Effects of alpha- and beta-adrenergic agonists on Trypanosoma cruzi interaction with host cells

We studied the effects of adrenergic agonists on the capacity of blood trypomastigote forms of Trypanosoma cruzi to associate with (i.e., bind and/or penetrate) host cells in vitro. The extent of T. cruzi association with mouse macrophages in the presence of the beta-adrenergic agonist L-isoproterenol was significantly decreased with respect to mock-treated controls. Similar results were obtained when the parasite was pretreated with L-isoproterenol and was then allowed to interact with untreated macrophages. In contrast, pretreatment of trypomastigotes with either L-phenylephrine or methoxamine-alpha-adrenergic agonists--enhanced their reactivity with macrophages. Interaction with a nonphagocytic host cell was also decreased and increased by parasite pretreatment with beta- and alpha-adrenergic agonists, respectively. The L-isoproterenol and L-phenylephrine effects were no longer detectable 2 and 3 hr after their removal, respectively, and were therefore reversible. Atenolol, a specific beta 1 adrenoreceptor blocker inhibited the L-isoproterenol effect, whereas butoxamine, a specific beta 2 blocker, did not. Thus, beta 1-like but not beta 2-like binding sites appeared to be expressed on T. cruzi. Both prazosin and yohimbine, preferential alpha 1- and alpha 2-receptor blockers, respectively, abolished the L-phenylephrine effect. The opposite effects of alpha- and beta-adrenergic agonists suggested that the infectivity of T. cruzi may be regulated by activation of surface components comparable to the adreno-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colonization of North America by Drosophila subobscura: Ecological analysis of three communities of drosophilids in California

We have carried out bimonthly collections of Drosophila species during a 16 month period in three localities of California (Eureka, Davis, and Gilroy). D. subobscura, which has colonized North America only a few years ago, is well established and has an annual abundance cycle similar to that observed in the Old World. The differences between the three localities account for 26.4% of the species diversity. Davis and Gilroy exhibit similarities attributable to their similar environmental conditions. Seasonality accounts for 46% and 50.4 % of the species diversity in Davis and Gilroy respectively, but only for 39.4% in Eureka. Differences between years explain only a small percentage of the total species diversity. A correspondence analysis of the association between species and months of collection shows ecological similarities between D. subobscura and the Nearctic members of the obscura species group.     

The Entamoeba histolytica Dnmt2 Homolog (Ehmeth) Confers Resistance to Nitrosative Stress

Nitric oxide (NO) has antimicrobial properties against many pathogens due to its reactivity as an S-nitrosylating agent. It inhibits many of the key enzymes that are involved in the metabolism and virulence of the parasite Entamoeba histolytica through S-nitrosylation of essential cysteine residues. Very little information is available on the mechanism of resistance to NO by pathogens in general and by this parasite in particular. Here, we report that exposure of the parasites to S-nitrosoglutathione (GSNO), an NO donor molecule, strongly reduces their viability and protein synthesis. However, the deleterious effects of NO were significantly reduced in trophozoites overexpressing Ehmeth, the cytosine-5 methyltransferase of the Dnmt2 family. Since these trophozoites also exhibited high levels of tRNA^Asp methylation, the high levels suggested that Ehmeth-mediated tRNA^Asp methylation is part of the resistance mechanism to NO. We previously reported that enolase, another glycolytic enzyme, binds to Ehmeth and inhibits its activity. We observed that the amount of Ehmeth-enolase complex was significantly reduced in GSNO-treated E. histolytica, which explains the aforementioned increase of tRNA methylation. Specifically, we demonstrated via site-directed mutagenesis that cysteine residues 228 and 229 of Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth binding to enolase and for Ehmeth-mediated resistance to NO. These results indicate that Ehmeth has a central role in the response of the parasite to NO, and they contribute to the growing evidence that NO is a regulator of epigenetic mechanisms.     

Pre-release packing and chilling reduce host-searching ability of the parasitoid Diachasmimorpha longicaudata used in the augmentative control of tephritid flies

Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) is a solitary parasitoid used in augmentative releases to control Tephritidae (Diptera) fruit flies of economic importance. Pre-release process includes packing adult parasitoids in cages at high densities and expose them to a temperature of 2 ± 2 °C during 105 min. In this process, females’ antennae may be damaged resulting in a reduction in their host-searching ability and fecundity. Here we measured, for five consecutive days after chilling, the searching ability and fecundity of parasitoids with damaged (D) and undamaged (ND) antennae compared with parasitoids that were not chilled. Female individual responses to fruit infested by Anastrepha spp. was determined in an olfactometer. Latency in the response and latency in making a choice were recorded. Additionally, groups of 30 females were used to measure their ability to find hosts in infested fruit in the laboratory. Fecundity was determined by using artificial oviposition units with Anastrepha ludens (Loew) larvae. In the olfactometer test, ND had lower response than control females during the 1st and 2nd days after chilling. However, no difference in the response was observed between ND and D and the control females on the 5th day. Additionally, chilled females showed a longer latency of response to infested fruit than females of the control treatment when tested in groups. However, after a 24–48 h period, no difference between D and ND and control females was observed. Our results showed similar searching ability and fecundity among parasitoids of the three tested conditions at individual and at group levels. We conclude that pre-release chilling reduces female searching ability only for the first 1–2 days after chilling.     

