Palaeoclimatic changes resulted in range expansion and subsequent divergence in brown fur seals, Arctocephalus pusillus.

Past climatic change as a driving force of marine diversification is still largely unclear, particularly for Southern Hemisphere species. Here, we present a case using the brown fur seal, Arctocephalus pusillus, assessing the geographical structure and demographic history using mitochondrial and nuclear data. Results show the two previously defined subspecies (one from Australia and the other from southern Africa) are phylogeographically distinct. Migration analyses based on nuclear data suggest the absence of migrants among the two genetically close assemblages. The demographic history of A. pusillus is characterized by a glacial population expansion (approx. 18 kya) in the southern African lineage, which coincides with time estimates of population expansion of prey species of seals. Approximate Bayesian calculations support an eastward dispersal event during the Last Glacial Maximum when sea levels were lower, followed by a postglacial divergence event, approximately 13 kya. The demographic history of the brown fur seal in the Southern Oceans provides support that recent palaeoclimatic changes could have facilitated expansions in some marine species and that postglacial sea-level rise may have acted as a dispersal barrier for species mostly confined to continental shelves.

Soil phosphorus dynamics following land application of unsaturated and partially saturated red mud and water treatment residuals.

The secondary use of P-sorbing industrial by-products as a fertilizer or soil conditioner is gaining increased attention, particularly in light of diminishing reserves of rock phosphate traditionally used to manufacture P fertilizer. This study examined applications of red mud (RM) and water treatment residuals (WTR) at two levels of P saturation (i.e. 'as received' and partially saturated) in a soil incubation and runoff plot study. When incubated with soils ranging in texture and initial P concentration, P-sorbing residuals that were less enriched with P decreased water-extractable soil P (WEP) concentration to a greater extent than more P saturated residuals. In contrast to WTR treatments, not all of the RM applications decreased soil WEP concentrations below those of the control soils. The runoff study investigated soil P dynamics when partially P-saturated RM and WTR's were surface applied to grass plots at 2 t ha-1 on Day 0, followed by three rainfall simulations (7 cm h-1 for 30 min, Days 2, 7 and 28) and at 3 t ha-1 on Day 70 followed by two more rainfall simulations (Days 77 and 96). Application of residuals at these rates did not significantly increase dissolved reactive P (DRP) in runoff compared with unamended controls during the study. Forage cuttings taken 90 days after the first rainfall simulation indicated that nutrient uptake was not compromised by the application of the residuals. Overall results indicate that WTRs may be a more suitable soil amendment than RM residuals given their greater ability to reduce soil WEP across a range of soils without simultaneously increasing Mehlich-3 extractable soil P concentrations above the upper threshold limit (150 mg P kg-1), and their minimal impact on plant nutrient uptake.

The use of inert carriers in regulatory biodegradation tests of low density poorly water-soluble substances.

Many poorly water-soluble compounds fail regulatory ready biodegradation tests as the method of test material preparation limits the bioavailability of the chemical. The recognised method for delivery of poorly soluble materials into biodegradability tests consists of coating test material inside the test vessel or onto inert substrates (i.e., glass cover slide, boiling beads, filter paper, or Teflon stir bar) that are placed inside the vessels. Volatile solvents are often used to augment this process. Although these substrates work fairly well for delivering many poorly soluble materials into biodegradability tests, they have not been effective in keeping low density, poorly water-soluble substances in the test medium. Soon after medium is added to the test vessels, these chemicals break loose from the substrates and float on the surface where they have limited contact with micro-organisms in the test medium. Hence, there is a reduced potential for measuring substantial biodegradability in the test. This paper describes the work undertaken to establish a standard method of adding low density, poorly water-soluble substances into test vessels of biodegradability studies to ensure these materials remain in contact with micro-organisms in the test medium. The substances are prepared for testing by adsorption onto silica gel followed by dispersion into the culture medium. This method of delivery may provide greater intra- and inter-laboratory consistency in biodegradability test results for low density, poorly water-soluble substances and it may more closely mimic the probable transport and fate of these substances in the environment.

Structure and function of pectic enzymes: virulence factors of plant pathogens.

The structure and function of Erwinia chrysanthemi pectate lysase C, a plant virulence factor, is reviewed to illustrate one mechanism of pathogenesis at the molecular level. Current investigative topics are discussed in this paper.

Structure of a plant cell wall fragment complexed to pectate lyase C.

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(1-->4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein-oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the beta elimination cleavage of polygalacturonic acid.

Calmodulin stabilizes an amphiphilic alpha-helix within RC3/neurogranin and GAP-43/neuromodulin only when Ca2+ is absent.

