Oxidative stress in blood in Alzheimer's disease and mild cognitive impairment: a meta-analysis.

Abnormal oxidative stress is an established feature of Alzheimer's disease, but clinical trials aiming to reduce oxidative stress have not yet proven an effective therapy for dementia patients. The purpose of this review is to systematically analyze available data describing markers of oxidative stress and antioxidants in blood from subjects with Alzheimer's disease or those with mild cognitive impairment to highlight potential interactions between peripheral redox changes and central nervous system pathology and contribute to the design of future clinical study. PubMed, SCOPUS and Web of Science were systematically queried to collect studies which have evaluated markers of oxidative stress, levels of antioxidants, copper, transferrin and ceruloplasmin levels in blood from subjects with Alzheimer's disease and matched controls. After application of quality measures, results were aggregated in a random effects analysis. We found that markers of lipid peroxidation are elevated in blood in Alzheimer's disease and in mild cognitive impairment, copper metabolism is dysregulated and total antioxidant capacity is decreased. While surprisingly none of the major antioxidative enzymes are significantly decreased, non-enzymatic antioxidants in blood (particularly uric acid, vitamins A, E and C, α- and ß-carotene) are significantly decreased. There is significant oxidative damage in peripheral blood early in the process of neurodegeneration. We propose that clinical studies assessing cognitive outcomes after antioxidant therapy tailor interventions to individual patients' deficiencies and confirm an improvement in an appropriate serological marker of oxidative stress. This strategy may be most effectively applied in a clinical trial of primary prevention.

White Matter Disease and Outcomes of Mechanical Thrombectomy for Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: The increased severity of white matter disease is associated with worse outcomes and an increased rate of intracerebral hemorrhage in patients with ischemic stroke undergoing thrombolytic treatment. However, whether white matter disease is associated with outcomes in patients undergoing endovascular treatment remains unclear. MATERIALS AND METHODS: In this prespecified exploratory analysis of our prospective multi-institutional study that enrolled consecutive adult patients with anterior circulation ischemic stroke undergoing endovascular treatment from November 2017 to September 2018, we compared the following outcomes between patients with none-to-minimal (van Swieten score, 0-2) and moderate-to-severe (van Swieten score, 3-4) white matter disease using logistic regression: 90-day mRS 3-6, death, intracerebral hemorrhage, successful recanalization, and early neurologic recovery. RESULTS: Of the 485 patients enrolled in the Blood Pressure after Endovascular Stroke Therapy (BEST) study, 389 had white matter disease graded (50% women; median age, 68 years; range, 58-79 years). A van Swieten score of 3-4 (n = 74/389, 19%) was associated with a higher rate of 90-day mRS of 3-6 (45% versus 18%; adjusted OR, 2.73; 95% CI, 1.34-5.93; P = .008). Although the death rate was higher in patients with van Swieten scores of 3-4 (26% versus 15%), the adjusted likelihood was not significantly different (adjusted OR, 1.14; 95% CI, 0.56-2.26; P = .710). Ordered regression revealed a shift toward worse mRS scores with increasing van Swieten scores (adjusted common OR, 3.04; 95% CI, 1.93-4.84; P < .001). No associations between white matter disease severity and intracerebral hemorrhage, successful recanalization, and early neurologic recovery were observed. CONCLUSIONS: Moderate-to-severe white matter disease is associated with worse outcomes in patients undergoing endovascular treatment without a significant increase in hemorrhagic complications. Studies comparing patients with and without endovascular treatment are necessary to determine whether the benefit of endovascular treatment is attenuated with greater white matter disease.

The evolution of bacterial transformation: sex with poor relations.

Bacteria are the only organisms known to actively take up DNA and recombine it into their genomes. While such natural transformation systems may provide many of the same benefits that sexual reproduction provides eukaryotes, there are important differences that critically alter the consequences, especially when recombination's main benefit is reducing the mutation load. Here, analytical and numerical methods are used to study the selection of transformation genes in populations undergoing deleterious mutation. Selection for transformability depends on the shape of the fitness function against mutation. If the fitness function is linear, then transformation would be selectively neutral were it not for the possibility that transforming cells may take up DNA that converts them into nontransformable cells. If the selection includes strong positive (synergistic) epistasis, then transformation can be advantageous in spite of this risk. The effect of low quality DNA (from selectively killed cells) on selection is then studied analytically and found to impose an additional cost. The limited data available for real bacterial populations suggest that the conditions necessary for the evolution of transformation are unlikely to be met, and thus that DNA uptake may have some function other than recombination of deleterious mutations.

A refined 3-dimensional QSAR of cytochrome P450 2C9: computational predictions of drug interactions.

