Absence of Transitory [Ca2+]I Flux During Early In Vitro Metacyclogenesis of Trypanosoma cruzi1

ABSTRACT. The phorbol ester TPA (phorbol 12-myristate 13-acetate) substitutes for CO₂ as an agonist for transforming Trypanosoma cruzi epimastigotes to the metacyclic trypomastigote stage in a starvation medium consisting of phosphate buffered saline + 10 mM proline, 10 mM sodium acetate and 0.035% NaHCO₃. Since TPA is thought to stimulate protein kinase C by mimicking the activity of the secondary messenger diacylglycerol, the above result suggested that T. cruzi metacyclogenesis could be activated by a Ca^2+-dependent protein kinase C signal induction pathway. Accordingly, cytosolic calcium flux ([Ca²⁺]_i) in epimastigotes, activated with 5% CO₂ or TPA (10^-7 M), was measured with the Ca²⁺ molecular probe, fluo-3AM. In addition, [Ca²⁺]_i was measured in cells incubated with putative metacyclogenic agonists (e.g. proline, glutamate, bioamines, ionophores and catecholamines). None of the compounds studied, except for EGTA, affected cytosolic Ca²⁺ levels. Control assays with 11 μM thapsigargin, which mobilizes noncytoplasmic Ca²⁺ stores by inhibiting endoplasmic reticulum Ca²⁺-ATPase. validated our fluorometric assay procedure. Although thapsigargin significantly increases cytoplasmic Ca²⁺ fluorescence, it has no effect on transformation. The protein kinase C inhibitors staurosporine, H-7 and HA 1004 were tested for their effect on T. cruzi metacyclogenesis. Low concentrations of staurosporine and HA 1004 significantly elevated Pent strain transformation while H-7 had no effect on Peru strain metacyclogenesis. Inhibitor H-7 did significantly depress CL transformation. the results indicate that induction of T. cruzi metacyclic trypomastigote formation by CO₂ and TPA is not accompanied by changes in cytosolic Ca²⁺ and do not provide supporting evidence for participation of a protein kinase C-mediated phosphoinositide cascade in metacyclogenesis.     

Phagocytosis and Encapsulation: Cellular Immune Responses in Arthropoda

The least understood aspects of the cellular immune reactionsof arthropods are the earliest events: the initial recognitionof foreignness, and the resulting changes in hemocyte behaviorand morphology. There are indications that the recognition ofa surface as foreign is based primarily upon its electrostaticcharge, but more specific criteria may be utilized in some situations.Phagocytes are capable of recognizing foreignness without theintervention of soluble opsonins but, in some arthropods, thereis in vitro evidence that opsonins can increase the efficiencyof phagocytosis. It has been hypothesized, on the basis of ultrastructuralevidence that encapsulation, a multicellular immune response,is induced when labile hemocytes rupture upon encountering aforeign object. The released products may promote the formationof a sheath of ameboid hemocytes around the object. This hypothesisis now supported by the results of experiments performed onan in vitro encapsulation system. This system may prove usefulin the purification of encapsulation—promoting factors;in determining the mechanism of their release from hemocytes;and in investigating the possibility that the various cellularimmune reactions of arthropods have a common underlying mechanism.     

Transitions in cuticular composition across a hybrid zone: historical accident or environmental adaptation?

Two subspecies of the grasshopper Chorthippus parallelus form a hybrid zone in the Pyrenees. Transitions across the zone, including changes in mating signals and reproductive isolation have been intensively studied. Cuticular pheromones have been identified as likely mate recognition signals. Since the major role of the cuticle is in waterproofing, environmental adaptation of cuticular composition has the interesting potential to generate assortative mating as an incidental by-product. We describe the pattern of variation in cuticular hydrocarbon blend in four transects through the hybrid zone. We find no evidence for a previously observed displaced cline in one blend component. There were differences between subspecies but these varied among transects and were small compared with variation between transects. We examined environmental variation within one transect and found a correlation between vegetation and cuticular composition, suggesting that environment influences the constitution of the cuticle, and hence natural selection may interact with mating signals in this species.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 193−201.     

Naturally Occurring Regenerative Differentiation of Xylem Around Adventitious Roots in Luffa cylindrica Seedlings

Regeneration of xylem induced by adventitious root formationin the hypocotyl of Luffa cylindrica Roem. seedlings is described.This naturally occurring form of xylem regeneration involvesthe formation of a bypass of regenerated tracheary elementsaround a root without external severance of the vascular strands.The regeneration of xylem around an adventitious root is polarand is very similar in its developmental pattern to the well-knownxylem regeneration induced by wounding vascular strands. Adventitious root formation, Luffa cylindrica Roem, regenerated tracheary elements, vascular differentiation, xylem regeneration     

Intracellular Localization of LRV1 in Infected Leishmania Cells and Epitope Conservation of the Coat Protein

ABSTRACT. Polyclonal antibodies were raised against a recombinant fragment of the coat protein of LRV1-1 to determine the epitope conservation of the coat protein among LRV1 isolates, and the intracellular localization of LRV1 particles in promastigote cells of Leishmania braziliensis . Western blot analysis showed that specific epitopes of the coat protein are highly conserved among isolates from different geographic areas. Using indirect immunofluorescence assays LRV1 viral particles were observed as fluorescent granules, limited to the cytoplasm and with no apparent association to the host organelles or the cell membrane, characteristic of a persistent, non-infectious virus.