Environmental Stress, Bottom-up Effects, and Community Dynamics: Integrating Molecular-Physiological and Ecological Approaches

Environmental stress and nutrient/productivity models predict the responses of community structure along gradients of physical conditions and bottom-up effects. Although both models have succeeded in helping to understand variation in ecological communities, most tests have been qualitative. Until recently, two roadblocks to more quantitative tests in marine environments have been a lack of (1) inexpensive, field-deployable technology for quantifying (e.g.) temperature, light, salinity, chlorophyll, and productivity, and (2) methods of quantifying the sub-organismal mechanisms linking environmental conditions to their ecological expression. The advent of inexpensive remote-sensing technology, adoption of molecular techniques such as quantification of heat-shock proteins and RNA:DNA ratios, and the formation of interdisciplinary alliances between ecologists and physiologists has begun to overcome these roadblocks. An integrated eco-physiological approach focuses on the determinants of: distributional limits among microhabitat patches and along (local-scale) environmental gradients (e.g., zonation); among-site (mesoscale) differences in community pattern; and geographic (macroscale) differences in ecosystem structure. These approaches promise new insights into the physiological mechanisms underlying variation in processes such as species interactions, physical disturbance, survival and growth. Here, we review two classes of models for community dynamics, and present examples of ecological studies of these models in consumer-prey systems. We illustrate the power of new molecular tools to characterize the sub-organismal responses of some of the same consumers and prey to thermal stress and food concentration. Ecological and physiological evidence tends to be consistent with model predictions, supporting our argument that we are poised to make major advances in the mechanistic understanding of community dynamics along key environmental gradients.     

Significance of GABAergic systems in the action of improgan, a non-opioid analgesic

Improgan is the prototype drug from a new class of non-opioid analgesics chemically related to histamine and histamine antagonists, but the mechanism of action of these compounds has not been identified. Because several classes of analgesics act in the brain by reducing GABAergic inhibition of endogenous pain-relieving circuits, and because the activity of these substances is abolished by the GABA(A) agonist muscimol, the present study assessed the effects of muscimol on improgan antinociception in rats. Intracerebroventricular (icv) improgan (80 microg) and morphine (20 microg) both induced 80-100% of maximal analgesic responses on the tail flick test 10 to 30 min later. However, muscimol pretreatment (0.5 microg, icv) completely eliminated the antinociceptive activity of both compounds. Since improgan in vitro lacks activity at opioid and GABA(A) receptors, these findings: 1) confirm earlier literature showing that muscimol inhibits morphine analgesia, and 2) suggest that improgan activates a supraspinal, descending analgesic pathway, possibly via inhibition of GABAergic transmission. Since muscimol is the first compound discovered which inhibits improgan analgesia, muscimol will be a useful tool for the further characterization of this new class of pain-relieving substances.     

Validation of Trypanosoma brucei trypanothione synthetase as drug target

In trypanosomes, the parasite-specific thiol trypanothione [T(SH)2] fulfills various functions, the best established being detoxification of H2O2 and organic hydroperoxides and ribonucleotide reduction. Recently, a trypanothione synthetase (Tb-TryS) gene from Trypanosoma brucei was isolated and the heterologously expressed Tb-TryS catalyzed the entire synthesis of T(SH)2 from glutathione (GSH) and spermidine in vitro. To confirm the in situ function of the complex Tb-TryS activities and to evaluate the importance of T(SH)2 metabolism in T. brucei, TryS suppression by double-stranded RNA interference was performed. Knockdown of TryS led to depletion of both T(SH)2 and glutathionylspermidine (Gsp) and accumulation of GSH, while concomitantly impairment of viability and arrest of proliferation were observed. TryS-downregulated cells displayed a significantly increased sensitivity to H2O2 and tert.-butyl hydroperoxide. These data verify the hypothesis that in T. brucei, a single enzyme synthesizes the spermidine-conjugated thiols (Gsp and T(SH)2) and further confirms the significance of trypanothione in the defense against oxidative stress and the maintenance of viability and proliferation in unstressed parasites.     

Ontogeny of the mandarinfish <Emphasis Type="Italic">Siniperca chuatsi</Emphasis> (Perciformes: Sinipercidae) reared in aquarium

Early-stage morphologies of the mandarinfish Siniperca chuatsi are described on the basis of an ontogenetic series of reared specimens in an aquarium. Spherical eggs (diameter 1.70–1.82mm) with a single oil globule (0.40–0.48mm) were free-demersal and easily floated when agitated. Hatching occurred about 3 days after spawning at about 24°C. Newly hatched larvae (3.8–4.2mm in notochord length: NL) had many melanophores on the yolk sac. After reaching ca. 5.5mm NL (8–9+19–20=27–28 myomeres), larvae had almost completely absorbed the yolk, possessed a large mouth and sharp teeth, and were starting to prey on other fish larvae. Three large preopercle spines appeared at ca. 5.5mm NL, five spines by ca. 8.5mm NL, and eight by ca. 21mm in standard length (SL). The interopercle bore a single spine at ca. 8.5mm NL and two spines at ca. 13.5mm SL. A single spine appeared at the supracleithrum and another at the opercle at ca. 10mm SL. Dorsal fin spines and pelvic, anal, and caudal fins were completed at ca. 10mm SL. Dorsal fin rays and pectoral fins were completed at ca. 13.5mm SL. Four ontogenetic characters (free-demersal eggs, large jaws with large teeth, conspicuous head spination, and precocious completion of dorsal fin spines) are rare among freshwater percoids.     

