Breeding system,pollination and demography in the rare granite endemic shrub Verticordia staminosa ssp. staminosa in south‐west Western Australia

Abstract Verticordia staminosa C. Gardner & A. S. George staminosa (Myrtaceae) is a rare granite endemic shrub that grows in discrete subpopulations on one isolated rock outcrop in a remnant of native vegetation in the Western Australian wheat belt. Key considerations in assessing the risk of extinction for rare plant species in fragmented landscapes are the reproductive dependence on a pollinator, breeding system, importance of seeds in demography, and regeneration niche. The present study determined the extent to which these factors constrain population growth in V. staminosa ssp. staminosa. Measurements across nine subpopulations on the breeding system, pollinator activity, rates of flowering, pollination and seed production, seedling demography, mature plant mortality and size‐class structure were undertaken over three consecutive years. The study species has a mixed mating system with similar rates of pollen tube development and fertilization observed in self‐, cross‐ and open‐pollinated flowers. Floral morphology, orientation and the concentration and volume of nectar produced suggest some degree of specialization associated with pollination by birds, which were occasionally seen visiting flowers. However, feral honey bees were the most commonly observed flower visitor and they seem to have replaced honeyeaters as the primary pollinator. Honey‐bee abundance increased with subpopulation size. However, rates of pollination and the subsequent proportion of flowers that produced viable seeds were independent of subpopulation size. Germination and seedling emergence occurred each winter but were greatest in the wettest winter. Recruitment was heavily biased towards individuals growing in or over cracks/fissures in the rock. Over the 3‐year study, recruitment exceeded mortality. A relatively unspecialized flower and mixed mating system have buffered the taxon against the effects of pollinator disruption. Seed production does not constrain population growth. The environmental variables of climate and suitable establishment crevices appear to be the major constraints to population growth.     

A catalytic loop within Pseudomonas aeruginosa exotoxin A modulates its transferase activity.

Mutagenesis techniques were used to replace two loop regions within the catalytic domain of Pseudomonas aeruginosa exotoxin A (ETA) with functionally silent polyglycine loops. The loop mutant proteins, designated polyglycine Loops N and C, were both less active than the wild-type enzyme. However, the polyglycine Loop C mutant protein, replaced with the Gly(483)-Gly(490) loop, showed a much greater loss of enzymatic activity than the polyglycine Loop N protein. The former mutant enzyme exhibited an 18,000-fold decrease in catalytic turnover number (k(cat)), with only a marginal effect on the K(m) value for NAD(+) and the eukaryotic elongation factor-2 binding constant. Furthermore, alanine-scanning mutagenesis of this active-site loop region revealed the specific pattern of a critical region for enzymatic activity. Binding and kinetic data suggest that this loop modulates the transferase activity between ETA and eukaryotic elongation factor-2 and may be responsible for stabilization of the transition state for the reaction. Sequence alignment and molecular modeling also identified a similar loop within diphtheria toxin, a functionally and structurally related class A-B toxin. Based on these results and the similarities between ETA and diphtheria toxin, we propose that this catalytic subregion represents the first report of a diphthamide-specific ribosyltransferase structural motif. We expect these findings to further the development of pharmaceuticals designed to prevent ETA toxicity by disrupting the stabilization of the transition state during the ADP-ribose transfer event.     

Uptake and stimulation-evoked release of tritiated neurotransmitters from slices of the rat rostral ventral medulla containing the nucleus raphe magnus: implications for pathways controlling nociception

1. The rostral ventral medulla plays a central role in the integration of nociceptive control. 2. Slices of this area of the brainstem may be labelled with tritiated noradrenaline, dopamine, serotonin, GABA and choline. 3. Uptake was greatest for noradrenaline and dopamine, GABA was intermediate and serotonin and choline were poorly accumulated. 4. Conditions for the release of all transmitter candidates except acetylcholine were established using either potassium or electrical stimulation and release was proven to be calcium dependent. 5. Electrophysiological and microinjection data are at variance with the commonly assumed actions of noradrenaline and dopamine and can be rationalized by the presence of a GABA interneuron integrating nociceptive input to the nucleus raphe magnus.     

The INO1 promoter of Saccharomyces cerevisiae includes an upstream repressor sequence (URS1) common to a diverse set of yeast genes.

The INO1 promoter of Saccharomyces cerevisiae includes a copy of an upstream repression sequence (URS1; 5'AGCCGCCGA 3') observed in the promoters of several unrelated yeast genes. Expression of INO1-lacZ and CYC1-lacI'Z, activated by the INO1 UASINO, is significantly decreased by the INO1 URS1.     

