Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton [1-4]. In the context of stable adhesion [1], cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis [2]. Here actin polymerization has been proposed to drive cell surfaces together [5], although F-actin reorganization also occurs as cell contacts mature [6]. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere [7]. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.

TACE inhibition: a new approach to treating inflammation

Clinical trials showing the benefits of reducing the effects of TNF-alpha in rheumatoid arthritis have highlighted the key role of the cytokine TNF-alpha in this inflammatory condition. A new approach to reducing the effects of TNF-alpha is to decrease its synthesis by inhibiting TNF-alpha converting enzyme with GW3333. In rat models of arthritis, GW3333 has some beneficial effects. Further longer-term studies of GW3333 in animal models are required to determine whether its benefit is maintained. TACE inhibition may represent a new approach to treating inflammation.

The lateral line canal sensory organs of the Epaulette Shark (Hemiscyllium ocellatum)

Examination of the lateral line canals in the Epaulette Shark reveals a much more differentiated sensory system than previously reported from any elasmobranch. Two main types of lateral line canals are found. In one type rounded patches of sensory epithelia are separated by elevations of the canal floor. The other type is a straight canal without restrictions and with an almost continuous sensory epithelium. In addition, we found epithelia (type A) with very long apical microvilli on the supporting cells. These microvilli reach beyond the stereovilli of the hair cells. Another type (B) of sensory epithelium has short microvilli on the supporting cells. In this latter type of epithelium the stereovilli of the hair cells are comparatively tall and reach out beyond the supporting cell microvilli. New hair cells are found widely in both types of sensory epithelia. These always occur as single cells, unlike those described in teleost lateral line canal sensory epithelia where new hair cells seem to form in pairs. Dying hair cells are also widespread, indicating a continuous turnover of hair cells.

Spatial and temporal changes in multiple hormone groups during lateral bud release shortly following apex decapitation of chickpea (Cicer arietinum) seedlings

Although the co-ordination of promotive root-sourced cytokinin (CK) and inhibitory shoot apex-sourced auxin (IAA) is central to all current models on lateral bud dormancy release, control by those hormones alone has appeared inadequate in many studies. Thus it was hypothesized that the IAA : CK model is the central control but that it must be considered within the relevant timeframe leading to lateral bud release and against a backdrop of interactions with other hormone groups. Therefore, IAA and a wide survey of cytokinins (CKs), were examined along with abscisic acid (ABA) and polyamines (PAs) in released buds, tissue surrounding buds and xylem sap at 1 and 4 h after apex removal, when lateral buds of chickpea are known to break dormancy. Three potential lateral bud growth inhibitors, IAA, ABA and cis-zeatin 9-riboside (ZR), declined sharply in the released buds and xylem following decapitation. This is in contrast to potential dormancy breaking CKs like trans-ZR and trans-zeantin 9-riboside 5'phosphate (ZRMP), which represented the strongest correlative changes by increasing 3.5-fold in xylem sap and 22-fold in buds. PAs had not changed significantly in buds or other tissues after 4 h, so they were not directly involved in the breaking of bud dormancy. Results from the xylem and surrounding tissues indicated that bud CK increases resulted from a combination synthesis in the bud and selective loading of CK nucleotides into the xylem from the root.

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [H-3]zeatin riboside ([H-3]ZR), a water-soluble blue dye or [H-3]H2O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within I h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [H-3]ZR and of blue dye in the same bud position was increased 3- to 10-fold relative to intact plants, whereas content of [H-3]H2O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [H-3]H2O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [H-3]ZR injected at physiological concentrations. Within 1 h, 80-95% of [H-3]ZR was converted to other active CKs (mainly zeatin riboside-5'phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O-acetylZR, O-acetylZRMP and a compound correlated with sites of high CK-concentrations) and inactive catabolites (adenosine, adenine, 5'AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.

