Differential effects of endothelin peptides in the systemic and pulmonary vascular beds of the cat

The cardiovascular and pulmonary responses to vasoactive intestinal contractor (VIC) were compared with those of endothelin (ET)-1, ET-2, ET-3 and sarafotoxin 6b (S6b) and the mechanism by which ET-1 alters vascular resistance was investigated in the hindquarters vascular bed of the cat. In a manner similar to ET-1 and ET-2, VIC at a dose of 0.3 nmol/kg i.v. produced increases in pulmonary arterial pressure (PAP) and biphasic changes in systemic arterial pressure (AP), systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR). The biphasic changes were characterized by initial decreases followed by increases. In contrast, ET-3 and S6b at doses of 0.3 nmol/kg i.v. produced mainly decreases in AP and SVR, increases in PAP, and biphasic changes in PVR. A monocyclic ET-1 analog and the ET-1 C-terminal hexapeptide fragment produced no effect on AP, SVR, PAP and PVR at doses of 30–100 nmol/kg i.v. ET-1 at a dose of 0.3 nmol i.a. produced a biphasic change in hindquarters perfusion pressure. The initial vasodilation and secondary vasoconstriction were not modified by a variety of blocking agents, whereas the vasoconstrictor response was significantly reduced by infusion of nimodipine, a calcium entry blocking agent. Results of the present study indicate that VIC, a peptide specific to the mouse gastrointestinal tract, elicits cardiovascular responses that are similar to those elicited by ET-1 and ET-2. The present results indicate that responses to these novel peptides are complex and while the mechanism of action remains uncertain, these data indicate that structural differences among the peptides confer differences in biological activity.     

Immunogenicity of a non-class I MHC expressing murine tumor transfected with the influenza virus hemagglutinin or murine interleukin-2 genes

Summary The transfection of murine SP1 tumor cells with the hemagglutinin (HA) gene of influenza virus results, after fluorescent-activated cell sorting (FACS), in the selection of high-HA-expressing cell lines called H4A and H4B. Both lines fail to grow in syngeneic animals at doses that result in 100% tumor take of non-transfected tumor cells. Both grow in immunosuppressed mice. SP1 and H4A or H4B cells express few class I major histocompatibility complex (MHC) antigens but do express class II IA^k antigens. H4A or H4B cells engender a cytotoxic T lymphocyte (CTL) response but cannot protect against a challenge with SP1 cells. This CTL response is inhibited by anti-CD4 but not anti-CD8 antibodies. Using FACS, we were able to select a population (called H5AK5) with high class-I MHC antigen expression. Like H4A and H4B, H5AK5 cells fail to grow in syngeneic animals but do grow in immunosuppressed mice. However, unlike H4A or H4B, H5AK5 can induce protection against a challenge with 1 × 10⁵ SP1 cells. These studies indicate that the immunogenicity ofHA-transfected SP1 cells may correlate with the cell-surface expression of class II MHC antigens. However, HA-expressing SP1 cells seem able to induce a protective response against a parent SP1 cell challenge only if they also express class I MHC antigens. This view is supported by the observations that SP1 cells expressing murine interleukin-2 do not express class I MHC antigens, fail to grow in syngeneic animals, do grow in immunosuppressed mice but do not protect against a challenge with parental SP1 cells.This work was supported by The Clayton Fund, The Sid W. Richardson Foundation and PHS grants CA 39853 and 41525. Toshiyuki Itaya is a visiting scientist supported by the Smith Education Fund of the Department of Cell Biology. Troy Fiesinger is a summer research investigator sponsored by The University of Texas M. D. Anderson Cancer Center Summer Program for College Students     

Searching behaviour and cereal aphid consumption by Bembidion lampros and Pterostichus cupreus, in relation to temperature and prey density

Maximum consumption rates were determined for two carabids, Bembidion lampros Herbst. and Pterostichus cupreus L., feeding on the cereal aphid Rhopalosiphum padi L. at different temperatures in the laboratory. Mean daily consumption increased with increasing temperature for both species, B. lampros consuming a maximum of 16 1–3 instar nymphs and 9 apterous adult aphids at 25°C. P. cupreus was particularly voracious and consumed 125 apterous adult R. padi per day at 20°C. The behaviour of both species was analysed by video filming starved beetles, maintained at different constant temperatures, in arenas sown with spring barley. The behavioural components (1) still; (2) run/walk; (3) search and (4) confrontation were identified and were common to both species. P. cupreus was more active over the temperature range tested; B. lampros was inactive under 10°C. The proportion of time spent searching, number of plants searched, and velocity increased with increasing temperature for both species. When observed in similar arenas seeded with R. padi colonies, individuals of P. cupreus significantly increased their time spent searching in arenas with increasing aphid density. Following discovery of an aphid colony, individuals climbed and searched the host plant and its nearest neighbours. Plants in aphid free arenas were rarely climbed. B. lampros was not observed climbing in either aphid free arenas or in arenas with increasing aphid densities, and did not significantly increase its time spent searching in response to increased prey density. The few B. lampros that found aphids caught them walking on the soil surface. The relative efficiences of these two carabids as predators of R. padi are discussed, and the results are compared with similar studies elsewhere with predators of Sitobion avenae on winter wheat.

