P1 clones from Drosophila melanogaster as markers to study the chromosomal evolution of Muller's A element in two species of the obscura group of Drosophila

Thirty P1 clones from the X chromosome (Muller's A element) of Drosophila melanogaster were cross-hybridized in situ to Drosophila subobscura and Drosophila pseudoobscura polytene chromosomes. An additional recombinant phage Dsuby was also used as a marker. Twenty-three (77%) of the P1 clones gave positive hybridization on D. pseudoobscura chromosomes bat only 16 (53%) did so with those of D. subobscura. Eight P1 clones gave more than one hybridization signal on D. pseudoobscura and/or D. subobscura chromosomes. All P1 clones and Dsuby hybridized on Muller's A element (X chromosome) of D. subobscura. In contrast, only 18 P1 clones and Dsuby hybridized on Muller's D element (XR chromosomal arm) of D. pseudoobscura; 4 additional P1 clones hybridized on Muller's D element (XR chromosomal arm) of this species and the remaining P1 clone gave on hybridization signal on each arm of the X chromosome. This latter clone may contain one breakpoint of a pericentric inversion that may account for the interchange of genetic material between Muller's A and D elements in D. pseudoobscura. In contrast to the rare interchange of genetic material between chromosomal elements, profound differences in the order and spacing of markers were detected between D. melanogaster, D. pseudoobscura and D. subobscura. In fact, the number of chromosomal segments delimited by identical markers and conserved between pairwise comparisons is small. Therefore, extensive reorganization within Muller's A element has been produced during the divergence of the three species. Rough estimates of the number of cytologically detectable inversions contributing to differentiation of Muller's A element were obtained. The most reliable of these estimates is that obtained from the D. pseudoobscura and D. melanogaster comparison since a greater number of markers have been mapped in both species. Tentatively, one inversion breakpoint about every 200 kb has been produced and fixed during the divergence of D. pseudoobscura and D. melanogaster.     

EFFECTS OF COMPETITION AND DISTURBANCE ON THE RESPROUTING PERFORMANCE OF THE MEDITERRANEAN SHRUB ERICA MULTIFLORA L. (ERICACEAE)

Two field experiments were designed to evaluate the importance of competition, fire, repeated disturbance, and their interactions on the vegetative and reproductive performance of the Mediterranean shrub Erica multiflora over a 2.5-yr period. In a burn experiment, fire was applied to the ground-level stumps of previously clipped 13-yr-old plants with a propane torch and competition was diminished by removal of neighboring plants. Fire resulted in a reduction of sprout vigor and biomass of flowers; mature neighbors also reduced E. multiflora sprout vigor and flowering. The interaction between fire and competition was nonsignificant. In a stand burned by a wildfire we studied the effects of regenerating neighbors on target plants by removing all neighbors or only Quercus coccifera, the most dominant species in the burned stand. In this stand we also simulated herbivory by repeatedly clipping the sprouts of E. multiflora. Regenerating neighbors did not affect target plant sprout vigor after the wildfire, but did cause a decrease in the biomass of flowers per plant. Survival decreased after repeated clipping but was not affected by neighborhood treatment. The results suggest that the importance of competition on resprouting vigor was temporally variable. Variables related to plant size rather than species determined competitive superiority: resprouting neighbors did not affect resprouting performance of target plants, but mature neighbors did. In nature, fire may directly reduce vegetative and reproductive biomass by the heating effect. But it may have an indirect positive effect on biomass, by reducing competition among plants. Frequent disturbances that removed aboveground biomass of E. multiflora had a detrimental effect on target plant survival independent of neighborhood effect.     

Banding patterns of the chromosomes ofCercopithecus neglectus: Comparison withMiopithecus talapoin andErythrocebus patas

The G, Q, and C bands and the location of the nucleolar organizing regions (NORs) of the chromosomes of two male Cercopithecus neglectusare described. The diploid number of the species is 2n =62. Comparison with the karyotypes of Miopithecus talapoin (2n =54), and Erythrocebus patas (2n =54)showed the presence of total banding homeology for only 10 chromosome pairs.     

