Sustained release of locally delivered celecoxib provides pain relief for osteoarthritis: a proof of concept in dog patients.

OBJECTIVE: Drug delivery platforms that allow for gradual drug release after intra-articular administration have become of much interest as a treatment strategy for osteoarthritis (OA). The aim of this study was to investigate the safety and efficacy of an intra-articular sustained release formulation containing celecoxib (CXB), a cyclooxygenase-2 (COX-2) selective inhibitor. METHODS: Amino acid-based polyesteramide microspheres (PEAMs), a biodegradable and non-toxic platform, were loaded with CXB and employed in two in vivo models of arthritis: an acute inflammatory arthritis model in rats (n = 12), and a randomized controlled study in chronic OA dog patients (n = 30). In parallel, the bioactivity of sustained release of CXB was evaluated in monolayer cultures of primary dog chondrocytes under inflammatory conditions. RESULTS: Sustained release of CXB did not alleviate acute arthritis signs in the rat arthritis model, based on pain measurements and synovitis severity. However, in OA dog patients, sustained release of CXB improved limb function as objective parameter of pain and quality of life based on gait analysis and owner questionnaires. It also decreased pain medication dependency over a 2-month period and caused no adverse effects. Prostaglandin E2 levels, a marker for inflammation, were lower in the synovial fluid of CXB-treated dog OA patients and in CXB-treated cultured dog chondrocytes. CONCLUSION: These results show that local sustained release of CXB is less suitable to treat acute inflammation in arthritic joints, while safe and effective in treating pain in chronic OA in dogs.

Charging gold nanoparticles in ZnO by electric fields.

Controlling the plasmon resonance frequency of metal nanostructures holds promise for both fundamental and applied research in optics. The plasmon resonance frequency depends on the number of free electrons in the metal. By adding or removing electrons to a metal nano-object, the plasmon resonance frequency shifts. In this study we indirectly change the number of free electrons in gold nanoparticles by applying an electrical potential difference over a heterostructure consisting of a ZnO layer with embedded gold nanoparticles. The potential difference induces shifts of defect energy levels in the ZnO by the electric field. This results in an exchange of electrons between particles and matrix which in turn modifies the gold nanoparticle plasmon properties. The positive charge shifts the ZnO optical absorption peak from 377 nm to 386 nm and shifts the nanoparticle plasmon from 549 nm to 542 nm. This electro-optical effect is a promising way to obtain fast optical switching in a solid state composition.

Implicit and explicit affective associations towards cannabis use in patients with recent-onset schizophrenia and healthy controls.

BACKGROUND: Cannabis use is common in patients with recent-onset schizophrenia and this is associated with poor disease outcome. More insight in the cognitive-motivational processes related to cannabis use in schizophrenia may inform treatment strategies. The present study is the first known to compare implicit and explicit cannabis associations in individuals with and without psychotic disorder. METHOD: Participants consisted of 70 patients with recent-onset psychotic disorder and 61 healthy controls with various levels of cannabis use. Three Single-Category Implicit Association Tests (SC-IAT) were used to assess 'relaxed', 'active' and 'negative' implicit associations towards cannabis use. Explicit expectancies of cannabis use were assessed with a questionnaire using the same words as the SC-IAT. RESULTS: There were no differences in implicit associations between patients and controls; however, patients scored significantly higher on explicit negative affect expectancies than controls. Both groups demonstrated strong negative implicit associations towards cannabis use. Explicit relaxed expectancies were the strongest predictors of cannabis use and craving. There was a trend for implicit active associations to predict craving. CONCLUSIONS: The findings indicate that patients suffering from schizophrenia have associations towards cannabis similar to controls, but they have stronger negative explicit cannabis associations. The strong negative implicit associations towards cannabis could imply that users of cannabis engage in a behaviour they do not implicitly like. Explicit relaxing expectancies of cannabis might be an important mediator in the continuation of cannabis use in patients and controls.

Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C' scalar couplings (3hbJNC').

The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein chymotrypsin inhibitor 2. Dihedral angle restraints for the phi and psi angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond 3hbJNC' coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.

Liver transplantation for hepatocellular carcinoma in patients without underlying liver disease: a systematic review.

