Invasion thresholds for fungicide resistance: deterministic and stochastic analyses

Fungicide resistance is an important practical problem, but one that is poorly understood at the population level. Here we introduce a simple nonlinear model for fungicide resistance in botanical epidemics which includes the dynamics of the chemical control agent and the host population, while also allowing for demographic stochasticity in the host-parasite dynamics. This provides a mathematical framework for analysing the risk of fungicide resistance developing by including the parameters for the amount applied, longevity and application frequency of the fungicide. The model demonstrates the existence of thresholds for the invasion of the resistant strain in the parasite population which depend on two quantities: the relative fitness of the resistant strain and the effectiveness of control. This threshold marks a change from definite elimination of the resistant strain below the threshold to a finite probability of invasion which increases above the threshold. The fungicide decay rate, the amount of fungicide applied and the period between applications affect the effectiveness of control and, consequently, they influence whether or not resistance develops and the time taken to achieve a critical frequency of resistance. All three parameters are amenable to control by the grower or by coordinating the activity of a population of growers. Providing crude estimates of the effectiveness of control and relative fitness are available, the results can be used to predict the consequences of changing these parameters for the risk of invasion and the proportion of sites at which this might be expected to occur. Although motivated for fungicide resistance, the model has broader application to herbicide, antibiotic and antiviral resistance. The modelling approach and results are discussed in the context of resistance to chemical control in general.     

Purification and characterization of a novel haemagglutinin from Chlorella pyrenoidosa

We previously identified a strong haemagglutination activity in the freshwater unicellular green alga, Chlorella pyrenoidosa. Here, we sought to purify and characterize the haemagglutinin associated with this activity. Ammonium sulfate precipitation, gel filtration on sephacryl S-200 and DEAE-Sepharose ion-exchange chromatography were used to purify the haemagglutinin, which was designated CPH (Chlorella pyrenoidosa haemagglutinin). The molecular weight of CPH was estimated as 58 kDa by SDS-PAGE and 60 kDa by gel filtration of the native protein, indicating that this haemagglutinin exists as a monomer. The haemagglutinin activity of CPH was inhibited by glycoproteins, especially yeast mannan, but not by monosaccharides or disaccharides, indicating that CPH is carbohydrate-specific. In addition to the composition of CPH shown to be rich in glycine and acidic amino acids, heamagglutinating activity of CPH was insensitive to variations in pH or the presence of divalent cations, and atomic force microscopy revealed that the protein is rod-shaped. These results indicate that the characteristics of CPH are consistent with its identification as a haemagglutinin, and suggest that CPH may be a viable candidate for applications in a variety of biomedical fields.     

Enzymatic and Molecular Biochemical Characterizations of Human Neutrophil Elastases and a Cathepsin G-like Enzyme

Human neutrophil elastase (HNE, EC 3. 4. 21. 37) is a causative factor of inflammatory diseases, including emphysema and rheumatoid arthritis. Enzymatic characterization is important for the development of new drugs involved in the regulation of this enzyme. In this study, we investigated the enzymatic and biochemical properties of five different elastolytic enzymes, with a molecular mass between 24 kDa and 72 kDa. Three elastases, molecular masses of 27, 29, 31 kDa, might be elastase isozymes that have the same NH₂-terminal amino acid sequences of Ile-Val-Gly-Gly-Arg-Arg-Ala. The 24-kDa enzyme, which showed the identical NH₂-terminal amino acid sequences to elastase, was a degraded fragment of native elastase. The elastolytic activity was conserved at the 6/7 domain of the NH₂-terminal region. The inhibitory characteristics of PMSF, DipF were the same as those of native elastases. The 72-kDa molecule, which showed elastolytic activity, might be a trimer formed between native elastases (31 kDa and 29 kDa) and a cathepsin G-like enzyme, which did not show elastolytic activity but enhanced the elastolytic activity of neutrophil elastase. Although this cathepsin G-like enzyme showed weak cathepsin G activity, it has distinguishable NH₂-terminal sequences of Ile-Val-Gly-Gly-Ser-Arg-Ala- from those of elastase or cathepsin G. The potentiation of elastolytic activity could be a result of the trimerization of native elastase with a cathepsin G-like enzyme, and was then weakly inhibited by serine protease inhibitors, such as PMSF, DipF. Therefore, we suggest the cathepsin G-like enzyme to be a novel enzyme, which has an important role in the development of inflammation.     

