Modification of an algal culture medium for sustained growth of a saxitoxin-producing isolate of Alexandrium minutum

In order to study saxitoxin (STX) production bymicro-algae in the laboratory, a defined algal culture medium which supports optimum growth over a longtime-period is a requirement. In the development of such a medium, a number of modifications were made to a standard algal culture medium (GP) and growth of a STX-producing isolate of Alexandrium minutum in the different formulations was assessed by measuring maximum cell densities and mean generation times (MGT). All experiments were carried out under controlled conditions in an aerobic atmosphere with increased CO₂. Whilst maximum cell densities in the different modifications were similar, the MGT was significantly shortened by the addition of Tris buffer and the trace metals strontium, selenium and molybdenum. Replacement of natural with artificial seawater and removal of soil extract did not adversely affect algal growth. Five of the six media formulations supported the growth of A. minutumover a 9-month period. This revised version was published online in August 2006 with corrections to the Cover Date.     

The genetic effective and adult census size of an Australian population of tiger prawns (Penaeus esculentus)

This study compares estimates of the census size of the spawning population with genetic estimates of effective current and long-term population size for an abundant and commercially important marine invertebrate, the brown tiger prawn (Penaeus esculentus). Our aim was to focus on the relationship between genetic effective and census size that may provide a source of information for viability analyses of naturally occurring populations. Samples were taken in 2001, 2002 and 2003 from a population on the east coast of Australia and temporal allelic variation was measured at eight polymorphic microsatellite loci. Moments-based and maximum-likelihood estimates of current genetic effective population size ranged from 797 to 1304. The mean long-term genetic effective population size was 9968. Although small for a large population, the effective population size estimates were above the threshold where genetic diversity is lost at neutral alleles through drift or inbreeding. Simulation studies correctly predicted that under these experimental conditions the genetic estimates would have non-infinite upper confidence limits and revealed they might be overestimates of the true size. We also show that estimates of mortality and variance in family size may be derived from data on average fecundity, current genetic effective and census spawning population size, assuming effective population size is equivalent to the number of breeders. This work confirms that it is feasible to obtain accurate estimates of current genetic effective population size for abundant Type III species using existing genetic marker technology.     

Affinities of Shiga toxins 1 and 2 for univalent and oligovalent Pk-trisaccharide analogs measured by electrospray ionization mass spectrometry

The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B(5) and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.     

Diaphragms and flooding survival

Why don't the gas spaces of submerged organs of wetland plants flood extensively when damaged? In addressing this intriguing question, Soukup et al . (pp. 71–75 in this issue) report on the role of rhizome diaphragms as barriers to flooding in Phragmites australis . This should prompt some reappraisal of the ways in which flooding resistance can be realized, even perhaps in undamaged organs.
Most emergent wetland macrophytes have an abundance of interconnected internal gas space, much of it in the form of large voids transversely partitioned at intervals by perforated cellular plates termed diaphragms. Functionally, it provides a low-resistance pathway for internal oxygen transport to support the respiratory needs of submerged and buried organs (Armstrong, 1979; Armstrong et al ., 1988; Crawford, 1992) and facilitates carbon dioxide removal. However, it does more than this, since it enables oxygen to be released from the root to where it can support aerobic microbial activity in otherwise anaerobic sediments, and phytotoxin immobilization or destruction (Armstrong et al ., 1992; Begg et al ., 1994; Gilbert & Frenzel, 1998). This oxygen release is regarded by some as a valuable aid to effluent purification by constructed wetlands. Perhaps a less desirable property of this gas-space provision is its recently discovered role in enhancing the emissions of greenhouse gases such as methane from wetlands (Brix et al ., 1992; Chanton & Whiting, 1996; Crutzen, 1991; Dacey & Klug, 1979).     

Simulations on the Primitive Electrolyte Environment of a High Charge-density Polyelectrolyte. A Sampling Problem and its Solution

Abstract

We show that the classical Metropolis Monte Carlo (MMC) algorithm converges very slowly when applied to the primitive electrolyte environment for a high charge-density polyelectrolyte. This slowness of convergence, which is due to the large density inhomogeneity around the polyelectrolyte, produces noticeable errors in the ion distribution functions for MMC runs of 1.3 × 10⁶ trial steps started from nonequilibrium distributions. We report that an algorithm which we call DSMC (for density-scaled Monte Carlo) overcomes this problem and provides relatively rapid convergence in this application. We suggest that DSMC should be well-suited for other Monte Carlo simulations on physical systems where large density inhomogeneities occur.     

Bimodal effects of luteinizing hormone and role of androgens in modifying superovulatory responses of rats to infusion with purified porcine follicle-stimulating hormone

Prepubertal (28-30 days old) female rats were infused s.c. over a 60-h period with a purified porcine pituitary follicle-stimulating hormone (FSH) preparation having FSH specific activity 8.4 times that of NIH-FSH-S1 and luteinizing hormone (LH) specific activity less than 0.005 times that of NIH-LH-S1, based on radioreceptor assays. When the FSH infusion rate of this preparation was increased over the range of 0.5-2 units/day (mg NIH-FSH-S1 equivalent), an all-or-none response was observed, with the threshold dose for superovulation being between 1 and 2 units/day. Eleven of twelve rats receiving the 2 units/day dose ovulated a mean +/- SEM of 67 +/- 8 oocytes on the morning of the third day after the beginning of FSH infusion. Addition of human chorionic gonadotrophin (hCG), as a source of LH activity, to a subthreshold (1 U/day) FSH infusion rate resulted in 20% of rats ovulating at an hCG dosage of 50 mIU/day; increasing the hCG infusion to 200 mIU/day concomitant with the subthreshold FSH infusion rate increased ovulation rate to a mean of 69 +/- 8/rat, with 100% of rats ovulating. To determine the effect of varying both FSH infusion rates and LH:FSH ratios, FSH was infused at several rates, with hCG added to give varying hCG:FSH ratios for each FSH infusion rate. Administration of hCG alone was ineffective in causing ovulation except at the highest infusion rates. Adding hCG to FSH to reach a ratio of 0.2 IU hCG/U FSH significantly increased the superovulatory response to an intermediate, 1 U/day FSH dose, but not to the low, 0.5 U/day dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteomic profiling of metalloprotease activities with cocktails of active-site probes

Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a 'cocktail' to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems.