Effects of trout on the diel periodicity of drifting in baetid mayflies

Some benthic invertebrates in streams make frequent, short journeys downstream in the water column (=drifting). In most streams there are larger numbers of invertebrates in the drift at night than during the day. We tested the hypothesis that nocturnal drifting is a response to avoid predation from fish that feed in the water column during the day. We surveyed diel patterns of drifting by nymphs of the mayfly Baetis coelestis in several streams containing (n=5) and lacking (n=7) populations of rainbow trout, Oncorhynchus mykiss. Drifting was more nocturnal in the presence of trout (85% of daily drift occurred at night) than in their absence (50% of daily drift occurred at night). This shift in periodicity is due to reduced daytime drifting in streams with trout, because at a given nighttime drift density, the daytime drift density of B. coelestis was lower in streams occupied by trout than in troutless streams. Large size classes of B. coelestis were underrepresented in the daytime drift in trout streams compared to nighttime drift in trout streams, and to both day and night drift in troutless streams. Differences in daytime drift density between streams with and without trout were the result of differences in mayfly drift behaviour among streams because predation rates by trout were too low to significantly reduce densities of drifting B. coelestis. We tested for rapid (over 3 days) phenotypic responses to trout presence by adding trout in cages to three of the troutless streams. Nighttime drifting was unaffected by the addition of trout, but daytime drift densities were reduced by 28% below cages containing trout relative to control cages (lacking trout) placed upstream. Drift responses were measured 15 m downstream of the cages suggesting that mayflies detected trout using chemical cues. Overall, these data support the hypothesis that infrequent daytime drifting is an avoidance response to fish that feed in the water column during the day. Avoidance is more pronounced in large individuals and is, at least partially, a phenotypic response mediated by chemical cues.     

Wound-Associated Competency Factors Are Required for the Proximal Cell Responses of Soybean to the Phytophthora sojae Wall Glucan Elicitor

Intact soybean (Glycine max L. [Merr.]) tissues show distinct proximal and distal cell responses to the Phytophthora sojae (Kauf. and Gerde.) wall glucan elicitor. Proximal cells respond with accumulations of glyceollin and phenolic polymers, whereas distal cells respond with an increase of isoflavone conjugates. Comparison of the activities of the P. sojae glucan in the classical cut cotyledon and a cotyledon infiltration assay suggests that the proximal, but not the distal, responses to elicitor require tissue wounding. Washing the surface of cut cotyledons prior to elicitor treatment also greatly diminishes the proximal responses, which can be restored in a dose-dependent manner by prior treatment of the washed cells with wound exudate from cut "donor" cotyledons. Thus, discrete wound-associated factors, which we term elicitation competency factors, are required for the proximal cell response to the glucan elicitor. The wound factors induce a competent state that is transient in nature. Maximal elicitor response is seen 2 to 3 h after wounding, and cells become elicitor nonresponsive after 4 h. Competency is markedly affected by the age of tissues; cotyledons become more inherently competent as they approach senescence. The time course of attainment of the competent state and its duration are strongly affected by light and temperature. Since the wound-associated competency factors can also be obtained from washings of hypersensitive lesions, we hypothesize that similar competency factors may be released from hypersensitively dying cells in incompatible infections. This event may program the immediately surrounding cells to make them competent for the proximal defense responses.     

Receptor interactions of the position 4 side chains of angiotensin II analogues: Importance of aromatic ring quadrupole

