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R Melton

Publications and source records attributed to R Melton.

At least 19 recordsLinked to original sources
PubMed2004

A gene cluster for secondary metabolism in oat: implications for the evolution of metabolic diversity in plants.

The evolution of the ability to synthesize specialized metabolites is likely to have been key for survival and diversification of different plant species. Oats (Avena spp.) produce antimicrobial triterpenoids (avenacins) that protect against disease. The oat beta-amyrin synthase gene AsbAS1, which encodes the first committed enzyme in the avenacin biosynthetic pathway, is clearly distinct from other plant beta-amyrin synthases. Here we show that AsbAS1 has arisen by duplication and divergence of a cycloartenol synthase-like gene, and that its properties have been refined since the divergence of oats and wheat. Strikingly, we have also found that AsbAS1 is clustered with other genes required for distinct steps in avenacin biosynthesis in a region of the genome that is not conserved in other cereals. Because the components of this gene cluster are required for at least four clearly distinct enzymatic processes (2,3-oxidosqualene cyclization, beta-amyrin oxidation, glycosylation, and acylation), it is unlikely that the cluster has arisen as a consequence of duplication of a common ancestor. Although clusters of paralogous genes are common in plants (e.g., gene clusters for rRNA and specific disease resistance), reports of clusters of genes that do not share sequence relatedness and whose products contribute to a single selectable function are rare [Gierl, A. & Frey, M. (2001) Planta 213, 493-498]. Taken together, our evidence has important implications for the generation of metabolic diversity in plants.

Acylation↗
PubMed2002

A saponin-detoxifying enzyme mediates suppression of plant defences.

Plant disease resistance can be conferred by constitutive features such as structural barriers or preformed antimicrobial secondary metabolites. Additional defence mechanisms are activated in response to pathogen attack and include localized cell death (the hypersensitive response). Pathogens use different strategies to counter constitutive and induced plant defences, including degradation of preformed antimicrobial compounds and the production of molecules that suppress induced plant defences. Here we present evidence for a two-component process in which a fungal pathogen subverts the preformed antimicrobial compounds of its host and uses them to interfere with induced defence responses. Antimicrobial saponins are first hydrolysed by a fungal saponin-detoxifying enzyme. The degradation product of this hydrolysis then suppresses induced defence responses by interfering with fundamental signal transduction processes leading to disease resistance.

Anti-Infective Agents↗
PubMed2001

A new class of oxidosqualene cyclases directs synthesis of antimicrobial phytoprotectants in monocots.

Many plants synthesize antimicrobial secondary metabolites as part of their normal program of growth and development, often sequestering them in tissues where they may protect against microbial attack. These include glycosylated triterpenoids (saponins), natural products that are exploited by man for a variety of purposes including use as drugs [Hostettmann, K. & Marston, A. (1995) Saponins (Cambridge Univ. Press, Cambridge, U.K.)]. Very little is known about the genes required for the synthesis of this important family of secondary metabolites in plants. Here we show the novel oxidosqualene cyclase AsbAS1 catalyzes the first committed step in the synthesis of antifungal triterpenoid saponins that accumulate in oat roots. We also demonstrate that two sodium azide-generated saponin-deficient mutants of oat, which define the Sad1 genetic complementation group, are defective in the gene encoding this enzyme and provide molecular genetic evidence indicating a direct link between AsbAS1, triterpenoid saponin biosynthesis, and disease resistance. Orthologs of AsbAS1 are absent from modern cereals and may have been lost during selection, raising the possibility that this gene could be exploited to enhance disease resistance in crop plants.

Amino Acid Sequence↗
PubMed2000

Solution structure of the catalytic domain of human collagenase-3 (MMP-13) complexed to a potent non-peptidic sulfonamide inhibitor: binding comparison with stromelysin-1 and collagenase-1.

The full three-dimensional structure of the catalytic domain of human collagenase-3 (MMP-13) complexed to a potent, sulfonamide hydroxamic acid inhibitor (CGS 27023) has been determined by NMR spectroscopy. The results reveal a core domain for the protein consisting of three alpha-helices and five beta-sheet strands with an overall tertiary fold similar to the catalytic domains of other matrix metalloproteinase family members. The S1' pocket, which is the major site of hydrophobic binding interaction, was found to be a wide cleft spanning the length of the protein and presenting facile opportunity for inhibitor extension deep into the pocket. Comparison with the reported X-ray structure of collagenase-3 showed evidence of flexibility for the loop region flanking the S1' pocket in both NMR and X-ray data. This flexibility was corroborated by NMR dynamics studies. Inhibitor binding placed the methoxy phenyl ring in the S1' pocket with the remainder of the molecule primarily solvent-exposed. The binding mode for this inhibitor was found to be similar with respect to stromelysin-1 and collagenase-1; however, subtle comparative differences in the interactions between inhibitor and enzyme were observed for the three MMPs that were consistent with their respective binding potencies.

