bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells ofDrosophila melanogaster
Authors: Kimura Ken-ichi , Kodama Akitoshi , Hayasaka Yosihiro , Ohta Takumi
Published: March 2004

At the last step of metamorphosis in Drosophila, the wing epidermal cells are removed by programmed cell death during the wing spreading behavior after eclosion. The cell death was accompanied by DNA fragmentation demonstrated by the TUNEL assay. Transmission electron microscopy revealed that this cell death exhibited extensive vacuoles, indicative of autophagy. Ectopic expression of an anti-apoptotic gene, p35, inhibited the cell death, indicating the involvement of caspases. Neck ligation and hemolymph injection experiments demonstrated that the cell death is triggered by a hormonal factor secreted just after eclosion. The timing of the hormonal release implies that the hormone to trigger the death might be the insect tanning hormone, bursicon. This was supported by evidence that wing cell death was inhibited by a mutation of rickets, which encodes a G-protein coupled receptor in the glycoprotein hormone family that is a putative bursicon receptor. Furthermore, stimulation of components downstream of bursicon, such as a membrane permeant analog of cAMP, or ectopic expression of constitutively active forms of G proteins or PKA, induced precocious death. Conversely, cell death was inhibited in wing clones lacking G protein or PKA function. Thus, activation of the cAMP/PKA signaling pathway is required for transduction of the hormonal signal that induces wing epidermal cell death after eclosion.
Distinct and collaborative roles ofDrosophilaEXT family proteins in morphogen signalling and gradient formation
Authors: Han Chun , Belenkaya Tatyana Y. , Khodoun Marat , Tauchi Miyuki , Lin Xinda , Lin Xinhua
Published: March 2004

Heparan sulfate proteoglycans (HSPG) have been implicated in regulating the signalling activities of secreted morphogen molecules including Wingless (Wg),Hedgehog (Hh) and Decapentaplegic (Dpp). HSPG consists of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The formation of HS GAG chains is catalyzed by glycosyltransferases encoded by members of the EXT family of putative tumor suppressors linked to hereditary multiple exostoses. Previous studies in Drosophila demonstrated that tout-velu (ttv), the Drosophila EXT1, is required for Hh movement. However, the functions of other EXT family members are unknown. We have identified and isolated the other two members of the Drosophila EXT family genes, which are named sister of tout-velu (sotv) and brother of tout-velu(botv), and encode Drosophila homologues of vertebrate EXT2 and EXT-like 3 (EXTL3), respectively. We show that both Hh and Dpp signalling activities, as well as their morphogen distributions, are defective in cells mutant for ttv, sotv or botv in the wing disc. Surprisingly, although Wg morphogen distribution is abnormal in ttv, sotv and botv, Wg signalling is only defective in botv mutants or ttv-sotv double mutants, and not in ttv nor sotv alone, suggesting that Ttv and Sotv are redundant in Wg signalling. We demonstrate further that Ttv and Sotv form a complex and are co-localized in vivo. Our results, along with previous studies on Ttv, provide evidence that all three Drosophila EXT proteins are required for the biosynthesis of HSPGs, and for the gradient formation of the Wg, Hh and Dpp morphogens. Our results also suggest that HSPGs have two distinct roles in Wg morphogen distribution and signalling.
PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization
Authors: Salustri Antonietta , Garlanda Cecilia , Hirsch Emilio , De Acetis Marika , Maccagno Alessia , Bottazzi Barbara , Doni Andrea , Bastone Antonio , Mantovani Giovanna , Peccoz Paolo Beck , Salvatori Giovanni , Mahoney David J. , Day Anthony J. , Siracusa Gregorio , Romani Luigina , Mantovani Alberto
Published: March 2004

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3–/– mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3–/– mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-α-trypsin inhibitor is normal in Ptx3–/– cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor α-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.
Cell movements controlled by the Notch signalling cascade during foregut development inDrosophila
Authors: Fuss Bernhard , Josten Frank , Feix Maritta , Hoch Michael
Published: March 2004

Notch signalling is an evolutionarily conserved cell interaction mechanism,the role of which in controlling cell fate choices has been studied extensively. Recent studies in both vertebrates and invertebrates revealed additional functions of Notch in proliferation and apoptotic events. We provide evidence for an essential role of the Notch signalling pathway during morphogenetic cell movements required for the formation of the foregut-associated proventriculus organ in the Drosophila embryo. We demonstrate that the activation of the Notch receptor occurs in two rows of boundary cells in the proventriculus primordium. The boundary cells delimit a population of foregut epithelial cells that invaginate into the endodermal midgut layer during proventriculus morphogenesis. Notch receptor activation requires the expression of its ligand Delta in the invaginating cells and apical Notch receptor localisation in the boundary cells. We further show that the movement of the proventricular cells is dependent on the short stop gene that encodes the Drosophila plectin homolog of vertebrates and is a cytoskeletal linker protein of the spectraplakin superfamily. short stop is transcriptionally activated in response to the Notch signalling pathway in boundary cells and we demonstrate that the localisation of the Notch receptor and Notch signalling activity depend on short stop activity. Our results provide a novel link between the Notch signalling pathway and cytoskeletal reorganisation controlling cell movement during the development of foregut-associated organs.
Computational model of dynein-dependent self-organization of microtubule asters
Authors: Cytrynbaum E. N. , Rodionov V. , Mogilner A.
Published: March 2004

