Identification and Characterisation of the RalA-ERp57 Interaction: Evidence for GDI Activity of ERp57

RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro. These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57’s active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H₂O₂ inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor (RalGDI).     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Cyclical changes in the renin-angiotensin-aldosterone system during the menstrual cycle of the baboon (Papio hamadryas)

Abstract: This study characterizes the renin-angiotensin-aldosterone system during the normal menstrual cycle in the baboon. Ten animals received a daily dose of an ACE inhibitor or placebo in a randomized blind cross-over design. Data were obtained during the mid-follicular and early luteal phases of normal non-pregnant menstrual cycles. All examinations and blood collections were performed with ketamine sedation: 7–kg by im injection. Blood pressure was recorded by sphygmomanometer. Serum ACE activity was measured by spectrophotometry. Aldosterone (ALDO), angiotensin I (AI), and angiotensin II (AII) were measured by radioimmunoassay. Plasma renin activity (PRA) was measured by AI generation. The renin-angiotensin-aldosterone system was found to be activated in the follicular phase and suppressed during the luteal phase of the normal non-pregnant menstrual cycle in the baboon.     

Allelopathy as a competitive strategy in persistent thickets of Lantana camara L. in three Australian forest communities

Field experiments were established to assess possible allelopathic suppression by Lantana camara L. of two indigenous tree species. The design allowed comparison of allelopathic effects with density-dependent resource competition effects. Fire and its role in competitive interactions was included as an experimental treatment. Allelopathic responses were measured in L. camara thickets by germinating and growing Alectryon subcinereus (A. Gray) Radlk. in dry rainforest ecotones (Macleay River) or Cryptocarya rigida (Meissner) in warm temperate rainforest and wet sclerophyll forest (Lake Macquarie) at 10, 20 and 30 seedlings m^-2, where L. camara was either physically removed (LR), burnt (LB), or cut and left in place (LT). Germination for both species increased significantly by completely removing L. camara (LR) whereas burning (LB) was significant only for C. rigida. Seedling growth for both species was negatively related to increasing density when all L. camara was removed (LR) but was positively related in the other two treatments (LB and LT). C. rigida seedling biomass increased 47.4% (1.75%2.58 g) and 68.6% (1.98%2.95 g) with increasing seedling density for LT and LB respectively and decreased 23.2% (2.93–2.25 g) for LR. A. subcinereus seedling biomass increased 29.7% (1.95–2.53 g) and 34.7% (2.25–3.03 g) with increasing seedling density for LT and LB respectively and decreased 27.9% (3.30–2.38 g) for LR. Phytotoxin dilution effects were inferred in LT and LB rather than density-dependent intraspecific competition, whereas the reverse was true for LR. Seedling biomass for C. rigida resulting from potential phytotoxin dilution at high seedling density was not significantly different from the response of LR at low seedling density but, for A. subcinereus, the phytotoxin dilution response was significantly less than LR at low seedling density. Moderately intense fire (LB) was not significantly different from the LT treatment at both locations, emphasising that moderate to low intensity fires should not be used to control existing invasions of L. camara. Competitive strategies for invasive populations are identified that may modify succession following disturbance, thereby allowing thicket formation and long-term persistence to affect community dynamics. Such strategies need to be recognised in managing natural communities, particularly for biodiversity conservation.Nomenclature: Harden (1990).     

Blood volume measurement in baboons: simultaneous estimation of red cell volume and plasma volume

A method is described for frequent sequential blood volume estimation in baboons using 32P for red cell volume measurements and 125I-albumin for simultaneous plasma volume measurements. Values for red cell, plasma, and total blood volumes are reported. Close correlations of the volumes to bodyweight were demonstrated. Circulatory half-lives of the isotopes, determined from disappearance curves, confirmed their suitability for serial measurements in these baboons.     

