Chemical-Scale Studies on the Role of a Conserved Aspartate in Preorganizing the Agonist Binding Site of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor and related Cys-loop receptors are ligand-gated ion channels that mediate fast synaptic transmission throughout the central and peripheral nervous system. A highly conserved aspartate residue (D89) that is near the agonist binding site but does not directly contact the ligand plays a critical part in receptor function. Here we probe the role of D89 using unnatural amino acid mutagenesis coupled with electrophysiology. Homology modeling implicates several hydrogen bonds involving D89. We find that no single hydrogen bond is essential to proper receptor function. Apparently, the side chain of D89 establishes a redundant network of hydrogen bonds; these bonds preorganize the agonist binding site by positioning a critical tryptophan residue that directly contacts the ligand. Earlier studies of the D89N mutant led to the proposal that a negative charge at this position is essential for receptor function. However, we find that receptors with neutral side chains at position 89 can function well, if the side chain is less perturbing than the amide of asparagine (nitro or keto groups allow function) or if a compensating backbone mutation is introduced to relieve unfavorable electrostatics

Probing the role of the cation–π interaction in the binding sites of GPCRs using unnatural amino acids

We describe a general application of the nonsense suppression methodology for unnatural amino acid incorporation to probe drug–receptor interactions in functional G protein-coupled receptors (GPCRs), evaluating the binding sites of both the M2 muscarinic acetylcholine receptor and the D2 dopamine receptor. Receptors were expressed in Xenopus oocytes, and activation of a G protein-coupled, inward-rectifying K^+ channel (GIRK) provided, after optimization of conditions, a quantitative readout of receptor function. A number of aromatic amino acids thought to be near the agonist-binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at W6.48 of the D2 receptor establishes a cation–π interaction between the agonist dopamine and W6.48, suggesting a reorientation of W6.48 on agonist binding, consistent with proposed “rotamer switch” models. Interestingly, no comparable cation–π interaction was found at the aligning residue in the M2 receptor

Reduced ratio of protective versus proinflammatory cytokine responses to commensal bacteria in HLA-B27 transgenic rats

Germ-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T- or B- cell-depleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-γ, IL-12, TNF, IL-10 and TGF-β production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-γ, IL-12 and TNF than did non-TG MLN cells. In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-β. T cell depletion abolished IFN-γ and decreased IL-12 production, but did not affect IL-10 and TGF-β production. Conversely, neither IL-10 nor TGF-β was produced in cultures of B cell-depleted MLN. In addition, CD4+ T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-γ when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-β, but not IFN-γ, IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-γ-producing CD4+ T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation

Concentrations of Pro-Inflammatory Cytokines Are Not Associated with Senescence Marker p16INK4a or Predictive of Intracellular Emtricitabine/Tenofovir Metabolite and Endogenous Nucleotide Exposures in Adults with HIV Infection

As the HIV-infected population ages, the role of cellular senescence and inflammation on co-morbid conditions and pharmacotherapy is increasingly of interest. p16INK4a expression, a marker for aging and senescence in T-cells, is associated with lower intracellular concentrations of endogenous nucleotides (EN) and nucleos(t)ide reverse transcriptase inhibitors (NRTIs). This study expands on these findings by determining whether inflammation is contributing to the association of p16INK4a expression with intracellular metabolite (IM) exposure and endogenous nucleotide concentrations

Transplantation of human fetal biliary tree stem/progenitor cells into two patients with advanced liver cirrhosis.

Efforts to identify cell sources and approaches for cell therapy of liver diseases are ongoing, taking into consideration the limits recognized for adult liver tissue and for other forms of stem cells. In the present study, we described the first procedure of via hepatic artery transplantation of human fetal biliary tree stem cells in patients with advanced cirrhosis.MethodsThe cells were immune-sorted from human fetal biliary tree by protocols in accordance with current good manufacturing practice (cGMP) and extensively characterized. Two patients with advanced cirrhosis (Child-Pugh C) have been submitted to the procedure and observed through a 12 months follow-up.ResultsThe resulting procedure was found absolutely safe. Immuno-suppressants were not required, and the patients did not display any adverse effects correlated with cell transplantation or suggestive of immunological complications. From a clinical point of view, both patients showed biochemical and clinical improvement during the 6 month follow-up (Table1), and the second patient maintained a stable improvement for 12 months.ConclusionThis report represents proof of the concept that the human fetal biliary tree stem cells are a suitable and large source for cell therapy of liver cirrhosis. The isolation procedure can be carried out under cGMP conditions and, finally, the infusion procedure is easy and safe for the patients. This represents the basis for forthcoming controlled clinical trials

Lifestyle impacts on the aging-associated expression of biomarkers of DNA damage and telomere dysfunction in human blood: Measuring the influence of lifestyle on aging

Cellular aging is characterised by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and that activation of DNA damage responses. There is some evidence the DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging associated expression of serum markers of DNA damage (CRAMP, EF-1α, Stathmin, n-acetyl-glucosaminidase, and chitinase) in comparison to other described markers of cellular aging (p16INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age-independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age-independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16INK4a expression and negatively with telomere length in peripheral blood T-lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging

Effect of metal Ions (Ni2+, Cu2+ and Zn2+) and water coordination on the structure of L-phenylalanine, L-tyrosine, L-tryptophan and their zwitterionic forms

Methods of quantum chemistry have been applied to double-charged complexes involving the transition metals Ni2+, Cu2+ and Zn2+ with the aromatic amino acids (AAA) phenylalanine, tyrosine and tryptophan. The effect of hydration on the relative stability and geometry of the individual species studied has been evaluated within the supermolecule approach. The interaction enthalpies, entropies and Gibbs energies of nine complexes Phe•M, Tyr•M, Trp•M, (M = Ni2+, Cu2+ and Zn2+) were determined at the Becke3LYP density functional level of theory. Of the transition metals studied the bivalent copper cation forms the strongest complexes with AAAs. For Ni2+and Cu2+ the most stable species are the NO coordinated cations in the AAA metal complexes, Zn2+cation prefers a binding to the aromatic part of the AAA (complex II). Some complexes energetically unfavored in the gas-phase are stabilized upon microsolvation

Effect of Cytotoxic Chemotherapy on Markers of Molecular Age in Patients With Breast Cancer

Senescent cells, which express p16 INK4a, accumulate with aging and contribute to age-related pathology. To understand whether cytotoxic agents promote molecular aging, we measured expression of p16 INK4a and other senescence markers in breast cancer patients treated with adjuvant chemotherapy