Structure-activity studies of modified citrus limonoids as antifeedants for Colorado potato beetle larvae, Leptinotarsa decemlineata

Limonin and ten structurally modified limonins were evaluated as antifeedants against 4th instar larvae of the Colorado potato beetle, Leptinotarsa decemlineata Say, in no-choice leaf dics assays. The epoxide and furan groups were shown to be essential structural requirements for high antifeedant activity.

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Résumé Le pouvoir phagodissuasif de la limonine et de 10 limonines dont la structure avait été modifiée a été évalué sur des disques de feuilles de Solanum tuberosum offerts à des larves du 4ème stade de L. decemlineata. Aucune activité dissuasive n'ayant été observée avec le tétrahydrolimonine ou le déoxylimonine, l'anneau furane et le groupe époxy sont donc nécessaires pour obtenir cette activité. Ni la réduction de la fonction 7-kéto, ni la rupture de l'anneau A de la limonine n'ont eu d'effêt sur cette activité. La réduction des fonctions 16-carbonyl et 7-kéto de la limonine ont diminué l'activité, mettant en évidence une action possible de l'anneau D-lactone dans l'activité phagodissuasive. Tandis que la déépoxydation de la limonine entraîne une perte d'activité totale, une restauration partielle de cette activité a pu être obtenu par une réduction ultérieure de la fonction 7-kéto.

     

Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors

We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc     

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. We have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [3H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified approximately 100-kDa steroid-binding subunit was eluted from gel slices and subjected to enzymatic digestion. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [3H]dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the [3H]dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with [3H]dexamethasone 21-mesylate and [35S]methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of [3H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, our data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate.(ABSTRACT TRUNCATED AT 250 WORDS)     

A binary vector for transferring genomic libraries to plants.

The transformation of mutant plants with a complete recombinant library derived from wild-type DNA followed by assay of transformed plants for complementation of the mutant phenotype is a promising method for the isolation of plant genes. The small genome of Arabidopsis thaliana is a good candidate for attempting this so-called shotgun transformation. We present the properties of an A. thaliana genomic library cloned in a binary vector, pC22. This vector, designed to introduce genomic libraries into plants, contains the oriV of the Ri plasmid pRiHR1 by which it replicates perfectly stably in Agrobacterium. Upon transfer of the library from E. coli to A. tumefaciens large differences in transfer efficiencies of individual recombinant clones were observed. There is a direct relation between transfer efficiency and stability of the recombinant clones both in E. coli and A. tumefaciens. The stability is independent of the insert size, but seems to be related to the nature of the insert DNA. The feasibility of shotgun transformation and problems of statistical sampling are discussed.     

Degradation without apparent change in size of molybdate-stabilized nonactivated glucocorticoid-receptor complexes in rat thymus cytosol

We have investigated the stability of the [3H]dexamethasone 21-mesylate-labeled nonactivated glucocorticoid-receptor complex in rat thymus cytosol containing 20 mM sodium molybdate. Cytosol complexes were analyzed under nondenaturing conditions by gel filtration chromatography in the presence of molybdate and under denaturing conditions by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. When analyzed under nondenaturing conditions, complexes from fresh cytosol and from cytosol left for 2 h at 3 degrees C eluted from gel filtration as a single peak of radioactivity with a Stokes radius of approximately 7.7 nm, suggesting that no proteolysis of the complexes had occurred in either cytosol. When analyzed under denaturing conditions, however, whereas the fresh cytosol gave a receptor band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr approximately 90,000 (corresponding to the intact complex), the cytosol that had been left for 2 h at 3 degrees C gave only a fragment (Mr approximately 50,000). This fragment, just as the intact complex, could be thermally activated to a DNA-binding form. Proteolysis of the receptor could be blocked by preparing the cytosol in the presence of EGTA, leupeptin, or a heat-stable factor present in the cytosol of rat liver and WEHI-7 mouse thymoma cells. From these results we conclude: (i) 20 mM molybdate does not protect the nonactivated glucocorticoid-receptor complex present in rat thymus cytosol against proteolysis under conditions which are commonly used for cell-free labeling of the receptor, and (ii) the demonstration of a Stokes radius of approximately 8 nm for the nonactivated glucocorticoid-receptor complex is not sufficient to indicate that the receptor complex is present in its intact form.     