UDP-N-acetyl-D-galactosamine as a donor substrate for the glycosyltransferase encoded by the B gene at the human blood group ABO locus

The properties of the enzyme in the serum of blood group B individuals that catalyses the transfer of small amounts of N-acetyl-D-galactosamine to H-active precursor structures were compared with those of the blood group B gene-associated alpha-(1----3)-D-galactosyltransferase and with the blood group A gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases in the serum of blood group A1 and A2 individuals. The biosynthetic products formed by the enzyme in B serum were identical with the A-active structures synthesised by the A1 and A2 gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases but the enzyme differed from the A1 and A2 transferases in its apparent Km for UDP-N-acetyl-D-galactosamine, its heat susceptibility, its failure to bind to Sepharose 4B, and its adsorption to H-active sites on group O red cell ghosts under conditions which bind the B transferase but fail to adsorb the A1 and A2 transferases. The correlation between the levels of alpha-(1----3)-D-galactosyltransferase and alpha-(1----3)-N-acetyl-D-galactosaminyltransferase activities in all the group B serum samples tested, the maintenance of the same ratio of activities after successive cycles of binding to group O red cell ghosts, the retention of the ability to convert blood group O to A-active cells after treatment of the serum with Sepharose 4B, and the failure to detect any comparable activity in group O serum samples tested under the same conditions indicated that the enzyme in group B serum that utilises UDP-N-acetyl-D-galactosamine to make blood group A-active structures is the B gene-associated alpha-(1----3)-D-galactosyltransferase.     

Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes

The use of I50 (concentration of inhibitor required for 50% inhibition) for enzyme or drug studies has the disadvantage of not allowing easy comparison among data from different laboratories or under different substrate conditions. Modifications of the Michaelis-Menten equation for treatment of inhibitors can allow both the determination of the type of inhibition (competitive, noncompetitive, and uncompetitive) and the Ki for the inhibitor. For competitive and uncompetitive inhibitors when the assay conditions are [S] = Km, then Ki = I50/2. For different conditions of [S] there is a divergence between competitive and uncompetitive inhibitors that may be used to identify the type of inhibitor. The equation for Ki also differs. For noncompetitive inhibitors the Ki = I50 and this relationship is valid with changing [S]. The equations developed require a single substrate, reversible-type inhibitors, and kinetics of the Michaelis-Menten type. Examples of the use of the equations are illustrated with experimental data from scientific publications.     

Effect of an ntrC mutation on amino acid or urea utilization and on nitrogenase switch-off in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a nitrogen-fixing bacterium that grows well with ammonium chloride or sodium nitrate as alternative single nitrogen sources but that grows more slowly with L-alanine, L-serine, L-proline, or urea. The ntrC mutant strain DCP286A was able to utilize only ammonium or urea of these nitrogen sources. The addition of 1 mmol.L-1 ammonium chloride to the nitrogen-fixing wild-type strain inhibited nitrogenase activity rapidly and completely. Urea was a less effective inhibitor; approximately 20% of nitrogenase activity remained 40 min after the addition of 1 mmol x L-1 urea. The effect of the ntrC mutation on nitrogenase inhibition (switch-off) was studied in strain DCP286A containing the constitutively expressed gene nifA of H. seropedicae. In this strain, nitrogenase inhibition by ammonium was completely abolished, but the addition of urea produced a reduction in nitrogenase activity similar to that of the wild-type strain. The results suggest that the NtrC protein is required for assimilation of nitrate and the tested amino acids by H. seropedicae. Furthermore, NtrC is also necessary for ammonium-induced switch-off of nitrogenase but is not involved in the mechanism of nitrogenase switch-off by urea.     

Maspin and tumor metastasis

For most cancer cell types, the acquisition of metastatic activity leads to clinically incurable disease. Improvements in surgery and radiotherapy, and the development of new chemotherapeutic agents or their use in new combinations, have, so far, only incrementally improved patient survival. Despite the obvious importance of metastasis, the process remains incompletely characterized at the molecular and biochemical levels. Tumor metastasis is a complex process and requires multiple cellular functions over time. From cellular invasion, extravasation from the primary tumor, intravasation to the secondary organs, to successful colonization, tumor cells utilize many cellular or biochemical mechanisms to complete the metastatic spread. During the process of metastasis, there are consistent changes in gene expression. Studies of genes that are reduced or silenced have yielded surprising insights into in vivo mechanisms of regulating tumor metastasis. This review describes a tumor suppressor gene, Maspin, which is often silenced in cancer cells and exhibits suppressing activity against tumor growth and metastasis. Maspin has been shown to be involved in processes that are important to both tumor growth and metastasis such as cell invasion, angiogenesis, and more recently apoptosis. Hence, many efforts have been devoted to deciphering the molecular mechanism of maspin. While some insights have come from the protease inhibitory effect of maspin, more perceptive results on how maspin may function in suppressing tumor metastasis have come from studies of gene manipulation, protein interactions and global protein profiling.