Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: modulation of innate immune system and variations in carbohydrate structure

The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2-/- and TLR4-/-) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-β, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages.     

The mammalian CHORD-containing protein melusin is a stress response protein interacting with Hsp90 and Sgt1

Melusin is a mammalian muscle specific CHORD containing protein capable of activating signal transduction pathways leading to cardiomyocytes hypertrophy in response to mechanical stress. To define melusin function we searched for molecular partners possibly involved in melusin dependent signal transduction. Here we show that melusin and heat shock proteins are co-regulated. Moreover, melusin directly binds to Hsp90, a ubiquitous chaperone involved in regulating several signaling pathways. In addition, melusin interacts with Sgt1, an Hsp90 binding molecule. Melusin does not behave as an Hsp90 substrate but rather as a chaperone capable to protect citrate synthase from heat induced aggregation. These results describe melusin as a new component of the Hsp90 chaperone machinery.     

Development of an online biosensor for in situ monitoring of chlorine dioxide gas disinfection efficacy

A prototype bioluminescence-based biosensor was designed and constructed to evaluate the antimicrobial efficacy of chlorine dioxide (ClO₂) gas under various treatment conditions. The biosensor consisted of a bioluminescent bioreporter (Pseudomonas fluorescens 5RL), an optical transducer (photomultiplier tube), and a light-tight chamber housing, the bioreporter and the transducer. The bioluminescent recombinant P. fluorescens 5RL in the biosensor allowed for online monitoring of bioluminescence during ClO₂ gas disinfection. Experiments were performed to evaluate the effects of the two key physical parameters associated with ClO₂ disinfection: relative humidity (40, 60, 80%) and ClO₂ gas concentration (0.5, 1.0, 1.6, 2.1 mg/l) on the bioreporter. Results showed that increasing concentrations of ClO₂ gas corresponded to a faster decrease in luminescence. The rates of luminescence decrease from P. fluorescens 5RL, and the log reduction time (LRT, time required to obtain 1-log reduction in luminescence) were calculated for each treatment tested. The LRT values of luminescence were 103, 78, 53, and 35 s for 0.5, 1.0, 1.6, and 2.1 mg/l of ClO₂ gas treatment, respectively, at 78% relative humidity. The gas concentration which caused a tenfold change in LRT (z value) for luminescence of P. fluorescens 5RL was 3.4 mg/l of ClO₂. The prototype biosensor showed potential for many applications, such as monitoring real-time microbial inactivation and understanding parameters that influence the efficacy of gaseous decontamination procedures. This paper is journal article # 2008-18279 of the Purdue University Agricultural Research Program.     

Microbial Community Analysis of Soils Contaminated with Lead, Chromium and Petroleum Hydrocarbons

The impact on the microbial community of long-term environmental exposure to metal and organic contamination was investigated. Twenty-four soil samples were collected along a transect dug in soils contaminated with road paint and paint solvents, mainly toluene. Chemical analysis along the transect revealed a range from high to low concentrations of metals (lead and chromium) and organic solvent compounds. Principal components analysis of microbial community structure based on denaturing gradient gel electrophoresis of the V3 region of the 16S rRNA gene and fatty acid methyl esters derived from phospholipids (phospholipid fatty acid analysis) showing samples with similar fingerprints also had similar contaminant concentrations. There was also a weak positive correlation between microbial biomass and the organic carbon concentration. Results indicated that microbial populations are present despite some extreme contaminant levels in this mixed-waste contaminated site. Nucleotide sequence determination of the 16S rRNA gene indicated the presence of phylogenetically diverse bacteria belonging to the α-, β-, γ-, and δ-Proteobacteria, the high and low G + C Gram-positive bacteria, green nonsulfur, OP8, and others that did not group within a described division. This indicates that soils contaminated with both heavy metals and hydrocarbons for several decades have undergone changes in community composition, but still contain a phylogenetically diverse group of bacteria (including novel phylotypes) that warrant further investigation.     

Salmonella from gopher tortoises (Gopherus polyphemus) in south Georgia

From 2002 to 2006, gopher tortoises (Gopherus polyphemus) were collected at Moody Air Force Base, Lowndes/Lanier counties, Georgia, USA, and opportunistically surveyed for the presence of Salmonella species. Four of 155 (2.6%) cloacal swabs collected from 80 tortoises were positive for the presence of Salmonella enterica, and the following serovars were identified: Give, Hartford, Javiana, and Luciana. Female tortoises (5%) were infected at a rate similar to male tortoises (5%). All isolates were obtained from adult tortoises (n = 73); subadults (n = 7) were all negative. Each isolated serovar is a potential human pathogen, suggesting appropriate precautions should be emphasized when handling these animals.     

Integrated stratigraphy and 40Ar/39Ar chronology of early Middle Miocene sediments from DSDP Leg 42A,Site 372 (Western Mediterranean)

