Limb Regeneration in the Fiddler Crab, Uca pugilator: Hormonal and Growth Factor Control

This paper summarizes recent work on various aspects of hormonalcontrol of regeneration in the crustacean, Uca pugilator. Hormonalcontrol in this crab is effected by means of the crustacteansteroid hormones, the ecdysteroids. New evidence is presentedsupporting a role for the retinoid hormones, all-trans retinoicacid and 9-cis retinoic acid, in the control of regenerationin these animals. The possible role of fibroblast growth factorsin organization of the limb blastema is explored and the similaritiesbetween vertebrate and invertebrate control of regenerationare discussed.     

Limb Regeneration in the Fiddler Crab, Uca pugilator: Histological, Physiological and Molecular Considerations

This paper summarizes our recent work on the histological, physiologicaland molecular aspects of limb regeneration in the fiddler crabUca pugilator and new information is presented on mitotic activityin the blastema during the first days of blastemal organization.We also report for the first time the localization of vertebrategrowth factor immunoreactivity (FGF 2 and FGF 4) in the regeneratingblastema. In the first part of this paper we review recent histologicalfindings concerning the physical events that accompany autotomyof limbs and propose a new function for the autotomy membrane—thetethering of the regenerating pedal nerve to the walls of thecoxa. In the second part of the paper we review our recent findingson the identification and characterization of the Uca ecdysteroidreceptor (UpEcR, and its potential dimer partner, the retinoid-X-receptor,UpRXR). Using Uca-specific antibody probes raised in our lab,we have been able to identify specific cells in the early blastemathat express receptor proteins. The regenerating limb of thefiddler crab is responsive to both steroids and retinoids andmRNA for steroid and retinoid receptors are expressed in theregenerating limb buds during all stages of regeneration. TheDNA and deduced amino acid sequences of the ecdysteroid receptoris very similar to the sequences of insect EcRs, while the retinoidreceptor is similar to insect protein (ultraspiracle) in theDNA-binding domain, but closer to vertebrate RXRs in the ligandbinding domain     

Seasonal and Lunar Variation in the Emergence Time of a Population of Uca lactea annulipes (Milne-Edwards, 1837) at a Shore in Kuwait

This study monitored the endogenous emergence time of the fiddler crab Uca lactea annulipes (Milne-Edwards, 1837) in the field, for the first time, at an intertidal shore in Kuwait, from 1997 to 2001. The results revealed a significant cyclic change in the median emergence time as the season progressed from winter, through spring and summer, to autumn (.44, 1.29, 3.12, and 1.1?h prior to the dead-low tide, respectively). The data also revealed a significant shift in the median emergence time according to moon phase (2.27?h at new moon versus 2.56?h at full moon prior to the dead-low tide). (Author correspondence: l.almusawi@ropme.org)     

Adaptations for feeding on rock surfaces and sandy sediment by the fiddler crabs (Brachyura: Ocypodidae) Uca tetragonon(Herbst, 1790) and Uca vocans(Linnaeus, 1758)

Two species of fiddler crab, Uca tetragonon(Herbst, 1790) and Uca vocans(Linnaeus, 1758), which belong to the subgenus Gelasimus, dwell on rocky shores and muddy–sandy tidal flats, respectively, in Phuket Is., Thailand. We investigated their feeding ecology in relation to the morphology of their feeding organs: minor food-handling chelipeds and maxillipeds. U. tetragononfed chiefly on rocks covered by filamentous green algae. U. vocansfed on the emerged sand and in shallow water along the shoreline and in pools. While feeding, both crabs made sand pellets beneath their mouthparts and discarded them, indicating that they divided the matter scooped up with their minor chelipeds into edible and inedible fractions by using the maxillipeds in the water passing through their buccal cavity. The morphology of maxillipeds hardly differed between the two species, which means that both species are flotation-feeders. The morphology of their minor chelipeds, however, differed: the tips of the dactyl and pollex were flat in U. tetragononand pointed in U. vocans.When the minor cheliped was closed, U. tetragononhad a hemispherical space in the distal one-fourth of the gape, which was closed by the framing keratin layers and a few setae of the dactyl and pollex. On the other hand, U. vocanshad an ellipsoidal space in the distal half of the gape. We consider these morphological characters to be adaptations to the different feeding substrates for retaining more food-laden sediment. We discuss the role of the setae on the minor chelipeds on the basis of the morphological differences between populations of U. tetragononin Phuket Is. and East Africa where the crab inhabits muddy–sandy tidal flats.     

