Investigating the in vivo activity of the DeaD protein using protein-protein interactions and the translational activity of structured chloramphenicol acetyltransferase mRNAs

Here, we report the use of an in vivo protein-protein interaction detection approach together with focused follow-up experiments to study the function of the DeaD protein in Escherichia coli. In this method, functions are assigned to proteins based on the interactions they make with others in the living cell. The assigned functions are further confirmed using follow-up experiments. The DeaD protein has been characterized in vitro as a putative prokaryotic factor required for the formation of translation initiation complexes on structured mRNAs. Although the RNA helicase activity of DeaD has been demonstrated in vitro, its in vivo activity remains controversial. Here, using a method called sequential peptide affinity (SPA) tagging, we show that DeaD interacts with certain ribosomal proteins as well as a series of other nucleic acid binding proteins. Focused follow-up experiments provide evidence for the mRNA helicase activity of the DeaD protein complex during translation initiation. DeaD overexpression compensates for the reduction of the translation activity caused by a structure placed at the initiation region of a chloramphenicol acetyltransferase gene (cat) used as a reporter. Deletion of the deaD gene, encoding DeaD, abolishes the translation activity of the mRNA with an inhibitory structure at its initiation region. Increasing the growth temperature disrupts RNA secondary structures and bypasses the DeaD requirement. These observations suggest that DeaD is involved in destabilizing mRNA structures during translation initiation. This study also provides further confirmation that large-scale protein-protein interaction data can be suitable to study protein functions in E. coli.     

Glomerular morphometry in biopsy evaluation of minimal change disease, membranous glomerulonephritis, thin basement membrane disease and Alport's syndrome

OBJECTIVE: To assess the role of glomerular morphometry in biopsy evaluation in renal disorders in addition to conventional diagnostic procedures. STUDY DESIGN: The study includes 10 cases each of minimal change disease (MCD), idiopathic membranous glomerulonephritis (idiopathic MGN), thin basement membrane disease (TBMD) and Alport's syndrome. Renal biopsies for normal study were obtained from age- and sex-matched autopsy cases without any renal disorder, confirmed histologically and ultrastructurally. Glomerular morphometry was performed by semiautomatic procedure using Quantimet-600 image analysis system (Leica, Cambridge, United Kingdom). RESULTS: Morphometric findings revealed significant increase in glomerular "diameter and area" and "tuft diameter and area" in patients of idiopathic MGN, but no significant difference was found in patients of MCD, TBMD and Alport's syndrome. Evaluation of glomerular volume fractions revealed a decrease in capillary space volume fraction and an increase in "membranes and mesangial matrix" volume fraction in patients with idiopathic MGN. Significant decrease in capillary space volume fraction was also observed in patients of MCD. Patients with Alport's syndrome showed variable changes. CONCLUSION: Glomerular morphometry could be considered as an adjunct to the diagnostic armamentarium of light microscopy, immunofluorescence and electron microscopy because it provides deep insight into quantitative parameters.     

Poly-(3-hexylthiophene) self-assembled monolayer based cholesterol biosensor using surface plasmon resonance technique

Cholesterol oxidase (ChOx) has been covalently immobilized onto 1-fluoro-2-nitro-4-azidobenzene (FNAB) modified poly-(3-hexylthiophene) (P3HT) self-assembled monolayer (SAM) onto gold coated glass plates. These ChOx/FNAB/P3HT/Au bio-electrodes have been characterized using contact angle (CA) measurements, UV-vis spectroscopy, electrochemical impedance technique, cyclic voltammetric technique and atomic force microscopic (AFM) technique, respectively. The ChOx/FNAB/P3HT/Au bio-electrodes were utilized for the estimation of cholesterol concentration in standard solutions using surface plasmon resonance (SPR) technique. It is shown that this SPR biosensor has linearity from 50 to 500 mg/dl of cholesterol in solution with detection limit of 50 mg/dl, sensitivity of 1.0 4 m degrees /(mg dl), reusability of around 15 times and a shelf-life of about 10 weeks when stored at 4 degrees C.     

Rapid method for detection of Salmonella in milk by surface plasmon resonance (SPR)

The Plasmonic surface plasmon resonance (SPR) device was used to develop a rapid, simple and specific immunoassay for detection of Salmonella in milk. Rapid detection of Salmonella contamination is a major challenge for the food industry. Salmonella contamination is well known in all foods including pasteurised milk. The SPR assay was developed as a sandwich model using a polyclonal antibody against Salmonella as capture and detection antibody. Milk spiked with Salmonella typhimurium cells, killed by thimerosal (1%, w/w) treatment was used. Using the Plasmonic SPR assay it was possible to detect S. typhimurium down to a concentration of 1.25 x 10(5) cells ml(-1) in both milk and buffer system. The results obtained are comparable with existing, approved rapid Salmonella detection techniques. No negative effects on the sensitivity of the assay are encountered due to the milk matrix. Hence, no sample preparation or clean-up steps are required. The sample volume requirement for the assay is only 10 microl. Using the assay S. typhimurium was detected in milk within 1h, whereas the cultural techniques require 3-4 days for presumptive positive isolates and further time for confirmation. The rapid tests require at least 24h for the results. The Plasmonic SPR device operates on the Kretschmann configuration and is a cuvette-based system with the advantage of having eight channels on one single SPR chip.     