Polar arrays of microtubules play many important roles in the cell. Normally, such arrays are organized by a centrosome anchoring the minus ends of the microtubules, while the plus ends extend to the cell periphery. However, ensembles of molecular motors and microtubules also demonstrate the ability to self-organize into polar arrays. We use quantitative modeling to analyze the self-organization of microtubule asters and the aggregation of motor-driven pigment granules in fragments of fish melanophore cells. The model is based on the observation that microtubules are immobile and treadmilling, and on the experimental evidence that cytoplasmic dynein motors associated with granules have the ability to nucleate MTs and attenuate their minus-end dynamics. The model explains the observed sequence of events as follows. Initially, pigment granules driven by cytoplasmic dynein motors aggregate to local clusters of microtubule minus ends. The pigment aggregates then nucleate microtubules with plus ends growing toward the fragment boundary, while the minus ends stay transiently in the aggregates. Microtubules emerging from one aggregate compete with any aggregates they encounter leading to the gradual formation of a single aggregate. Simultaneously, a positive feedback mechanism drives the formation of a single MT aster – a single loose aggregate leads to focused MT nucleation and hence a tighter aggregate which stabilizes MT minus ends more effectively leading to aster formation. We translate the model assumptions based on experimental measurements into mathematical equations. The model analysis and computer simulations successfully reproduce the observed pathways of pigment aggregation and microtubule aster self-organization. We test the model predictions by observing the self-organization in fragments of various sizes and in bi-lobed fragments. The model provides stringent constraints on rates and concentrations describing microtubule and motor dynamics, and sheds light on the role of polymer dynamics and polymer-motor interactions in cytoskeletal organization.
Regions of human kidney anion exchanger 1 (kAE1) required for basolateral targeting of kAE1 in polarised kidney cells: mis-targeting explains dominant renal tubular acidosis (dRTA)
Authors: Toye Ashley M. , Banting George , Tanner Michael J. A.
Published: March 2004

Distal renal tubular acidosis (dRTA) is characterised by defective acid secretion by kidney α-intercalated cells. Some dominantly inherited forms of dRTA result from anion exchanger 1 (AE1) mutations. We have developed a stably transfected cell model for the expression of human kidney AE1 (kAE1) and mutant kAE1 proteins in MDCKI cells. Normal kAE1 was delivered to the plasma membrane of non-polarised cells and to the basolateral membrane of polarised cells. The AE1 N-glycan was processed to a complex form. Surprisingly, expression of kAE1 increased the permeability of the paracellular barrier of polarised MDCKI monolayers. All dominant dRTA mutations examined altered the targeting of kAE1 in MDCKI cells. The mutant proteins kAE1(R589H), kAE1(S613F) and kAE1(R901Stop) were retained in the ER in non-polarised cells, but the kAE1(R901Stop) protein was also present in late endosomes/lysosomes. The complex N-glycan of kAE1(R901Stop) was larger than that of normal kAE1. In polarised cells, the mutant kAE1(R901Stop) was mis-targeted to the apical membrane, while the kAE1(R589H) and kAE1(S613F) mutants did not reach the cell surface. These results demonstrate that dominant dRTA mutations cause aberrant targeting of kAE1 in polarised kidney cells and provide an explanation for the origin of dominant dRTA. Our data also demonstrate that the 11 C-terminal residues of kAE1 contain a tyrosine-dependent basolateral targeting signal that is not recognised by μ1B-containing AP-1 adaptor complexes. In the absence of the N-terminus of kAE1, the C-terminus was not sufficient to localise kAE1 to the basolateral membrane. These results suggest that a determinant within the kAE1 N-terminus co-operates with the C-terminus for kAE1 basolateral localisation.
Foxj1 regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells
Authors: Gomperts Brigitte N. , Gong-Cooper Xiulan , Hackett Brian P.
Published: March 2004

The forkhead box transcription factor Foxj1 is required for cilia formation and left-right axis determination. To define the role of Foxj1 in ciliogenesis, microarray analysis was performed to identify differentially expressed genes in the pulmonary epithelium of foxj1+/+ and foxj1-/- mice. In the absence of Foxj1, the expression of calpastatin, an inhibitor of the protease calpain, decreased. RNase protection confirmed the decrease in calpastatin expression and decreased calpastatin was detected in the proximal pulmonary epithelium of foxj1-/- mice by immunohistochemistry. No change was detected in the expression of calpain 2 in the pulmonary epithelium by western blot or immunohistochemistry. By western blot and immunofluorescence, ezrin, a substrate for calpain, was also found to decrease in the pulmonary epithelium of foxj1-/- mice. No change in ezrin gene expression was found by RT-PCR. A decrease in ezrin binding phosphoprotein-50 (EBP-50) was also detected by immunofluorescence in the foxj1-/- mouse pulmonary epithelium. Immunoelectron microscopy demonstrated ezrin associated with the basal bodies of cilia in the pulmonary epithelium. Treatment of tracheal explants from foxj1-/- mice with a calpain inhibitor resulted in a partial reappearance of cilia observed in these mice. Additionally, following treatment of foxj1-/- tracheal explants with calpain inhibitor, basal bodies were observed in an apical location along with relocalization of ezrin and EBP-50. Regulation of calpain activity by calpastatin thus provides a mechanism for regulating the anchoring of basal bodies to the apical cytoskeleton in ciliated cells. In the absence of Foxj1, decreased calpastatin expression with decreased ezrin and EBP-50 results in an inability of basal bodies to anchor to the apical cytoskeleton and subsequent failure of axonemal formation.
β3 integrin phosphorylation is essential for Arp3 organization into leukocyte αVβ3-vitronectin adhesion contacts
Authors: Chandhoke Surjit K. , Williams Matthew , Schaefer Erik , Zorn Linda , Blystone Scott D.
Published: March 2004

