Cellular recognition of trimyristoylated peptide or enterobacterial lipopolysaccharide via both TLR2 and TLR4

Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.     

Biological activity of ruthenium nitrosyl complexes

Nitric oxide plays an important role in various biological processes, such as neurotransmission, blood pressure control, immunological responses, and antioxidant action. The control of its local concentration, which is crucial for obtaining the desired effect, can be achieved with exogenous NO-carriers. Coordination compounds, in particular ruthenium(III) and (II) amines, are good NO-captors and -deliverers. The chemical and photochemical properties of several ruthenium amine complexes as NO-carriers in vitro and in vivo have been reviewed. These nitrosyl complexes can stimulate mice hippocampus slices, promote the lowering of blood pressure in several in vitro and in vivo models, and control Trypanosoma cruzi and Leishmania major infections, and they are also effective against tumor cells in different models of cancer. These complexes can be activated chemically or photochemically, and the observed biological effects can be attributed to the presence of NO in the compound. Their efficiencies are explained on the basis of the [Ru^IINO⁺]³⁺/[Ru^IINO⁰]²⁺ reduction potential, the specific rate constant for NO liberation from the [RuNO]²⁺ moiety, and the quantum yield of NO release.     

An archaeal protein with homology to the eukaryotic translation initiation factor 5A shows ribonucleolytic activity

To identify proteins that are involved in RNA degradation and processing in Halobacterium sp. NRC-1, we purified proteins with RNA-degrading activity by classical biochemical techniques. One of these proteins showed strong homology to the eukaryotic initiation factor 5A (eIF-5A) and was accordingly named archaeal initiation factor 5A (aIF-5A). Eukaryotic IF-5A is known to be involved in mRNA turnover and to bind RNA. Hypusination of eIF-5A is required for sequence-specific binding of RNA. This unique post-translational modification is restricted to Eukarya and Archaea. The exact function of eIF-5A in RNA turnover remained obscure. Here we show for the first time that aIF-5A from Halobacterium sp. NRC-1 exhibits RNA cleavage activity, preferentially cleaving adjacent to A nucleotides. Detectable RNA binding could be shown for aIF-5A purified from Halobacterium sp. NRC-1 but not from Escherichia coli, while both proteins possess RNA cleavage activity, indicating that hypusination of aIF-5A is required for RNA binding but not for its RNA cleavage activity. Furthermore, we show that the hypusinated form of eIF-5A also shows RNase activity while the unmodified protein does not. Charged amino acids in the N-terminal domain of aIF-5A as well as in the C-terminal domain, which is highly similar to the cold shock protein A (CspA), an RNA chaperone of E. coli, are important for RNA cleavage activity. Moreover our results reveal that activity of aIF-5A depends on its oligomeric state.     

The Erwin equation of biodiversity: From little steps to quantum leaps in the discovery of tropical insect diversity

Almost 40 years ago, Terry L. Erwin published a seemingly audacious proposition: There may be as many as 30 million species of insects in the world. Here, we translate Erwin's verbal argument into a diversity-ratio model—the Erwin Equation of Biodiversity—and discuss how it has inspired other biodiversity estimates. We categorize, describe the assumptions for, and summarize the most commonly used methods for calculating estimates of global biodiversity. Subsequent diversity-ratio extrapolations have incorporated parameters representing empirical insect specialization ratios, and how insect specialization changes at different spatial scales. Other approaches include macroecological diversity models and diversity curves. For many insect groups with poorly known taxonomies, diversity estimates are based on the opinions of taxonomic experts. We illustrate our current understanding of insect diversity by focusing on the six most speciose insect orders worldwide. For each order, we compiled estimates of the (a) maximum estimated number of species, (b) minimum estimated number of species, and (c) number of currently described species. By integrating these approaches and considering new information, we believe an estimate of 5.5 million species of insects in the world is much too low. New molecular methodologies (e.g., metabarcoding and NGS studies) are revealing daunting numbers of cryptic and previously undescribed species, at the same time increasing our precision but also uncertainty about present estimates. Not until technologies advance and sampling become more comprehensive, especially of tropical biotas, will we be able to make robust estimates of the total number of insect species on Earth.     

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.     

Variability in reproductive traits in Jatropha curcas L. accessions during early developmental stages under warm subtropical conditions

Variability in floral, fruit, and seed characteristics, and oil content of 15 accession of Jatropha curcas during early development were assessed during two flowering periods in south Florida subtropical climate. The two flowering periods had leaf flushing in March. Field evaluation using 18 quantitative traits showed significant variation among accessions. The number of female flowers and female : male flower ratio ranged from 1 to 15 and 1 : 8.8 to 1 : 67.8, respectively. Fruit set by natural pollination was 89 and 66% during the first (1st) and second (2nd) flowering periods, respectively. A higher number of female‐type inflorescences were observed during summer. There were significant differences in seed traits, except for number of seeds per fruit. Accession TREC 31 had the highest individual seed dry weight and 100‐seed weight (0.83 g and 79.7 g, respectively). The oil content varied from 19.30% to 35.62%. Seed dry weight had positive correlation with seed fresh weight, seed length, seed thickness, seed width, and 100‐seed weight, but negative correlation with oil content. Based on the cluster analysis using 15 morphological traits, jatropha accessions were grouped into five main clusters and accessions from different geographic regions grouped together in a cluster. Principal component analyses (PCA) revealed morphological variation. The first three components explained 73.5% of the total variation and seed dry weight, 100‐seed weight, total flowers per inflorescence, male flowers per inflorescence and fruit set can be used to distinguish accessions. The PCA also indicated that flowering traits were more influenced by seed origin while seed traits were affected by flowering spans. Although evaluations were performed in plants during the juvenile phase, accessions TREC 31 and TREC 55 had superior averages for almost all characters evaluated. These results provide a preliminary assessment of the high variability in jatropha accessions evaluated and their potential for use in breeding and genetic improvement programs.     

Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation

A multichaperone nucleosome-remodeling complex that contains the H1 linker histone chaperone nuclear autoantigenic sperm protein (NASP) has recently been described. Linker histones (H1) are required for the proper completion of normal development, and NASP transports H1 histones into nuclei and exchanges H1 histones with DNA. Consequently, we investigated whether NASP is required for normal cell cycle progression and development. We now report that without sufficient NASP, HeLa cells and U2OS cells are unable to replicate their DNA and progress through the cell cycle and that the NASP(-/-) null mutation causes embryonic lethality. Although the null mutation NASP(-/-) caused embryonic lethality, null embryos survive until the blastocyst stage, which may be explained by the presence of stored NASP protein in the cytoplasm of oocytes. We conclude from this study that NASP and therefore the linker histones are key players in the assembly of chromatin after DNA replication.     

Nebulization of liposomal rh-Cu/Zn-SOD with a novel vibrating membrane nebulizer

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.