A triad of interacting group (TyrOH? His$ \underline\ominus$O₂C) in angiotensin II (ANG II) has been postulated to create the tyrosinate anion pharmacophore (tyanophore) responsible for receptor activation/triggering (Biochim. Biophys. Acta 1991, 1065, 21). In the present study we investigated the effects on bioactivity of substituting the Tyr⁴ residue in [Sar¹]ANG II with other anionic or electronegative amino acids, and with a number of aromatic amino acids lacking a hydroxyl group. [Sar¹ Nva(δ-OH)⁴]ANG II, [Sar¹ Nva(δ-OCH₃)⁴]ANG II, [Sar¹ Met⁴]ANG II, [Sar¹ Gln⁴]ANG II, [Sar¹ Glu⁴]ANG II and [Sar¹ DL -Alg⁴]ANG II had agonist activities in the rat isolated uterus assay of 4, 3, 19, 10, > 0.1 and > 0.1%, respectively, of that of ANG II. [Sar¹ Nal⁴]ANG II, [Sar¹ Pal⁴]ANG II, [Sar¹ DL -Phg(4′-F)⁴]ANG II, [Sar¹ Phe(4′-F)⁴]ANG II, [Sar¹ Phe(F₅)⁴]ANG II and [Sar¹ His⁴]ANG II had agonist activities of 4.5, 7, < 0.1, 0.2, 1 and 0.6%, respectively. All peptides investigated were devoid of measurable antagonist activity except [Sar¹] Phe(4′-F)⁴ ANG II (pA₂ = 7.7). These findings illustrate that anionic or electronegative aliphatic side chains replacing tyrosinate at position 4 can partially activate the angiotension receptor. For ANG II analogues containing an aromatic amino acid other than Tyr at position 4, ligand binding and agonist activity are not dependent on the electronegativity or dipole moment of the aromatic ring, or on the ability of the 4′ ring substituent to accept a proton. Modelling based on ab initio calculations of aromatic ring multipoles illustrate that the apparent binding affinity (PA₂) of ANG II analogues is associated with a perpendicular electrostatic interaction of the position 4 aromatic ring with a receptor-based group. In addition, intramolecular interactions providing for the conformation of the ligand as it approaches its receptor appear to have a role in determining agonist vs antagonist activity.     

Current issues in the chemistry of cytochromec oxidase

Some contemporary issues relevant to the chemistry of mammalian cytochromec oxidase are discussed. These include the optical properties of heme A and the spectroscopic consequences of the differences in side-chain substitution compared to heme B; a common fallacy concerning the electrostatic exchange interaction between cytochromea ₃ and Cu_B; the question of the number and location of the copper components of the enzyme; and the mode of binding of ligands such as cyanide and azide.     

Transport kinetics of maltotriose in strains ofSaccharomyces

Summary Maltotriose transport was studied in two brewer's yeast strains, an ale strain 3001 and a lager strain 3021, using laboratory-synthesized¹⁴C-maltotriose. The maltotriose transport systems preferred a lower pH (pH 4.3) to a higher pH (pH 6.6). Two maltotriose transport affinity systems have been indentified. The high affinity system hasK _m values of 1.3 mM for strain 3021 and 1.4 mM for strain 3001. The low affinity competitively inhibited by maltose and glucose withK _i values of 58 mM and 177 mM. respectively, for strain 3021, and 55 mM and 147 mM, respectively, for strain 3001. Cells grown in maltotriose and maltose had higher maltotriose and maltose transport rates, and cells grown in glucose had lower maltortriose and maltose transport rates. Early-logarithmic phase cells transported glucose faster than either maltose or maltotriose. Cells harvested later in the growth phase had increased maltotriose and maltose transport activity. Neither strain exhibited significant differences with respect to maltose and maltotriose transport activity.     

Packaging capacity and stability of human adenovirus type 5 vectors.

Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population.     

Brefeldin A reversibly blocks early but not late protein transport steps in the yeast secretory pathway.

We have found that brefeldin A (BFA) inhibited the growth of an ise1 mutant of Saccharomyces cerevisiae. Genetic complementation and mapping studies demonstrated that ise1 was allelic to erg6, a gene required for the biosynthesis of the principal membrane sterol of yeast, ergosterol. Treatment of ise1 cells with BFA resulted in an immediate block in protein transport through the secretory pathway. Vacuolar carboxypeptidase Y (CPY) and the secreted pheromone alpha-factor accumulated as both the core glycosylated (ER) and alpha 1,6 mannosylated (early Golgi) forms in drug-treated cells. The modification of alpha-factor with alpha 1,6 mannose in BFA-treated cells did not appear to result from retrograde transport of the alpha 1,6 mannosyl-transferase into the ER. We found that transport of CPY from medial and late Golgi compartments to the vacuole was unaffected by BFA, nor was secretion of alpha 1,3 mannosylated alpha-factor or invertase blocked by BFA. The effects of BFA on the secretory pathway were also reversible after brief exposure (< 40 min) to the drug. We suggest that the primary effect of BFA in S. cerevisiae is restricted to the ER and the alpha 1,6 mannosyltransferase compartment of the Golgi complex.     

Rational design and PCR-based synthesis of an artificial Schizophyllum commune xylanase gene.

A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure. Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage. The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E. coli genes. The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.