Antimicrobial activity of the Naja atra cathelicidin and related small peptides

We have identified an 11-residue pattern (KR(F/A)KKFFKK(L/P)K), which we have named the ATRA motif, within the sequence of the Chinese cobra (Naja atra) cathelicidin. A series of 11-residue peptides (ATRA-1, -2, -1A and -1P) were designed to probe the significance of the conserved residues within the ATRA motif, and their contributions to antimicrobial performance. The antimicrobial activities of the peptides were assessed against Escherichia coli K12 strain and Aggregatibacter actinomycetemcomitans Y4. ATRA-1 and -1A, demonstrated potencies comparable to that of N. atra cathelicidin. Structural examination by circular dichroism of the four short peptides suggested the significance of specific amino acid positions within the motif by their contribution to helicity. The results of these studies indicate that short peptides derived from the repeated ATRA motif from the N. atra cathelicidin can demonstrate both low toxicity against host cells and high antimicrobial activity against the gram-negative bacteria used in this study. They constitute novel, effective antimicrobial peptides that are much shorter (and thus less expensive to produce) than the natural cathelicidins, and they may represent new templates for therapeutic drug development.     

Adherence to Chemoprophylaxis and Plasmodium falciparum Anti-Circumsporozoite Seroconversion in a Prospective Cohort Study of Dutch Short-Term Travelers

Background

We conducted a prospective study in a cohort of short-term travelers assessing the incidence rate of anti-circumsporozoite seroconversion, adherence to chemoprophylaxis, symptoms of malaria during travel, and malaria treatment abroad.

Methods

Adults were recruited from the travel clinic of the Public Health Service Amsterdam. They kept a structured daily travel diary and donated blood samples before and after travel. Blood samples were serologically tested for the presence of Plasmodium falciparum anti-circumsporozoite antibodies.

Results

Overall, the incidence rate (IR) of anti-circumsporozoite seroconversion was 0.8 per 100 person-months. Of 945 travelers, 620 (66%) visited high-endemic areas and were advised about both chemoprophylaxis and preventive measures against mosquito bites. Most subjects (520/620 = 84%) took at least 75% of recommended prophylaxis during travel. Travel to Africa, use of mefloquine, travel duration of 14–29 days in endemic areas, and concurrent use of DEET (N,N-diethyl-meta-toluamide) were associated with good adherence practices. Four travelers without fever seroconverted, becoming anti-circumsporozoite antibody-positive. All four had been adherent to chemoprophylaxis; two visited Africa, one Suriname, one India. Ten subjects with fever were tested for malaria while abroad and of these, three received treatment. All three were adherent to chemoprophylaxis and tested negative for anti-circumsporozoite antibodies.

Conclusion

Travel to Africa, using mefloquine, travel duration of 14–29 days in endemic areas, and use of DEET were associated with good adherence to chemoprophylaxis. The combination of chemoprophylaxis and other preventive measures were sufficient to protect seroconverting travelers from clinical malaria. Travelers who were treated for malaria abroad did not seroconvert.     

An analysis of CAF-1-interacting proteins reveals dynamic and direct interactions with the KU complex and 14-3-3 proteins

CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly.     

Recipient’s Genetic R702W NOD2 Variant Is Associated with an Increased Risk of Bacterial Infections after Orthotopic Liver Transplantation

Introduction

Orthotopic liver transplantation (OLT) is accompanied by a significant postoperative infection risk. Immunosuppression to prevent rejection increases the susceptibility to infections, mainly by impairing the adaptive immune system. Genetic polymorphisms in the lectin complement pathway of the donor have recently been identified as important risk determinants of clinically significant bacterial infection (CSI) after OLT. Another genetic factor involved in innate immunity is NOD2, which was reported to be associated with increased risk of spontaneous bacterial peritonitis in cirrhotic patients.

Methods

We assessed association of three genetic NOD2 variants (R702W, G908R and 3020insC) with increased risk of CSI after OLT. 288 OLT recipient-donor pairs from two tertiary referral centers were genotyped for the three NOD2 variants. The probability of CSI in relation to NOD2 gene variants was determined with cumulative incidence curves and log-rank analysis.

