Snake cathelicidin from Bungarus fasciatus is a potent peptide antibiotics

Background

Cathelicidins are a family of antimicrobial peptides acting as multifunctional effector molecules of innate immunity, which are firstly found in mammalians. Recently, several cathelicidins have also been found from chickens and fishes. No cathelicidins from other non-mammalian vertebrates have been reported.

Principal Findings

In this work, a cathelicidin-like antimicrobial peptide named cathelicidin-BF has been purified from the snake venoms of Bungarus fasciatus and its cDNA sequence was cloned from the cDNA library, which confirm the presence of cathelicidin in reptiles. As other cathelicidins, the precursor of cathelicidin-BF has cathelin-like domain at the N terminus and carry the mature cathelicidin-BF at the C terminus, but it has an atypical acidic fragment insertion between the cathelin-like domain and the C-terminus. The acidic fragment is similar to acidic domains of amphibian antimicrobial precursors. Phylogenetic analysis revealed that the snake cathelicidin had the nearest evolution relationship with platypus cathelicidin. The secondary structure of cathelicidin-BF investigated by CD and NMR spectroscopy in the presence of the helicogenic solvent TFE is an amphipathic α-helical conformation as many other cathelicidins. The antimicrobial activities of cathelicidin BF against forty strains of microorganisms were tested. Cathelicidin-BF efficiently killed bacteria and some fungal species including clinically isolated drug-resistance microorganisms. It was especially active against Gram-negative bacteria. Furthermore, it could exert antimicrobial activity against some saprophytic fungus. No hemolytic and cytotoxic activity was observed at the dose of up to 400 µg/ml. Cathelicidin-BF could exist stably in the mice plasma for at least 2.5 hours.

Conclusion

Discovery of snake cathelicidin with atypical structural and functional characterization offers new insights on the evolution of cathelicidins. Potent, broad spectrum, salt-independent antimicrobial activities make cathelicidin-BF an excellent candidate for clinical or agricultural antibiotics.     

Using non-smooth models to determine thresholds for microbial pest management

Journal of Mathematical Biology - Releasing infectious pests could successfully control and eventually maintain the number of pests below a threshold level. To address this from a mathematical...     

A proteasomal stress response: pre-treatment with proteasome inhibitors increases proteasome activity and reduces neuronal vulnerability to oxidative injury

We report here that exposure to low concentrations of proteasome inhibitors (e.g. 10-100 nm MG-132, 0.1-3 nm epoxomicin or 10-30 nm clasto-lactacystin beta-lactone) resulted in an enhancement, rather than an inhibition, of proteasome activity in cultured neocortical neurons. Size-fractionation chromatography confirmed that the enhanced peptide cleavage activity was associated with proteasome-sized complexes. This sub toxic exposure reduced neuronal death caused by subsequent exposure to oxidative stress (100-200 microm H(2)O(2) for 30 min, 24-h exposure to 100 microm paraquat or 7.5 microm menadione), but did not alter vulnerability to excitotoxicity (5-min exposure to 30-100 microm NMDA or 24 exposure to 12 microm NMDA). Sub toxic proteasome inhibitor exposure caused an increase in levels of proteasome core subunit proteins and mRNAs, but not in levels of potentially cytoprotective heat shock proteins (hsp70, hsp90 and hsp40). The neuroprotective effects of proteasome inhibitor pre-treatment were blocked by coapplication of proteasome inhibitors during the oxidative insult. These findings support a model in which sublethal proteasome inhibition induces neurons to increase proteasome activity and promotes resistance to oxidative injury and suggests that enhancement of proteasome activity is a potential therapeutic target for diseases in which oxidative stress has been implicated.     

Relationships and Hybridization among Smilax china and Its Affinities: Evidence from Allozyme Data

Smilax china L. is a widespread species in China with different ploidy levels. It is morphologically similar to S. davidiana, S. trinervula, and S. glauco-china. In this study, the chromosome number and the variation in allozyme patterns of eight enzyme systems with 25 alleles in 11 populations of S. china and three affinitive species were investigated. The allozyme data, together with morphological and cytological data, suggest that S. glauco-china is not closely related to the other taxa investigated. The diploid species S. davidiana and S. trinervula are involved as ancestor species and share great introgressions with S. china. In S. china, populations from Guilin and Guiyang are allotetraploid; their diploid progenitors probably are diploid populations of S. china and S. trinervula. The results suggest this species arose from multiple origins.     

Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis

YopE of Yersinia pseudotuberculosis inactivates three members of the small RhoGTPase family (RhoA, Rac1 and Cdc42) in vitro and mutation of a critical arginine abolishes both in vitro GTPase-activating protein (GAP) activity and cytotoxicity towards HeLa cells, and renders the pathogen avirulent in a mouse model. To understand the functional role of YopE, in vivo studies of the GAP activity in infected eukaryotic cells were conducted. Wild-type YopE inactivated Rac1 as early as 5 min after infection whereas RhoA was down regulated about 30 min after infection. No effect of YopE was found on the activation state of Cdc42 in Yersinia-infected cells. Single-amino-acid substitution mutants of YopE revealed two different phenotypes: (i) mutants with significantly lowered in vivo GAP activity towards RhoA and Rac1 displaying full virulence in mice, and (ii) avirulent mutants with wild-type in vivo GAP activity towards RhoA and Rac1. Our results show that Cdc42 is not an in vivo target for YopE and that YopE interacts preferentially with Rac1, and to a lesser extent with RhoA, during in vivo conditions. Surprisingly, we present results suggesting that these interactions are not a prerequisite to establish infection in mice. Finally, we show that avirulent yopE mutants translocate YopE in about sixfold higher amount compared with wild type. This raises the question whether YopE's primary function is to sense the level of translocation rather than being directly involved in downregulation of the host defence.     

The Development and Application of the Two Real-Time RT-PCR Assays to Detect the Pathogen of HFMD

Large-scale Hand, Foot, and Mouth Disease (HFMD) outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs). Among them, human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1–2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID₅₀/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells). The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11) by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.     

Expression of HAb18G in non-small lung cancer and characterization of activation, migration, proliferation, and apoptosis in A549 cells following siRNA-induced downregulation of HAb18G

VSL#3 probiotics can be effective on induction and maintenance of the remission of clinical ulcerative colitis. However, the mechanisms are not fully understood. The aim of this study was to examine the effects of VSL#3 probiotics on dextran sulfate sodium (DSS)-induced colitis in rats. Acute colitis was induced by administration of DSS 3.5 % for 7 days in rats. Rats in two groups were treated with either 15 mg VSL#3 or placebo via gastric tube once daily after induction of colitis; rats in other two groups were treated with either the wortmannin (1 mg/kg) via intraperitoneal injection or the wortmannin + VSL#3 after induction of colitis. Anti-inflammatory activity was assessed by myeloperoxidase (MPO) activity. Expression of inflammatory related mediators (iNOS, COX-2, NF-κB, Akt, and p-Akt) and cytokines (TNF-α, IL-6, and IL-10) in colonic tissue were assessed. TNF-α, IL-6, and IL-10 serum levels were also measured. Our results demonstrated that VSL#3 and wortmannin have anti-inflammatory properties by the reduced disease activity index and MPO activity. In addition, administration of VSL#3 and wortmannin for 7 days resulted in a decrease of iNOS, COX-2, NF-κB, TNF-α, IL-6, and p-Akt and an increase of IL-10 expression in colonic tissue. At the same time, administration of VSL#3 and wortmannin resulted in a decrease of TNF-α and IL-6 and an increase of IL-10 serum levels. VSL#3 probiotics therapy exerts the anti-inflammatory activity in rat model of DSS-induced colitis by inhibiting PI3K/Akt and NF-κB pathway.     

Optimization of a Saccharomyces cerevisiae fermentation process for production of a therapeutic recombinant protein using a multivariate bayesian approach

Various approaches have been applied to optimize biological product fermentation processes and define design space. In this article, we present a stepwise approach to optimize a Saccharomyces cerevisiae fermentation process through risk assessment analysis, statistical design of experiments (DoE), and multivariate Bayesian predictive approach. The critical process parameters (CPPs) were first identified through a risk assessment. The response surface for each attribute was modeled using the results from the DoE study with consideration given to interactions between CPPs. A multivariate Bayesian predictive approach was then used to identify the region of process operating conditions where all attributes met their specifications simultaneously. The model prediction was verified by twelve consistency runs where all batches achieved broth titer more than 1.53 g/L of broth and quality attributes within the expected ranges. The calculated probability was used to define the reliable operating region. To our knowledge, this is the first case study to implement the multivariate Bayesian predictive approach to the process optimization for the industrial application and its corresponding verification at two different production scales. This approach can be extended to other fermentation process optimizations and reliable operating region quantitation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1095–1105, 2012     

Biotic and abiotic stress responses through calcium-dependent protein kinase (CDPK) signaling in wheat (Triticum aestivum L.)

