Structural and functional studies of the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system: implications for the design of novel antimicrobial peptides

Toxin-antitoxin (TA) systems play important roles in bacterial physiology, such as multidrug tolerance, biofilm formation, and arrest of cellular growth under stress conditions. To develop novel antimicrobial agents against tuberculosis, we focused on VapBC systems, which encompass more than half of TA systems in Mycobacterium tuberculosis. Here, we report that theMycobacterium tuberculosis VapC30 toxin regulates cellular growth through both magnesium and manganese ion-dependent ribonuclease activity and is inhibited by the cognate VapB30 antitoxin. We also determined the 2.7-Å resolution crystal structure of the M. tuberculosis VapBC30 complex, which revealed a novel process of inactivation of the VapC30 toxin via swapped blocking by the VapB30 antitoxin. Our study on M. tuberculosis VapBC30 leads us to design two kinds of VapB30 and VapC30-based novel peptides which successfully disrupt the toxin-antitoxin complex and thus activate the ribonuclease activity of the VapC30 toxin. Our discovery herein possibly paves the way to treat tuberculosis for next generation.     

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.     

Inhibitory role of E2F-1 in the regulation of tumor suppressor p53 during DNA damage response

Appropriate regulation of DNA damage response is pivotal for maintaining genome stability. p53 as well as E2F-1 plays a critical role during DNA damage response, however, the physiological significance of their interaction has been elusive. In the present study, we found that E2F-1 has an inhibitory effect on p53 during adriamycin (ADR)-mediated DNA damage response. Upon ADR exposure, p53 and E2F-1 were markedly induced at protein and mRNA levels in p53-procifient U2OS and HCT116 cells, and formed a stable complex as examined by co-immunoprecipitation experiments. Of note, chromatin immunoprecipitation (ChIP) experiments revealed that ADR-mediated induction coincides with the efficient recruitment of p53 and E2F-1 onto the promoters of p53-target genes, such as p21(WAF1) and BAX. Subsequent RT-PCR and luciferase reporter assays demonstrated that E2F-1 strongly attenuates p53-dependent transactivation of p53-target genes. Importantly, siRNA-mediated knockdown of E2F-1 stimulated apoptosis in response to ADR, which was associated with an accelerated response of p21(WAF1) and BAX. Collectively, our present findings suggest that E2F-1 participates in p53-mediated DNA damage response and might have a checkpoint function to limit overactive p53.     

Resin gathering in neotropical resin bugs (Insecta: Hemiptera: Reduviidae): Functional and comparative morphology

Apiomerini (Reduviidae: Harpactorinae) collect plant resins with their forelegs and use these sticky substances for prey capture or maternal care. These behaviors have not been described in detail and morphological structures involved in resin gathering, transfer, and storage remain virtually undocumented. We here describe these behaviors in Apiomerus flaviventris and document the involved structures. To place them in a comparative context, we describe and document leg and abdominal structures in 14 additional species of Apiomerini that represent all but one of the 12 recent genera in the tribe. Based on these morphological data in combination with the behavioral observations on A. flaviventris, we infer behavioral and functional hypotheses for the remaining genera within the tribe Apiomerini. Setal abdominal patches for resin storage are associated with maternal care so far only documented for species of Apiomerus. Based on the occurrence of these patches in several other genera, we propose that maternal care is widespread within the tribe. Ventral abdominal glands are widespread within female Apiomerini. We propose that their products may prevent hardening of stored resins thus providing long‐term supply for egg coating. Judging from the diverse setal types and arrangements on the front legs, we predict six different behavioral patterns of resin gathering within the tribe. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Maintenance of CMV-specific CD8+ T cell responses and the relationship of IL-27 to IFN-γ levels with aging