An integrated magneto-biostratigraphic framework is presented for Middle Miocene sediments of DSDP Site 372 located in the Western Mediterranean. Detailed biostratigraphic analysis shows a nearly complete sequence of early Middle Miocene calcareous plankton bioevents in the Mediterranean, including the LCO (Last Common Occurrence) of the nannofossil Sphenolithus heteromorphus which has been astronomically dated in the Ras il Pellegrin (RIP) section on Malta Island [Abels, H.A., Hilgen, F.J., Krijgsman, W., Kruk, R.W., Raffi, I., Turco, E., Zachariasse, W.J., 2005. Long-period orbital control on middle Miocene global cooling: integrated stratigraphy and astronomical tuning of the Blue Clay Formation on Malta, Paleoceanography, 20, PA4012. doi: 10.1029/2004PA001129. 11 pp]. Thermal demagnetization of discrete samples revealed a characteristic low-temperature component with dual polarities despite a weak paleomagnetic signal. The resultant magnetostratigraphic record, combined with the calcareous plankton biostratigraphy, is straightforwardly correlated to the geomagnetic polarity time scale (CK95) of Cande and Kent [Cande, S.C., Kent, D.V., 1995. Revised calibration of the Geomagnetic Polarity Time Scale for the Late Cretaceous and Cenozoic, J. Geophys. Res., 100, 6093–6095] and the Astronomical Tuned Neogene Time Scale (ATNTS04) of Lourens et al. [Lourens, L.J., Hilgen, F.J., Laskar, J., Shackleton, N.J., Wilson, D., 2004. The Neogene Period. In: Gradstein F.M., Ogg J.G., Smith A.G. (Eds.), A Geologic Time Scale, Cambridge Univ. Press, pp. 409–440]. The subchrons recorded in Site 372 succession range from C5Br up to C5AAr. To confirm the magnetostratigraphic calibration, ⁴⁰Ar/³⁹Ar dating was performed on feldspar of two volcanic ash layers. The radio-isotopic dating indicates a younger age for these two ash layers compared to the magnetostratigraphic calibrated ages according to the CK95 and ATNTS04 age models. However, if the astronomically calibrated age of 28.21 ± 0.04 Ma is used for the Fish Canyon standard (FCs), the age for the older ash layer exactly matches its ATNTS04 age. Ages for bioevents were calculated assuming constant sedimentation rates between magnetostratigraphic age-tie points. The S. heteromorphus LCO has an age of 13.54 Ma and 13.63 Ma according to CK95 and ATNTS04, respectively, which is consistent with the astronomical tuned age of 13.65 Ma determined at RIP section [Abels, H.A., Hilgen, F.J., Krijgsman, W., Kruk, R.W., Raffi, I., Turco, E., Zachariasse, W.J., 2005. Long-period orbital control on middle Miocene global cooling: integrated stratigraphy and astronomical tuning of the Blue Clay Formation on Malta, Paleoceanography, 20, PA4012. doi:10.1029/2004PA001129. 11 pp]. We therefore conclude that the magnetostratigraphic calibration of DSDP Site 372 is correct and that, for the time being, this site can be considered as a reference section for the early Middle Miocene of the Mediterranean region.     

Potential risk of biologic pollution associated to the introduction of <i>Pinus radiata</i> in grassland areas

Afforestation is a recommended practice to mitigate global warming. However, their implementation may generate undesirable impacts, mostly if exotic species are used. Plantations of Pinus radiata D Don in Ventania (Bs. As., Argentina) soils showed notorious increments of extractable P (Pe), which could affect the dynamic of this element as well as the degree of phosphorus saturation (GSP_Bray). The objectives of this study were: i) to quantify the GSP_Bray in Mollisols afforested with P. radiata comparing the results with those coming from adjacent, natural grassland areas (base line); ii) to evaluate the potential environmental risk induced by afforestation through the identification of a change point (PC) in the GSP_Bray indicative of a phosphate leaching increment. Treatments included mature stands of P. radiata (TB) and adjacent areas with natural grassland vegetation (TP). Samples were taken at 0-15; 15-30 and 30-45 cm soil depth, and texture, pH, total organic carbon (COT), Pe, soluble reactive phosphorus (PSR), phosphorus sorption index (ISP) and GSP_Bray were determined. The results showed a significant acidification in TB and an increase in the COT stock, indicating an additional atmospheric CO₂ sequestration by the trees. The Pe and PSR values were notoriously higher in TB, and they were reflected in a significant increment in the GSP_Bray with respect to TP. The detection of a significant PC in the GSP_Bray-PSR regression indicates higher chances of phosphate leaching in the forest stands, which could reach water courses, lakes and artificial reservoirs promoting their eutrophication. Because of the potential environmental pollution risk of biologic origin derived from the afforestation with P. radiata in Mollisols areas, their inclusion in clean development practices must be reconsidered.     

Association of Microbial Community Composition and Activity with Lead, Chromium, and Hydrocarbon Contamination

Microbial community composition and activity were characterized in soil contaminated with lead (Pb), chromium (Cr), and hydrocarbons. Contaminant levels were very heterogeneous and ranged from 50 to 16,700 mg of total petroleum hydrocarbons (TPH) kg of soil⁻¹, 3 to 3,300 mg of total Cr kg of soil⁻¹, and 1 to 17,100 mg of Pb kg of soil⁻¹. Microbial community compositions were estimated from the patterns of phospholipid fatty acids (PLFA); these were considerably different among the 14 soil samples. Statistical analyses suggested that the variation in PLFA was more correlated with soil hydrocarbons than with the levels of Cr and Pb. The metal sensitivity of the microbial community was determined by extracting bacteria from soil and measuring [³H]leucine incorporation as a function of metal concentration. Six soil samples collected in the spring of 1999 had IC₅₀ values (the heavy metal concentrations giving 50% reduction of microbial activity) of approximately 2.5 mM for CrO₄²⁻ and 0.01 mM for Pb²⁺. Much higher levels of Pb were required to inhibit [¹⁴C]glucose mineralization directly in soils. In microcosm experiments with these samples, microbial biomass and the ratio of microbial biomass to soil organic C were not correlated with the concentrations of hydrocarbons and heavy metals. However, microbial C respiration in samples with a higher level of hydrocarbons differed from the other soils no matter whether complex organic C (alfalfa) was added or not. The ratios of microbial C respiration to microbial biomass differed significantly among the soil samples (P < 0.05) and were relatively high in soils contaminated with hydrocarbons or heavy metals. Our results suggest that the soil microbial community was predominantly affected by hydrocarbons.