Calcium-dependent protein kinases (CDPKs) sense the calcium concentration changes in plant cells and play important roles in signaling pathways for disease resistance and various stress responses as indicated by emerging evidences. Among the 20 wheat CDPK genes studied, 10 were found to respond to drought, salinity and ABA treatments. Consistent with previous observations, one CDPK gene was shown to respond to multiple abiotic stresses in wheat suggesting that CDPKs could be converging points for multiple signaling pathways. Among the 12 wheat CDPK genes that were responsive to Blumeria graminis tritici (Bgt) infection or the treatment of hydrogen peroxide (H₂O₂), eight also responded to abiotic stresses, suggesting a cross-talk between biotic and abiotic stress signaling pathways. Phylogenetic analysis indicated that some of these genes were closely related to CDPKs from other species, whose functions have been partially studied, suggesting similar functions wheat CDPK genes. Combining the up-to-date knowledge of CDPK functions and our observations, a model was developed to project the possible roles of wheat CDPK genes in the signaling of biotic and abiotic stress responses.Key words: CDPK, calcium, kinase, stress response, disease resistance, signal transduction, wheatSessile plants have developed sophisticated signaling pathways to deal with dramatic environmental changes that may affect their normal growth, such as pathogen attack, drought, and cold. Calcium is a universal secondary messenger that responds to these stimuli. The fluctuation in cytosolic Ca²⁺ levels can be sensed by calcium-dependent protein kinases (CDPKs), which will modify the phosphorylation status of substrate proteins.^1–3 Accumulating evidence indicate that CDPKs mediate biotic and abiotic stress signaling pathways.^4–7 For example, overexpression of the rice CDPK gene OsCDPK7 provides cold, salt, and drought tolerance for the transgenic rice plants, demonstrating the potential of CDPK engineering to generate stress tolerance enhanced crops.^8,9In wheat, 10 out of 14 CDPK genes appeared to respond to abiotic stresses including drought, NaCl, as well as ABA stimulus (Fig. 1A).¹⁰ Five CDPKs (TaCPK4, 6, 9, 10 and 18) were particularly interesting since they could respond to at least two of the three treatments, among which the expression level of TaCPK9 was enhanced under all three treatments suggesting that TaCPK9 is the point where multiple signaling pathways cross. In wheat, TaCPK4 responded to both ABA treatment and NaCl stress (Fig. 1A). Interestingly, its best Arabidopsis homologs AtCPK4 and AtCPK11, as suggested by a Neighbor-Joining phylogenetic analysis (Fig. 1B), have been postulated as two important positive regulators in CDPK/calcium-mediated ABA signaling pathways.¹¹ Such a correlation strongly supports the idea that TaCPK4 is a good candidate in wheat for ABA signaling. Figure 1A also shows that one wheat CDPK gene could respond to multiple abiotic stresses suggesting that CDPKs are converging points for multiple signaling pathways. On the other hand, multiple CDPKs were involved in single stress response. It is however not clear how these CDPKs are organized in one signaling pathway.Open in a separate windowFigure 1The roles of wheat CDPKs in abiotic and biotic stress responses. (A) One CDPK gene responded to multiple abiotic stresses and multiple CDPKs were required for single stress response. (B) Phylogenetic relationship of wheat CDPKs with functionally studied CDPKs from barley (HvCPKs), Arabidopsis (AtCPKs), and potato (StCDPKs) that are known to be involved in ABA signaling, oxidative burst regulation and defense to powdery mildew pathogenesis. (C) A model depicting CDPK-mediated signaling pathways under biotic and abiotic treatments in wheat (see text for details). Dotted lines with a question mark indicate unknown intermediate steps.Regarding the roles of CDPKs in defense reactions, 12 TaCPKs were found to be responsive to either Blumeria graminis tritici (Bgt) infection or H₂O₂ treatment. The response to H₂O₂ was investigated because cytosolic calcium influx and reactive oxygen species, such as H₂O₂ are known to be implicated in both plant innate immunity and abiotic stresses.^12–17 Among these CDPK genes, five responded to both treatments (Group II) whereas the ones that responded to Bgt infection (Group I) or H₂O₂ treatment (Group III) were four and three respectively. The differential expression patterns suggest different functional modes of these CDPK genes. Involvement of CDPK genes in plant defense response has been shown in multiple species.^5,7 Recently, two barley CDPK paralogs (HvCDPK3 and HvCDPK4) were found to play antagonistic roles during the early phase of powdery mildew pathogenesis.⁵ The close similarity between wheat CDPK genes (TaCPK2 and TaCPK5, Fig. 1B) with these two barley genes may suggest their potential roles in wheat powdery mildew resistance. Surprisingly, we did not detect the responsiveness of TaCPK5 to wheat Bgt infection, indicating the divergence of CDPK functions in these two members of Triticeae family. Recently, one potato (Solanum tuberosum) CDPK gene StCDPK5 has been shown to be directly involved in regulating oxidative burst via phosphorylation of the NADPH oxidase StRBOHB.¹⁸ In light of the close relationship of TaCPK2 with HvCDPK5 and StCDPK5 (Fig. 1B), we speculate that TaCPK2 could be associated with both biotic and abiotic stress response signaling pathways and therefore play multiple roles in wheat.A model was proposed in Figure 1C regarding the positions of wheat CDPK genes in signaling pathways for biotic and abiotic responses. The hypothesis depicted four different roles of wheat CDPK genes: (1) Group I genes that respond only to Bgt infection may, like potato StCDPK5, render defense response through an oxidase like NADPH oxidase that generates increased amount of H₂O₂;¹⁸ (2) At one aspect, Group II genes may participate in defense response in a manner similar to Group I genes; (3) On the other hand, since Group II genes also respond to H₂O₂ treatment directly, an auto-regulation circuit was proposed, which eventually joins the oxidase pathway; (4) Group III CDPK genes and some remaining CDPK genes are considered to be mainly involved in abiotic stress responses. The model positioned CDPKs both upstream and downstream of H₂O₂, presenting a complicated wiring of the signaling pathway network involving wheat CDPKs. Future biochemical, genetic, and transgenic analyses may help elucidate the genuineness of such a rather early model for the functions of wheat CDPK genes.