Analysis of foot-and-mouth disease virus internalization events in cultured cells

It has been demonstrated that foot-and-mouth disease virus (FMDV) can utilize at least four members of the alpha(V) subgroup of the integrin family of receptors in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the betaG-betaH loop of VP1. While there have been extensive studies of virus-receptor interactions at the cell surface, our understanding of the events during viral entry into the infected cell is still not clear. We have utilized confocal microscopy to analyze the entry of two FMDV serotypes (types A and O) after interaction with integrin receptors at the cell surface. In cell cultures expressing both the alphaVbeta3 and alphaVbeta6 integrins, virus adsorbed to the cells at 4 degrees C appears to colocalize almost exclusively with the alphaVbeta6 integrin. Upon shifting the infected cells to 37 degrees C, FMDV capsid proteins were detected within 15 min after the temperature shift, in association with the integrin in vesicular structures that were positive for a marker of clathrin-mediated endocytosis. In contrast, virus did not colocalize with a marker for caveola-mediated endocytosis. Virus remained associated with the integrin until about 1 h after the temperature shift, when viral proteins appeared around the perinuclear region of the cell. By 15 min after the temperature shift, viral proteins were seen colocalizing with a marker for early endosomes, while no colocalization with late endosomal markers was observed. In the presence of monensin, which raises the pH of endocytic vesicles and has been shown to inhibit FMDV replication, viral proteins were not released from the recycling endosome structures. Viral proteins were not observed associated with the endoplasmic reticulum or the Golgi. These data indicate that FMDV utilizes the clathrin-mediated endocytosis pathway to infect the cells and that viral replication begins due to acidification of endocytic vesicles, causing the breakdown of the viral capsid structure and release of the genome by an as-yet-unidentified mechanism.     

Identification of antigens of Colombian Giardia duodenalis isolates recognized by total IgG and subclasses

Antigen profiles were described for Giardia duodenalis cysts and trophozoites that are recognized by IgG and its anti-G. dudodenalis subclasses (IgG1, IgG2, IgG3, IgG4). Antigens were identified by Western blot from G. duodenalis cyst and trophozoite isolates. Cysts and trophozoites were each subjected to protein separation by SDS-PAGE. The proteins were then transferred to nitrocellulose membranes by electroimmunoblot, and their antigenicity was determined by exposing them to sera from patients with confirmed diagnosis of G. duodenalis infection. The antigen-antibody reaction was revealed by specific alkaline phosphatase antibody conjugates against IgG, IgG2, IgG3, IgG4: bands were visualized by addition of the substrate 5-bromo-4-chloro-3-indolyl-phosphate and the stain nitro blue tetrazolium. The bands were read and analyzed by linear regression using Quantity One software. Thirty two antigens were simultaneously recognized by total IgG anti-G. duodenalis in the cyst and trophozoite stages. The antigens varied in molecular weight from 22 to 185 kDa. Nineteen antigens were identified by both IgG, and IgG3 anti-G duodenalis, with molecular weights ranging from 42 to 180 kDa. IgG2 and IgG4 did not identify any antigen in either stage. The antigens of molecular weights 27, 30, 31, 33, 45, 49, 57, 78, 89 and 170 kDa are shared with G. duodenalis isolates from other geographical regions of Colombia. The recognition of cyst and trophozoite antigens of Colombian G. duodenalis isolates by IgG, IgG1 and IgG3 anti-G. duodenalis suggested that they are involved in the induction of the host immune response.     

Severe bone changes in a case of Hutchinson-Gilford syndrome

The Hutchinson-Gilford progeria syndrome (HGPS) is a very rare, but well known inherited condition of uncertain etiology in which features of premature and accelerated aging are mixed with those of delayed maturity and immaturity. Appearance at birth and birth weight are usually normal but growth typically slows after 1 year. All organ systems undergo degeneration to such an extent that the patient resembles an old man or woman. Short stature, micrognatia, alopecia, sculptured nose, prominent scalp veins, loss of subcutaneous fat, prominent joints, hyperlipidemia and early arteriosclerosis characterize the syndrome. Skeletal compromise includes hypoplasia and dysplasia, persistent open fontanelles, severe osteolysis and pathological fractures. There are no intellectual deficits in patients with this syndrome, and intelligence is unaffected. The life span in progeria is shortened by early arteriosclerosis. In this case, we review the characteristics of the severe osteolytic compromise in distal arms and limbs and bone deformities in a case of an 8-year-old girl, who was admitted to our hospital with short stature and loss of hair. On examination, the child had the major clinical criteria for HGPS as well as severe alterations in osteogenesis, including craniofacial disproportion, short and sculptured nose, delayed dentition, severe scoliosis, clavicular deformity and asymmetrical and hypoplastic arms and legs. Generalized osteopenia and severe osteolytic compromise in distal extremities were found by X-ray examination. In summary, we report the case of an 8-year-old girl who meets the diagnostic criteria for HGPS with severe involvement of her bones and joints with a review of the current literature and a possible therapeutic approach.     

Evolution of energy metabolism and its compartmentation in Kinetoplastida

Kinetoplastida are protozoan organisms that probably diverged early in evolution from other eukaryotes. They are characterized by a number of unique features with respect to their energy and carbohydrate metabolism. These organisms possess peculiar peroxisomes, called glycosomes, which play a central role in this metabolism; the organelles harbour enzymes of several catabolic and anabolic routes, including major parts of the glycolytic and pentosephosphate pathways. The kinetoplastid mitochondrion is also unusual with regard to both its structural and functional properties.     

