Age-related disease association of endogenous γ-H2AX foci in mononuclear cells derived from leukapheresis.

The phosphorylated form of histone H2AX (γ-H2AX) forms immunohistochemically detectable foci at DNA double strand breaks. In peripheral blood mononuclear cells (PBMCs) derived from leukapheresis from patients enrolled in the Baltimore Longitudinal Study of Aging, γ-H2AX foci increased in a linear fashion with regards to age, peaking at ~57 years. The relationship between the frequency of γ-H2AX foci and age-related pathologies was assessed. We found a statistically significant (p = 0.023) 50% increase in foci in PBMCs derived from patients with a known history of vitamin D deficiency. In addition, there were trends toward increased γ-H2AX foci in patients with cataracts (34% increase, p<0.10) and in sleep apnea patients (44%, p<0.10). Among patients ≥57 y/o, we found a significant (p = 0.037) 36% increase in the number of γ-H2AX foci/cell for patients with hypertension compared to non-hypertensive patients. Our results support a role for increased DNA damage in the morbidity of age-related diseases. γ -H2AX may be a biomarker for human morbidity in age-related diseases.

Syntheses of alpha- and beta-C-glucopyranosyl serines from a common intermediate.

A versatile synthesis leading to either C-linked alpha- or beta-glucopyranosyl serines is presented from a common, advanced synthetic intermediate. Cyclization of the penultimate carbinol onto the alkene and methanolysis of the lactone yields selectively the alpha-linkage. A transposition of these last steps leads to the beta-linked isomer selectively.

Probing the binding site of the A1 adenosine receptor reengineered for orthogonal recognition by tailored nucleosides.

His272 (7.43) in the seventh transmembrane domain (TM7) of the human A3 adenosine receptor (AR) interacts with the 3' position of nucleosides, based on selective affinity enhancement at a H272E mutant A3 AR (neoceptor) of 3'-ureido, but not 3'-OH, adenosine analogues. Here, mutation of the analogous H278 of the human A1 AR to Ala, Asp, Glu, or Leu enhanced the affinity of novel 2'- and 3'-ureido adenosine analogues, such as 10 (N6-cyclopentyl-3'-ureido-3'-deoxyadenosine), by >100-fold, while decreasing the affinity or potency of adenosine and other 3'-OH adenosine analogues. His278 mutant receptors produced a similar enhancement regardless of the charge character of the substituted residue, implicating steric rather than electrostatic factors in the gain of function, a hypothesis supported by rhodopsin-based molecular modeling. It was also demonstrated that this interaction was orientationally specific; i.e., mutations at the neighboring Thr277 did not enhance the affinity for a series of 2'- and 3'-ureido nucleosides. Additionally, H-bonding groups placed on substituents at the N6 or 5' position demonstrated no enhancement in the mutant receptors. These reengineered human A1 ARs revealed orthogonality similar to that of the A3 but not the A2A AR, in which mutation of the corresponding residue, His278, to Asp did not enhance nucleoside affinity. Functionally, the H278D A1 AR was detectable only in a measure of membrane potential and not in calcium mobilization. This neoceptor approach should be useful for the validation of molecular modeling and the dissection of promiscuous GPCR signaling.