Limited flexibility of the inter-protofilament bonds in microtubules assembled from pure tubulin

The superposition of the regular arrangement of tubulin subunits in microtubules gives rise to moiré patterns in cryo-electron micrographs. The moiré period can be predicted from the dimensions of the tubulin subunits and their arrangement in the surface lattice. Although the average experimental moiré period is usually in good agreement with the theoretical one, there is variation both within and between microtubules. In this study, we addressed the origin of this variability. We examined different possibilities, including artefacts induced by the preparation of the vitrified samples, and variations of the parameters that describe the microtubule surface lattice. We show that neither flattening nor bending of the microtubules, nor changes in the subunit dimensions, can account for the moiré period variations observed in 12 and 14 protofilament microtubules. These can be interpreted as slight variations, in the range –0.5 Å to +0.9 Å, of the lateral interactions between tubulin subunits in adjacent protofilaments. These results indicate that the inter-protofilament bonds are precisely maintained in microtubules assembled in vitro from pure tubulin. The fact that the moiré period is not affected by bending indicates that the local interactions between tubulin subunits are sufficiently stiff to accommodate large deformations of the microtubule wall.     

Morphology of the meniscotemporal part of the temporomandibular joint and its biomechanical implications

The temporomandibular joint is a diarthrosis. It has classically been referred to as being a condylar, and functionally as a double condylar joint. We have used the moiré topographical technique to study the morphology of the pars suprameniscalis or meniscotemporalis of the joint. The findings show that this pars suprameniscalis could be classified as a saddle joint. The biomechanical implications are also discussed.     

Detection of stimulated back muscle contractions by moiré topography

The ability to treat scoliosis via surface stimulated trunk muscle contractions is now being evaluated at several treatment centers. In order to make biomechanical analysis of the procedure, so that the technique can be used optimally, data are needed to quantify the muscle contractions and structural changes by different electrode locations. This paper presents the use of a modified shadow moiré technique to quantify geometric changes resulting from electrical stimulation applied to the surface of the back in a healthy subject.     

An application of the moiré method to three-dimensional measurements of the occlusal aspects of molars

Moiré photographs of the occlusal surface of human molars were made by means of a specially designed apparatus for moiré contourography. Since molars show contour lines with equal intervals, three-dimensional measurement of the complicated relief of the occlusal surface is possible. The positioning of molar teeth for making the photographs and the technique of the moiré photography for small objects were presented. The three-dimensional measuring method of the molar surface and its use for anthropological purpose were discussed.     

Observations on graphing paleozoological data: Suggestions for better graphs

Paleozoologists have long used graphs of diverse styles to describe, analyze, and summarize their data. Some of these graphs provide excellent visual representations of complex data and are readily deciphered. Other graph styles require close study to be interpreted. Ease of visual decoding of information contained in a graph – graph perception – varies from graph style to graph style. Historical instances of graphing paleozoological data indicate some difficult to decipher graph styles have been used for at least a century. Graphs with three-dimensions, moiré effects, superimposed lines, or segmented bars, or which demand simultaneous decipherment of position and magnitude, are ill-advised. Temporal trends in data are best graphed following the principle of superposition such that data from old material is graphed at the bottom and data from younger material is graphed at the top of the diagram.     

Selection of a subtilisin-hyperproducing Bacillus in a highly structured environment

Mutants that secrete increased amounts of enzyme into a selection medium can be efficiently enriched from large populations of mutagenized microorganisms during growth in hollow fibers. Under these conditions, each colony grows in its own microenvironment and cross-feeding between neighboring colonies is limited. We applied the technique to B. subtilis carrying a plasmid-encoded protease gene. The plasmid was subjected to random mutagenesis and clones secreting up to fivefold-increased amounts of enzyme were selected using a medium containing bovine serum albumin as the sole nitrogen source. Received: 22 May 1997 / Received revision: 21 October 1997 / Accepted: 7 November 1997     

Measuring energy expenditure in birds using bolus injections of 13C-labelled Na-bicarbonate

The ¹³C-labelled Na-bicarbonate technique uses stable isotopes to measure energy expenditure in birds. After administration, the isotopes reach equilibrium within the body's bicarbonate pools at a fast rate due to the small size of the bicarbonate pool in relation to CO₂ flux. This technique is therefore ideal for measuring energy expenditure over short-term activities. The major advantage of this technique is that it can be applied without the animal having to wear a respirometry mask or being enclosed in a respirometry chamber. Despite the technique's suitability for use in birds and other animals, there have been few studies that have used it to date and so its potential is not fully understood. Here we discuss the methodology and review previous applications.     

