Resonance Raman analysis of a fluorescently labeled oligonucleotide forming a very stable hairpin

An oligodeoxynucleotide has been synthesized, which mimics an ``antigene' oligonucleotide with a polypyrimidic stretch on its 5′ side and is protected on its 3′ side against nucelases by a naturally forming and very stable hairpin, 5′GCGAAGC3′. The in vitro degradation of the resulting oligonucleotide d(5′TTCTCGCGAAGC3′) has already been studied by fluorescence resonance energy transfer (FRET) (Réfrégiers et al. 1996, J Biomol Struct Dyn 14: 365 – 371). The technique required the grafting of fluorophores at both ends of the oligonucleotide. In the present work we have compared the hairpin formed in the presence and in the absence of such fluorophores. This was achieved by the study of the Raman spectra (excitation at 257 nm) of the oligodeoxynucleotides H, which forms the hairpin (5′TTCTCGCGAAGC3′), and a con-trol C (5′TTCTCCGGAAGC3′) which is unable to form the hairpin. Resonance Raman spectroscopy with 257 nm excitation greatly favors the resonance of purines and therefore the study of the 3′ part of the oligonucleotides. The difference spectrum obtained from resonance Raman spectra of C and H showed marker peaks specific for hairpin formation. The search for these marker peaks in difference spectra involving the Raman spectrum of H labeled by fluorophores and either C or H proved that the fluorophores do not modify the structure of the hairpin but only the vibrations of the two terminal bases on which the fluorophores are grafted. The use of such labeling is then justified in order to allow oligonucleotides protected by a hairpin on their 3′ side to be studied by fluorescence spectroscopy. Received: 13 December 1996 / Accepted: 7 April 1997     

Sex differences in the rate of fatigue development and recovery

Background

Many musculoskeltal injuries in the workplace have been attributed to the repetitive loading of muscle and soft tissues. It is not disputed that muscular fatigue is a risk factor for musculoskeltal injury, however the disparity between gender with respect to muscular fatigability and rate of recovery is not well understood. Current health and safety guidelines do not account for sex differences in fatiguability and may be predisposing one gender to greater risk. The purpose of this study was to quantify the sex differences in fatigue development and recovery rate of lower and upper body musculature after repeated bouts of sustained isometric contractions.

Methods

Twenty-seven healthy males (n = 12) and females (n = 15) underwent bilateral localized fatigue of either the knee extensors (male: n = 8; female: n = 8), elbow flexors (male: n = 8; female: n = 10), or both muscle groups. The fatigue protocol consisted of ten 30-second sub-maximal isometric contractions. The changes in maximum voluntary contraction (MVC), electrically evoked twitches, and motor unit activation (MUA) were assessed along with the ability to control the sustained contractions (SLP) during the fatigue protocol using a mixed four-factor repeated measures ANOVA (gender × side × muscle × time) design with significance set at p < 0.05.

Results

There was a significant loss of MVC, MUA, and evoked twitch amplitude from pre- to post-fatigue in both the arms and legs. Males had greater relative loss of isometric force, a higher rate of fatigue development, and were less capable of maintaining the fatiguing contractions in the legs when compared to the females.

Conclusion

The nature of the induced fatigue was a combination of central and peripheral fatigue that did not fully recover over a 45-minute period. The results appear to reflect sex differences that are peripheral, and partially support the muscle mass hypothesis for explaining differences in muscular fatigue.

     

Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.     

A microsatellite genetic linkage map for Xiphophorus

Interspecies hybrids between distinct species of the genus Xiphophorus are often used in varied research investigations to identify genomic regions associated with the inheritance of complex traits. There are 24 described Xiphophorus species and a greater number of pedigreed strains; thus, the number of potential interspecies hybrid cross combinations is quite large. Previously, select Xiphophorus experimental crosses have been shown to exhibit differing characteristics between parental species and among the hybrid fishes derived from crossing them, such as widely differing susceptibilities to chemical or physical agents. For instance, genomic regions harboring tumor suppressor and oncogenes have been identified via linkage association of these loci with a small set of established genetic markers. The power of this experimental strategy is related to the number of genetic markers available in the Xiphophorus interspecies cross of interest. Thus, we have undertaken the task of expanding the suite of easily scored markers by characterization of Xiphophorus microsatellite sequences. Using a cross between Xiphophorus maculatus and X. andersi, we report a linkage map predominantly composed of microsatellite markers. All 24 acrocentric chromosome sets of Xiphophorus are represented in the assembled linkage map with an average intergenomic distance of 7.5 cM. Since both male and female F1 hybrids were used to produce backcross progeny, these recombination rates were compared between "male" and "female" maps. Although several genomic regions exhibit differences in map length, male- and female-derived maps are similar. Thus Xiphophorus, in contrast to zebrafish, Danio rerio, and several other vertebrate species, does not show sex-specific differences in recombination. The microsatellite markers we report can be easily adapted to any Xiphophorus interspecies and some intraspecies crosses, and thus provide a means to directly compare results derived from independent experiments.     

