Evolutionary maintenance of stigma-height dimorphism in Narcissus papyraceus (Amaryllidaceae)

Stigma-height dimorphism is a sexual polymorphism in which plant populations are composed of two floral morphs that differ significantly in style length but not anther position. The morphs exhibit approach and reverse herkogamy, floral designs that in most species typically occur as monomorphic conditions. We investigated the floral biology of stigma-height dimorphism in the Mediterranean geophyte Narcissus papyraceus (Amaryllidaceae) in an effort to understand the evolutionary forces maintaining stylar polymorphism. Our survey of 66 populations in Spain, Portugal, and Morocco indicated that 56% were dimorphic with the long-styled morph at an average frequency of 0.79. The remaining 44% of populations sampled were monomorphic for the long-styled morph. In dimorphic populations there was a significant positive relation between population size and the frequency of the short-styled morph. Controlled pollinations demonstrated that N. papyraceus is self-sterile with no significant differences in female fertility between intra- and intermorph crosses. Prior self-pollination reduced seed set in flowers that were subsequently cross-pollinated. Estimates of mating patterns using allozyme markers in eight populations indicated that N. papyraceus is largely outcrossing (mean t(m) = 0.81) with no significant differences between monomorphic and dimorphic populations or style morphs. Stigma-height dimorphism in N. papyraceus is maintained in populations by insect-mediated cross-pollination with biased morph ratios and stylar monomorphism likely resulting from the combined influence of the inheritance of the polymorphism, morph-specific differences in assortative mating and founder effects.     

Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS

Whereas considerable progress has been made in understanding the molecular mechanisms of axon guidance across the midline, it is still unclear how the axonal trajectories of longitudinal pioneer neurons, which never cross the midline, are established. Here we show that longitudinal glia of the embryonic Drosophila CNS direct formation of pioneer axon pathways. By ablation and analysis of glial cells missing mutants, we demonstrate that glia are required for two kinds of processes. Firstly, glia are required for growth cone guidance, although this requirement is not absolute. We show that the route of extending growth cones is rich in neuronal cell bodies and glia, and also in long processes from both these cell types. Interactions between neurons, glia and their long processes orient extending growth cones. Secondly, glia direct the fasciculation and defasciculation of axons, which pattern the pioneer pathways. Together these events are essential for the selective fasciculation of follower axons along the longitudinal pathways.     

Effect of protein kinase A activity on the association of ADP-ribosylation factor 1 to golgi membranes

The small GTP-binding protein ADP-ribosylation factor 1 (ARF1) is an essential component of the molecular machinery that catalyzes the formation of membrane-bound transport intermediates. By using an in vitro assay that reproduces recruitment of cytosolic proteins onto purified, high salt-washed Golgi membranes, we have analyzed the role of cAMP-dependent protein kinase A (PKA) on ARF1 incorporation. Addition to this assay of either pure catalytic subunits of PKA (C-PKA) or cAMP increased ARF1 binding. By contrast, ARF1 association was inhibited following C-PKA inactivation with either PKA inhibitory peptide or RIIalpha as well as after cytosol depletion of C-PKA. C-PKA also stimulated recruitment and activation of a recombinant form of human ARF1 in the absence of additional cytosolic components. The binding step could be dissociated from the activation reaction and found to be independent of guanine nucleotides and saturable. This step was stimulated by C-PKA in an ATP-dependent manner. Dephosphorylated Golgi membranes exhibited a decreased ability to recruit ARF1, and this effect was reverted by addition of C-PKA. Following an increase in the intracellular level of cAMP, ARF proteins redistributed from cytosol to the perinuclear Golgi region of intact cells. Collectively, the results show that PKA exerts a key regulatory role in the recruitment of ARF1 onto Golgi membranes. In contrast, PKA modulators did not affect recruitment of beta-COP onto Golgi membranes containing prebound ARF1.     

Growth-altering effects of sodium hypochlorite in cultured human dermal fibroblasts

Sodium hypochlorite, the most widely used antimicrobial active chlorine compound in chemical disinfection, is little used as an antiseptic in clinical practice. This study aimed to assess the capacity of hypochlorite to alter human dermal fibroblast growth in vitro in relation to the concentration and exposure time. Effects of decreasing concentrations of hypochlorite (0.5%-0.00025%) on fibroblast adherence capacity and proliferation, according to varying exposure times and fetal calf serum (FCS) concentrations were investigated combining XTT assay, which provides cytochemical quantification of metabolically-active cell number, and total cell protein content, an indirect method for assessing substrate-adhered cell number. Initial cytotoxicity was produced at 0.0075% hypochlorite within contact time of two hours, provoking concentration-dependent cell detachment. From 0.1% upwards, NaOCl exerted a profound cytotoxic effect on fibroblasts. At later stages (4 h) and concentrations > or = 0.01% hypochlorite produced dose-dependent mitochondrial dysfunction: cell survival progressively diminished from 71% to 10%. Cytotoxic effects were not significantly affected by exposure-time periods, probably because maximum chlorine is released within the first four hours. Hypochlorite concentrations from 0.005% to 0.00025% were found to have no inhibitory effects on cell growth; in fact, they appear to exhibit the opposite effect. Increments in protein content found after 24 h exposure ranged from 30% to 120% above control values. Hypochlorite is highly cytotoxic for fibroblasts at concentrations > or = 0.01% provoking concentration-dependent loss of cell adherence capacity and mitochondrial dysfunction. In contrast, a mitogenic effect was observed with concentrations < or = 0.005% which supports NaOCl as a source growth-promoting activity in cultured human fibroblasts. Hypochlorite proved to be a highly reactive molecule which inhibits or stimulates cell division according to the concentration.     

