PubMed Health⌕ Search

Biomedical subjects

B Oppert

Publications and source records attributed to B Oppert.

At least 19 recordsLinked to original sources
PubMed2006

Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion.

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.

Animals↗
PubMed2006

Diversity of digestive proteinases in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae.

The spectrum of Tenebrio molitor larval digestive proteinases was studied in the context of the spatial organization of protein digestion in the midgut. The pH of midgut contents increased from 5.2-5.6 to 7.8-8.2 from the anterior to the posterior. This pH gradient was reflected in the pH optima of the total proteolytic activity, 5.2 in the anterior and 9.0 in the posterior midgut. When measured at the pH and reducing conditions characteristic of each midgut section, 64% of the total proteolytic activity was in the anterior and 36% in the posterior midgut. In the anterior midgut, two-thirds of the total activity was due to cysteine proteinases, whereas the rest was from serine proteinases. In contrast, most (76%) of the proteolytic activity in the posterior midgut was from serine proteinases. Cysteine proteinases from the anterior were represented by a group of anionic fractions with similar electrophoretic mobility. Trypsin-like activity was predominant in the posterior midgut and was due to one cationic and three anionic proteinases. Chymotrypsin-like proteinases also were prominent in the posterior midgut and consisted of one cationic and four anionic proteinases, four with an extended binding site. Latent proteinase activity was detected in each midgut section. These data support a complex system of protein digestion, and the correlation of proteinase activity and pH indicates a physiological mechanism of enzyme regulation in the gut.

Animals↗
PubMed2006

Digestive proteinases of the larger black flour beetle, Cynaeus angustus (Coleoptera: Tenebrionidae).

Digestion in the larger black flour beetle, Cynaeus angustus (LeConte), was studied to identify new control methods for this pest of stored grains and grain products. The physiological pH of the larval gut, as measured with extracts in water, was approximately 6.1, and the pH for optimal hydrolysis of casein by gut extracts was 6.2 when buffers were reducing. However, under non-reducing conditions, hydrolysis of casein and synthetic serine proteinase substrates was optimal in alkaline buffer. Three major proteinase activities were observed in zymograms using casein or gelatin. Caseinolytic activity of C. angustus gut extracts was inhibited by inhibitors that target aspartic and serine proteinase classes, with minor inhibition by a cysteine proteinase inhibitor. In particular, soybean trypsin and trypsin/chymotrypsin inhibitors were most effective in reducing the in vitro caseinolytic activity of gut extracts. Based on these data, further studies are suggested on the effects of dietary soybean inhibitors of serine proteinases, singly and in combination with aspartic and cysteine proteinase inhibitors, on C. angustus larvae. Results from these studies can be used to develop new control strategies to prevent damage to grains and stored products by C. angustus and similar coleopteran pests.

Animals↗
PubMed2006

Cloning and characterization of protease inhibitor-like cDNAs from the Hessian fly mayetiola destructor (SAY).

Analysis of transcriptomes from the salivary glands and midgut of Hessian fly larvae Mayetiola destructor (say) identified a set of diverse cDNAs that encode proteins with a relatively high percentage (over 10%) of cysteinyl residues. Structural comparison of these putative proteins with known sequences in GenBank revealed that the positions of the cysteinyl residues in the identified proteins were highly conserved within a family of proteinase inhibitors despite very little overall sequence similarity. Phylogenetic analysis sorted this set of cDNAs into five different groups. To determine if these cDNAs indeed encode proteinase inhibitors, recombinant proteins were generated with two cDNAs from two different groups. Biochemical analysis of the recombinant proteins against commercial and insect gut proteinases demonstrated that the recombinant proteins are strong proteinase inhibitors with different specificities. Northern blot and real-time PCR analysis revealed that the different genes were expressed at different developmental stages and in different tissues. The overall results indicated that M. destructor contains a complex of genes that code for proteinase inhibitors which may regulate proteinase activities in different regulatory pathways. The GenBank accession numbers for the cDNAs in this paper were DQ232690 to DQ232718.

Amino Acid Sequence↗
PubMed2005

A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae.

A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.

Amino Acid Sequence↗
PubMed2005

Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: purification and characterization of a trypsin-like proteinase.

A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55 degrees C, pH optimum at 8.5, and K(m) value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0-9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N-terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50-72% identity with other insect trypsin-like proteinases, and 44-50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.

Amino Acid Sequence↗
PubMed2005

Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae).

Increasing levels of inhibitors that target cysteine and/or serine proteinases were fed to Tribolium castaneum larvae, and the properties of digestive proteinases were compared in vitro. Cysteine proteinases were the major digestive proteinase class in control larvae, and serine proteinase activity was minor. Dietary serine proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine proteinase inhibitors, there was a dramatic shift from primarily cysteine proteinases to serine proteinases in the proteinase profile of the midgut. Moreover, a combination of cysteine and serine proteinase inhibitors in the diet prevented this shift from cysteine proteinase-based digestion to serine proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.

Animals↗
PubMed2003

Effects of proteinase inhibitors on digestive proteinases and growth of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae).