A ligand-based model is reported that predicts the Ki values for cytochrome P450 2C9 (CYP2C9) inhibitors. This CoMFA model was used to predict the affinity of 14 structurally diverse compounds not in the training set and appears to be robust. The mean error of the predictions is 6 microM. The experimentally measured Ki values of the 14 compounds range from 0.1 to 48 microM. Leave-one-out cross-validated partial least-squares gives a q2 value of between 0.6 and 0.8 for the various models which indicates internal consistency. Random assignment of biological data to structure leads to negative q2 values. These models are useful in that they establish a pharmacophore for binding to CYP2C9 that can be tested with site-directed mutagenesis. These models can also be used to screen for potential drug interactions and to design compounds that will not bind to this enzyme with high affinity.

Triazolam substrate inhibition: evidence of competition for heme-bound reactive oxygen within the CYP3A4 active site.

In human liver microsomes, triazolam is principally metabolized by CYP3A4 to form two metabolites, 1'-hydroxytriazolam (1'OHTz) and 4-hydroxytriazolam (4OHTz). The velocity of 1'OHTz formation was found to decrease at higher triazolam concentrations (>200 microM), indicative of "substrate inhibition". Coincubation of [(14)C]triazolam with authentic metabolite standards of either 1'OHTz or 4OHTz up to 30 microM did not significantly inhibit the rate of [(14)C]1'OHTz formation. The effects of secondary compounds on triazolam oxidation were shown to be product-specific, producing either activation or inhibition depending on the triazolam metabolite monitored. When human liver microsomes were supplemented with exogenous human cytochrome b(5), it was observed that substrate inhibition was attenuated and the resulting increase in 1'OHTz formation, relative to control (nonsupplemented) incubations, corresponded to a decrease in the ratio of 4OHTz to 1'OHTz. In contrast, when cofactor (e.g., 100 microM NADPH) was rate limiting, the metabolite ratio (4OHTz/1'OHTz) was markedly increased over the entire substrate concentration range (0.5-1000 microM). To explain these kinetic observations, a two-site binding model is proposed in which triazolam is hypothesized to bind within the CYP3A4 active site in spatially distinct orientations, which may lead to the formation of either the 1'-hydroxytriazolam or 4-hydroxytriazolam. Differential inhibition/activation is consistent with this two-site model and substrate inhibition is hypothesized to result from competition between the two sites for reactive oxygen.

Topological alteration of the CYP3A4 active site by the divalent cation Mg(2+).

Phenyldiazene reacted with lymphoblast-expressed CYP3A4 to give a stable phenyl-iron complex that could be induced to rearrange in situ producing approximately equal amounts of four N-phenyl-protoporphyrin IX isomers (N(B):N(A):N(C):N(D), 01:01:02:02). In the presence of 10 mM MgCl(2), the formation profile of the protoporphyrin isomers was markedly altered compared with control, favoring the N(A) isomer (N(B):N(A):N(C):N(D), 01:34:01:02). In addition, an investigation of MgCl(2) effects on CYP3A4-mediated metabolism of triazolam revealed that 10 mM MgCl(2) increased the apparent K(m) of triazolam 4-hydroxylation from 83 to 173 microM and reduced the V(max) for the reaction from 3.4 to 2.4 min(-1). Moreover, when the reaction kinetics of the oxidation of pyrene by CYP3A4 was examined in the absence of MgCl(2), it was found that the substrate-velocity curve was best approximated by a sigmoidal velocity curve (Hill coefficient 1.7 +/- 0.1). However, when the reaction was conducted in the presence of 10 mM MgCl(2), the resulting pyrene kinetics was not sigmoidal but rather biphasic (Hill coefficient 0.80 +/- 0.07). Based on the current results, it appears that CYP3A4 is conformationally sensitive to its in vitro environment and parameters, such as the presence of a divalent magnesium, can have a measurable effect on active site topography and consequently catalytic activity.

Covalent alteration of the CYP3A4 active site: evidence for multiple substrate binding domains.

The inhibition of CYP3A4-mediated oxidation of triazolam and testosterone was assessed in the presence of a selection of known CYP3A4 substrates and inhibitors. Under experimental conditions where the Michaelis-Menten model predicts substrate-independent inhibition ([S] = K(m)), results yielded substrate-dependent inhibition. Moreover, when the same experimental design was extended to a group of structurally similar flavonoids it was observed that flavanone, flavone, 3-hydroxyflavone, and 6-hydroxyflavone (10 microM) activated triazolam metabolism, but inhibited testosterone hydroxylation. In additional studies, residual CYP3A4 activity toward testosterone and triazolam hydroxylation was measured after pretreatment with the CYP3A4 mechanism based inhibitor, midazolam. After midazolam preincubation, CYP3A4 6 beta-hydroxylase activity was reduced by 47% while, in contrast, triazolam hydroxylation was reduced by 75%. These results provide physical evidence, which supports the hypothesis that the active site of CYP3A4 contains spatially distinct substrate-binding domains within the enzyme active site.

Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. Purification, partial characterization, and substrate specificity.

The enzyme N-succinyl-L-diaminopimelic acid desuccinylase from Escherichia coli has been purified 7,100-fold to apparent homogeneity. The enzyme is part of the diaminopimelic acid-lysine pathway in bacteria and catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to produce L-diaminopimelic acid and succinate. The enzyme exists as a mixture of dimeric and tetrameric species of identical subunits of molecular weight approximately 40,000. Activity was completely abolished following dialysis of the enzyme against metal chelators. Cobalt(II) and zinc were effective in restoring the activity. The apparent affinities of the apoenzyme for cobalt and zinc were similar (Kd values near 1 microM) and the cobalt enzyme was 2.2-fold more active than the zinc enzyme. The Km and turnover number for the hydrolysis of the natural substrate, N-succinyl-L-diaminopimelic acid, were 0.4 mM and 16,000 min-1, respectively. The substrate specificity of the enzyme was defined by preparing a number of substrate analogues that systematically lack the various functional groups present in the molecule. These studies show that the enzyme is highly specific for the natural substrate. These properties of N-succinyl-L-diaminopimelic acid desuccinylase and the fact that the enzyme is essential for bacterial growth make it an ideal target for the development of inhibitors with potential antibacterial activity.

Hematopoietic progenitors and aging: alterations in granulocytic precursors and responsiveness to recombinant human G-CSF, GM-CSF, and IL-3.

BACKGROUND: Changes either in the number or in the responsiveness of hematopoietic progenitors may be a major factor accounting for age-related changes in stimulus driven hematopoiesis. METHODS: To test these hypotheses, we compared the relative proportions and the responsiveness of CD34+ bone marrow cells from healthy young (20-30 yrs) and healthy elderly (70-80 yrs) volunteers to G-CSF, GM-CSF, and IL-3 in an in vitro marrow culture system. RESULTS: There was no age-related difference either in the proportion of CD34+ marrow cells or in the proportion of a more mature CD34+ subset, defined as CD34+, CD33+ cells. Maximal colony formation by CD34+ cells stimulated with a combination of G-CSF, GM-CSF, and IL-3 was similar in the two groups, but the dose-response studies with individual growth factors revealed a 2-fold decrease in sensitivity of the elderly subjects' cells to G-CSF (p < .01). CONCLUSIONS: Aging has little impact on the marrow content of early precursors of the neutrophil lineage. There is, however, a significant difference in the in vitro proliferative response of these cells to the lineage specific growth factor G-CSF. This alteration may account for the greater propensity in elderly populations for the development of neutropenia with severe infections and chemotherapy.

Mechanism-based inactivation of cytochrome P450 2B1 by 7-ethynylcoumarin: verification of apo-P450 adduction by electrospray ion trap mass spectrometry.

7-Ethynylcoumarin was synthesized as a potential mechanism-based inhibitor, and it was found to be an effective inactivator of 7-ethoxy-4-(trifluoromethyl)coumarin (7EFC) O-deethylation catalyzed by purified, reconstituted P450 2B1. In contrast, 7-ethynylcoumarin demonstrated minimal inactivation of P450 2A6-mediated 7-hydroxycoumarin formation. The inactivation of P450 2B1 demonstrated pseudo-first-order kinetics and was NADPH- and inhibitor-dependent. The maximal rate constant for the inactivation of 2B1 was 0.39 min(-)(1) at 30 degrees C, and thus, the time required to inactivate 50% of the P450 2B1 that was present (t(1/2)) was 1.8 min. The estimated concentration which led to half-maximal inactivation (K(I)) was 25 microM. No protection from inactivation was seen in the presence of nucleophiles (glutathione and sodium cyanide), an iron chelator (deferroxamine), or superoxide dismutase and catalase. Addition of the substrate (7EFC) protected P450 2B1 from inactivation, in a concentration-dependent manner. The partition ratio for P450 2B1 was 25; i.e., the number of metabolic events was 25-fold higher than the number of inactivating events. Incubations of 7-ethynylcoumarin with P450 2B1 for 10 min resulted in an 80% loss in enzymatic activity, while 90% of the ability to form a reduced-CO complex remained. This activity loss was not recovered following dialysis, indicative of irreversible inactivation. Covalent attachment of the entire inhibitor and oxygen to apo-P450 2B1, in a 1:1 ratio, was shown via electrospray ion trap mass spectrometry. This method also verified the absence of modification to the heme or the cytochrome P450 reductase. Taken together, the characterization of the inhibition seen with P450 2B1 and 7-ethynylcoumarin was consistent with all of the criteria required to distinguish a mechanism-based inactivator. In addition, electrospray ion trap mass spectrometry has the potential to be applied to protein adducts above and beyond those associated with the mechanism-based inactivation of cytochrome P450s.