Arginine deprivation and tumour cell death: Arginase and its inhibition

Arginase treatment of cell cultures reduced arginine in the medium to ~ micromolar levels within 5–30 min, and proved as effective as arginine-free medium (AFM) prepared by formulation. The enzyme was heat stable and as active at pH 7.2 as at pH 9.9. It persisted in culture for at least 3 days with only a small diminution in its speed of action, and still actively destroyed arginine after 6 days, since arginine supplementation failed to rescue viable cells.Addition of L-norvaline, an inhibitor of arginase, rescued cells from arginase-induced deprivation. Its efficacy at low concentrations was short-lived (probably < 1 day), while at higher concentrations it did not appear to inhibit completely the enzyme. However, L-norvaline at these same levels also slowed the growth of positive non-enzyme treated controls receiving the normal arginine levels. Thus the difference in this growth indicated that arginase was more inhibitory than cursory examination of initial kinetic data suggested. It also agreed with the inhibition of arginase in the ornithine assay used to measure biochemically enzyme activity. We conclude that norvaline partially but not completely antagonises arginase activity, which allows cell rescue in a dose-dependent manner between 0.4 and 4 mM, but cannot be used above about 2 mM without exhibiting a general non-specific interference of cell growth of its own, although no evidence of cell toxicity was observed in either AFM or arginine-containing medium. L-ornithine, the product of arginase that inhibits the enzyme by a feedback mechanism, had no inhibitory effect on arginase over a similar concentration range.     

Plasma 3-nitrotyrosine and outcome in neonates with severe bronchopulmonary dysplasia after inhaled nitric oxide

Plasma protein levels of 3-nitrotyrosine and 3-chlorotyrosine were measured by LC-MS/MS at 0 and 72 h after the initiation of inhaled nitric oxide (INO) at 20 ppm in 22 prematurely born infants with clinically documented bronchopulmonary dysplasia. Infants were classified at the time of hospital discharge as either "off mechanical ventilation," "on mechanical ventilation," or "expired/organ failure." These outcomes were tested for association with changes in plasma levels of 3-nitrotyrosine and 3-chlorotyrosine and selected clinical risk factors. Infants whose 3-nitrotyrosine levels decreased over the 72 h period were more likely to wean off of mechanical ventilation (p =.03). There was no significant association between changes in 3-chlorotyrosne levels and outcome. After controlling for other variables, an odds ratio of 8.3 (95% CI: 1.3-54.4) for improved outcomes was observed if the 3-nitrotyrosine levels decreased. These data suggest that nitrative and oxidative stress may be related to the severity of lung disease and, consequentially, the overall outcome in this select group of infants with severe bronchopulmonary dysplasia.     

The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development

The anthocyanin and proanthocyanidin (PA) biosynthetic pathways share common intermediates until leucocyanidin, which may be used by leucoanthocyanidin dioxygenase (LDOX) to produce anthocyanin, or the enzyme leucoanthocyanidin reductase (LAR) to produce catechin, a precursor of PA. The Arabidopsis mutant tannin deficient seed 4 (tds4-1) has a reduced PA level and altered pattern PA accumulation. We identified the TDS4 gene as LDOX by complementation of the tds4-1 mutation either with a cosmid encoding LDOX or a 35S:LDOX construct. Independent Arabidopsis lines with a T-DNA insertion in the LDOX gene had a similar phenotype, and one was allelic to tds4-1. The seed phenotype of ban tds4 double mutants showed that LDOX precedes BANYULS (BAN) in the PA pathway, confirming recent biochemical characterisation of BAN as an anthocyanidin reductase. Double mutant analysis was also used to order the other TDS genes. Analysis of the PA intermediates in tds4-1 revealed three dimethylaminocinnamaldehyde (DMACA) reacting compounds that accumulated in extracts from developing seeds. Analysis of Arabidopsis PA and its precursors indicates that Arabidopsis, unlike many other plants, exclusively uses the epicatechin and not the catechin pathway to PA. Transmission electron microscopy (TEM) showed that the pattern observed when seeds of tds4 were stained with DMACA was a result of the accumulation of PA intermediates in the cytoplasm of endothelial cells. Fluorescent marker dyes were used to show that tds4 endothelial cells had multiple small vacuoles, instead of a large central vacuole as observed in the wild types (WT). These results show that in addition to its established role in the formation of anthocyanin, LDOX is also part of the PA biosynthesis pathway.     

Synthesis and SAR evaluation of oxadiazolopyrazines as selective Haemophilus influenzae antibacterial agents

The parallel synthesis and antibacterial activity of 5-hydroxy[1,2,5] oxadiazolo[3,4-b]pyrazines is reported. The compounds were synthesized by condensing diaminofurazan with alpha-keto acids to give a variety of aryl-substituted analogues. Halogenated phenyl groups at C-6 give rise to the greatest Haemophilus influenzae antibacterial activity.     

Synthesis of the first examples of A-C8/C-C2 amide-Linked pyrrolo[2,1-c][1,4]benzodiazepine dimers

We report the synthesis of novel A-C8/C-C2 amide-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers (4a and 4b) via convergent routes. These compounds lack the potent DNA interstrand cross-linking ability and resultant pronounced cytotoxicity of the known A-C8/A-C8' linked dimers (e.g., 2a-b).     

Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1

Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2.     

Mean residence time of molecules diffusing in a cell bounded by a semi-permeable membrane: Monte Carlo simulations and an expression relating membrane transition probability to permeability

 The rapid exchange of water across erythrocyte membranes is readily measured using an NMR method that entails doping a suspension of cells with a moderately high concentration of Mn²⁺ and measuring the rate of transverse relaxation of the nuclear magnetisation. Analysis of the data yields an estimate of the rate constant for membrane transport, from which the membrane permeability can be determined. It is assumed in the analysis that the efflux rate of the water is solely a function of the rate of membrane permeation and that the time it takes for intracellular water molecules to diffuse to the membrane is relatively insignificant. The limits of this assumption were explored by using random-walk simulations of diffusion in cells modelled as parallel planes, spheres, and biconcave discs. The rate of membrane transport was specified in terms of a transition probability but it was not initially clear what the relationship should be between this parameter and the diffusional membrane permeability P _d. This relationship was derived and used to show that the mean residence time for a water molecule is determined by P _d when the diffusion coefficient is above a certain threshold value; it is determined by the distance to the membrane below that value. Received: 7 January 2000 / Revised version: 4 April 2000 / Accepted: 4 April 2000