Lateral epicondylalgia: a musculoskeletal physiotherapy perspective

Tennis elbow or lateral epicondylalgia (LE) is a challenging musculoskeletal condition to treat. This is largely due to the lack of research-based evidence of the clinical efficacy of the myriad of treatment approaches espoused in the literature. In view of this, successful rehabilitation of LE is based on choosing treatments that address the physical impairments found during clinical examination. The primary physical impairment in LE is a deficit in grip strength predominately due to pain and its consequences on motor function. Hence the mainstay of successful management of this condition is therapeutic exercise, providing it is not pain provocative. Adjunctive procedures such as manipulative therapy and sports taping techniques have recently been shown to provide substantial initial pain relief. Early relief of pain in the rehabilitation program helps accelerate recovery and most importantly motivates the client to persist with the therapeutic exercise program. The manipulative therapy and taping treatments presented in this masterclass warrant consideration in the clinical best practice management of LE, and serve as a model for other similar musculoskeletal conditions. (C) 2003 Elsevier Science Ltd. All rights reserved.

Hypoalgesia induced by elbow manipulation in lateral epicondylalgia does not exhibit tolerance

Previous studies have demonstrated that the initial hypoalgesic effect of spinal manipulative therapy was not antagonized by naloxone and did not exhibit tolerance with repeated applications. The implication is that endogenous opioid mechanisms of pain relief are probably not at play in spinal manipulative therapy. The role of endogenous opioid peptides in manipulation of the peripheral joints has not been investigated. The aim of this study was to evaluate whether the initial hypoalgesic effect of a peripheral manipulative technique (mobilization-with-movement treatment for the elbow) demonstrated a tolerance to repeated applications (ie, reduction in magnitude of effect over repeated applications). Twenty-four participants with unilateral chronic lateral epicondylalgia participated in the study. A repeated measures study was conducted to examine the effect of repeated applications of the mobilization-with-movement treatment for the elbow on 6 separate treatment occasions at least 2 days apart. Pain-free grip strength and pressure pain threshold were chosen as the pain-related outcome measures. Changes in the percent maximum possible effect scores of measures of hypoalgesia were evaluated across the 6 treatment sessions by using linear trend analysis. The results showed no significant difference for the hypoalgesic effect of the treatment technique between sessions (P >.05). This peripheral manipulative therapy treatment technique appeared to have a similar effect profile to previously studied spinal manipulative therapy techniques, thereby contributing to the body of knowledge that indicates that manipulative therapy most likely induces a predominant non-opioid form of analgesia.

An electromyographic analysis of the deep cervical flexor muscles in performance of craniocervical flexion

Background and Purpose. This study evaluated an electromyographic technique for the measurement of muscle activity of the deep cervical flexor (DCF) muscles. Electromyographic signals were detected from the DCF, sternocleidomastoid (SCM), and anterior scalene (AS) muscles during performance of the craniocervical flexion (CCF) test, which involves performing 5 stages of increasing craniocervical flexion range of motion-the anatomical action of the DCF muscles. Subjects. Ten volunteers without known pathology or impairment participated in this study. Methods. Root-mean-square (RMS) values were calculated for the DCF, SCM, and AS muscles during performance of the CCF test. Myoelectric signals were recorded from the DCF muscles using bipolar electrodes placed over the posterior oropharyngeal wall. Reliability estimates of normalized RMS values were obtained by evaluating intraclass correlation coefficients and the normalized standard error of the mean (SEM). Results. A linear relationship was evident between the amplitude of DCF muscle activity and the incremental stages of the CCF test (F=239.04, df=36, P<.0001). Normalized SEMs in the range 6.7% to 10.3% were obtained for the normalized RMS values for the DCF muscles, providing evidence of reliability for these variables. Discussion and Conclusion. This approach for obtaining a direct measure of the DCF muscles, which differs from those previously used, may be useful for the examination of these muscles in future electromyographic applications.

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific micro-domains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent micro-domain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.

