Identification of bacterial plant pathogens using multilocus polymerase chain reaction/electrospray ionization-mass spectrometry.

Polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as "TIGER") utilizes PCR with broad-range primers to amplify products from a wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses allows for calculations of base compositions for the broad-range PCR products, which can then be compared to a database for identification. PCR/ESI-MS has the benefits of PCR in sensitivity and high-throughput capacity, but also has the distinct advantage of being able to detect and identify organisms with no prior characterization or sequence data. Existing broad range PCR primers, designed with an emphasis on human pathogens, were tested for their ability to amplify DNA of well characterized phytobacterial strains, as well as to populate the existing PCR/ESI-MS bacterial database with base counts. In a blinded panel study, PCR/ESI-MS successfully identified 93% of unknown bacterial DNAs to the genus level and 73% to the species/subspecies level. Additionally, PCR/ESI-MS was capable of detecting and identifying multiple bacteria within the same sample. The sensitivity of PCR/ESI-MS was consistent with other PCR based assays, and the specificity varied depending on the bacterial species. Preliminary tests with real life samples demonstrate a high potential for using PCR/ESI-MS systems for agricultural diagnostic applications.

Carboxypeptidase G2 rescue in patients with methotrexate intoxication and renal failure.

The methotrexate (MTX) rescue agent carboxypeptidase G2 (CPDG2) rapidly hydrolyses MTX to the inactive metabolite DAMPA (4-[[2,4-diamino-6-(pteridinyl)methyl]-methylamino]-benzoic acid) and glutamate in patients with MTX-induced renal failure and delayed MTX excretion. DAMPA is thought to be an inactive metabolite of MTX because it is not an effective inhibitor of the MTX target enzyme dihydrofolate reductase. DAMPA is eliminated more rapidly than MTX in these patients, which suggests a nonrenal route of elimination. In a phase II study (May 1997-March 2002), CPDG2 was administered intravenously to 82 patients at a median dose of 50 U kg(-1) (range 33-60 U kg(-1)). Eligible patients for this study had serum MTX concentrations of >10 microM at 36 h or >5 microM at 42 h after start of MTX infusion and documented renal failure (serum creatinine > or =1.5 times the upper limit of normal). Immediately before CPDG2 administration, a median MTX serum level of 11.93 microM (range 0.52-901 microM) was documented. Carboxypeptidase G2 was given at a median of 52 h (range 25-178 h) following the start of an MTX infusion of 1-12 g m(-2) 4-36 h(-1) and resulted in a rapid 97% (range 73-99%) reduction of the MTX serum level. Toxicity related to CPDG2 was not observed. Toxicity related to MTX was documented in about half the patients; four patients died despite CPDG2 administration due to severe myelosuppression and septic complications. In conclusion, administration of CPDG2 is a well-tolerated, safe and a very effective way of MTX elimination in delayed excretion due to renal failure.

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.

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.

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.

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.

Factors influencing condom use and STD acquisition among African American college women.

To identify factors that influence condom use and the risk of acquiring sexually transmitted diseases (STDs) among African American college women, the authors surveyed a sample of 123 sexually experienced undergraduate women at a state university. The college women who were older, had initiated sex earlier, or had more recent sexual partners were more likely than others in the study to report a history of an STD. The findings have implications for prevention and intervention programs for African American college women.

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.

Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy.

A novel prodrug activation system, endogenous in human tumor cells, is described. A latent enzyme-prodrug system is switched on by a simple synthetic, small molecule co-substrate. This ternary system is inactive if any one of the components is absent. CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is an antitumor prodrug that is activated in certain rat tumors via its 4-hydroxylamine derivative to a potent bifunctional alkylating agent. However, human tumor cells are resistant to CB 1954 because they are unable to catalyze this bioactivation efficiently. A human enzyme has been discovered that can activate CB 1954, and it has been shown to be commonly present in human tumor cells. The enzyme is NQO2 [NAD(P)H quinone oxidoreductase 2], but its activity is normally latent, and a nonbiogenic co-substrate such as NRH [nicotinamide riboside (reduced)] is required for enzymatic activity. There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH. Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as cosubstrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically accessible and suitable for further pharmaceutical development. NQO2 activity appears to be related to expression of NQO1 (DT-diaphorase), an enzyme that is known to have a favorable distribution toward certain human cancers. NQO2 is a novel target for prodrug therapy and has a unique activation mechanism that relies on a synthetic co-substrate to activate an apparently latent enzyme. Our findings may reopen the use of CB 1954 for the direct therapy of human malignant disease.

