Microsatellite DNA analysis of spatial and temporal population structuring of <Emphasis Type="Italic">Phragmites australis</Emphasis> along the Hudson River Estuary

Phragmites australis is a perennial grass that has invaded wetlands of the northeastern United States over the past century. The Hudson River Estuary and surrounding watersheds are no exception in that populations of P. australis have spread dramatically along its shores and tributaries in the past 40 years. Recent studies have shown that genetically variable populations of P. australis can spread by seed dispersal in addition to clonal mechanisms. It is important to characterize the genetic variation of Hudson River populations as part of a management strategy for this species to determine the mechanisms by which its spreads and colonizes new habitats, particularly those with frequent anthropogenic disturbances. The goals of this study were to quantify levels of genetic variation and structuring in Hudson River populations of P. australis using microsatellite DNA analysis. A total of 354 culms of P. australis were collected from nine locations ranging from Albany, New York to Staten Island, New York in the summers of 2004 (N = 174) and 2011 (N = 180). Microsatellite data from eight loci indicated that the Hudson River Estuary has some of the highest levels of genetic variation of all U. S. Atlantic Coast regions containing P. australis. Gene diversity (Hs) across all loci in the 2004 collection was 0.45 (±0.02) and that of the 2011 collection was 0.47 (±0.07). Patches within sample sites were rarely monoclonal and had multiple genetic phenotypes. Moran’s Identity tests indicated that individuals within a patch were closely related, whereas little genetic relatedness was evident among individuals from sample sites >1 km apart. Spatial structuring was also not evident in autospatial correlation and principle coordinate analyses. These findings suggest that genetic diversity is maintained within stands by sexual reproduction and that seeds are important in dispersal of P. australis across the Hudson River Estuary. Ample habitats are available for establishment of new Phragmites stands due to high levels of anthropogenic disturbance from populations living along the Estuary. Wildlife managers should focus on monitoring habitats that provide seedbed for Phragmites and promote land use practices that prevent soil disturbance and establishment of new stands.     

Allosteric effect of ATP on Na(+),K(+)-ATPase conformational kinetics

The kinetics of the E2 --> E1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >or=50 microM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E2:E2 --> E2:E1 --> E1:E1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E2:E2 --> E2:E1 and E2:E1 --> E1:E1 transitions were found to be 10.4 (+/-1.0) and 0.49 (+/-0.02) s-1, respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/-0.3) s-1. It was found that ATP acting at a high affinity site (Kd approximately 0.25 microM), stimulated the reverse reaction, E1ATP --> E2ATP, in addition to its known allosteric low affinity (Kd approximately 71 microM) stimulation of the forward reaction, E2ATP --> E1ATP.     

Orientational polarisability of lipid membrane surfaces

Here we present a fluorescence method based on the Stokes shift of the voltage-sensitive dye di-8-ANEPPS to quantify the orientational polarisability of lipid membrane surfaces, i.e. the polarisability due to molecular reorientation. Di-8-ANEPPS is already an established probe of membrane dipole potential. Its use, therefore, as a probe of both the dipole potential and orientational polarisability allows a direct comparison of these two properties in an identical region of the lipid bilayer. We applied the new technique on phosphatidylcholine vesicles to study the effects of different degrees of hydrocarbon saturation and of the incorporation of cholesterol and some of its oxidized derivatives. We found that lipids with unsaturated chains had a lower orientational polarisability than those with saturated chains. This could be explained by a reduction in membrane dipole potential as a result of a decrease in lipid packing density. Cholesterol derivatives were found to either increase or decrease the orientational polarisability depending on their molecular structure. The varying effects could be explained by antagonistic effects of the dipole potential and membrane order, which are both changed to varying degrees by the cholesterol derivatives and which lead to increases and decreases in orientational polarisability, respectively.     

Nonparametric estimation of age-at-onset distributions from censored kin-cohort data

We present a nonparametric estimator of genotype-specific age-at-onsetdistributions from kin-cohort data. Standard error calculationsare derived and the methodology is illustrated through an analysisof the influence of mutations of the Parkin gene on Parkinson'sdisease. Semiparametric efficiency considerations are brieflydiscussed.     

