Transmural gradients of cardiac myofiber shortening in aortic valve stenosis patients using MRI tagging

Aortic valve stenosis impairs subendocardial perfusion with a risk of irreversible subendocardial tissue damage. A likely precursor of damage is subendocardial contractile dysfunction, expressed by the parameter TransDif, which is defined as epicardial minus endocardial myofiber shortening, normalized to the mean value. With the use of magnetic resonance tagging in two short-axis slices of the left ventricle (LV), TransDif was derived from LV torsion and contraction during ejection. TransDif was determined in healthy volunteers (control, n = 9) and in patients with aortic valve stenosis before (AVSten, n = 9) and 3 mo after valve replacement (AVRepl, n = 7). In the control group, TransDif was 0.00 +/- 0.14 (mean +/- SD). In the AVSten group, TransDif increased to 0.96 +/- 0.62, suggesting impairment of subendocardial myofiber shortening. In the AVRepl group, TransDif decreased to 0.37 +/- 0.20 but was still elevated. In eight of nine AVSten patients, the TransDif value was elevated individually (P < 0.001), suggesting that the noninvasively determined parameter TransDif may provide important information in planning of treatment of aortic valve stenosis.     

Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer

Computerized measuring devices are needed to assess canine semen quality objectively both for research and practical purposes. As internal image settings may influence the results considerably, the effect of different technical settings and semen processing on the parameters assessed by the Hamilton-Thorne Ceros 12.1 semen analyzer (HTR Ceros 12.1) was investigated. The frame rate (15, 30 or 60 frames/s) significantly (P<0.05) influenced most of the measured motility characteristics in experiment 1 while no differences in the motility parameters were found using a different sampling duration (0.5 or 1 s, i.e. 30 or 60 frames scanned) in experiment 2. In experiment 3, an increase in sperm velocity (VAP, VSL, VCL), in linearity and in the percentage of motile and rapidly moving spermatozoa was observed with increasing sperm concentrations (25 x 10(6), 50 x 10(6) or 100 x 10(6) ml(-1)). In experiment 4, a clear effect of the diluent used was visible with higher velocity parameters (VAP, VSL, VCL) and higher percentages of motile, progressive and rapid spermatozoa for semen samples diluted in Hepes-TALP or prostatic fluid in comparison with physiological saline or egg-yolk-Tris extender. In experiment 5, significant (P<0.01) and high correlations were found between the conventional dog semen analysis methods and HTR Ceros 12.1 measurements (n=97 semen samples) for the sperm concentration (r=0.91), the motility (r=0.74) and the progressive motility (r=0.84). In experiment 6, the ejaculates from 21 proven, fertile dogs were compared with the ejaculates of a population (N: 11) of young beagles (1.5 years) but no significant differences in HTR Ceros 12.1 measurements were found between the two groups. Based on our results, diluting dog semen samples to 50 x 10(6) ml(-1) with physiological saline solution and scanning 30 frames at a frame rate of 60 frames/s (i.e. a scanning time of 0.5 s), are the set-up parameters proposed to obtain objective and standardized canine semen motility results using the HTR Ceros 12.1.     

Predominant <Emphasis Type="Italic">Bacillus</Emphasis> spp. in Agricultural Soil under Different Management Regimes Detected via PCR-DGGE

A PCR system for studying the diversity of species of Bacillus and related taxa directly from soil was developed. For this purpose, a specific 24-bp forward primer located around position 110 of the 16S ribosomal RNA gene was designed and combined with a reverse bacterial primer located at the end of the gene. The specificity of this PCR system for bacilli and related taxons was confirmed on the basis of tests with diverse strains as well as with soil DNA. Analysis of a soil DNA derived clone library showed that the amplified fragments affiliated exclusively with sequences of gram-positive bacteria, with up to 95% of the sequences originating from putative Bacillus species. In particular, sequences affiliated to those of B. mycoides, B. pumilus, B. megaterium, B. thuringiensis, and B. firmus, as well as to related taxa such as Paenibacillus, were obtained. A minority, i.e., less than 6%, of the clones affiliated with other gram-positive bacteria, such as Arthrobacter spp., Frankia spp., and uncultured gram-positives. The amplified fragments were used as templates for a second PCR using bacterial 16S rDNA primers, yielding PCR products of about 410 bp, which were separated by denaturing gradient gel electrophoresis (DGGE). Amplicons indicating Bacillus spp. were found in the gel between 45% and roughly 60% denaturant, whereas those representing other, high-G+C% bacteria, were localized in gel regions with denaturant concentrations exceeding about 60%, thus allowing the distinction between these two groups of sequences. We applied this system to compare the group-specific diversity in bacterial communities in an agricultural soil under different regimes, i.e., permanent grassland, grassland recently turned to arable land, and arable land under agricultural rotation. Differences in the Bacillus-related community structures between the treatments were clearly detected. Higher diversities, as judged by Shannon–Weaver indices calculated on the basis of the molecular profiles, were consistently observed in the permanent grassland and the grassland turned into arable land, as compared to the arable land.     

