Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants.

The human cytomegalovirus UL80 open reading frame encodes protease and assembly protein from its N- and C-terminal regions, respectively. We reported previously that a 30-kDa protease is derived by autoproteolytic processing of a polyprotein which is the translation product of the entire UL80 open reading frame (E. Z. Baum, G. A. Bebernitz, J. D. Hulmes, V. P. Muzithras, T. R. Jones, and Y. Gluzman, J. Virol. 67:497-506, 1993). Three autoproteolytic cleavage sites within the UL80 polyprotein were characterized; site 143 is within the protease domain and inactivates the protease. In this article, we report (i) expression analyses of UL80 in infected cells, including the processing kinetics of the UL80 polyprotein; (ii) the existence of an additional cleavage site (site 209) within the protease domain of the UL80 polyprotein; and (iii) the effect of mutagenesis at each of the cleavage sites upon proteolytic activity and steady-state levels of the UL80 processing products. During the course of infection, UL80 polyprotein processing begins at cleavage site 643 and follows at sites 256 and 143. Cleavage at site 643 and/or 256 within the polyprotein is not a prerequisite for efficient protease activity, since all three proteases (85-, 80-, and 30-kDa proteins) were equally active in cleaving the assembly protein precursor to its mature form. Inhibition of cleavage at site 143 resulted in a three- to sixfold increase in the steady-state level of the 30-kDa protease, supporting the hypothesis that cleavage at this site may represent a mechanism by which cytomegalovirus regulates the level of active protease.     

Inverse electrocardiographic transformations: dependence on the number of epicardial regions and body surface data points

The inverse problem of electrocardiography, the computation of epicardial potentials from body surface potentials, is influenced by the desired resolution on the epicardium, the number of recording points on the body surface, and the method of limiting the inversion process. To examine the role of these variables in the computation of the inverse transform, Tikhonov's zero-order regularization and singular value decomposition (SVD) have been used to invert the forward transfer matrix. The inverses have been compared in a data-independent manner using the resolution and the noise amplification as endpoints. Sets of 32, 50, 192, and 384 leads were chosen as sets of body surface data, and 26, 50, 74, and 98 regions were chosen to represent the epicardium.The resolution and noise were both improved by using a greater number of electrodes on the body surface. When 60% of the singular values are retained, the results show a trade-off between noise and resolution, with typical maximal epicardial noise levels of less than 0.5% of maximum epicardial potentials for 26 epicardial regions, 2.5% for 50 epicardial regions, 7.5% for 74 epicardial regions, and 50% for 98 epicardial regions. As the number of epicardial regions is increased, the regularization technique effectively fixes the noise amplification but markedly decreases the resolution, whereas SVD results in an increase in noise and a moderate decrease in resolution. Overall the regularization technique performs slightly better than SVD in the noise-resolution relationship.There is a region at the posterior of the heart that was poorly resolved regardless of the number of regions chosen. The variance of the resolution was such as to suggest the use of variable-size epicardial regions based on the resolution.     

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.     

The benefit to some minnows of spawning in the nests of other species

Synopsis Fishes that act as nest associates spawn simultaneously with nest-building hosts and then abandon their eggs. The proposed benefit for this behavior is increased brood survivorship, arising from the physical environment provided by the nest or the parental care provided by the host. Field and enclosure experiments indicated that associates benefit from the parental care provided by the host, and not from the physical environment provided by the nests of hosts. This information, along with the effect of nest association on host reproductive success, is necessary before the nature of this nesting symbiosis can be characterized.     

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.     

Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA.

A transformation scheme for Cryptococcus neoformans to yield high-frequency, integrative events was developed. Adenine auxotrophs from a clinical isolate of C. neoformans serotype A were complemented by the cryptococcal phosphoribosylaminoimidazole carboxylase gene (ade2) with a biolistic DNA delivery system. Comparison of two DNA delivery systems (electroporation versus a biolistic system) showed notable differences. The biolistic system did not require linear vectors and transformed each auxotrophic strain at similar frequencies. Examination of randomly selected transformants by biolistics showed that 15 to 40% were stable, depending on the recipient auxotroph, with integrative events identified in all stable transformants by DNA analysis. Although the ade2 cDNA copy transformed at a low frequency, DNA analysis found homologous recombination in each of these transformants. DNA analysis of stable transformants receiving genomic ade2 revealed ectopic integration in a majority of cases, but approximately a quarter of the transformants showed homologous recombination with vector integration or gene replacement. This system has the potential for targeted gene disruption, and its efficiency will also allow for screening of DNA libraries within C. neoformans. Further molecular strategies to study the pathobiology of this pathogenic yeast are now possible with this transformation system.