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1.
Vinaya Kumar Golla Eulàlia Sans-Serramitjana Karunakar Reddy Pothula Lorraine Benier Jayesh Arun Bafna Mathias Winterhalter Ulrich Kleinekathöfer 《Biophysical journal》2019,116(2):258-269
Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores. 相似文献
2.
Valentina A. Spiteri James Doutch Robert P. Rambo Jayesh Gor Paul A. Dalby Stephen J. Perkins 《Biophysical journal》2021,120(9):1814-1834
The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is composed of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are conserved in the Fc region in IgG; however, their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. After deglycosylation using peptide:N-glycosidase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s020,w of IgG1 decreased from 6.45 S by 0.16–0.27 S. This change was attributed to the reduction in mass after glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. Although the radius of gyration (RG) was unchanged, the cross-sectional radius of gyration (RXS-1) increased by 0.1 nm, and the commonly occurring distance peak M2 of the distance distribution curve P(r) increased by 0.4 nm. These changes revealed that the Fab-Fc separation in IgG1 was perturbed after deglycosylation. To explain these changes, atomistic scattering modeling based on Monte Carlo simulations resulted in 123,284 and 119,191 trial structures for glycosylated and deglycosylated IgG1 respectively. From these, 100 x-ray and neutron best-fit models were determined. For these, principal component analyses identified five groups of structural conformations that were different for glycosylated and deglycosylated IgG1. The Fc region in glycosylated IgG1 showed a restricted range of conformations relative to the Fab regions, whereas the Fc region in deglycosylated IgG1 showed a broader conformational spectrum. These more variable Fc conformations account for the loss of binding to the Fcγ receptor in deglycosylated IgG1. 相似文献
3.
Steven A. Harris Bhumit A. Patel Adrian Gospodarek Jayesh Desai Alejandro de Janon Gutiérrez Edita Botonjic-Sehic Mark Brower Nuno D. S. Pinto 《Biotechnology progress》2021,37(5):e3187
Protein concentration determination is a necessary in-process control for the downstream operations within biomanufacturing. As production transitions from batch mode to an integrated continuous bioprocess paradigm, there is a growing need to move protein concentration quantitation from off-line to in-line analysis. One solution to fulfill this process analytical technology need is an in-line index of refraction (IoR) sensor to measure protein concentration in real time. Here the performance of an IoR sensor is evaluated through a series of experiments to assess linear response, buffer matrix effects, dynamic range, sensor-to-sensor variability, and the limits of detection and quantitation. The performance of the sensor was also tested in two bioprocessing scenarios, ultrafiltration and capture chromatography. The implementation of this in-line IoR sensor for real-time protein concentration analysis and monitoring has the potential to improve continuous bioprocess manufacturing. 相似文献
4.
Ruodan Nan Stuart Tetchner Elizabeth Rodriguez Po-Jung Pao Jayesh Gor Imre Lengyel Stephen J. Perkins 《The Journal of biological chemistry》2013,288(26):19197-19210
The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular
degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of
complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3.
During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor
H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical
ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100
μm zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble
oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with
zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions
showed that putative weak zinc binding sites with different capacities exist in all five proteins,
in agreement with experiments. Factor H forms large oligomers in >10 μm zinc. In
contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much
reduced at 60 μm zinc and even more so at >100 μm zinc. The
removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc.
Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment
epithelial deposits in the retina as well as reducing the progression to advanced age-related
macular degeneration in higher risk patients. 相似文献
5.