The physiology of the gut lumen of the red flour beetle, T. castaneum, was studied to determine the conditions for optimal protein hydrolysis. Although the pH of gut lumen extracts from T. castaneum was 6.5, maximum hydrolysis of casein by gut proteinases occurred at pH 4.2. The synthetic substrate N-alpha-benzoyl-DL-arginine-rho-nitroanilide was hydrolyzed by T. castaneum gut proteinases in both acidic and alkaline buffers, whereas hydrolysis of N-succinyl-ala-ala-pro-phe rho-nitroanilide occurred in alkaline buffer. Inhibitors of T. castaneum digestive proteinases were examined to identify potential biopesticides for incorporation in transgenic seed. Cysteine proteinase inhibitors from potato, Job's tears, and sea anemone (equistatin) were effective inhibitors of in vitro casein hydrolysis by T. castaneum proteinases. Other inhibitors of T. castaneum proteinases included leupeptin, L-trans-epoxysuccinylleucylamido [4-guanidino] butane (E-64), tosyl-L-lysine chloromethyl ketone, and antipain. Casein hydrolysis was inhibited weakly by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor (Kunitz). The soybean trypsin inhibitor had no significant effect on growth when it was bioassayed alone, but it was effective when used in combination with potato cysteine proteinase inhibitor. In other bioassays with single inhibitors, larval growth was suppressed by the cysteine proteinase inhibitors from potato, Job's tears, or sea anemone. Levels of inhibition were similar to that observed with E-64, although the moles of proteinaceous inhibitor tested were approximately 1000-fold less. These proteinaceous inhibitors are promising candidates for transgenic seed technology to reduce seed damage by T. castaneum.

Animals↗
PubMed2002

Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae).

The cigarette beetle, Lasioderma serricorne (Fabricius), is a common pest of stored foods. A study of digestive proteinases in L. serricorne was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. Optimal casein hydrolysis by luminal proteinases of L. serricorne was in pH 8.5-9.0 buffers, although the pH of luminal contents was slightly acidic. Results from substrate and inhibitor analyses indicated that the primary digestive proteinases were serine proteinases. The most effective inhibitors of caseinolytic hydrolysis were from soybean (both Bowman Birk and Kunitz), with some inhibition by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin. Casein zymogram analysis identified at least eight proteolytic activities. Activity blot analyses indicated one major proteinase activity that hydrolysed the trypsin substrate N-alpha-benzoyl-L-arginine rho-nitroanilide, and three major proteinase activities that hydrolysed the chymotrypsin substrate N-succinyl ala-ala-pro-phe rho-nitroanilide. The absence of cysteine, aspartic, and metallo proteinases in L. serricorne digestion was evidenced by the lack of activation by thiol reagents, alkaline pH optima, and the results from class-specific proteinase inhibitors. The data suggest that protein digestion in L. serricorne is primarily dependent on trypsin- and chymotrypsin-like proteinases.

Animals↗
PubMed2001

Different mechanisms of resistance to Bacillus thuringiensis toxins in the indianmeal moth.

Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of (125)I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688(s)) and two resistant (198(r) and Dpl(r)) to Bacillus thuringiensis. Toxicological studies showed that the 198(r) colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl(r) colony was 4-fold more resistant to protoxin versus toxin. Binding results with (125)I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dpl(r) colony showed a drastic reduction in binding affinity (60-fold higher K(d)), although they had similar concentrations of binding sites. Insects from the 198(r) colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher K(d) and ca. three-fold-lower R(t) compared with the 688(s) colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198(r) colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473-23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensis resistance genes occurring in natural populations.

Animals↗
PubMed2000

cDNAs of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins.

Aminopeptidase N has been reported to be a Bacillus thuringiensis (Bt) Cry1A toxin-binding protein in several lepidopteran insects. cDNAs of aminopeptidase-like proteins from both Bt-susceptible RC688s and Bt-resistant HD198r strains of the Indianmeal moth, Plodia interpunctella, were cloned and sequenced. They contain 3345 and 3358 nucleotides, respectively, and each has a 3048 bp open reading frame that encodes 1016 amino acids. Putative protein sequences include 10 potential glycosylation sites and a zinc metal binding site motif of HEXXH, which is typical of the active site of zinc-dependent metallopeptidases. Sequence analysis indicated that the deduced protein sequences are most similar to an aminopeptidase from Heliothis virescens with 62% sequence identity and highly similar to three other lepidopteran aminopeptidases from Plutella xylostella, Manduca sexta, Bombyx mori with sequence identities of 51-52%. Four nucleotide differences were observed in the open reading frames that translated into two amino acid differences in the putative protein sequences. Polymerase chain reaction (PCR) confirmed an aminopeptidase gene coding difference between RC688s and HD198r strains of P. interpunctella in the PCR amplification of a specific allele (PASA) using preferential primers designed from a single base substitution. The gene mutation for Asp185-->Glu185 was also confirmed in two additional Bt-resistant P. interpunctella strains. This mutation is located within a region homologous to the conserved Cry1Aa toxin binding regions from Bombyx mori and Plutella xylostella. The aminopeptidase-like mRNA expression levels in the Bt-resistant strain were slightly higher than those in the Bt-susceptible strain. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF034483 for susceptible strain RC688s and AF034484 for resistant strain HD198r).

