We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation "seen" by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.
We had previously demonstrated that the cyc- mutant of S49 wild-type lymphoma cells both desensitizes and undergoes a sequestration-internalization of the beta-receptor in response to short-term treatment with adrenaline. The cyc- mutant of S49 wild-type lymphoma cells lacks the alpha s subunit of the stimulatory coupling protein Ns, but has fully functional Ni, the inhibitory component of the regulatory complex. This suggested that functional Ns was not required for desensitization. To examine the role of Ni in desensitization, both S49 wild-type and cyc- cells were treated with islet-activating protein under conditions that led to over 85% attenuation of Ni function in S49 wild-type cells and approx. 50% attenuation of Ni function in cyc- cells. This treatment had no effect on the adrenaline-induced desensitization of adenylate cyclase or the sequestration event measured by the apparent movement of beta-adrenergic receptors to a light-vesicle fraction. Further, the desensitization event, which occurs before the sequestration event, observable only in intact cells, was also not altered by islet-activating-protein pretreatment of S49 wild-type cells. The data suggest that a functional Ni is not required for desensitization in the S49 lymphoma cells.
A method is described for the assay of N-(phosphonacetyl)-L-aspartic acid in plasma or urine, based on inhibition of partially purified aspartate carbamoyltransferase from rat liver. Concentrations of N-(phosphonacetyl)-L-aspartic acid as low as 0.1 microgram/ml can be detected. Plasma disappearance and urinary excretion curves from one patient are shown as examples of the application of the method.
Dendritic cells (DCs) modulate B-cell survival and differentiation, mainly through production of growth factors such as B lymphocyte stimulator (BLyS; also known as "B-cell factor belonging to the tumor necrosis factor family" [BAFF]). We have recently shown that, in human immunodeficiency virus (HIV)-infected individuals with rapid and those with classic disease progression, B-cell dysregulations were associated with increased BLyS expression in plasma and by blood myeloid DCs (mDCs), in contrast to aviremic HIV-infected individuals with slow disease progression (also known as "elite controllers"). In previous work with transgenic mice expressing HIV genes, B-cell dysregulations were concomitant with altered mDCs and dependent on HIV negative factor (Nef). We now report that HIV Nef is detected early after infection and despite successful therapy in plasma and BLyS-overexpressing blood mDCs of HIV-infected rapid and classic progressors, whereas it is low to undetectable in aviremic slow progressors. In vitro, HIV Nef drives monocyte-derived DCs toward BLyS overexpression through a process involving STAT1. Importantly, this is counteracted in the presence of all-trans retinoic acid. Nef thus contributes to high BLyS proinflammatory profiles in HIV-infected individuals.
Since elevated levels of circulating complexes have been noted to occur in the sera of patients with post-streptococcal sequelae, the possibility that these complexes contained streptococcal antigens within the complex was investigated. Sera from these patients were precipitated with polyethylene glycol to extract a fraction rich in these complexes, which was then injected into rabbits. The rabbit sera were then reacted with both cellular and extracellular fractions obtained from streptococcal strains associated with either acute post-streptococcal nephritis (APSGN) or acute rheumatic fever (ARF) by using immunoelectrophoresis and ELISA techniques. The data demonstrate that both ARF and APSGN complexes contain streptococcal an- tigens. However, APSGN complexes react uniquely to certain extracellular antigens present in those strains associated with nephritis, while ARF complexes react specifically to certain streptococcal extracellular antigens excreted by strains associated with rheumatic fever. Neither of the two groups of complexes appear to contain streptococcal antigens related to any cellular antigens derived from the group A streptococcus. Ad- ditionally, a rabbit serum immunized with streptococcal extracellular products reacted directly with complexes
The human Herpesvirus type-6 (HHV-6) has been implicated in multiple sclerosis (MS). Valacyclovir is an antiviral agent with an excellent safety profile. A two-year placebo-controlled, double-blind study was conducted to (1) ascertain if high-dose, prolonged treatment with valacyclovir would be safe and (2) observe if valacyclovir would delay the progression of MS clinically or by magnetic resonance imaging (MRI).Fifty-eight patients were stratified as to severity and randomly assigned to receive valacyclovir (3000 mg/day) or placebo for a period of two years. Patients were followed clinically over the two-year period by means of the Expanded Disability Status Scale (EDSS), the Ambulation Index (AI) and brain MRI scans. Patients underwent routine lab studies every three months. Patients continued on the medication for two years unless they had a sustained progression or repeated exacerbations.No patient discontinued the study due to side effects or toxicity. In Relative Ranking of Progression, time to first attack, attack rate, and time to withdrawal there were trends (but not statistically significant) toward drug effect over placebo in the Severe clinical category. MRI evaluation showed no significant drug effect.Although not statistically significant, positive trends were detected for acyclovir by clinical measures, but not by MRI.
The molecular basis for the effects of 4 beta-phorbol 12-myristate 13-acetate (PMA) on adenylyl cyclase activation was examined using site-directed mutants of the hamster beta-adrenergic receptor (beta AR) expressed in L cells. Phorbol ester activation of protein kinase C (PKC) in L cells transfected with wild-type beta AR caused at least three discernible effects on adenylyl cyclase activity, each with an EC50 of 20 to 50 nM, (i) a 2-3-fold increase in the Kact for epinephrine stimulation, (ii) a 2-3-fold increase in the maximal level (Vmax) of hormonal stimulation, and (iii) a decrease in the Gi-mediated inhibition of forskolin stimulation. Deletion from the beta AR of amino acid residues 259-262, which removes one of the two consensus sites for phosphorylation by PKC, eliminated (greater than 90%) the PMA-induced increase in the Kact, whereas the PMA-induced increase in the Vmax and loss of Gi-mediated inhibition were not affected by the deletion. Neither deletion of the other PKC consensus site in the beta AR (residues 343-348) nor truncation of the Ser/Thr-rich C-terminal domain (residues 354-418) affected the PMA-induced changes in adenylyl cyclase. The effects of PMA on Gi-mediated inhibition and the Vmax closely mimicked the action of islet-activating protein, consistent with a direct effect of PMA-activated PKC on Gi. In contrast, the effects on the Kact appear to be receptor specific. These results demonstrate that the consensus site for phosphorylation by PKC, found in the third intracellular loop of the beta AR, is required for the PMA-induced increase in the Kact for epinephrine stimulation. Use of L cells transfected with D(259-262)beta AR allowed the characterization of the postreceptor effects of PMA without interference from receptor-level effects.