Activation of RAT erythrocyte phosphofructokinase by AMP and by non-physiological concentrations of Cyclic AMP

Summary Cyclic AMP (300µ m) activates phosphofructokinase from dialyzed haemolysates of mature rat erythrocytes. The main conclusions are: a) Cyclic AMP, at pH 7.1 and low concentrations of fructose-6-phosphate, is able to reverse the inhibition produced by different amounts of ATP (up to 1.5mm). b) The cyclic nucleotide is a positive allosteric effector of the enzyme as shown by the displacement of sigmoidal fructose-6-phosphate saturation curve to hyperbolic kinetics in the presence of inhibitory concentrations (1.5mm) of ATP. c) Cyclic AMP has no significant influence as deinhibitor of phosphofructokinase either at pH 7.1 and non-inhibitory levels (0.25mm) of ATP or at pH 8.1 and inhibitory (1.5mm) of non-inhibitory (0.25mm) concentrations of ATP. Similar conclusions were obtained with 300µ m AMP but not at a lower concentration (3µ m) with both nucleotides.The comparison of cyclic AMP results with those obtained under similar concentrations of AMP suggest that cyclic AMP is really only an in vitro modulator of the enzyme from rat erythrocytes, presumably at an AMP regulatory site, since non-physiological concentrations are required to act as deinhibitor.     

Analysis of the activation process of porcine procarboxypeptidase B and determination of the sequence of its activation segment

The molecular events which lead to the proteolytic transformation of porcine procarboxypeptidase B (PCPB) in carboxypeptidase B (CPB) have been determined. Among pancreatic and other tested proteinases, trypsin is the only one capable of generating carboxypeptidase B activity from the zymogen, in vitro. In the first step of this process, trypsin produces cleavage at the boundary between the activation region and the CPB region. Subsequently, a definite sequence of cleavages occurs at the C-terminal end of the released activation segment of 95 residues, giving rise to characteristic intermediates and to a proteolytically resistant activation fragment of 81 residues. In this process, the newly formed CPB participates in the quick-trimming of the released activation peptides. Only a single CPB species is formed in the activation process. This fact and the inability of the released activation peptides to inhibit CPB--and, therefore, their inability to slow down the kinetics of appearance of CPB activity--are two important characteristics differentiating between the activation processes of procarboxypeptidases A and B. The sequence of the 95 residues (MW = 12,835) of the activation region of porcine PCPB has also been deduced, largely from the information obtained by Edman degradation of its fragments and in part by considerations of homology with the rat precursor. The porcine PCPB activation region contains a high percentage of acidic residues, lacks cysteines, methionines, and side-chain posttranslational modifications, and presents a low but significant homology (31%) with the corresponding sequence of porcine procarboxypeptidase A.     

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal.     

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.     

Assessing the Environmental Impact of Water Consumption by Energy Crops Grown in Spain

The environmental impact of the water consumption of four typical crop rotations grown in Spain, including energy crops, was analyzed and compared against Spanish agricultural and natural reference situations. The life cycle assessment (LCA) methodology was used for the assessment of the potential environmental impact of blue water (withdrawal from water bodies) and green water (uptake of soil moisture) consumption. The latter has so far been disregarded in LCA. To account for green water, two approaches have been applied: the first accounts for the difference in green water demand of the crops and a reference situation. The second is a green water scarcity index, which measures the fraction of the soil‐water plant consumption to the available green water. Our results show that, if the aim is to minimize the environmental impacts of water consumption, the energy crop rotations assessed in this study were most suitable in basins in the northeast of Spain. In contrast, the energy crops grown in basins in the southeast of Spain were associated with the greatest environmental impacts. Further research into the integration of quantitative green water assessment in LCA is crucial in studies of systems with a high dependence on green water resources.