Grassland responses to multiple disturbances on the New England Tablelands in NSW,Australia

Disturbances have played a significant role in shaping vegetation patterns following European settlement and agricultural development in Australia, particularly over the last 100 years. However, little is known about the temporal dynamics of plant communities in relation to disturbances and their interactions. In this study we examined the response and recovery of temperate grassland communities to disturbance, using a multi-factorial experiment involving grazing exclusion (absent and present), fire (absent and present), soil cultivation (none, 5 and 20 cm) and soil amendment (none, fertiliser and fertiliser plus clover seeds) on the New England Tablelands in NSW, Australia. Temporal patterns of plant communities were analysed using detrended correspondence analysis for eight surveys over 24 months. Vegetation patterns at each survey were analysed using principal components analysis. The effects of treatments on malleability (Bray–Curtis dissimilarity) of plant communities were analysed using a linear mixed model, and the relationships between malleability and plant species groups were depicted using a generalised additive model and further analysed using a linear model. Perennial native grasses and a non-native forb (Hypochaeris radicata) initially dominated the vegetation, but after the disturbances H. radicata and other non-native species dominated. Compositional changes from the initial states were greatest in the first spring (7 months after treatment application), and then the vegetation tended to recover towards its original state. Soil cultivation resulted in the greatest deviation in community composition, followed by soil amendment, fire, with grazing exclusion the least. The recovery process and rate of recovery varied with treatment reflecting the dominance of soil cultivation and its interactions with other forms of disturbances. Soil amendment and grazing exclusion tended to reduce the effects of soil cultivation. Malleability was negatively related to perennial native grasses; positively to other non-native species, annual native grasses and perennial native sedges/rushes; and negatively to H. radicata when its cover was below 18%, but positively when above 18%. The degree of malleability reflected the high resilience of the vegetation to disturbance, and was mainly due to the recovery of perennial native grasses and H. radicata. This resilience demonstrated that the small-scale disturbances did not cause vegetation to cross an ecological threshold and that the present vegetation is resilient to common disturbances occurring at small scales. The results also suggest (1) that the present vegetation has developed mechanisms to adapt to these disturbances, (2) the importance of disturbance scale or (3) that the ecological threshold had already been crossed and the present vegetation is in a degraded state compared with its original state before the end of the 19th century.     

The chromosome replication machinery of the archaeon Sulfolobus solfataricus

In the three domains of life, the archaea, bacteria, and eukarya, there are two general lineages of DNA replication proteins: the bacterial and the eukaryal/archaeal lineages. The hyperthermophilic archaeon Sulfolobus solfataricus provides an attractive model for biochemical study of DNA replication. Its relative simplicity in both genomic and biochemical contexts, together with high protein thermostability, has already provided insight into the function of the more complex yet homologous molecules of the eukaryotic domain. Here, we provide an overview of recent insights into the functioning of the chromosome replication machinery of S. solfataricus, focusing on some of the relatively well characterized core components that act at the DNA replication fork.     

Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest

DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Ter sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. In support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the RTP-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences. From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork.     

Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E. coli

The Escherichia coli replication terminator TerB was inserted in its two alternate orientations into a Bacillus subtilis fork-arrest assay plasmid. After transferring these new plasmids into B. subtilis, which could overproduce the E. coli terminator protein Tus, it was shown that the E. coli Tus-TerB complex could cause polar replication fork arrest, albeit at a very low level, in B. subtilis. A new B. subtilis-E. coli shuttle plasmid was designed to allow the insertion of either the Terl (B. subtilis) or TerB (E. coli) terminator at the same site and in the active orientation in relation to the approaching replication fork generated in either organism. Fork-arrest assays for both terminator-containing plasmids replicating in both organisms which also produced saturating levels of either the B. subtilis terminator protein (RTP) or Tus were performed. The efficiency of the Tus-TerB complex in causing fork arrest was much higher in E. coli than in B. subtilis. The efficiency of the B. subtilis RTP-Terl complex was higher in B. subtilis than in E. coli, but the effect was significantly less. Evidently a specificity feature in E. coli operates to enhance appreciably the fork-arrest efficiency of a Tus-Ter complex. The specificity effect is of less significance for an RTP-Ter complex functioning in B. subtilis.