Myles Horton Jayesh Modi Shiel K. Patel Andrew M. Demchuk Mayank Goyal Michael D. Hill Shelagh B. Coutts 《PloS one》2013,8(6)
Background
TIA and minor stroke have a high risk of recurrent stroke. Abnormalities on CT/CTA and MRI predict recurrent events in TIA and minor stroke. However there are many other imaging abnormalities that could potentially predict outcome that have not been assessed in this population. Also the definition of recurrent events used includes deterioration due to stroke progression or recurrent stroke and whether imaging is either of these is not known.Aims
To improve upon the clinical, CT/CTA and MRI parameters that predict recurrent events after TIA and minor stroke by assessing further imaging parameters. Secondary aim was to explore predictors of stroke progression versus recurrent stroke.Methods
510 consecutive TIA and minor stroke patients had CT/CTA and most had MRI. Primary outcome was recurrent events (stroke progression or recurrent stroke) within 90 days. Further imaging parameters were assessed for prediction of recurrent events (combined outcome of stroke progression and recurrent stroke). We also explored predictors of symptom progression versus recurrence individually.Results
36 recurrent events (36/510, 7.1% (95% CI: 5.0–9.6)) including 19 progression and 17 recurrent strokes. On CT/CTA: white matter disease, prior stroke, aortic arch focal plaque≥4 mm, or intraluminal thrombus did not predict recurrent events (progression or recurrent stroke). On MRI: white matter disease, prior stroke, and microbleeds did not predict recurrent events. Parameters predicting the individual outcome of symptom progression included: ongoing symptoms at initial assessment, symptom fluctuation, intracranial occlusion, intracranial occlusion or stenosis, and the CT/CTA metric. No parameter was strongly predictive of a distinct recurrent stroke.Conclusions
There was no imaging parameter that could improve upon our original CT/CTA or MRI metrics to predict the combined outcome of stroke progression or a recurrent stroke after TIA and minor stroke. We are better at using imaging to predict stroke progression rather than recurrent stroke. 相似文献6.
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8.
Lee YC Flora R McCafferty JA Gor J Tsaneva IR Perkins SJ 《Journal of molecular biology》2003,333(4):677-682
In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells. 相似文献
9.
Hickery MS Bayliss MT Dudhia J Lewthwaite JC Edwards JC Pitsillides AA 《The Journal of biological chemistry》2003,278(52):53063-53071
Cartilage glycosaminoglycan (GAG) synthesis and composition, upon which its structural integrity depends, varies with age, is modified by anabolic and catabolic stimuli, and is regulated by UDP-glucuronate availability. However, how such stimuli, prototypically represented by transforming growth factor-beta1 (TGF-beta1) and IL-1alpha, modify GAG synthesis during aging of normal human articular cartilage is not known. Using explants, we show that chondroitin sulfate (CS):total GAG ratios decrease, whereas C6S:C4S ratios increase with cartilage maturation, and that chondrocytes in the cartilage mid-zone, but not the superficial or deep zones, exhibit uridine 5'-diphosphoglucose dehydrogenase (UDPGD) activity, which is also increased in mature cartilage. We also show that IL-1alpha treatment reduces both total GAG and CS synthesis, decreases C6S:C4S ratios (less C6S), but fails to modify chondrocyte UDPGD activity at all ages. On the other hand, TGF-beta1 increases total GAG synthesis in immature, but not mature, cartilage (stimulates CS but not non-CS), age-independently decreases C6S:C4S (more C4S), and increases chondrocyte UDPGD activity in a manner inversely correlated with age. Our findings show that TGF-beta1, but not IL-1alpha, modifies matrix synthesis such that its composition more closely resembles "less mature" articular cartilage. These effects of TGF-beta1, which appear to be restricted to periods of skeletal immaturity, are closely associated although not necessarily mechanistically linked with increases in chondrocyte UDPGD activity. The antianabolic effects of IL-1alpha are, on the other hand, likely to be independent of any direct modification in UDPGD activity and manifest equally in human cartilage of all ages. 相似文献
10.
Stem cells in veterinary medicine--attempts at regenerating equine tendon after injury 总被引:1,自引:0,他引:1
Stem cells have evoked considerable excitement in the animal-owning public because of the promise that stem cell technology could deliver tissue regeneration for injuries for which natural repair mechanisms do not deliver functional recovery and for which current therapeutic strategies have minimal effectiveness. This review focuses on the current use of stem cells within veterinary medicine, whose practitioners have used mesenchymal stem cells (MSCs), recovered from either bone marrow or adipose tissue, in clinical cases primarily to treat strain-induced tendon injury in the horse. The background on why this treatment has been advocated, the data supporting its use and the current encouraging outcome from clinical use in horses treated with bone-marrow-derived cells are presented together with the future challenges of stem-cell therapy for the veterinary community. 相似文献