Surface charges of the membrane and cell adhesion substances determine the structural integrity of hair bundles from the inner ear of fish

Summary The hairs (stereocilia = stereovilli) of sensory cells from the inner ear of vertebrates are interconnected by several types of connectors, whose role is unknown. They appear to stabilize the hair bundle mechanically, and may be directly involved in mechano-electric transduction. Our transmission electron-microscopical investigation of sensory epithelia from two species of fish (Rutilus rutilus, Scardinius erythrophthalmus, both Leuciscidae) has shown that not only the connectors but also the surface charges of the membrane are important factors for determining the shape of the hair bundle and the spatial interrelation of the stereovilli. A reduction of the ionic strength in the medium leads to an increase in distance between the stereovilli. This may be the result of an extension of the spread of the surface potential of the membrane at low ionic strength. The connectors are not broken by the increase in distance between the stereovilli. They are EDTA (ethylene-diamine-tetra-acetic-acid) resistant as are some cell adhesion molecules such as N-CAM (nerve-cell adhesion molecule) and protein A from Dictyostelium discoideum. The connectors do not prevent polycation-induced fusion of adjacent stereovillar membranes.     

N-cadherin and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces

Receptor-mediated interactions between neurons and astroglia are likely to play a crucial role in the growth and guidance of CNS axons. Using antibodies to neuronal cell surface proteins, we identified two receptor systems mediating neurite outgrowth on cultured astrocytes. N-cadherin, a Ca2(+)-dependent cell adhesion molecule, functions prominently in the outgrowth of neurites on astrocytes by E8 and E14 chick ciliary ganglion (CG) neurons. beta 1-class integrin ECM receptor heterodimers function less prominently in E8 and not at all in E14 neurite outgrowth on astrocytes. The lack of effect of integrin beta 1 antibodies on E14 neurite outgrowth reflects an apparent loss of integrin function, as assayed by E14 neuronal attachment and process outgrowth on laminin. N-CAM appeared not to be required for neurite outgrowth by either E8 or E14 neurons. Since N-cadherin and integrin beta 1 antibodies together virtually eliminated E8 CG neurite outgrowth on cultured astrocytes, these two neuronal receptors are probably important in regulating axon growth on astroglia in vivo.     

A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer.

The cardiac troponin T pre-mRNA contains an exonic splicing enhancer that is required for inclusion of the alternative exon 5. Here we show that enhancer activity is exquisitely sensitive to changes in the sequence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the level of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-linking and immunoprecipitation indicated that only four (SRp30a, SRp40, SRp55, and SRp75) of six essential splicing factors known as SR proteins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vitro competition of SR protein splicing activity and UV cross-linking demonstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer strength. Thus, a subset of SR proteins interacts directly with the exonic enhancer to promote inclusion of a poorly defined alternative exon. Independent regulation of the levels of SR proteins may, therefore, contribute to the developmental regulation of exon inclusion.     

Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium.

The gene for the penicillinase from B. licheniformis has been cloned in a functional stat on a 1.5 kb DNA fragment and its nucleotide sequence has been determined. A sequence of 307 amino acid residues is infered for the penicillinase precursor. Of these 34 amino acids precede the sequence of the secreted form of the enzyme. This peptide extension shows the features of a signal for secretion and also provides the hydrophobic anchor for the membrane-bound form of the enzyme.     

Exploiting nonsystematic covariate monitoring to broaden the scope of evidence about the causal effects of adaptive treatment strategies

In studies based on electronic health records (EHR), the frequency of covariate monitoring can vary by covariate type, across patients, and over time, which can limit the generalizability of inferences about the effects of adaptive treatment strategies. In addition, monitoring is a health intervention in itself with costs and benefits, and stakeholders may be interested in the effect of monitoring when adopting adaptive treatment strategies. This paper demonstrates how to exploit nonsystematic covariate monitoring in EHR‐based studies to both improve the generalizability of causal inferences and to evaluate the health impact of monitoring when evaluating adaptive treatment strategies. Using a real world, EHR‐based, comparative effectiveness research (CER) study of patients with type II diabetes mellitus, we illustrate how the evaluation of joint dynamic treatment and static monitoring interventions can improve CER evidence and describe two alternate estimation approaches based on inverse probability weighting (IPW). First, we demonstrate the poor performance of the standard estimator of the effects of joint treatment‐monitoring interventions, due to a large decrease in data support and concerns over finite‐sample bias from near‐violations of the positivity assumption (PA) for the monitoring process. Second, we detail an alternate IPW estimator using a no direct effect assumption. We demonstrate that this estimator can improve efficiency but at the potential cost of increase in bias from violations of the PA for the treatment process.     

Appraisal Tools for Clinical Practice Guidelines: A Systematic Review

Introduction

Clinical practice guidelines can improve healthcare processes and patient outcomes, but are often of low quality. Guideline appraisal tools aim to help potential guideline users in assessing guideline quality. We conducted a systematic review of publications describing guideline appraisal tools in order to identify and compare existing tools.

