Sex-dependent co-occurrence of hypoxia and ß-amyloid plaques in hippocampus and entorhinal cortex is reversed by long-term treatment with ubiquinol and ascorbic acid in the 3 × Tg-AD mouse model of Alzheimer's disease.

Structural and functional abnormalities in the cerebral microvasculature have been observed in Alzheimer's disease (AD) patients and animal models. One cause of hypoperfusion is the thickening of the cerebrovascular basement membrane (CVBM) due to increased collagen-IV deposition around capillaries. This study investigated whether these and other alterations in the cerebrovascular system associated with AD can be prevented by long-term dietary supplementation with the antioxidant ubiquinol (Ub) stabilized with Kaneka QH P30 powder containing ascorbic acid (ASC) in a mouse model of advanced AD (3 × Tg-AD mice, 12 months old). Animals were treated from prodromal stages of disease (3 months of age) with standard chow without or with Ub + ASC or ASC-containing vehicle and compared to wild-type (WT) mice. The number of ß-amyloid (Aß) plaques in the hippocampus and entorhinal cortex was higher in female than in male 3 × Tg-AD mice. Extensive regions of hypoxia were characterized by a higher plaque burden in females only. This was abolished by Ub + ASC and, to a lesser extent, by ASC treatment. Irrespective of Aß burden, increased collagen-IV deposition in the CVBM was observed in both male and female 3 × Tg-AD mice relative to WT animals; this was also abrogated in Ub + ASC- and ASC-treated mice. The chronic inflammation in the hippocampus and oxidative stress in peripheral leukocytes of 3 × Tg-AD mice were likewise reversed by antioxidant treatment. These results provide strong evidence that long-term antioxidant treatment can mitigate plasma oxidative stress, amyloid burden, and hypoxia in the AD brain parenchyma.

Effect of dietary fat modification on subcutaneous white adipose tissue insulin sensitivity in patients with metabolic syndrome.

SCOPE: To determine whether the insulin resistance that exists in metabolic syndrome (MetS) patients is modulated by dietary fat composition. METHODS AND RESULTS: Seventy-five patients were randomly assigned to one of four diets for 12 wk: high-saturated fatty acids (HSFAs), high-MUFA (HMUFA), and two low-fat, high-complex carbohydrate (LFHCC) diets supplemented with long-chain n-3 (LFHCC n-3) PUFA or placebo. At the end of intervention, the LFHCC n-3 diet reduced plasma insulin, homeostasis model assessment of insulin resistance, and nonsterified fatty acid concentration (p < 0.05) as compared to baseline Spanish habitual (BSH) diet. Subcutaneous white adipose tissue (WAT) analysis revealed decreased EH-domain containing-2 mRNA levels and increased cbl-associated protein gene expression with the LFHCC n-3 compared to HSFA and HMUFA diets, respectively (p < 0.05). Moreover, the LFHCC n-3 decreased gene expression of glyceraldehyde-3-phosphate dehydrogenase with respect to HMUFA and BSH diets (p < 0.05). Finally, proteomic characterization of subcutaneous WAT identified three proteins of glucose metabolism downregulated by the LFHCC n-3 diet, including annexin A2. RT-PCR analysis confirmed the decrease of annexin A2 (p = 0.027) after this diet. CONCLUSION: Our data suggest that the LFHCC n-3 diet reduces systemic insulin resistance and improves insulin signaling in subcutaneous WAT of MetS patients compared to HSFA and BSH diets consumption.

Identification and characterization of human archaemetzincin-1 and -2, two novel members of a family of metalloproteases widely distributed in Archaea.

Systematic analysis of degradomes, the complete protease repertoires of organisms, has demonstrated the large and growing complexity of proteolytic systems operating in all cells and tissues. We report here the identification of two new human metalloproteases that have been called archaemetzincin-1 (AMZ1) and archaemetzincin-2 (AMZ2) to emphasize their close relationship to putative proteases predicted by bioinformatic analysis of archaeal genomes. Both human proteins contain a catalytic domain with a core motif (HEXXHXXGX3CX4CXMX17CXXC) that includes an archetypal zinc-binding site, the methionine residue characteristic of metzincins, and four conserved cysteine residues that are not present at the equivalent positions of other human metalloproteases. Analysis of genome sequence databases revealed that AMZs are widely distributed in Archaea and vertebrates and contribute to the defining of a new metalloprotease family that has been called archaemetzincin. However, AMZ-like sequences are absent in a number of model organisms from bacteria to nematodes. Phylogenetic analysis showed that these enzymes have undergone a complex evolutionary process involving a series of lateral gene transfer, gene loss, and genetic duplication events that have shaped this novel family of metalloproteases. Northern blot analysis showed that AMZ1 and AMZ2 exhibit distinct expression patterns in human tissues. AMZ1 is mainly detected in liver and heart whereas AMZ2 is predominantly expressed in testis and heart, although both are also detectable at lower levels in other tissues. Both human enzymes were produced in Escherichia coli, and the purified recombinant proteins hydrolyzed synthetic substrates and bioactive peptides, demonstrating that they are functional proteases. Finally, these activities were abolished by inhibitors of metalloproteases, providing further evidence that AMZs belong to this catalytic class of proteolytic enzymes.

Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-D-arginine.

Polyarginine-containing peptides represent potent inhibitors of furin, a mammalian endoprotease that plays an important role in metabolism, activation of pathogenic toxins, and viral proliferation. The therapeutic use of D-polyarginines is especially interesting because they are not cleaved by furin and possess inhibitory potency almost equal to L-polyarginines. In this study we attempted to determine the important elements within polyarginines that contribute to effective inhibition. Structure-function analyses of polyarginine peptides showed that inhibition by polyarginine-containing peptides appeared to depend on the total number of basic charges of the positively charged inhibitors bound to the negatively charged substrate binding pocket; peptide positioning did not appear to be rigorously determined. Screening of L- and D-decapeptide positional scanning combinatorial peptide libraries indicated a preference for basic residues in nearly all positions, similar to previous results with hexapeptide libraries. Length and terminal modification studies showed that the most potent D-polyarginine tested was nona-D-arginine (D9R) amide with a K(i) of 1.3 nm. D9R amide was shown to protect RAW264.7 cells against anthrax toxemia with an IC(50) of 3.7 microm. Because of its high stability, specificity, low toxicity, small molecular weight, and extremely low K(i) against furin, D9R amide or its derivatives may represent promising compounds for therapeutic use.