Infestation of maize by Prostephanus truncatus initiated by male-produced pheromone

Delta traps baited with maize cobs, which were infested each with one male Prostephanus truncatus (Horn) (Col.: Bostrichidae), were distributed in southern Benin and collected after one, two, three and four weeks. The numbers of P. truncatus caught during the different trapping periods were not significantly different. Sixty-four percent of the trapped P. truncatus were females. Females attracted during the one-week trapping period produced a mean of 6.9 progeny during the seven days. The sex ratio of the progeny was 1:1. Trap catches with the infested cobs were on average 13 times lower than catches with 2 mg of the artificial pheromone. Estimation of P. truncatus densities in a maize store at the beginning of the storage period (based on laboratory data) revealed that small initial numbers of P. truncatus, possibly attracted by a single male, sufficed to initiate high infestation rates later in the storage season.     

Potential hosts of Prostephanus truncatus (Coleoptera: Bostrichidae) among native and agroforestry trees in Kenya

This study was carried out to establish the extent to which an invasive stored products pest, Prostephanus truncatus Horn exploits wood as a feeding and breeding resource in Kenya. Twenty seven out of 84 native and agroforestry trees and shrubs supported the breeding of P. truncatus under laboratory conditions. Adult survival (<1% to 18%) for 8 weeks was recorded on 51 tree species that did not support breeding. The breeding success of this beetle varied widely between host tree species and showed no obvious trends based on tree systematics or wood hardness. Studies of P. truncatus survival on two host tree species found that the greatest increase in the P. truncatus population occurred on stem sapwood, whilst the smallest population increase was on twigs. Breeding also varied greatly from season to season; studies on ten selected host species showed that reproduction was often greatest in wood samples collected and tested during the dry season in September. The implications of these findings for the management of P. truncatus infestations in farm stored maize are discussed.     

Preparative two-dimensional polyacrylamide gel electrophoresis of rat liver ribosomal proteins and determination of their amino acid compositions

A maize root fraction which inactivates nitrate reductase has been shown to have protease activity which can be measured by the hydrolysis of azocasein. This inactivating enzyme was also found to inactivate yeast tryptophan synthase. Yeast proteases A and B, which inactivate this latter enzyme, also gave a specific inactivation of the maize nitrate reductase. The maize root inactivating enzyme, like yeast protease B, degraded casein, and was inhibited by phenylmethylsulphonyl fluoride. A partially-purified yeast inhibitor prevented catalysis by the yeast proteases and maize root inactivating enzyme, but purified yeast inhibitors were without effect on the latter protein. The level of nitrate reductase-inactivating activity, and associated azocasein-degrading activity, increased with age of the maize root. Evidence was obtained for a heat stable inhibitor which maintained them in an inactive state, especially in the young root tip cells.     

A cysteine protease from maize isolated in a complex with cystatin

We recently purified a latent but SDS-activated protease complex (40, 15- or 13-kDa proteins) from maize [Yamada et al. (1998) Plant Cell Physiol. 39: 106]. Here, we revealed that the complex was composed of a cysteine protease (40 kDa) and a cystatin, cysteine protease inhibitor (15- or 13-kDa). This is the first report on the isolation of a complex consisting of a cystatin and a target cysteine protease from plants. Cloning of the cysteine protease revealed that it had low homology (25-30%) to other maize cysteine proteases cloned to date but was highly homologous to other plant cysteine proteases such as rice oryzain alpha (84%) and the homologs (50-80%). The cysteine protease expressed in Escherichia coli showed the same substrate and inhibitor specificities as the protease of the complex, demonstrating that the isolated cDNA clone exactly encodes the protease of the complex. The protease expressed in E. coli itself was active but not latent, probably because it was not bound to cystatin. It is most likely that in vitro activation of the protease complex by SDS is caused by the release of bound cystatin. The mRNA of protease was expressed in various tissues except for seeds.     