Integrins play a pivotal role in self-regulated hematopoietic adhesion and migration. Leukocyte αVβ3 integrin-mediated adhesion to vitronectin requires protein kinase C activation and phosphorylation on tyrosine 747 of the β3 cytoplasmic tail. We have previously shown that β3 phosphorylation is required for Rho activation. In this study, an antibody specific to phosphorylated β3 tyrosine 747 was used to localize phosphorylated αVβ3 in vitronectin adhesive structures. Early adhesion contacts containing phosphorylated β3 preceded actin stress fiber formation. β3 phosphorylation decreased progressively throughout the course of adhesion coincident with the appearance of actin stress fibers. Time-dependent increases in colocalization of β3 with tyrosine 402 phosphorylated Pyk2 in similar adhesive structures was observed, providing evidence for downstream signaling complex formation. Surprisingly, Arp3 organized into similar adhesion contacts in cells expressing wild-type β3 but not in those expressing a nonphosphorylatable mutant of β3, suggesting that β3 phosphorylation is required for sequestration of Arp3 to adhesion complexes. Suppression of actin stress fiber formation by an inhibitor to Rho kinase disrupted Arp3 organization while prolonging β3 phosphorylation throughout the adhesion time course. These data confirm a requirement for β3 phosphorylation in αVβ3-mediated adhesion to vitronectin and suggest that β3 phosphorylation permits signaling complex assembly at the adhesion site necessary for actin stress fiber formation in leukocytes.
Isoform-specific differences in rapid nucleocytoplasmic shuttling cause distinct subcellular distributions of 14-3-3σ and 14-3-3ζ
Authors: van Hemert Martijn J. , Niemantsverdriet Maarten , Schmidt Thomas , Backendorf Claude , Spaink Herman P.
Published: March 2004

Nucleocytoplasmic transport of proteins plays an important role in the regulation of many cellular processes. Differences in nucleocytoplasmic shuttling can provide a basis for isoform-specific biological functions for members of multigene families, like the 14-3-3 protein family. Many organisms contain multiple 14-3-3 isoforms, which play a role in numerous processes, including signalling, cell cycle control and apoptosis. It is still unclear whether these isoforms have specialised biological functions and whether this specialisation is based on isoform-specific ligand binding, expression regulation or specific localisation. Therefore, we studied the subcellular distribution of 14-3-3σ and 14-3-3ζ in vivo in various mammalian cell types using yellow fluorescent protein fusions and isoform-specific antibodies. 14-3-3σ was mainly localised in the cytoplasm and only low levels were present in the nucleus, whereas 14-3-3ζ was found at relatively higher levels in the nucleus. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the 14-3-3 proteins rapidly shuttle in and out of the nucleus through active transport and that the distinct subcellular distributions of 14-3-3σ and 14-3-3ζ are caused by differences in nuclear export. 14-3-3σ had a 1.7× higher nuclear export rate constant than 14-3-3ζ, while import rate constants were equal. The 14-3-3 proteins are exported from the nucleus at least in part by a Crm1-dependent, leptomycin B-sensitive mechanism. The differences in subcellular distribution of 14-3-3 that we found in this study are likely to reflect a molecular basis for isoform-specific biological specialisation.
The stroma as a crucial target in rat mammary gland carcinogenesis
Authors: Maffini Maricel V. , Soto Ana M. , Calabro Janine M. , Ucci Angelo A. , Sonnenschein Carlos
Published: March 2004

A complex network of interactions between the stroma, the extracellular matrix and the epithelium drives mammary gland development and function. Two main assumptions in chemical carcinogenesis of the mammary gland have been that carcinogens induce neoplasia by causing mutations in the DNA of the epithelial cells and that the alterations of tissue architecture observed in neoplasms are a consequence of this primary mutational event. Here, we use a rat mammary tissue recombination model and the chemical carcinogen N-nitrosomethylurea (NMU) to determine whether the primary target of the carcinogen is the epithelium, the stroma or both tissue compartments. Mammary epithelial cells were exposed in vitro either to the carcinogen or vehicle before being transplanted into the cleared fat pads of rats exposed to carcinogen or vehicle. We observed that neoplastic transformation of these mammary epithelial cells occurred only when the stroma was exposed in vivo to NMU, regardless of whether or not the epithelial cells were exposed to the carcinogen. Mammary epithelial cells exposed in vitro to the carcinogen formed phenotypically normal ducts when injected into a non-treated stroma. Mutation in the Ha-ras-1 gene did not correlate with initiation of neoplasia. Not only was it often found in both cleared mammary fat pads of vehicle-treated animals and intact mammary glands of untreated animals, but it was also absent in some tumors. Our results suggest that the stroma is a crucial target of the carcinogen and that mutation in the Ha-ras-1 gene is neither necessary nor sufficient for tumor initiation.
EGFR signaling to p120-catenin through phosphorylation at Y228
Authors: Mariner Deborah J. , Davis Michael A. , Reynolds Albert B.
Published: March 2004