An Efficient Transformation Method to Regenerate a High Number of Transgenic Plants Using a New Embryogenic Line of Medicago truncatula cv. Jemalong

A simple and efficient regeneration–transformation method was established to obtain transgenic plants of the model legume Medicago truncatula cv. Jemalong. This method takes advantage of a new highly embryogenic line (M9-10a) isolated in our laboratory. Leaflets of in vitro grown M9-10a plants were co-cultured with Agrobacterium tumefaciens EHA105. Plasmid constructs containing the oat arginine decarboxylase gene, Adc and the GUS reporter gene (p35SAdc–Gus) or ELIP-like drought stress protein 22 (DSP22) encoding gene from Craterostigma plantagineum (p35SDsp22) were used. Both constructs include the nptII gene as selection marker. Embryogenic calli (100–97%) were obtained on embryo induction medium containing 100 mg l ^–1 kanamycin and 500 mg l^–1 carbenicillin. Using a two-fold increase in kanamycin concentration, instead of 50 mg l^–1 usually used, we reduced the number of emerging false kanamycin-resistant (Kan^R) embryos, which is an important improvement to the method, making it less laborious and very efficient. Isolation of late torpedo/cotyledonary-stage embryos to lower carbenicillin/agar media reduced secondary embryogenesis and prevents hyperhydricity, improving embryo conversion. Primary transformants (T₀) were regenerated within 3–4 months and those that were able to root in a 50 mg l^–1 kanamycin medium were transferred to the greenhouse to produce seeds. Southern blot hybridisation analysis confirmed the integration of either the Adc or Dsp22 transgenes in the genome of the T₀ transformants. Detection of -glucuronidase (GUS) activity in Adc–Gus T₀ plants demonstrated the expression of the inserted transgene. In average, 1–2 independent transgenic lines are obtained per Kan^R embryogenic callus, independently of the plasmid construct used for transformation. Inheritance of the transgenes is shown to be stable in the T₁ generation.Both authors contributed equally to this work.     

POSA: Perl Objects for DNA Sequencing Data Analysis

Background  

Capillary DNA sequencing machines allow the generation of vast amounts of data with little hands-on time. With this expansion of data generation, there is a growing need for automated data processing. Most available software solutions, however, still require user intervention or provide modules that need advanced informatics skills to allow implementation in pipelines.     

Biochemical basis for depressed serum retinol levels in transthyretin-deficient mice

Transthyretin (TTR) acts physiologically in the transport of retinol in the circulation. We previously reported the generation and partial characterization of TTR-deficient (TTR(-)) mice. TTR(-) mice have very low circulating levels of retinol and its specific transport protein, retinol-binding protein (RBP). We have examined the biochemical basis for the low plasma retinol-RBP levels. Cultured primary hepatocytes isolated from wild type (WT) and TTR(-) mice accumulated RBP in their media to an identical degree, suggesting that RBP was being secreted from the hepatocytes at the same rate. In vivo experiments support this conclusion. For the first 11 h after complete nephrectomy, the levels retinol and RBP rose in the circulations of WT and TTR(-) mice at nearly identical rates. However, human retinol-RBP injected intravenously was more rapidly cleared from the circulation (t(12) = 0.5 h for TTR(-) versus t(12) >6 h for WT) and accumulated faster in the kidneys of TTR(-) compared with WT mice. The rate of infiltration of the retinol-RBP complex from the circulation to tissue interstitial fluids was identical in both strains. Taken together, these data indicate that low circulating retinol-RBP levels in TTR(-) mice arise from increased renal filtration of the retinol-RBP complex.     

Dwarfs and Giants: Cannibalism and Competition in Size-Structured Populations

Cannibals and their victims often share common resources and thus potentially compete. Smaller individuals are often competitively superior to larger ones because of size-dependent scaling of foraging and metabolic rates, while larger ones may use cannibalism to counter this competition. We study the interplay between cannibalism and competition using a size-structured population model in which all individuals consume a shared resource but in which larger ones may cannibalize smaller conspecifics. In this model, intercohort competition causes single-cohort cycles when cannibalism is absent. Moderate levels of cannibalism reduce intercohort competition, enabling coexistence of many cohorts. More voracious cannibalism, in combination with competition, produces large-amplitude cycles and a bimodal population size distribution with many small and few giant individuals. These coexisting "dwarfs" and "giants" have very different life histories, resulting from a reversal in importance of cannibalism and competition. The population structure at time of birth determines whether individuals suffer severe cannibalism, with the few survivors reaching giant sizes, or whether they suffer intense intracohort competition, with all individuals remaining small. These model results agree remarkably well with empirical data on perch population dynamics. We argue that the induction of cannibalistic giants in piscivorous fish is a population-dynamic emergent phenomenon that requires a combination of size-dependent cannibalism and competition.     

Oxygenated monoterpenoids from badea (Passiflora quadrangularis) fruit pulp

The new monoterpenoids (2E)-2,6-dimethyl-2,5-heptadienoic acid, (2E)-2,6-dimethyl-2,5-heptadienoic acid beta-D-glucopyranosyl ester, (5E)-2,6-dimethyl-5,7-octadiene-2,3-diol, and (3E)-3,7-dimethyl-3-octene-1,2,6,7-tetrol were isolated from the fruit pulp of Passiflora quadrangularis along with the known 2,5-dimethyl-4-hydroxy-3(2H)-furanone beta-D-glucopyranoside.