Mechanism of action and cloning of epoxide hydrolase from the cabbage looper, Trichoplusia ni

The majority of the JH III epoxide hydrolase activity in last stadium day 3 (gate 1) wandering Trichoplusia ni was membrane bound with approximately 9% of the activity found in the cytosol. Both the microsomal and cytosolic JH epoxide hydrolases were stable, retaining 30% of their original activity after incubation at 4 degrees C for 15 days. 18O-labeled water underwent enzyme catalyzed regioselective addition to the least substituted C10 position of JH III. In multiple turnover reactions with JH epoxide hydrolase in 97.9% 18O-labeled water, only 91.3% 18O incorporation was observed. This is consistent with an SN2 reaction likely involving a carboxylate in the active site of JH epoxide hydrolase. The DNA amplification cloning of a fragment of a putative T. ni epoxide hydrolase is reported. The deduced amino acid sequence shares 67% similarity to the rat microsomal epoxide hydrolase.     

Follow up and maintenance of implant supported prostheses: a comparison of 20 complete mandibular overdentures and 20 complete mandibular fixed cantilever prostheses

Two independently selected groups of 20 patients who were edentulous in te mandible, were followed up on a regular basis over a 5-year period following restoration with a lower implant stabilised prosthesis and conventional upper denture. Significantly more treatment was required by those who received complete mandibular overdentures than those provided with complete fixed mandibular prostheses. In addition to adjustment for the relief of denture trauma to the mucosa, more mechanical problems arose with overdenture implant prostheses.     

Analogs of 3-hydroxy-1H-1-benzazepine-2,5-dione: structure-activity relationship at N-methyl-D-aspartate receptor glycine sites

A series of aromatic and azepine ring-modified analogs of 3-hydroxy-1H-1-benzazepine-2,5-dione (HBAD) were synthesized and evaluated as antagonists at NMDA receptor glycine sites. Aromatic ring-modified HBADs were generally prepared via a Schmidt reaction with substituted 2-methoxynaphthalene-1,4-diones followed by demethylation. Electrophilic aromatic substitution of benzazepine 3-methyl ethers gave 7-substituted analogs. The preparation of multiply substituted 2-methoxynaphthalene-1,4-diones was effected via Diels-Alder methodology utilizing substituted butadienes with 2-methoxybenzoquinones followed by aromatization. Structural modifications, such as elimination of the aromatic ring, removal of the 3-hydroxyl group, and transfer of the hydroxyl group from C-3 to C-4, were also studied. An initial evaluation of NMDA antagonism was performed using a [3H]MK801 binding assay. HBADs demonstrating NMDA antagonist activity as indicated by inhibition of [3H]MK801 binding were further evaluated employing a [3H]-5,7-dichlorokynurenic acid (DCKA) glycine site binding assay. Selected HBADs were characterized for functional antagonism of NMDA and AMPA receptors using electrophysiological assays in Xenopus oocytes and cultured rat cortical neurons. Antagonist potency of HBADs showed good correlation between the different assay systems. HBADs substituted at the 8-position possessed the highest potency with the 8-methyl (5), 8-chloro (6), and 8-bromo (7) analogs being the most active. For HBAD 6, the IC50 in [3H]-DCKA binding assays was 0.013 microM and the Kb values for antagonism of NMDA receptors in oocytes (NR1a/2C) and cortical neurons were 0.026 and 0.048 microM, respectively. HBADs also antagonized AMPA-preferring non-NMDA receptors expressed in oocytes but at a lower potency than corresponding inhibition of NMDA receptors. HBADs demonstrating a high potency for NMDA glycine sites showed the highest steady-state selectivity index relative to AMPA receptors. Substitution at the 6-, 7-, and 9-positions generally reduced or eliminated glycine site affinity. Moving the hydroxyl group from C-3 to C-4 reduced receptor affinity, and potency was eliminated by the removal of the aromatic ring or the hydroxyl group. These data indicate that the HBAD series has specific structural requirements for high receptor affinity. With the exception of substitution at C-8, modified HBADs generally have a lower affinity at NMDA receptor glycine sites than the parent compound 3. Mouse maximum electroshock-induced seizure studies show that the three HBADs selected for testing have in vivo potency with the 6,8-dimethyl analog (52) being the most potent (ED50 = 3.9 mg/kg, iv).     