The alpha4 isoform of the Na,K-ATPase is expressed in the germ cells of the testes.

In addition to the three isoforms of the catalytic subunit of the Na, K-ATPase originally identified (alpha1, alpha2, and alpha3), a fourth alpha polypeptide (alpha4) has recently been found in mammalian cells. This novel alpha-subunit of the Na,K-ATPase is selectively expressed in male gonadal tissues. In the testes, alpha4 is functionally active and comprises approximately half of the Na, K-ATPase activity of the organ. At present, the pattern of expression of the alpha4 polypeptide within the cells of the male gonad is unknown. By in situ hybridization, immunocytochemistry, and the ouabain inhibition profile of Na,K-ATPase activity, we show that the alpha4-subunit is expressed in the germ cells of rat testes. The highest amounts of the isoform are found in spermatozoa, where it constitutes two thirds of the Na,K-ATPase activity of the gametes. The other Na pump present in the cells is the ubiquitously expressed alpha1 polypeptide. The characteristic localization of alpha4 in the gonad is further supported by the drastic reduction of the polypeptide in mice that are infertile as a consequence of arrest in maturation of the germ cells. In addition, GC-1spg cells, a murine cell line derived from testis spermatogonia, also contain the Na, K-ATPase alpha4 polypeptide. However, the level of expression of the isoform in these cells is much lower than in the spermatozoa, a fact that may depend on the limited ability of the GC-1spg cells to differentiate in vitro. The particular expression of the Na,K-ATPase alpha4 isoform we encounter and the specific enzymatic properties of the polypeptide suggests its importance for ionic homeostasis of the germ cells of the testes.

A health department perspective on the Guide to Community Preventive Services.

PIP: This article discusses some of the public health practice needs that would be met by the Guide to Clinical Preventive Services (GCPS) and highlights some of the challenges that lie ahead in using the recommendations to actually improve the health of the population and communities. Overall, the discussions indicate that the Guide would be useful across public health settings from academia to health departments. It is noted that it would be the first comprehensive, evidence-based evaluation of community preventive services. In addition, the focus of the Guide on economic evaluation would prove to be extremely helpful to decision-makers in both public health and managed care settings in which fixed (or shrinking) budgets have become the norm. However, in terms of challenges, the implementation of the Guide is seen as the most difficult process, despite its clear and compelling recommendations. A number of initiatives are being developed and carried out that would support the dissemination and implementation of the Guide.^ieng

Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.

Developments with targeted enzymes in cancer therapy.

Cancer therapy based on the delivery of enzymes to tumour sites has advanced in several directions since antibody-directed enzyme/prodrug therapy was first described. It has been shown that methoxypolyethylene glycol (MPEG) can be used to deliver enzyme to a variety of solid tumours. MPEG-enzyme conjugates show reduced immunogenicity and may allow repeated treatment with enzymes of bacterial origin. Enzyme delivery to tumours by polymers can be used to convert a low toxicity prodrug to a potent cytotoxic agent. An example of such a prodrug is CB1954, which can be activated by a human enzyme in the presence of a cosubstrate. Tumour-located enzymes can also be used in conjunction with a combination of antimetabolites and rescue agents. The rescue agent protects normal tissue but is degraded at cancer sites by the enzyme, thus deprotecting the tumour and allowing prolonged antimetabolite action.

Compromised disease resistance in saponin-deficient plants.