Efficacy of Xylocoris flavipes (Reuter) (Het., Anthocoridae) to suppress Prostephanus truncatus (Horn) (Col., Bostrichidae) in traditional maize stores in southern Togo

In the course of three storage periods (1988–90) the predacious insects that occur in traditional maize stores in southern Togo were observed and their relationship to the introduced pest Prostephanus truncatus was investigated. The bug Xylocoris flavipes , a predator on a wide range of post-harvest pests, was the only one observed regularly and in higher numbers. Laboratory trials with P. truncatus showed that the larvae were preyed on, and no preference for any one of the three instars was detected. On average, 7.3 pest larvae were killed by larvae or adults of the predator. In glass jar trials on loose maize X. flavipes reduced the numbers of P. truncatus by 57.6% after 8 weeks. The losses and damage were reduced by 50 and 17.6%, respectively. Observations in traditional maize stores indicated a tendency to decreasing populations of X. flavipes when populations of P. truncatus increased, presumably due to the unfavourable environmental conditions created by the pest. Therefore, it is concluded that X. flavipes does not play an important role in the control of this pest.     

Identification of serine proteases from Leishmania braziliensis

Leishmania (V) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary leishmaniasis (cutaneous and mucosal). The drugs of choice used in leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory infections. Among the promising new targets for anti-protozoan chemotherapy are the proteases. In this study, serine proteases were partially purified from aqueous, detergent and extracellular extracts of Leishmania braziliensis promastigotes by aprotinin-agarose affinity chromatography. By zymography, the enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified proteases exhibited esterase activity against Nalpha-benzoyl-L-arginine ethyl ester hydrochloride and Nalpha-p-tosyl-L-arginine methyl ester hydrochloride (serine protease substrates) and optimal activity at pH 8. 0. Proteases purified from the aqueous and extracellular extracts were effectively inhibited by benzamidine (trypsin inhibitor) and those from the detergent extract were inhibited by N-tosyl-L-phenyl-alanine chloromethyl ketone (chymotrypsin inhibitor) indicating that all these enzymes are serine proteases. These findings indicate that L. braziliensis serine proteases display some biochemical similarities with L. amazonensis serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a serine protease from Leishmania braziliensis.     

Studies on predation of Prostephanus truncatus (Horn) (Col., Bostrichidae) and Sitophilus zeamais Mots. (Col., Curculionidae) at different densities on maize by Teretriosoma nigrescens Lewis (Col., Histeridae)

Laboratory experiments using whole cobs were conducted to examine the effect of varying densities of the larger grain borer, Prostephanus truncatus and the maize weevil, Sitophilus zeamais on rate of population increase by the histerid beetle, Teretriosoma nigrescens , a predator primarily of P. truncatus . Densities of all species of insects were determined at the end of the experiment, and an electrophoretic analysis of gut content was conducted on larval and adult T. nigrescens sampled during the experiments. Results indicated that T. nigrescens has a strong preference for P. truncatus and densities of T. nigrescens were associated only with densities of P. truncatus . The maize weevil played little role as an alternative prey or in interfering with T. nigrescens reproduction.     

A novel slow-tight binding serine protease inhibitor from eastern oyster (Crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus

A serine protease inhibitor was purified from plasma of the eastern oyster, Crassostrea virginica. The inhibitor is a 7609.6 Da protein consisting of 71 amino acids with 12 cysteine residues that are postulated to form 6 intra-chain disulfide bridges. Sequencing of the cloned cDNA identified an open reading frame encoding a polypeptide of 90 amino acids, with the 19 N-terminal amino acids forming a signal peptide. No sequence similarity with known proteins was found in sequence databases. The protein inhibited the serine proteases subtilisin A, trypsin and perkinsin, the major extracellular protease of the oyster protozoan parasite, Perkinsus marinus, in a slow binding manner. The mechanism of inhibition involves a rapid binding of inhibitor to the enzyme to form a weak enzyme-inhibitor complex followed by a slow isomerization to form a very tight binding enzyme-inhibitor complex. The overall dissociation constants K(i) with subtilisin A, perkinsin and trypsin were 0.29 nM, 13.7 nM and 17.7 nM, respectively. No inhibition of representatives of the other protease classes was detected. This is the first protein inhibitor of proteases identified from a bivalve mollusk and it represents a new protease inhibitor family. Its tight binding to subtilisin and perkinsin suggests it plays a role in the oyster host defense against P. marinus.     