Efficiency of wear and decalcification technique for estimating the age of estuarine dolphin <Emphasis Type="Italic">Sotalia guianensis</Emphasis>

Most techniques used for estimating the age of Sotalia guianensis (van Bénéden, 1864) (Cetacea; Delphinidae) are very expensive, and require sophisticated equipment for preparing histological sections of teeth. The objective of this study was to test a more affordable and much simpler method, involving of the manual wear of teeth followed by decalcification and observation under a stereomicroscope. This technique has been employed successfully with larger species of Odontoceti. Twenty-six specimens were selected, and one tooth of each specimen was worn and demineralized for growth layers reading. Growth layers were evidenced in all specimens; however, in 4 of the 26 teeth, not all the layers could be clearly observed. In these teeth, there was a significant decrease of growth layer group thickness, thus hindering the layers count. The juxtaposition of layers hindered the reading of larger numbers of layers by the wear and decalcification technique. Analysis of more than 17 layers in a single tooth proved inconclusive. The method applied here proved to be efficient in estimating the age of Sotalia guianensis individuals younger than 18 years. This method could simplify the study of the age structure of the overall population, and allows the use of the more expensive methodologies to be confined to more specific studies of older specimens. It also enables the classification of the calf, young and adult classes, which is important for general population studies.     

Measurement of scapular kinematics with the moiré fringe projection technique

The aim of this study was to investigate the applicability of the moiré fringe projection technique (MFPT) to quantify the scapular motions relative to the thorax. This system was composed of a LCD projector, a digital photographic camera, and a microcomputer. To automatically obtain the scapular profiles, the phase shifting method was combined with the MFPT. Four fringes were projected on the scapula and four on the reference planes. By the simple subtraction of the reference values from the scapular phase maps, the map due to the moiré fringes could be digitally obtained. After the phase decoding, the tridimensional (3D) profiles were obtained without prior information about the samples and the calculations of the scapular kinematics were carried out using dedicated software. On average, the movements of lateral rotation ranged from ?1.8±6.1° to ?26.5±3.5°; the protraction from 28.4±4.7° to 27.7±6.8°, and the posterior tilt from ?6.4±7.8° to ?21.7±6.1°, during the arm elevation in the scapular plane performed by six healthy subjects. For the test–retest reliability, the intra-class correlation coefficients ranged from 0.92 to 0.997 and the maximum estimated error was 0.8%. The MFPT allowed the scapular 3D measurements to be obtained in a digital and non-invasive manner. The main advantages compared with other existing systems were its ease in implementation, the use of standard optical components, and its possible clinical applications.     

Impact of C60 Adsorption on Surface Plasmon Polaritons on Self-Assembled Ag(111) Islands on Si(111)

The influence of C₆₀ adsorption on the properties of surface plasmon polaritons on small Ag islands is discussed. Under illumination with UV light as well as under illumination with femtosecond laser pulses, a decrease of the photoemission yield with increasing C₆₀ coverage is observed. With angular resolved measurements, changes of the band structure during deposition are studied. Based on these experiments, an increase of the work function with increasing coverage is measured. In two photon photoemission, the surface plasmons are imaged as a periodic moiré pattern, the wavelength of which changes because of a modified effective surface dielectric function. Our findings imply that the wavelength of the plasmon wave becomes shorter as a result. Finally, a decrease of the intensity of the moiré pattern maxima compared with the intensity of the first maximum with increasing C₆₀ coverage has been observed. Accordingly, the damping of the plasmon wave becomes stronger.     

Etudein situ de l'influence de l'humidité et de la teneur en nitrate d'un sol dunaire sur l'accumulation et la réduction du nitrate chez l'oyat (Ammophila arenaria L.)

Résumé L'étude du fonctionnement du cycle de l'azote dans les milieux dunaires des c?tes méditerranéennes fran?aises a conduit à analyser le comportement de l'oyat,Ammophila arenaria, au cours de son développement en réponse aux variations de l'humidité et de la teneur en nitrate du sol. Les mesures d'activité nitrate réductase et de la teneur en nitrate dans les feuilles ont été effectuées simultanément avec les mesures des teneurs en eau et en nitrate du sol. Cette plante a des activités nitrate réductase ne dépassant pas 0,27 μmoles h⁻¹ g⁻¹ matière fra?che, qui n'ont pu être mesuréesin vivo qu'en présence de nitrate exogène dans le milieu d'incubation. Il appara?t que la plante réduit ou accumule préférentiellement le nitrate selon que l'on se situe en période humide ou sèche. Pour des valeurs voisines ou inférieures à 2% d'humidité dans les vingt premiers centimètres de sol, l'activité nitrate réductase décro?t et des quantités importantes de nitrate s'accumulent (169 μg N.NO₃.g⁻¹ matière sèche). Dans ces conditions, le nitrate dans la solution du sol peut atteindre une concentration élevée (6 meq.l⁻¹). Inversement, au-delà de 2% d'humidité le nitrate endogène s'épuise et l'activité nitrate réductase augmente. La régulation de l'assimilation et de l'accumulation du nitrate par l'oyat dépend de son alimentation hydrique et done des quantités de nitrate absorbées à partir de la solution du sol et véhiculées dans la sève brute.