Epidermal growth factor receptor (EGFR) signals to p120ctn (p120), implying a role for EGFR in modulating cell-cell adhesion in epithelial tissues. p120 controls cadherin turnover, and may have other roles that modulate cadherin adhesiveness. To clarify the role for EGFR and other tyrosine kinases in regulating p120 function, we have generated and characterized a new phosphospecific antibody to p120 Y228, as well as a novel siRNA-based reconstitution system for analyzing roles of individual p120 phosphorylation events. In A431 cells, epidermal growth factor induced striking p120 phosphorylation at Y228. Y228-phosphorylated p120 localized to adherens junctions and lamellipodia, and was significantly enhanced in cells around the colony periphery. A screen of carcinoma cell lines revealed that some contain unusually high steady state levels of Y228 phosphorylation, suggesting that disregulated kinase activity in tumors may affect adhesion by constitutive cross talk to cadherin complexes. Despite these observations, mutation of Y228 and other prominent Src-associated p120 phosphorylation sites did not noticeably reduce the ability of E-cadherin to assemble junctions and induce compaction of cultured cells. Although A431 cells display significant activation of both EGFR and Src kinases, our data suggest that these account for only a fraction of the steady state activity that targets p120 Y228, and that Src family kinases are not necessary intermediates for epidermal growth factor-induced signaling to p120 Y228.
Laminin 5 deposition regulates keratinocyte polarization and persistent migration
Authors: Frank Diane E. , Carter William G.
Published: March 2004

Repair of wounded epidermis requires both keratinocyte migration and deposition of laminin 5 over exposed dermal collagen. To understand the coupling between leading cell migration and laminin 5 deposition, we developed a novel migration assay using time-lapse microscopy. We demonstrate that in migrating, human keratinocytes the deposition of laminin 5 promoted `processive migration', characterized by stable cell polarization that was tightly coupled to persistent, linear migration in the absence of a chemotactic gradient. Processive migration required deposition of laminin 5, which was restricted to the rear of the polar cell. Integrin α3β1 interacted with these laminin 5 deposits at contact sites that did not require actin-dependent cross-linking. Further, we show that the migrating cells switched adhesion by integrin α2β1 on collagen at the front of the cell to integrin α3β1 on exogenous laminin 5 at the rear of the cell. Along with this switch of integrin usage was the removal of collagen from sites under the cell that precisely correlated with deposition of laminin 5. Processive migration was blocked with suppressors of microtubule dynamics (nocodazole and taxol) or rottlerin, a PKC-δ inhibitor. These drugs were also shown to block deposition of laminin 5 but, surprisingly, constitutive secretion was unimpaired, suggesting deposition was a regulated event. Thus, at the front of the cell, the leading lamellipodium was stabilized through integrin interactions in focal complexes with the exogenous substratum. However, at the rear of the cell, stable cell polarization and linear migration was promoted by laminin 5 deposits and integrin α3β1.
Ganglioside GM1 levels are a determinant of the extent of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus
Authors: Pang Hao , Le Phuong U. , Nabi Ivan R.
Published: March 2004

Cholera toxin is associated with caveolae and raft domains in various cell types and previous studies have shown that cholera toxin can be internalized by caveolae/raft-dependent endocytosis as well as by other pathways. We undertook the study of cholera toxin endocytosis in CaCo-2 and HeLa cells. CaCo-2 cells do not express detectable levels of caveolin and, relative to HeLa cells, also present significantly reduced expression of ganglioside GM1, the cholera toxin receptor, that remains Triton X-100 insoluble. Amongst the HeLa cell population, caveolin expression is constant, however, GM1 expression is highly variable. Cholera toxin is internalized to the Golgi apparatus via a caveolae/raft-dependent pathway sensitive to methyl-β-cyclodextrin and genistein in high-GM1-expressing HeLa cells but not in low-GM1 HeLa cells or in CaCo-2 cells. Limited cholera toxin endocytosis to endosomes sensitive to neither methyl-β-cyclodextrin nor genistein is also observed in all cells and corresponds to a non-caveolae/raft endocytic pathway. Increasing cell-associated GM1 by adding GM1 to the cell media of both HeLa and CaCo-2 cells selectively enhances the methyl-β-cyclodextrin-, genistein-sensitive delivery of cholera toxin to the Golgi apparatus but not to endosomes. GM1 expression levels are therefore a selective determinant of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus and variable expression of GM1 between cells can impact on the endocytosis and choice of pathway followed by cholera toxin.
Membrane-associated HB-EGF modulates HGF-induced cellular responses in MDCK cells
Authors: Singh Amar B. , Tsukada Toshiaki , Zent Roy , Harris Raymond C.
Published: March 2004

