Extracellular potassium can simulate the role of serum as an activator of uridine uptake by quiescent hamster cells in culture

Replacement of ~100 mM of sodium chloride in the extracellular medium of quiescent hamster fibroblasts (Nil 8 and BHK cells) by potassium chloride causes an increase in the rate of uridine uptake. This increase is identical with that achieved by addition of 10% serum to the same cultures. The effects of serum and KCl are not additive. The dependence of the rate of uridine uptake on extracellular KCl concentration is of a sigmoid nature. The time course of the activation process is similar to that of serum activation of uridine uptake in the same cells. The high rate of uridine uptake persists for at least 30 min after return to an extracellular medium containing a high concentration of sodium.     

Risk assessment of heavy metal and metalloid toxicity through a contaminated vegetable (Cucurbita maxima) from wastewater irrigated area: A case study for a site-specific risk assessment in Jhang,Pakistan

The present research was conducted in district Jhang, Pakistan, to evaluate the concentration of metals/metalloids in soil and pumpkin (Cucurbita maxima) irrigated with domestic wastewater. Data revealed that the levels of metals and metalloids in soil samples from two different sites were below the safe limits except Cd, whereas, in the vegetable, the concentrations of As, Se, Ni, Mo, Pb, Mn, and Cu were above the safe limits. The levels of 12 metals and metalloids in the soil were ranged between 0.14 to 22.76 mg/kg at site-I and 0.16 to 22.13 mg/kg at site-II. The levels of these metals in the vegetable were found 0.35 to 61.13 mg/kg at site-I and 0.31 to 53.63 mg/kg at site-II. The transfer factor at both sites was highest for As and Co. The pollution load index recorded for Se, Cu, Cd, Mo, Pb, and Co was greater than 1. The daily intake of As, Mn, and Mo was above the oral reference dose, which reflects that the intake of pumpkin is not safe for the inhabitants of the selected sites. The control measures should be taken to phytoextract heavy metals and metalloids from polluted sites so as to reduce the health risks.     

Subtractive Proteomics and Immuno-informatics Approaches for Multi-peptide Vaccine Prediction Against Klebsiella oxytoca and Validation Through In Silico Expression

International Journal of Peptide Research and Therapeutics - Klebsiella oxytoca is a gram-negative bacterium. It is opportunistic in nature and causes hospital acquired infections....     

LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l -carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.     

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA‐specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N‐terminal sequence was found in two selected peptides: F4‐2 (KPRSMLH) and F5‐4 (KPRSVSG). The peptide F5‐4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5‐4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage‐derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative analysis of the methods used for finding surface energy to investigate protein interaction behavior on chromatographic supports

Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.     

MdfA, an interesting model protein for studying multidrug transport

The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each can recognize and expel a broad spectrum of chemically unrelated drugs from the cell. Despite extensive research for many years, the actual mechanism of multidrug transport is still largely unknown. In addition to general questions dealing with energy coupling, the molecular view of substrate recognition by Mdrs is generally obscure. This mini-review describes structural and functional properties of the Escherichia coli Mdr, MdfA, and discusses the possibility that this transporter may serve as a model for studying the multidrug recognition phenomenon and the mechanism of multidrug transport.     

Membrane topology of the multidrug transporter MdfA: complementary gene fusion studies reveal a nonessential C-terminal domain

The hydrophobicity profile and sequence alignment of the Escherichia coli multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters. According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition. To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA. Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA. Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport.     

New prospects in studying the bacterial signal recognition particle pathway

In vivo and in vitro studies have suggested that the bacterial version of the mammalian signal recognition particle (SRP) system plays an essential and selective role in protein biogenesis. The bacterial SRP system consists of at least two proteins and an RNA molecule (termed Ffh, FtsY and 4.5S RNA, respectively, in Escherichia coli). Recent evidence suggests that other putative bacterial-specific SRP components may also exist. In vitro experiments confirmed the expected basic features of the bacterial SRP system by demonstrating interactions among the SRP components themselves, between them and ribosomes, ribosome-linked hydrophobic nascent polypeptides or inner membranes. The availability of a conserved (and essential) bacterial SRP version has facilitated the implementation of powerful genetic and biochemical approaches for studying the cascade of events during the SRP-mediated targeting process in vivo and in vitro as well as the three-dimensional structures and the properties of each SRP component and complex.