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In situ study of the influence of dune soil humidity and nitrate contents on the nitrate accumulation and reduction in marram (Ammophila arenaria L.)

Summary The search for a better understanding of the nitrogen cycle in the sand dunes of french mediterranean beaches have driven us to study the habits ofAmmophila arenaria. In this way, we have followed its responses to different soil water potentials and nitrate contents of the soil during its development. Nitrate reductase activities and nitrate contents of leaves have been measured simultaneously with water and nitrate contents of soil. This plant has a slight nitrate reduction activity, not more than 0,27 μmoles h⁻¹ g⁻¹ fresh matter, which could be measuredin vivo only by the addition of nitrate to the incubation media. It seems that the plant reduces or else it stores, in a preferential way the nitrate whether it is in a humid or dry period. If humidity values are near or below 2%, the marram diminishes its assimilation activity and stores important amounts of nitrate (169 μg N−NO₃ ⁻.g⁻¹ dry matter). In these conditions the nitrate can be highly concentrated in the soil solutions (6 meq.l⁻¹). Opposite, above 2% of humidity, the nitrate reductase activity decreases and the endogenous nitrate is consumed. The regulation of the assimilation and storage of nitrate by the marram is mainly a function of the soil humidity and so, of the amount of nitrate taken from the soil solution and carried into the xylem.

     

A missense mutation in Col1A1 in a Jewish Israeli patient with mild osteogenesis imperfecta, detected by DGGE

Osteogenesis imperfecta (OI) underlies germline mutations in either Col1A1 or Col1A2. Here we describe, for the first time, the use of the denaturing gradient gel electrophoresis (DGGE) technique for mutation analysis of the Col1A1 gene. By employing this technique, we identified a point mutation in a young Jewish Israeli patient with mild OI. The missense mutation, a G to A alteration at position 888, result in a Gly to Arg substitution at codon 79. Furthermore, the patient’s mother, who was clinically labeled as OI based solely on the fact that she has blue sclera, was found not to carry this mutation in two different tissues. We suggest that blue sclera alone should not be used as a parameter for the diagnosis of OI, and that DGGE can be effectively used for mutation analysis of the Col1A1 gene. Received: 13 March 1997 / Accepted: 26 June 1997     

Carboxyterminal telopeptide of type I collagen,ICTP, as a marker of matrix degradation in neonatal mouse calvarial bones,in vitro

Bone resorption,in vitro, is often measured as the release of prelabelled⁴⁵Ca from neonatal mouse calvarial bones, or from fetal rat long bones. In this report we describe a technique to measure the breakdown of bone-matrix,in vitro. We also describe a new way to dissect neonatal mouse calvarial bones, in order to obtain large amounts of bone samples.Twelve bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 culture medium in serum-free conditions in 0.5 cm² multiwell culture dishes. Matrix degradation after treatment with parathyroid hormone was assessed by measuring the amount of carboxyterminal telopeptide of type I collagen (ICTP) by RIA. The data on matrix degradation was compared to the release of prelabelled⁴⁵Ca from neonatal mouse calvarial bones. We found that the dose-responses for parathyroid hormone-induced release of prelabelled⁴⁵Ca and ICTP were identical.In conclusion: RIA-analysis of the ICTP-release is an easy and accurate method to measure degradation of bone-matrix,in vitro. Furthermore, the new dissection technique, described in this report, makes it easy to obtain large amounts of bone samples and thus to perform extensive experiments, e.g. dose-responses for agents that enhance bone resorption.     

Moiré patterns: an accurate technique for determination of the locus of the centres of rotation

This study describes an accurate technique for the determination of the centre of rotation of small angles. The moiré fringe method localizes the centre of rotation by defining two primary fringes, each of which is found by the intersection of three lines. The primary fringes intersect at the centre of rotation at 90 degrees to each other, the angle least likely to produce an error in measurement. By utilizing joints with known centres of rotation, we have found that the method is extremely accurate and reproducible to within 2 mm of the real centre for angular changes as small as 3 degrees. This technique is useful in evaluating whether a joint is a simple hinge, i.e. rotating about a single axis of rotation or whether the joint moves about a changing axis of rotation referred to as a locus or centrode.     

