Changes in nuclear phenotypes following cold shock in Panstrongylus megistus (Burmeister).
(17/268)
The nuclear phenotypes of Malpighian tubule epithelial cells of 5th instar male nymphs of the blood-sucking insect Panstrongylus megistus were studied immediately after a short (1 h) cold shock at 0 degrees C, and 10 and 30 days later. The objective was to compare the responses to a cold shock with those known to occur after hyperthermia in order to provide insight into the cellular effect of cold in this species. Nuclei which usually exhibited a conspicuous Y chromosome chromocenter were the most frequent phenotype in control and treated specimens. Phenotypes in which the heterochromatin was unravelled, or in which there was nuclear fusion or cell death were more abundant in the shocked specimens. Most of the changes detected have also been found in heat-shocked nymphs, except for nuclear fusion which generates giant nuclei and which appeared to be less effective or necessary than that elicited after heat shock. Since other studies showed that a short cold shock does not affect the survival of more than 14% of 5th instar nymphs of P. megistus with domestic habit and can induce tolerance to a prolonged cold shock, heat shock proteins proteins are probably the best candidates for effective protection of the cells and the insects from drastic damage caused by low temperature shocks. (+info)
Tissue-specific autoregulation of Drosophila suppressor of forked by alternative poly(A) site utilization leads to accumulation of the suppressor of forked protein in mitotically active cells.
(18/268)
The Suppressor of forked protein is the Drosophila homolog of the 77K subunit of human cleavage stimulation factor, a complex required for the first step of the mRNA 3'-end-processing reaction. We have shown previously that wild-type su(f) function is required for the accumulation of a truncated su(f) transcript polyadenylated in intron 4 of the gene. This led us to propose a model in which the Su(f) protein would negatively regulate its own accumulation by stimulating 3'-end formation of this truncated su(f) RNA. In this article, we demonstrate this model and show that su(f) autoregulation is tissue specific. The Su(f) protein accumulates at a high level in dividing tissues, but not in nondividing tissues. We show that this distribution of the Su(f) protein results from stimulation by Su(f) of the tissue-specific utilization of the su(f) intronic poly(A) site, leading to the accumulation of the truncated su(f) transcript in nondividing tissues. Utilization of this intronic poly(A) site is affected in a su(f) mutant and restored in the mutant with a transgene encoding wild-type Su(f) protein. These data provide an in vivo example of cell-type-specific regulation of a protein level by poly(A) site choice, and confirm the role of Su(f) in regulation of poly(A) site utilization. (+info)
Coordinating cell fate and morphogenesis in Drosophila renal tubules.
(19/268)
Using the renal tubules of Drosophila as an example, we explore how cell specification leads to the morphogenetic movements that underlie the generation of tissue architecture. Taking two stages of development, we show first that the tubule cells are allocated by signalling between the endodermal and ectodermal compartments of the posterior gut. Activation of the Wnt pathway patterns the ectodermal anlage, resulting in the expression of tubule genes in a subset of cells and their eversion from the hindgut to form the tubule primordia. We argue that early gene expression directs these morphogenetic movements but not the complete programme of tubule differentiation. In the second example we show that the allocation of the mitogenic tip cell lineage in each tubule is required not only for the normal pattern of cell division but also for the stereotyped three-dimensional arrangement of the mature tubules. Analysis of mutants in which the tip cell lineage is misspecified reveals that both daughters of the tip cell progenitor are required for the tubules to navigate through the body cavity, so that the distal tips locate in their characteristic positions. We show that the regulator of Rac, Myoblast city is essential for this second morphogenetic process. (+info)
Chloride channels in apical membrane patches of stellate cells of Malpighian tubules of Aedes aegypti.
