Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.
Over 35 million people worldwide are reported to suffer from Alzheimer's disease (AD). With healthcare advances and marked increase in life expectancy, there is an ever-increasing incidence of AD. Longevity and the continuous improvement of peri-operative medicine, reducing mortality and morbidity, have led to an exponential increment in surgery and, as a consequence, a larger number of aged patients are undergoing surgery. While anesthetics are indispensable clinical tools and generally considered safe and effective, in some situations there is a growing concern about the potential neurotoxicity of these agents. Particularly among the elderly, a number of cases of post-operative cognitive decline (POCD), both short-term and longterm, have been globally reported. It is argued, and justifiably so (with regard to cardiac surgery in particular, having several risk factors which could result in cognitive decline), that it is not possible to dissociate the effects of surgery and anesthesia. However, significant reports of cognitive decline on follow up of patients undergoing non-cardiac, prolonged surgical procedures under general anesthesia have resulted in several scientific groups focusing greater attention on the possible neurotoxic effect of anesthetics in POCD.The overwhelming response to the call for articles for this supplemental issue is proof enough of the common note of concern and urgency shared by the scientific community to scrutinize the possible toxic effects (if any) of anesthesia in POCD (the long-term form simulating the clinical and molecular mechanisms involved in AD), particularly in the aged population. Scientists, anesthesiologists, neurologists, neuropsychologists and surgeons have expressed their expert views on various aspects of the pathophysiology of AD, and the role of anesthetics as a possible risk factor. These efforts, when viewed as a whole, will inevitably stimulate rethinking on the subject.The introductory article by Dr. Finder provides a panoramic view of the classic and recent literature on the complex cellular and molecular mechanisms underlying AD, supported by subsequent articles which focus on the more recent observations in specific areas of molecular research. Amyloid-β (Aβ) plaques were considered the pathogenic species in AD; however, accumulating evidence suggests that plaques could represent final waste deposits, with the oligomeric intermediates representing the key toxic players since the severity of cognitive deficits in AD correlates with the level of oligomers in the brain but not with the total Aβ burden. The proposed neurotoxic effects of Aβ oligomers are synaptic failure, membrane disruption with Ca 2+ influx, mitochondrial failure and oxidative stress, and recruitment of cellular factors or activation of cellular processes such as apoptosis and inflammation.Aβ has a natural role in many functions of the nervous system. There is evidence that Aβ is part of the innate immune system of the brain and natural antibodies against oligomeric, fibrillar Aβ and plaques ...
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