Site-directed mutagenesis of Lys36 in human thioredoxin: the highly conserved residue affects reduction rates and growth stimulation but is not essential for the redox protein's biochemical or biological properties.

Previous studies have demonstrated that a recombinant form of the human redox protein thioredoxin can stimulate the growth rate of Swiss 3T3 murine fibroblasts and that this ability to promote cellular proliferation was dependent upon a redox-active form. A site-directed mutagenesis study of the highly conserved Lys36 adjacent to the two active site cysteines of thioredoxin was performed to determine whether the basic residue was essential for the biochemical and mitogenic properties of human thioredoxin. Two mutants were generated in which the lysine residue was replaced with either glutamic acid (K36E) or leucine (K36L).

Micrometer- and nanometer-sized polymeric light-emitting diodes.

A method for the fabrication of micrometer-and submicrometer-sized polymeric light-emitting diodes is presented. Such diodes have a variety of applications. Light sources of dimensions around 100 nanometers are required for subwavelength, near-field optical microscopy.

Advances with phospholipid signalling as a target for anticancer drug development.

The phosphatidylinositol-3-kinases (PtdIns-3-kinase) are a family of enzymes involved in the control of cell replication. One member of the family, the mammalian p110/p85 PtdIns-3-kinase, is a potential target for anticancer drug development because of its role as a component of growth factor and oncogene activated signalling pathways. There are a number of inhibitors of this PtdIns-3-kinase, the most potent being wortmannin (IC50 4 nM).

A multiwell assay for inhibitors of phosphatidylinositol-3-kinase and the identification of natural product inhibitors.

A convenient and reliable multisample assay for the screening of inhibitors of the growth factor signalling enzyme phosphatidylinositol-3-kinase (PtdIns-3-K) has been developed. Four natural product inhibitors of Ptdlns-3-K have been identified with IC50 values for hypericin 0.18 microM, emodin 3.

Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase.

Phosphatidylinositol-3-kinase is an important enzyme for intracellular signaling. The microbial product wortmannin and some of its analogues have been shown to be potent inhibitors of phosphatidylinositol-3-kinase. The 50% inhibitory concentration for inhibition by wortmannin is 2 to 4 nM.

Site-directed mutagenesis of active site cysteines in human thioredoxin produces competitive inhibitors of human thioredoxin reductase and elimination of mitogenic properties of thioredoxin.

Thioredoxin and thioredoxin reductase comprise a redox system ubiquitous in all organisms. To better understand the thiol chemistry of the mammalian thioredoxin-thioredoxin reductase redox system, mutants of human thioredoxin were produced by site-directed mutagenesis in which the two active site cysteines were replaced by serine residues, individually (C32S and C35S) and in combination (C32S/C35S). C35S and C32S/C35S were found to be competitive inhibitors of the reduction of human thioredoxin by human thioredoxin reductase with Ki values of 1.

Biochemical, structural, and biological properties of human thioredoxin active site peptides.

The human redox protein thioredoxin is an autocrine growth factor for some cancer cells. Redox activity is essential for this function but other required structural features of thioredoxin are not known. Two 8-mer peptides (I and II) and one 14-mer peptide (III) were designed based on the amino acid sequence of the redox active site of thioredoxin.

The thioredoxin/thioredoxin reductase redox system and control of cell growth.

Thioredoxin is a redox protein that is important for a variety of intracellular functions, possibly including regulation of transcription factor activity. We have shown that human thioredoxin has the same predicted amino acid sequence as adult T-cell-derived leukemic cell growth factor. Recombinant human thioredoxin stimulates the proliferation of Swiss murine 3T3 fibroblasts with an EC50 of 100 nM and the proliferation of a number of human cancer cells.

Inhibition of the signalling enzyme phosphatidylinositol-3-kinase by antitumor ether lipid analogues.

Phosphatidylinositol-3-kinase (PtdIns-3-kinase) is an enzyme found associated with many growth factor receptor protein tyrosine kinases and oncogene protein tyrosine kinases. PtdIns-3-kinase appears to be important for mitogenesis and the malignant transformation of cells. The antitumor ether lipid analogue, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), was found to be an inhibitor of Swiss mouse 3T3 fibroblast and bovine brain PtdIns-3-kinases.

