Electron donors for biological sulfate reduction

Biological sulfate reduction is widely used for treating sulfate-containing wastewaters from industries such as mining, tannery, pulp and paper, and textiles. In biological reduction, sulfate is converted to hydrogen sulfide as the end product. The process is, therefore, ideally suited for treating metal-containing wastewater from which heavy metals are simultaneously removed through the formation of metal sulfides. Metal sulfide precipitates are more stable than metal hydroxides that are sensitive to pH change. Theoretically, conversion of 1 mol of sulfate requires 0.67 mol of chemical oxygen demand or electron donors. Sulfate rich wastewaters are usually deficient in electron donors and require external addition of electron donors in order to achieve complete sulfate reduction. This paper reviews various electron donors employed in biological sulfate reduction. Widely used electron donors include hydrogen, methanol, ethanol, acetate, lactate, propionate, butyrate, sugar, and molasses. The selection criteria for suitable electron donors are discussed.     

The congenital bicuspid aortic valve can experience high-frequency unsteady shear stresses on its leaflet surface

The bicuspid aortic valve (BAV) is a common congenital malformation of the aortic valve (AV) affecting 1% to 2% of the population. The BAV is predisposed to early degenerative calcification of valve leaflets, and BAV patients constitute 50% of AV stenosis patients. Although evidence shows that genetic defects can play a role in calcification of the BAV leaflets, we hypothesize that drastic changes in the mechanical environment of the BAV elicit pathological responses from the valve and might be concurrently responsible for early calcification. An in vitro model of the BAV was constructed by surgically manipulating a native trileaflet porcine AV. The BAV valve model and a trileaflet AV (TAV) model were tested in an in vitro pulsatile flow loop mimicking physiological hemodynamics. Laser Doppler velocimetry was used to make measurements of fluid shear stresses on the leaflet of the valve models using previously established methodologies. Furthermore, particle image velocimetry was used to visualize the flow fields downstream of the valves and in the sinuses. In the BAV model, flow near the leaflets and fluid shear stresses on the leaflets were much more unsteady than for the TAV model, most likely due to the moderate stenosis in the BAV and the skewed forward flow jet that collided with the aorta wall. This additional unsteadiness occurred during mid- to late-systole and was composed of cycle-to-cycle magnitude variability as well as high-frequency fluctuations about the mean shear stress. It has been demonstrated that the BAV geometry can lead to unsteady shear stresses under physiological flow and pressure conditions. Such altered shear stresses could play a role in accelerated calcification in BAVs.     

A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana

Piriformospora indica, a basidiomycete of the Sebacinaceae family, promotes the growth, development and seed production of a variety of plant species. Arabidopsis plants colonized with the fungus produce 22% more seeds than uncolonized plants. Deactivating the Arabidopsis single-copy gene DMI-1, which encodes an ion carrier required for mycorrihiza formation in legumes, does not affect the beneficial interaction between the two symbiotic partners. We used cellular and molecular responses initiated during the establishment of the interaction between P. indica and Arabidopsis roots to isolate mutants that fail to respond to the fungus. An ethylmethane sulfonate mutant (Piriformospora indica-insensitive-2; pii-2), and a corresponding insertion line, are impaired in a leucine-rich repeat protein (At1g13230). The protein pii-2, which contains a putative endoplasmic reticulum retention signal, is also found in Triton X-100-insoluble plasma membrane microdomains, suggesting that it is present in the endoplasmic reticulum/plasma membrane continuum in Arabidopsis roots. The microdomains also contain an atypical receptor protein (At5g16590) containing leucine-rich repeats, the message of which is transiently upregulated in Arabidopsis roots in response to P. indica. This response is not detectable in At1g13230 mutants, and the protein is not detectable in the At1g13230 mutant microdomains. Partial deactivation of a gene for a sphingosine kinase, which is required for the biosynthesis of sphingolipid found in plasma membrane microdomains, also affects the Arabidopsis/P. indica interaction. Thus, pii-2, and presumably also At5g16590, two proteins present in plasma membrane microdomains, appear to be involved in P. indica-induced growth promotion and enhanced seed production in Arabidopsis thaliana.     