Amino Acid Sequence↗
PubMed2000

cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the indianmeal moth, Plodia interpunctella.

Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two trypsin-like proteinases, which is found in both the Bt-susceptible and -resistant strains of the Indianmeal moth. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF064525 for the RC688 strain and AF064526 for HD198).

Amino Acid Sequence↗
PubMed1999

Protease interactions with bacillus thuringiensis insecticidal toxins

The microbe Bacillus thuringiensis (Bt) produces crystals that contain insecticidal crystal proteins (ICPs) used to control many major pests. ICPs are degraded by proteases from a variety of sources, including those endogenous to the bacterium, those purified from animals and plants, or those found in insects. Proteases in the bacterium function in protein metabolism during sporulation; in some cases they hydrolyze ICPs. Insect proteases are implicated in Bt toxin specificity, mode of action and insect adaptation to Bt. This review describes the current knowledge of protease interactions with ICPs with special emphasis on the role of proteases in insect resistance to Bt toxins. Arch. Copyright 1999 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Journal Article↗
PubMed1998

Spore coat protein synergizes bacillus thuringiensis crystal toxicity for the indianmeal moth

Spores from Bacillus thuringiensis serovars kurstaki and entomocidus synergized crystal protein toxicity for larvae of the Indianmeal moth (Plodia interpunctella). Preparations of spore-crystal mixtures of either serovar were more toxic for the larvae than either purified spores or crystals alone (based on dry weight). Spores lost 53% of their toxicity for the Indianmeal moth after 2 h of UV-irradiation, but remained partially toxic (28%) even after 4 h of irradiation. Spore coat protein was toxic for the Indianmeal moth and was synergistic with B. thuringiensis serovar kurstaki HD-1 crystal protein. Enhanced toxicity of the combined spore-crystal preparation was attributed to a combination of crystal and spore coat protein, and included the effects of spore germination and resulting septicemia in the larval hemolymph. Ultraviolet irradiation of spores reduced the toxicity from septicemia but not the synergism caused by spore coat protein. The potencies of spore-crystal preparations must be carefully evaluated on the basis of contributions from all three factors.

Journal Article↗
PubMed1997

Proteinase-mediated insect resistance to Bacillus thuringiensis toxins.

Two Bacillus thuringiensis (Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins. The absence of this enzyme is genetically linked to larval survival on Bt-treated diets. When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs.

Animals↗
PubMed1997

Sequencing, analysis and expression in Escherichia coli of a gene encoding a 15 kDa Cryptosporidium parvum protein.

A previous paper presented data on a cDNA sequence encoding a protein associated with the AIDS related pathogen, Cryptosporidium parvum. However, the position of the start codon was uncertain, and the 5' end was continuous, lending doubt about the size and complete sequence of the final protein product. Herein we present the complete gene sequence and conclude the predicted size of the putative protein to be 16.2 kDa.

Amino Acid Sequence↗
PubMed1997

cDNAs for a chymotrypsinogen-like protein from two strains of Plodia interpunctella.

Gut proteinases are involved in the solubilization and activation of insecticidal toxins produced by Bacillus thuringiensis and may also be involved in resistance development. Approximately threefold lower chymotrypsin-like enzyme activity was observed in a Bt(entomocidus)-resistant strain of the Indianmeal moth, Plodia interpunctella, than that in the Bt-susceptible strain. Because chymotrypsin-like proteinases are involved in Bt protoxin activation in P. interpunctella, we compared cDNA sequences, mRNA expression levels, and genomic DNA for chymotrypsin-like enzymes in Bt-susceptible and Bt-resistant strains of P. interpunctella. To isolate cDNA coding for chymotrypsinogen-like proteinases, a probe was developed using polymerase chain reaction (PCR) amplification of a cDNA library from the Bt-susceptible strain using a vector primer and a degenerate primer corresponding to a conserved sequence in the active site of serine proteinases. This probe was used to screen cDNA libraries from resistant and susceptible strains. Predicted amino acid sequences from cDNA clones of each strain share similarity with sequences of chymotrypsin-like proteinases and are most similar to a chymotrypsin-like proteinase from the tobacco hornworm, Manduca sexta. cDNAs for putative chymotrypsinogen-like proteins, from both Bt-susceptible and Bt-resistant strains of P. interpunctella share an identical open reading frame of 846 nucleotides. The encoded proteins contain amino acid sequence motifs of serine proteinase active sites, disulfide-bridge cysteine residues, and both zymogen activation and signal peptides. A difference between these cDNAs was observed only in the untranslated region where a substitution of guanine for adenine occurred in the Bt-resistant strain. Southern and Northern blotting analyses indicated that there are no major differences in chymotrypsinogen-like genomic organization and mRNA expression in the two strains. These data suggest that chymotrypsinogen-like proteinase genes and their transcription are similar in the Bt-susceptible and Bt-resistant strains of P. interpunctella.

Amino Acid Sequence↗