The protein inhibitor of calcium-dependent proteases: purification from bovine heart and possible mechanisms of regulation

A bovine heart protein which specifically inhibits calcium-dependent proteases has been purified to near homogeneity. The purified inhibitor had a Stokes radius of 6.8 nm estimated by gel filtration and a molecular weight of 145,000 estimated by sodium dodecyl sulfate-gel electrophoresis. There is evidence that it may be a glycoprotein. The inhibitor could be phosphorylated by bovine heart cyclic AMP-dependent protein kinase, and its inhibitory effect on Peak II (high-calcium-requiring) protease was modestly increased. However, no other phosphorylating or dephosphorylating conditions significantly influenced its activity. The inhibitor was not hydrolyzed by calcium-dependent proteases, but it was very sensitive to proteolytic inactivation by trypsin or proteases present in a lysosomal fraction from rat heart. Thus, proteolysis may represent a mechanism for decreasing the activity of the inhibitor in different physiologic or pathologic conditions.     

Digestive amylase from the larger grain borer, Prostephanus truncatus Horn

A combination of ion-exchange chromatography, preparative electrophoresis and gel filtration chromatography allowed a 1209-fold purification of one of the two major digestive alpha-amylases from larvae of the larger grain borer, Prostephanus truncatus Horn. The purified enzyme showed a molecular mass of 60.2 kDa, an isoelectric point of 4.7 and an optimal pH for activity of 6.0. The enzyme was heat labile and it was recognized by proteinaceous inhibitors from amaranth seeds (Amaranthus hypochondriacus), whereas extracts from maize (Zea mays) and tepary bean (Phaseolus acutifolius) produced very low inhibition. When the enzyme was measured at different stages of development, maximal activity was found in the second instar larvae. Activity drastically decreased to a very low level during the pupae stage and increased again at the adult stage. A zymogram of the different developmental stages showed two main bands of alpha-amylase activity, which almost disappeared at the pupae stage to increase again during the adult stage, revealing a new, smaller band. This new band may be required for a better adaptation of the adult insect to its new environment.     

Purification and characterization of proteolytic enzymes from normal and opaque-2Zea mays L. developing endosperms

Purification and characterization of proteases from developing normal maize endosperm and high lysine opaque-2 maize endosperm have been carried out with a view to understand their role in storage protein modification. At day 15, normal maize endosperm had two types of proteolytic enzymes, namely, protease I and protease II, while at day 25 protease n disappeared and in place protease III appeared. However, in opaque-2 maize endosperm at both the stages only one type of enzyme (protease I) was present. These proteases had many properties in common-optimum pH and temperature were respectively, 5.7and 40°C; their activity was inhibited to the extent of 75 –93 % by p-chloromercuribenzoate; trypsin inhibitor inhibited the activity more at early stages of endosperm development; all proteases cleaved synthetic substrates p-tosyl-L-arginine methylesler and N-benzoyl-L-tyrosine ethyl ester and poly-L-glutamic acid. TheKm values of day 15 and 25 normal maize endosperm proteases ranged from 2.73–3.30, while for opaque-2 maize endosperm protease I it was 3.33 mg azocasein per ml assay medium. These enzymes, however, differed with respect to proteolytic activity towards poly-L-lysine. Only normal maize endosperm protease III at day 25 followed by protease II at day 15 showed high activity towards this homopolypeptide suggesting thereby their role in determining the quality of normal maize endosperm protein. Part of Ph.D. thesis submitted by the first author     

Characterization of a highly stable trypsin-like proteinase inhibitor from the seeds of Opuntia streptacantha (O. streptacantha Lemaire)

A trypsin inhibitor from Opuntia streptacantha Lemaire (Prickly pear) seeds was purified and characterized. Of several proteases tested, this inhibitor showed specificity to trypsin-like enzymes. The major inhibitor present in these seeds showed distinctive characteristics, most notably a low molecular weight of 4.19 kDa, as determined by MALDI TOF, and an unusually high thermal stability, retaining most of the activity after heating the sample 1 h to 120 °C with a pressure of 1 kg/cm². Its complete amino acid sequence was obtained through mass spectrometry, this establishing presence a blocked N-terminal region. When comparing its sequence in the MEROPS database for peptidases and peptidase inhibitors, it showed 34.48% identity with a serine-proteinase inhibitor from the I15 family.     

Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds.

A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysine-aspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima.     