Inhibition of protein tyrosine phosphatase by the antitumor agent gallium nitrate.

Protein tyrosine phosphatases (PTPases) play an important role in regulating cell growth and transformation. We report that the antitumor agent gallium nitrate is a potent inhibitor (concentration producing 50% inhibition, 2-6 microM) of detergent-solubilized cellular membrane PTPase from Jurkat human T-cell leukemia cells and HT-29 human colon cancer cells. This is the first report of a selective, small molecule drug inhibitor of PTPase.

Improved results in zone 2 flexor tendon injuries with a modified technique of immediate controlled mobilization.

The results following primary and delayed primary repair in zone 2 flexor tendon injuries were evaluated in 85 fingers of 79 patients using immediate controlled mobilization post-operatively. In 31 patients a conventional Kleinert technique was used. In the remaining 48 patients a modified technique was used with rubber band traction to all fingers instead of only to the injured one.

Biodegradation of 4-nonylphenol in seawater and sediment.

Biodegradation of 14C-labelled nonylphenol at the concentration 11 microg litre (-1) in seawater has been estimated by collection and quantification of the formed labelled carbon dioxide. Initially degradation was very slow but when the microorganisms had become adapted, after four weeks at 11 degrees C, the degradation rate increased rapidly and after 58 days about 50% of 14C from NP was found in the CO2 fraction. In the presence of sediment the initial degradation rate was high and did not increase after longer incubation.

Ultrasonic characterization of mycelial morphology as a fractal structure.

Preliminary measurements of scattering of ultrasound from filamentous microorganisms in aqueous-medium suspensions are reported. The data are used to characterize the structuring (morphology) between the organisms quantitatively by modelling the morphology in terms of fractal structures and obtaining the fractal dimension as the characteristic parameter. The fractal dimension is calculated from the slope of a log-log plot of scattered intensity versus the scattering vector magnitude.

Nonperturbative diffraction tomography via Gauss-Newton iteration applied to the scattering integral equation.

A nonperturbational inverse scattering solution for the scattering integral equation (SIE) is presented. The numerical discretization of the SIE is performed by the moment method (MM) using sinc basis functions. Previous algorithms using the alternating variable (AV) nonlinear iteration with algebraic reconstruction technique (ART) solution of the linearizations are shown to diverge for high contrast/large size acoustic scatterers.

Hydroperoxide-induced broncho- and vasoconstriction in the isolated rat lung.

The effects of different hydroperoxides on lung mechanics and perfusate flow rate and their mechanisms of action were studied in isolated perfused rat lungs. The administration of hydrogen peroxide, t-butyl hydroperoxide, cumene hydroperoxide, linoleic acid hydroperoxide, and linoleic acid ethylester hydroperoxide (0.1-2 mM) to the perfusate caused a marked decrease in lung compliance, conductance, and perfusate flow rate, with constriction strength of t-butyl hydroperoxide greater than hydrogen peroxide greater than cumene hydroperoxide greater than linoleic acid ethylester hydroperoxide greater than linoleic acid hydroperoxide.

Enhanced mobilization of intracellular Ca2+ induced by halothane in hepatocytes isolated from swine susceptible to malignant hyperthermia.

Halothane, in a dose-dependent manner, induced the release of intracellular Ca2+ in hepatocytes prepared from swine. The magnitude of the release induced by halothane was greater for hepatocytes prepared from animals susceptible to malignant hyperthermia (MH) than for those from normal swine. Two different methods were used to ascertain the release of Ca2+ induced by halothane: 1) the release of 45Ca2+ from nonmitochondrial stores of saponin-permeabilized hepatocytes was measured; and 2) changes in luminescence from intact hepatocytes loaded with the Ca2(+)-sensitive photoprotein aequorin were recorded.

Mechanisms of hydroperoxide-induced broncho- and vasoconstriction in isolated and perfused rat lung.

The mechanisms of hydroperoxide-induced broncho- and vasoconstriction were investigated in the perfused and ventilated rat lung. Hydrogen peroxide (500 microM), tertiary butylhydroperoxide (500 microM) and arachidonic acid (100 microM) induced similar profiles of broncho- and vasoconstriction which could be prevented by the inhibitor of cyclooxygenase, diclofenac (100 microM) but not by nordihydroguaiaretic acid (5 and 25 microM), an inhibitor of lipoxygenase. The hydroperoxides also caused a time-dependent increase in the levels of thromboxane and prostacycline, products of cyclooxygenase.

