Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Research into the Rhizobium/Leguminosae symbiosis in Latin America

More than 60 institutions and 100 researchers were involved in Rhizobium research in 1978 in Latin America. Half of these researchers were located in Argentina and Brazil. Research activity and the application of research findings vary widely among countries. Problems that plague research include 1) inadequate training of research personnel and insufficient attention paid to the Rhizobium/Legume symbiosis at agriculture schools; 2) poorly-established research priorities that do not sufficiently weigh the immediate needs for the farmers such as the identification of limiting environmental factors (e.g. nutritional deficiencies), techniques for smallscale inoculant production, and quality control of available inoculants; 3) isolation of the researchers and a lack of adequate library support; 4) poorly integrated research teams (e.g. in many institutes researchers are either microbiologists with no agricultural background or agronomists lacking microbiological training); and 5) insufficient dissemination of research findings. Problems with inoculant production and control include 1) a local dependence on national or imported inoculants rather than on locally-selected strains, 2) poor inoculant quality control which results in low inoculation success rates and subsequent discredit to the inoculation practice, and 3) high prices for inoculants. Extension problems include 1) lacking or deficient legume-promotion programs by government agencies, 2) poor contact between research and extension workers, and 3) administrators, leaders, extension workers and agronomists working in the field that lack adequate knowledge of the Rhizobium/Legume symbiosis. Immediate measures to foster extension and legume promotion programs and informal and/or official quality control are needed in Argentina, Uruguay, Brazil, Mexico, and probably Colombia. Countries where combined efforts should primarily be directed toward stimulating research and extension include Peru, Venezuela, Costa Rica, and Chile. In Ecuador, Paraguay, Bolivia, Nicaragua, Honduras, Guatemala, the Dominican Republic and Panama, priority should be given to research. Colombia should also be included in this group as national research institutions need to be strengthened. Table 2 lists these priorities more fully.     

Reliability of FEV1/FEV6 to Diagnose Airflow Obstruction Compared with FEV1/FVC: The PLATINO Longitudinal Study

QUESTION

A 6-second spirometry test is easier than full exhalations. We compared the reliability of the ratio of the Forced expiratory volume in 1 second/Forced expiratory volume in 6 seconds (FEV₁/FEV₆) to the ratio of the FEV₁/Forced vital capacity (FEV₁/FVC) for the detection of airway obstruction.

METHODS

The PLATINO population-based survey in individuals aged 40 years and over designed to estimate the prevalence of post-Bronchodilator airway obstruction repeated for the same study participants after 5–9 years in three Latin-American cities.

RESULTS

Using the FEV₁/FVC<Lower limit of normal (LLN) index, COPD prevalence apparently changed from 9.8 to 13.2% in Montevideo, from 9.7 to 6.0% in São Paulo and from 8.5 to 6.6% in Santiago, despite only slight declines in smoking prevalence (from 30.8% to 24.3%). These changes were associated with differences in Forced expiratory time (FET) between the two surveys. In contrast, by using the FEV₁/FEV₆ to define airway obstruction, the changes in prevalence were smaller: 9.7 to 10.6% in Montevideo, 8.6 to 9.0% in São Paulo, and 7.5 to 7.9% in Santiago. Changes in the prevalence of COPD with criteria based on FEV₁/FVC correlated strongly with changes in the FET of the tests (R² 0.92) unlike the prevalence based on a low FEV₁/FEV₆ (R² = 0.40).

CONCLUSION

The FEV₁/FEV₆ is a more reliable index than FEV₁/FVC because FVC varies with the duration of the forced exhalation. Reporting FET and FEV₁/FEV₆<LLN helps to understand differences in prevalence of COPD obtained from FEV₁/FVC-derived indices.     

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2′-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent K_m of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.     

