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.     

Structure and function of Y chromosomal DNA

The sequence organization of four different families of Y chromosomal repetitive DNA is characterized at three levels of spatial extension along the Y chromosome of Drosophila hydei. At the lowest level of resolution, DNA blot analysis of Y chromosomal fragments of different lengths and in situ hybridization experiments on metaphase chromosomes demonstrate the clustering of each particular sequence family within one defined region of the chromosome. At a higher level of resolution, family specific repeats can be detected within these clusters by crosshybridization within 10–20 kb long continuous stretches of cloned DNA in EMBL3 phages. At the highest level of resolution, detailed sequence analysis of representative subclones about 1 kb in length reveals a satellite-like head to tail arrangement of family specific degenerated subrepeats as the building scheme common to all four families. Our results provide the first comparative sequence analysis of three novel families of repetitive DNA on the long arm of the F chromosome of D. hydei. Additional data are presented which support the existence of two related subfamilies of repetitive DNA on the short arm of the Y chromosome.     

Hydroxynitrile lyases: Functions and properties

Plant hydroxynitrile lyases (Hnl) have attracted the attention of bioorganic scientists for more than 90 years. However, the most important increase in knowledge of this class of enzymes has only arisen in the recent decade. The industrial application of these enzymes as biocatalysts for the synthesis of enantiomerically pure α-cyanohydrins may be responsible for the growing interest in this area.
The Hnls are involved in the catabolic degradation of cyanogenic glycosides, releasing HCN which serves as defense agent against herbivores and microbial attack, or as a nitrogen source. Hydroxynitrile lyases from various plant families appear to represent a new example of enzymes that originated from the convergent evolution of different precursor proteins. The enzymes have been classified into non-FAD- and FAD-containing proteins. FAD-containing enzymes have been isolated exclusively from the Rosaceae, whereas the FAD-independent Hnls, which are more heterogenous in structure, have been characterized from various plant families (Poaceae, Euphorbiaceae, Linaceae, Olacaceae. Filitaceae). The aim of this review is to present a general survey of the natural function and localization of this class of enzymes and a comprehensive summary of the biochemical and genetic data of the isolated proteins.     

Direct observation of phytoplankton, TEP and aggregates on polycarbonate filters using brightfield microscopy

There has been little use of standard (i.e. non-inverted) microscopesfor observing and counting phytoplankton in filtered water samplesusing brightfield white light illumination due to light interferencefrom the filters. If filters are placed on newly designed frostedslides (Cyto-clear, Poretics Corp.), however, phytoplanktoncan be viewed directly on the surfaces of polycarbonate filtersunder brightfield illumination. Lake and seawater samples wereused to show that samples stained with alcian blue (to identifythe presence of paniculate polysaccharides) and analyzed withwhite light can also be simultaneously stained with fluorochromes(i.e. DAPI and acridine orange) for additional examination ofthe sample using fluorescent techniques. Black filters, whichare necessary for epifluorescent techniques, did not interferewith brightfield viewing. Using double staining techniques,we found that transparent exopolymer particles (TEP) recentlydiscovered in marine systems are also present in lakes. Notall aggregates in the fresh and seawater systems absorbed thealcian blue stain, however, indicating that not all amorphousparticles in these systems are rich in negatively charged polysaccharides.     

The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

Using real-world data to predict health outcomes—The prediction design: Application and sample size planning

The gold standard for investigating the efficacy of a new therapy is a (pragmatic) randomized controlled trial (RCT). This approach is costly, time-consuming, and not always practicable. At the same time, huge quantities of available patient-level control condition data in analyzable format of (former) RCTs or real-world data (RWD) are neglected. Therefore, alternative study designs are desirable. The design presented here consists of setting up a prediction model for determining treatment effects under the control condition for future patients. When a new treatment is intended to be tested against a control treatment, a single-arm trial for the new therapy is conducted. The treatment effect is then evaluated by comparing the outcomes of the single-arm trial against the predicted outcomes under the control condition. While there are obvious advantages of this design compared to classical RCTs (increased efficiency, lower cost, alleviating participants’ fear of being on control treatment), there are several sources of bias. Our aim is to investigate whether and how such a design—the prediction design—may be used to provide information on treatment effects by leveraging external data sources. For this purpose, we investigated under what assumptions linear prediction models could be used to predict the counterfactual of patients precisely enough to construct a test and an appropriate sample size formula for evaluating the average treatment effect in the population of a new study. A user-friendly R Shiny application (available at: https://web.imbi.uni-heidelberg.de/PredictionDesignR/ ) facilitates the application of the proposed methods, while a real-world application example illustrates them.     

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.