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Résumé Les taux maximum de consommation de R. padi L. à différentes températures ont été déterminés au laboratoire chez deux carabes, B. lampros Herbstet P. cupreus L. La consommation moyenne a augmenté avec la température chez les deux espèces, B. lampros consommant un maximum de 15,9 larves des stades 1 à 3 et 9,1 pucerons adultes aptères, à 25°C. P. cupreus a été particulièrement vorace et a consommé 125,3 adultes aptères par jour à 20°C. Le comportement des deux espèces a été observé en filmant en vidéo des carabes à jeun, maintenus à différentes températures constantes, dans des enceintes semées en orge de printemps. Des éléments du comportement, communs aux deux espèces, ont été définis: 1) immabilité, 2) marche et course, 3) recherche, 4) affrontement. P. cupreus a été plus actif à toutes les températures, B. lamprosa été inactif au-dessous de 10°C. La part de temps consacrée à la recherche, le nombre de plantes prospectées, et la vitesse ont augmenté avec la température chez les deux espèces. Dans des enceintes similaires colonisées par R. padi, P. cupreus a significativement augmenté le temps consacré à la recherche dans les enceintes, parallèlement à l'augmentation de la densité des pucerons. Après la découverte d'une colonie de pucerons, P. cupreus escalade et prospecte la plante et ses voisines immédiates; tandis que les plantes des enceintes sans pucerons sont rarement escaladées. B. lampros n'a pas été observé escaladant des plantes d'enceintes avec ou sans pucerons, et il n'a pas accru son temps de prospection en fonction de la densité de pucerons. Les quelques B. lampros qui ont capturé des pucerons l'ont fait lorsque ceux-ci marchaient sur la surface du sol. La discussion a porté sur l'efficacité relative des deux carabes comme prédateurs de R. padi, et les résultats ont été comparés à ceux d'études du même type, menées ailleurs, avec des prédateurs de Sitobion avenae sur blé d'hiver.

     

Spectroscopic studies on cadmium (II)- and cobalt(II)-substituted metallothionein from the crab Cancer pagurus. Evidence for one additional low-affinity metal-binding site

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal-free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)-MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6-MT by paramagnetic Co(II) ions enabled the geometry of the metal-binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6-MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal-binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal-thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two-step process. Furthermore, the spectroscopic features in both Cd(II)- and Co(II)-metal derivatives above the full metal occupancy of six suggest the existence of one additional metal-binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7-MT in the gel filtration studies demonstrate the low metal-binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal-thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.     

Kinetic evidence for a reversible isomerization of pig muscle glyceraldehyde-3-phosphate dehydrogenase in its crystallization medium

Ammonium sulfate, a typical component of crystallization media of proteins, stabilizes an inactive conformation of pig muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, in the presence of ammonium sulfate the reconstitution of the catalytically active holoenzyme from the apoenzyme and NAD is not instantaneous, as in the case of enzymes from Bacillus stearothermophilus and the Mediterranean lobster Palinurus vulgaris. With pig muscle enzyme, at pH 6.0, the time course of formation of the characteristic Racker band can be monitored by a rapid mixing stopped flow technique. Activation follows a single exponential curve with a rate constant independent of the concentration of both NAD and protein and, therefore, appears to be limited by a slow protein isomerization (k = 7 +/- 2 s-1). Accordingly, when the apoenzyme is simultaneously exposed to NAD and either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate, the ensuing reactions (the redox and the acylation steps, respectively) are kinetically limited by the same protein isomerization. At pH 7.0 and 8.0, however, two among the four active sites react with NAD at an unmeasurably high rate, while the other two sites behave as they do at pH 6.0. When the pig muscle apoenzyme is preincubated and allowed to react with either glyceraldehyde 3-phosphate or 1,3-bisphosphoglycerate before the rapid mixing with NAD, both the redox reaction and the NAD-dependent activation of apo-acyl-enzyme toward arsenolysis become unmeasurably fast. Similarly, when the sulfate in the medium is replaced by ions such as phosphate and citrate, the reconstitution of the active holoenzyme is practically instantaneous. Thus, the slow protein isomerization observed in the presence of sulfate and abolished by competing substrates and anions is diagnostic of a structural state of the pig muscle apoenzyme, which is induced by sulfate ions bound within the enzyme active site.     

