Assessment of Heavy Metals and Metalloids in Solanum tuberosum and Pisum sativum Irrigated with Urban Wastewater in the Suburbs of Sargodha City,Pakistan

Dietary exposure to heavy metals (viz., Ni, As, Fe, Cr, Mn, Co, Mo, Cu, Zn, Se, Cd, and Pb) has been recognized as a potential hazard to human health. This study investigates the level of contamination at two different sites in Pakistan, one irrigated with canal water (Site-I) and the other with urban wastewater (Site-II). At Site-II, irrigation with wastewater resulted in a significant increase in heavy metals and metalloids in soil and a subsequent build-up in two vegetables selected for study (Solanum tuberosum [potato] and Pisum sativum [pea]). Results showed that mean concentrations of heavy metals and metalloids in soil at Site-I were lower than those of Site-II. Mean concentrations of As and Cd in soil at both sites and for both vegetables were found above maximum permissible levels, while for both vegetables As at both sites and Cd, Mo, and Pb exceeded the suggested maximum levels for vegetables. High levels of some metals in the soils and vegetables could be due to unnecessary use of fertilizers and disposable water for irrigating the soils and the environmental cues prevalent in the areas, such as presence of ions that may bind the metals, often play an important role in uptake.     

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 ??-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.     

Removal of chromium on Polyalthia longifolia leaves biomass

Adsorption is an environmental friendly process for removal and/or recovery of heavy metals from wastewater. In recent years, it has been substantiated as a popular technique to treat industrial waste effluents, with significant advantages. In this work, batchwise removal of chromium (III) ions from water by Polyalthia longifolia leaves was studied as a function of adsorbent dose, pH, contact time, and agitation speed. Surface characteristics of the leaves were evaluated by recording IR spectra. The Langmuir, Freundlich, and Temkin adsorption isotherms were employed to explain the sorption process. It was found that one gram of leaves can remove 1.87 mg of trivalent chromium when working at pH 3.0. It has been concluded that Polyalthia longifolia leaves can be used as cost-effective and benign adsorbents for removal of Cr(III) ions from wastewater.     

Proteomic approaches to identifying carbonylated proteins in brain tissue

Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.     

Identification of haptoglobin and apolipoprotein A-I as biomarkers for high altitude pulmonary edema

We have investigated the plasma proteome using 2D gel electrophoresis and matrix-assisted laser desorption/ionization tandem time of flight from patients with high altitude pulmonary edema (HAPE). A complete proteomic analysis was performed on 20 patients with HAPE and ten healthy sea level controls. In total, we have identified 25 protein spots in human plasma and found that 14 of them showed altered changes in HAPE patients, which mainly were acute phase proteins (APPs), compliment components, and apolipoproteins among others. Among the APPs, haptoglobin α2 chain, haptoglobin β chain, transthyretin, and plasma retinol binding precursor showed overexpression in HAPE patients as compared to controls. To validate the result of proteomic analysis, two proteins were selected for enzyme-linked immunosorbent assay and Western blotting analysis. Our data conclusively shows that two proteins, haptoglobin and apolipoprotein A-I are upregulated in plasma of HAPE patients. These proteins may provide a fast and effective control of inflammatory damage until the subsequent mechanisms can begin to operate. Taken together, our findings further support the hypothesis that inflammatory response system is linked to the pathophysiology of HAPE.     

Defining species in Tulasnella by correlating morphology and nrDNA ITS-5.8S sequence data of basidiomata from a tropical Andean forest

The genus Tulasnella comprises important orchid mycobionts. Molecular phylogenetic studies on nrITS-5.8S sequences of Tulasnella species previously isolated from mycorrhizas of epiphytic orchids from a tropical Andean forest showed genomic variability among clones which was difficult to interpret as intra- or interspecific variations or to correlate with described Tulasnella species. To improve this situation, we collected basidiomata of Tulasnella in an Andean forest, studied part of the sequences of fungal ribosomal genes and correlated molecular data with the morphology of the specimens. Within five basidiomata displaying slight morphological variability, we found inter-specimen nrITS1-5.8S-ITS2 variability corresponding to proportional differences of less than 1% except for one clone with 5.1% divergence. Results indicate that the slightly variable basidiomata should be considered as one species, which is morphologically tentatively assigned to the Tulasnella pruinosa complex. However, comparison of nrITS1-5.8S-ITS2 sequences, including sequences of T. pruinosa from other origins, indicate that Tulasnella sp. is only distantly related to the T. pruinosa specimens included in the analyses. Sequences of all morphologically similar and taxonomically well-identified species are required to decide whether the basidiomata analyzed in the present study represent a new species. The new sequences are rather similar to sequences obtained previously from mycorrhizae of epiphytic orchids of the same area indicating mycorrhizal potential of this fungus.     

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest¹, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association ^{2, 3}. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells ². The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions ^4-7. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease ⁷. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).Download video file.^{(77M, mov)}