Entamoeba histolytica: electron-dense granule secretion, collagenase activity and virulence are altered in the cytoskeleton mutant BG-3

HM-1:IMSS, a pathogenic strain of Entamoeba histolytica, and its mutant BG-3, Identified by resistance to cytochalasin D, were tested for their capacity to: (i) secrete electron-dense granules; (ii) adhere and digest native type I collagen gels; and (iii) produce liver abscesses in new-born hamsters. The results demonstrate that the mutant has low adherence to collagen, low electron-dense granule secretion and collagenolytic activity, and low capacity to produce liver lesions in vivo, compared with the parental strain HM1:IMSS.     

Activity of maize transglutaminase overexpressed in Escherichia coli inclusion bodies: an alternative to protein refolding

Transglutaminases (TGases) catalyze protein post-translational modification by ε-(γ-glutamyl) links and covalent polyamine conjugation. In plants, this enzyme is poorly characterized and only the maize plastidial TGase gene (tgz) has been cloned. The tgz gene (Patent WWO03102128) had been subcloned and overexpressed in Escherichia coli cells, and the recombinant protein (TGZp) was present mainly in inclusion bodies (IB) fraction. In this work, after overexpression of TGZ15p and SDS-PAGE IB fraction analysis, bands about 65 and 56 kDa were obtained. Western blot, alkylation and MALDI-TOF/TOF analyses indicated that the 56 kDa band corresponded to a truncated sequence from the native TGZ15p (expected MW 65 kDa), by elimination of a chloroplast signal peptide fragment during expression processing. So that large-scale protein production and protein crystallization can be applied, we characterized the TGZ15p enzyme activity in the IB protein fraction, with and without refolding. Results indicate that it presented the biochemical characteristics of other described TGases, showing a certain plant-substrate preference. Solubilization of the IB fraction with Triton X-100 as nondenaturing detergent yielded active TGZ without the need for refolding, giving activity values comparable to those of the refolded protein, indicating that this is a valuable, faster way to obtain TGZ active protein.     

Purification and in vitro refolding of maize chloroplast transglutaminase over-expressed in Escherichia coli

In contrast to mammalian transglutaminases (TGs), plant members of the superfamily are poorly characterized. In order to produce pure and active TG for its functional and structural studies, variants of maize chloroplast transglutaminase (TGZ, Patent WWO03102128) were sub-cloned into a pET28 vector and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were present mainly as insoluble inclusion bodies. The TGZ4p variant with four B-type repeats (M _r∼55 kDa), was affinity purified from urea-solubilized inclusion bodies. TGZ4p was refolded by rapid dilution in a Ca²⁺- and guanidine-containing buffer. Active TGZ4p shows the general catalytic characteristics described for other TGs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biochemical and genetic characterization of l-glutamate transport and utilization in Salmonella typhimurium LT-2 mutants

Two systems for l-glutamate transport were found in Salmonella typhimurium LT-2 GltU⁺ (glutamate utilization) mutants. The first one is similar to the glt system previously described in Escherichia coli; by transductional analysis the structural gene, gltS, coding for the transport protein was located at minute 80 of the chromosome as part of the operon gltC-gltS, and its regulator, the gltR gene, near minute 90; the gltS gene product transports both l-glutamate and l-aspartate, is sodium independent, and is -hydroxyaspartate sensitive. The second transport system, whose structural gene was called gltF and is located at minute 0, was l-glutamate specific, sodium independent, and -methylglutamate sensitive. Two aspartase activities occurred in S. typhimurium LT-2: the first one was present only in the GltU⁺ mutants, had a pH 6.4 optimum, was essential for both l-glutamate and l-aspartate metabolism, and mapped at minute 94, close to the ampC gene. The second one had a pH 7.2 optimum, could be induced by several amino acids, and thus may have a general role in nitrogen metabolism.     

Charge transfer mediated by nigericin in black lipid membranes

Nigericin, in the concentration range (10^–6M or higher) at which it uncouples intact mitochondria, was found to increase the conductance of black lipid membranes (BLM) by several orders of magnitude. The dependence of the membrane conductance on pH and K⁺ concentration suggests a mechanism for the transfer of charge mediated by this ionophore based on a mobile dimer with both nigericin molecules protonated and complexed with one K⁺. This charged complex accounts for the uncoupling effect observed in intact mitochondria.A preliminary communication of this work was presented at the International School on Mitochondria: Biogenesis, Structure and Function. Mexico City, June 1975.     

Dynamical vs. judgmental comparison: hysteresis effects in motion perception

Perceptual comparison was investigated by gradually varying the relative length of two apparent motion paths, and independently determining when an initial percept was lost during the course of attribute change and when an alternative percept emerged. Dynamical comparison was indicated by a range of attribute values for which perception was bistable. Within this range, a percept that lost stability was immediately replaced by an alternative percept. Judgmental comparison was indicated by a range of attribute values for which perception was uncertain. When an initial percept was lost, an alternative percept did not immediately emerge because the alternatives being compared could not be distinguished. Differences in the effects of random noise on dynamical vs. judgmental comparison were demonstrated with computational simulations, and implications are discussed for motion energy models and solutions to the motion correspondence problem.     

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.