Topsin-M: the new benomyl for mycorrhizal-suppression experiments

The fungicide benomyl was the most commonly used biocide for both field and greenhouse experiments in which arbuscular mycorrhizal fungal (AMF) suppression is desired. Unfortunately benomyl is no longer manufactured and therefore is not available for experimental use and no fungicide has been proposed as a successful alternative for experimentally suppressing mycorrhizal fungi. In this study we examined the potential for the fungicide Topsin M (topsin) to suppress mycorrhizal symbiosis in both field and greenhouse experiments. Topsin reduced AMF colonization of the obligately mycotrophic, warm-season grass Andropogon gerardii with a large and significant reduction in plant biomass production. Topsin reduced AMF colonization of the facultatively mycotrophic, cool-season grass Pascopyron smithii but did not significantly reduce biomass production. Fertilization with nitrogen and phosphorus was able to compensate for reductions in biomass due to the application of fungicide because biomass production of plants that received topsin fungicide was not significantly different from fertilized controls not receiving topsin. While we are not advocating that topsin fungicide is a universal mechanism for mycorrhizal-suppressed controls, in systems where benomyl was found to be successful topsin appears to be a useful, available and successful alternative.     

An isozyme of the NADP-malic enzyme of a CAM plant, Aloe arborescens, with variation on conservative amino acid residues

In Aloe arborescens, an obligate CAM plant, Western analysis detected three major isoforms of NADP-malic enzyme (NADP-ME), 72kDa with a pI of 6.0, 65kDa with a pI of 5.6 and 65kDa with a pI of 5.5. Among them, the 65kDa protein with a pI of 5.5 was leaf-specific, and the 65kDa protein with a pI of 5.6 was found only in roots, whereas the 72kDa protein was uniformly detected in both organs. Activity staining indicated enzyme activity of both 65kDa NADP-MEs but little activity of the 72kDa protein. A cDNA clone encoding a leaf-abundant NADP-ME, AME1, was isolated. Deduced amino acid sequence of AME1 showed a high degree of homology to known NADP-MEs, but it was also found that AME1 contained substitutions on five conservative amino acid residues, some of which have been predicted to be important for their enzyme activity. Transgenic rice carrying the aloe AME1 gene efficiently produced an additional 65kDa protein with a pI of 5.5 as an active NADP-ME. These results indicate that AME1 corresponds to the leaf-specific 65kDa NADP-ME, which may be involved in CAM photosynthesis. It was also shown that substitutions of these conservative amino acid residues identified in AME1 still allowed it to give enzyme activity.     

Reduced fungicide applications improve insect pest control in grapevine

Agricultural intensification is a major driver of biodiversity decline in many species including arthropods. This may also affect important ecosystem services such as natural pest regulation. Traditional grapevine varieties rely on a high number of fungicide applications, which can be greatly reduced in novel fungus resistant cultivars. Additionally, in contrast to the traditional trellis system, the semi-minimal pruned hedge offers a structurally more diverse habitat for arthropods. We investigated natural control of the grapevine pest Lobesia botrana ([Denis & Schiffermüller], 1775) (Lepidoptera: Tortricidae) with artificial inoculation of grape bunches, egg baits, camera surveillance and beatsheet sampling of predators. Reduced fungicide applications enhanced densities of L. botrana predators along with increased egg predation and reduced damage of inoculated grape bunches. Minimal pruning did not have a clear effect. Improved pest control potential adds to other advantages of reduced fungicide applications. Planting fungus-resistant varieties should be augmented, potentially also in other crop systems that rely heavily on fungicides.     

The effect of strobilurins on leaf gas exchange, water use efficiency and ABA content in grapevine under field conditions

Strobilurins are one of the most important classes of agricultural fungicides. In addition to their anti-fungal effect, strobilurins have been reported to produce simultaneous effects in plant physiology. This study investigated whether the use of strobilurin fungicide improved water use efficiency in leaves of grapevines grown under field conditions in a Mediterranean climate in southern Spain. Fungicide was applied three times in the vineyard and measurements of leaf gas exchange, plant water status, abscisic acid concentration in sap ([ABA]), and carbon isotope composition in leaves were performed before and after applications. No clear effect on stomatal conductance, leaf water potential and intrinsic water use efficiency was found after three fungicide applications. ABA concentration was observed to increase after fungicide application on the first day, vanishing three days later. Despite this transient effect, evolution of [ABA] matched well with the evolution of leaf carbon isotope ratio, which can be used as a surrogate for plant water use efficiency. Morning stomatal conductance was negatively correlated to [ABA]. Yield was enhanced in strobilurin treated plants, whereas fruit quality remained unaltered.     