Regulation of parathyroid hormone type 1 receptor dynamics, traffic, and signaling by the Na+/H+ exchanger regulatory factor-1 in rat osteosarcoma ROS 17/2.8 cells

The effects of the expression of the Na+/H+ exchanger regulatory factor-1 (NHERF1) on the distribution, dynamics, and signaling properties of the PTH type 1 receptor (PTH1R) were studied in rat osteosarcoma cells ROS 17/2.8. NHERF1 had a dramatic effect on the subcellular distribution of PTH1R, promoting a substantial relocation of the receptor to regions of the plasma membrane located in very close proximity to cytoskeletal fibers. Direct interactions of NHERF1 with the PTH1R and the cytoskeleton were required for these effects, because they were abolished by 1) PTH1R mutations that impair NHERF1 binding, and 2) NHERF1 mutations that impair binding to the PTH1R or the cytoskeleton. NHERF1 reduced significantly the diffusion of the PTH1R by a mechanism that was also dependent on a direct association of NHERF1 with the PTH1R and the cytoskeleton. NHERF1 increased ligand-dependent production of cAMP and induced ligand-dependent rises in intracellular calcium. These effects on calcium were due to increased calcium uptake, as they were blocked by calcium channel inhibitors and by the addition of EGTA to the medium. These calcium effects were abolished by protein kinase A inhibition but phospholipase C inhibition was without effect. Based on these analyses, we propose that, in ROS cells, the presence of NHERF1 induces PTH-dependent calcium signaling by a cAMP-mediated mechanism that involves local protein kinase A-dependent activation of calcium channels.     

Effect of aluminium on the growth of 34 plant species: A summary of results obtained in low ionic strength solution culture

The results from many experiments conducted over 5 years to determine the tolerance of 34 plant species (87 cultivars) to aluminium (Al) are summarised. All experiments were conducted in a temperature-controlled glasshouse using a low-ionic-strength solution culture technique. The activity of Al³⁺ (M) at which top yields were reduced by 50% (Al_RY50) was determined for each cultivar.The species Bromus wildenowii, Cynosurus cristatus, Hordeum vulgare, Triticum aestivum (cvs Warigal, Scout, Sonora-63), Avena byzantina, Arabidopsis thaliana, Lycopersicon esculentum and Nicotiana plumbaginifolia were all very sensitive to Al (Al_RY50<1). The species Poa pratense, Lolium perenne (NZ-derived cultivars), Lotus corniculatus, Avena sativa (cvs West, Carbeen, Camellia and Coolabah), Triticum aestivum (cvs Cardinal and Waalt), Allium cepa and Asparagus officinalis were sensitive to Al (Al_RY50 1–2).The pasture grass species Lolium perenne (Australian and European and derived cultivars), Lolium hybridum and Lolium multiflorum, Dactylis glomerata (Apanui and Kara), Phalaris aquatica, Festuca arundinacea and the pasture legumes species Trifolium pratense, Trifolium repens and Trifolium subterraneum were all moderately sensitive to Al (Al_RY50 2–5). Other species that were also moderately sensitive included Triticum aestivum (cvs Atlas-66, BH146, and Carazinho), Avena sativa (cvs Swan and Blackbutt), Avena Strigosa, Petunia x and Phaseolus vulgaris (cvs Red Kidney, Black Turtle and Haricot).The most tolerant species (Al_RY50>5) were (in order of increasing tolerance) Phaseolus vulgaris (cvs Tendergreen, The Prince and Yatescrop), Cucurbita maxima, Dactylis glomerata (cv Wana), Paspalum dilatatum, Lotus pedunculatus, Ehrharta calycina, Medicago sativa, Holcus lanatus, Festuca rubra, Phaseolus lunatus and Agrostis tenuis. Agrostis tenuis was at least twice as tolerant as the next most tolerant species (Al_RY50>30 compared to 15.6).     

The Effect of pH and Plaque on the Uptake of Cu and Mn in Phragmites australis(Cav.) Trin ex. Steudel

The physico-chemical properties of iron oxyhydroxide plaquesformed on the roots of Phragmites australis under field andlaboratory conditions were determined using electron microscopyand energy dispersive spectrometry (EDS) analysis. Plaques werepresent as an amorphous coating on roots with an uneven distribution.They were shown to be composed of iron in both the field andlaboratory, and phosphorus was found to be adsorbed onto thesurface of plaques formed in the laboratory. The uptake of copperand manganese in the presence and absence of plaque was investigatedunder two different pH regimes. Concentrations of Cu were lowerin the shoots of P. australis in the presence of plaque (565mg kg^-1) than when it was absent (1400 mg kg^-1), under growthconditions of higher pH (6.0). The adsorption of Cu and Mn ontothe plaque surface was not the mechanism by which plaque reducedthe uptake of other metals. Alternatively, the plaque may simplyact as a physical barrier. Under conditions of lower pH (3.5)the activity of hydrogen ions at the root surface interferedwith the movement of metals into the root and masked any potentialeffect of iron plaque. Copyright 2000 Annals of Botany Company Phragmites australis, iron oxyhydroxide plaque, pH, manganese, copper, EDS, SEM, wetlands     

Isolation and characterization of a diuretic peptide from Acheta domesticus. Evidence for a family of insect diuretic peptides.

A diuretic peptide (Acheta-DP) has been isolated from extracts of whole heads of the house cricket, Acheta domesticus. The native peptide increases both cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro to an extent comparable with those responses obtained with supra-maximal amounts of crude extracts of corpora cardiaca. The primary structure of Acheta-DP was established as a 46-residue amidated peptide: TGAQSLSIVAPLDVLRQRLMNELNRRRMRELQGSRIQQNRQLLTSI-NH2. Acheta-DP has 41% sequence identity with a diuretic peptide isolated from Manduca sexta, providing direct evidence for the presence of a family of diuretic peptides in insects.