Results

The R702W NOD2 variant in the recipient was associated with CSI after OLT. Eight out of 15 (53.3%) individuals with a mutated genotype compared to 80/273 (29.3%) with wild type genotype developed CSI (p=0.027, univariate cox regression), illustrated by a higher frequency of CSI after OLT over time (p=0.0003, log rank analysis). Multivariate analysis (including the donor lectin complement pathway profile) showed independence of this R702W NOD2 association from other risk factors (HR 2.0; p=0.04). The other NOD2 variants, G908R and 3020insC, in the recipient were not associated with CSI. There was no association with CSI after OLT for any of the NOD2 variants in the donor.

Conclusion

The mutated NOD2 R702W genotype in the recipient is independently associated with an increased risk of bacterial infections after liver transplantation, indicating a predisposing role for this genetic factor impairing the recipient’s innate immune system.     

Aminophospholipid translocase in the plasma membrane of Friend erythroleukemic cells can induce an asymmetric topology for phosphatidylserine but not for phosphatidylethanolamine

The ATP-dependent translocation of phospholipids in the plasma membrane of intact Friend erythroleukemic cells (FELCs) was studied in comparison with that in the membrane of mature murine erythrocytes. This was done by following the fate of radiolabeled phospholipid molecules, previously inserted into the outer monolayer of the plasma membranes by using a non-specific lipid transfer protein. The transbilayer equilibration of these probe molecules was monitored by treating the cells--under essentially non-lytic conditions--with phospholipases A2 of different origin. Rapid reorientations of the newly introduced aminophospholipids in favour of the inner membrane leaflet were observed in fresh mouse erythrocytes; the inward translocation of phosphatidylcholine (PC) in this membrane proceeded relatively slow. In FELCs, on the other hand, all three glycerophospholipids equilibrated over both halves of the plasma membrane very rapidly, i.e. within 1 h; nevertheless, an asymmetric distribution in favour of the inner monolayer was only observed for phosphatidylserine (PS). Lowering the ATP-level in the FELCs caused a reduction in the rate of inward translocation of both aminophospholipids, but not of that of PC, indicating that this translocation of PS and phosphatidylethanolamine (PE) is clearly ATP-dependent. Hence, the situation in the plasma membrane of the FELC is rather unique in a sense that, though an ATP-dependent translocase is present and active both for PS and PE, its activity results in an asymmetric distribution of PS, but not of PE. This remarkable situation might be the consequence of the fact that, in contrast to the mature red cell, this precursor cell still lacks a complete membrane skeletal network.     

Only seed size matters for germination in different populations of the dimorphic Tragopogon pratensis subsp. pratensis (Asteraceae)

Many studies have focused on the ecology of seed dimorphism, the production of two seed types by a single plant. Morphology and seed size are usually correlated, but how morphology affects germination percentage and seedling growth is poorly understood. Here we explicitly separate these effects for nine populations of the dimorphic species Tragopogon pratensis subsp. pratensis. Larger seeds yielded higher germination percentages, yet seed morphology had no additional direct effect on germination. Neither seed size nor seed morphology affected seedling growth. Neither germination nor seedling growth varied among populations, but seed head varied significantly. Results show that germination is mainly controlled by seed size rather than by seed morphology. This study is one of the few to distinguish explicitly between seed size and seed morphology effects on ecological characteristics and suggests that seed dimorphism may exert its ecological effects predominantly through its correlated size.     

Novel ATP competitive MK2 inhibitors with potent biochemical and cell-based activity throughout the series

Optimization of our previously described pyrrolopiperidone series led to the identification of a new benzamide sub-series, which exhibits consistently high potency in biochemical and cell-based assays throughout the series. Strong inhibition of LPS-induced production of the cytokine TNFα is coupled to the regulation of HSP27 phosphorylation, indicating that the observed cellular effects result from the inhibition of MK2. X-ray crystallographic and computational analyses provide a rationale for the high potency of the series.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Structural changes of yellow Cameleon domains observed by quantitative FRET analysis and polarized fluorescence correlation spectroscopy

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.