In MDCK cells, hepatocyte growth factor/scatter factor (HGF/SF) induces epithelial cell dissociation, scattering, migration, growth and formation of branched tubular structures. By contrast, these cells neither scatter nor form tubular structures in response to the epidermal growth factor (EGF) family of growth factors. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors and is synthesized as a membrane-associated precursor molecule (proHB-EGF). ProHB-EGF is proteolytically cleaved to release a soluble ligand (sHB-EGF) that activates the EGF receptor. Although recent studies suggest possible physiological functions, the role of proHB-EGF remains largely undefined. Using MDCK cells stably expressing proHB-EGF, a noncleavable deletion mutant of proHB-EGF or soluble HB-EGF, we show that epithelial cell functions differ depending on the form of HB-EGF being expressed. Expression of noncleavable membrane-anchored HB-EGF promoted cell-matrix and cell-cell interactions and decreased cell migration, HGF/SF-induced cell scattering and formation of tubular structures. By contrast, expression of soluble HB-EGF induced increased cell migration, decreased cell-matrix and cell-cell interactions and promoted the development of long unbranched tubular structures in response to HGF/SF. These findings suggest that HB-EGF can not only modulate HGF/SF-induced cellular responses in MDCK cells but also that membrane-bound HB-EGF and soluble HB-EGF give rise to distinctly different effects on cell-cell and cell-extracellular matrix interactions.
Chemical defense of an opilionid (Acanthopachylus aculeatus)
Authors: Eisner Thomas , Rossini Carmen , González Andrés , Eisner Maria
Published: March 2004

The opilionid Acanthopachylus aculeatus was shown to produce a defensive secretion containing quinones (2,3-dimethyl-1,4-benzoquinone,2,5-dimethyl-1,4-benzoquinone and 2,3,5-trimethyl-1,4-benzoquinone),confirming the findings reported nearly a half century ago in a classic study. The mechanism by which the opilionid puts the secretion to use is described. When disturbed, the animal regurgitates enteric fluid, which it conveys by intercoxal clefts to the anterolateral corners of the carapace, where the two gland openings are situated. It then injects some of its quinonoid secretion into the fluid, and conveys the mixed liquid along the length of its flanks by way of two special channels. Such a discharge mechanism may be widespread among opilionids of the family Gonyleptidae (suborder Laniatores), to which A. aculeatus belongs. In a bioassay based on a scratch reflex in decapitated cockroaches (Periplaneta americana) the liquid effluent of A. aculeatus was shown to be potently irritating. Use of the effluent was demonstrated to protect the opilionid against ants (Formica exsectoides). Wolf spiders (Lycosa ceratiola) were shown to be minimally affected by the effluent (they showed little response when the fluid was added to their mouthparts as they fed on mealworms, their normal laboratory prey), although they proved to be aversive to mere contact with the opiliond itself, and to reject the animal without inducing it to discharge. A. aculeatus may therefore contain distasteful factors besides its glandular products.
Mitochondria-rich cell activity in the yolk-sac membrane of tilapia(Oreochromis mossambicus) larvae acclimatized to different ambient chloride levels
Authors: Lin Li-Yih , Hwang Pung-Pung
Published: March 2004

Mitochondria-rich cells (MRCs) in the yolk-sac membrane of tilapia(Oreochromis mossambicus) larvae were examined by Na+/K+-ATPase immunocytochemistry and vital staining for glycoproteins following acclimation to high (7.5–7.9 mmol l–1), normal (0.48–0.52 mmol l–1) or low (0.002–0.007 mmol l–1) ambient Cl–levels. With a combination of concanavalin-A (Con-A)–Texas-Red conjugate staining (larvae exposed to the dye in vivo in the water) and a monoclonal antibody raised against Na+/K+-ATPase, MRCs were easily recognized and presumed to be active when Con-A-positive (i.e. with their apical membrane in contact with the water) or inactive when Con-A-negative. The proportion of active cells gradually increased during a 48-h acclimation to low-Cl– medium but decreased during acclimation to high-Cl– medium. Total densities of MRCs did not change when ambient chloride levels were altered. Furthermore, in live larvae exposed to changes in ambient Cl–, yolk-sac MRCs,vitally stained with DASPEI and subsequently traced in time, did not significantly alter turnover. The polymorphism of the apical membrane compartment of the MRCs represents structural modification of the active MRCs. Yolk-sac pavement cells labeled with the membrane marker FM1-43 (fluorescent lipophilic tracer) were shown to cover active MRCs in larvae transferred from normal to high ambient Cl– levels, thereby inactivating the MRCs.
Metabolism of the sub-Antarctic caterpillar Pringleophaga marioni during cooling, freezing and thawing
Authors: Sinclair Brent J. , Klok C. Jaco , Chown Steven L.
Published: March 2004

Although general models of the processes involved in insect survival of freezing exist, there have been few studies directly investigating physiological processes during cooling, freezing and thawing, without which these models remain hypothetical. Here, we use open-flow respirometry to investigate the metabolism of the freeze-tolerant sub-Antarctic caterpillar Pringleophaga marioni Viette (Lepidoptera: Tineidae) during cooling,freezing and thawing and to compare animals exposed to non-lethal(–5.8°C) and lethal (–6.0°C, after which caterpillars are moribund for several days, and –18°C, after which caterpillars are completely unresponsive) freezing stress. We found a large decrease in metabolic rate (that is not associated with freezing) at–0.6±0.1°C and calculated a Q10 of 2.14×103 at this breakpoint. This breakpoint is coincident with the critical thermal minimum (CTmin) and is hypothesised to be a metabolic manifestation of the latter, possibly a failure of the Na+/K+-ATPase pump. This provides a plausible link between processes at the cellular level and observations of the action of the CTmin at tissue and whole-organism levels. Caterpillars froze at –4.6±0.1°C and had detectable metabolism when frozen. Post-thaw, metabolic rates were lower than pre-freezing measurements. Post-thaw metabolic rates did not differ between temperatures that did and did not kill the caterpillars, which suggests that mortality may be a result of a breakdown in processes at the organismal,rather than cellular, level of organisation.
Independent effects of heart–head distance and caudal blood pooling on blood pressure regulation in aquatic and terrestrial snakes
Authors: Seymour Roger S. , Arndt Joachim O.
Published: March 2004

