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.

Two-dimensional gel electrophoretic analysis of cell lysates from Brucella abortus 2308 and the isogenic hfq mutant Hfq3 revealed that the RNA binding protein Hfq (also known as host factor I or HF-I) is required for the optimal stationary phase production of the periplasmic Cu,Zn superoxide dismutase SodC. An isogenic sodC mutant, designated MEK2, was constructed from B. abortus 2308 by gene replacement, and the sodC mutant exhibited much greater susceptibility to killing by O 2 ؊ generated by pyrogallol and the xanthine oxidase reaction than the parental 2308 strain supporting a role for SodC in protecting this bacterium from O 2 ؊ of exogenous origin. The B. abortus sodC mutant was also found to be much more sensitive to killing by cultured resident peritoneal macrophages from C57BL6J mice than 2308, and the attenuation displayed by MEK2 in cultured murine macrophages was enhanced when these phagocytes were treated with gamma interferon (IFN-␥). The attenuation displayed by the B. abortus sodC mutant in both resting and IFN-␥-activated macrophages was alleviated, however, when these host cells were treated with the NADPH oxidase inhibitor apocynin. Consistent with its increased susceptibility to killing by cultured murine macrophages, the B. abortus sodC mutant also displayed significant attenuation in experimentally infected C57BL6J mice compared to the parental strain. These experimental findings indicate that SodC protects B. abortus 2308 from the respiratory burst of host macrophages. They also suggest that reduced SodC levels may contribute to the attenuation displayed by the B. abortus hfq mutant Hfq3 in the mouse model.The Brucella spp. are gram-negative facultative intracellular pathogens capable of infecting a wide variety of mammalian hosts. Brucella abortus is the etiological agent of bovine brucellosis, an infection that leads to spontaneous abortion and infertility (10). Human brucellosis presents as a debilitating febrile illness commonly referred to as Malta Fever or undulant fever (59), and an active infection is characterized by fever, malaise, chills, night sweats, and anorexia. Human brucellosis is a zoonotic disease; therefore, the incidence of disease in humans is directly related to its occurrence in animals (43). Although considered mainly an occupational hazard in many countries, human brucellosis remains a significant problem where the disease is endemic in food animals and raw milk or other unpasteurized dairy products are still consumed.Brucella spp. are not found free living, nor are they commensal organisms (23). The preferred ecological niche for the brucellae is within the phagosomal compartment of host macrophages, and the capacity of this organism to establish and maintain chronic infections is dependent upon its ability to survive and replicate within these phagocytic cells (48). Experimental evidence indicates that the production of reactive oxygen intermediates (ROIs) represents one of the primary mechanisms utilized by host macrophages for limiting the intracellular replic...

We test Lorentz invariance by searching for a time-dependent quadrupole splitting of Zeeman levels in 21 Ne. A component at twice the Earth's sidereal frequency would suggest a preferred direction whichaffects the local physics of the nucleus. The technique employs polarized 21 Ne and 3 He gases produced by spin exchange with laser optically pumped Rb. Both species are contained in the same glass cell; 3 He provides magnetometry and a monitor of systematic effects. Our data produce an upper limit (1

The UDP-MurNAc-pentapeptide is transferred to the outer face of the cell membrane by a lipid carrier and incorporated along with UDP-N-acetylglucosamine into the cell wall structure. The synthesis of other types of peptidoglycan precursors was demonstrated a few years ago in the context of several studies concerning vancomycin resistance. Vancomycin and other glycopeptide antibiotics can bind to the DAla-D-Ala terminus of pentapeptide-containing precursors by hydrogen bonding, thereby effectively blocking polymerization and preventing further cross-linking reactions (7, 41). Investigations of the molecular basis of vancomycin resistance started with strains of Enterococcus faecium and Enterococcus faecalis which showed inducible resistance to high levels of vancomycin and teicoplanin, another glycopeptide antibiotic. Examination of enzymes involved in cell wall synthesis in the resistant bacteria indicated that resistance to vancomycin was due to the synthesis of a novel type of peptidoglycan in which the terminal D-alanine residue was replaced by D-lactate, resulting in a drastic reduction of affinity for vancomycin (2,4,12,25,36). Two enzymes designated VanH and VanA are required for the synthesis of this alternative precursor (5). VanH is an ␣-ketoacid dehydrogenase that reduces pyruvate to D-lactate The synthesis of another type of peptidoglycan precursor has been described for Enterococcus gallinarum, which expresses inducible resistance to low levels of vancomycin but is susceptible to teicoplanin. In this case, the modified precursor terminates in D-serine instead of D-lactate (9). This feature results from the presence of another variant D-Ala-D-Ala ligase accepting D-serine (18).The genera Lactobacillus, Leuconostoc, and Pediococcus comprise strains and species constitutively resistant to vancomycin (15,20,33,39,46,49). Recently, peptidoglycan precursors from several of these lactic acid bacteria were analyzed. In Pediococcus pentosaceus and Lactobacillus casei (9, 26), the exclusive presence of a terminal D-lactate has been demonstrated. This presence could result from the action of a ligase which preferentially or exclusively catalyzes the synthesis of a D-Ala-D-Lac depsipeptide, as was suggested by Elisha and Courvalin (19). Analysis of Leuconostoc mesenteroides extracts identified a precursor that also terminates in D-lactate, but with an additional branched L-alanine In this paper, we report that the wild-type strain Lactobacillus plantarum NCIMB8826 is naturally resistant to high levels of vancomycin and teicoplanin and exclusively produces Dlactate-ending peptidoglycan precursors. We describe the construction of a strain defective for both D-and L-LDH, resulting in drastically reduced production of both isomers of lactate. We show that this alteration leads to the synthesis of a new type of precursor ending with D-alanine in addition to the usual muramyl depsipentapeptide observed in the wild-type strain,