31 research outputs found
Two rapid assays for screening of patulin biodegradation
ArtÃculo sobre distintos ensayos para comprobar la biodegradación de la patulinaThe mycotoxin patulin is produced by the blue
mould pathogen Penicillium expansum in rotting apples
during postharvest storage. Patulin is toxic to a wide range
of organisms, including humans, animals, fungi and bacteria.
Wash water from apple packing and processing
houses often harbours patulin and fungal spores, which can
contaminate the environment. Ubiquitous epiphytic yeasts,
such as Rhodosporidium kratochvilovae strain LS11 which
is a biocontrol agent of P. expansum in apples, have the
capacity to resist the toxicity of patulin and to biodegrade
it. Two non-toxic products are formed. One is desoxypatulinic
acid. The aim of the work was to develop rapid,
high-throughput bioassays for monitoring patulin degradation
in multiple samples. Escherichia coli was highly
sensitive to patulin, but insensitive to desoxypatulinic acid.
This was utilized to develop a detection test for patulin,
replacing time-consuming thin layer chromatography or
high-performance liquid chromatography. Two assays for patulin degradation were developed, one in liquid medium
and the other in semi-solid medium. Both assays allow the
contemporary screening of a large number of samples. The
liquid medium assay utilizes 96-well microtiter plates and
was optimized for using a minimum of patulin. The semisolid
medium assay has the added advantage of slowing
down the biodegradation, which allows the study and isolation
of transient degradation products. The two assays are
complementary and have several areas of utilization, from
screening a bank of microorganisms for biodegradation
ability to the study of biodegradation pathways
Calcium modulates force sensing by the von Willebrand factor A2 domain
von Willebrand factor (VWF) multimers mediate primary adhesion and aggregation of platelets. VWF potency critically depends on multimer size, which is regulated by a feedback mechanism involving shear-induced unfolding of the VWF-A2 domain and cleavage by the metalloprotease ADAMTS-13. Here we report crystallographic and single-molecule optical tweezers data on VWF-A2 providing mechanistic insight into calcium-mediated stabilization of the native conformation that protects A2 from cleavage by ADAMTS-13. Unfolding of A2 requires higher forces when calcium is present and primarily proceeds through a mechanically stable intermediate with non-native calcium coordination. Calcium further accelerates refolding markedly, in particular, under applied load. We propose that calcium improves force sensing by allowing reversible force switching under physiologically relevant hydrodynamic conditions. Our data show for the first time the relevance of metal coordination for mechanical properties of a protein involved in mechanosensing
Granular Assembly of α-Synuclein Leading to the Accelerated Amyloid Fibril Formation with Shear Stress
α-Synuclein participates in the Lewy body formation of Parkinson's disease. Elucidation of the underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop a controlling strategy toward the disease, but also to apply the protein fibrils for future biotechnology. Discernable homogeneous granules of α-synuclein composed of approximately 11 monomers in average were isolated in the middle of a lag phase during the in vitro fibrillation process. They were demonstrated to experience almost instantaneous fibrillation during a single 12-min centrifugal membrane-filtration at 14,000×g. The granular assembly leading to the drastically accelerated fibril formation was demonstrated to be a result of the physical influence of shear force imposed on the preformed granular structures by either centrifugal filtration or rheometer. Structural rearrangement of the preformed oligomomeric structures is attributable for the suprastructure formation in which the granules act as a growing unit for the fibril formation. To parallel the prevailing notion of nucleation-dependent amyloidosis, we propose a double-concerted fibrillation model as one of the mechanisms to explain the in vitro fibrillation of α-synuclein, in which two consecutive concerted associations of monomers and subsequent oligomeric granular species are responsible for the eventual amyloid fibril formation
Chronic kidney disease after liver, cardiac, lung, heart–lung, and hematopoietic stem cell transplant
Patient survival after cardiac, liver, and hematopoietic stem cell transplant (HSCT) is improving; however, this survival is limited by substantial pretransplant and treatment-related toxicities. A major cause of morbidity and mortality after transplant is chronic kidney disease (CKD). Although the majority of CKD after transplant is attributed to the use of calcineurin inhibitors, various other conditions such as thrombotic microangiopathy, nephrotic syndrome, and focal segmental glomerulosclerosis have been described. Though the immunosuppression used for each of the transplant types, cardiac, liver and HSCT is similar, the risk factors for developing CKD and the CKD severity described in patients after transplant vary. As the indications for transplant and the long-term survival improves for these children, so will the burden of CKD. Nephrologists should be involved early in the pretransplant workup of these patients. Transplant physicians and nephrologists will need to work together to identify those patients at risk of developing CKD early to prevent its development and progression to end-stage renal disease
The P2X1 receptor and platelet function
Extracellular nucleotides are ubiquitous signalling molecules, acting via the P2 class of surface receptors. Platelets express three P2 receptor subtypes, ADP-dependent P2Y1 and P2Y12 G-protein-coupled receptors and the ATP-gated P2X1 non-selective cation channel. Platelet P2X1 receptors can generate significant increases in intracellular Ca2+, leading to shape change, movement of secretory granules and low levels of αIIbβ3 integrin activation. P2X1 can also synergise with several other receptors to amplify signalling and functional events in the platelet. In particular, activation of P2X1 receptors by ATP released from dense granules amplifies the aggregation responses to low levels of the major agonists, collagen and thrombin. In vivo studies using transgenic murine models show that P2X1 receptors amplify localised thrombosis following damage of small arteries and arterioles and also contribute to thromboembolism induced by intravenous co-injection of collagen and adrenaline. In vitro, under flow conditions, P2X1 receptors contribute more to aggregate formation on collagen-coated surfaces as the shear rate is increased, which may explain their greater contribution to localised thrombosis in arterioles compared to venules within in vivo models. Since shear increases substantially near sites of stenosis, anti-P2X1 therapy represents a potential means of reducing thrombotic events at atherosclerotic plaques