Changes in orientation in a gravitational field markedly alter the patterns of blood pressure and flow in animals, especially tall or long ones such as giraffes or snakes. Vertical orientation tends to reduce blood flow and pressure in the head for two major reasons. First, the increased vertical blood column above the heart creates a gravitational hydrostatic pressure against which the heart must work. Second, expansion of dependent vessels in the lower extremities causes blood pooling and reduces return of venous blood to the heart, thereby lowering flow and pressure. For most animals, it is difficult to separate these two effects, but snakes offer the possibility of bending the animal in the region of the heart and manipulating the two ends of the body independently. We studied baroregulatory responses in terrestrial pythons (Liasis fuscus) and aquatic file snakes (Acrochordus arafurae) by tilting only the front or rear parts and then the whole animal. Changes in head blood pressure during partial tilts added up to the change during full tilt. The vertical distance to the head had twice as much influence on head blood pressure than did blood pooling in the pythons and four times as much in file snakes. This accounts for the cephalad location of the heart in terrestrial species compared with aquatic ones.
Nitric oxide control of the dorsal aorta and the intestinal vein of the Australian short-finned eel Anguilla australis
Authors: Jennings Brett L. , Broughton Brad R. S. , Donald John A.
Published: March 2004

This study investigated the mechanisms by which nitric oxide (NO) regulates the dorsal aorta and the intestinal vein of the Australian short-finned eel Anguilla australis. NADPH diaphorase histochemistry and immunohistochemistry using a mammalian endothelial nitric oxide synthase (NOS)antibody could not demonstrate NOS in the endothelium of either blood vessel;however, NOS could be readily demonstrated in the endothelium of the rat aorta that was used as a control. Both blood vessels contained NADPH diaphorase positive nerve fibres and nerve bundles, and immunohistochemistry using a neural NOS antibody showed a similar distribution of neural NOS immunoreactivity in the perivascular nerves. In vitro organ bath physiology showed that a NO/soluble guanylyl cyclase (GC) system is present in the dorsal aorta and the intestinal vein, since the soluble GC inhibitor oxadiazole quinoxalin-1 (ODQ; 10–5 mol l–1)completely abolished the vasodilatory effect of the NO donor, sodium nitroprusside (SNP; 10–4 mol l–1). In addition, nicotine (3×10–4 mol l–1)mediated a vasodilation that was not affected by removal of the endothelium. The nicotine-mediated dilation was blocked by the NOS inhibitor, Nω-nitro-l-arginine (l-NNA;10–4 mol l–1), and ODQ(10–5 mol l–1). More specifically, the neural NOS inhibitor, Nω-propyl-l-arginine(10–5 mol l–1), significantly decreased the dilation induced by nicotine (3×10–4 mol l–1). Furthermore, indomethacin (10–5 mol l–1) did not affect the nicotine-mediated dilation,suggesting that prostaglandins are not involved in the response. Finally, the calcium ionophore A23187 (3×10–6 mol l–1) caused an endothelium-dependent dilation that was abolished in the presence of indomethacin. We propose the absence of an endothelial NO system in eel vasculature and suggest that neurally derived NO contributes to the maintenance of vascular tone in this species. In addition,we suggest that prostaglandins may act as endothelially derived relaxing factors in A. australis.
Effect of β-adrenergic stimulation on the relationship between membrane potential, intracellular [Ca2+] and sarcoplasmic reticulum Ca2+ uptake in rainbow trout atrial myocytes
Authors: Llach Anna , Huang Jingbo , Sederat Franklin , Tort Lluis , Tibbits Glen , Hove-Madsen Leif
Published: March 2004