Localization and quantification of net fluxes of H+ along maize roots by combined use of pH-indicator dye videodensitometry and H+-selective microelectrodes

Two methods for measuring proton fluxes along intact maize roots grown with NH ₄ ⁺ or NO ₃ ⁻ at pH 6.5 were compared. Videodensitometric measurement of changes in a pH-indicator dye by video camera was used to map pH around roots and determine the amounts of protons released by various root regions. This method was compared with potentiometric determination of the concentration of H⁺ in the unstirred layer at the root surface using ion-selective microelectrodes. With NH ₄ ⁺ the roots released large amounts of H⁺ in preferential regions where the rate of flux can reach 1.4 or even 2.5 nmol m⁻¹ s⁻¹. Videodensitometry indicated a first region of root acidification in the subapical zone, but this was more difficult to localize with microelectrodes. With NO₃ ⁻ both methods showed that the roots released small amounts of H⁺ and that the apical region took up H⁺ in the first 10 mm then sometimes released H⁺ over the following 10 mm of root. The H⁺ flux profiles obtained by both methods were in good agreement in terms of both order of magnitude of the fluxes and spatial differences along the root. These results suggest that videodensitometry, which is easier to use than potentiometry, can be used to screen different plant species or cultivars under various experimental conditions. The microelectrode technique is indispensable, however, for studying the underlying mechanisms of net H⁺ fluxes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the message^{for review see 1}. However, it''s now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates¹. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others². Codon bias is organism as well as tissue specific^2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs⁴. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes^5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein folding^{for review see 9}. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding^1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems^6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.     

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Background

Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrP^Sc, a protease-resistant misfolded isoform of the cellular prion protein PrP^C. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrP^Sc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrP^Sc in BSE-affected cattle therefore remains unknown.

Methodology/Principal Findings

We report here that PrP^Sc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP^Sc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrP^Sc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrP^Sc in blood was not substantiated in the BSE-affected cattle examined.

Conclusions/Significance

The distribution of PrP^Sc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrP^Sc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.     

Utilisation du15N2 pour estimer la fixation d'azote et sa repartition chez les legumineuses

Resumé Les auteurs proposent une méthode d'utilisation du¹⁵N₂ pour l'étude de la fixation d'azote dans les associations complexes légumineuses-Rhizobium cultivées sur sol. La procédure consiste, à marquer l'atmosphère du sol à l'aide de¹⁵N₂ et à calculer la quantité d'azote total fixé pendant cette période. Les premiers résultats obtenus sur des haricots et des trèfles démontrent qu'après seulement 7 h d'incubation, des quantités significatives de¹⁵N sont mesurées dans les plantes permettant ainsi de déterminer précisément la valeur du rapport C₂H₄/N₂ qui a été établi entre 2,6 et 3,1 dans les conditions de l'expérience. Sur de longues périodes, ces mêmes quantités se sont avérées suffisantes pour suivre la dynamique de l'azote des nodules vers les organes reproducteurs de plants de haricots.

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Use of¹⁵N to estimate dinitrogen fixation and distribution in legumes

Summary A method for use of¹⁵N₂ in the study of dinitrogen fixation by complex legumes-Rhizobium associations grown on soil is proposed. The procedure consists in labelling the soil atmosphere with¹⁵N₂ during short periods of time, measuring¹⁵N enrichment in the plants and calculating the total nitrogen fixed during this period. The first results obtained with bean and clover plants demonstrate that after only 7 h of incubation, significative amounts of¹⁵N are measured in the plants to allow precise determination of C₂H₄/N₂ ratio which ranged between 2.6 and 3.1 in this experiment. Over longer periods of time, such amounts are meaningfull to follow the pattern of N dynamic from the nodules to the reproductive organs of bean plants.

     

Determination of specific protein kinase activities using phosphorus-33

The immune complex kinase assay is the most widely applied method to assess the catalytic activity of protein tyrosine kinases. It offers the advantage that the activity of a single selected enzyme can be determined, and that the enzyme activity can be normalized for the amount of enzyme in a parallel immunoblotting experiment. Here, we describe the use of the recently introduced isotope phosphorus-33 for the protein kinase assay. The lower energy of ³³P, compared with the traditionally applied ³²P, allows the simultaneous examination of the amount of enzyme with ¹²⁵I-labeled antibodies. By analysing one and the same sample for both kinase activity and protein amount, the variation between parallel processed samples is avoided. Using this method, specific kinase activities can be calculated with high precision. The assay is particularly useful for the detection of cytokine and growth factor-induced activation of kinases, as changes in enzyme amounts by subcellular relocalization can be distinguished.