Kinesin Motor Enzymology: Chemistry,Structure, and Physics of Nanoscale Molecular Machines

Biophysical Reviews - Molecular motors are enzymes that convert chemical potential energy into controlled kinetic energy for mechanical work inside cells. Understanding the biophysics of these...     

Purification of sarcoplasmic reticulum vesicles through their loading with calcium phosphate

A technique of purification of sarcoplasmic reticulum was devised through selective loading of the vesicular material with calcium phosphate. In presence of amount of disposable calcium lower than the maximum accumulation capacity of the total vesicular population, we have defined conditions of loading which allow the selection by centrifugation. The results described in this work show that about 30% of the starting material can be isolated as a vesicular population homogenous on the stand of the amount of accumulated cation. The purification is achieved by the removal of calcium by dissociation of the precipitate. Freeze-fracture electron microscopy studies show that the more active fraction when freed of calcium phosphate precipitate displays smooth convex (EF_s) and particulated concave (PF_p) fracture planes. It has been verified that the purification described in this work allows the removal of all the inactive material. The rate of calcium uptake of the selected preparation is about twice as large as that displayed by the starting material. The structural homogeneity of this material and the increase in the activity are good evidence for the purity of the selected sarcoplasmic reticulum vesicles.     

Withdrawal of inhaled corticosteroids in individuals with COPD - a systematic review and comment on trial methodology

Inhaled corticosteroids (ICS) reduce COPD exacerbation frequency and slow decline in health related quality of life but have little effect on lung function, do not reduce mortality, and increase the risk of pneumonia. We systematically reviewed trials in which ICS have been withdrawn from patients with COPD, with the aim of determining the effect of withdrawal, understanding the differing results between trials, and making recommendations for improving methodology in future trials where medication is withdrawn. Trials were identified by two independent reviewers using MEDLINE, EMBASE and CINAHL, citations of identified studies were checked, and experts contacted to identify further studies. Data extraction was completed independently by two reviewers. The methodological quality of each trial was determined by assessing possible sources of systematic bias as recommended by the Cochrane collaboration. We included four trials; the quality of three was adequate. In all trials, outcomes were generally worse for patients who had had ICS withdrawn, but differences between outcomes for these patients and patients who continued with medication were mostly small and not statistically significant. Due to data paucity we performed only one meta-analysis; this indicated that patients who had had medication withdrawn were 1.11 (95% CI 0.84 to 1.46) times more likely to have an exacerbation in the following year, but the definition of exacerbations was not consistent between the three trials, and the impact of withdrawal was smaller in recent trials which were also trials conducted under conditions that reflected routine practice. There is no evidence from this review that withdrawing ICS in routine practice results in important deterioration in patient outcomes. Furthermore, the extent of increase in exacerbations depends on the way exacerbations are defined and managed and may depend on the use of other medication. In trials where medication is withdrawn, investigators should report other medication use, definitions of exacerbations and management of patients clearly. Intention to treat analyses should be used and interpreted appropriately.     

Use of spatiotemporal analysis of laboratory submission data to identify potential outbreaks of new or emerging diseases in cattle in Great Britain

Background  

New and emerging diseases of livestock may impact animal welfare, trade and public health. Early detection of outbreaks can reduce the impact of these diseases by triggering control measures that limit the number of cases that occur. The aim of this study was to investigate whether prospective spatiotemporal methods could be used to identify outbreaks of new and emerging diseases in scanning surveillance data. SaTScan was used to identify clusters of unusually high levels of submissions where a diagnosis could not be reached (DNR) using different probability models and baselines. The clusters detected were subjected to a further selection process to reduce the number of false positives and a more detailed epidemiological analysis to ascertain whether they were likely to represent real outbreaks.     

The herd,a source of flexibility for livestock farming systems faced with uncertainties?

'Adapt to endure' has become a necessity in agriculture, but the means to do so remain largely undefined. The aim of this literature review is to analyse how the herd contributes to a livestock farming system's capacity to adapt to a changing world and evolve when the future is uncertain. We identify six categories of elements linked to the herd, called 'sources of flexibility', that are used to manage perturbation. The first three are: using the animal's adaptive capacities, using the diversity of species and breeds and combining the diversity of animal products. The last three are: organising the mobility of animals and livestock farmers, juggling the herd numbers and mastering the balance between productivity and herd survival. These sources of flexibility are described in the literature by studying the different ways in which they are used. For example, the 'juggle herd numbers' source is described by volume, categories of animals, type of transfer, such as births, purchases or gifts, and timing of use, especially linked to the timing of the perturbation. Identified studies also compare or rank sources and analyse the connections between them. The flexibility framework (management science) is used for this analysis according to the levels of organisation of a livestock farming system: a strategic level referring to long-term options and to the capacity to modify the system structure, and an operational level referring to adjustment decisions during the productive cycle, the presence or absence of intervention by the livestock farmer, and the time scales involved. We conclude that the decision to use one or another source (in terms of modalities, alternatives, scheduling and combinations) is made according to the production objectives, the structural means, the type/frequency/intensity of perturbations and the context/environment. Consequently, the flexibility of a livestock farming system cannot be assessed in absolute terms. Enhancing flexibility needs management of all elements and scales involved (and not only the herd), and requires diversity to be organised at different scales.     