Sub-lethal concentration of arsenic interferes with the proliferation of hepatocytes and induces in vivo apoptosis in Clarias batrachus L

We studied the hepatocellular alterations induced by sub-lethal concentrations (0.50 muM) of arsenic in Indian catfish Clarias batrachus L. Sub-lethal arsenic exposure altered serum aspartate aminotransferase and alkaline phosphatase levels and brought about significant changes in different serum biochemical parameters. Arsenic exposure reduced total hepatocyte protein content and suppressed the proliferation of hepatocytes in a time-dependent manner. Routine histological studies on liver documented arsenic-induced changes characterized by dilated sinusoids, formation of intracellular edema, megalocytosis, vacuolation and appearance of hepatic cells with distorted nuclei. Transmission electron microscopy of hepatocytes further revealed hyperplasia and hypertrophy of mitochondria, development of dilated rough endoplasmic reticulum and changes in peroxisome size with duration of arsenic exposure. Degeneration of mitochondrial cristae and condensation of chromatin was also evident in arsenic-exposed hepatocytes. A significant number of hepatocytes isolated from arsenic-exposed fish stained with annexin V and demonstrated DNA ladder characteristic of apoptosis. Single-cell gel electrophoresis of exposed hepatocytes also revealed the development of comets usually seen in apoptotic cells. Using specific inhibitors it was determined that the arsenic-induced apoptosis of hepatocytes was caspase-mediated, involving the caspase 3 pathway.     

Autoproteolysis of rat p67 generates several peptide fragments: the N-terminal fragment, p26, is required for the protection of eIF2alpha from phosphorylation

Eukaryotic initiation factor 2-associated glycoprotein, p67, plays important roles in the regulation of eIF2alpha phosphorylation and thus maintains cell growth and proliferation. The p67 sequence can be divided into two segments, the N-terminal segment of amino acids 1-107 (p26) and the downstream segment of amino acids 108-480 (p52). Comparison of the amino acid sequences of p67 from lower to higher organisms suggests that there is a progressive addition of several unique domains at the N-terminus of p67, and these unique domains, which are present in p26, play important roles in the modulation of eIF2alpha phosphorylation in mammalian cells. To test the hypothesis that the p26 segment is generated from p67 due to its autoproteolysis and whether p26 is required for the protection of eIF2alpha from phosphorylation, we have analyzed the time-dependent cleavage of functionally active rat recombinant p67 purified from either baculovirus-infected insect cells or transiently transfected mammalian cells. We noticed a regulated cleavage of p67 that generates several peptides along with the most stable p26 and p52 fragments. The p52 fragment has a low level of autoproteolysis activity that possibly increases the autoproteolysis of full-length p67. This activity could not be inhibited by a serine protease inhibitor, PMSF, but could be inhibited by a cocktail of protease inhibitors that includes bestatin, leupeptin, E64, AEBSF, and aprotinin. To provide evidence that the fragmentation of p67 is not due to the presence of any contaminant protease(s), we fractionated purified rat p67 with molecular sieve, anion exchange, and cation exchange chromatographic steps performed in the presence of different K+ ion concentrations. Our data show that the extent of cleavage of p67 into different fragments is higher in the presence of 0.75 M K+ ion and in samples stored at -80 degrees C. Under parallel conditions, p67's mutants, D251A and D262A, exhibited very little to no cleavage, whereas the H231E mutant exhibited extensive cleavage that generated a large amount of p26 fragment. The p26 fragment exhibited protection of eIF2alpha phosphorylation both in vivo and in vitro. Altogether, our data provide evidence that rat p67 has autoproteolytic activity that generates p26, which is required to block eIF2alpha from phosphorylation.     

Conformational variability of Gly-Gly segments in peptides: A comparison of the crystal structures of an acyclic pentapeptide and an octapeptide

The crystal structure of an acyclic pentapeptide, Boc-Gly-Gly-Leu-Aib-Val-OMe, reveals an extended conformation for the Gly-Gly segment, in contrast to the helical conformation determined earlier in the octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-OMe [I. L. Karle, A. Banerjee, S. Bhattacharjya, and P. Balaram [1996] Biopolymers, Vol. 38, pp. 515–526). The pentapeptide crystallizes in space group P2₁ with one molecule in the asymmetric unit. The cell parameters are: a = 10.979(2) Å, b = 9.625(2) Å, c = 14.141(2) Å, and β = 96.93(1)°, R = 6.7% for 2501 reflections (I > 3σ(I)). The Gly-Gly segment is extended (ϕ₁ = −92°, ψ₁ = −133°, ϕ₂ = 140°, ψ₂ = 170°), while the Leu-Aib segment adopts a type II β-turn conformation (ϕ₃ = −61°, ψ₃ = 130°, ϕ₄ = 71°, ψ₄ = 6°). The observed conformation for the pentapeptide permits rationalization of a structural transition observed for the octapeptide in solution. An analysis of Gly-Gly segments in peptide crystal structures shows a preference for either β-turn or extended conformations. © 1997 John Wiley & Sons, Inc.     