Methods

Among others we searched MEDLINE, EMBASE and the Cochrane Database of Systematic Reviews from 1995 to May 2011 for relevant primary and secondary publications. We also handsearched the reference lists of relevant publications.On the basis of the available literature we firstly generated 34 items to be used in the comparison of appraisal tools and grouped them into thirteen quality dimensions. We then extracted formal characteristics as well as questions and statements of the appraisal tools and assigned them to the items.

Results

We identified 40 different appraisal tools. They covered between three and thirteen of the thirteen possible quality dimensions and between three and 29 of the possible 34 items. The main focus of the appraisal tools were the quality dimensions “evaluation of evidence” (mentioned in 35 tools; 88%), “presentation of guideline content” (34 tools; 85%), “transferability” (33 tools; 83%), “independence” (32 tools; 80%), “scope” (30 tools; 75%), and “information retrieval” (29 tools; 73%). The quality dimensions “consideration of different perspectives” and “dissemination, implementation and evaluation of the guideline” were covered by only twenty (50%) and eighteen tools (45%) respectively.

Conclusions

Most guideline appraisal tools assess whether the literature search and the evaluation, synthesis and presentation of the evidence in guidelines follow the principles of evidence-based medicine. Although conflicts of interest and norms and values of guideline developers, as well as patient involvement, affect the trustworthiness of guidelines, they are currently insufficiently considered. Greater focus should be placed on these issues in the further development of guideline appraisal tools.     

The influence of phylogeny and ecology on root,shoot and plant ionomes of 14 native Brazilian species

The ionome is the elemental composition of a living organism, its tissues, cells or cell compartments. The ionomes of roots, stems and leaves of 14 native Brazilian forest species were characterised to examine the relationships between plant and organ ionomes and the phylogenetic and ecological affiliations of species. The null hypothesis that ionomes of Brazilian forest species and their organs do not differ was tested. Concentrations of mineral nutrients in roots, stems and leaves were determined for 14 Brazilian forest species, representing seven angiosperm orders, grown hydroponically in a complete nutrient solution. The 14 species could be differentiated by their ionomes and the partitioning of mineral nutrients between organs. The ionomic differences between the 14 species did not reflect their phylogenetic relationships or successional ecology. Differences between shoot ionomes and root ionomes were greater than differences in the ionome of an organ when compared among genotypes. In conclusion, differences in ionomes of species and their organs reflect a combination of ancient phylogenetic and recent environmental adaptations.     

Agonist-dependent AT(4) receptor internalization in bovine aortic endothelial cells

Recent studies have characterized a specific binding site for the C-terminal 3-8 fragment of angiotensin II (Ang IV). In the present study we looked at the internalization process of this receptor on bovine aortic endothelial cells (BAEC). Under normal culture conditions, BAEC efficiently internalized (125)I-Ang IV as assessed by acid-resistant binding. Internalization of (125)I-Ang IV was considerably decreased after pretreatment of cells with hyperosmolar sucrose or after pretreatment of BAEC with inhibitors of endosomal acidification such as monensin or NH(4)Cl. About 50% of internalized (125)I-Ang IV recycled back to the extracellular medium during a 2 h incubation at 37 degrees C. (125)I-Ang IV remained mostly intact during the whole process of internalization and recycling as assessed by thin layer chromatography. As expected, internalization of (125)I-Ang IV was completely abolished by divalinal-Ang IV, a known AT(4) receptor antagonist. Interestingly, (125)I-divalinal-Ang IV did not internalize into BAEC. These results suggest that AT(4) receptor undergoes an agonist-dependent internalization and recycling process commonly observed upon activation of functional receptors.     

Kinin B1 receptor antagonists with multi-enzymatic resistance properties

The kinin B, receptor has been implicated in a variety of pathological states; therefore, potent, selective, and specific antagonists with prolonged duration of action in vivo are needed. Using R-715 (AcLys[D-beta-Nal(7),Ile(8)] desArg9BK) as a template, new peptides containing alpha-MePhe in position 5, Oic in position 2, and AcOrn instead of AcLys at the N-terminal were prepared and tested for their antagonist potency, their selectivity, and their specificity for the kinin B1 receptor. In vitro metabolic stabilities toward aminopeptidase M (from human plasma), aminopeptidase P (from human platelets), and angiotensin-converting enzyme (purified from rabbit lung) were also investigated. The results of this study indicate that the three modifications applied separately are as well tolerated as they are when present conjointly in the template R-715. Indeed, pA2 values of R-715 (ranging from 8.40 to 8.5) do not differ significantly from the analogues R-954 and R-955 (both ranging from 8.4 to 8.6) when measured at kinin B1 receptors from rabbit aortas and human umbilical veins. Moreover, the chemical modifications utilized in the peptides R-954 and R-955 have provided resistance against aminopeptidases M and P, as well as the angiotensin-converting enzyme, unlike the early (e.g., Lys[Leu8]desArg9BK) and more recent (e.g., R-715, B-9858) generations of B, receptor antagonists. Ongoing in vivo assays will validate the assumption that the analogues R-954 and R-955 have a prolonged duration of action.     

Regulation of Alternative Polyadenylation by U1 snRNPs and SRp20

Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3′-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3′-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3′-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3′ and 5′ splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3′-terminal exons and perhaps in the communication between polyadenylation and splicing.