A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap Mushroom Coprinopsis cinerea

The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Data-Limited Population-Status Evaluation of Two Coastal Fishes in Southern Angola Using Recreational Catch Length-Frequency Data

Excessive truncation of a population’s size structure is often identified as an important deleterious effect of exploitation, yet the effect on population persistence of size-structure truncation caused by exploitation is often not quantified due to data limitations. In this study, we estimate changes in eggs per recruit (EPR) using annual length-frequency samples over a 9 year period to assess persistence of the two most important recreational fishes in southern Angola: west coast dusky kob (Argyrosomus coronus) and leerfish (Lichia amia). Using a length- and age-structured model, we improve on an existing method to fit this type of model to length-frequency data and estimate EPR. The objectives of the methodological changes are to add flexibility and robustness to the approach for assessing population status in data-limited situations. Results indicate that dusky kob presents very low levels of EPR (5%-10% of the per recruit reproductive capacity in the absence of fishing) in 2013, whereas large inter-annual variability in leerfish estimates suggest caution must be applied when drawing conclusions about its exploitation status. Using simulated length frequency data with known parameter values, we demonstrate that recruitment decline due to overexploitation leads to overestimation of EPR values. Considering the low levels of EPR estimated for the study species, recruitment limitation is not impossible and true EPR values may be even lower than our estimates. It is, therefore, likely that management action, such as the creation of Marine Protected Areas, is needed to reconstitute the west coast dusky kob population.     

A pertussis toxin-sensitive G-protein mediates some aspects of insulin action in BC3H-1 murine myocytes.

The involvement of G-proteins in the insulin signal transduction system has been studied in detail using the murine BC3H-1 myocyte system. Pertussis toxin (PT) treatment, previously shown to attenuate some of the metabolic effects of insulin in this cell line (Luttrell, L.M., Hewlett, E.L., Romero, G., and Rogol, A.D. (1988) J. Biol. Chem. 263, 6134-6141), abolished insulin-induced generation of diacylglycerol and inositolglycan mediators with no effects on either the autophosphorylation of the insulin receptor or the phosphorylation of the major endogenous substrates for insulin-stimulated tyrosine kinase activity (pp185 and pp42-45). In vitro ADP-ribosylation and immunoblotting studies suggest that the major PT substrate is a 40-kDa protein of the G alpha family. This protein band did not exhibit detectable tyrosine phosphorylation upon stimulation of either intact cells or cell membranes with insulin. In the presence of low concentrations of GTP, insulin treatment of isolated myocyte plasma membranes resulted in a small (30-40%) but significant stimulation of GTP hydrolysis. This effect was best observed in the presence of small concentrations of sodium dodecyl sulfate. The rate of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to BC3H-1 membranes was also significantly increased in the presence of insulin. The effects of insulin on GTP hydrolysis and GTP gamma S binding were found to be dependent on the concentration of insulin. These effects were not detected in plasma membranes prepared from PT-pretreated BC3H-1 myocytes. In contrast, pretreatment with the B (inactive) subunit of PT did not alter the response of myocyte membranes to insulin. High affinity binding of [125I]iodoinsulin to myocyte plasma membranes was reduced by 60-70% in the presence of guanine nucleotides. Similar effects on insulin binding were produced by PT pretreatment of the cells. In contrast, adenine nucleotides had no effect on insulin binding. Scatchard analysis of the binding data showed that the observed effects of guanine nucleotides and PT on insulin binding resulted either from a reduction in the number of high affinity insulin binding sites or from a significant reduction of the affinity of insulin for its receptor. Low affinity binding sites did not appear to be affected by either guanine nucleotides nor PT pretreatment. These results provide substantial evidence suggestive of a noncovalent interaction between the insulin receptor and a regulatory G-protein system during the process of insulin signaling.     

Study of Poaceae phenology in a Mediterranean climate. Which species contribute most to airborne pollen counts?

The present study sought to determine which of the common Poaceae species in the study area contribute most to the Poaceae pollen season curve, and to determine the phenological behaviour of the species studied. The different floral phenophases in thirty-three Poaceae species common in and around the city of Córdoba (SW Iberian Peninsula) were checked periodically over the period 2004–2006. Results showed that longer phenological ranges were recorded in the coolest and wettest year, and shorter ranges in the warmest and driest year. Moreover, ranges varied as a function of altitude: populations in lower-lying areas flowered earlier than those at higher altitudes. The results, taken in conjunction with the findings of preliminary research into potential pollen production, showed that probably only four of the Poaceae species studied—Dactylis glomerata, Lolium rigidum, Trisetaria panicea and Vulpia geniculata—were major contributors to the Poaceae airborne pollen curve.     

Hormone Effects on the Membrane Potential and on Sucrose-Induced Depolarization of Young Citrus Leaves

The effect of IAA, GA₃ and ABA on transmembrane potential difference(Em) and on sucrose-induced depolarization has been studiedin young Citrus leaves. The addition of any of these hormonesto the perfusion solution (short-term experiments) did not affectEm or sucrose-induced depolarization. Hormonal treatments ofyoung leaves on the tree resulted, after 4 to 16 days (long-termexperiments), in an increase of Em for GA₃- and ABA-treatedleaves, while in IAA-treated ones no hyperpolarization was found.Only in ABA treated leaves this membrane hyperpolarization couldbe related to an enhancement of sucrose uptake. (Received April 28, 1992; Accepted September 21, 1992)     

A rapid extraction method for detecting aflatoxin producing isolates

A rapid extraction method for screening aflatoxin producing potential ofAspergillus flavus group isolates is described. The method is performed using a moist wheat medium with ca. five infected grains extracted with 2 mL of chloroform, and using thin layer chromatography. This method was proved with 95A. flavus isolates from animal feeds.     

Regulated nitrate transport in the cyanobacterium Anacystis nidulans.

Intracellular accumulation of nitrate, indicative of the operation of an active nitrate transport system, has been measured in intact cells of the cyanobacterium Anacystis nidulans. The ability of the cells to accumulate nitrate was effectively hindered by either ammonium addition or selective inhibition of CO2 fixation by DL-glyceraldehyde, with the effect of either compound being prevented by previously blocking ammonium assimilation. The results support the contention that nitrate utilization in cyanobacteria is regulated at the level of nitrate transport through the concerted action of ammonium assimilation and CO2 fixation.