Amino Acid Sequence↗
PubMed2000

Effects of targeted replacement of the tomatinase gene on the interaction of Septoria lycopersici with tomato plants.

Many plants produce constitutive antifungal molecules belonging to the saponin family of secondary metabolites, which have been implicated in plant defense. Successful pathogens of these plants must presumably have some means of combating the chemical defenses of their hosts. In the oat root pathogen Gaeumannomyces graminis, the saponin-detoxifying enzyme avenacinase has been shown to be essential for pathogenicity. A number of other phytopathogenic fungi also produce saponin-degrading enzymes, although the significance of these for saponin resistance and pathogenicity has not yet been established. The tomato leaf spot pathogen Septoria lycopersici secretes the enzyme tomatinase, which degrades the tomato steroidal glycoalkaloid alpha-tomatine. Here we report the isolation and characterization of tomatinase-deficient mutants of S. lycopersici following targeted gene disruption. Tomatinase-minus mutants were more sensitive to alpha-tomatine than the wild-type strain. They could, however, still grow in the presence of 1 mM alpha-tomatine, suggesting that nondegradative mechanisms of tolerance are also important. There were no obvious effects of loss of tomatinase on macroscopic lesion formation on tomato leaves, but trypan blue staining of infected tissue during the early stages of infection revealed more dying mesophyll cells in leaves that had been inoculated with tomatinase-minus mutants. Expression of a defense-related basic beta-1,3 glucanase gene was also enhanced in these leaves. These differences in plant response may be associated with subtle differences in the growth of the wild-type and mutant strains during infection. Alternatively, tomatinase may be involved in suppression of plant defense mechanisms.

Avena↗
PubMed1999

Design and synthesis of thiol containing inhibitors of matrix metalloproteinases.

A series of thiol containing derivatives was prepared. Several of these compounds were found to inhibit matrix metalloproteinases 1, 3, and 9 with selectivity towards 3 and 9. Compounds 15, 20, and 22 were administered to rats orally at 75 mumol/kg. Drug levels of compounds 20 and 22 in the plasma were found to exceed the IC50 values for MMP 3 and 9 four hours after administration.

Animals↗
PubMed1998

Solution structure of the catalytic domain of human stromelysin-1 complexed to a potent, nonpeptidic inhibitor.

The full three-dimensional structure of the catalytic domain of human stromelysin-1 (SCD) complexed to a novel and potent, nonpeptidic inhibitor has been determined by nuclear magnetic resonance spectroscopy (NMR). To accurately mimic assay conditions, the structure was obtained in Tris buffer at pH 6.8 and without the presence of organic solvent. The results showed that the major site of enzyme-inhibitor interaction occurs in the S1' pocket whereas portions of the inhibitor that occupy the shallow S2' and S1 pockets remained primarily solvent exposed. Because this relatively small inhibitor could not deeply penetrate stromelysin's long narrow hydrophobic S1' pocket, the enzyme was found to adopt a dramatic fold in the loop region spanning residues 221-231, allowing occupation of the solvent-accessible S1' channel by the enzyme itself. This remarkable conformational fold at the enzyme binding site resulted in constriction of the S1' loop region about the inhibitor. Examination of the tertiary structure of the stromelysin-inhibitor complex revealed few hydrogen-bonding or hydrophobic interactions between the inhibitor and enzyme that can contribute to overall binding energy; hence the resultant compact structure may in part account for the relatively high potency exhibited by this inhibitor.

Amino Acid Sequence↗
PubMed1998

Inhibition of interleukin-1alpha-induced cartilage oligomeric matrix protein degradation in bovine articular cartilage by matrix metalloproteinase inhibitors: potential role for matrix metalloproteinases in the generation of cartilage oligomeric matrix protein fragments in arthritic synovial fluid.

OBJECTIVE: To determine whether matrix metalloproteinases (MMPs) degrade cartilage oligomeric matrix protein (COMP) to produce fragments similar to those found in synovial fluid (SF) from patients with arthritis. METHODS: COMP fragments were generated in vitro by treating (a) bovine articular cartilage with interleukin-1alpha (IL-1alpha), (b) purified bovine COMP with MMPs, and (c) articular cartilage with MMPs. The fragments generated in each case were analyzed by Western blot, using an antibody to the C-terminal heptadecapeptide of COMP. RESULTS: IL-1alpha stimulation of cartilage resulted in a fragmentation of COMP, which was inhibited by MMP inhibitors CGS 27023A and BB-94. Isolated, recombinant MMPs rapidly degraded purified COMP, as well as COMP residing in cartilage. Several COMP fragments produced in vitro had similar electrophoretic mobility to those in SF of patients with arthritis. CONCLUSION: MMPs may contribute to the COMP fragments found in vivo. Quantitation of MMP-specific fragments may be useful in the evaluation of MMP inhibitors in patients with arthritis.