An EM-based Semi-Parametric Mixture Model Approach to the Regression Analysis of Competing-Risks Data

We consider a mixture model approach to the regression analysis of competing-risks data. Attention is focused on inference concerning the effects of factors on both the probability of occurrence and the hazard rate conditional on each of the failure types. These two quantities are specified in the mixture model using the logistic model and the proportional hazards model, respectively. We propose a semi-parametric mixture method to estimate the logistic and regression coefficients jointly, whereby the component-baseline hazard functions are completely unspecified. Estimation is based on maximum likelihood on the basis of the full likelihood, implemented via an expectation-conditional maximization (ECM) algorithm. Simulation studies are performed to compare the performance of the proposed semi-parametric method with a fully parametric mixture approach. The results show that when the component-baseline hazard is monotonic increasing, the semi-parametric and fully parametric mixture approaches are comparable for mildly and moderately censored samples. When the component-baseline hazard is not monotonic increasing, the semi-parametric method consistently provides less biased estimates than a fully parametric approach and is comparable in efficiency in the estimation of the parameters for all levels of censoring. The methods are illustrated using a real data set of prostate cancer patients treated with different dosages of the drug diethylstilbestrol. Copyright (C) 2003 John Wiley Sons, Ltd.

The modeling of turbulent reactive flows based on multiple mapping conditioning

A new modeling approach-multiple mapping conditioning (MMC)-is introduced to treat mixing and reaction in turbulent flows. The model combines the advantages of the probability density function and the conditional moment closure methods and is based on a certain generalization of the mapping closure concept. An equivalent stochastic formulation of the MMC model is given. The validity of the closuring hypothesis of the model is demonstrated by a comparison with direct numerical simulation results for the three-stream mixing problem. (C) 2003 American Institute of Physics.

A partial velocity approach to subcycling structural dynamics

Subcycling, or the use of different timesteps at different nodes, can be an effective way of improving the computational efficiency of explicit transient dynamic structural solutions. The method that has been most widely adopted uses a nodal partition. extending the central difference method, in which small timestep updates are performed interpolating on the displacement at neighbouring large timestep nodes. This approach leads to narrow bands of unstable timesteps or statistical stability. It also can be in error due to lack of momentum conservation on the timestep interface. The author has previously proposed energy conserving algorithms that avoid the first problem of statistical stability. However, these sacrifice accuracy to achieve stability. An approach to conserve momentum on an element interface by adding partial velocities is considered here. Applied to extend the central difference method. this approach is simple. and has accuracy advantages. The method can be programmed by summing impulses of internal forces, evaluated using local element timesteps, in order to predict a velocity change at a node. However, it is still only statistically stable, so an adaptive timestep size is needed to monitor accuracy and to be adjusted if necessary. By replacing the central difference method with the explicit generalized alpha method. it is possible to gain stability by dissipating the high frequency response that leads to stability problems. However. coding the algorithm is less elegant, as the response depends on previous partial accelerations. Extension to implicit integration, is shown to be impractical due to the neglect of remote effects of internal forces acting across a timestep interface. (C) 2002 Elsevier Science B.V. All rights reserved.

Designing a 161-element Ku-band microstrip reflectarray of variable size patches using an equivalent unit cell waveguide approach

An equivalent unit cell waveguide approach (WGA) to designing 4 multilayer microstrip reflectarray of variable size patches is presented. In this approach, a normal incidence of a plane wave on an infinite periodic array of radiating elements is considered to obtain reflection coefficient phase curves for the reflectarray's elements. It is shown that this problem is equivalent to the problem of reflection of the dominant TEM mode in a waveguide with patches interleaved by layers of dielectric. This waveguide problem is solved using a field matching technique and a method of moments (MoM). Based on this solution, a fast computer algorithm is developed to generate reflection coefficient phase curves for a multilayer microstrip patch reflectarray. The validity of the developed algorithm is tested against alternative approaches and Agilent High Frequency Structure Simulator (HFSS). Having confirmed the validity of the WGA approach, a small offset feed two-layer microstrip patch array is designed and developed. This reflectarray is tested experimentally and shows good performance.

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, intergrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations. (C) 2003 Elsevier Science Ltd. All rights reserved.