Separation of Neuropeptides by HPLC: Evaluation of Different Supports, with Analytical and Preparative Applications to Human and Porcine Neurophysins, β-Lipotropin, Adrenocorticotropic Hormone, and β-Endorphin

The separation of 38 hypophyseal or hypothalamic peptides and proteins by reversed-phase HPLC is described. The efficacy of several supports is discussed: large-pore reversed-phase silica supports turned out to be most effective in the separation of neuropeptides and proteins. The use of high sodium phosphate and phosphoric acid in the eluent resulted in high yields and better separation. On the basis of this procedure, beta-lipotropin, beta-endorphin, and adrenocorticotropic hormone were prepared from porcine pituitary glands, and human neurophysins from human pituitary posterior lobes. Moreover, this system is used for the analysis of the perifusate of pituitary posterior lobes, showing the release of micro-heterogeneous neurophysins.     

Perfusion of Immobilized Isolated Nerve Terminals as a Model for the Regulation of Transmitter Release: Release of Different, Endogenous Transmitters, Repeated Stimulation, and High Time Resolution

To study the release of neurotransmitters, i.e., the recruitment of transmitters for release and the regulation of the release process, isolated nerve terminals (synaptosomes) of the rat forebrain were immobilized in Sephadex gel inside a perfusion chamber. In this way, the following were achieved: (a) A very limited pressure stress was exerted on the synaptosomes, so that these remained viable for long periods (greater than 30 min) inside the chamber and did not elute from the chamber, which allowed long-term experiments with repeated stimulations; (b) estimation of the release of various endogenous transmitters, both in a Ca(2+)-dependent (exocytotic) and Ca(2+)-independent manner; (c) a step-like stimulation with depolarizing agents (rise time, 3-4 s) and a high time resolution (600-ms sampling); and (d) negligible reuptake of transmitter into the terminals or extracellular breakdown. It is concluded that this perfusion setup helps to provide new insights in the presynaptic stimulus-secretion coupling, co-transmission, and the exo-endocytosis cycle.     

Morphological and behavioural adaptations to the rocky substrate by the fiddler crab Uca panamensis (Stimpson, 1859): preference for feeding substratum and feeding mechanism

The fiddler crab Uca panamensis (Stimpson, 1959) inhabits rocky shores. We examined its preference for feeding substratum—sand or rock—and its manner of feeding. The crab made its burrow in the sand among rocks but preferred to feed on rocks. The feeding time decreased as the distance between the burrow and the rock increased. We consider this to be a result of exclusive interaction among the crabs because they defended their feeding area on the rocks against others.The crab wetted a small area of rock with water held in the branchial chambers before and during feeding. It pinched up the wetted surface in the minor chelipeds, which have bundles of setae on the posterior tips of the dactyl and pollex, and put the material into its buccal cavity. It never expelled sand pellets while feeding on rock, which indicates that it swallowed the food particles directly, without sorting. The bundles of setae retained water by capillary attraction, which suggests that they capture the suspended fine food particles scraped from the rock. The wetting action may prevent the fine materials from dispersing. We consider that morphological alteration of the minor chelipeds, the application of water from the branchial chambers, and direct swallowing permit the fiddler crab to feed on fine materials attached to rocks.     

Reaction of [Pt(Gly-Gly-N,N',O)I]- with the N-acetylated dipeptide L-methionyl-L-histidine: selective platination of the histidine side chain by intramolecular migration of the platinum(II) complex

The reaction of the monofunctional [Pt(Gly-Gly-N,N′,O)I]⁻ complex, in which Gly-Gly is the dipeptide glycyl-glycine coordinated through two nitrogen and oxygen atoms, with the N-acetylated dipeptide l-methionyl-l-histidine (MeCOMet-His) studied by ¹H NMR spectroscopy. All reactions were carried out in 50 mM phosphate buffer at pD 7.4 and at 25 °C. In the initial stage of the reaction, the platinum(II) complex forms the kinetically favored [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-S)]⁻ complex, with unidentate coordination of the MeCOMet-His dipeptide through the sulfur atom of the methionine residue. In the second stage of the reaction, complete intramolecular migration of the [Pt(Gly-Gly-N,N′,O)] unit from the sulfur to the N3 nitrogen atom of imidazole was observed and a new platinum(II)-peptide complex, [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-N3)]⁻ was formed. In comparison with previous results obtained for the reaction of [Pt(dien)Cl]⁺ with different methionine- and histidine-containing peptides, this migration reaction was sufficiently fast and strongly selective to the N3 atom of the imidazole ring of the histidine side chain. This study is an important step in the development of new platinum(II) complexes for selective covalent modification of peptides and proteins.     