(20/268)
Stellate cells of Aedes aegypti Malpighian tubules were investigated using patch-clamp methods to probe the route of transepithelial Cl(-) secretion. Two types of Cl(-) channel were identified in excised, inside-out apical membrane patches. The first Cl(-) channel, type I, had a conductance of 24 pS, an open probability of 0.816+/-0.067, an open time of 867+/-114 ms (mean +/- s.e.m., four patches) and the selectivity sequence I(-)>Cl(-)(much greater than) isethionate>gluconate. The I(-)/Cl(-)>>isethionate>gluconate. The I(-)Cl(-) permeability ratio was 1.48, corresponding to Eisenman sequence I. The type I Cl(-) channel was blocked by 2,2'-iminodibenzoic acid (DPC) and niflumic acid (2-[3-(trifluoromethyl)anilo]nicotinic acid). The removal of Ca(2+) from the Ringer's solution on the cytoplasmic side had no effect on channel activity. The second Cl(-) channel, type II, had a conductance of 8 pS, an open probability of 0.066+/-0.021 and an open time of 7.53+/-1.46 ms (mean +/- s.e.m., four patches). The high density and halide selectivity sequence of the type I Cl(-) channel is consistent with a role in transepithelial Cl(-) secretion under control conditions, but it remains to be determined whether these Cl(-) channels also mediate transepithelial Cl(-) secretion under diuretic conditions in the presence of leucokinin. (+info)
The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules.
(21/268)
The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol x l(-1). The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl- concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol x l(-1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane. (+info)
Energizing epithelial transport with the vacuolar H(+)-ATPase.
(22/268)
The Ussing model has long provided the conceptual foundation for understanding epithelial transport mechanisms energized by the Na(+)-K(+)-ATPase. Plasma membranes may also use the vacuolar (V-type) H(+)-ATPase as the primary energy source of membrane and epithelial transport. A pure electrogenic pump, the V-type H(+)-ATPase energizes not only membranes it inhabits but also other transport pathways via electrical coupling. (+info)
Analysis of epithelial K(+) transport in Malpighian tubules of Drosophila melanogaster: evidence for spatial and temporal heterogeneity.
(23/268)
Transport of K(+) by the lower, main and distal segments of the Malpighian tubules of Drosophila melanogaster was analyzed using self-referencing K(+)-selective microelectrodes. Transport properties of the Malpighian tubules of Drosophila melanogaster change along their length. Self-referencing ion-selective (SeRIS) microelectrode measurements (relative to the bath concentration of 20 mmoll(-1)) showed a 1% reduction (P<0.05) of [K(+)] in the unstirred layer adjacent to the main segment of the Malpighian tubules, confirming secretion of K(+) from the bath to the tubule lumen. Conversely, SeRIS measurements showed a 0.7% increase (P<0.05) in [K(+)] in the unstirred layer adjacent to the lower segment of Malpighian tubules, confirming reabsorption of K(+) from the luminal fluid to the bath. Measurements using SeRIS also showed that the distal segment neither secreted nor reabsorbed K(+). There was pronounced spatial heterogeneity in K(+) transport by the lower segment and the main segment; not all morphologically similar cells participated equally in K(+) transport, nor did all main segment cells respond equally to stimulation of K(+) transport by cyclic AMP. Pronounced temporal heterogeneity in K(+) reabsorption by the lower Malpighian tubules was also observed. We suggest that this reflects periodic reduction in K(+) reabsorption due to retention of fluid within the lower segment when the ureter contracts. (+info)
Excretory role of the midgut in larvae of the tobacco hornworm, Manduca sexta (L.).
(24/268)
Caterpillars of Manduca sexta use two distinct transport mechanisms for the excretion of dyes. One pump (Type A) has a high affinity for acid (anionic) dyes and occurs in the midgut and medial Malpighian tubules. Acid dyes accumulate rapidly in the lumen of the midgut while the Malpighian tubules appear to play only a minor role in the excretion of these dyes. The other pump (Type B) excretes basic (cationic) dyes and is located primarily in the proximal Malpighian tubules. Evidence is presented that hippuric acid competes with acid dyes for excretion by both midgut and Malpighian tubules. After the final-instar larva purges its gut the ability of the midgut and Malpighian tubules to excrete dyes gradually decreases. Sixty hours after the purge only the Malpighian tubules retain some dye excreting activity. (+info)