Diversity of bacteria associated with natural aphid populations

The bacterial communities of aphids were investigated by terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments generated by PCR with general eubacterial primers. By both methods, the gamma-proteobacterium Buchnera was detected in laboratory cultures of six parthenogenetic lines of the pea aphid Acyrthosiphon pisum and one line of the black bean aphid Aphis fabae, and one or more of four previously described bacterial taxa were also detected in all aphid lines except one of A. pisum. These latter bacteria, collectively known as secondary symbionts or accessory bacteria, comprised three taxa of gamma-proteobacteria (R-type [PASS], T-type [PABS], and U-type [PAUS]) and a rickettsia (S-type [PAR]). Complementary analysis of aphids from natural populations of four aphid species (A. pisum [n = 74], Amphorophora rubi [n = 109], Aphis sarothamni [n = 42], and Microlophium carnosum [n = 101]) from a single geographical location revealed Buchnera and up to three taxa of accessory bacteria, but no other bacterial taxa, in each aphid. The prevalence of accessory bacterial taxa varied significantly among aphid species but not with the sampling month (between June and August 2000). These results indicate that the accessory bacterial taxa are distributed across multiple aphid species, although with variable prevalence, and that laboratory culture does not generally result in a shift in the bacterial community in aphids. Both the transmission patterns of the accessory bacteria between individual aphids and their impact on aphid fitness are suggested to influence the prevalence of accessory bacterial taxa in natural aphid populations.     

Secondary and tertiary structure changes of reconstituted LmrA induced by nucleotide binding or hydrolysis. A fourier transform attenuated total reflection infrared spectroscopy and tryptophan fluorescence quenching analysis

LmrA, a membrane protein of Lactococcus lactis, extrudes amphiphilic compounds from the inner leaflet of the cytoplasmic membrane, using energy derived from ATP hydrolysis. A combination of total reflection Fourier transform infrared spectroscopy, (2)H/H exchange, and fluorescence quenching experiments was used to investigate the effect of nucleotide binding and/or hydrolysis on the structure of LmrA reconstituted into proteoliposomes. These measurements allowed us to describe secondary structure changes of LmrA during the catalytic cycle. The structure of LmrA is enriched in beta-sheet after ATP binding, and the protein recovers its initial secondary structure after ATP hydrolysis, when P(i) has been released. (2)H/H exchange and fluorescence quenching studies indicate that the protein undergoes two distinct tertiary structure changes during the hydrolysis process. Indeed, the protein alone is poorly accessible to the aqueous medium but adopts a more accessible conformation when ATP hydrolysis takes place. After ATP hydrolysis, but when P(i) is still associated with the protein, the accessibility is intermediate between these two states.     

Superoxide prevents nitric oxide-mediated suppression of helper T lymphocytes: decreased autoimmune encephalomyelitis in nicotinamide adenine dinucleotide phosphate oxidase knockout mice

NO, which suppresses T cell proliferation, may be inactivated by superoxide (O2-) due to their strong mutual affinity. To examine this possibility, preactivated Th clones were cocultured with stimulated macrophages. PMA neutralized the inhibitory activity of NO, which was dependent on extracellular O2- production. In contrast, macrophages from p47phox -/- (pKO) mice, which lack functional NADPH oxidase, retained their NO-dependent inhibition of T cell proliferation upon stimulation with PMA, indicating that NADPH oxidase is the major source of NO-inactivating O2- in this system. To examine the NO-O2- interaction in vivo, the role of NADPH oxidase in experimental autoimmune encephalomyelitis was studied in pKO mice. No clinical or histological signs were observed in the pKO mice. Neither a bias in Th subsets nor a reduced intensity of T cell responses could account for the disease resistance. Although spleen cells from pKO mice proliferated poorly in response to the immunogen, inhibition of NO synthase uncovered a normal proliferative response. These results indicate that NO activity may play a critical role in T cell responses in pKO mice and that in normal spleens inhibition of T cell proliferation by NO may be prevented by simultaneous NADPH oxidase activity.     

