Effect of organic solvents on the activity and stability of an extracellular protease secreted by the haloalkaliphilic archaeon <Emphasis Type="Italic">Natrialba magadii</Emphasis>

The effect of various organic solvents on the activity and stability of an extracellular protease produced by the haloalkaliphilic archaeon Natrialba magadii was tested. This protease was active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propylenglycol, and dioxane. Among the solvents tested, DMSO, propylenglycol, and glycerol were effective in preserving enzyme stability in suboptimal NaCl concentrations. The stabilizing effect of DMSO on this haloalkaliphilic protease was more efficient at pH 8 than at pH 10, suggesting that DMSO may not substitute for salt to allow halophilic proteins to withstand the effect of high pH values. These results show that Nab. magadii extracellular protease is a solvent tolerant enzyme and suggest a potential application of this haloalkaliphilic protease in aqueous-organic solvent biocatalysis.     

Calcium Ions Promote Superoxide Dismutase 1 (SOD1) Aggregation into Non-fibrillar Amyloid: A LINK TO TOXIC EFFECTS OF CALCIUM OVERLOAD IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)?*

Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca²⁺ dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca²⁺. We show that at physiological pH, Ca²⁺ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca²⁺ boosts the onset of SOD1 aggregation. In agreement, Ca²⁺ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca²⁺ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca²⁺-induced aggregates, thus indicating that Ca²⁺ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca²⁺ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca²⁺ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.     

c-Mos and cyclin B/cdc2 connections during Xenopus oocyte maturation.

Fully-grown G2 arrested Xenopus oocytes can be induced to enter and progress into meiotic cell cycle by progesterone stimulation. This process is termed oocyte maturation. An early response to progesterone is the synthesis of the onco-protein c-Mos, defined as the candidate initiator of Xenopus oocyte maturation, which triggers the MAPK cascade, MPF activation and promotes CSF activity. Here we review our current knowledge on the synthesis, activation and functions of c-Mos in connection with MPF activation during maturation. We also discuss our recent results concerning the dispensability of cyclin B degradation in meiosis I-meiosis II transition and the stabilization of c-Mos through its direct phosphorylation by cyclin B/cdc2.     

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one (n = 31 recipients) or two (n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127=17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo (P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 microg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.     

Treatment of industrial wastewater with two-stage constructed wetlands planted with Typha latifolia and Phragmites australis

Industrial wastewater treatment comprises several processes to fulfill the discharge permits or to enable the reuse of wastewater. For tannery wastewater, constructed wetlands (CWs) may be an interesting treatment option. Two-stage series of horizontal subsurface flow CWs with Phragmites australis (UP series) and Typha latifolia (UT series) provided high removal of organics from tannery wastewater, up to 88% of biochemical oxygen demand (BOD₅) (from an inlet of 420 to 1000 mg L⁻¹) and 92% of chemical oxygen demand (COD) (from an inlet of 808 to 2449 mg L⁻¹), and of other contaminants, such as nitrogen, operating at hydraulic retention times of 2, 5 and 7 days. No significant (P < 0.05) differences in performance were found between both the series. Overall mass removals of up to 1294 kg COD ha⁻¹ d⁻¹ and 529 kg BOD₅ ha⁻¹ d⁻¹ were achieved for a loading ranging from 242 to 1925 kg COD ha⁻¹ d⁻¹ and from 126 to 900 kg BOD₅ ha⁻¹ d⁻¹. Plants were resilient to the conditions imposed, however P. australis exceeded T. latifolia in terms of propagation.     

Symptoms of nitrogen saturation in an aggrading forested watershed in western Maryland

The objective of this study was to evaluate the nitrogen (N) biogeochemistry of an 18–22 year old forested watershed in western Maryland. We hypothesized that this watershed should not exhibit symptoms of N saturation. This watershed was a strong source of nitrate (NO₃ ⁻) to the stream in all years, with a mean annual export of 9.5 kg N ha⁻¹ year⁻¹ and a range of 4.4–18.4 kg N ha⁻¹ year⁻¹. During the 2001 and 2002 water years, wet deposition of inorganic N was 9.0 kg N ha⁻¹ year⁻¹ and 6.3 kg N ha⁻¹ year⁻¹, respectively. Watershed N export rates in 2001 and 2002 water years were 4.2 kg N ha⁻¹ year⁻¹ and 5.3 kg N ha⁻¹ year⁻¹, respectively. During the wetter water years of 2003 and 2004, the watershed exported 15.0 kg N ha⁻¹ year⁻¹ and 18.4 kg N ha⁻¹ year⁻¹, rates that exceeded annual wet deposition of N by a factor of two (7.5 kg N ha⁻¹ year⁻¹ in 2003) and three (5.5 kg N ha⁻¹ year⁻¹ in 2004). Consistent with the high rates of N export, were high concentrations (2.1–3.3%) of N in foliage, wood (0.3%) and fine roots, low C:N ratios in the forest floor (17–24) and mineral soil (14), high percentages (83–96%) of the amount of mineralized N that was nitrified and elevated N concentrations (up to 3 mg N l⁻¹) in soil solution. Although this watershed contained a young aggrading forest, it exhibited several symptoms of N saturation commonly observed in more mature forests.     

Functional analysis of the protein machinery required for transport of lipopolysaccharide to the outer membrane of Escherichia coli

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.     

Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic Escherichia coli Infection in Developing World: Defining the Biomarker,Antibody and Method

Background

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.

Methodology

First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.

Principal findings

EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.

Conclusion

RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.