Liver resection is the treatment of choice for hepatocellular carcinoma (HCC) occurring in the absence of underlying chronic liver disease. Orthotopic liver transplantation (OLT) is reserved for patients with unresectable disease but remains controversial. The aim of this study was to review the published literature on OLT for HCC in patients without coexisting chronic liver disease. A Medline-based search identified 126 patients reported in 16 papers over the last 32 years. One third had fibrolamellar HCC (FL-HCC), and two thirds had non-FL-HCC. Recurrence data were given in 55 patients of whom 27 had tumor recurrence. Seventy-five percent of the recurrences occurred within the first 2 years after OLT, although recurrences were reported up to 72 months after OLT for FL-HCC. The 5-year survival rate was greater in patients who underwent transplantation for FL-HCC than for non-FL-HCC (39.4% and 11.2%, respectively). There was insufficient information available to determine the influence of tumor size, distribution, stage, and vascular invasion on survival, although most patients in whom tumor characteristics were specified had advanced disease. This study indicates that FL-HCC carcinoma is a more favorable indication for OLT than non-FL-HCC in patients without underlying liver disease, although more detailed prognostic information is required to improve patient selection.

PC12 variants deficient in norepinephrine transporter mRNA have wild type activities of several other related transporters.

Wild type PC12 pheochromocytoma cells express a Na(+)-dependent norepinephrine transporter that operates in the uptake of catecholamines. In addition to the previously described Na(+)-dependent system A for the uptake of alpha-amino-isobutyric acid and system Gly for glycine, we have identified two other Na(+)-dependent transporter systems for amino acid uptake in these cells: 1) system beta for beta-alanine and taurine; and 2) a system for creatine. Uptake of alpha-amino-isobutyric acid, glycine, beta-alanine, and creatine is not affected in some PC12 variants that were previously shown to be deficient in catecholamine uptake and to have decreased levels of norepinephrine transporter mRNA. We have isolated two PC12 cDNA clones that are essentially identical in sequence to recently reported cDNAs for rat brain taurine and creatine transporters, respectively, and a third cDNA that appears to code for a novel transporter. mRNAs for these three transporters are present at wild type levels in those variants that express no or little norepinephrine transporter mRNA. These results support the notion that the expression of catecholamine reuptake transporters may be particularly susceptible to down-regulation.

Differential expression of transporters for norepinephrine and glutamate in wild type, variant, and WNT1-expressing PC12 cells.

Wild type PC12 pheochromocytoma cells express a Na(+)-dependent norepinephrine transporter that operates in the uptake of catecholamines, including dopamine. This transporter is not expressed in two spontaneously occurring flat cell variants of PC12 or in two other flat cell variants whose phenotype was induced by expression of the Wnt-1 oncogene. However, each of the flat cell variants, including those that express Wnt-1, exhibit a Na(+)-dependent, Cl(-)-independent glutamate/aspartate transporter activity that is not present in wild type PC12 cells. The flat cell variants took up glycine by a Na(+)-dependent process as well as did wild type cells. All of the flat cell variants have decreased levels of norepinephrine transporter mRNA but normal levels of glycine transporter mRNA. Glutamate/aspartate transporter mRNA was detected only in the variants that exhibited glutamate/aspartate transporter activity, and the nucleotide sequence of a partial glutamate/aspartate transporter cDNA from these cells demonstrated that it was the glial form of the transporter that was expressed. These variants were more sensitive than was wild type PC12 to alanosine, a toxic aspartate analog that enters cells by a transporter-mediated system such as the glutamate/aspartate transporter; however, these variants were as sensitive as wild type cells to another toxic aspartate analog, N-(phosphonacetyl)-L-aspartic acid, which is believed to enter cells by endocytosis. We suggest that the Wnt-1 gene product, or a homolog, may be involved in glial differentiation and that the mechanisms that alter the expression of the norepinephrine and glutamate/aspartate transporters in wild type and variant PC12 cells may also operate to regulate neurotransmitter transporter expression in vivo.

The tryptophan synthase bienzyme complex transfers indole between the alpha- and beta-sites via a 25-30 A long tunnel.

