The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ⌬F508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ⌬F508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ⌬F508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.The maturation of polytopic multidomain membrane proteins is a complex process that requires the proper folding and assembly of individual domains to form a functional complex (1). These processes may be tightly coupled and occur simultaneously or may proceed in a hierarchical fashion. In addition, these processes may proceed in either a co-or post-translational manner (2, 3). The unique nature of these proteins often requires chaperone systems to promote the proper interactions both within and across multiple protein domains. Perturbations that alter the structures of the individual domains or that alter the interactions of these multi-domain complexes are recognized by the cellular quality control (QC) machines, which ultimately target the newly synthesized protein for maturation or degradation.Studies of the cystic fibrosis transmembrane conductance regulator (CFTR), 5 the protein whose loss results in cystic fibrosis (CF) have provided insight into the folding of polytopic membrane proteins (4). CFTR is a member of the ABC-transporter family of proteins and is composed of five distinct domains; two transmembrane domains, TMD1 and TMD2; two nucleotide binding domains, NBD1 and NBD2; and a regulatory domain, R (4). The most common CF-causing mutation, the deletion of phenylalanine 508 (⌬F508), is located in the N-terminal cytoplasmic NBD1 (5-9). This single amino acid deletion results in a dramatic reduction of mature, plasma membrane resident CFTR. The immature protein is arrested in an intermediate conformational state that is recognized by the cellular quality control machinery and targeted for degradation by the ubiquitin-proteasome system (10 -13). Previous work has shown that the ⌬F508 CFTR can be "rescued" by a variety of treatments; tha...

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.Key words: Anandamide; Cannabis; Cannabinoid receptor; Marijuana porcine brain and found to be a lipid of novel structure [7]. Anandamide displayed specific binding to the CBI receptor and inhibited a prototypical twitch response in mouse vas deferens. Anandamide has also been shown to induce similar behavioral [8,9], pharmacological [10,11], and signal transduction effects [12] as classical cannabinoid agonists, but high concentrations were required to induce these effects. Levels of anandamide were first estimated to occur at 0.4 pmol/g (133 pg/g) in whole porcine brain [7], and recently quantitated in porcine and bovine brain at 173 pmol/g (60 ng/g) and 101 pmol/g (35 ng/g) respectively [13]. A recent study reports levels of anandamide in rat testis to be considerably lower (6 pmol/ g, 2.1 ng/g) [14]. However, anandamide has never been isolated from human tissue or fluids. Furthermore, levels of anandamide have not been measured in regions of rat brain or in tissues such as spleen where CB2 receptors have been shown to be expressed at high levels. Studies of anandamide distribution should help elucidate the physiologic role of anandamide as a cannabimimetic eicosanoid and possibly broader functions. In this study we report the isolation and quantitation of anandamide by liquid chromatography/mass spectrometry in various tissues and fluids from postmortem human and rat.

In the National Heart, Lung and Blood Institute Type Il Coronary Intervention Study, patients with Type lI hyperlipoproteinemia and coronary artery disease (CAD) were placed on a lowfat, low-cholesterol diet and then were randomly allocated to receive either 6 g cholestyramine four times daily or placebo. This double-blind study evaluated the effects of cholestyramine on the progression of CAD as assessed by angiography. Diet alone reduced the low-density lipoprotein cholesterol 6% in both groups. After randomization, low-density lipoprotein cholesterol decreased another 5% in the placebo group and 26% in the cholestyramine-treated group. Coronary angiography was performed in 1 16 patients before and after 5 years of treatment. CAD progressed in 49% (28 of 57) of the placebotreated patients vs 32% (19 of 59) of the cholestyramine-treated patients (p < .05). When only definite progression was considered, 35% (20 of 57) of the placebo-treated patients vs 25% (15 of 59) of the cholestyramine-treated patients exhibited definite progression; the difference was not statistically significant. However, when this analysis was performed with adjustment for baseline inequalities of risk factors, effect of treatment was more pronounced. Of lesions causing 50% or greater stenosis at baseline, 33% of placebo-treated and 12% of cholestyramine-treated patients manifested lesion progression (p < .05). Similar analyses with other end points (percent of baseline lesions that progressed, lesions that progressed to occlusion, lesions that regressed, size of lesion change, and all cardiovascular end points) all favored the cholestyramine-treated group, but were not statistically significant. Thus, although the sample size does not allow a definitive conclusion to be drawn, this study suggests MethodsStudy design. The study design has been described extensively elsewhere. 1 Briefly, patients were screened with two sets of eligibility criteria: elevated levels of low-density lipoprotein (LDL) cholesterol and angiographic evidence of CAD. Patients underwent coronary angiography if LDL cholesterol after 1 month of therapy with low-cholesterol, low-fat diet3 was in the

Effects of different salts (NaCl, MgC12, CaC12, GdmC1, NaBr, NaC104, NaH2PO4, Na2S04) on the stability of the ubiquitin molecule at pH 2.0 have been studied by differential scanning calorimetry, circular dichroism, and Tyr fluorescence spectroscopies. It is shown that all of the salts studied significantly increase the thermostability of the ubiquitin molecule, and that this stabilization can be interpreted in terms of anion binding. Estimated thermodynamic parameters of binding for C1-show that this binding is relatively weak (Kd = 0.15 M) and is characterized by a negative enthalpy of -15 kJ/mol per site. Particularly surprising was the observed stabilizing effect of GdmCl through the entire concentration range studied (0.01-2 M), however, to a lesser extent than stabilization by NaCl. This stabilizing effect of GdmCl appears to arise from the binding of C1-ions. Analysis of the observed changes in the stability of the ubiquitin molecule in the presence of GdmCl can be adequately described by combining the thermodynamic model of denaturant binding with C1-binding effects.

The generation of a second-harmonic signal at a flat unbonded interface between two solids has been observed. This signal is caused by passage of a longitudinal acoustic wave across the interface. The harmonic amplitude depends upon the pressure applied normal to the interface and is largest close to zero pressure, as expected theoretically. The effect has also been used to detect the presence of microcracks on the surface of Al 2024, developed during fatigue.