Analysis of chimeric RGS proteins in yeast for the functional evaluation of protein domains and their potential use in drug target validation

For the identification of regulators of G-protein signaling (RGS) modulators, previously, we developed a luciferase based yeast pheromone response (YPhR) assay to functionally investigate RGS4 (K.H. Young, Y. Wang, C. Bender, S. Ajit, F. Ramirez, A. Gilbert, B.W. Nieuwenhuijsen, in: D.P. Siderovski (Ed.), Meth. Enzymol. 389 Regulators of G_protein Signaling, Part A, 2004.). To extend the diversity of this assay, additional RGS proteins were evaluated for functional complementation in a RGS (sst2Delta) knockout yeast strain. For RGS proteins that did not function in their native form, a series of chimeric constructs were generated with the N terminus of RGS4 fused in frame with the partial or full-length RGS cDNA of interest. RGS4 N terminus fused to either full-length or the C terminus of RGS7 successfully complemented sst2Delta. On the contrary, the RGS7N/RGS4C chimera (N terminus of RGS7 in frame with RGS domain of RGS4) was not effective, showing that N terminus of RGS4 helps in targeting. RGS10 exists as two splice variants, differing only by 8 amino acids (aa) in the N terminus, being either 168 aa (RGS10S), or 174 aa (RGS10). While RGS10 was functional in yeast, RGS10S required the presence of the N terminus of RGS4 for its activity. Although the same RGS4 N terminus domain was present in chimeras generated, the GTPase accelerating protein (GAP) function observed was not similar, suggesting differences in the RGS domain function. In conclusion, the use of RGS4 N terminus chimeric constructs enabled us to develop a selectivity assay for different RGS proteins.     

Calreticulin transacetylase catalyzed modification of the TNF-α mediated pathway in the human peripheral blood mononuclear cells by polyphenolic acetates

Polyphenols, coumarin (1,2-benzopyrone) and chromone (1,4-benzopyrone), are naturally occurring constituent of variety of plant species. They have attracted immense interest because of their diverse pharmacological activities. Not much was known about biological activities of acetyl derivative (polyphenolic acetates) of parent polyphenols. In previous investigations, we have conclusively established calreticulin transacetylase catalyzed activation of endothelial nitric oxide synthase (eNOS) by polyphenolic acetates. In the present work, calreticulin transacetylase of human peripheral blood mononuclear cells was characterized with respect to specificity for various polyphenolic acetates and its role in the activation of TNF-α induced nitric oxide synthase (iNOS). Peripheral blood mononuclear cells incubated with a model polyphenolic acetate, 7,8-diacetoxy-4-methylcoumarin (DAMC), along with l-arginine caused activation of NOS. The incubation of peripheral blood mononuclear cells with TNF-α and DAMC resulted in increased production of NO as compared to TNF-α alone. This increased NO production was attenuated by l-Nω-nitro-l-arginine methyl ester (l-NAME), a well known non-specific inhibitor of NOS, and 1400W (N-[3-(aminomethyl) benzyl] acetamidine), a specific inhibitor of human iNOS. These results substantiate the CRTAase catalyzed activation of iNOS. Further, expression of NOS isoforms by semi-quantitative PCR and real-time RT-PCR confirms the preponderance of iNOS in TNF-α treated peripheral blood mononuclear cells over the untreated one. It was also observed that polyphenolic acetates inhibit TNF-α mediated release of IL-6 from peripheral blood mononuclear cells.     