Animals↗
PubMed1995

Bioactive conformation of stromelysin inhibitors determined by transferred nuclear Overhauser effects.

The transferred nuclear Overhauser effect has been used to determine the biologically active conformations of two stromelysin inhibitors. Both inhibitors used in this study were hydroxamic acids generated via chemical synthesis. These structures, representing the conformation of each inhibitor bound to stromelysin, superimposed with excellent agreement. The study also provided information on the shape and orientation of the S2' and S1' pockets of the enzyme relative to thermolysin. Comparisons were made between stromelysin and thermolysin inhibitors to critically examine thermolysin as a template for stromelysin-inhibitor design. The enzyme-bound conformations of these stromelysin inhibitors were determined for use as a template in conformationally restricted drug design.

Computer Simulation↗
PubMed1995

Taking training seriously: a policy statement on public health training by the Joint Council of Governmental Public Health Agencies.

Continuing education and on-the-job training in public health has suffered serious neglect over the past years. As a result, many state and local public health entities have failed to keep step with emerging challenges facing the public's health--challenges that require new and innovative approaches to prevention and treatment. In this article, the Joint Council of Governmental Public Health Agencies seeks to affirm the importance of public health training by examining barriers to training; identifying areas in which training should be emphasized; providing examples of successful training programs; and recommending ways in which public health leaders can revitalize training programs.

Education, Continuing↗
PubMed1991

Elevation of synovial plasminogen activator activity after injection of interleukin-1 alpha into rabbit knee joint.

We investigated production of plasminogen activator (PA) and cartilage degradation induced by injection of recombinant human interleukin-1 (rhIL-1 alpha) in rabbit knees. Rabbits were injected intra-articularly (i.a.) with 100 ng rhIL-1 alpha and necropsied at 0, 3, 6, 18 and 54 h and synovial lavage and articular cartilage were collected. PA activity in the joint lavage was measured using Z-Lys-thiobenzyl ester as a substrate. Cartilage degradation was assessed by quantitating sulfated glycosaminoglycan (S-GAG) to hydroxyproline (Hyp) and appearance of keratan sulfate (KS) in synovial lavage by and ELISA. The PA activity in the lavage of IL-1 injected knees at 3, 6, and 18 h was elevated 8 to 10 fold compared to vehicle controls. At 54 h the activity declined to approximately one third of that seen at the earlier time points. KS in the joint lavage was highest at 18 h, suggesting proteoglycan degradation. The maximal loss of cartilage proteoglycan (S-GAG/Hyp) occurred by 54 h. These observations demonstrate that i.a. injection of IL-1 stimulated the production of PA activity within the rabbit joint. Since elevation of PA activity is followed by cartilage degradation, we investigated effect of anti-inflammatory agents on PA activity and cartilage degradation in this model. We found that triamcinolone, indomethacin and dexamethasone were able to suppress PA activity but not the cartilage degradation. These observations suggest that in this model of cartilage degradation suppression of PA is not sufficient to inhibit cartilage degradation.

Animals↗
PubMed1984

Structure of an immunodominant epitope of the circumsporozoite surface protein of Plasmodium knowlesi.

Previous studies have shown that the immunodominant region of the circumsporozoite surface (CS) protein of Plasmodium knowlesi is contained within a tandemly repeated dodecapeptide: Gln-Ala-Gln-Gly-Asp-Gly-Ala-Asn-Ala-Gly-Gln-Pro. We show here that the CS protein epitopes reacting with six monoclonal antibodies raised against the intact parasite are represented in a synthetic tandem repeat of this dodecapeptide. The specificity of four of these antibodies was studied further by preparing synthetic peptides corresponding to overlapping regions of the repeats and measuring their ability to inhibit the specific interaction between the antibodies and CS proteins. We find that three antibodies have very similar patterns of reactivity with this series of peptides and that they define an epitope of eight amino acids (Gly-Asp-Gly-Ala-Asn-Ala-Gly-Gln) within the dodecapeptide. The remaining antibody probably recognizes a configurational epitope formed by a tandem repeat of the dodecapeptide.

Animals↗