No abstract

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91 phox ) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91 phox /NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca 2؉ . However, the significance of Ca 2؉ binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca 2؉ ionophore that induces Ca 2؉ influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca 2؉ binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca 2؉ binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.Photosynthetic plants have developed various mechanisms to cope with oxidative stress, such as the production of antioxidants and enzymes that scavenge reactive oxygen species (ROS).3 Plants are also equipped with mechanisms for producing ROS in response to internal and external stimuli. ROS production is induced during many physiological processes, including stress responses, cell growth, hormonal responses, stomatal closure, and disease resistance (see Refs. 1-4 and references therein).ROS production is induced in plants in response to recognition of pathogenic signals, such as pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) or elicitors. Elicitor-induced ROS production is preceded by a rapid increase in the cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) (5-7) and is inhibited both by Ca 2ϩ chelators such as EGTA and BAPTA, and by Ca 2ϩ channel blockers such as La 3ϩ (6,8). The overexpression of rice two-pore channel 1 (OsTPC1), which is a putative voltage-gated Ca 2ϩ channel, enhanced elicitor-induced ROS production (9). Elicitor-induced ROS production is also inhibited by diphenylene iodonium (DPI), which is known to inhibit NADPH oxidase activity (6, 10). NADPH oxidase activity in the microsomal membrane fraction from tomato and tobacco was activated by adding Ca 2ϩ in vitro (11), suggesting that elicitor-induced ROS production by plant NADPH oxidase might be dependent on Ca 2ϩ . In mammalian phagocytes, ROS production is mediated by the NADPH-dependent phagocytic oxidase (phox) complex, which consists of the catalytic subunit gp91 phox /NADPH oxidase (NOX) 2, together with the regulatory subunits p22 phox , p40 phox , p47 phox , p67 phox , and the small GTP-binding protein Rac (12). In...

No abstract

Porphyromonas gingivalis fimbriae are critical for the promotion of bacterial infection. The fimA gene encoding fimbrillin, a subunit of fimbriae, has been classified into five genotypes (types I to V) based on their nucleotide sequences. Using a fimA type-specific PCR assay, our previous study demonstrated a close relationship between P. gingivalis possessing type II and type IV fimA genes and adult periodontitis. In that study, some clinical specimens were found to be positive for both types I- and II- fimA specific primers, likely due to the coexistence of two clonal types or a single clone of an unknown genotype in the samples. In the present study, we cloned a new variant of the fimA gene, designated as type Ib fimA, from P. gingivalis HG1691. The nucleotide sequence of the cloned fimA gene showed a 97.1% homology with that of type I fimA, indicating it as a clonal variant of type I fimA. Organisms with type Ib fimA were detected in 13.5% of periodontitis patients and in 2.9% of periodontal healthy adults. Statistical analysis revealed a strong relationship between periodontitis and specific fimA types such as type Ib [odds ratio (OR) 6.51], type II (OR 77.8), and type IV (OR 7.54). Moreover, type Ib fimA-organisms were also found to be related to periodontitis in Down's syndrome (OR 1.91) and mentally disabled populations (OR 4.00). These findings suggest that P. gingivalis with type Ib fimA is closely associated with the progression of periodontitis, similar to organisms with type II and IV fimA.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

No abstract

The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA-RNA hybrid Escherichia coil RNase H complex. Sitedirected mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out. A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively. In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme. The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model. Moreover, the specific cleavage of the RNA strand in DNA-RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes. A mechanism of enzymatic action is proposed.

To investigate a possible peroral route of infective endocarditis (IE), the occurrence of staphylococci in the oral cavity was examined using saliva and supragingival plaque specimens from 56 systemically and periodontally healthy adults aged 22-43 years old (27.1±5.3). Nine Staphylococcus species and 334 isolates were identified. In saliva, the total occurrence rate was 83.9 % and the total number of bacteria was 10 2 -10 4 c.f.u. ml "1 . Staphylococcus aureus was the most frequent species (46.4 %), followed by Staphylococcus epidermidis (41.1 %) and others (Staphylococcus hominis, Staphylococcus warneri, Staphylococcus intermedius, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus lugdunensis and Staphylococcus gallinarum, isolation frequencies ranging in order from 12.5 to 1.8 %). A similar isolation tendency was observed in supragingival plaque, with a total occurrence rate of 73.2 % and amounts of bacteria ranging from 10 2 to 10 5 c.f.u. g "1. Four common Staphylococcus species (S. aureus, S. epidermidis, S. lugdunensis and S. hominis) were isolated from nasal swab samples taken from the oral staphylococci-positive subjects. Genotyping of all 18 combinations of oral-and nasal-derived isolates by PFGE indicated that identical clones or close relatives were commonly distributed in these two cavities. Since the provision of micro-organisms from the nasal cavity was shown and occurrence rates in the oral cavity were adequate, these results suggest a possible peroral route of staphylococcal IE, as in cases of viridans streptococcal IE.