Two neuronal protein kinase C substrates, RC3/neurogranin and GAP-43/neuromodulin, preferentially bind to calmodulin (CaM) when Ca2+ is absent. We examine RC3.CaM and GAP-43.CaM interactions by circular dichroism spectroscopy using purified, recombinant RC3 and GAP-43, sequence variants of RC3 displaying qualitative and quantitative differences in CaM binding affinities, and overlapping peptides that cumulatively span the entire amino acid sequence of RC3. We conclude that CaM stabilizes a basic, amphiphilic alpha-helix within RC3 and GAP-43 under physiological salt concentrations only when Ca2+ is absent. This provides structural confirmation for two binding modes and suggests that CaM regulates the biological activities of RC3 and GAP-43 through an allosteric, Ca(2+)-sensitive mechanism that can be uncoupled by protein kinase C-mediated phosphorylation. More generally, our observations imply an alternative allosteric regulatory role for the Ca(2+)-free form of CaM.

Improving a rural hospital's peer review process.

Quality monitoring in a small rural hospital is a challenge on many fronts. The case study presented here illustrates a quality monitoring failure and analyzes the contributing factors. Based on their experience, the authors suggest methods to improve small rural hospital quality monitoring.

Mutational and biophysical studies suggest RC3/neurogranin regulates calmodulin availability.

RC3/neurogranin is a forebrain-enriched, postnatal-onset, thyroid hormone-dependent, protein kinase C substrate of dendritic spines that interacts with calmodulin. These characteristics suggest a prominent role within the Ca(2+)-mediated second messenger cascades associated with neonatal synaptogenesis and adult neural plasticity. To understand the molecular interactions between RC3 and calmodulin, we characterized recombinant RC3 and four sequence variants: Ser-36-->Ala, Ser-36-->Asp, Ser-36-->Lys, and Phe-37-->Trp. Interactions between CaM and variant Phe-37-->Trp can be monitored by fluorescence spectroscopy, allowing us to determine, by competitive assays, the relative affinities of the wild-type and variant proteins for calmodulin. The effects of salt and Ca2+ on the rank order of these affinities permit partial dissection of hydrophobic, ionic, and structural components of the RC3-CaM interaction and suggest that it is bimodal. We demonstrate that RC3 binds preferentially to CaM when Ca2+ is absent and that the addition of a negative charge to residue 36 is sufficient to disrupt all detectable RC3-CaM interactions. We propose a model wherein a Ca(2+)-"sensitive," bimodal interaction between RC3 and CaM regulates the transduction of postsynaptic Ca2+ fluxes into physiological responses through the modulation of Ca2+/CaM availability.

Rapid purification, site-directed mutagenesis, and initial characterization of recombinant RC3/neurogranin.

RC3/Neurogranin is a postnatal-onset, forebrain-specific, thyroid hormone-regulated, protein kinase C (PKC) substrate that binds calmodulin (CaM) and accumulates in dendritic spines. We bacterially expressed and purified RC3 and, for comparison, GAP-43/neuromodulin to near homogeneity using relatively simple procedures. We then raised antisera against recombinant RC3 that does not crossreact with GAP-43 and is suitable for immunohistochemical analysis of brain slices. We also constructed over 30 RC3 sequence variants by PCR-mediated, site-directed mutagenesis, and purified four of these to near homogeneity. The elution profiles displayed by RC3 and sequence variants during purification on CaM-Sepharose columns suggest that two different affinity forms of the RC3.CaM complex coexist when Ca2+ is absent and that GAP-43.CaM interactions are far more sensitive to salt than those that occur between recombinant RC3 and CaM. Variant proteins in which serine 36 was changed failed to serve as a substrate for PKC, implicating this as the target residue.

Estimating the severity of osteoarthritis with magnetic resonance spectroscopy.

This study explores the possibility of estimating the severity of osteoarthritis in the elderly through magnetic resonance measurements of proton relaxation times in articular cartilage. Twenty-five paired samples were cut from eight cartilage specimens. The specimens had been taken in the normal course of knee surgery from patients aged 60 years or older. The reciprocal of the T1 relaxation time (1/T1) was found to have a negative association with the histological-histochemical grade of the severity of osteoarthritis. A revised, weighted version of the Mankin-Dorfman severity scale yielded stronger correlations with 1/T1 and with percent water. A nonlinear regression equation expressing osteoarthritis as a function of 1/T1 explained 38% of the variation in the severity of osteoarthritis as measured by the new scale. A superior methodology for estimating the severity of osteoarthritis on the basis of contour maps of the magnetic resonance response is suggested.