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51.
For many proteins, compact conformations are known to accumulate in advance of the rate-limiting step in folding. To understand the nature and significance of these early conformational events, we employed ultrarapid mixing methods to fully characterize the kinetics of folding of the 57-residue B1 domain of protein G. Continuous-flow fluorescence measurements exhibit a major exponential phase on the submillisecond time scale (600-700 micros), which is followed by a slower phase with a denaturant-dependent time constant (2-30 ms) observable by conventional stopped-flow measurements. The combined kinetic traces quantitatively account for the total change in Trp 43 fluorescence upon folding, including the previously unresolved 'burst phase' signal. The denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state mechanism, U right harpoon over left harpoon I right harpoon over left harpoon N, where I is a productive intermediate with native-like fluorescence properties. The relatively slow rate and exponential time course of the initial folding phase indicates that a substantial free energy barrier is encountered during chain condensation, resulting in a partially organized ensemble of states distinct from the initial unfolded conformations. 相似文献
52.
Progress in understanding dynamic aspects of protein folding relies on the continuing development of methods for obtaining more detailed structural information on the transient conformational ensembles that often appear within microseconds of initiating refolding. Advances in rapid mixing and other time-resolved spectroscopic methods have made it possible to explore some of the earliest stages of folding, including the initial formation of compact states, which is determined by the presence of a sequence-specific kinetic barrier, as well as the 'downhill' folding kinetics after the rate-limiting barrier has been crossed. 相似文献
53.
Chachadi Vishwanath B. Pujari Radha Shastry Padma Swamy Bale M. Inamdar Shashikala R. 《Glycoconjugate journal》2020,37(2):251-261
Glycoconjugate Journal - Sclerotium rolfsii lectin (SRL) exerts apoptotic effect against various cancer cells and an antitumor activity on mice with colon and breast cancer xenografts. The current... 相似文献
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Barkur S Shastry 《Journal of biomedical science》2010,17(1):69
Retinopathy of prematurity (ROP) is a vascular vitreoretinopathy that affects infants with short gestational age and low birth-weight.
The condition is a multifactorial disease and is clinically similar to familial exudative vitreoretinopathy (FEVR), which
is a bilateral hereditary eye disorder affecting full-term infants. Both of them are characterized by the abnormal vessel
growth in the vitreous that can lead to vitreoretinal traction, retinal detachment and other complications resulting in blindness.
Despite the recent advances in diagnosis and treatment, ROP remains a major cause of childhood blindness in developed countries.
The etiology of pathogenesis of advanced ROP is currently unknown. In the past, many causative factors such as length of time
exposed to supplemental oxygen, excessive ambient light exposure and hypoxia have been suggested but evidence for these as
independent risk factors in recent years is not compelling. It is not clear why ROP in a subset of infants with low birth-weight
progresses to a severe stage (retinal detachment) despite timely intervention whereas in other infants with similar clinical
characteristics ROP regresses spontaneously. Recent research with candidate gene approach, higher concordance rate in monozygotic
twins and other clinical and experimental animal studies, suggest a strong genetic predisposition to ROP besides environmental
factors such as prematurity. Three genes, which are involved in the Wnt signaling pathway, are mutated in both FEVR and in
a small percentage of ROP disorder. However, none of the genetic factors identified thus far in ROP, account for a substantial
number of patient population. Future studies involving genomics, bioinformatics and proteomics may provide a better understanding
of the pathophysiology and management of ROP. 相似文献
56.
Prasad V Chandele A Jagtap JC Sudheer Kumar P Shastry P 《Free radical biology & medicine》2006,41(3):431-442
Reactive oxygen species (ROS) and caspases 8, 9, and 3 are reported to be crucial players in apoptosis induced by various stimuli. Recently, caspase 2 has been implicated in stress-induced apoptosis but the exact mechanism remains unclear. In this study, we report that ROS generation led to activation of caspase 2 during beta-carotene-induced apoptosis in the human leukemic T cell line Molt 4. The apoptosis progressed by simultaneous activation of caspases 8 and 9, and a cross talk between these initiator caspases was mediated by the proapoptotic protein Bid. Inhibition of caspases 2, 8, 9, and 3 independently suppressed the caspase cascade. The kinetics and function of caspase 2 were similar to those of caspase 3, suggesting its role as an effector caspase. Interestingly, beta-carotene-induced apoptosis was caspase 2 dependent but caspase 3 independent. The study also revealed cleavage of the antiapoptotic protein BclXL as an important event during apoptosis, which was regulated by ROS. The mechanistic studies identify a functional link between ROS and the caspase cascade involving caspase 2 and cleavage of BclXL. The interdependence of caspases 8, 9, 2, and 3 in the cascade provides evidence for the presence of an extensive feedback amplification loop in beta-carotene-induced apoptosis in Molt 4 cells. 相似文献
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G?ker Arpa? Shankar Shastry William?O. Hancock Erkan Tüzel 《Biophysical journal》2014,107(8):1896-1904
Intracellular cargo transport frequently involves multiple motor types, either having opposite directionality or having the same directionality but different speeds. Although significant progress has been made in characterizing kinesin motors at the single-molecule level, predicting their ensemble behavior is challenging and requires tight coupling between experiments and modeling to uncover the underlying motor behavior. To understand how diverse kinesins attached to the same cargo coordinate their movement, we carried out microtubule gliding assays using pairwise mixtures of motors from the kinesin-1, -2, -3, -5, and -7 families engineered to have identical run lengths and surface attachments. Uniform motor densities were used and microtubule gliding speeds were measured for varying proportions of fast and slow motors. A coarse-grained computational model of gliding assays was developed and found to recapitulate the experiments. Simulations incorporated published force-dependent velocities and run lengths, along with mechanical interactions between motors bound to the same microtubule. The simulations show that the force-dependence of detachment is the key parameter that determines gliding speed in multimotor assays, while motor compliance, surface density, and stall force all play minimal roles. Simulations also provide estimates for force-dependent dissociation rates, suggesting that kinesin-1 and the mitotic motors kinesin-5 and -7 maintain microtubule association against loads, whereas kinesin-2 and -3 readily detach. This work uncovers unexpected motor behavior in multimotor ensembles and clarifies functional differences between kinesins that carry out distinct mechanical tasks in cells. 相似文献
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