Biochemical characterization of a low molecular weight aspartic protease inhibitor from thermo-tolerant Bacillus licheniformis: Kinetic interactions with Pepsin

The present article reports a low molecular weight aspartic protease inhibitor, API, from a newly isolated thermo-tolerant Bacillus licheniformis. The inhibitor was purified to homogeneity as shown by rp-HPLC and SDS-PAGE. API is found to be stable over a broad pH range of 2–11 and at temperature 90 °C for 2 1/2 h. It has a Mr (relative molecular mass) of 1363 Da as shown by MALDI-TOF spectra and 1358 Da as analyzed by SDS-PAGE .The amino acid analysis of the peptide shows the presence of 12 amino acid residues having Mr of 1425 Da. The secondary structure of API as analyzed by the CD spectra showed 7% α-helix, 49% β-sheet and 44% aperiodic structure. The Kinetic studies of Pepsin–API interactions reveal that API is a slow-tight binding competitive inhibitor with the IC₅₀ and K_i values 4.0 nM and (3.83 nM–5.31 nM) respectively. The overall inhibition constant K_i? value is 0.107 ± 0.015 nM. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I ? (k₄, k₅) EI ? (k₆, k₇) EI?. Rate constant k₆ = 2.73 ± 0.32 s^− 1 reveals a fast isomerization of enzyme–inhibitor complex and very slow dissociation as proved by k₇ = 0.068 ± 0.009 s^− 1. The Rate constants from the intrinsic tryptophanyl fluorescence data is in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI?.     

Proteomic profiling of high glucose primed monocytes identifies cyclophilin A as a potential secretory marker of inflammation in type 2 diabetes

Hyperglycemia is widely recognized to be a potent stimulator of monocyte activity, which is a crucial event in the pathogenesis of atherosclerosis. We analyzed the monocyte proteome for potential markers that would enhance the ability to screen for early inflammatory status in Type 2 diabetes mellitus (T2DM), using proteomic technologies. Monocytic cells (THP-1) were primed with high glucose (HG), their protein profiles were analyzed using 2DE and the downregulated differentially expressed spots were identified using MALDI TOF/MS. We selected five proteins that were secretory in function with the help of bioinformatic programs. A predominantly downregulated protein identified as cyclophilin A (sequence coverage 98%) was further validated by immunoblotting experiments. The cellular mRNA levels of cyclophilin A in various HG-primed cells were studied using qRT-PCR assays and it was observed to decrease in a dose-dependent manner. LC-ESI-MS was used to identify this protein in the conditioned media of HG-primed cells and confirmed by Western blotting as well as ELISA. Cyclophilin A was also detected in the plasma of patients with diabetes. We conclude that cyclophilin A is secreted by monocytes in response to HG. Given the paracrine and autocrine actions of cyclophilin A, the secreted immunophilin could be significant for progression of atherosclerosis in type 2 diabetes. Our study also provides evidence that analysis of monocyte secretome is a viable strategy for identifying candidate plasma markers in diabetes.     

In vitro hemodynamic investigation of the embryonic aortic arch at late gestation

This study focuses on the dynamic flow through the fetal aortic arch driven by the concurrent action of right and left ventricles. We created a parametric pulsatile computational fluid dynamics (CFD) model of the fetal aortic junction with physiologic vessel geometries. To gain a better biophysical understanding, an in vitro experimental fetal flow loop for flow visualization was constructed for identical CFD conditions. CFD and in vitro experimental results were comparable. Swirling flow during the acceleration phase of the cardiac cycle and unidirectional flow following mid-deceleration phase were observed in pulmonary arteries (PA), head-neck vessels, and descending aorta. Right-to-left (oxygenated) blood flowed through the ductus arteriosus (DA) posterior relative to the antegrade left ventricular outflow tract (LVOT) stream and resembled jet flow. LVOT and right ventricular outflow tract flow mixing had not completed until approximately 3.5 descending aorta diameters downstream of the DA insertion into the aortic arch. Normal arch model flow patterns were then compared to flow patterns of four common congenital heart malformations that include aortic arch anomalies. Weak oscillatory reversing flow through the DA junction was observed only for the Tetralogy of Fallot configuration. PA and hypoplastic left heart syndrome configurations demonstrated complex, abnormal flow patterns in the PAs and head-neck vessels. Aortic coarctation resulted in large-scale recirculating flow in the aortic arch proximal to the DA. Intravascular flow patterns spatially correlated with abnormal vascular structures consistent with the paradigm that abnormal intravascular flow patterns associated with congenital heart disease influence vascular growth and function.