Entamoeba histolytica:Identification of Functional Gsand GiProteins as Possible Signal Transduction Elements in the Interaction of Trophozoites with Fibronectin

Soid-Raggi, L. G., Torres-Maárquez, M. E., and Meza, I. 1998.Entamoeba histolytica:Identification of functional G_sand G_iproteins as possible signal transduction elements in the interaction of trophozoites with fibronectin.Experimental Parasitology90, 262–269. Trophozoites ofEntamoeba histolyticaadhere to several components of the extracellular matrix. Binding is mediated by specific receptors identified in the parasite surface. Interaction of trophozoites with FN induces the formation of special adhesion structures that are dynamic cytoskeleton membrane complexes and facilitate both adhesion and substrate degradation. The process requires activation of signaling pathways in which PLC, IP₃, Ca²⁻, and PKC participate. These observations, and recent experiments showing increments in cAMP in the trophozoites during the interaction with FN, suggest that FN receptors in the amebic surface could be coupled to G-proteins. We report here that trophozoite plasma membrane peptides of 92, 49, 42, 37, and 21 kDa are ADP-ribosylated byVibrio choleraeandBordetella pertussistoxins. Three of them are also recognized by antibodies prepared against the α-subunit of G_s-and G_i-proteins. Adenylyl cyclase activity detected in isolated membranes was strongly stimulated by treatment with the toxins. Forskolin (an agonist of the enzyme) and FN also induced increments in the enzymatic activity. Live amebas incubated with the toxins showed enhanced adhesion to FN substrates and a striking reorganization of polymerized actin. The actin rearrangement is reminiscent of the one induced by either forskolin or dibutyril cyclic AMP treatment. Our present data show the presence and the functionality of G_s- and G_i-like proteins and their apparent activation duringin vitrointeraction of amebas with FN and complement previous observations indicating the operation of signal transduction mechanisms inE. histolytica.     

Occluding Junctions in MDCK Cells: Modulation of Transepithelial Permeability by the Cytoskeleton

In MDCK cell monolayers the opening and resealing of occluding junctions can be induced by removal and restoration of calcium to the external medium. The overall changes in permeability of the occluding junctions in the monolayer can be monitored by the drop and recovery of the total transepithelial electrical resistance. We have investigated the effects of cytochalasin B (CB) on this process. When CB is added to sealed monolayers there is a gradual drop in the electrical resistance across the monolayer. This drop is accompanied by a slow disorganization of the microfilament pattern of these cells, including a disturbance of a ring of cortical microfilaments that is normally associated with the junctions. Cells in open monolayers treated with CB will not reseal and have an altered filament distribution. These cells do not have a continuous cortical ring. We have used a voltage scanning technique that uses a microelectrode to measure the resistance at selected points along the junction which surrounds a single cell. In untreated, closed monolayers, the junction is heterogeneous with alternating points of high and low conductance. In closed monolayers treated with CB, although there are low conductance points, we have observed an increased frequency of high conductance points that correlates with the change in the overall conductance. The frequency of high conductance points along the junction and the overall conductance both increase with time of exposure to CB. In an effort to understand the molecular basis for the permeability changes induced by EGTA and CB, we have looked for differences in the protein components of the cell membranes of open, closed, and CB-treated MDCK monolayers. This was done by radioiodinating the surface membrane proteins under control and experimental conditions that bring about permeability changes. No significant differences in the labeled protein patterns were found under these conditions. These results suggest that the permeability changes involve only a structural rearrangement of membrane components. In addition we have observed that about 36% of the surface label remains bound to the insoluble cytoskeletons obtained from cells in control and experimental conditions that alter the permeability of the tight junctions. The iodinated proteins attached to the CS include polypeptides with M_r of ≥ 120K daltons as well as peptides with M_r = 56K, 50K, 36K, and 18K daltons.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.     

Extracellular matrix-induced signaling in Entamoeba histolytica: its role in invasiveness

Entamoeba histolytica trophozoites can invade the mucosa of the host large intestine to cause disease. Here, Isaura Meza describes recent work indicating that the trophozoites respond to the binding of extracellular matrix proteins by activating receptor-mediated signal transduction pathways. Activation of phosphokinase C or adenylyl cyclase induces protein phosphorylation, actin gene expression and reorganization of the actin cytoskeleton to form adhesive structures that promote interaction with the substrate and the release of proteases. The release of proteases at the sites of contact, with the subsequent degradation of the substrate and generation of chemotactic peptides, facilitates locomotion and dissemination of the invading trophozoites.     

Streptidine, a metabolic derivative produced after administration of streptomycin in vivo, is vestibulotoxic in rats

Streptomycin is the treatment of choice in developing countries for patients suffering from tuberculosis or other infectious diseases. However, it produces incapacitating vestibular symptoms whose onset is delayed and gradual. This observation led to the notion that a streptomycin metabolic derivative and not the antibiotic itself is the damaging agent for the inner ear. To study further the existence of this ototoxic metabolite, chronic treatment with streptomycin or its putative derivative streptidine was carried out in young male Long Evans rats. The presence of streptomycin or streptidine in the blood of animals of either experimental group was assessed by high performance liquid chromatography and analysis of swimming behavior was used to evaluate vestibular damage. Features of the sensory epithelium and quantification of hair cells were attained in sections of the utricular organ of all groups by light microscopy. After 25, 35 and 45 days of treatment with streptomycin, a metabolite with the same chromatographic properties as the streptidine standard run in parallel was identified in the blood of rats. Concentrations of the metabolite were 2.26 microg/ml on the 25th day and around 8.0 microg/ml in both the 35th and the 45th day of treatment, while streptomycin was below its detection level at either period. In streptidine-treated rats, the concentration of this compound was 1.0, 1.84 and 4.94 microg/ml on the 25th, 35th and 45th treatment days, respectively. Treatment with either streptomycin or streptidine resulted in similar abnormal swimming patterns and histological alterations of the utricular epithelium. Loss of hair cells was roughly equivalent even though streptidine was administered in a dose 90% lower than streptomycin. The gradual appearance of streptidine as a metabolic derivative of the antibiotic in the blood of rats or the administration of this compound alone, causing similar functional and structural vestibular deterioration seen in streptomycin-treated animals, supports the notion that streptidine is a potential contributor to ototoxicity after prolonged antibiotic administration.     