The bacterial tryptophan synthase bienzyme complexes (with subunit composition alpha 2 beta 2) catalyze the last two steps in the biosynthesis of L-tryptophan. For L-tryptophan synthesis, indole, the common metabolite, must be transferred by some mechanism from the alpha-catalytic site to the beta-catalytic site. The X-ray structure of the Salmonella typhimurium tryptophan synthase shows the catalytic sites of each alpha-beta subunit pair are connected by a 25-30 A long tunnel [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Since the S. typhimurium and Escherichia coli enzymes have nearly identical sequences, the E. coli enzyme must have a similar tunnel. Herein, rapid kinetic studies in combination with chemical probes that signal the bond formation step between indole (or nucleophilic indole analogues) and the alpha-aminoacrylate Schiff base intermediate, E(A-A), bound to the beta-site are used to investigate tunnel function in the E. coli enzyme. If the tunnel is the physical conduit for the transfer of indole from the alpha-site to the beta-site, then ligands that block the tunnel should also inhibit the rate at which indole and indole analogues from external solution react with E(A-A). We have found that when D,L-alpha-glycerol 3-phosphate (GP) is bound to the alpha-site, the rate of reaction of indole and nucleophilic indole analogues with E(A-A) is strongly inhibited. These compounds appear to gain access to the beta-site via the alpha-site and the tunnel, and this access is blocked by the binding of GP to the alpha-site. However, when small nucleophiles such as hydroxylamine, hydrazine, or N-methylhydroxylamine are substituted for indole, the rate of quinonoid formation is only slightly affected by the binding of GP. Furthermore, the reactions of L-serine and L-tryptophan with alpha 2 beta 2 show only small rate effects due to the binding of GP. From these experiments, we draw the following conclusions: (1) L-Serine and L-tryptophan gain access to the beta-site of alpha 2 beta 2 directly from solution. (2) The small effects of GP on the rates of the L-serine and L-tryptophan reactions are due to GP-mediated allosteric interactions between the alpha- and beta-sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric effects acting over a distance of 20-25 A in the Escherichia coli tryptophan synthase bienzyme complex increase ligand affinity and cause redistribution of covalent intermediates.

The reactions of L-histidine (L-His) and L-tryptophan (L-Trp) with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase are introduced as probes both of beta-subunit catalysis and of ligand-mediated alpha-beta allosteric interactions. Binding of DL-alpha-glycerol 3-phosphate (GP), an analogue of 3-indole-D-glycerol 3'-phosphate (IGP), to the alpha-catalytic site increases the affinity of alpha 2 beta 2 for L-His 4.5-fold and the affinity for L-Trp 17-fold and brings about a redistribution of beta-bound intermediates that favors the quinonoids derived from each amino acid. Inorganic phosphate (Pi) (presumably via binding to the alpha-catalytic site) influences the distribution of L-His intermediates as does GP. Previous binding studies [Heyn, M. P., & Weischet, W. O. (1975) Biochemistry 14, 2962-2968] indicate that when the phosphoryl group subsite of the alpha-catalytic site is occupied by GP or Pi, a high-affinity indole subsite is induced at the alpha-catalytic site. Interaction of benzimidazole (BZ), an analogue of indole, with this site also shifts the distribution of beta-bound L-His intermediates in favor of the L-His quinonoid. In the absence of Pi or GP, BZ interacts primarily at the beta-catalytic site and competes with L-His for the beta-subunit indole subsite. Since L-His and GP (or Pi) are substrate analogues and L-Trp is the physiological product, these allosteric effects likely take place with the natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

L-serine analogues form Schiff base and quinonoidal intermediates with Escherichia coli tryptophan synthase.

Substrate analogues of L-serine have been found that react with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase. Upon reaction with alpha 2 beta 2, the analogues glycine, L-histidine, L-alanine, and D-histidine form chemical intermediates derived from reaction with enzyme-bound pyridoxal 5'-phosphate with characteristic UV-visible spectral bands. The spectra of the products of the glycine, L-histidine, and L-alanine reactions with alpha 2 beta 2 contain contributions from the external aldimine, the quinonoid species, and other intermediates along the catalytic pathway. Just as previously reported for the reaction of L-serine with beta 2 [Goldberg, M. E., York, S., & Stryer, L. (1968) Biochemistry 7, 3662-3667], the reactions of glycine, L-histidine, and L-alanine with the beta 2 form of tryptophan synthase yield spectra with no contributions from catalytic intermediates beyond the external aldimine. The kinetics of intermediate formation and comparisons of the time courses for the exchange of alpha-1H for solvent 2H catalyzed by alpha 2 beta 2 or beta 2 were found to be consistent with these assignments. Intermediates further along the tryptophan synthase catalytic pathway are stabilized to a greater degree in the alpha 2 beta 2 complex than in the beta 2 species alone. This observation strongly suggests that the association of alpha and beta subunits to form the native alpha 2 beta 2 species lowers the activation energies for the interconversion of the external aldimine with chemical species further along the catalytic path.(ABSTRACT TRUNCATED AT 250 WORDS)