Neuroglobin: enzymatic reduction and oxygen affinity

Neuroglobin (Ngb) is a hexacoordinate globin expressed in the nervous system of vertebrates, involved in neuroprotection. O₂ equilibrium measurements on mouse Ngb yielded significantly different P₅₀ values, ranging from ∼2 torr to ∼10 torr. By a kinetic approach minimizing the effects of protein autoxidation, we measured P₅₀ = 2.2 torr at 20 °C. As predicted from the structure, O₂ binds to the Y44D Ngb mutant more quickly (k = 2.2 s⁻¹ vs 0.15 s⁻¹) and with slightly higher affinity (P₅₀ = 1.3 torr) than wild-type. In addition, we introduced a novel reduction protocol for metNgb based on NADH:flavorubredoxin oxidoreductase (FlRd-red) from Escherichia coli, a candidate for the Ngb reducing activity recently identified in E. coli extracts. Interestingly, E. coli FlRd-red shares sequence similarity with the FAD-binding domain of the human apoptosis-inducing factor, a finding which may have unexpected significance with reference to the mechanism of neuroprotection by Ngb.     

AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death.

In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (Gln-Val-Val-Ala-Gly). Recombinant A. thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified. AtCYS1 inhibits the catalytic activity of papain (Kd = 4.0 x 10-2 micro m, at pH 7.0 and 25 degrees C), generally taken as a molecular model of cysteine proteases. The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex. AtCYS1 is constitutively expressed in roots and in developing siliques of A. thaliana. In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A. thaliana suspension cultured cells and in transgenic tobacco plants. The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Potentiality of the cox1 gene in the taxonomic resolution of soil fungi

We explored the potential of the cox1 gene in the species resolution of soil fungi and compared it with the nuclear internal transcribed spacer (ITS) and small subunit (SSU)-rDNA. Conserved primers allowing the amplification of the fungal cox1 gene were designed, and a total of 47 isolates of Zygomycota and Ascomycota were investigated. The analysis revealed a lack of introns in >90% of the isolates. Comparison of the species of each of the six studied genera showed high interspecific sequence polymorphisms. Indeed, the average of nucleotide variations (4.2–11%) according to the genus, due mainly to the nucleotide substitutions, led to the taxonomic resolution of all the species studied regarding both ITS and SSU-rDNA, in which <88% were discriminated. The phylogenetic analysis performed after alignment of the cox1 gene across distant fungal species was in accordance with the well-known taxonomic position of the species studied and no overlap was observed between intra- and interspecific variations. These results clearly demonstrated that the cox1 sequences could provide good molecular markers for the determination of the species composition of environmental samples and constitute an important advance to study soil fungal biodiversity.     

A transport model for the (Na+/K+)ATPase in liposomes including the (Na+/K+)-channel function

(Na+/K+)ATPase liposomes of various degrees of reconstitution are formed by varying the amount of phosphatidylcholine added to the soluble (Na+/K+)ATPase before vesicles are formed by cholate removal. In the presence of ATP, the reconstituted sodium pump effectuates (Na+/K+) antiport. In the absence of ATP, the reconstituted sodium pump forms a (Na+/K+) channel. The stable plateaus formed by (1) the active Na+ transport, (2) the active K+ transport, (3) the 'passive' Na+ flux, and (4) the 'passive' K+ flux are determined in the optimally and the partially reconstituted liposomes. The activities of all four vectorial functions vary in a tightly correlated fashion, suggesting that they are mediated by the same transport-active configuration of (Na+/K+)ATPase. A transport model which includes the active and the passive (Na+/K+) fluxes mediated by the sodium pump in liposomes is outlined.     

Emergence of Respiratory Streptococcus agalactiae Isolates in Cystic Fibrosis Patients

Streptococcus agalactiae is a well-known pathogen for neonates and immunocompromized adults. Beyond the neonatal period, S. agalactiae is rarely found in the respiratory tract. During 2002–2008 we noticed S. agalactiae in respiratory secretions of 30/185 (16%) of cystic fibrosis (CF) patients. The median age of these patients was 3–6 years older than the median age CF patients not harboring S. agalactiae. To analyze, if the S. agalactiae isolates from CF patients were clonal, further characterization of the strains was achieved by capsular serotyping, surface protein determination and multilocus sequence typing (MLST). We found a variety of sequence types (ST) among the isolates, which did not substantially differ from the MLST patterns of colonizing strains from Germany. However serotype III, which is often seen in colonizing strains and invasive infections was rare among CF patients. The emergence of S. agalactiae in the respiratory tract of CF patients may represent the adaptation to a novel host environment, supported by the altered surfactant composition in older CF patients.