MRX8, the conserved mitochondrial YihA GTPase family member,is required for de novo Cox1 synthesis at suboptimal temperatures in Saccharomyces cerevisiae

The synthesis of Cox1, the conserved catalytic-core subunit of Complex IV, a multisubunit machinery of the mitochondrial oxidative phosphorylation (OXPHOS) system under environmental stress, has not been sufficiently addressed. In this study, we show that the putative YihA superfamily GTPase, Mrx8, is a bona fide mitochondrial protein required for Cox1 translation initiation and elongation during suboptimal growth condition at 16°C. Mrx8 was found in a complex with mitochondrial ribosomes, consistent with a role in protein synthesis. Cells expressing mutant Mrx8 predicted to be defective in guanine nucleotide binding and hydrolysis were compromised for robust cellular respiration. We show that the requirement of Pet309 and Mss51 for cellular respiration is not bypassed by overexpression of Mrx8 and vice versa. Consistently the ribosomal association of Mss51 is independent of Mrx8. Significantly, we find that GTPBP8, the human orthologue, complements the loss of cellular respiration in Δmrx8 cells and GTPBP8 localizes to the mitochondria in mammalian cells. This strongly suggests a universal role of the MRX8 family of proteins in regulating mitochondrial function.     

Kaolin and Jasmonic acid improved cotton productivity under water stress conditions

Drought is one of the most emerging threat that causes a severe reduction in cotton plant growth and development. Being cotton is a major cash crop has great threat to prevailing drought events in Pakistan. A field experiment was conducted in Kharif season 2018 at Research Area of MNS-University of Agriculture, Multan, Pakistan to assess the role of foliar applied kaolin and jasmonic acid on vegetative growth, gas exchange and reproductive traits of cotton under normal irrigated and artificial water deficit conditions. The experiment was laid -out in a factorial randomized complete block design with split – split plot arrangement. Main plots were allocated for irrigation levels, sub-plots for two -cotton genotypes viz. NIAB − 878 and SLH − 19 while sub – sub plots for treatments of kaolin and Jasmonic acid. Water deficit stress was created by skipping irrigation at flowering for 21 days. Foliar sprays of Kaolin (5%, w/v) and Jasmonic acid (100 μM) were applied alone or in combination at 60 days after planntinon both to normal irrigated and water-stresse skip irrigation while irrigation water alone was sprayed in control plots. Both cotton genotypes responded variably to normal irrigated and skip conditions. Skipping irrigation for up to 21 days at flowering caused a significant decrease in leaf relative water content, SPAD values, net photosynthetic rate and seed cotton yield in both the genotypes. Seed cotton yield showed an overall decline of 24.7% in skip over Normal irrigated crop. The genotype NIAB − 878 produced maximum seed cotton yield of 3.304 Mg ha⁻¹ in normal that dropped to 2.579 Mg ha⁻¹ in skip, thus showing an average decline of 21.9 %. Similarly, SLH − 19 produced 2.537 Mg ha⁻¹ seed cotton under normal that dropped to 1.822 Mg ha⁻¹ in skip, showing an average decline of 28.2%. The Application of Kaolin and JA Jasmonic acid, either applied individually or in combination, improved vegetative and reproductive development of both cotton varieties in normal and skip regimes. However, combined kaolin and Jasmonic Acid application proved to be more beneficial in terms of seed cotton production and other parameters studied.     

Technological and economic potential of poly(lactic acid) and lactic acid derivatives

Abstract: Lactic acid has been an intermediate-volume specialty chemical (world production ∼ 40,000 tons/yr) used in a wide range of food processing and industrial applications. Lactic acid has the potential of becoming a very large volume, commodity-chemical intermediate produced from renewable carbohydrates for use as feedstocks for biodegradable polymers, oxygenated chemicals, plant growth regulators, environmentally friendly 'green' solvents, and specialty chemical intermediates. The recent announcements of new development-scale plants for producing lactic acid and polymer intermediates by major U.S. companies, such as Cargill, Ecochem (DuPont/ConAgra), and Archer Daniels Midland, attest to this potential.
In the past, efficient and economical technologies for the recovery and purification of lactic acid from crude fermentation broths and the conversion of lactic acid to the chemical or polymer intermediates had been the key technology impediments and main process cost centers. The development and deployment of novel separations technologies, such as electrodialysis (ED) with bipolar membranes, extractive distillations integrated with fermentation, and chemical conversion, can enable low-cost production with continuous processes in large-scale operations. The use of bipolar ED can virtually eliminate the salt or gypsum waste produced in the current lactic acid processes. Thus, the emerging technologies can use environmentally sound processes to produce environmentally useful products from lactic acid. The process economics of some of these processes and products can also be quite attractive. In this paper, the recent technical advances in lactic and polyactic acid processes are discussed. The economic potential and manufacturing cost estimates of several products and process options are presented. The technical accomplishments at Argonne National Laboratory (ANL) and the future directions of this program at ANL are discussed.