Hybrid plants preserve unique genetic variation in the St Helena endemic trees <Emphasis Type="Italic">Commidendrum rotundifolium</Emphasis> DC Roxb. and <Emphasis Type="Italic">C. spurium</Emphasis> (G.Forst.) DC

The island of St Helena in the South Atlantic Ocean has a rich endemic flora, with 10 endemic genera and 45 recognised endemic species. However, populations of most endemic species have undergone dramatic reductions or extinction due to over-exploitation, habitat destruction and competition from invasive species. Consequently, endemic species are likely to have lost genetic variation, in some cases to extreme degrees. Here, the entire extant wild populations and all planted trees in seed orchards, of two critically endangered species in the endemic genus Commidendrum (Asteraceae), C. rotundifolium and C. spurium, were sampled to assess levels of genetic variation and inbreeding. Six new microsatellite loci were developed from next-generation sequence data, and a total of 190 samples were genotyped. Some seed orchard trees contained alleles from both wild C. rotundifolium and C. spurium indicating they could be hybrids and that some backcrossing may have occurred. Some of these trees were more similar to C. rotundifolium than C. spurium both genetically and morphologically. Importantly, allelic variation was detected in the putative hybrids that was not present in wild material. C. rotundifolium is represented by just two individuals one wild and one planted and C. spurium by seven, therefore the seed orchard trees comprise an important part of the total remaining genetic diversity in the genus Commidendrum.     

Effect of active muscle mass during ischemic exercise on peak lower leg vascular conductance.

Uncertainty exists as to whether a period of passive arterial occlusion (PAO) or ischemic exercise (IE) results in peak lower leg vascular conductance (LVC). This uncertainty is due to the different body positions, active muscle mass, and occlusion times used for PAO or IE. The purpose of this study was to examine whether 10 min of PAO elicits a similar LVC compared with ischemic dorsiflexion (IDF), ischemic plantar flexion (IPF), and ischemic plantar-dorsiflexion (IPDF). Ten subjects (5 women, 27 +/- 9 yr, 68 +/- 3 kg) were studied on 3 days over 1 wk in a semireclined position with the right foot attached to an isokinetic dynamometer. Mean arterial pressure (Finapres) and lower leg blood flow (LBF, venous occlusion plethysmography) were measured at rest and after PAO and IE. PAO was administered randomly on 1 of the 3 days and before IE. IE protocols consisted of maximal isokinetic dorsiflexion and/or plantar flexion at 120 and 60 degrees/s, respectively. In a second experiment, an additional eight subjects (4 women, 29 +/- 12 yr, 77 +/- 12 kg) were studied to examine the effect of isokinetic speed during IDF on peak LBF and LVC. Peak LVC (ml.min(-1).100 ml(-1).mmHg(-1)) was similar among IPF (0.590 +/- 0.16), IPDF (0.532 +/- 0.17), and PAO (0.511 +/- 0.18), and significantly lower after IDF (0.334 +/- 0.15). No differences in peak LBF and LVC were observed after IDF using different isokinetic speeds. We conclude that 10 min of PAO, IPF, and IPDF performed in a similar posture are adequate stimuli to elicit peak LVC.     

Non-disaccharide-based mechanisms of protection during drying

Few tissues or organisms can survive the removal of nearly all their intra and extracellular water. These few have developed specialized adaptations to protect their cellular components from the damage caused by desiccation and rehydration. One mechanism, common to almost all such organisms, is the accumulation of disaccharides within cells and tissues at the onset of dehydration. This adaptation has been extensively studied and will not be considered in this review. It has become increasingly clear that true desiccation tolerance is likely to involve several mechanisms working in concert; thus, we will highlight several other important and complimentary adaptations found especially in the dehydration-resistant tissues of higher plants. These include the scavenging of reactive oxygen species, the down-regulation of metabolism, and the accumulation of certain amphiphilic solutes, proteins, and polysaccharides.     

Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament

RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.     

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.     

Feeding preference of the sand flies Lutzomyia umbratilis and L. spathotrichia (diptera: Psychodidae, Phlebotominae) in an urban forest patch in the city of Manaus, Amazonas, Brazil

Precipitin tests were performed on blood meals of 199 sand flies (161 Lutzomyia umbratilis, 34 L. spathotrichia, two Lutzomyia of group shannoni, one L. anduzei) in a non-flooded upland forest on the Campus of the Universidade Federal do Amazonas. This is the second largest forest fragment in an urban setting in Brazil. Results on L. umbratilis, which is considered to be the principal leishmaniasis vector in this region, indicated rodents as its predominant blood source in contrast to previous reports in which blood meal analysis indicated that this species fed principally on Xenarthra (particularly sloths).     

In vitro development following one-step dilution of OPS-vitrified porcine blastocysts

The objective of this experiment was to compare the in vitro survival and hatching rates of OPS-vitrified porcine blastocysts obtained after conventional (three-step dilution) or direct (one-step dilution) warming procedures. Expanded blastocysts were collected by laparotomy from weaned crossbred sows (n=7) on Day 6 of the cycle (D0: onset of estrus). Vitrification was performed as described by Berthelot et al. [Cryobiology 41 (2000) 116] using 17% (v/v) ethylene glycol and 17% (v/v) dimethyl-sulfoxide in the second vitrification medium. Conventional warming was carried out by plunging straws containing embryos in 800 microl of TCM199 Hepes containing 20% new born calf serum (TCM-NBCS) and 0.13 M sucrose for 1 min. Embryos were then transferred to another well with the same medium for 5 min, washed in TCM-NBCS with 0.075 M sucrose for 5 min and transferred to TCM-NBCS for 5 min. In one-step dilution, embryos were placed in 400 microl TCM-NBCS containing 0.13 M sucrose. To evaluate in vitro development, embryos warmed by conventional (n=59) or direct (n=58) procedures were cultured for 96 h. Non-vitrified embryos were used as controls (n=20). No significant (P>0.05) differences were observed in the in vitro development of vitrified and non-vitrified embryos. The survival and hatching rates obtained by three-step dilution (84.8 and 71.2%, respectively) and one-step dilution (86.2 and 74.1%, respectively) procedures were not different (P>0.05). The average diameter of expanded blastocysts from each donor was significantly different (P<0.001) among embryo donors. The embryo diameter or the interactions among the factors evaluated did not affect (P>0.05) the embryo survival and hatching of the vitrified/warmed blastocysts. However, the donor of embryos had a significant effect (P<0.001) on these parameters, confirming previous experiments. This experiment shows that porcine embryo vitrification and one-step dilution are promising procedures to be used under field conditions. However, the good results obtained in vitro must be confirmed also by in vivo experiments.     

Specific detection of the toxigenic species Fusarium proliferatum and F. oxysporum from asparagus plants using primers based on calmodulin gene sequences

Fusarium proliferatum and Fusarium oxysporum are the causal agents of a destructive disease of asparagus called Fusarium crown and root rot. F. proliferatum from asparagus produces fumonisin B1 and B2, which have been detected as natural contaminants in infected asparagus plants. Polymerase chain reaction (PCR) assays were developed for the rapid identification of F. proliferatum and F. oxysporum in asparagus plants. The primer pairs are based on calmodulin gene sequences. The PCR products from F. proliferatum and F. oxysporum were 526 and 534 bp long, respectively. The assays were successfully applied to identify both species from the vegetative part of the plants.