Oxidative Stress, Hypoxia, and Ischemia-Like Conditions Increase the Release of Endogenous Amino Acids by Distinct Mechanisms in Cultured Retinal Cells

Abstract: The aim of this study was to elucidate the mechanisms by which retinal cells release endogenous amino acids in response to ascorbate/Fe²⁺-induced oxidative stress, as compared with chemical hypoxia or ischemia. In the absence of stimulation, oxidative stress increased the release of aspartate, glutamate, taurine, and GABA only when Ca²⁺ was present. Under hypoxia or ischemia, the release of aspartate, glutamate, glycine, alanine, taurine, and GABA increased mainly by a Ca²⁺-independent mechanism. The increased release observed in N -methyl- d -glucamine⁺ medium suggested the reversal of the Na⁺-dependent amino acid transporters. Upon oxidative stress, the release of aspartate, glutamate, and GABA, occurring through the reversal of the Na⁺-dependent transporters, was reduced by about 30%, although the release of taurine was enhanced. An increased release of [³H]arachidonic acid and free radicals seems to affect the Na⁺-dependent transporters for glutamate and GABA in oxidized cells. All cell treatments increased [Ca²⁺]_i (1.5 to twofold), although no differences were observed in membrane depolarization. The energy charge of cells submitted to hypoxia or oxidative stress was not changed. However, ischemia highly potentiated the reduction of the energy charge, as compared with hypoglycemia or hypoxia alone. The present work is important for understanding the mechanisms of amino acid release that occur in vivo upon oxidative stress, hypoxia, or ischemia, frequently associated with the impairment of energy metabolism.     

Predation by xanthid crabs on early post-settlement gastropods: the role of prey size, prey density, and habitat complexity

Small predators in marine benthic communities create a hazardous environment for newly settled invertebrates, especially for the smallest individuals. To explore the effects of predation on a newly settled gastropod, queen conch (Strombus gigas Linnaeus), by a xanthid crab (Micropanope sp.), prey size, prey density, and habitat complexity were manipulated in five laboratory experiments. All crabs >3.1 mm CW killed all conch <2 mm SL when individual crabs (<14 mm carapace width (CW)) were offered individual conch that were 2–35 days old after metamorphosis (1.2–8.8 mm shell length (SL)). Only 10% of the crabs >5.0 mm CW, however, killed conch that were >5.0 mm SL, suggesting that conch may reach a size refuge from xanthid crabs at 5 mm SL. Furthermore, when given a choice, crabs (4.8 mm CW) preferred smaller conch (2.0 mm SL) to larger (3.7 mm SL), suggesting that 1 week of additional growth in shell length is advantageous to survivorship. Proportional mortality decreased as conch density increased when crabs were offered conch at seven different densities (two to 96 individuals). Crabs proved to be effective predators regardless of the amount of seagrass structure provided in a microcosm experiment, and could consume two conch in 10 s. The high densities of xanthid crabs that occur in the wild, their effectiveness as predators, and their large appetites point to the important role that small predators may potentially play in structuring the population dynamics of their small prey immediately after settlement.     

Release of choline in the isolated heart, an indicator of ischemic phospholipid degradation and its protection by ischemic preconditioning: no evidence for a role of phospholipase D

The release of choline as a water-soluble product of phospholipid hydrolysis was measured in the perfusate of rat hearts to monitor ischemic membrane degradation and its protection by ischemic preconditioning (IPC). Hearts were subjected to global ischemia (GI; 30 min of no-flow) followed by 60 min of reperfusion. To induce IPC, GI was preceded by four no-flow episodes of 5 min each. Deleterious consequences of GI and reperfusion, namely coronary flow reduction, incidence of arrhythmias and release of cardiac troponin T, were significantly attenuated by IPC. The release of choline increased during reperfusion in a biphasic manner: a first phase peaked immediately after GI and was followed by a second, delayed phase indicating choline release caused during reperfusion. Only the second phase was blocked by both IPC and by AACOCF3 (5 microM), an inhibitor of cytosolic phospholipase A2. The activity of phospholipase D (PLD) was unchanged after GI or IPC or GI plus IPC. In conclusion, choline release into heart perfusate was found to be a useful real-time indicator of phospholipid degradation caused by GI and by reperfusion and its protection by IPC. The results supplement previous observations on the accumulation of fatty acids in the phospholipid pool. There was no evidence for PLD activation by GI or IPC.     