Long depolarizations cause a steady tonic contraction and induce sarcoplasmic reticulum (SR) Ca2+-uptake in trout atrial myocytes. Simultaneous measurements of cytosolic [Ca2+]([Ca2+]i) and whole membrane current showed an elevated[Ca2+]i throughout the depolarization. Rapid caffeine(Caf) applications at –80 mV before and after a long depolarization were used to determine SR Ca2+ loading and its dependency on membrane potential and [Ca2+]i during depolarization. Following a 10 s depolarization, the maximal SR Ca2+ load was 597 μmol l–1 and loading was half-maximal at –12 mV. Theβ-adrenergic agonist isoproterenol (ISO) did not affect the maximal SR Ca2+ loading but shifted the potential for half-maximal loading by–26 mV. Following a 3 s depolarization, the maximal SR Ca2+uptake rate (V̇max) was 418μmol l–1 s–1 in control conditions. ISO did not affect V̇max, but significantly lowered the average free Ca2+ transient during the depolarization and shifted the K0.5 for the relationship between SR Ca2+ uptake and [Ca2+]i from 1.27 in control to 0.8 μmol l–1 with ISO. Following repetitive 200 ms depolarizations, ISO increased the l-type Ca2+current (ICa) amplitude by 91±29% and the peak Ca2+ transient by 41±10%, and decreased the half life of the Ca2+ transient from 151±12 to 111±6 ms. Using the relationship between [Ca2+]i and SR Ca2+uptake to calculate the total SR Ca2+ uptake during a Ca2+ transient elicited by a 200 ms depolarization, a significant increase in the SR Ca2+ uptake from 37±6 μmol l–1 in control to 68±4 μmol l–1with ISO was seen. When normalized to the total Ca2+ transport the contribution of the SR was not significantly different in the absence(35±6%) or presence of ISO (41±4%). Exposure of cells to ISO and low extracellular [Ca2+] increased ICa by 67±40%(N=5) but significantly reduced SR Ca2+ uptake at membrane potentials above –30 mV. Together, these results suggest that (i) ISO has a stimulatory effect on the SR Ca2+ pump that may contribute to the faster decay of the Ca2+ transient, and (ii) the relative contribution of the SR to the Ca2+ removal during relaxation is not altered by ISO in trout atrial myocytes.
Contribution of eye retraction to swallowing performance in the northern leopard frog,Rana pipiens
Authors: Levine Robert P. , Monroy Jenna A. , Brainerd Elizabeth L.
Published: March 2004

Most anurans retract and close their eyes repeatedly during swallowing. Eye retraction may aid swallowing by helping to push food back toward the esophagus, but this hypothesis has never been tested. We used behavioral observations, cineradiography, electromyography and nerve transection experiments to evaluate the contribution of eye retraction to swallowing in the northern leopard frog, Rana pipiens. Behavioral observations of frogs feeding on 1.5 cm long crickets reveal a high degree of variability in eye retraction and swallowing. Eye retraction can occur bilaterally or unilaterally, and both swallowing movements and eye retraction can occur separately as well as together. During swallowing, cineradiography shows that the eyes and associated musculature retract well into the oropharynx and appear to make contact with the prey item. This contact appears to help push the prey toward the esophagus, and it may also serve to anchor the prey for tongue-based transport. Electromyographic recordings confirm strong activity in the retractor bulbi muscles during eye retraction. After bilateral denervation of the retractor bulbi, frogs maintain the ability to swallow but show a 74% increase in the number of swallows required per cricket (from a mean of 2.3 swallows to a mean of 4.0 swallows per cricket). Our results indicate that, in Rana pipiens feeding on medium-sized crickets, eye retraction is an accessory swallowing mechanism that assists the primary tongue-based swallowing mechanism.
Passive tools for enhancing muscle-driven motion and locomotion
Authors: Minetti Alberto E.
Published: March 2004

Musculo–skeletal systems and body design in general have evolved to move effectively and travel in specific environments. Humans have always aspired to reach higher power movement and to locomote safely and fast, even through unfamiliar media (air, water, snow, ice). For the last few millennia,human ingenuity has led to the invention of a variety of passive tools that help to compensate for the limitations in their body design. This Commentary discusses many of those tools, ranging from halteres used by athletes in ancient Greece, to bows, skis, fins, skates and bicycles, which are characterised by not supplying any additional mechanical energy, thus retaining the use of muscular force alone. The energy cascade from metabolic fuel to final movement is described, with particular emphasis on the steps where some energy saving and/or power enhancement is viable. Swimming is used to illustrate the efficiency breakdown in complex locomotion, and the advantage of using fins. A novel graphical representation of world records in different types of terrestrial and aquatic locomotion is presented, which together with a suggested method for estimating their metabolic cost (energy per unit distance), will illustrate the success of the tools used.
Stick insect locomotion in a complex environment: climbing over large gaps
Authors: Blaesing Bettina , Cruse Holk
Published: March 2004

In a complex environment, animals are challenged by various types of obstacles. This requires the controller of their walking system to be highly flexible. In this study, stick insects were presented with large gaps to cross in order to observe how locomotion can be adapted to challenging environmental situations. Different approaches were used to investigate the sequence of gap-crossing behaviour. A detailed video analysis revealed that gap-crossing behaviour resembles modified walking behaviour with additional step types. The walking sequence is interrupted by an interval of exploration, in which the insect probes the gap space with its antennae and front legs. When reaching the gap, loss of contact of an antenna with the ground does not elicit any observable reactions. In contrast, an initial front leg step into the gap that often follows antennal `non-contact' evokes slowing down of stance velocity. An ablation experiment showed that the far edge of the gap is detected by tactile antennal stimulation rather than by vision. Initial contact of an antenna or front leg with the far edge of the gap represents a `point of no return', after which gap crossing is always successfully completed. Finally,flow chart diagrams of the gap-crossing sequence were constructed based on an ethogram of single elements of behaviour. Comparing flow charts for two gap sizes revealed differences in the frequency and succession of these elements,especially during the first part of the sequence.
Take-off mechanics in hummingbirds (Trochilidae)
Authors: Tobalske Bret W. , Altshuler Douglas L. , Powers Donald R.
Published: March 2004

