Transglutaminase-catalyzed modifications of SV-IV, a major protein secreted from the rat seminal vesicle epithelium

One of the major proteins secreted from the rat seminal vesicle epithelium, namely SV-IV, was shown to act in vitro as acyl donor and acceptor substrate for transglutaminase from both guinea pig liver and rat anterior prostate secretory fluid. Electrophoretic and chromatographic experiments indicated that the enzyme catalyzed the formation of multiple modified forms of SV-IV. In the absence of small Mr amines, transglutaminase was able to produce at least six different molecular forms of the protein, half of which possessed an Mr higher than that of native SV-IV. These findings suggested that a variable number of intermolecular, and perhaps intramolecular, crosslinks were formed between one or both glutamine residues and one or more lysine residues occurring in the SV-IV polypeptide chain. In addition, at least three modified forms of the protein were produced by transglutaminase in the presence of high concentrations of spermidine, thus indicating the formation of different (gamma-glutamyl)polyamine derivatives of SV-IV. Rabbit uteroglobin and rat anterior prostate secretory protein(s) were also shown to be able to covalently bind spermidine in the presence of the enzyme. The possible biological significance of transglutaminase-mediated modifications of SV-IV, as well as of other proteins occurring in the mammal seminal fluid, are discussed.     

The plant activation of m-phenylenediamine by Tradescantia clone 03 and clone 4430 cells in liquid suspension culture

In this study, we expanded the use of the genus Tradescantia to investigate the plant activation of promutagens and further refine the methodology of the plant cell/microbe coincubation assay. Liquid suspension cell cultures of Tradescantia clone 03 and Tradescantia clone 4430 were used to activate the promutagen m-phenylenediamine into a mutagenic compound which was detected by Salmonella typhimurium strain TA98 in the plant cell/microbe coincubation assay. Optimum treatment parameters were established for both plant cell lines. Optimum was defined as the lowest concentration or shortest time period that provided consistently positive results and high rates of revertants. Preliminary experiments with both cell lines defined 2.5 mumoles m-phenylenediamine per plate as the optimum concentration to be used in the determination of the optimal coincubation period and the optimal concentration of plant cells. These experiments also determined the optimal physiological stage at which both clones should be used in the coincubation assay. Differences were found in the optimal of coincubation (1h for clone 03, 2 h for clone 4430) and growth stage (mid-log for clone 03, mid- to late-log for clone 4430). Similar activation responses were seen for both clones when the concentration of plant cells (mg/ml) was varied. Under optimized conditions, clone 03 cells demonstrated an approximately 10% higher activation response than clone 4430.     

The Pyrexia transient receptor potential channel mediates circadian clock synchronization to low temperature cycles in Drosophila melanogaster

Circadian clocks are endogenous approximately 24 h oscillators that temporally regulate many physiological and behavioural processes. In order to be beneficial for the organism, these clocks must be synchronized with the environmental cycles on a daily basis. Both light : dark and the concomitant daily temperature cycles (TCs) function as Zeitgeber (‘time giver’) and efficiently entrain circadian clocks. The temperature receptors mediating this synchronization have not been identified. Transient receptor potential (TRP) channels function as thermo-receptors in animals, and here we show that the Pyrexia (Pyx) TRP channel mediates temperature synchronization in Drosophila melanogaster. Pyx is expressed in peripheral sensory organs (chordotonal organs), which previously have been implicated in temperature synchronization. Flies deficient for Pyx function fail to synchronize their behaviour to TCs in the lower range (16–20°C), and this deficit can be partially rescued by introducing a wild-type copy of the pyx gene. Synchronization to higher TCs is not affected, demonstrating a specific role for Pyx at lower temperatures. In addition, pyx mutants speed up their clock after being exposed to TCs. Our results identify the first TRP channel involved in temperature synchronization of circadian clocks.     

Glutaredoxin-2 Is Required to Control Proton Leak through Uncoupling Protein-3

Glutathionylation has emerged as a key modification required for controlling protein function in response to changes in cell redox status. Recently, we showed that the glutathionylation state of uncoupling protein-3 (UCP3) modulates the leak of protons back into the mitochondrial matrix, thus controlling reactive oxygen species production. However, whether or not UCP3 glutathionylation is mediated enzymatically has remained unknown because previous work relied on the use of pharmacological agents, such as diamide, to alter the UCP3 glutathionylation state. Here, we demonstrate that glutaredoxin-2 (Grx2), a matrix oxidoreductase, is required to glutathionylate and inhibit UCP3. Analysis of bioenergetics in skeletal muscle mitochondria revealed that knock-out of Grx2 (Grx2^−/−) increased proton leak in a UCP3-dependent manner. These effects were reversed using diamide, a glutathionylation catalyst. Importantly, the increased leak did not compromise coupled respiration. Knockdown of Grx2 augmented proton leak-dependent respiration in primary myotubes from wild type mice, an effect that was absent in UCP3^−/− cells. These results confirm that Grx2 deactivates UCP3 by glutathionylation. To our knowledge, this is the first enzyme identified to regulate UCP3 by glutathionylation and is the first study on the role of Grx2 in the regulation of energy metabolism.     