Saponins are glycosylated plant secondary metabolites found in many major food crops [Price, K. R., Johnson, I. T. & Fenwick, G. R. (1987) CRC Crit. Rev. Food Sci. Nutr. 26, 27-133]. Because many saponins have potent antifungal properties and are present in healthy plants in high concentrations, these molecules may act as preformed chemical barriers to fungal attack. The isolation of plant mutants defective in saponin biosynthesis represents a powerful strategy for evaluating the importance of these compounds in plant defense. The oat root saponin avenacin A-1 fluoresces under ultraviolet illumination [Crombie, L., Crombie, W. M. L. & Whiting, D. A. (1986) J. Chem. Soc. Perkins 1, 1917-1922], a property that is extremely rare among saponins. Here we have exploited this fluorescence to isolate saponin-deficient (sad) mutants of a diploid oat species, Avena strigosa. These sad mutants are compromised in their resistance to a variety of fungal pathogens, and a number of lines of evidence suggest that this compromised disease resistance is a direct consequence of saponin deficiency. Because saponins are widespread throughout the plant kingdom, this group of secondary metabolites may have general significance as antimicrobial phytoprotectants.

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.

Targeting enzymes to cancers--new developments.

Two methods of using tumour located enzymes have been described. These are antibody directed enzyme prodrug therapy (ADEPT) and macromolecule directed enzyme prodrug therapy (MDEPT), where the tumour located enzyme converts a non-toxic prodrug into a cytotoxic drug at tumour sites. The alternative use of tumour located enzymes is to inactivate rescue agents that protect cells from antimetabolite action, and is described as 'Antimetabolite with inactivation of rescue agent at cancer sites' (AMIRACS). The leakiness of tumour blood vessels and poor lymphatic drainage allows enzymes to be targeted to many cancers by attachment to polymeric macromolecules (MDEPT), as well as to antibodies and antibody fragments (ADEPT). To avoid systemic toxicity, enzyme activity in blood and normal tissues must be very low before giving a prodrug or rescue agent. Antibodies directed against the enzyme component of macromolecular conjugates have proved to be very efficient at clearing normal tissues. Human enzymes which are over expressed by cancer cells can be exploited particularly if they require co-factors or co-substrates, either in situ or targeted to extracellular sites. Bacterial enzymes have advantages in specificity but require some form of immunological control in view of their immunogenicity. Prodrugs which generate drugs with very short half lives are desirable, and have been developed, including one which has a differential toxicity between prodrug and the active drug of 1000 to 10,000 fold. The range of antimetabolites available for AMIRACS was initially restricted to inhibitors of dihydrofolate reductase but has been greatly extended by the introduction of inhibitors of other enzymes. The limitations of these systems are discussed.

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.

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.

Heterologous expression of Septoria lycopersici tomatinase in Cladosporium fulvum: effects on compatible and incompatible interactions with tomato seedlings.

The anti-fungal, steroidal, glycoalkaloid saponin, alpha-tomatine, is present in uninfected tomato plants in substantial concentrations, and may contribute to the protection of tomato plants against attack by phytopathogenic fungi. In general, successful fungal pathogens of tomato are more resistant to alpha-tomatine in vitro than fungi that do not infect this plant. For a number of tomato pathogens, this resistance has been associated with the ability to detoxify alpha-tomatine through the action of enzymes known as tomatinases. In contrast, the biotrophic tomato pathogen Cladosporium fulvum is sensitive to alpha-tomatine and is unable to detoxify this saponin. This paper describes the effects of heterologous expression of the cDNA encoding tomatinase from the necrotroph Septoria lycopersici in two different physiological races of C. fulvum. Tomatinase-producing C. fulvum transformants showed increased sporulation on cotyledons of susceptible tomato lines. They also caused more extensive infection of seedlings of resistant tomato lines. Thus, alpha-tomatine may contribute to the ability of tomato to restrict the growth of C. fulvum in both compatible and incompatible interactions.