An endonuclease fromCaenorhabditis elegans: Partial purification and characterization

A deoxyribonuclease was partially purified from the free-living nematodeCaenorhabditis elegans. The DNase functioned as an endonuclease and introduced both single-strand nicks and double-strand breaks into DNA. The enzyme hydrolyzed double-stranded DNA seven times more rapidly than single-stranded DNA. DNase activity was not affected by the addition of divalent cations below 1mm but was inhibited at higher ionic concentrations. In addition, the enzyme was not inhibited in the presence of 10mm EDTA. The enzyme was inhibited by salt concentrations greater than 20mm. Three independent mutations in thenuc-1 gene were shown to reduce nuclease activity to less than 1% of that seen in wild-type organisms. This work was supported by National Institutes of Health Grant AG03161 and a TCU Research Foundation Grant. Some stocks used in these experiments were obtained from theCaenorhabditis Genetics Center, which is supported by Contract NOI-AG-9-2113 between the NIH and the curators of the University of Missouri.     

Herbal medicine among the miskito of Eastern Nicaragua

Medicinal plants identified by Miskito informants in Awastara, Nicaragua, were collected in the field. They are listed and botanically identified in this paper. Particularly interesting among the collection of 23 plant species are those used to cure snakebite and athlete’s foot, as observed in the field.     

The phosphate group of 3-phosphoglycerate accounts for conformational changes occurring on binding to 3-phosphoglycerate kinase. Enzyme inhibition and thiol reactivity studies

Steady-state kinetic study of the inhibition of 3-phosphoglycerate kinase reaction by the substrate analogues D-glycerol 3-phosphate, 2-phosphoglycolate, tartronate and malonate revealed competition with respect to 3-phosphoglycerate. D-Glycerate had no detectable inhibitory effect. The data indicate that (a) the phosphate of 3-phosphoglycerate plays an essential role in the formation of its complex with the enzyme and, taking into account the relatively strong binding of 3-phosphoglycerate, (b) the two charged groups of the substrate might cause a synergic interaction with the protein. The carboxyl-lacking D-glycerol 3-phosphate is a non-competitive inhibitor with respect to MgATP, while all the investigated carboxyl-containing inhibitors compete for MgATP binding. The inhibitory analogues of 3-phosphoglycerate reduce the reactivity of both the two fast-reacting and the five slow-reacting thiol groups of the enzyme molecule. In the case of the fast-reacting thiols the effect is specifically associated with the presence of a ligand's phosphate group. Similarly mainly the phosphate-containing nucleotides and analogues slow down significantly the reaction rate of the fast-reacting thiols, while adenosine is less effective and the competitive inhibitor adenine has no effect at all. MgADP has an especially dramatic effect as compared to MgATP, in line with the known X-ray structural data. The fast-reacting thiols are of particular interest, since their reactivity is possibly controlled by ligand-induced conformational changes. This is shown by the similar ligand protection against alkylation irrespective of the reagent's electrostatic charge (iodoacetamide or iodoacetate) and also by the similar substrate-binding properties of carboxamidomethylated and the unmodified enzyme.     

Molecular Genetic Characterization of a Locus That Contains Duplicate Adh Genes in DROSOPHILA MOJAVENSIS and Related Species

Drosophila mojavensis and other species of the mulleri subgroup contain a duplicate gene encoding the enzyme alcohol dehydrogenase (ADH). Studies on the genetic relationship of the two genes using electrophoretic variants show them to be closely linked. We have cloned a 13.5-kb fragment of D. mojavensis DNA into the lambda vector, Charon 30. This fragment contains both Adh genes separated by approximately 2 kb of DNA. The clone hybridized to a single position on chromosome 3 in D. mojavensis following in situ hybridization. It is likely that the genes are tandemly arranged in the genome. One of the two genes shows a complexity in its structure that suggests the close linkage of a pseudogene or part of a gene. The structure of the Adh locus in five species of the mulleri subgroup have been compared by constructing restriction maps of genomic DNA. Two of these species D. arizonensis and D. mojavensis express Adh-1 in the ovaries; the others do not. In comparing these species it is evident that there has been one or two insertions into the region between the Adh genes. It is possible that one of these structural changes is related to the change in Adh tissue-specific expression that has occurred during the evolution of these species.