Roles of basic residues and salt-bridge interaction in a vacuolar H-pumping pyrophosphatase (AVP1) from Arabidopsis thaliana

To investigate the possible role of basic residues in H⁺ translocation through vacuolar-type H⁺-pumping pyrophosphatases (V-PPases), conserved arginine and lysine residues predicted to reside within or close to transmembrane domains of an Arabidopsis thaliana V-PPase (AVP1) were subjected to site-directed mutagenesis. One of these mutants (K461A) exhibited a “decoupled” phenotype in which proton-pumping but not hydrolysis was inhibited. Similar results were reported previously for an E427Q mutant, resulting in the proposal that E427 might be involved in proton translocation. However, the double mutant E427K/K461E has a wild type phenotype, suggesting that E427 and K461 form a stabilising salt bridge, but that neither residue plays a critical role in proton translocation.     

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular fungal pegs within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.     

Postmortem Changes in Rat Brain: Studies on Membrane-Bound Enzymes and Receptors

The relationship between the stability of potential neurochemical markers and autolysis time was studied at 4 degrees C and 25 degrees C using postmortem brain samples from two rat strains. In general, qualitatively similar results were obtained with either N/Nih or Sprague-Dawley rats; however, quantitative differences were often observed, particularly in regard to benzodiazepine receptor changes. For every enzyme activity or binding property examined, no significant change was found when brains were kept at 4 degrees C for up to 72 h prior to freezing at -70 degrees C. Na,K-ATPase and low-affinity Ca-ATPase activities were also stable in brains kept at 25 degrees C for up to 72 h. Mg-ATPase activity was reduced in brains kept at 25 degrees C for 24 and 48 h. [3H]Guanidinoethylmercaptosuccinic acid [( 3H]GEMSA) binding to enkephalin convertase in the cytosol was not significantly changed in brains kept at 25 degrees C; however, a small increase was seen for [3H]GEMSA binding to the membrane fraction at 24, but not 48 and 72 h postmortem. [3H]Quinuclidinyl benzilate [( 3H]QNB) binding to muscarinic cholinergic receptors decreased in brains kept at 25 degrees C for 72 h. Opioid receptor binding also decreased in brains kept at 25 degrees C. Using [3H]2-D-alanine-5-D-leucine enkephalin to label delta opioid receptors, a statistically significant decrease in binding was observed as early as 6 h postmortem, and was completely abolished after 72 h at 25 degrees C. In contrast, [3H]naloxone binding was unchanged after 24 h at 25 degrees C, but was decreased after 48 and 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmentally acquired PKA localisation in mouse oocytes and embryos

Localisation of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs) is known to coordinate localised signalling complexes that target cAMP-mediated signalling to specific cellular sub-domains. The cAMP PKA signalling pathway is implicated in both meiotic arrest and meiotic resumption, thus spatio-temporal changes in PKA localisation during development may determine the oocytes response to changes in cAMP. In this study we aim to establish whether changes in PKA localisation occur during oocyte and early embryo development.Using fluorescently-labelled PKA constructs we show that in meiotically incompetent oocytes PKA is distributed throughout the cytoplasm and shows no punctuate localisation. As meiotic competence is acquired, PKA associates with mitochondria. Immature germinal vesicle (GV) stage oocytes show an aggregation of PKA around the GV and PKA remains co-localised with mitochondria throughout oocyte maturation. After fertilisation, the punctuate, mitochondrial distribution was lost, such that by the 2-cell stage there was no evidence of PKA localisation. RT-PCR and Western blotting revealed two candidate AKAPs that are known to be targeted to mitochondria, AKAP1 and D-AKAP2. In summary these data show a dynamic regulation of PKA localisation during oocyte and early embryo development.     

Reproductive seasonality and the effect of the GnRH agonist deslorelin as a contraceptive in captive male Black Flying-foxes (Pteropus alecto)

Effective contraception would enhance genetic management of captive Pteropus species, which typically breed well in captivity. Male reproductive seasonality was monitored (15-mo interval) in captive P. alecto (6 controls and 5 treated with 4.7 mg deslorelin). In untreated males, there were seasonal changes in testicular volume, body weight and testosterone secretion; testicular volume and body weight peaked in February and March, respectively, whereas testosterone concentration remained >5 ng/ml before rising (P < 0.001) to 24.9 ± 3.6 ng/ml (mean ± SEM) in April. However, there was no corresponding change in sperm quality, and seminal vesicle gland (SVG) secretions remained present in ejaculates. In treated males, testosterone concentration had an initial ‘flare’ response (mean ± SEM peak: 19.95 ± 3.27 ng/ml) before declining (P < 0.001) by 32 d to basal levels, where it remained. In these males, there was reduced sperm motility after 1 mo (P < 0.001) and the absence of SVG secretions after 4 mo. However, aspermic ejaculates were first recorded 5 mo post-treatment. At 10 mo after treatment, spermatogenesis was still disrupted, when membrane-intact, but non-motile sperm were present in two individuals. Motile sperm were first recovered from one of these males 13 mo after deslorelin treatment. We concluded that captive P. alecto males: (a) had seasonal reproductive changes in testicular volume, body weight and testosterone secretion; (b) produced motile, membrane-intact sperm and SVG secretions throughout the year; and (c) had a rapid decline in testosterone concentration and consequent suppression of testicular function for at least 5 mo following deslorelin administration.     

Computational methods for generating models of denatured and partially folded proteins

Partially folded and denatured proteins can give important insights into protein folding, misfolding, and aggregation. Such non-native states of proteins are however very difficult to characterise in detail as they are dynamic, heterogeneous systems comprising of ensembles of interconverting conformers. This article describes methods that produce models for non-native proteins in atomic detail. A variety of molecular dynamics based protocols are discussed together with some recent procedures that include restraints from experimental data. These models provide an important framework for interpreting experimental data from studies of non-native states using nuclear magnetic resonance spectroscopy, fluorescence, circular dichroism, and small angle scattering techniques.