The Effects of an entomopathogenic fungus, Beauveria bassiana (balsamo) vuillemin (hyphomycetes), on Prostephanus truncatus (horn) (Col.: Bostrichidae), Sitophilus zeamais motschulsky (Col.: Curculionidae), and grain losses in stored maize in the Benin Republic

A fungal entomopathogen, Beauveria bassiana, was used to treat maize ears placed in traditional grain stores against Prostephanus truncatus in a field experiment conducted from September 1997 to March 1998 in the Benin Republic, West Africa. Treatments included oil-based spray with and without conidia, maize stored with and without the husk, and stores with and without artificial infestation. Additional treated ears kept in insect-proof cages under field conditions were sampled weekly and exposed to insects to estimate the virulence and persistence of the pathogen during the storage season. P. truncatus densities were significantly lower in treatments that included conidia, although densities were high in all artificially infested treatments and grain losses were severe. The effect of the pathogen was modeled with an exponential decay function and incorporated in a published P. truncatus simulation model. The effects of hypothetical pathogens with different virulence and persistence characteristics were evaluated in terms of insect density and percentage grain loss.     

Isolation and characterization of a novel proteinase inhibitor from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus).

A proteinase inhibitor (designated as TMI) was isolated and purified from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus) by using successive chromatographies which included Sephadex G-100, DEAE-Sephacel chromatographies, and C(4) reverse-phase HPLC. The purified inhibitor was shown to be a homogeneous protein with a molecular mass of about 47 or 36 kDa in the presence or absence of a reducing agent, beta-mercaptoethanol. The inhibitor decreases in molecular mass by about 23% with N-linked neuraminidase treatment, suggesting that it is a glycoprotein. Further enzymatic analyses indicated that this inhibitor possesses strong inhibitory activities toward three zinc-dependent metalloproteinases and not fibrinogenolytic serine proteases previously isolated from the venom of the same snake species with an IC(50) of about 0.2-1.1 microM. Its IC(50) value was approximately three orders of magnitude more effective than those of the tripeptide inhibitors we previously purified from the crude venom of the same snake (Biochem. Biophys. Res. Commun. 248, 562-568 (1998)). The purified inhibitor showed stronger inhibitory action against caseinolytic activities of crude venoms from closely related species of Taiwan habu than those from unrelated species. N-terminal sequence analysis showed that its sequence is distinctly different from sequences of those serum inhibitors reported for other snake species in the literature. Based on inhibition susceptibility and primary structures of various snake protease inhibitors, it is suggested that this novel inhibitor isolated from the serum of Taiwan habu may be a unique self-defense protein factor mainly for protection against envenomation from snakes of the same genus.     

Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes

Photorhabdus luminescens secretes both high molecular weight insecticidal toxin complexes and also a range of extracellular proteases into culture broth. Previous studies by others have suggested that insecticidal activity of the broth is associated with these proteases. However, by gene cloning and targeted knock-out, we have previously shown that oral insecticidal activity is associated with high molecular weight 'toxin complexes' (Tc) encoded by toxin complex or tc genes. Here we further clarify this distinction by biochemically separating the protease fractions away from the oral insecticidal activity of the Tc proteins. We purified three distinct protease fractions from the broth: one consisting of a single species of 55 kDa and two of several putatively related species of approximately 40 kDa. All of these clearly separate from the oral insecticidal activity associated with the high molecular weight Tc proteins and also show no effect on insect weight gain following injection into the haemocoel. Here we examine the substrate preferences and inhibitor profiles of these protease fractions and discuss their relationship with those previously described from other P. luminescens strains and phase variants.     

Electrophoretic analysis of the prey spectrum of Teretrius nigrescens (Lewis) (Col., Histeridae), a predator of Prostephanus truncatus (Horn) (Col., Bostrichidae), in Mexico, Honduras, and Benin

Abstract: Extensive collections of Teretrius nigrescens in Mexico, Honduras, and Benin, were electrophoretically analysed to elucidate the prey spectrum of the predator. Both polyacrylamide gel electrophoresis and cellulose acetate electrophoresis were used. Beetles were sampled with pheromone traps, using the synthetic aggregation pheromone of Prostephanus truncatus , and directly from farmers' maize stores. The proportion of electrophoretically detected prey protein from adult T. nigrescens in pheromone traps was low: of the 1108 specimens analysed, only in 34 cases, prey protein could be clearly identified. More frequently, prey protein was detected in adult T. nigrescens sampled in maize stores in Mexico, Honduras, and Benin, with 87 samples showing distinct prey bands of the total 1214 predators analysed. Of the 241 T. nigrescens larvae sampled in maize stores in Benin, 136 showed distinct bands of prey protein. In all samples, P. truncatus was the most frequently detected prey species. The second most often identified prey species was Sitophilus zeamais . The results are discussed with regard to various methods for prey spectrum analysis and specifically the biology of T. nigrescens .     