Protein reactions with methyl and ethyl vinyl sulfones

Disulfide bonds of bovine serum albumin and wool were reduced byn-tributylphosphine to sulfhydryl groups that were then modified by methyl or ethyl vinyl sulfone in a nucleophilic addition reaction toS-(-ethylsulfonylmethyl)-l-cysteine andS(-ethylsulfonylethyl)-l-cysteine, respectively. The reductive alkylation was carried out either simultaneously, with both the reducing and alkylating agents present in the reaction mixture, or sequentially, with the reduced proteins first isolated before alkylation. Amino acid analysis studies showed that authentic, syntheticS-(-ethylsulfonylethyl)-l-cysteine eluted as a well-resolved peak after serine but that the peak associated with the corresponding methyl derivative overlapped the corresponding peak due to threonine. The extent of alkylation of the sulfhydryl groups of cysteine, -NH₂ of lysine, and NH groups of the imidazole ring of histidine was also measured by amino acid analysis. The results show that alkyl vinyl sulfones have a strong chemical affinity for protein functional groups.     

A slow wave frequency complex of the canine small intestine during the fasting state

The electrical activity of the duodenum and proximal jejunum was studied in conscious healthy dogs implanted with unipolar silver electrodes. A computerized method was used for the calculation of the mean frequency of the slow wave for each consecutive minute of the electromyographic signal. A "slow wave frequency complex" was identified in the fasted animals. It was characterized by an increase of the mean frequency of the slow wave which ranged, from one dog to another, between 1 and 3 cycles/min. The complex lasted about 30 min. It consisted of two distinct phases: a phase of increasing frequency of the slow wave which lasted about one-third of the total duration of the complex and a phase of progressive return of the frequency to its precomplex value. Each phase III of the migrating myoelectric complex occurring in both the duodenum and the jejunum was associated with one slow wave frequency complex. The phase III began a few minutes before the start of the slow wave frequency complex and ended a few minutes before the slow wave frequency reached its maximum. Ectopic phase IIIs which occurred in the jejunum but not in the duodenum were not associated with slow wave frequency complexes. The slow wave frequency complex was never seen in the fed dogs.     

Antibody Response and Protection Induced by Immunization with Smooth and Rough Strains in Experimental Salmonellosis

The antibody response of mice to a smooth strain of Salmonella typhimurium was shown previously to be extremely rapid and potent. As measured by the complement-mediated bactericidal reaction, it was also found to be highly specific as well as reproducible. Experiments which studied the effects of antigen type (live or heat-killed), antigen dose, and the route of immunization indicated that the most rapid and highest antibody response was achieved with live, smooth organisms injected by the intraperitoneal route. Living vaccines of rough strains of either S. typhimurium or S. enteritidis induced antibodies directed against the corresponding smooth organisms. The response to the rough strains was apparently due to antibody production rather than to the simple release of preformed natural antibody. The duration of protection conferred by the rough strain vaccines was closely correlated with the endotoxic content of the immunizing strain. Smooth heat-killed vaccines and a rough live vaccine protected against homologous but not heterologous challenge. In contrast, immunization with a smooth live vaccine protected mice against both homologous and heterologous challenge infections. Protection was not due to a local effect in the peritoneal cavity, since mice were also protected against subcutaneous challenge. The secondary antibody response, induced in immunized animals by the virulent challenge infection, was demonstrated to be rapid and potent, and hence a factor to be considered in protection.     

Tricuspid Valve Replacement in Rheumatic Heart Disease

The diagnosis of tricuspid valve disease is often difficult; the best treatment is not yet established. Twenty patients had tricuspid valve replacements at St. Thomas''s Hospital as part of multiple valve replacement procedures. The hospital mortality was 25%, most deaths being due to a low cardiac output causing hepatorenal failure. Preoperative cardiac cachexia had a fatal outcome in all cases. Except in two instances, surviving patients returned to a satisfactory level of activity.     

Preferential synthesis of actin during early development of the slime mold Dictyostelium discoideum

The changes in protein synthesis during differentiation of the cellular slime mold Dictyostelium were studied by SDS-polyacrylamide gel electrophoresis. Total cell protein was analyzed following a 2-hr pulse-label. It was found that during the preaggregation stage, comprising the first third of the developmental cycle, a single major band accounts for more than 20% of the total labeled protein on the gel. This species was produced in at least 5–10-fold lower amounts, relative to total cell protein synthesis, in vegetative cells and in later developing stages. Actin was purified from vegetative cells and was found to correspond to the major band in several respects. The discovery of a single protein being synthesized in such quantity at a specific developmental stage provides a powerful tool for the isolation of a specific messenger RNA molecule and for an intensive study of all the factors involved in regulating protein synthesis in a eukaryotic organism.