Effect of habitable space on substrate selection by Paragnetina media (Walker) (Plecoptera: Perlidae)

A biological measure of space available within substrates was used as an index to examine substrate selection by the stonefly nymph Paragnetina media (Walker). Physical measures, such as total surface area of substrate, have not worked well in the past in explaining distribution of aquatic invertebrates. Although analysis of habitable space within substrate did not explain selection completely, the technique provided a precise measure and might be a more rigorous means by which substrate selection could be examined.     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Characterization of membrane-bound MgATPases, purified by aqueous two-phase partitioning, from young sugar beet roots

Membrane-bound MgATPase activity from roots of young sugar beet ( Beta vulgaris L. cv. Monohill) was investigated in a membrane fraction purified by partition in an aqueous polymer two-phase system. After two steps of "washing" with fresh bottom phase (rich in dextran), the polyethylene glycol rich top phase (U₃) was practically free of mitochondrial membranes (cytochrome oxidase), and the remaining MgATPase activity showed high substrate specificity for ATP. An optimum for the MgATPase activity was found at pH 7. The activation by Na⁺ or K⁺ was strongest on the acid side without any observable shift in pH optimum. Oligomycin had no effect, but vanadate strongly inhibited the U₃ MgATPase and the K⁺ activation was lost. The complex activation pattern achieved by varying the Na⁺/K⁺ ratio at constant total concentration was interpreted as a synergistic (Na⁺+ K⁺)-activation. The U₃ fraction MgATP-ase activity showed a 4-fold increase in the presence of 0.01% Triton X-100 implying that the MgATPase activity is located in vesicles of which 75% or more are sealed with the ATP binding site on the inside. Comparison with the properties of plasma membrane. ATPases from other plants indicated that the U₃ fraction MgATPase was mainly of plasma membrane origin.     

Efficient separation of subfossil Chironomidae from lake sediments

Electron transport system (ETS) activity, CO₂ evolution, O₂ consumption, N₂-fixation (C₂H₂ reduction) and methanogenesis were appropriately measured in aerobic and anaerobically incubated sediment at 4, 10 and 20 ° C to better characterize these activities under different incubation conditions. ETS activity was always higher in the aerobically incubated sediment at all three incubation temperatures, whereas (C₂H₂ reduction was always greater in the anaerobic sediment. Carbon dioxide evolution was detected only in the aerobic sediment at 10 and 20 ° C but not at 4 ° C. Methane evolution in anaerobic sediment increased gradually with an increase in the incubation temperature.     

Internal P loading in lakes: A different approach to its evaluation

P release rates in five lakes were estimated from a mass balance approach and from laboratory tests. The influence of various environmental parameters on P release has been considered. The internal P loading can contribute phosphorus to overlying waters at levels comparable to the external sources (from 30 to 100%). When considering the recovery from eutrophication of the investigated lakes, the nutrient release from sediments is of critical importance.     

Temporal fluctuations of silver,copper and zinc in the bivalve Macoma balthica at five stations in South San Francisco Bay

Concentrations of Cu, Ag and Zn were measured in the soft tissues of the estuarine bivalve Macoma balthica in South San Francisco Bay at near-monthly intervals for periods of two to three years at four stations, and eight years at a metal-enriched station. The amplitude and frequency of fluctuations differed among stations and among metals. Fluctuations were greatest at stations with the greatest metal enrichment and with the least dilution and flushing of wastes. A consistent seasonal pattern of fluctuation in Cu and Ag concentrations was evident in M. balthica at the metal-enriched station. These seasonal changes in tissue metal concentrations appeared to be affected by metal inputs, hydrologic processes that may affect both metal concentrations and bioavailability, and seasonal changes in the weight of the bivalve. The contributions of each of these interacting factors could not be determined quantitatively. At the metal-enriched station significant variation in the amplitude of seasonal fluctuations was also evident from year to year. Interpretation of metal concentrations in bivalves from estuaries will require careful consideration of the processes which affect metal dynamics in these complex environments.     

Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

Cross-linked collagen surface for cell culture that is stable,uniform, and optically superior to conventional surfaces

Summary A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed. This work was supported by the Leopold Schepp Foundation, the Dysautonomia Foundation, National Institutes of Health Grants NS14768 and NS11237, and Institutional Core Grant HD06276.     

Alterations in the structure of the electrical double layer adjacent to a charged membrane arising from electromagnetically induced changes in the activity of membrane bound enzymes

Electromagnetic fields of very low amplitude have been reported to influence a number of cellular functions. Many of these effects have a high degree of frequency specificity. Herein it is suggested that some of these reported results could be explained by a fieldinduced alteration in the enzymic activity of integral membrane proteins. It is shown that such a field-induced transition from an initial nonequilibrium steady-state to a final nonequilibrium steady-state can lead to an alteration in the concentration profiles of those charged species in the cell's ambient electrolyte that comprise the so-called electrical double layer. Examples of variations in the concentration profiles of those ions that react with a membrane-bound enzyme, as well as nonreacting ionic species, are given. The modulation of such effects by systematic variations in extracellular pH and ionic strength is discussed.