Changes in luminal pH caused by calcium release in sarcoplasmic reticulum vesicles.

Fast (milliseconds) Ca2+ release from sarcoplasmic reticulum is an essential step in muscle contraction. To electrically compensate the charge deficit generated by calcium release, concomitant fluxes of other ions are required. In this study we investigated the possible participation of protons as counterions during calcium release. Triad-enriched sarcoplasmic reticulum vesicles, isolated from rabbit fast skeletal muscle, were passively loaded with 1 mM CaCl2 and release was induced at pCa = 5.0 and pH = 7.0 in a stopped-flow fluorimeter. Accompanying changes in vesicular lumen pH were measured with a trapped fluorescent pH indicator (pyranin). Significant acidification (approximately 0.2 pH units) of the lumen occurred within the same time scale (t(1/2) = 0.75 s) as calcium release. Enhancing calcium release with ATP or the ATP analog 5'-adenylylimidodiphosphate (AMPPNP) produced >20-fold faster acidification rates. In contrast, when calcium release induced with calcium with or without AMPPNP was blocked by Mg2+, no acidification of the lumen was observed. In all cases, rate constants of luminal acidification corresponded with reported values of calcium release rate constants. We conclude that proton fluxes account for part (5-10%) of the necessary charge compensation during calcium release. The possible relevance of these findings to the physiology of muscle cells is discussed.     

Structural differences between the repertoires of mouse and human germline genes and their evolutionary implications

 Although human and mouse antibodies are similar when one considers their diversification strategies, they differ in the extent to which kappa and lambda light chains are present in their respective variable light chain repertoires. While the Igk-V germline genes are preponderant in mice (95% or more), they comprise only 60% in humans. This may account for differences in the structural repertoire encoded in the Igk-V germline genes of these species. However, this subject has not been properly investigated, partially because a systematic structural characterization of the mouse Igk-V germline genes has not been undertaken. In the present study we compiled all available information on mouse Igk-V germline genes to characterize their structural repertoire. As expected, comparison with the structural repertoire of human Igk-V germline genes indicates differences. The most interesting is that the mouse Igk-V germline gene repertoire is more diverse in structural terms than its human counterpart: the mouse encodes seven canonical structure classes (combination of canonical structures in L1 and L3). In contrast, the human encodes only four. Analysis of the evolutionary relationships of human and mouse Igk-V germline genes led us to propose that the difference reflects a strategy of mice to compensate for the small lambda chain contribution to the repertoire of their variable light chains. Received: 1 June 1997 / Revised: 6 October 1997     

Prenatal Stress Down-Regulates Reelin Expression by Methylation of Its Promoter and Induces Adult Behavioral Impairments in Rats

Prenatal stress causes predisposition to cognitive and emotional disturbances and is a risk factor towards the development of neuropsychiatric conditions like depression, bipolar disorders and schizophrenia. The extracellular protein Reelin, expressed by Cajal-Retzius cells during cortical development, plays critical roles on cortical lamination and synaptic maturation, and its deregulation has been associated with maladaptive conditions. In the present study, we address the effect of prenatal restraint stress (PNS) upon Reelin expression and signaling in pregnant rats during the last 10 days of pregnancy. Animals from one group, including control and PNS exposed fetuses, were sacrificed and analyzed using immunohistochemical, biochemical, cell biology and molecular biology approaches. We scored changes in the expression of Reelin, its signaling pathway and in the methylation of its promoter. A second group included control and PNS exposed animals maintained until young adulthood for behavioral studies. Using the optical dissector, we show decreased numbers of Reelin-positive neurons in cortical layer I of PNS exposed animals. In addition, neurons from PNS exposed animals display decreased Reelin expression that is paralleled by changes in components of the Reelin-signaling cascade, both in vivo and in vitro. Furthermore, PNS induced changes in the DNA methylation levels of the Reelin promoter in culture and in histological samples. PNS adult rats display excessive spontaneous locomotor activity, high anxiety levels and problems of learning and memory consolidation. No significant visuo-spatial memory impairment was detected on the Morris water maze. These results highlight the effects of prenatal stress on the Cajal-Retzius neuronal population, and the persistence of behavioral consequences using this treatment in adults, thereby supporting a relevant role of PNS in the genesis of neuropsychiatric diseases. We also propose an in vitro model that can yield new insights on the molecular mechanisms behind the effects of prenatal stress.