T lymphocyte insensitivity to corticosteroids in chronic obstructive pulmonary disease

Background

There are increased numbers of activated lymphocytes in the lungs of chronic obstructive pulmonary disease (COPD) patients. The clinical benefits of corticosteroids in COPD patients are limited. Our hypothesis is that lymphocytes play a role in this corticosteroid insensitivity.

Objectives

To investigate the effects of the corticosteroid dexamethasone on lung lymphocyte cytokine production from patients with COPD compared to controls.

Methods

Cultured airway lymphocytes obtained by bronchoscopy from healthy non-smokers (HNS), smokers (S) and COPD patients were stimulated with phytohaemagglutinin (PHA) & phorbol myristate acetate (PMA), +/- dexamethasone. Supernatants were assayed for interleukin (IL)-2 and interferon (IFN)γ. Immunofluoresence was used to analyse changes in CD8 glucocorticoid receptor (GRα and GRβ) expression.

Results

The inhibition of PHA/PMA stimulated IFNγ production by dexamethasone was reduced in COPD patients compared to HNS (p < 0.05 at concentrations from 0.1-1 μM). There was also a significant reduction (p < 0.05) in the mean inhibitory effect at 1 μM in COPD patients (54.1%) compared to smokers (72.1%), and in smokers compared to HNS (85.5%). There was a numerically reduced effect of dexamethasone on IL-2 production that did not reach statistical significance. There was no difference in GRα and GRβ expression in follicular CD8 cells between COPD patients (50.9% and 30.4% respectively) and smokers (52.9% and 29.7% respectively).

Conclusions

IFNγ production from COPD airway lymphocytes is corticosteroid insensitive. This phenomenon may be important in the poor clinical response often observed with corticosteroids.     

LRP-1--CD44, a new cell surface complex regulating tumor cell adhesion

The low-density lipoprotein receptor-related protein 1 (LRP-1) is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. In the field of cancer, LRP-1-mediated endocytosis was first associated with antitumor properties. However, recent results suggested that LRP-1 may coordinate the adhesion-deadhesion balance in malignant cells to support tumor progression. Here, we observed that LRP-1 silencing or RAP (receptor-associated protein) treatment led to accumulation of CD44 at the tumor cell surface. Moreover, we evidenced a tight interaction between CD44 and LRP-1, not exclusively localized in lipid rafts. Overexpression of LRP-1-derived minireceptors indicated that the fourth ligand-binding cluster of LRP-1 is required to bind CD44. Labeling of CD44 with EEA1 and LAMP-1 showed that internalized CD44 is routed through early endosomes toward lysosomes in a LRP-1-dependent pathway. LRP-1-mediated internalization of CD44 was highly reduced under hyperosmotic conditions but poorly affected by membrane cholesterol depletion, revealing that it proceeds mostly via clathrin-coated pits. Finally, we demonstrated that CD44 silencing abolishes RAP-induced tumor cell attachment, revealing that cell surface accumulation of CD44 under LRP-1 blockade is mainly responsible for the stimulation of tumor cell adhesion. Altogether, our data shed light on the LRP-1-mediated internalization of CD44 that appeared critical to define the adhesive properties of tumor cells.     

Defunct brain stem cardiovascular regulation underlies cardiovascular collapse associated with methamphetamine intoxication

Background  

Intoxication from the psychostimulant methamphetamine (METH) because of cardiovascular collapse is a common cause of death within the abuse population. For obvious reasons, the heart has been taken as the primary target for this METH-induced toxicity. The demonstration that failure of brain stem cardiovascular regulation, rather than the heart, holds the key to cardiovascular collapse induced by the pesticide mevinphos implicates another potential underlying mechanism. The present study evaluated the hypothesis that METH effects acute cardiovascular depression by dampening the functional integrity of baroreflex via an action on brain stem nuclei that are associated with this homeostatic mechanism.