A combined CaMKII inhibition and mineralocorticoid receptor antagonism via eplerenone inhibits functional deterioration in chronic pressure overloaded mice

In the diseased and remodelled heart, increased activity and expression of Ca^2+/calmodulin‐dependent protein kinase II (CaMKII), an excess of fibrosis, and a decreased electrical coupling and cellular excitability leads to disturbed calcium homeostasis and tissue integrity. This subsequently leads to increased arrhythmia vulnerability and contractile dysfunction. Here, we investigated the combination of CaMKII inhibition (using genetically modified mice expressing the autocamtide‐3‐related‐peptide (AC3I)) together with eplerenone treatment (AC3I‐Epler) to prevent electrophysiological remodelling, fibrosis and subsequent functional deterioration in a mouse model of chronic pressure overload. We compared AC3I‐Epler mice with mice only subjected to mineralocorticoid receptor (MR) antagonism (WT‐Epler) and mice with only CaMKII inhibition (AC3I‐No). Our data show that a combined CaMKII inhibition together with MR antagonism mitigates contractile deterioration as was manifested by a preservation of ejection fraction, fractional shortening, global longitudinal strain, peak strain and contractile synchronicity. Furthermore, patchy fibrosis formation was reduced, potentially via inhibition of pro‐fibrotic TGF‐β/SMAD3 signalling, which related to a better global contractile performance and a slightly depressed incidence of arrhythmias. Furthermore, the level of patchy fibrosis appeared significantly correlated to eplerenone dose. The addition of eplerenone to CaMKII inhibition potentiates the effects of CaMKII inhibition on pro‐fibrotic pathways. As a result of the applied strategy, limiting patchy fibrosis adheres to a higher synchronicity of contraction and an overall better contractile performance which fits with a tempered arrhythmogenesis.     

Soil nutrient dissimilarity and litter nutrient limitation as major drivers of home field advantage in riparian tropical forests

Decomposition is a key process driving carbon and nutrient cycling in ecosystems worldwide. The home field advantage effect (HFA) has been found to accelerate decomposition rates when litter originates from “home” when compared to other (“away”) sites. It is still poorly known how HFA plays out in tropical, riparian forests, particularly in forests under restoration. We carried out three independent reciprocal litter transplant experiments to test how litter quality, soil nutrient concentrations, and successional stage (age) influenced HFA in tropical riparian forests. These experimental areas formed a wide gradient of soil and litter nutrients, which we used to evaluate the more general hypothesis that HFA varies with dissimilarity in soil nutrients and litter quality. We found that HFA increased with soil nutrient dissimilarity, suggesting that litter translocation uncouples relationships between decomposers and litter characteristics; and with litter N:P, indicating P limitation in this system. We also found negative HFA effects at a site under restoration that presented low decomposer ability, suggesting that forest restoration does not necessarily recover decomposer communities and nutrient cycling. Within each of the independent experiments, the occurrence of HFA effects was limited and their magnitude was not related to forest age, nor soil and litter quality. Our results imply that HFA effects in tropical ecosystems are influenced by litter nutrient limitation and soil nutrient dissimilarity between home and away sites, but to further disentangle major HFA drivers in tropical areas, a gradient of dissimilarity between litter and soil properties must be implemented in future experimental designs.     

Cloning of the Aspergillus niger gene encoding α-l-arabinofuranosidase A

Using l-arabitol as an inducer, simple induction conditions were established that resulted in high-level expression of -l-arabinofuranosidase A by an Aspergillus niger d-xylulose kinase mutant strain. These conditions were adapted to construct a cDNA expression library from which an -l-arabinofuranosidase A cDNA clone was isolated using specific antiserum. The corresponding gene encoding -l-arabinpfuranosidase A (abfA) was isolated from a genomic library and cloned into a high copy plasmid vector. By co-transformation of uridine auxotrophic mutants lacking orotidine-5-phosphate decarboxylase activity, the afbA gene was introduced both in A. niger and A. nidulans, using the A. niger pyrA gene as selection marker. The identity of the abfA gene was confirmed by overexpression of the gene product by A. niger and A. nidulans transformants, upon growth using sugar beet pulp as the carbon source.