Use of the two-hybrid system to identify Rab-interacting proteins

The yeast two-hybrid system has been useful for identifying many partners and effectors of small GTPases of the Rab family. We describe here such a screen using Rab6, a protein involved in the regulation of intracellular transport at the level of the Golgi apparatus, as bait.     

Epoxidation of cottonseed oil by aqueous hydrogen peroxide catalysed by liquid inorganic acids

The kinetics of epoxidation of cottonseed oil by peroxyacetic acid generated in situ from hydrogen peroxide and glacial acetic acid in the presence of liquid inorganic acid catalysts were studied. It was possible to obtain up to 78% relative conversion to oxirane with very less oxirane cleavage by in situ technique. The rate constants for sulphuric acid catalysed epoxidation of cottonseed oil were in the range 0.39-5.4 x 10(-6)L mol(-1)s(-1) and the activation energy was found to be 11.7 kcal mol(-1). Some thermodynamic parameters such as enthalpy, entropy, and free energy of activation were determined to be of 11.0 kcal mol(-1), -51.4 cal mol(-1)K(-1) and 28.1 kcal mol(-1), respectively. The order of effectiveness of catalysts was found to be sulphuric acid>phosphoric acid>nitric acid>hydrochloric acid. Acetic acid was found to be superior to formic acid for the in situ cottonseed oil epoxidation.     

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia

Background

Artemisinin and its derivatives have been used for falciparum malaria treatment in China since late 1970s. Monotherapy and uncontrolled use of artemisinin drugs were common practices for a long period of time. In vitro tests showed that the susceptibility of Plasmodium falciparum to artemisinins was declining in China. A concern was raised about the resistance to artemisinins of falciparum malaria in the country. It has been reported that in vitro artemisinin resistance was associated with the S769N mutation in the PfATPase6 gene. The main purpose of this study was to investigate whether that mutation has occurred in field isolates from China.

Methods

Plasmodium falciparum field isolates were collected in 2006–2007 from Hainan and Yunnan provinces, China. A nested PCR-sequencing assay was developed to analyse the genotype of the PfATPase6 S769N polymorphism in the P. falciparum field isolates.

Results

The genotyping results of six samples could not be obtained due to failure of PCR amplification, but no S769N mutation was detected in any of the 95 samples successfully analysed.

Conclusion

The results indicate that the S769N mutation in the PfATPase6 gene is not present in China, suggesting that artemisinin resistance has not yet developed, but the situation needs to be watched very attentively.     

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum

The cytosolic coat-protein complex COP-I interacts with cytoplasmic 'retrieval' signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.     

ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1‐GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1‐GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R≈35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1‐GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1‐GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1‐GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.     

Improving guideline adherence for cardiac rehabilitation in the Netherlands

Background

In 2004, the Netherlands Society of Cardiology released the current guideline on cardiac rehabilitation. Given its complexity and the involvement of various healthcare disciplines, it was supplemented with a clinical algorithm, serving to facilitate its implementation in daily practice. Although the algorithm was shown to be effective for improving guideline adherence, several shortcomings and deficiencies were revealed. Based on these findings, the clinical algorithm has now been updated. This article describes the process and the changes that were made.

Methods

The revision consisted of three phases. First, the reliability of the measurement instruments included in the 2004 Clinical Algorithm was investigated by evaluating between-centre variations of the baseline assessment data. Second, based on the available evidence, a multidisciplinary expert advisory panel selected items needing revision and provided specific recommendations. Third, a guideline development group decided which revisions were finally included, also taking practical considerations into account.

Results

A total of nine items were revised: three because of new scientific insights and six because of the need for more objective measurement instruments. In all revised items, subjective assessment methods were replaced by more objective assessment tools (e.g. symptom-limited exercise instead of clinical judgement). In addition, four new key items were added: screening for anxiety/depression, stress, cardiovascular risk profile and alcohol consumption.

Conclusion

Based on previously determined shortcomings, the Clinical Algorithm for Cardiac Rehabilitation was thoroughly revised mainly by incorporating more objective assessment methods and by adding several new key areas.     

Are cancer cells really softer than normal cells?

Solid tumours are often first diagnosed by palpation, suggesting that the tumour is more rigid than its surrounding environment. Paradoxically, individual cancer cells appear to be softer than their healthy counterparts. In this review, we first list the physiological reasons indicating that cancer cells may be more deformable than normal cells. Next, we describe the biophysical tools that have been developed in recent years to characterise and model cancer cell mechanics. By reviewing the experimental studies that compared the mechanics of individual normal and cancer cells, we argue that cancer cells can indeed be considered as softer than normal cells. We then focus on the intracellular elements that could be responsible for the softening of cancer cells. Finally, we ask whether the mechanical differences between normal and cancer cells can be used as diagnostic or prognostic markers of cancer progression.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.