Bradyrhizobium japonicum porphobilinogen synthase uses two Mg(II) and monovalent cations

Bradyrhizobium japonicum porphobilinogen synthase (B. japonicum PBGS) has been purified and characterized from an overexpression system in an Escherichia coli host (Chauhan, S., and O'Brian, M. R. (1995) J. Biol. Chem. 270, 19823-19827). B. japonicum PBGS defines a new class of PBGS protein, type IV (classified by metal ion content), which utilizes a catalytic MgA present at a stoichiometry of 4/octamer, an allosteric MgC present at a stoichiometry of 8/octamer, and a monovalent metal ion, K+. However, the divalent MgB or ZnB present in some other PBGS is not present in B. japonicum PBGS. Under optimal conditions, the Kd for MgA is <0.2 microM, and the Kd for MgC is about 40 microM. The response of B. japonicum PBGS activity to monovalent and divalent cations is mutually dependent and varies dramatically with pH. B. japonicum PBGS is also found to undergo a dynamic equilibrium between active multimeric species and inactive monomers under assay conditions, a kinetic characteristic not reported for other PBGSs. B. japonicum PBGS is the first PBGS that has been rigorously demonstrated to lack a catalytic ZnA. However, consistent with prior predictions, B. japonicum PBGS can bind Zn(II) (presumably as ZnA) at a stoichiometry of 4/octamer with a Kd of 200 microM; but this high concentration is outside a physiologically significant range.     

Association between the Rfp-Y haplotype and the incidence of Marek's disease in chickens

Certain haplotypes at the major histocompatibility (B) complex (Mhc) of the chicken provide an easily demonstrated influence on tumor formation following infections with Marek's disease virus (MDV). Recognition that there is a second histocompatibility complex of genes in the chicken, Rfp-Y, comprised of Mhc class I and class II genes, some of which are at least transcribed, evokes the question of whether this gene complex might also influence the outcome of MDV infections. To test this hypothesis, pedigree-hatched chicks in families from the original Rfp-Y-defining stock in which three Rfp-Y and two B system haplotypes are segregating were challenged with the RB1B strain of MDV. Birds with the Y3/Y3 genotype were found to have 2.3 times the risk of developing a tumor compared with birds with other Rfp-Y genotypes combined (P <0.02). Additionally, birds carrying the BR9/B11 genotype had 2.3 times the risk of tumor formation, relative to birds with the B11/B11 genotype (P <0.02). We found no evidence for an interaction between genotypes within the B and Rfp-Y systems. These data provide evidence that Rfp-Y haplotypes, as well as B haplotypes, can significantly influence the outcome of infection with MDV.     

Mottle on computed radiographs of the chest in pediatric patients

PURPOSE: To investigate the relationship between radiation exposure and perceived mottle at bedside pediatric chest examinations performed with screen-film and computed radiographic techniques. MATERIALS AND METHODS: In a pediatric intensive care unit, chest radiographs were obtained with both computed radiography (60 radiographs) and a 600-speed screen-film system (14 radiographs). The relative radiation exposure was estimated by using the sensitivity value obtained in the processing of each computed radiograph. Five radiologists assessed the mottle present in the computed radiographs and screen-film images. RESULTS: For computed radiographs, the perceived level of mottle was inversely related to radiation exposure. For the same radiation exposure, the perceived mottle on computed radiographs was significantly higher than that on screen-film images (P < .001 for small cassettes; P < .01 for large cassettes). CONCLUSION: Pediatric computed radiography of the chest requires approximately twice the exposure of a 600-speed screen-film system to attain the same level of mottle.