Proposal for a method to estimate nutrient shock effects in bacteria

ABSTRACT: BACKGROUND: Plating methods are still the golden standard in microbiology; however, some studies have shown that these techniques can underestimate the microbial concentrations and diversity. A nutrient shock is one of the mechanisms proposed to explain this phenomenon. In this study, a tentative method to assess nutrient shock effects was tested. FINDINGS: To estimate the extent of nutrient shock effects, two strains isolated from tap water (Sphingomonas capsulata and Methylobacterium sp.) and two culture collection strains (E. coli CECT 434 and Pseudomonas fluorescens ATCC 13525) were exposed both to low and high nutrient conditions for different times and then placed in low nutrient medium (R2A) and rich nutrient medium (TSA). The average improvement (A.I.) of recovery between R2A and TSA for the different times was calculated to more simply assess the difference obtained in culturability between each medium. As expected, A.I. was higher when cells were plated after the exposition to water than when they were recovered from high-nutrient medium showing the existence of a nutrient shock for the diverse bacteria used. S. capsulata was the species most affected by this phenomenon. CONCLUSIONS: This work provides a method to consistently determine the extent of nutrient shock effects on different microorganisms and hence quantify the ability of each species to deal with sudden increases in substrate concentration.     

Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection

Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H(+)ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.     

Diagnosis of Helicobacter pylori

The different invasive and noninvasive diagnostic tests for Helicobacter pylori have been applied mainly in emerging countries. Molecular methods have been developed, especially a test for detection of H. pylori and its clarithromycin resistance directly from stools. The long-term effects of eradication on histologic lesions have been studied in a meta-analysis and the prognostic value of post-treatment in gastric mucosa-associated lymphoid tissue lymphoma has been assessed. An operating link for gastritis assessment (the OLGA staging) has also been published. Attempts to simplify the urea breath test protocol have been made, and new stool antigen tests have been proposed and compared to those previously available.     

A new species of Laranda Walker 1869 (Orthoptera: Grylloidea, Phalangopsidae) from remnant patches of the Brazilian Atlantic Forest

The genus Laranda has six described species and is confined to South and Southeast of Brazil. We describe a new species and discuss the biology and distribution of the genus. The new species can be distinguished from its known congeners by the following characteristics: absence of yellow spots on pronotum and base of posterior tibiae; female copulatory papilla: sclerotization in dorsal view forming opposing acute angles, apical lobes narrow and small; male genitalia: pseudepiphallic median process short and wide; pseudepiphallic paramere with apex incurved and ectophallic fold surpassing apex of the parameres. The genus is distributed within the Atlantic Forest biome; the new species is found on tree trunks, as well as on forest leaf litter.     

Characterization and biotechnological application of an acid alpha-galactosidase from Tachigali multijuga Benth. seeds

Tachigali multijuga Benth. seeds were found to contain protein (364 mg g(-1)dwt), lipids (24 mg g(-1)dwt), ash (35 mg g(-1)dwt), and carbohydrates (577 mg g(-1)dwt). Sucrose, raffinose, and stachyose concentrations were 8.3, 3.0, and 11.6 mg g(-1)dwt, respectively. alpha-Galactosidase activity increased during seed germination and reached a maximum level at 108 h after seed imbibition. The alpha-galactosidase purified from germinating seeds had an M(r) of 38,000 and maximal activity at pH 5.0-5.5 and 50 degrees C. The enzyme was stable at 35 degrees C and 40 degrees C, but lost 79% of its activity after 30 min at 50 degrees C. The activation energy (E(a)) values for p-nitrophenyl-alpha-d-galactopyranoside (pNPGal) and raffinose were 13.86 and 4.75 kcal mol(-1), respectively. The K(m) values for pNPGal, melibiose, raffinose, and stachyose were 0.45, 5.37, 39.62 and 48.80 mM, respectively. The enzyme was sensitive to inhibition by HgCl(2), SDS, AgNO(3), CuSO(4), and melibiose. d-Galactose was a competitive inhibitor (K(i)=2.74 mM). In addition to its ability to hydrolyze raffinose and stachyose, the enzyme also hydrolyzed galactomannan.     

Trichothecene-induced cytotoxicity on human cell lines

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC₅₀). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100–1,000, and the corresponding IC₅₀ values were in the range from 2.2 nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900 nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC₅₀ values were 4.4–10.8 nmol/l for T-2 toxin, 7.5–55.8 mol/l for HT-2 toxin, 600–4,900 nmol/l for DON, 300–2,600 nmol/l for NIV, and 2.2–18.3 nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC₅₀ values ranging from 16.5 nmol/l (T-2 toxin) to 4,500 nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.     

Habitat Impoverishment and Egg Predation by <Emphasis Type="Italic">Alouatta caraya</Emphasis>

Species response to habitat loss, fragmentation, or alteration ranges from intolerance and extinction to tolerance and population growth. An ability to increase trophic niche breadth is likely to play a key role in promoting tolerance to habitat change in many species. Howlers (Alouatta spp.) are good examples of these tolerant species. Researchers have related their capacity to thrive well in disturbed habitats to their ability to exploit an eclectic vegetarian diet. Despite >50,000 h of observation of habituated free-ranging groups throughout the distribution of Alouatta, no case of intentional ingestion of animal matter has ever been observed. Here, we report an unexpected trophic niche broadening for free-ranging groups of black-and-gold howlers (Alouatta caraya) living in small (≤2 ha) impoverished habitat islands in the State of Rio Grande do Sul, Brazil. We studied 3 isolated social groups (15–17, 12–14, and 5 individuals) and observed 1 of them preying on birds’ nests. We recorded 19 events of egg-eating during 2274 h of observation in 1 group and 2 suspected cases in another. Our findings highlight the dietary flexibility that characterize howlers and contrast with the widely held view that they observe a strictly vegetarian diet.     

Anti-Inflammatory Thalidomide Improves Islet Grafts Survival and Functions in a Xenogenic Environment

Thalidomide possesses both anti-inflammatory and anti-angiogenic properties. This study investigates its potential application in islet transplantation with a xenogenic transplantation model. Transplantation was performed using C57Bl/6 mice and NMRI nu/nu mice as recipients of porcine islets. Moreover, islet graft vasculature and inflammation were investigated to identify the mechanisms of thalidomide action. In the immunocompetent environment of C57Bl/6 mice, a fast graft rejection was observed. The group treated with thalidomide 200 mg/kg BW per day achieved and maintained euglycemia in the complete observation period for 42 days. The treated mice had more functional islet graft mass with less leukocyte infiltration. The pro-inflammatory TNF-α and VEGF content in islet grafted kidneys was significantly lowered by the treatment. By comparison, thalidomide was not effective in improving graft survival in immunocompromised nude mice. It strongly inhibited the VEGF and TNF-α-induced endothelial proliferation of isolated pig islets in a dose dependent manner. The magnitude of thalidomide''s inhibitory effect was nearly identical to the effect of VEGF- receptor 2 inhibitor SU416 and anti-TNF-receptor 1 neutralizing antibody, and was reversed by sphingosine-1-phosphate. In conclusion, the anti-inflammatory effect of thalidomide improved islet graft survival and function in a transplantation model with a maximum immune barrier.