inability to determine, for multilocus fingerprinting probes, relatively important genetic information, such as the number of loci, the number of alleles, and the distribution of these alleles into specific loci. One of the most useful genetic parameters that could be derived from such data would be the average heterozygosity, which has traditionally been employed to measure the level of genetic variation within populations and to compare genetic variation among different loci. We derive here explicit formulas for both the estimation of average heterozygosity at multiple hypervariable loci and a maximum value for this estimate. These estimates are based upon the DNA restriction-pattern matrices that are typical for fingerprinting studies of humans and natural populations. For several empirical data sets from our laboratory, estimates of average and maximal heterozygosity are shown to be relatively close to each other. Furthermore, variances of these statistics based on simulation studies are relatively small. These observations, as well as consideration of the effect of missing alleles and alternate numbers of loci, suggest that the average heterozygosity can be accurately estimated using phenotypic DNA fingerprint patterns, because this parameter is relatively insensitive to the lack of certain genetic information.      

Dynamics of Sulfate-Reducing Microorganisms (dsrAB genes) in Two Contrasting Mudflats of the Seine Estuary (France)

By combining molecular biology and biochemical approaches, the dynamics of sulfate-reducing microorganisms (SRM) was investigated in the sediments of the Seine estuary (France). Both intertidal mixing-zone and freshwater mudflats were sampled during a 1-year period; the quantification of SRM was realized by using competitive polymerase chain reaction (PCR) based on dsrAB gene amplification, previously described by Leloup et al. (2004), and sulfate reduction rate (SRR) was determined via the SO₄² radiotracer method. Throughout the year, abundance of dsrAB genes and SRR were predominantly high in the top 15 cm of the sediment. A seasonal dynamic was observed; a predominance of activity was noted during the early summer, and seems to be mainly controlled by physical–chemical parameters (temperature and dissolved organic carbon concentration) and topographic evolution of the mudflat (erosion/deposit erosion).     

Differential impact of mouse Rad9 deletion on ionizing radiation-induced bystander effects

The cellular response to ionizing radiation is not limited to cells irradiated directly but can be demonstrated in neighboring "bystander" populations. The ability of mouse embryonic stem (ES) cells to express a bystander effect and the role of the radioresistance gene Rad9 were tested. Mouse ES cells differing in Rad9 status were exposed to broad-beam 125 keV/ microm 3He alpha particles. All populations, when confluent, demonstrated a dose-independent bystander effect with respect to cell killing, and the Rad9-/- genotype did not selectively alter that response or cell killing after direct exposure to this high-LET radiation. In contrast, relative to Rad9+/+ cells, the homozygous mutant was sensitive to direct exposure to alpha particles when in log phase, providing evidence of a role for Rad9 in repair of potentially lethal damage. Direct exposure to alpha particles induced an increase in the frequency of apoptosis and micronucleus formation, regardless of Rad9 status, although the null mutant showed high spontaneous levels of both end points. All populations demonstrated alpha-particle-induced bystander apoptosis, but that effect was most prominent in Rad9-/- cells. Minimal alpha-particle induction of micronuclei in bystander cells was observed, except for the Rad9-/- mutant, where a significant increase above background was detected. Therefore, the Rad9 null mutation selectively sensitizes mouse ES cells to spontaneous and high-LET radiation-induced bystander apoptosis and micronucleus formation, but it has much less impact on cell killing by direct or bystander alpha-particle exposure. Results are presented in the context of defining the function of Rad9 in the cellular response to radiation and its differential effects on individual bystander end points.     

Experimental evaluation of the relationship between lethal or non-lethal virulence and transmission success in malaria parasite infections

Background

Theoretical studies suggest that direct and indirect selection have the potential to cause substantial evolutionary change in female mate choice. Similarly, sexual selection is considered a strong force in the evolution of male attractiveness and the exaggeration of secondary sexual traits. Few studies have, however, directly tested how female mate choice and male attractiveness respond to selection. Here we report the results of a selection experiment in which we selected directly on female mating preference for attractive males and, independently, on male attractiveness in the guppy, Poecilia reticulata. We measured the direct and correlated responses of female mate choice and male attractiveness to selection and the correlated responses of male ornamental traits, female fecundity and adult male and female survival.

Results

Surprisingly, neither female mate choice nor male attractiveness responded significantly to direct or to indirect selection. Fecundity did differ significantly among lines in a way that suggests a possible sexually-antagonistic cost to male attractiveness.

Conclusions

The opportunity for evolutionary change in female mate choice and male attractiveness may be much smaller than predicted by current theory, and may thus have important consequences for how we understand the evolution of female mate choice and male attractiveness. We discuss a number of factors that may have constrained the response of female choice and male attractiveness to selection, including low heritabilities, low levels of genetic (co)variation in the multivariate direction of selection, sexually-antagonistic constraint on sexual selection and the "environmental covariance hypothesis".

     

Dissecting out the Complex Ca2+-Mediated Phenylephrine-Induced Contractions of Mouse Aortic Segments

L-type Ca²⁺ channel (VGCC) mediated Ca²⁺ influx in vascular smooth muscle cells (VSMC) contributes to the functional properties of large arteries in arterial stiffening and central blood pressure regulation. How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (V_m) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca²⁺ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca²⁺ release and the tonic contraction determined by Ca²⁺ influx. The latter component involves both Ca²⁺ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca²⁺ influx by small variations of the VSMC V_m causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca²⁺ release from non-contractile sarcoplasmic reticulum Ca²⁺ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca²⁺ release form contractile or non-contractile Ca²⁺ stores with concomitant Ca²⁺ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.     

Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

Haptoglobin (Hp) is an acute-phase protein that is produced by the liver to capture the iron that is present in the blood circulation, thus avoiding its accumulation in the blood. Moreover, Hp has been detected in a wide variety of tissues, in which it performs various functions. In addition, this protein is considered a potential biomarker in many diseases, such as cancer, including ovarian carcinoma; however, its participation in the cancerous processes has not yet been determined. The objective of this work was to demonstrate the expression of Hp and its receptor CCR2 in the ovarian cancer cells and its possible involvement in the process of cell migration through changes in the rearrangement of the actin cytoskeleton using western blot and wound-healing assays and confirming by confocal microscopy. Ovarian cancer cells express both Hp and its receptor CCR2 but only after exposure to ascitic fluid, inducing moderated cell migration. However, when the cells are exposed to exogenous Hp, the expression of CCR2 is induced together with drastic changes in the actin cytoskeleton rearrangement. At the same time, Hp induced cell migration in a much more efficient manner than did ascitic fluid. These effects were blocked when the CCR2 synthetic antagonist RS102895 was used to pretreat the cells. These results suggest that Hp-induced changes in the cell morphology, actin cytoskeleton structure, and migration ability of tumor cells, is possibly “preparing” these cells for the potential induction of the metastatic phenotype.