Distinct osmo-sensing protein kinase pathways are involved in signalling moderate and severe hyper-osmotic stress

Plant growth is severely affected by hyper-osmotic salt conditions. Although a number of salt-induced genes have been isolated, the sensing and signal transduction of salt stress is little understood. We provide evidence that alfalfa cells have two osmo-sensing protein kinase pathways that are able to distinguish between moderate and extreme hyper-osmotic conditions. A 46 kDa protein kinase was found to be activated by elevated salt concentrations (above 125 mM NaCl). In contrast, at high salt concentrations (above 750 mM NaCl), a 38 kDa protein kinase, but not the 46 kDa kinase, became activated. By biochemical and immunological analysis, the 46 kDa kinase was identified as SIMK, a member of the family of MAPKs (mitogen-activated protein kinases). SIMK is not only activated by NaCl, but also by KCl and sorbitol, indicating that the SIMK pathway is involved in mediating general hyper-osmotic conditions. Salt stress induces rapid but transient activation of SIMK, showing maximal activity between 8 and 16 min before slow inactivation. When inactive, most mammalian and yeast MAPKs are cytoplasmic but undergo nuclear transloca- tion upon activation. By contrast, SIMK was found to be a constitutively nuclear protein and the activity of the kinase was not correlated with changes in its intra-cellular compartmentation, suggesting an intra-nuclear mechanism for the regulation of SIMK activity.     

Enzymological properties of poly(ADP-ribose)polymerase: characterization of automodification sites and NADase activity.

We have characterized the effect of poly(ADP-ribose) polymerase automodification on the enzyme's activities, which include poly(ADP-ribose) synthesis and NADase activity. The apparent Km of the enzyme for NAD+ during polymer synthesis is higher than the one measured for alternate NADase activity. Furthermore, we have found that there are 28 automodification sites, in contrast to the 15 sites (postulated to be on the 15 glutamic acids) reported to be present in the automodification domain. For the first time, we show that some of these acceptor sites are outside the reported automodification domain (15 kDa); we demonstrate automodification in the NAD+ binding domain (55.2 kDa) and the DNA binding domain (42.5 kDa). We have analyzed the relationship between the number of sites modified on poly(ADP-ribose) polymerase and its effect on the polymerization activity and its alternate NADase activity. Automodification greatly altered both enzyme activities, decreasing both polymer synthesis and alternate NADase activity.     

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.     

ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact

Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA+ (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.     

Identification,purification and characterization of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle plasma membrane

Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatinase: MMP-2; E.C. 3.4.24.24) as revealed by immunoblot studies of its plasma membrane suspension with polyclonal MMP-2 antibody. In this report, we described the purification and partial characterization of MMP-2 in the plasma membrane fraction of the smooth muscle. MMP-2 has been purified from plasma membrane fraction of bovine pulmonary artery smooth muscle to homogeneity using a combination of purification steps. Heparin sepharose purified preparation of 72 kDa progelatinase is composed of two distinct population of zymogens: a 72 kDa progelatinase tightly complexed with TIMP-2 (an ambient tissue inhibitor of metalloprotease in the smooth muscle plasma membrane), and a native 72 kDa progelatinase free of any detectable TIMP-2. The homogeneity of the native 72 kDa progelatinase form is demonstrated by SDS-PAGE under non-reducing condition, non-denaturing native gel electrophoresis. The purified TIMP-2 free proenzyme electrophoresed as a single band of 72 kDa which could be activated by APMA with the formation of 62 and 45 kDa active species. The proenzyme is activated poorly by trypsin but not by plasmin. The purified 72 kDa progelatinase is stable at aqueous solution and does not spontaneously autoactivate. The purified 72 kDa gelatinase exhibited properties that are typical of MMP-2 obtained from other sources. These are: (i) its activity is dependent on the divalent cation, Ca+2, and is inhibited by EDTA, EGTA and 1:1 0-phenanthroline; (ii) it was inhibited by a, macroglobulin but not by the inhibitors of serine, cysteine, thiol, aspartic proteinases and calpains; (iii) it was found to be inhibited by TIMP-2, the specific inhibitor of MMP-2; (iv) like MMP-2, obtained from other sources, its major substrates were found to be collagens (type IV and V) and gelatins (type I, IV and V). Additionally, the purified MMP-2 degrades Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH (dinitrophenyl labelled peptide), a well known synthetic substrate for the MMP-2.     

