La práctica otorrinolaringológica

O editor Antonio Virgili traballa ca. 1850-192

Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae.

The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase similar to those found in other bacteria. In order to obtain sufficient protein for biophysical studies, we aimed to overproduce the recombinant dihaem protein in Escherichia coli. Initial expression experiments indicated that the NapB signal peptide was not cleaved by the leader peptidase of the host organism. Apocytochrome was formed under aerobic, semi-aerobic and anaerobic growth conditions in either Luria--Bertani or minimal salts medium. The highest amounts of apo-NapB were produced in the latter medium, and the bulk was inserted into the cytoplasmic membrane. The two haem groups were covalently attached to the pre-apocytochrome only under anaerobic growth conditions, and with 2.5 mM nitrite or at least 10 mM nitrate supplemented to the minimal salts growth medium. In order to obtain holocytochrome, the gene sequence encoding mature NapB was cloned in-frame with the E. coli ompA (outer membrane protein A) signal sequence. Under anaerobic conditions, NapB was secreted into the periplasmic space, with the OmpA signal peptide being correctly processed and with both haem c groups attached covalently. Unless expressed in the DegP-protease-deficient strain HM125, some of the recombinant NapB polypeptides were N-terminally truncated as a result of proteolytic activity. Under aerobic growth conditions, co-expression with the E. coli ccm (cytochrome c maturation) genes resulted in a higher yield of holocytochrome c. The pure recombinant NapB protein showed absorption maxima at 419, 522 and 550 nm in the reduced form. The midpoint reduction potentials of the two haem groups were determined to be -25 mV and -175 mV. These results support our hypothesis that the Nap system fulfils a nitrate-scavenging role in H. influenzae

Dehydroascorbate Influences the Plant Cell Cycle through a Glutathione-Independent Reduction Mechanism

Glutathione is generally accepted as the principal electron donor for dehydroascorbate (DHA) reduction. Moreover, both glutathione and DHA affect cell cycle progression in plant cells. But other mechanisms for DHA reduction have been proposed. To investigate the connection between DHA and glutathione, we have evaluated cellular ascorbate and glutathione concentrations and their redox status after addition of dehydroascorbate to medium of tobacco (Nicotiana tabacum) L. cv Bright Yellow-2 (BY-2) cells. Addition of 1 mm DHA did not change the endogenous glutathione concentration. Total glutathione depletion of BY-2 cells was achieved after 24-h incubation with 1 mm of the glutathione biosynthesis inhibitor l-buthionine sulfoximine. Even in these cells devoid of glutathione, complete uptake and internal reduction of 1 mm DHA was observed within 6 h, although the initial reduction rate was slower. Addition of DHA to a synchronized BY-2 culture, or depleting its glutathione content, had a synergistic effect on cell cycle progression. Moreover, increased intracellular glutathione concentrations did not prevent exogenous DHA from inducing a cell cycle shift. It is therefore concluded that, together with a glutathione-driven DHA reduction, a glutathione-independent pathway for DHA reduction exists in vivo, and that both compounds act independently in growth control

Exposure of Arabidopsis thaliana to excess Zn reveals a Zn-specific oxidative stress signature.

Zinc (Zn) is an essential micronutrient for plants, but accumulation of excess Zn causes oxidative stress, even though the element is not redox-active. An oxidative stress signature, consisting of multiple oxidative stress related parameters, is indicative of disturbance of redox homeostasis and signaling, but has not been determined after exposure to excess Zn. To reveal general and Zn-specific effects, changes in oxidative stress related gene expression, enzyme activities and metabolites were determined after 24h exposure of Arabidopsis thaliana plants to a concentration range of 0, 100, 250 or 500μM excess ZnS