The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel

Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli. Each subunit contains 16 beta-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop. We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology. Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant. These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations. The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage- dependent gating processes. We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating.      

Studies on granuloma formation in Syrian hamsters experimentally infected with Schistosoma mansoni

Juvenile rats are more susceptible to the acute toxicity of the phosphorothionate insecticides parathion and chlorpyrifos than are adult rats. Developmental changes in brain acetylcholinesterase and hepatic aliesterase (carboxylesterase), cytochrome P450, and the P450-mediated metabolism of these two phosphorothionate insecticides were investigated in male Sprague-Dawley rats. Specific activities of acetylcholinesterase in cerebral cortex, but not medulla oblongata, and of liver aliesterases increased with age, indicating the presence of both more target esterases and more protective esterases, respectively, in the adult compared to the juvenile animal. Sensitivity of the brain acetylcholinesterase to inhibition by paraoxon and chlorpyrifosoxon, as measured by IC50 values, did not change significantly with age, whereas the hepatic aliesterase sensitivity to inhibition decreased with age. Progressive increases in activities of P450-mediated activation (desulfuration) (6- to 14-fold) and detoxication (dearylation) (2- to 4-fold), as well as concentrations of P450 (7-fold) and protein (2-fold), were observed between neonate and adult hepatic microsomes. Microsomal pentoxyresorufin O-dealkylase activity followed a developmental pattern similar to desulfuration and dearylation, displaying a 16-fold increase between neonates and adults. However, microsomal ethoxyresorufin O-deethylase activity increased until 21 days of age, displaying a 16-fold increase, then decreased in adulthood to a level 10-fold higher than neonates. These results indicate that target enzyme sensitivity is not responsible for age-related toxicity differences, nor is the potential for hepatic bioactivation, whereas lower levels of hepatic aliesterase-mediated protection and P450-mediated dearylation probably contribute significantly to the greater sensitivity of juveniles to phosphorothionate toxicity.     

MODELING THE SOLIDIFICATION OF FOUNDRY CASTINGS

A comprehensive technique is described for performing the simulation of the solidification of foundry castings. A finite element method (FEM) mesh is produced for the pattern of the part, using geometric modeling techniques. Results are presented to demonstrate selection rules for the mesh size. The size of the heat transfer problem is reduced by replacing the FEM mesh in the mold material by appropriate boundary conditions on the surface of the casting. The boundary conditions are found automatically by a program which relates local surface curvature on the part to a pre-calculated library of solutions. Methods for ensuring that all of the latent heat of fusion is included are presented, and comparisons are made between different solution methods.     

Marginal leakage of amalgam restorations pretreated with various liners

PURPOSE: To evaluate and compare the microleakage of amalgam restorations lined with four different liners. MATERIALS AND METHODS: Cylindrical occlusal cavities were prepared in extracted human molars and the teeth were then divided into four groups and lined with one of four liners: Amalcoden, AM (glass-metal ionomer), FujiDuet, FD (glass ionomer), Amalgambond, AB (adhesive resin), or copal varnish, CV. Each cavity was then restored with a high-copper amalgam alloy and aged for 7 days in 37 degrees C distilled water. The specimens were then thermocycled for 300 cycles followed by immersion in 0.5% basic fuchsin dye solution for 24 hours. The teeth were then embedded in resin, sectioned, and dye penetration at the tooth/amalgam interface evaluated microscopically at x 100 and scored from 0 to 4 (0 = no leakage and 4 = greatest leakage). RESULTS: The median values were: AM = 1.0; FD = 0.5; AB = 1.1; and CV = 4.0. Statistical analysis using Kruskal-Wallis and Mann-Whitney U indicated no significant differences between AM and AB or FD and AB. CV was however, significantly worse at P < 0.01.     

A safe high-yield technique for cutting needle biopsy of the lung in patients with diffuse lung disease

The approach to the diagnosis and management of patients with diffuse infiltrative lung disease (DILD) is controversial. The results of transbronchial biopsy are often unsatisfactory. The role of open lung biopsy is highly variable. Percutaneous cutting needle biopsy (CNB) is not recommended because of its reported high morbidity/mortality relative to its low diagnostic yield. We report a technique for CNB with a high diagnostic yield and a low morbidity and no mortality in 228 patients with DILD over the past 23 years. METHODS: The salient features of the technique for CNB are as follows: the anesthetic needle does not enter the pleural space; a Franklin Silverman needle is inserted into the intercostal space posteriorly at outer one-third of chest wall; the biopsy is performed with the breath held at normal end expiration; the plane of pleural space is broken with sudden insertion of needle 8 to 15 cm into lung; and the pathway of the needle is maintained parallel to the lateral chest wall. RESULTS: A diagnosis was established in 129 of 145 biopsies (89%) performed by a trained operator (A.H.N.). There were 36 pneumothoraces (25%), four minimal hemoptyses (3%), and two chest tube placements (1%). There were no deaths (0%). CONCLUSION: With meticulous attention to technique and careful selection of patients, the procedure offers a relatively safe alternative to open lung or thoracoscopic lung biopsy in patients with DILD.     

Anti-plasmodial and anti-trypanosomal activity of synthetic naphtho[2,3-b]thiopen-4,9-quinones

Naphtho[2,3-b]thiophen-4,9-quinone and five derivatives were prepared using the Friedel-Crafts reaction and tandem-lithiation of aromatic diethylamides. These quinones were evaluated for their trypanocidal and anti-plasmodial activities by their effects on: (1) growth of epimastigote forms of Trypanosoma cruzi in vitro, (2) lysis of trypomastigote forms of T. cruzi in murine blood, (3) growth of Plasmodium falciparum in vitro, and (4) inhibition of the recombinant enzyme trypanothione reducatase. The parent compound, naphtho[2,3-b]thiophen-4,9-quinone (3a), was among the most active quinone tested in vitro against P. falciparum at 0.2 microM. However, it was inactive against P. berghei-infected mice treated with 2.3 mmol/kg daily for 5 days. Most of the quinones prepared were active against T. cruzi epimastigotes in culture but exhibited weak activity at 4 degrees C against trypomastigotes in murine blood as well against the enzyme trypanothione reducatase. Further structural modifications will be necessary to improve the in vivo activity of the naphthothiophenquinones.