Correlation of treponemicidal activity in normal human serum with the presence of IgG antibody directed against polypeptides of Treponema phagedenis biotype Reiter and Treponema pallidum, Nichols strain

Normal human serum (NHS) was shown to have complement-dependent treponemicidal activity against both Treponema pallidum and Treponema phagedenis biotype Reiter (TPR) by employing in vitro-in vivo neutralization and TPR plaque assays, respectively. The molecular basis of NHS treponemicidal activity was studied by immunoblot analysis in conjunction with treponemicidal assays. Five major T. pallidum polypeptide bands (47kDa, 35kDa, 33kDa doublet, and 30 kDa) and three major TPR polypeptide bands (47kDa and 33kDa doublet) bound IgG present in NHS. Absorption of NHS with TPR completely removed both TPR and T. pallidum treponemicidal activity; corresponding immunoblots demonstrated a significant removal of IgG antibody against all three TPR polypeptide bands as well as four T. pallidum polypeptide bands (30kDa, 33kDa doublet, and 35kDa). In contrast, T. pallidum absorption of NHS was found to remove treponemicidal activity against T. pallidum but not TPR; corresponding Western blots showed the complete removal of IgG antibody against all but one T. pallidum polypeptide band (47kDa) but no detectable loss in IgG antibody against the TPR polypeptides. These results suggest that antibody in NHS generated against nonpathogenic, indigenous treponemes is responsible for the T. pallidum treponemicidal activity. Furthermore, the treponemicidal activity against T. pallidum correlated with the presence of IgG antibody against T. pallidum polypeptides of 30kDa, 35kDa, and a 33kDa doublet.     

GTPase and transglutaminase are associated in the secretion of the rat anterior prostate.

We have found that in the secretion of rat anterior prostate, a hydrolyzing activity on GTP is present with a high affinity for the substrate; ATP, GDP, and ADP are not substrates for enzymatic activity. At the same time we have shown that GTP is a negative modulator for the well-known type IV transglutaminase activity present in the prostatic secretion. The hydrolyzing activity on GTP appears to be due to two molecular species: a high-molecular-weight GTPase, having electrophoretical mobility higher than 100 kDa, and a low-molecular-weight GTPase, of about 30 kDa. The two enzymatic activities are associated in the prostatic secretion with the transglutaminase (type IV). We describe an experimental procedure to separate them.     

Description of Complex Forms of a Porin in Bacteroides fragilis and Possible Implication of this Protein in Antibiotic Resistance

Periodic surveys of antibiotic susceptibility patterns among anaerobes have emphasized that new mechanisms of resistance have emerged, especially in the Bacteroides fragilis group. Resistance to the combination of amoxicillin and clavulanic acid among some imipenem-susceptible Bacteroides fragilis strains has been associated with modifications in outer membrane protein electrophoretic patterns with the loss of some porin-like proteins. Porins are outer membrane proteins that play a major part in membrane permeability; if they are under-expressed, they can be responsible for antibiotic resistance. In a previous work, we isolated one outer membrane protein of 45 kDa from Bacteroides fragilis and showed its porin activity. In the present study, we aim to isolate the different complex forms of this protein and to underline their possible role in antibiotic resistance. We therefore compared the electrophoretic patterns of the outer membrane proteins of several strains of Bacteroides fragilis. Although these patterns are similar to each other, some proteins, especially those of high molecular weight, are less visible in the samples heated before electrophoresis. We targeted these high molecular weight proteins (which appeared sensitive to heat) and isolated them by electro-elution. We thus identified two high molecular weight proteins (210 and 130/135 kDa) which seemed to be components of a complex including the 45 kDa outer membrane protein formerly identified by us as a porin protein. Their porin activities were tested by the swelling assay of proteoliposomes which showed that the 210 kDa protein behaved like the 45 kDa protein whereas the 130/135 kDa protein had less porin activity. Furthermore, swelling assays with antibiotic solutions made it possible to compute the role of this protein complex in antibiotic resistance.     

