Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

Molecular mechanisms of oxidative stress-related neonatal jaundice

Oxidative stress is a pathological condition characterized by an overload of oxidant products, named free radicals, which are not well counteracted by antioxidant systems. Free radicals induce oxidative damage to many body organs and systems. In neonatal red blood cells, free-radical mediated-oxidative stress leads to eryptosis, a suicidal death process of erythrocytes consequent to alteration of cell integrity. Neonatal red blood cells are targets and at the same time generators of free radicals through the Fenton and Haber-Weiss reactions. Enhanced eryptosis in case of oxidative stress damage may cause anemia if the increased loss of erythrocytes is not enough compensated by enhanced new erythrocytes synthesis. The oxidative disruption of the red cells may cause unconjugated idiopathic hyperbilirubinemia in neonates. High levels of bilirubin are recognized to be dangerous for the central nervous system in newborns, however, many studies have highlighted the antioxidant function of bilirubin. Recently, it has been suggested that physiologic concentration of bilirubin correlates with higher antioxidant status while high pathological bilirubin levels are associated with pro-oxidants effects. The aim of this educational review is to provide an updated understanding of the molecular mechanisms underlying erythrocyte oxidant injury and its reversal in neonatal idiopathic hyperbilirubinemia.     

The effect of salinity on the reproduction of coastal submerged macrophytes in experimental communities

The effects of salinity on the reproduction of coastal submerged macrophyte species were studied on samples of communities from six seasonal marshes in two outdoor experiments performed in autumn and in spring. The submerged macrophyte communities were submitted to five different salinity levels (0, 1, 2, 4 and 6 g/1 Cl^?1). In a companion paper (Grillas, van Wijck & Bonis 1993) three groups of species were distinguished on the basis of their biomass production over the salinity range 0 to 6 g/1 Cl^?1: (1) glycophytes (non-salt-tolerant species), (2) salt-tolerant species and (3) halo-phytes. This part of the study describes the impact of salinity on the reproduction of the individual species during the two experiments. The species differ in their capacity to reproduce in the autumn; only Zannichelliapedunculata and Tolypella hispánica were able to produce fruits in that season. For all species reproduction was greater in spring and strongly correlated with biomass, except for Chara canescens. Differences in reproductive effort over the salinity range amplified the halophytic nature of Ruppia marítima and Chara canescens and the intolerance of Callitriche truncata and Chara contraria. For the other species, reproductive effort did not differ significantly over the salinity range. Regarding the effect of salinity on biomass and reproductive effort of individual species, there were large differences in the total weight of propagules produced at the community level and in the relative contribution of individual species. The resulting quantitative changes in the species composition of the seed bank could affect the structure of the communities by their effects on the establishment and survival of species populations.     

Ecological and behavioral consequences of digestion in frugivorous animals

The postingestional effect of seed size and mass and nutrient composition on fruit profitability are reviewed. It is emphasized that profitability results from the interaction between fruit characteristics and the physiological and morphological traits of frugivores. The processes by which frugivores regurgitate or defecate seeds and the differential processing of the nutrient rich pulp and ballast in fruit are strongly dependent on the interaction between frugivore gut morphology and seed size. Euphonias that lack a functional gizzard defecate seeds, whereas tanagers that have a delete a functional gizzards regurgitate seeds. Some frugivores separate seeds from pulp and exocarp in the gizzard. It is hypothesized that the gizzard plays an important role in determining the postigestional fate of different pulp components. Although fruit nutrient content is often invoked as a determinant of frugivore feeding choices, studies that rely on proximal nutrient analysis, have often failed to find clear nutrient composition-preference correlations. It is argued that a partial reason for this failure is that proximal nutrient analysis ignores the complexities of fruit digestion. The ecological and physiological correlates of lipid and sugar assimilation are used to identify the limitations of traditional proximal nutrient analyses in fruit-frugivore studies. We suggest that recognizing the intricacies of the digestive characteristics of frugivores may reveal a much richer patterning in the interaction of frugivores with plants than has been previously hypothesized.     