Influence of White Clover Mosaic Potexvirus Infection on the Endogenous Cytokinin Content of Bean

The cytokinin content in the primary leaves of bean (Phaseolus vulgaris) was monitored for 10 d after inoculation with white clover mosaic potexvirus. The cytokinins were isolated, purified, separated by high-performance liquid chromatography, and quantified by radioimmunoassay. The cytokinins detected at the time of inoculation (d 0) were: (a) the free bases, zeatin (Z), dihydrozeatin (DZ), and isopentenyladenine; (b) the riboside, DZ riboside (DZR); (c) the O-glucosides of DZ, DZR, and Z riboside; (d) the nucleotides, Z riboside-5′-monophosphate and isopentenyladenosine-5′-monophosphate; and (e) trace amounts of Z-9-glucoside and DZ-9-glucoside. During the 10 d after inoculation with white clover mosaic potexvirus, marked quantitative changes in this cytokinin profile were observed. The concentration of the free bases and DZR decreased, accompanied by an increase in the 9-glucosides and the nucleotides. Virus titer increased rapidly 3 d after inoculation, attaining a maximum level at d 5. This increase coincided with the increases in the 9-glucosides and the nucleotides. We propose that the decline in the cytokinin free bases and riboside may allow the increase of virus titer in bean and lead to the senescence of infected leaves.     

The localization and expression of the class II starch synthases of wheat.

The starch granules of hexaploid wheat (Triticum aestivum) contain a group of three proteins known as SGP-1 (starch granule protein-1) proteins, which have apparent molecular masses of 100, 108, and 115 kD. The nature and role of these proteins has not been defined previously. We demonstrate that these polypeptides are starch synthases that are present in both the starch granule and the soluble fraction at the early stages of wheat endosperm development, but that are exclusively granule bound at mid and late endosperm development. A partial cDNA clone encoding a fragment of the 100-kD protein was obtained by screening a wheat endosperm cDNA expression library using monoclonal antibodies. Three classes of cDNA were subsequently isolated from a wheat endosperm cDNA library by nucleic acid hybridization and were shown to encode the 100-, 108-, and 115-kD proteins. The cDNA sequences are highly homologous to class II starch synthases and have the highest homology with the maize SSIIa (starch synthase IIa) gene. mRNA for the SGP-1 proteins was detected in the leaf, pre-anthesis florets, and endosperm of wheat and is highly expressed in the leaf and in the grain during the early to mid stages of development. We discuss the roles of the SGP-1 proteins in starch biosynthesis in wheat.     

The relationship between the rate of starch synthesis, the adenosine 5′-diphosphoglucose concentration and the amylose content of starch in developing pea embryos

Mutations that reduced the rate of starch synthesis in pea (Pisum sativum L.) embryos through effects on enzymes on the pathway from sucrose to adenosine 5′-diphosphoglucose (ADPglucose) also led to a reduction in the amylose content of the starch of developing embryos. Evidence is presented that this relationship between rate of synthesis and the composition of starch is due to the fact that amylopectin-synthesising isoforms of starch synthase have higher affinities for ADPglucose than the amylose-synthesising isoform. First, developing mutant embryos (rb, rug3 and rug4 mutants) displayed both reduced amylose contents in their starches and reduced ADPglucose contents relative to wild-type embryos. Second, incubation of detached, wild-type embryos for 6 h at high and low glucose concentrations resulted in differences in both ADPglucose content and the relative rates of amylose and amylopectin synthesis. At 0.25 M glucose both ADPglucose content and the proportion of synthesised starch that was amylose were about twice as great as at 25 μM glucose. Third, S _0.5 values for soluble (amylopectin-synthesising) starch synthases in developing embryos were several-fold lower than that for granule-bound (amylose synthesising) starch synthase. Estimates of the expected amylose contents of the starch of the mutant embryos, based on the reduction in their ADPglucose contents and on the S _0.5 values of the starch synthases, were very similar to the measured amylose contents. The implications of these results for the determination of starch composition are discussed. Received: 6 February 1999 / Accepted: 22 May 1999     

Development of in vitro assays for the detection of botulinum toxins in foods

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bioassay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bioassay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin of the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bioassay. It is envisaged that such assays will prove realistic alternatives to animal-based tests.