A Novel Opioid Mechanism Seems to Modulate Phagocytosis in Tetrahymena

ABSTRACT. We have previously reported that a β-endorphin-like substance inhibits phagocytosis in Tetrahymena perhaps by a mu-like opioid receptor. We now report a further characterization of the elements involved in the signal transduction mechanism of this opioid. Affinity chromatography followed by immunoblots of both intracellular extracts and extracellular medium reveal the presence of two main proteins of 64 and 75 kDa. These molecular weights are much higher than that of any known opioid peptide or precursor protein and suggest that we may be dealing with either a novel opioid or with proteins that by chance cross-react with anti-β-endorphin antibody. Nevertheless, when the biological activity of these proteins was tested it was found that they had an effect similar to that of mammalian β-endorphin, namely inhibition of phagocytosis by a naloxone-reversible mechanism. We have probed a size-selected Tetrahymena library with a pro-opiomelanocortin probe and have obtained several positive clones; the sequencing of their inserts should establish whether we are dealing with a bona fide member of the opioid family. Another aspect we have been studying is the G-proteins which appear to be involved in the modulation of phagocytosis. We have found, by means of Western blotting (using an antibody against the conserved GTP-binding region of the α-subunit), two bands of 51 and 59 kDa; no α-subunit of 59 kDa had been reported previously and may represent a novel G-protein. In spite of these differences, the opioid signal transduction mechanism appears to remarkably resemble that present in more complex organisms.     

Two-chain structure of T-suppressor factor: Antigen-specific T-suppressor factor occurs as a single molecule and as separate antigen-binding and I-J+ parts,both of which are required for biological activity

Antigen-specific T-suppressor factor (TsF), which acts at the expression stage of the contact sensitivity reaction, was produced by culturing the lymphoid cells of mice injected with picryl-sulphonic (trinitrobenzenesulphonic) acid and then painted with picryl chloride. Supernatant activity was found around 50–60 and 90 kDa on Sephadex gel filtration. The activity at 50–60 kDa was due to two separate (or readily separable) molecules, one antigen binding and the other bearing I-J determinants as shown by affinity chromatography on insolubilized antigen and anti-I-J. These two separate molecules were inactive alone but complemented each other and may be designated as the variable chain of TsF (TsF_v) and the I-J⁺ chain. The use of gel filtration and sequential absorption of individual pools on anti-I-J antibody followed by antigen, together with a complementation assay, also showed a TSF_v chain at 30–40 kDa and an I-J⁺ chain at 20–30 kDa. The higher-molecular-weight activity around 90 kDa was due to a single molecule which was both antigen binding and I-J⁺. This molecule dissociated on treatment with the reducing agent dithioerythritol followed by alkylation into two separate chains, one antigen binding and the other I-J⁺, both of which were required for activity. There was a requirement for genetic matching between the antigen-binding chain (TsF_v) and the I-J⁺ chain for biological activity. These data support a two-chain model of TsF in which TsF_v and the I-J⁺ chain occur as a single disulphide-bonded molecule around 90 kDa, or as two separate (or readily separable) chains of lower molecular weight which were inactive alone but complemented each other.     

Matrilysin (MMP‐7) cleaves C‐type lectin domain family 3 member A (CLEC3A) on tumor cell surface and modulates its cell adhesion activity

Matrilysin (MMP‐7) plays important roles in tumor progression. Previous studies have suggested that MMP‐7 binds to tumor cell surface and promotes their metastatic potential. In this study, we identified C‐type lectin domain family 3 member A (CLEC3A) as a membrane‐bound substrate of MMP‐7. Although this protein is known to be expressed specifically in cartilage, its message was found in normal breast and breast cancer tissues as well as breast and colon cancer cell lines. Because few studies have been done on CLEC3A, we overexpressed its recombinant protein in human cancer cells. CLEC3A was found in the cell membrane, extracellular matrix (ECM), and culture medium of the CLEC3A‐expressing cells. CLEC3A has a basic sequence in the NH₂‐terminal domain and showed a strong heparin‐binding activity. MMP‐7 cleaved the 20‐kDa CLEC3A protein, dividing it to a 15‐kDa COOH‐terminal fragment and an NH₂‐terminal fragment with the basic sequence. The 15‐kDa fragment no longer had heparin‐binding activity. Treatment of the CLEC3A‐expressing cells with MMP‐7 released the 15‐kDa CLEC3A into the culture supernatant. Furthermore, the 20‐kDa CLEC3A promoted cell adhesion to laminin‐332 and fibronectin substrates, but this activity was abrogated by the cleavage by MMP‐7. These results suggest that CLEC3A binds to heparan sulfate proteoglycans on cell surface, leading to the enhancement of cell adhesion to integrin ligands on ECM. It can be speculated that the cleavage of CLEC3A by MMP‐7 weakens the stable adhesion of tumor cells to the matrix and promotes their migration in tumor microenvironments. J. Cell. Biochem. 106: 693–702, 2009. © 2009 Wiley‐Liss, Inc.