3-Methoxytyramine Is the Major Metabolite of Released Dopamine in the Rat Frontal Cortex: Reassessment of the Effects of Antipsychotics on the Dynamics of Dopamine Release and Metabolism in the Frontal Cortex, Nucleus Accumbens, and Striatum by a Simple Two Pool Model

Abstract: 3-Methoxytyramine (3-MT) and 3,4-dihydroxyphenylacetic acid (DOPAC) rates of formation were used, respectively, to assess the dynamics of dopamine (DA) release and turnover in the rat frontal cortex, nucleus accumbens, and striatum. Assuming total (re)uptake and metabolism of released DA are relatively uniform among the three brain regions, a simplified two pool model was used to assess the metabolic fate of released DA. Under basal conditions, 3-MT formation was found to comprise >60% of total DA turnover (sum of 3-MT plus DOPAC rates of formation) in the frontal cortex, and not more than 15% in the nucleus accumbens and striatum. Haloperidol increased the 3-MT rate of formation to a greater extent in the frontal cortex than in the two other regions. Clozapine increased the 3-MT rate of formation in the frontal cortex and decreased it in the striatum. Both drugs increased DOPAC rate of formation in the frontal cortex and nucleus accumbens. It was elevated by haloperidol but not clozapine in the striatum. It is concluded that (1) O -methylation is a prominent step in the catabolism of DA in the frontal cortex under both physiological conditions and after acute treatment with antipsychotics, (2) 3-MT is the major metabolite of released DA in the frontal cortex and possibly also in the nucleus accumbens and striatum, (3) in contrast to the frontal cortex, most of the DOPAC in the nucleus accumbens and striatum appear to originate from intraneuronal deamination of DA that has not been released, (4) because presynaptic uptake and metabolism of DA give rise to DOPAC, whereas postsynaptic uptake and metabolism produced both DOPAC and 3-MT, the ratio of 3-MT to DOPAC rates of formation can be a useful index of reuptake inhibition.     

Novel [Ru(polypyridine)(CO)2Cl2] and [Ru(polypyridine)2(CO)Cl]-type complexes: Characterizing the effects of introducing azopyridyl ligands by electrochemical, spectroscopic and crystallographic measurements

The reaction of ruthenium carbonyl polymer ([Ru(CO)₂Cl₂]_n) with azopyridyl compounds (2,2′-azobispyridine; apy or 2-phenylazopyridine; pap) generated new complexes, [Ru(azo)(CO)₂Cl₂] (azo = apy, pap). [Ru(apy)(CO)₂Cl₂] underwent photodecarbonylation to give a chloro-bridged dimer complex, whereas the corresponding pap complex ([Ru(pap)(CO)₂Cl₂]) was not converted to a dimer. The reactions of the chloro-bridged dimer containing the bpy ligand (bpy = 2,2′-bipyridine) with either apy or pap resulted in the formation of mixed polypyridyl complexes, [Ru(azo)(bpy)(CO)Cl]⁺. The novel complexes containing azo ligands were characterized by various spectroscopic measurements including the determination of X-ray crystallographic structures. Both [Ru(azo)(CO)₂Cl₂] complexes have two CO groups in a cis position to each other and two chlorides in a trans position. The azo groups are situated cis to the CO ligand in [Ru(azo)(bpy)(CO)Cl]⁺. All complexes have azo N-N bond lengths of 1.26-1.29 Å. The complexes exhibited azo-based two-electron reduction processes in electrochemical measurements. The effects of introducing azopyridyl ligands to the ruthenium carbonyl complexes were examined by ligand-based redox potentials, stretching frequencies and force constants of CO groups and bond parameters around Ru-CO moieties.     

Interaction of N,N,N′,N′-tetramethyl-p-phenylenediamine with photosystem II as revealed by thermoluminescence: Reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the QB niche

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (μM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S₂Q_B⁻ and S₃Q_B⁻ (B-band), S₂Q_A⁻ (Q-band), and Y_D⁺Q_A⁻ (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y_n) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot-Kok model of the S-state transitions and binary oscillations of Q_B confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q_B niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q_B-site and to reduce the higher S-states of the Mn cluster.