Liver-targeting of primaquine-(poly-γ-glutamic acid) and its degradation in rat hepatocytes

We have synthesized poly-γ-glutamic acid (PGA) modified with a synthetic trivalent glyco-ligand (TriGalNAc) for the hepatocyte asialoglycoprotein receptor (ASGP-R). We investigated in vivo distribution of unmodified PGA and TriGalNAc-modified PGA (TriGalNAc-PGA) in mice after intravenous injection. Most of unmodified PGA administered was transported to the bladder over 20–80 min, suggesting a rapid excretion of unmodified PGA into urine. In contrast, TriGalNAc-PGA was found exclusively in the liver over the same period of time. We further synthesized TriGalNAc-PGA–primaquine conjugate (TriGalNAc-PGA–PQ), and investigated binding, uptake, and catabolism of the conjugate by rat hepatocytes. Our studies indicated that approximately 250 ng per million cells of the conjugate bound to one million rat hepatocytes at 0 °C, and approximately 2 μg per million cells of the conjugate was taken up over 7 h incubation at 37 °C. Furthermore, our results suggested that TriGalNAc-PGA–PQ was almost completely degraded over 24 h, and small degradation products were secreted into cell culture medium.The results described in this report suggest that the TriGalNAc ligand can serve as an excellent targeting device for delivery of PGA-conjugates to the liver hepatocytes, and rat hepatocytes possess sufficient capacity to digest PGA even modified with other substituents.     

Dynamics of Cattle Production in Brazil

Movement of livestock production within a country or region has implications for genetics, adaptation, well-being, nutrition, and production logistics, particularly in continental-sized countries, such as Brazil. Cattle production in Brazil from 1977 to 2011 was spatialized, and the annual midpoint of production was calculated. Changes in the relative production and acceleration of production were calculated and spatialized using ARCGIS®. Cluster and canonical discriminant analyses were performed to further highlight differences between regions in terms of cattle production. The mean production point has moved from the Center of Minas Gerais State (in the southeast region) to the North of Goiás State (in the Midwest region). This reflects changes in environmental factors, such as pasture type, temperature and humidity. Acceleration in production in the northern region of Brazil has remained strong over the years. More recently, “traditional” cattle-rearing regions, such as the south and southeast, showed a reduction in growth rates as well as a reduction in herd size or internal migration over the period studied. These maps showed that this movement tends to be gradual, with few regions showing high acceleration or deceleration rates.     

Comparative genomics of grasses tolerant to aluminum

The family Poaceae includes over 10,000 species, among which are the most economically important cereals: maize, sorghum, rice, wheat, rye, barley, and oat. These cereals are very important components of human and animal food. Although divergence of the members of this family occurred about 40 million years ago, comparative genome analyses demonstrated that gene orders among species of this family remain largely conserved, which can be very useful for understanding their roles and evolution. Even with an intricate evolutionary history in which chromosome fragments, losses and duplications have to be considered at the ploidy level, grasses present a genetic model system for comparative genomics. The availability of mapped molecular markers, rice genome sequences and BAC and EST libraries from several grass species, such as rice, wheat, sorghum, and maize, facilitates biology and phylogeny studies of this group. The value of using information from different species in modern plant genetics is unquestionable, especially in the study of traits such as tolerance to aluminum in soils, which affects plant growth and development. Comparative genomic approaches to aluminum tolerance can identify genomic regions and genes responsible for aluminum tolerance in grasses.     

The influence of temperature and length of time of storage of frog mucus samples

Frozen, stored mucus has been extensively used for transport studies but there is no clear evidence of the influence that the temperature and length of time of storage may have on the results. We stored frog mucus samples at -20 and -80 degrees C and analysed them on days zero, 2, 10, 30 and 90. At each temperature, a sample was thawed, studied and refrozen on each of the study days, at the same time that one sample was thawed only on the study day. Displacement in a simulated cough machine and on the frog palate, as well as contact angle measurements, were determined for the mucus samples on each study day. Mucus cytologic analyses on each of the study days were done with special regard to neutrophil counts and cell integrity. Friedman analysis of variance did not show any difference between the different periods of storage and the two temperatures for any of the parameters studied. The medians for the relative transport velocity on the frog palate varied between 0.88 and 1.03, for the contact angle between 21 and 28 degrees, and for the displacement in the simulated cough machine between 58 and 95 mm over the 90 days of the experiment. There were no cytologic alterations compatible with cell degeneration. We conclude that the storage of frog mucus either at -20 or -80 degrees C for periods up to 90 days does not lead to any significant differences in mucus transportability.