Spectrum of Cav1.4 dysfunction in congenital stationary night blindness type 2

Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.     

Selective alteration of soil food web components by invasive giant goldenrod Solidago gigantea in two distinct habitat types

Apart from relatively well‐studied aboveground effects, invasive plant species will also impact the soil food web. So far, most research has been focusing on primary decomposers, while studies on effects at higher trophic levels are relatively scarce. Giant goldenrod Solidago gigantea, native to North America, is a widespread and common invasive species in most European countries. We investigated its impact on plant communities and on multiple trophic levels of the soil food web in two contrasting habitats: riparian zones and semi‐natural grasslands. In 30 pairs of invaded and uninvaded plots, floristic composition, pH, fungal biomass and the densities of 11 nematode taxa were determined by using a quantitative PCR‐based method. In the two habitats, the invader outcompeted both rare and dominant plant species. Belowground, S. gigantea invasion reduced pH, increased overall fungal biomass as well as the density of a single lineage of fungivorous nematodes, the family Aphelenchoididae. The densities of two other, phylogenetically distinct lineages of fungivorous nematodes, Aphelenchidae and Diphtherophoridae, were unaffected by the local increase in fungal biomass. Apparently this plant species induces a local asymmetric boost of the fungal community, and only Aphelenchoididae were able to benefit from this invader‐induced change. The alternative explanation – the results are explained by a subtle, S. gigantea‐induced 0.1–0.2 units decrease of pH – seems unlikely, as pH optima for nematode taxa are relatively broad. Thus, apart from readily observable aboveground effects, the invasive plant species S. gigantea affects fungal biomass as well as a specific part of the fungivorous nematode community in a soil type‐independent manner.     

A Monoclonal Antibody Selection for Immunohistochemical Examination of Lymphoid Tissues From Non-human Primates

Non-human primates (NHPs) offer valuable animal models for basic research into human diseases and for the preclinical validation of new therapeutics. Detailed in situ examination of the involved cell types using immunohistochemistry is often hampered by the lack of cross-reactive antibodies (Abs). In the current study, we have tested a large panel of monoclonal antibodies raised against human leukocyte differentiation and activation markers for cross-reactivity on cryosections of lymphoid tissue from six NHP species. In total, we have tested 130 Abs against 69 antigens expressed in tissues from one great ape species (chimpanzee/Pan troglodytes), two Old World species (rhesus macaque/Macaca mulatta and cynomolgus macaque/Macaca fascicularis), and three New World species (common marmoset/Callithrix jacchus, cotton-top tamarin/Saguinus oedipus, and owl monkey/Aotus triviogatus). We have found a large panel of cross-reactive Abs: 93 of 102 (91%) in chimpanzee, 97 of 125 (78%) in rhesus macaque, 70 of 109 (64%) in cynomolgus macaque, 69 of 116 (60%) in common marmoset, 40 of 81 (49%) in cotton-top tamarin, and 35 of 80 (44%) in owl monkey. The availability of a reliable panel of cross-reactive markers is important to gaining further insight into immunological processes in disease-affected tissues from NHP species. (J Histochem Cytochem 57:1159–1167, 2009)     

Membranes in motion

The Keystone Symposium on the Molecular Basis for Biological Membrane Organization and Dynamics held in January this year offered new insights into the molecular machines at work in cells. Topics included the machinery responsible for the dynamic shape of organelles, the budding and fusion of vesicular carriers, and the intricate sorting systems that ensure the correct delivery of cellular components.     

Homoplasy corrected estimation of genetic similarity from AFLP bands,and the effect of the number of bands on the precision of estimation

AFLP is a DNA fingerprinting technique, resulting in binary band presence–absence patterns, called profiles, with known or unknown band positions. We model AFLP as a sampling procedure of fragments, with lengths sampled from a distribution. Bands represent fragments of specific lengths. We focus on estimation of pairwise genetic similarity, defined as average fraction of common fragments, by AFLP. Usual estimators are Dice (D) or Jaccard coefficients. D overestimates genetic similarity, since identical bands in profile pairs may correspond to different fragments (homoplasy). Another complicating factor is the occurrence of different fragments of equal length within a profile, appearing as a single band, which we call collision. The bias of D increases with larger numbers of bands, and lower genetic similarity. We propose two homoplasy- and collision-corrected estimators of genetic similarity. The first is a modification of D, replacing band counts by estimated fragment counts. The second is a maximum likelihood estimator, only applicable if band positions are available. Properties of the estimators are studied by simulation. Standard errors and confidence intervals for the first are obtained by bootstrapping, and for the second by likelihood theory. The estimators are nearly unbiased, and have for most practical cases smaller standard error than D. The likelihood-based estimator generally gives the highest precision. The relationship between fragment counts and precision is studied using simulation. The usual range of band counts (50–100) appears nearly optimal. The methodology is illustrated using data from a phylogenetic study on lettuce.     

Determination of cyclophosphamide enantiomers in plasma by LC-MS/MS: Application to pharmacokinetics in breast cancer and lupus nephritis patients

This article describes the enantioselective analysis of cyclophosphamide (CPA) in human plasma using LC-MS/MS. CPA enantiomers were extracted from plasma using a mixture of ethyl acetate and chloroform (75:25, v/v). The enantiomers were separated on a Chiralcel(R) OD-R column, with the mobile phase consisting of a mixture of acetonitrile and water (75:25, v/v) plus 0.2% formic acid. The protonated ions and their respective product ions were monitored using two functions, 261 > 141 for CPA enantiomers and 189 > 104 for the internal standard (antipyrine). Recovery rates were higher than 95% and the quantification limit was 2.5-ng/ml plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of intra- and interassay precision and accuracy were less than 10%. The method was applied for the investigation of the enantioselective pharmacokinetics of CPA in a lupus nephritis patient treated with 1 g CPA infused over 2 h and in a breast cancer patient treated with 0.9 g infused over 1 h. No stereoselectivity in the pharmacokinetic parameters was observed for either patient. Clearance values of 2.63 and 2.93 l/h and of 3.36 and 3.61 l/h for (-)-(S) and (+)-(R)-CPA were obtained for the breast cancer and lupus nephritis patient, respectively.     

Sex‐specific food provisioning in a monomorphic seabird,the common guillemot Uria aalge: nest defence,foraging efficiency or parental effort?

Sexual differences in food provisioning rates of monomorphic seabirds are well known but poorly understood. Here, we address three hypotheses that attempt to explain female-biased food provisioning in common guillemots Uria aalge : (1) males spend more time in nest defence, (2) females have greater foraging efficiency, and (3) males allocate a greater proportion of foraging effort to self-maintenance. We found that males spent no more time with chicks than females but made longer trips and travelled further from the colony. There was extensive overlap between sexes in core foraging areas, indicating that females were not excluding males from feeding opportunities close to the colony. However, as a result of their longer trips, the total foraging areas of males were much greater than those of females. There was no difference between sexes in overall dive rate per hour at sea, in behaviour during individual dives or in a number of other measures of foraging efficiency including the frequency, depth and duration of dives and the dive: pause ratio during the final dive bout of each trip, which was presumably used by both sexes to obtain prey for the chick. These data strongly suggest that sexes did not differ in their ability to locate and capture prey. Yet males made almost twice as many dives per trip as females, suggesting that males made more dives than females for their own benefit. These results support the hypothesis that female-biased food provisioning arose from a difference between sexes in the allocation of foraging effort between parents and offspring, in anticipation of a prolonged period of male-only post-fledging care of the chick, and not from differences in foraging efficiency or time spent in nest defence.