Identification of a chitinase-modifying protein from Fusarium verticillioides: truncation of a host resistance protein by a fungalysin metalloprotease

Chitinase-modifying proteins (cmps) are proteases secreted by fungal pathogens that truncate the plant class IV chitinases ChitA and ChitB during maize ear rot. cmp activity has been characterized for Bipolaris zeicola and Stenocarpella maydis, but the identities of the proteases are not known. Here, we report that cmps are secreted by multiple species from the genus Fusarium, that cmp from Fusarium verticillioides (Fv-cmp) is a fungalysin metalloprotease, and that it cleaves within a sequence that is conserved in class IV chitinases. Protein extracts from Fusarium cultures were found to truncate ChitA and ChitB in vitro. Based on this activity, Fv-cmp was purified from F. verticillioides. N-terminal sequencing of truncated ChitA and MALDI-TOF-MS analysis of reaction products showed that Fv-cmp is an endoprotease that cleaves a peptide bond on the C-terminal side of the lectin domain. The N-terminal sequence of purified Fv-cmp was determined and compared with a set of predicted proteins, resulting in its identification as a zinc metalloprotease of the fungalysin family. Recombinant Fv-cmp also truncated ChitA, confirming its identity, but had reduced activity, suggesting that the recombinant protease did not mature efficiently from its propeptide-containing precursor. This is the first report of a fungalysin that targets a nonstructural host protein and the first to implicate this class of virulence-related proteases in plant disease.     

Alpha-2-macroglobulin-like protease inhibitor from the egg white of cuban crocodile (Crocodylus rhombifer)

A high molecular weight protease inhibitor was purified from the egg white of Cuban crocodile (Crocodylus rhombifer). It inhibited the casein hydrolyzing activity of trypsin, subtilisin and papain. Its native molecular weight was 730,000 and it consisted of four subunits of equal molecular weight, each pair of which were disulfide bonded. The amino acid composition, circular dichroic spectrum and electron micrographs of this protein are also presented. Upon incubation with trypsin this protein yielded a fragment of Mr = 80,000, similar in size to the one known to originate from alpha 2-macroglobulin under the same conditions. The molecular parameters of this protein and the broad inhibitory activity towards thiol and serine proteases with different substrate specificities suggest that it is a protein closely related to alpha 2-macroglobulin in mammalian serum. From its native molecular weight and amino acid composition we believe that this protein is also a reptilian counterpart of the avian ovomacroglobulin described by Miller and Feeney (3).     

Purification and cloning of a novel serine protease from the nematode-trapping fungus <Emphasis Type="Italic">Dactylellina varietas</Emphasis> and its potential roles in infection against nematodes

From the culture filtrate of the fungus Dactylellina varietas (syn. Dactylella varietas), an extracellular protease (designed Dv1) was purified by cation exchange and hydrophobic interaction chromatography. The purified protease showed a molecular mass of approximately 30 kDa and displayed an optimal activity at pH 8 and 60.5°C (more than 20 min). This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. However, its proteolytic activity was highly sensitive to the serine protease inhibitor Phenylmethylphonylfuoride (1 mM), indicating that it belongs to the serine-type peptidase group. This protease could immobilize the free-living nematodes Panagrellus redivivus and Caenorhabditis elegans and hydrolyze the purified cuticle of P. redivivus, suggesting it may play a role in infection against nematodes. The encoding gene of Dv1 and its promoter sequence were cloned using degenerate primers and the DNA walking technology. Its open-reading frame contains 1,224 base pairs and without any intron. The deduced amino-acid sequence shared low identity to serine proteases from other nematode-trapping fungi. Our report identified a novel pathogenic protease from the nematode-trapping fungus D. varietas, and the three-dimensional structure of this protease was predicted using the Swiss-Prot method. Jinkui Yang and Lianming Liang contributed equally to this work.