Platelet-derived growth factor blocks the increase in intracellular free Ca2+ caused by calcium ionophores and a volatile anesthetic agent in Swiss 3T3 fibroblasts without altering toxicity.

Platelet-derived growth factor (PDGF) produced an almost complete block of the increase in intracellular free Ca2+ concentration ([Ca2+]i) in Swiss 3T3 fibroblasts caused by the Ca2(+)-selective ionophores 4-bromo-A23187 and ionomycin, and by the volatile anesthetic agent halothane. The effect of PDGF was similar to the decreased [Ca2+]i response to Ca2(+)-ionophores produced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. There was no effect of PDGF or PMA on the acute or delayed toxicity of the Ca2(+)-ionophores to Swiss 3T3 cells, suggesting that the increase in [Ca2+]i is not the direct cause of toxicity of these agents.

Effects of some autacoids on breathing and perfusion flow in the isolated perfused rat lung.

The effects of three autacoids [5-hydroxytryptamine (5-HT), bradykinin and acetylcholine (Ach)) on breathing and pulmonary circulation were studied in the isolated perfused and ventilated rat lung after intravascular injection or intrabronchial instillation. 5-HT, bradykinin and Ach all caused a dose related reduction in breathing, after injection into the pulmonary artery, with Ach being the most potent. With regard to effects on the pulmonary circulation, a dose-related reduction in perfusate flow was noted with bradykinin and 5-HT with bradykinin being about 10 times more potent than 5-HT.

Bioaccumulation of 4-nonylphenol in marine animals--a re-evaluation.

The bioaccumulation of 4-nonylphenol (NP) has been studied in shrimps (Crangon grangon L.), mussels (Mytilus edulis L.) and sticklebacks (Gasterosteus aculeatus L.

Lethal and sublethal toxicity of 4-nonylphenol to the common mussel (Mytilus edulis L.).

The toxicity of nonylphenol to the common mussel (Mytilus edulis L.) has been determined in both semistatic and continuous flow test systems. The LC50 values obtained were for 96 h, 30 mg litre(-1); 360 h, 0.

A concave annular array design, based on phasor summation--Part II: Beam profiles and resultant images by B-scan and synthetic focus methods.

A brief review of the synthetic focusing method is given. The theoretical limit of resolution that may be achieved with the synthetic focusing methods is demonstrated using experimental results on a thin thread obtained with an annular array whose design was given in Part I. The advantage of large aperture arrays is illustrated by an in vitro reflection image of a dog artery, made with this array, that has at least four times the spatial resolution of present clinical B-scanners operating in the same frequency range.

A concave annular array design, based on phasor summation--Part I: Design methodology.

A detailed method for the design of annular arrays based on phasor summation is developed. The net phase shift across each annulus obtained by phasor summation is a very important factor in the design of an annular array, and determines the strength of the signal received. The method presented in this paper allows the designer to evaluate the trade-offs in parameters such as the strength of the signal from each annulus, the efficiency of each annulus, and the depth of focus that would be achievable.

Determination of the intracellular protein thiol distribution of hepatocytes using monobromobimane derivatisation of intact cells and isolated subcellular fractions.

The derivatisation of intact rat hepatocytes with monobromobimane resulted in rapid labelling of accessible protein thiols in several subcellular fractions. The derivatisation procedure did not cause acute cytotoxicity, nor did it alter the buoyant densities of the fractions or their gross protein compositions. Quantitation of the fluorescence irreversibly associated with the fractions demonstrated considerable intracellular heterogeneity in this pool of thiols.

Hydroperoxide and cigarette smoke induced effects on lung mechanics and glutathione status in rat isolated perfused and ventilated lungs.

The effects of t-butylhydroperoxide (TBH) and cigarette smoke on lung mechanics (CDYN and RL) and glutathione status (GSH) were studied using an isolated perfused and ventilated rat lung preparation. TBH (200, 400, 1000 microM) infused via the pulmonary circulation caused a dose-related bronchoconstriction. The lung GSH-levels were also significantly reduced.