We investigated whether healthy young (age ? 40) and elderly (age ? 65) people infected with cytomegalovirus (CMV) had similar levels of CD8⁺ T cell cytokine production and proliferation in response to an immunodominant CMV pp65 peptide pool given the role of CD8⁺ T cells in controlling viral infection and the association of CMV with immunosenescence. Plus, we determined the effects of aging and CMV-infectious status on plasma levels of IL-27, an innate immune cytokine with pro- and anti-inflammatory properties, as well as on its relationship to IFN-γ in that IL-27 can promote the production of IFN-γ. The results of our study show that young and elderly people had similar levels of CD8⁺ T cell proliferation, and IFN-γ and TNF-α production in response to CMV pp65 peptides. Plasma levels of IL-27 were similar between the two groups although CMV-infected young and elderly people had a trend toward increased levels of IL-27. Regardless of aging and CMV-infectious status, plasma levels of IL-27 correlated highly with plasma levels of IFN-γ. These findings suggest the maintenance of CMV pp65-specific CD8⁺ T cell proliferation and cytokine production with aging as well as the sustaining of circulatory IL-27 levels and its biological link to IFN-γ in young and elderly people irrespective of CMV infection.     

Altered regulation of renal nitric oxide and atrial natriuretic peptide systems in angiotensin II-induced hypertension

The present study was aimed to determine whether there is an altered role of local nitric oxide (NO) and atrial natriuretic peptide (ANP) systems in the kidney in association with the angiotensin (Ang) II-induced hypertension. Male Sprague-Dawley rats were used. Ang II (100 ng·min?1·kg?1) was infused through entire time course. Thirteenth day after beginning the regimen, kidneys were taken. The protein expression of NO synthase (NOS) and nitrotyrosine was determined by semiquantitative immunoblotting. The mRNA expression of components of ANP system was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclases were determined by the amount of cGMP generated in responses to sodium nitroprusside and ANP, respectively. There developed hypertension and decreased creatinine clearance in the experimental group. The protein expression of eNOS, nNOS and nitrotyrosine was increased in the cortex, while that of iNOS remained unaltered. The urinary excretion of NO increased in Ang II-induced hypertensive rats. The catalytic activity of soluble guanylyl cyclase was blunted in the glomerulus in Ang II-induced hypertensive rats. The mRNA expression of ANP was increased in Ang II-induced hypertensive rats. Neither the expression of NPR-A nor that of NPR-C was changed. The protein expression of neutral endopeptidase was decreased and the activity of particulate guanylyl cyclase was blunted in the glomerulus and papilla in Ang II-induced hypertensive rats. In conclusion, the synthesis of NO and ANP was increased in the kidney of Ang II-induced hypertension, while stimulated cGMP response was blunted. These results suggest desensitization of guanylyl cyclase in the kidney of Ang II-induced hypertensive rats, which may contribute to the associated renal vasoconstriction and hypertension.     

Slow and persistent increase of [Ca2+]c in response to ligation of surface IgM in WEHI-231 cells

WEHI-231 and Bal 17 B cell lines are representative models for immature and mature B cells, respectively. Their regulation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) was compared using fura-2 fluorescence ratiometry. The ligation of B cell antigen receptor (BCR) by anti-IgM antibody induced a slow but large increase of [Ca(2+)](c) in WEHI-231 cells while not in Bal 17 cells. The thapsigargin-induced store-operated Ca(2+) entry (SOCE) of Bal 17 cells reached a steady state which was blocked by 2-aminoethoxydiphenyl borate (2-APB). On the contrary, the thapsigargin-induced SOCE of WEHI-231 cells increased continuously, which was accelerated by 2-APB. The increase of [Ca(2+)](c) by BCR ligation was also enhanced by 2-APB in WEHI-231 cells while blocked in Bal 17 cells. The Mn(2+) quenching study showed that the thapsigargin-, or the BCR ligation-induced Ca(2+) influx pathway of WEHI-231 was hardly permeable to Mn(2+). The intractable increase of [Ca(2+)](c) may explain the mechanism of BCR-driven apoptosis of WEHI-231 cells, a well-known model of clonal deletion of autoreactive immature B cells.