A new approach to microtubule depolymerization

Microtubule thermal denaturation is treated in the framework of the Ising model. The partition function is calculated with a new iterative procedure which allows us to calculate values of the denaturation curves and enthalpy changes in excellent agreement with the experimental data, We also obtain the excess heat capacity due to polymerization, OCp.     

The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis,cell division and virulence in the mammalian host

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.     

A General Model for the Dynamics of the Cell Volume

The conservation of the cell volume within values compatible with the overall cell functions represents an ubiquitous property, shared by cells comprising the whole biological world. Water transport across membranes constitutes the main process associated to the dynamics of the cell volume, its chronic and acute regulations therefore represent crucial aspects of cell homeostasis. In spite of the biological diversity, the dynamics of the cell volume exhibits common basic features in the diverse types of cells. The purpose of this study is to show that there is a general model capable to describe the basic aspects of the dynamics of the cell volume. It is demonstrated here that the steady states of this model represent asymptotically stable configurations. As illustrations, several cases of non-polarized (i.e., symmetrical) and polarized (e.g., epithelial) cells performing water transport are shown here to represent particular cases of the general model. From a biological perspective, the existence of a general model for the dynamics of the cell volume reveals that, in spite of physiological and morphological peculiarities, there is a basic common design of the membrane transport processes. In view of its stability properties, this basic design may represent an ancestral property that has proven to be successful regarding the overall homeostatic properties of cells.     

Resistance testing of meningococci: the recommendations of the European Monitoring Group on Meningococci

One of the most controversial topics concerning Neisseria meningitidis is the definition of antimicrobial resistance. A process of harmonization, promoted by the European Monitoring Group on Meningococci (EMGM), is already in progress. There are several aspects of the MIC definition for meningococcal strains for which a consensus is needed, as follows. (1) What methods can be used? At present, both the Etest and agar dilution methods are recommended by the EMGM. Microdilution, also a recommended method, is not used across Europe. (2) What media produce the most reliable data? The use of Mueller-Hinton medium supplemented with 5% blood is recommended for the MIC determination of N. meningitidis by both Etest and agar dilution method. (3) What is the minimum set of antimicrobial agents to be tested? A minimum set should include rifampicin, ciprofloxacin (or another quinolone), penicillin, ampicillin and ceftriaxone. (4) What are the most suitable breakpoints for definition? There is no widely accepted robust evidence base for the definition of breakpoints. Although progress has been made in achieving a standardized methodology, we are still far from obtaining common breakpoints for antimicrobial resistance definition. These differences will imply inability for building a common epidemiologic figure about drug resistance.     

Scale and structure of time-averaging (age mixing) in terrestrial gastropod assemblages from Quaternary eolian deposits of the eastern Canary Islands

Quantitative estimates of time-averaging (age mixing) in gastropod shell accumulations from Quaternary (the late Pleistocene and Holocene) eolian deposits of Canary Islands were obtained by direct dating of individual gastropods obtained from exceptionally well-preserved dune and paleosol shell assemblages. A total of 203 shells of the gastropods Theba geminata and T. arinagae, representing 44 samples (= stratigraphic horizons) from 14 sections, were dated using amino acid (isoleucine) epimerization ratios calibrated with 12 radiocarbon dates. Most samples reveal a substantial variation in shell age that exceeds the error that could be generated by dating imprecision, with the mean within-sample shell age range of 6670 years and the mean standard deviation of 2920 years. Even the most conservative approach (Monte Carlo simulations with a non-sequential Bonferroni correction) indicates that at least 25% of samples must have undergone substantial time-averaging (e.g., age variations within those samples cannot be explained by dating imprecision alone). Samples vary in shell age structure, including both left-skewed (17 out of 44) and right-skewed distributions (26 out of 44) as well as age distributions with a highly variable kurtosis. Dispersion and shape of age distributions of samples do not show any notable correlation with the stratigraphic age of samples, suggesting that the structure and scale of temporal mixing is time invariant. The statistically significant multi-millennial time-averaging observed here is consistent with previous studies of shell accumulations from various depositional settings and reinforces the importance of dating numerous specimens per horizon in geochronological studies. Unlike in the case of marine samples, typified by right-skewed age distributions (attributed to an exponential-like shell loss from older age classes), many of the samples analyzed here displayed left-skewed distributions, suggestive of different dynamics of age mixing in marine versus terrestrial shell accumulations.     

Late Hauterivian coralline algae (Rhodophyta,Corallinales) from the Iberian Chain (E Spain). Taxonomy and the evolution of multisporangial reproductive structures

Upper Hauterivian reefal carbonates of the Llàcova Formation (Maestrat Basin, Iberian Chain, E Spain) contain Sporolithon phylloideum (Bucur and Dragastan) Tomás, Aguirre, Braga and Martín-Closas comb. nov. and Sporolithon rude (Lemoine) Ghosh and Maithy (1996). Moussavian et al. (1993) identified them as Parakymalithon phylloideum (Bucur and Dragastan) Moussavian 1987 and Archaeolithothamnium rude Lemoine 1925. The re-assessment of the type of P. phylloideum and additional material indicate that the diagnostic characters of the genus do not warrant separation from Sporolithon and the new combination Sporolithon phylloideum is proposed. The lectotype of Sporolithon rude presents sporangial cavities grouped in sori that can be merged originating a structure that resembles the multiporate tetrasporangial conceptacles of the Hapalidiaceae. We hypothesize that multiporate tetrasporangial conceptacles could have originated from the fusion of several sporangial cavities, suggesting a phylogenetic linkage between Sporolithaceae and Hapalidiaceae supported by other anatomical features, molecular phylogeny and the fossil record.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop 'maca' (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74-94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 microM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.     

Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.     

Characterization of the intracellular proteolytic cleavage of myocilin and identification of calpain II as a myocilin-processing protease

MYOC, a gene involved in different types of glaucoma, encodes myocilin, a secreted glycoprotein of unknown function, consisting of an N-terminal leucine-zipper-like domain, a central linker region, and a C-terminal olfactomedin-like domain. Recently, we have shown that myocilin undergoes an intracellular endoproteolytic processing. We show herein that the proteolytic cleavage in the linker region splits the two terminal domains. The C-terminal domain is secreted to the culture medium, whereas the N-terminal domain mainly remains intracellularly retained. In transiently transfected 293T cells, the cleavage was prevented by calpain inhibitors, such as calpeptin, calpain inhibitor IV, and calpastatin. Since calpains are calcium-activated proteases, we analyzed how changes in either intra- or extracellular calcium affected the cleavage of myocilin. Intracellular ionomycin-induced calcium uptake enhanced myocilin cleavage, whereas chelation of extracellular calcium by EGTA inhibited the proteolytic processing. Calpains I and II cleaved myocilin in vitro. However, in cells in culture, only RNA interference knockdown of calpain II reduced myocilin processing. Subcellular fractionation and digestion of the obtained fractions with proteinase K showed that full-length myocilin resides in the lumen of the endoplasmic reticulum together with a subpopulation of calpain II. These data revealed that calpain II is responsible for the intracellular processing of myocilin in the lumen of the endoplasmic reticulum. We propose that this cleavage might regulate extracellular interactions of myocilin, contributing to the control of intraocular pressure.