Deep intra‐island divergence of a montane forest endemic: phylogeography of the Puerto Rican frog Eleutherodactylus portoricensis (Anura: Eleutherodactylidae)

Aim Hypotheses proposed for lineage diversification of tropical montane species have rarely been tested within oceanic islands. Our goal was to understand how basin barriers and Pleistocene climatic fluctuations shaped the distribution of diversity in Eleutherodactylus portoricensis (Eleutherodactylidae), a frog endemic to the montane rain forests of Puerto Rico. Location The north‐eastern (Luquillo) and south‐eastern (Cayey) mountains of Puerto Rico. Methods We generated mitochondrial DNA (mtDNA) control region sequences (c. 565 bp) from 144 individuals of E. portoricensis representing 16 localities, and sequenced 646 bp of cytochrome b and 596 bp of nuclear DNA (nDNA) rhodopsin exon and intron 1 from a subset of individuals. We conducted a phylogenetic analysis on the mtDNA sequence data and explored population substructure with maximum parsimony networks, a spatial analysis of molecular variance, and pairwise F_ST analysis. Coalescent simulations were performed to test alternative models of population divergence in response to late Pleistocene interglacial periods. Historical demography was assessed through coalescent analyses and Bayesian skyline plots. Results We found: (1) two highly divergent groups associated with the disjunct Luquillo and Cayey Mountains, respectively; (2) a shallow mtDNA genetic discontinuity across the La Plata Basin within the Cayey Mountains; (3) phylogeographic congruence between nDNA and mtDNA markers; (4) divergence dates for both mtDNA and nDNA pre‐dating the Holocene interglacial (c. 10 ka), and nDNA suggesting divergence in the penultimate interglacial (c. 245 ka); and (5) historical demographic stability in both lineages. Main conclusions The low‐elevation Caguas Basin is a long‐term barrier to gene flow between the two montane frog populations. Measures of genetic diversity for mtDNA were similar in both lineages, but lower nDNA diversity in the Luquillo Mountains lineage suggests infrequent dispersal between the two mountain ranges and colonization by a low‐diversity founder population. Population divergence began prior to the Holocene interglacial. Stable population sizes over time indicate a lack of demonstrable demographic response to climatic changes during the last glacial period. This study highlights the importance of topographic complexity in promoting within‐island vicariant speciation in the Greater Antilles, and indicates long‐term persistence and lineage diversification despite late Pleistocene climatic oscillations.     

Betacyanins as phenol antioxidants. Chemistry and mechanistic aspects of the lipoperoxyl radical-scavenging activity in solution and liposomes

Reaction kinetics of betanin and its aglycone betanidin towards peroxyl radicals generated from the azo-initiated oxidation of methyl linoleate in methanol and of a heterogeneous aqueous/soybean phosphatidylcholine liposomal system were studied by monitoring formation of linoleic acid hydroperoxides and consumption of the pigments. Betanin was a weak retarder in methanol and an effective chain breaking antioxidant in the liposomal model, indicating that kinetic solvent effects and partition in lipid bilayers may affect its activity. Betanidin behaved as a chain terminating antioxidant in both models. Kinetic parameters characterizing peroxyl radical-scavenging activity showed that betanidin was more effective than betanin, in terms of both radical-scavenging rate constant and stoichiometric factor, with effectiveness of the same order as vitamin E under comparable conditions. Products identified by spectrophotometric and HPLC techniques indicated reaction of the glucose-substituted monophenol and ortho-diphenol moieties of betanin and betanidin, respectively, and suggested mechanisms of the antioxidant activity. Either betanin or betanidin incorporated in liposomes with α-tocopherol had additive effects, supporting partition of the pigments in the bilayer and lipoperoxyl radical reduction.     

Voluntary locomotor activity mitigates oxidative damage associated with isolation stress in the prairie vole (Microtus ochrogaster)

Organismal performance directly depends on an individual''s ability to cope with a wide array of physiological challenges. For social animals, social isolation is a stressor that has been shown to increase oxidative stress. Another physiological challenge, routine locomotor activity, has been found to decrease oxidative stress levels. Because we currently do not have a good understanding of how diverse physiological systems like stress and locomotion interact to affect oxidative balance, we studied this interaction in the prairie vole (Microtus ochrogaster). Voles were either pair housed or isolated and within the isolation group, voles either had access to a moving wheel or a stationary wheel. We found that chronic periodic isolation caused increased levels of oxidative stress. However, within the vole group that was able to run voluntarily, longer durations of locomotor activity were associated with less oxidative stress. Our work suggests that individuals who demonstrate increased locomotor activity may be better able to cope with the social stressor of isolation.