A CULTURE STUDY OF SALINITY RESPONSES IN ECOTYPES OF TWO ESTUARINE RED ALGAE1

Laboratory culture studies on the euryhalinity of Bostrychia radicans Montagne and Caloglossa leprieurii (Montagne) J. Agardh from the mouth and head of the Mullica River estuary, New Jersey, revealed both species probably have ecotypes whose growth patterns correlate with the salinity regime of their habitat in nature. Significant growth differences of tetrasporelings were determined in response to four salinities (5, 15, 25, 35%c) even after acclimation periods of the tetrasporophytes from 6 mo–2 yr in laboratory culture. However, one isolate of Bostrychia and both isolates of Caloglossa also demonstrated some capability for physiological adaptation to salinity changes although this was less significant statistically than their ecotypic response. It thus appears that certain euryhaline algae may consist of ecotypes, each of which has some capacity for physiological adaptation to salinity variations.     

Plurality of cyclic nucleotide phosphodiesterase in Spinacea oleracea: subcellular distribution,partial purification,and properties

An active cyclic nucleotide phosphodiesterase has been partially purified from the 100 000 g supernatant of a spinach homogenate. It precipitated at 20–40% saturation with (NH₄)₂SO₄ and was separated on a column of Sephadex G-200 into two major peaks of activity (peaks 1 and 2). Peak 1 (MW 5 × 10⁵) was resolved by column chromatography on DEAE-cellulose into 5 protein fractions; two of these (1_c and 1_m) exhibited cyclic nucleotide phosphodiesterase activity. Subcellular fractionation showed that the phosphodiesterase of highest specific activity is located in the peroxisomes but that an enzyme of relatively high specific activity also occurs in the chloroplast and Golgi fractions. The largest total activity was in the microsomes. Isoelectric focussing of chloroplast phosphodiesterase activity gave two bands corresponding to peaks 1_c and 2. Similar examination of the microsomal, peroxisomal and Golgi fractions showed phosphodiesterases corresponding to peaks 1_m and 2. Peak 1_c activity is greater towards purine 3′,5′-cyclic nucleotides than towards their 2′,3′-isomers; the converse is true of peak 1_m. Examination of the properties of 1_c and 1_m showed a number of other differences. The pH optimum of 1_c is 6.1 and that of 1_m is 4.9. Theophylline (0.1 mM) inhibited 1_c to a greater extent than it did 1_m; Ca²⁺ stimulated 1_c activity but had no effect on 1_m. Pre-incubation with trypsin inhibited 1_m activity whereas similar treatment of 1_c gave an initial 5-fold stimulation. Repeated freezing and thawing of preparations 1_c and 1_m also evoked a difference in response. These results were shown to be attributable to removal of an inhibitor from 1_c. Evidence is presented that an endogenous activator is also present.     

Microsphere-based protease assays and screening application for lethal factor and factor Xa.

BACKGROUND: Proteases regulate many biological pathways in humans and are components of several bacterial toxins. Protease studies and development of protease inhibitors do not follow a single established methodology and are mostly protease specific. METHODS: We have created recombinant fusion proteins consisting of a biotinylated attachment sequence linked to a GFP via a protease cleavage site to develop a multiplexable microsphere-based protease assay system. Using the proteases lethal factor and factor Xa, we performed kinetic experiments to determine optimal conditions for inhibitor screens and detect known inhibitors using the HyperCyt flow cytometry system. RESULTS: We have demonstrated specific cleavage of lethal factor and factor Xa substrates, optimized screening conditions for these substrates, shown specific inhibition of the proteases, and demonstrated high throughput detection of these inhibitors. CONCLUSIONS: The assay developed here is adaptable to any site-specific protease, compatible with high throughput flow cytometry systems, and multiplexable. Coupled with flow cytometry, which provides continuous time resolution and intrinsic resolution of free vs. bound fluorophores, this assay will be useful for high throughput screening of protease inhibitors in general and could simplify assays designed to determine protease mechanism.     

The ineffectiveness of insecticide seed coatings and planting-time soil insecticides as Diabrotica virgifera virgifera LeConte population suppressors

Abstract:  The effect of any management strategy on pest population levels must be researched and determinations need to be made as to how that strategy might work based on the control objectives. In certain areas of Europe, the objective is to contain or eradicate the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, population. In order to evaluate the impact of insecticide seed coatings and/or planting-time applications of insecticides as WCR population suppressors, plot trials and large field observations were carried out in Italy over a 5-year period. Larval, pupal and adult densities, along with root damage ratings, were estimated at different locations. Data from these studies revealed that the number of WCR adults emerging from untreated plots did not differ from the number of beetles emerging from those treated with insecticides, whether as seed coating or in-furrow applications. Both seed insecticide coatings (imidacloprid, fipronil, thiamethoxam, tefluthrin) and soil insecticides applied in-furrow (chlorpyrifos, diazinon, tefluthrin) did not reduce the number of beetles emerging from monoculture fields, either in plot trials or large field observations. Observations in the USA had previously shown that soil insecticides applied at planting time partially protected basal roots from economic damage, but did not reduce corn rootworm populations. Similarly, in Europe, it has been demonstrated that not only the application of soil insecticides at planting time but also insecticide seed coatings have no role in the containment and/or eradication of WCR. Although insecticide seed coatings and soil insecticides applied in-furrow may provide protection against economic damage to roots, these management strategies do not have an impact on WCR populations and therefore are useless in WCR containment and eradication programmes.     

Leaf anatomical changes in Populus trichocarpa, Quercus rubra, Pseudotsuga menziesii and Pinus ponderosa exposed to enhanced ultraviolet‐B radiation

Leaf anatomical characteristics are important in determining the degree of injury sustained when plants are exposed to natural and enhanced levels of ultraviolet-B (UV-B) radiation (280–320 nm). The degree to which leaf anatomy can adapt to the increasing levels of UV-B radiation reaching the earth's surface is poorly understood in most tree species. We examined four tree species, representing a wide range of leaf anatomical characteristics, to determine responses of leaf area, specific leaf weight, and leaf tissue parameters after exposure to ambient and enhanced levels of UV-B radiation. Seedlings were grown in a greenhouse with photosynthetically active radiation of 39 mol m^?2 day^?1 and under one of three daily irradiances of biologically effective UV-B radiation (UV-B_BE) supplied for 10 h per day: (1) approximate ambient level received at Pullman, Washington on June 21 (1 x ); two times ambient (2 x ), or three times ambient (3 x ). We hypothesized the response of each species to UV-B radiation would be related to inherent anatomical differences. We found that the conifers responded anatomically to nearly an equal degree as the broad-leaved trees, but that different tissues were involved. Populus trichocarpa, an indeterminate broadleaf species, showed significantly thicker palisade parenchyma in recently mature leaves at the 3 x level and in older leaves under the 2 x level. In addition, individual leaf area was generally greater with increased UV-B irradiance. Quercus rubra, a semi-determinate broadleaf species, exhibited significantly thicker palisade parenchyma at the 2 x and 3 x levels as compared to controls. Psuedotsuga menziesii, an evergreen coniferous species with bifacially flattened needles, and Pinus ponderosa, an evergreen coniferous species with a complete hypodermis, showed no significant change in leaf area or specific leaf weight under enhanced UV-B radiation. Epidermal thickness was unchanged in P. menziesii. However, P. ponderosa increased the thickness and number of hypodermal layers produced, presumably decreasing penetration of UV-B radiation into the leaf. We concluded that differences in inherent leaf anatomy of the four species examined are important in the responses to enhanced levels of UV-B radiation.