The role of the neutrophil and formed elements of the blood in an in vitro model of reperfusion injury

Using The globally ischaemic isolated guinea-pig heart we conducted studies to assess the role of activated neutrophils (PMNs) and the role of the endothelium in reperfusion injury. Reperfusion injury was induced by a 20 min period of global ischaemia followed by a 30 min reperfusion with Krebs' buffer supplemented with f-Met-Leu-Phe (fMLP) and heparinized blood. Ischaemia alone or blood alone resulted in a complete recovery in contractile function measured by developed pressure, fMLP (500 muM) and blood, administered to normoxic hearts did not affect contractile function. The combination of 100 muM fMLP and blood beginning at reperfusion and continuing for 30 min decreased the recovery in contractile function (max. 33 +/- 6% reovery) while buffer and 100 pM fMLP resulted in a complete recovery in function. In hearts infused with buffer and neutropenic blood incubated with 100 muM fMLP a complete recovery in function was observed. Isolated peritoneal neutrophils, 7-70 x 10(5) PMN/ min, incubated with 100 muM fMLP and Krebs' solution decreased contractile function in a concentration-related manner (max. 44 +/- 11% recovery). Platelets, plasma or red blood cells alone incubated with fMLP did not decrease recovery in developed pressure. Platelets and PMN incubated with 100 muM fMLP did not, while red blood cells and PMN did, elicit a reduction in recovery in contractile function (34 +/- 4% recovery). A 20 min period of global ischaemia destroys the functional integrity of the endothelium (response to Ach). Pre-treatment of the heart with sufficient H(2)O(2) to functionally damage the endothelium, followed by infusion of Krebs' solution supplemented with blood and 100 muM fMLP also elicited a reduction in recovery of contractile function (42 +/- 15% recovery). In summary, partially activated neutrophils play a major role in reperfusion injury and there exists a cooperativity between the RBC and PMN in this model.     

Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type1

Cytotoxic necrotizing factor type 1 (CNF1) from strains of pathogenic Escherichia coli induces in human epithelial HEp-2 cells, a profound reorganization of the actin cytoskeleton into prominent stress fibres and membrane ruffles. We report here that this process is associated with induction of phagocytic-like activity. CNF1-treated cells acquired the ability to ingest latex beads as well as non-invasive bacteria such as Listeria innocua, which were taken as a model system. Uptake of bacteria was similar to pathogen-induced phagocytosis, since L. innocua transformed with DNA coding for the pore-forming toxin listeriolysln O behaved, with respect to intracellular growth, like the invasive, pathogenic species L. monocytogenes. Our results raise the possibility that, in vivo, pathogenic CNF1 -producing E. coli may invade epithelia by this novel induced phagocytic-like mechanism.     

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.     

ROOT PROFILES AND COMPETITION BETWEEN THE INVASIVE,EXOTIC PERENNIAL,CARPOBROTUS EDULIS,AND TWO NATIVE SHRUB SPECIES IN CALIFORNIA COASTAL SCRUB

The mat-forming succulent, Carpobrotus edulis (Aizoaceae), surrounds and grows over many native plant species in California coastal communities. Two shrub species, Haplopappus ericoides and H. venetus var. sedoides, were found to have shallow root systems that occupied the same soil depths as those of C. edulis. In the presence of C. edulis, the normal rooting profiles of the shrubs were displaced downward, although partial overlap with C. edulis remained. Removal of C. edulis from around individuals of both shrub species resulted in higher predawn xylem pressure potentials in shrubs from the removal treatments as compared to controls, suggesting that the surrounding C. edulis was utilizing water that would otherwise have been available to these shrubs. In H. ericoides, predawn xylem pressure potentials of removal treatment shrubs remained higher than those of the controls throughout the remainder of the dry season even though these shrubs showed a marked increase in canopy area after removal of surrounding C. edulis. Removal of C. edulis from around H. venetus also initially led to higher predawn xylem pressure potentials in the removal shrubs as compared to the controls and increased production of new leaves. After this initial period the predawn xylem pressure potentials of the removal shrubs were not significantly different from those of controls, suggesting that the demand for water by the increased leaf areas eventually matched the enhanced water availability resulting from C. edulis removal. Also, the morphology of H. venetus shrubs changed to that more typical of unaffected individuals. Thus, C. edulis significantly affected not only the water relations of these two shrub species but also their shoot sizes and overall morphologies.     

Differentiation in sex investment by clones and populations of Daphnia

Dormancy is an ecological strategy by which organisms avoid stressful environments, but it also can have genetic consequences. Many facultative parthenogens shift from asexual to sexual reproduction to enter dormancy. Hence, conditions that favour dormancy are predicted to select for more sex, which should increase clonal diversity. We examined lake populations of Daphnia that face different ecological risks to remaining active year‐round, and quantified the extent to which they have differentiated in their investment in dormancy and sex. There was substantial genetic variation among populations and clones for sex induction and production of dormant eggs, and striking evidence of gender specialization. We also observed a positive association between the magnitudes of population‐level investment in dormancy and of variance among clones in sex induction. These results document an ecological gradient in dormancy that is manifest as a genetic gradient in clonal variation for the propensity to engage in sex.     

Exploiting the complete yeast genome sequence

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.