The effect of topical application of diethyl- -fluoroglutarate on the metabolism and electrical activity in vivo of cat cerebral cortex

Abstract— The effect of topical application of diethyl-α-fluoroglutarate on the primary response of cat cerebral cortex to thalamic stimulation was investigated, and samples of the involved tissue and of homologous contralateral control tissue were removed at appropriate times for biochemical analyses. Changes in electrocortical response were first noted 30–40 min after topical application of 10 μ1 (52.7 μmol) of the drug to the pericruciate gyrus. A rapid reduction in amplitude of the surface negative component of the primary response was observed initially, followed by amplitude reduction and rapid disappearance of the primary response throughout the cortical field within a few minutes after the change first observed at the surface. The effects were interpreted either as a direct action of the drug on the somadendritic membrane or an inhibition of the excitatory synaptic impingement. Analyses of the tissues removed at the time of maximum electrocortical response indicated profound metabolic changes, including depletion of energy reserves and several Krebs cycle intermediates. Large increases in tissue levels of glucose, glucose 6-phosphate, and pyruvate were found. Changes in amino acids comprised depletion of glutamate with increased tissue levels of aspartate, GABA, NH₃, threonine, serine and alanine. Tissues were also removed at 10 min after topical application of the drug but before the advent of electrocortical changes. Decreased levels of glutamate were associated with a rise in tissue aspartate. Tissue levels of the other amino acids were unchanged. Glucose, glucose 6-phosphate and pyruvate levels were increased, lactate and ATP levels were unchanged, and P-creatine, α-KG and malate levels were reduced. We believe that the observed pattern of changes represents responses of the cerebral cortex to either a block in the synthesis of glutamate or in oxidation of pyruvate, with consequent interference with oxidative and energy metabolism and eventual depression of cortical electrical activity.     

Flow cytometric quantification and characterization of intracellular protein aggregates in yeast

The sequestration of misfolded proteins into aggregates is an integral pathway of the protein quality control network that becomes particularly prominent during proteotoxic stress and in various pathologies. Methods for systematic analysis of cellular aggregate content are still largely limited to fluorescence microscopy and to separation by biochemical techniques. Here, we describe an alternative approach, using flow cytometric analysis, applied to protein aggregates released from their intracellular milieu by mild lysis of yeast cells. Protein aggregates were induced in yeast by heat shock or by chaperone deprivation and labeled using GFP- or mCherry-tagged quality control substrate proteins and chaperones. The fluorescence-labeled aggregate particles were distinguishable from cell debris by flow cytometry. The assay was used to quantify the number of fluorescent aggregates per μg of cell lysate protein and for monitoring changes in the cellular content and properties of aggregates, induced by stress. The results were normalized to the frequencies of fluorescent reporter expression in the cell population, allowing quantitative comparison. The assay also provided a quantitative measure of co-localization of aggregate components, such as chaperones and quality control substrates, within the same aggregate particle. This approach may be extended by fluorescence-activated sorting and isolation of various protein aggregates, including those harboring proteins associated with conformation disorders.     

L-Arginine restores splenocyte functions after trauma and hemorrhage potentially by improving splenic blood flow

Several studiesindicate that immune responses are markedly depressed early after onsetof hemorrhage. Decreased organ blood flow has been implicated in thepathophysiology of altered immune responses after trauma-hemorrhage. Inthis regard, administration ofL-arginine has been shown torestore depressed intestinal and hepatic blood flow aftertrauma-hemorrhage, probably due to provision of substrate forconstitutive nitric oxide synthase (cNOS). It remains unknown, however,whether administration ofL-arginine also amelioratesdepressed splenic blood flow and whether this agent has any salutaryeffects on depressed splenocyte functions after trauma-hemorrhage. Malerats underwent sham operation or laparotomy and were bled to andmaintained at a mean arterial blood pressure of 40 mmHg until 40% ofmaximum shed blood volume (MBV) was returned as Ringer lactate (RL).Hemorrhaged rats were then resuscitated with RL (4 times MBV over 1 h).During resuscitation, rats received 300 mg/kgL-arginine or saline (vehicle)intravenously; 4 h later, splenic blood flow, splenocyte proliferation,and splenocyte interleukin (IL)-2 and IL-3 were determined.Administration of L-arginineimproved depressed splenic blood flow and restored depressed splenocytefunctions after trauma-hemorrhage. Therefore, provision ofL-arginine during resuscitationafter trauma-hemorrhage should be considered a novel and safe approachfor improving splenic organ blood flow and depressed splenocytefunctions under such conditions.