Threshold policies control for predator-prey systems using a control Liapunov function approach

The stability of predator-prey models, in the context of exploitation of renewable resources, subject to threshold policies (TP) is studied in this paper using the idea of backstepping and control Liapunov functions (CLF) well known in control theory, as well as the concept of virtual equilibria. TPs are defined and analysed for different types of one and two species predator-prey models. The models studied are the single species Noy-Meir herbivore-vegetation model, in a grazing management context, as well as the Rosenzweig-MacArthur two species predator-prey model, in a fishery management context. TPs are shown to be versatile and useful in managing renewable resources, being simple to design and implement, and also yielding advantages in situations of overexploitation.     

Pupariation time as a source of variability in mating performance in mass-reared Anastrepha ludens (Diptera: Tephritidae)

The effect of time of pupariation on pupal weight and adult sexual competitiveness under field cage conditions was studied in mass-reared Anastrepha ludens (Loew) males. Larvae that took 72 h to pupariate after separation from diet resulted in lighter pupae than those that took 24 and 48 h. Wild pupae were heavier than the 48- and the 72-h pupae but not the 24-h pupae. Interestingly, no differences in mating performance were found between males of the 24- and 48-h pupae despite differences in pupal weight. In general, lower-than expected levels of mating compatibility between sterile and wild A. ludens resulted from the interaction of both strains as more homotypic pairs were observed. Discussion focuses on the effect of the mass-rearing process on male fruit fly mating performance.     

Effect of carbon source and sucrose concentration on growth and hexose accumulation of grape berries cultured in vitro

Using in vitro culture of isolated small berries of Vitis vinifera L. cv. Sultana, it was possible to study the effect of different carbon sources and sucrose concentration on fruit growth, hexose accumulation and soluble invertase activity during the first stage of berry development by eliminating the source tissue. Berries cultured in vitro lack stage III of berry development which is characterised by massive accumulation of water and sugars, and thereby berries reached only 30% of the weight of those grown in the plant. Sucrose and glucose were both good carbon sources for berry growth, while fructose was not as good. Berry growth, hexose accumulation and invertase activity increased as sucrose concentration increased up to 15% in the medium. Furthermore, the onset of hexose accumulation in cultured berries depended on the concentration of sucrose in the medium, starting earlier at higher concentrations. This revised version was published online in June 2006 with corrections to the Cover Date.     

A limited survey of aflatoxins and fumonisins in retail maizebased products in the UK using immunoassay detection

A total of 27 maize-based products destined for human consumption were collected from retail outlets within the city of Glasgow in the UK and were analysed for the presence of aflatoxins using immunoaftinity column chromatography with fluorescence detection and for fumonisins by competitive ELISA. Aflatoxins were detected at a trace level below 4 in eight (30%) of the 27 samples tested, no sample contained aflatoxins at a high level although one sample of sweetcorn did contain aflatoxins at a level of 5-10 Fumonisins were detected in eight (30%) of the samples at levels from 1 to 8mgkg^-1 and a further eight samples contained fumonisin at a level below 1 mgkg^-1 but above the detectable level. The highest concentration of fumonisins was found in a sample of fine corn meal at 8-12mgkg^-1.     

Rational Design of Antibiotic Treatment Plans: A Treatment Strategy for Managing Evolution and Reversing Resistance

The development of reliable methods for restoring susceptibility after antibiotic resistance arises has proven elusive. A greater understanding of the relationship between antibiotic administration and the evolution of resistance is key to overcoming this challenge. Here we present a data-driven mathematical approach for developing antibiotic treatment plans that can reverse the evolution of antibiotic resistance determinants. We have generated adaptive landscapes for 16 genotypes of the TEM β-lactamase that vary from the wild type genotype “TEM-1” through all combinations of four amino acid substitutions. We determined the growth rate of each genotype when treated with each of 15 β-lactam antibiotics. By using growth rates as a measure of fitness, we computed the probability of each amino acid substitution in each β-lactam treatment using two different models named the Correlated Probability Model (CPM) and the Equal Probability Model (EPM). We then performed an exhaustive search through the 15 treatments for substitution paths leading from each of the 16 genotypes back to the wild type TEM-1. We identified optimized treatment paths that returned the highest probabilities of selecting for reversions of amino acid substitutions and returning TEM to the wild type state. For the CPM model, the optimized probabilities ranged between 0.6 and 1.0. For the EPM model, the optimized probabilities ranged between 0.38 and 1.0. For cyclical CPM treatment plans in which the starting and ending genotype was the wild type, the probabilities were between 0.62 and 0.7. Overall this study shows that there is promise for reversing the evolution of resistance through antibiotic treatment plans.