Proteomics and functional analyses of pepper abscisic acid-responsive 1 (ABR1), which is involved in cell death and defense signaling

Abscisic acid (ABA) is a key regulator of plant growth and development, as well as plant defense responses. A high-throughput in planta proteome screen identified the pepper (Capsicum annuum) GRAM (for glucosyltransferases, Rab-like GTPase activators, and myotubularins) domain-containing ABA-RESPONSIVE1 (ABR1), which is highly induced by infection with avirulent Xanthomonas campestris pv vesicatoria and also by treatment with ABA. The GRAM domain is essential for the cell death response and for the nuclear localization of ABR1. ABR1 is required for priming cell death and reactive oxygen species production, as well as ABA-salicylic acid (SA) antagonism. Silencing of ABR1 significantly compromised the hypersensitive response but enhanced bacterial pathogen growth and ABA levels in pepper. High levels of ABA in ABR1-silenced plants antagonized the SA levels induced by pathogen infection. Heterologous transgenic expression of ABR1 in Arabidopsis thaliana conferred enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis infection. The susceptibility of the Arabidopsis ABR1 putative ortholog mutant, abr1, to these pathogens also supports the involvement of ABR1 in disease resistance. Together, these results reveal ABR1 as a novel negative regulator of ABA signaling and suggest that the nuclear ABR1 pool is essential for the cell death induction associated with ABA-SA antagonism.     

Bacterial model system for screening and determining optimal concentration of anti-caries natural extracts

In general, an antimicrobial test for screening anti-caries natural extracts was performed by measuring the minimum bactericidal concentration (MBC) against the type strains of mutans streptococci. However, it is unclear if the antimicrobial efficiency of natural extracts on the type strains of mutans streptococci is the same on the clinical strains. In this study, we introduced a bacterial model system for the screening of anti-caries and determining the optimal concentration of them to develop oral hygiene products for Korean populations.     

Development of Prevotella nigrescens-specific PCR primers based on the nucleotide sequence of a Pn23 DNA probe

A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases.     

Tracing of the Bile-chemotactic migration of juvenile Clonorchis sinensis in rabbits by PET-CT

Background

Adult Clonorchis sinensis live in the bile duct and cause clonorchiasis. It is known that the C. sinensis metacercariae excyst in the duodenum and migrate up to the bile duct through the common bile duct. However, no direct evidence is available on the in vivo migration of newly excysted C. sinensis juveniles (CsNEJs). Advanced imaging technologies now allow the in vivo migration and localization to be visualized. In the present study, we sought to determine how sensitively CsNEJs respond to bile and how fast they migrate to the intrahepatic bile duct using PET-CT.

Methodology/Principal Findings

CsNEJs were radiolabeled with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG). Rabbits with a gallbladder contraction response to cholecystokinin-8 (CCK-8) injection were pre-screened using cholescintigraphy. In these rabbits, gallbladders contracted by 50% in volume at an average of 11.5 min post-injection. The four rabbits examined were kept anesthetized and a catheter inserted into the mid duodenum. Gallbladder contraction was stimulated by injecting CCK-8 (20 ng/kg every minute) over the experiment. Anatomical images were acquired by CT initially and dynamic PET was then carried out for 90 min with a 3-min acquisition per frame. Twelve minutes after CCK-8 injection, about 3,000 ¹⁸F-FDG-labeled CsNEJs were inoculated into the mid duodenum through the catheter. Photon signals were detected in the liver 7–9 min after CsNEJs inoculation, and these then increased in the whole liver with stronger intensity in the central area, presenting that the CsNEJs were arriving at the intrahepatic bile ducts.

Conclusion

In the duodenum, CsNEJs immediately sense bile and migrate quickly with bile-chemotaxis to reach the intrahepatic bile ducts by way of the ampulla of Vater.     

First isolation of Streptococcus downei from human dental plaques

In this study, we isolated four bacterial strains grown on mitis-salivarius sucrose bacitracin agar. The strains had similar biochemical characteristics to biotypes I or II of mutans streptococci. The four isolates were identified as Streptococcus downei by 16S rDNA and dextranase gene (dex) sequencing as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dex. To our knowledge, this is the first report of the isolation and identification of S. downei from dental plaque in humans. The results suggest that S. downei can inhabit the human oral cavity.     

Suppressed cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with Toxoplasma gondii tachyzoites

Mechanisms of host immunosuppression after infection with Toxoplasma gondii are unclear. This study was performed to observe cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with live, nonreplicating (irradiated) RH tachyzoites on stimulation with concanavalin A (Con A) or lipopolysaccharide (LPS). For lymphocyte cultivation, 3 groups were prepared: coculture with live nonirradiated tachyzoites separated by a transwell (group T), live irradiated tachyzoites without a transwell (group R), and no tachyzoites (group C). Compared with group T, groups R and C, on stimulation with Con A, revealed significantly (P < 0.05) lower levels of interleukin-2 (IL-2) and IFN-gamma, but not IL-10. The levels of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were also significantly (P < 0.05) lower in groups R and C than in group T after stimulation with LPS. The results suggest that intracellular infection of murine splenic lymphocytes with T. gondii tachyzoites could impair their capacity to produce cytokine and immunoglobulin secretions.     

Mechanism of natural rifampin resistance of Streptomyces spp

In a previous phylogenetic study of the genus Streptomyces using the rpoB gene, N531, which stands for an aspargine residue in position 531 of RpoB instead of serine (S531), known to be associated with natural rifampin resistance in several organisms, was also observed in the RpoB of several Streptomyces species. To determine whether N531 is associated with the rifampin resistance of Streptomyces strains, we analyzed the rifampin minimum inhibitory concentrations (MICs) of 11 strains of the N531 RpoB type (putative rifampin resistant strains) and of 12 strains of the S531 RpoB type. (putative rifampin susceptible strains). In general, the N531 RpoB types showed higher MIC levels (16-128 microg/ml) than the S531 RpoB types (0-8 microg/ml). To determine the isolation frequencies of N531 RpoB types versus rifampin concentration, we applied screening methods involving different rifampin concentrations (0, 20 and 100 microg/ml) to Korean soils. Higher isolation frequencies of the N531 RpoB types were observed at the higher rifampin concentrations. In addition, during the course of this study we developed an allele specific PCR method to detect rifampin resistant Streptomyces strains. Our results strongly suggested that N531 might be involved in a major mechanism of natural rifampin resistance in strains of the genus Streptomyces.     

Prevalence of putative periodontopathogens in subgingival dental plaques from gingivitis lesions in Korean orthodontic patients

The objective of this study was to detect and compare the presence of periodontopathogens in the subgingival plaques of gingivitis lesions in adults who wore fixed orthodontic appliances, as opposed to adults who did not wear any orthodontic appliances. Thirty-six individuals participated in this study. Nineteen of these subjects did not wear any orthodontic appliances, and these subjects comprised the control group. The other 17 individuals had been wearing fixed orthodontic appliances for at least 3 months each. After a periodontal examination, we collected subgingival plaque samples from the gingivitis lesions of each patient. Using PCR based on 16S rDNA, we detected the presence of 6 putative periodontopathogenic species, Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia (formerly Bacteroides forsythus), Prevotella nigrescens, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. With regard to the presence of individual periodontopathogens, we found that T. forsythia, T. denticola, and P. nigrescens were significantly more common in the samples obtained from the orthodontic patients than in the samples obtained from the non-orthodontic patient controls. Our results indicate that the local changes associated with the wearing of fixed orthodontic appliances may affect the prevalence of periodontopathogens in subgingival dental plaques.