Tryparedoxin Peroxidase of Leishmania donovani: Molecular Cloning, Heterologous Expression, Specificity, and Catalytic Mechanism

Tryparedoxin peroxidase (TXNPx) of Trypanosomatidae is the terminal peroxidase of a complex redox cascade that detoxifies hydroperoxides by NADPH (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). A gene putatively coding for a peroxiredoxin-type TXNPx was identified in L. donovani and expressed in Escherichia coli to yield an N-terminally His-tagged protein (LdH6TXNPx). LdH6TXNPx proved to be an active peroxidase with tryparedoxin (TXN) 1 and 2 of Crithidia fasciculata as cosubstrates. LdH6TXNPx efficiently reduces H₂O₂, is moderately active with t-butyl and cumene hydroperoxide, but only marginally with linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide. The enzyme displays ping-pong kinetics with a k_cat of 11.2 s⁻¹ and limiting K_m values for t-butyl hydroperoxide and CfTXN1 of 50 and 3.6 μM, respectively. Site-directed mutagenesis confirmed that C52 and C173, as in related peroxiredoxins, are involved in catalysis. Exchanges of R128 against D and T49 against S and V, supported by molecular modelling, further disclose that the SH group of C52 builds the center of a novel catalytic triad. By hydrogen bonding with the OH of T49 and by the positive charge of R128 the solvent-exposed thiol of C52 becomes deprotonated to react with ROOH. Molecular models of oxidized TXNPx show C52 disulfide-bridged with C173′ that can be attacked by C41 of TXN2. By homology, the deduced mechanism may apply to most peroxiredoxins and complements current views of peroxiredoxin catalysis.     

ATP-stimulated hydrolysis of GTP by RecA protein: kinetic consequences of cooperative RecA protein-ATP interactions

The cooperativity of the single-stranded DNA dependent nucleoside triphosphatase activity of the recA protein was investigated by examining the influence of a good substrate (ATP) on the hydrolysis of a poor substrate (GTP). At pH 7.5 and 37 degrees C, both ATP and GTP are hydrolyzed with a turnover number of 17.5 min-1. The S0.5 for GTP (750 microM), however, is nearly 20-fold higher than the S0.5 for ATP (45 microM). Low concentrations of ATP activate the GTPase activity of the recA protein by lowering the S0.5 for GTP; in the presence of 50 microM ATP, the S0.5 for GTP is reduced from 750 microM to 200 microM. Concentrations of ATP greater than 50 microM result in competitive inhibition of the ATP-activated GTPase activity. Although GTP is a substrate for hydrolysis, it will not substitute for ATP as a high-energy cofactor in the standard recA protein promoted three-strand exchange reaction. To account for these results, a minimal kinetic model is presented in which ATP binding induces specific conformational changes in the recA protein that do not occur with GTP binding.     

Effects of ocean acidification on the early life history processes of the breadcrumb sponge Halichondria panicea

Ocean acidification (OA) is predicted to result in reduced survival, growth, reproduction, and overall biodiversity of marine invertebrates, and yet we lack information about the response to OA of some major groups of marine organisms. In particular, we know relatively little about how OA will impact temperate sponges, which will experience more extreme low pH conditions than tropical species. In this study, we quantified OA-induced changes in early life history patterns (larval mortality and condition, settlement rate, recruit survival, and size) in the non-calcifying breadcrumb sponge Halichondria panicea collected from a temperate intertidal site in the California Current Large Marine Ecosystem. Sponge larvae were exposed to OA conditions for 15 days, and early life history patterns were observed. Compared with baseline (“present”) conditions, larval mortality and settlement rates increased in the acidified treatment (“future”). This effect was restricted to larval stages; treatment had no effect on the growth and survival of recruits. This study is significant in that it shows that H. panicea may be particularly vulnerable to changes in ocean pH during the larval stage, which could ultimately reduce total sponge abundance by diminishing the number of larvae that survive to settlement.     

Pseudomonas cepacia lipase: Esterification reactions in AOT microemulsion systems

Summary The activity of purifiedPseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis(2ethylhexyl)sulfosuccinate sodium salt (AOT). The optima pH, T and water content (w_o) for the enzyme activity in this type of microemulsions have been determined. Studies on the effect of various fatty acids and alcohols on the enzyme specificity have shown a preference of this lipase for palmitic and caprylic acid as well as for propanol, while reactions involving cyclic alcohols can not be catalyzed at all. The differences on the behavior of this lipase as compared to other lipases studied in microemulsion systems as well as in other systems are discussed.     

Electron microscopy of frozen-hydrated bacteria.

Amorphous, unstained, frozen-hydrated sections of bacteria provide a faithful high-resolution image of procaryotic cells. Conventional preparation artifacts due to fixation, staining, and dehydration are nonexistent. Freezing damage is avoided by using glucose as a cryoprotectant. Cutting damage on frozen material is severe, but sectioning artifacts, being always related to the cutting direction, can be systematically recognized and thus taken into consideration. Geometry and density distribution of the bacterial envelope can be resolved to about 3 nm. The following main features have been observed. In Escherichia coli the inner and outer membranes have an approximately uniform density profile. The distance between the two membranes is constant, ca. 33 nm. In Staphylococcus aureus the cell wall is ca. 40 nm wide. It is bordered on the cytoplasmic side by an asymmetric 5.5-nm-wide bilayer. The bacterial nucleoid, clearly visible with conventional preparation methods, appears in exponentially growing bacteria as an ill-defined central region with approximately the same density as the rest of the cytoplasm. It becomes more clearly visible when bacteria are in the stationary phase, plasmolysed, fixed, or stained. We confirm that "mesosomes," hitherto quite often considered to be essential organelles in all procaryotes, are artifacts. They appear in large numbers during osmium fixation.