Exercise heart rates at different serum digoxin concentrations in patients with atrial fibrillation.

Heart rate at rest and during increasing workloads was measured in a double blind study of 12 patients with chronic atrial fibrillation when serum concentrations of digoxin were nil and at low and high therapeutic values. Twelve normal subjects were studied for comparison. The heart rate at all levels of exercise in most patients with atrial fibrillation was not adequately controlled by any serum digoxin concentration tested despite a reduction in heart rate with increasing serum digoxin concentrations. Control of the resting heart rate, even in patients with high serum digoxin concentrations, did not ensure adequate control of the heart rate during work rates equivalent to regular daily activities.     

Variation in mycorrhizal growth response influences competitive interactions and mechanisms of plant species coexistence

Plant species vary in their growth response to arbuscular mycorrhizal (AM) fungi, with responses ranging from negative to positive. Differences in response to AM fungi may affect competition between plant species, influencing their ability to coexist. We hypothesized that positively responding species, whose growth is stimulated by AM fungi, will experience stronger intraspecific competition and weaker interspecific competition in soil containing AM fungi, while neutrally or negatively responding species should experience weaker intraspecific and stronger interspecific competition. We grew Plantago lanceolata, which responds positively to AM fungi, and Bromus inermis, which responds negatively to AM fungi, in an additive response surface competition experiment that varied the total density and relative frequency of each species. Plants were grown in sterilized background soil that had been inoculated with whole soil biota, which includes AM fungi, or a microbial wash, that contained other soil microbes but no AM fungi, or in sterilized soil that contained no biota. The positively responding P. lanceolata was more strongly limited by intraspecific than interspecific competition when AM fungi were present. By contrast, the presence of AM fungi decreased the strength of intraspecific competition experienced by the negatively responding B. inermis. Because AM fungi are almost always present in soil, strong intraspecific competition in positively responding species would prevent them from outcompeting species that respond neutrally or negatively to AM fungi. The potential for increased intraspecific competition to offset growth benefits of AM fungi could, therefore, be a stabilizing mechanism that promotes coexistence among plant species.     

A morphometric analysis of Scottish Athyrium distentifolium: a contribution to the BAP

Summary

A morphologically distinct variety of Athyrium distentifolium called A. distentifolium var. flexile has been found only in Scotland. Research was undertaken for aUK Biodiversity Action Plan. To confirm that this taxon has a definitely recognisable morphology, a morphometric analysis was used on the range of characters used to define this variety. It showed that it can be clearly differentiated.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter

The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [³H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.     

A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

Corticotectal and corticostriatal projections from the frontal eye fields of the cat: an anatomical examination using WGA-HRP

Corticofugal projections from the frontal eye fields (FEF) are believed to access the superior colliculus (SC) directly (i.e., monosynaptically) and indirectly (i.e., multisynaptically) through the basal ganglia. The present results suggest that these two pathways are derived from largely segregated populations of corticofugal neurons. Furthermore, while the different subregions of the FEF from which these pathways originate have different termination patterns in the basal ganglia (i.e., striatum, ST), they share a common termination pattern in the SC. Injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the two major subdivisions of the FEF (presylvian and cruciate sulci) resulted in dense label in both the ST (bilaterally) and the SC (ipsilaterally). Corticostriatal labeling was found in the caudal part of the head of the caudate nucleus (heaviest ipsilaterally), with labeling from cruciate injections located ventromedial to that produced by presylvian injections. Only presylvian injections resulted in labeling in the putamen. Retrograde tracing experiments demonstrated that both presylvian and cruciate corticostriatal projections originated from neurons in lamina III and the upper aspects of lamina V. An additional but small group of presylvian corticostriatal projections was found in lamina VI. Corticotectal terminal labeling was restricted to the deep laminae of the SC and was derived exclusively from lamina V neurons in cortex. They differed from their corticostriatal counterparts in laminar/sub-laminar location and in soma sizes.     

MRI osteitis predicts cartilage damage at the wrist in RA: a three-year prospective 3T MRI study examining cartilage damage

Introduction

Cartilage damage impacts on patient disability in rheumatoid arthritis (RA). The aims of this magnetic resonance imaging (MRI) study were to investigate cartilage damage over three years and determine predictive factors.

Methods

A total of 38 RA patients and 22 controls were enrolled at t = 0 (2009). After 3 years, clinical and MRI data were available in 28 patients and 15 controls. 3T MRI scans were scored for cartilage damage, bone erosion, synovitis and osteitis. A model was developed to predict cartilage damage from baseline parameters.

Results

Inter-reader reliability for the Auckland MRI cartilage score (AMRICS) was high for status scores; intraclass correlation coefficient (ICC), 0.90 (0.81 to 0.95) and moderate for change scores (ICC 0.58 (0.24 to 0.77)). AMRICS scores correlated with the Outcome MEasures in Rheumatoid Arthritis Clinical Trials (OMERACT) MRI joint space narrowing (jsn) and X-Ray (XR) jsn scores (r =0.96, P < 0.0001 and 0.80, P < 0.0001, respectively). AMRICS change scores were greater for RA patients than controls (P = 0.06 and P = 0.04 for the two readers). Using linear regression, baseline MRI cartilage, synovitis and osteitis scores predicted the three-year AMRICS (R² = 0.67, 0.37 and 0.39, respectively). A multiple linear regression model predicted the three-year AMRICS (R² = 0.78). Baseline radial osteitis predicted increased cartilage scores at the radiolunate and radioscaphoid joints, P = 0.0001 and 0.0012, respectively and synovitis at radioulnar, radiocarpal and intercarpal-carpometacarpal joints also influenced three-year cartilage scores (P-values of 0.001, 0.04 and 0.01, respectively).

Conclusions

MRI cartilage damage progression is preceded by osteitis and synovitis but is most influenced by pre-existing cartilage damage suggesting primacy of the cartilage damage pathway in certain patients.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).