Initiating flight is challenging, and considerable effort has focused on understanding the energetics and aerodynamics of take-off for both machines and animals. For animal flight, the available evidence suggests that birds maximize their initial flight velocity using leg thrust rather than wing flapping. The smallest birds, hummingbirds (Order Apodiformes), are unique in their ability to perform sustained hovering but have proportionally small hindlimbs that could hinder generation of high leg thrust. Understanding the take-off flight of hummingbirds can provide novel insight into the take-off mechanics that will be required for micro-air vehicles. During take-off by hummingbirds, we measured hindlimb forces on a perch mounted with strain gauges and filmed wingbeat kinematics with high-speed video. Whereas other birds obtain 80–90% of their initial flight velocity using leg thrust,the leg contribution in hummingbirds was 59% during autonomous take-off. Unlike other species, hummingbirds beat their wings several times as they thrust using their hindlimbs. In a phylogenetic context, our results show that reduced body and hindlimb size in hummingbirds limits their peak acceleration during leg thrust and, ultimately, their take-off velocity. Previously, the influence of motivational state on take-off flight performance has not been investigated for any one organism. We studied the full range of motivational states by testing performance as the birds took off: (1) to initiate flight autonomously, (2) to escape a startling stimulus or (3) to aggressively chase a conspecific away from a feeder. Motivation affected performance. Escape and aggressive take-off featured decreased hindlimb contribution (46% and 47%,respectively) and increased flight velocity. When escaping, hummingbirds foreshortened their body movement prior to onset of leg thrust and began beating their wings earlier and at higher frequency. Thus, hummingbirds are capable of modulating their leg and wingbeat kinetics to increase take-off velocity.
Physiological, biochemical and morphological indicators of osmoregulatory stress in `California' Mozambique tilapia (Oreochromis mossambicus×O. urolepis hornorum) exposed to hypersaline water
Authors: Sardella Brian A. , Matey Victoria , Cooper Jill , Gonzalez Richard J. , Brauner Colin J.
Published: March 2004

The salinity tolerance of the `California' Mozambique tilapia(Oreochromis mossambicus × O. urolepis hornorum), a current inhabitant of the hypersaline Salton Sea in California, USA, was investigated to identify osmoregulatory stress indicators for possible use in developing a model of salinity tolerance. Seawater-acclimated (35 g l–1) tilapia hybrids were exposed to salinities from 35–95 g l–1, using gradual and direct transfer protocols, and physiological (plasma osmolality, [Na+],[Cl–], oxygen consumption, drinking rate, hematocrit, mean cell hemoglobin concentration, and muscle water content), biochemical(Na+, K+-ATPase) and morphological (number of mature,accessory, immature and apoptotic chloride cells) indicators of osmoregulatory stress were measured. Tilapia tolerated salinities ranging from 35 g l–1 to 65 g l–1 with little or no change in osmoregulatory status; however, in fish exposed to 75–95 g l–1 salinity, plasma osmolality, [Na+],[Cl–], Na+, K+-ATPase, and the number of apoptotic chloride cells, all showed increases. The increase in apoptotic chloride cells at salinities greater than 55 g l–1, prior to changes in physiological and biochemical parameters, indicates that it may be the most sensitive indicator of osmoregulatory stress. Oxygen consumption decreased with salinity, indicating a reduction in activity level at high salinity. Finally, `California' Mozambique tilapia have a salinity tolerance similar to that of pure Mozambique tilapia; however, cellular necrosis at 95 g l–1 indicates they may be unable to withstand extreme salinities for extended periods of time.
Ventilatory compensation of the alkaline tide during digestion in the snakeBoa constrictor
Authors: Andrade Denis V. , De Toledo Luis Felipe , Abe Augusto S. , Wang Tobias
Published: March 2004

The increased metabolic rate during digestion is associated with changes in arterial acid–base parameters that are caused by gastric acid secretion(the `alkaline tide'). Net transfer of HCl to the stomach lumen causes an increase in plasma HCO3– levels, but arterial pH does not change because of a ventilatory compensation that counters the metabolic alkalosis. It seems, therefore, that ventilation is controlled to preserve pH and not PCO2 during the postprandial period. To investigate this possibility, we determined arterial acid–base parameters and the metabolic response to digestion in the snake Boa constrictor, where gastric acid secretion was inhibited pharmacologically by oral administration of omeprazole.

The increase in oxygen consumption of omeprazole-treated snakes after ingestion of 30% of their own body mass was quantitatively similar to the response in untreated snakes, although the peak of the metabolic response occurred later (36 h versus 24 h). Untreated control animals exhibited a large increase in arterial plasma HCO3– concentration of approximately 12 mmol l–1, but arterial pH only increased by 0.12 pH units because of a simultaneous increase in arterial PCO2 by about 10 mmHg. Omeprazole virtually abolished the changes in arterial pH and plasma HCO3– concentration during digestion and there was no increase in arterial PCO2. The increased arterial PCO2 during digestion is not caused, therefore, by the increased metabolism during digestion or a lower ventilatory responsiveness to ventilatory stimuli during a presumably relaxed state in digestion. Furthermore, the constant arterial PCO2, in the absence of an alkaline tide, of omeprazole-treated snakes strongly suggests that pH rather than PCO2 normally affects chemoreceptor activity and ventilatory drive.