Effects of midgut-protein-preparative and ligand binding procedures on the toxin binding characteristics of BT-R1, a common high-affinity receptor in Manduca sexta for Cry1A Bacillus thuringiensis toxins.

The identity of the physiologically important Cry1A receptor protein(s) in the lepidopteran Manduca sexta has been a matter of dispute due to the multiple proteins which bind the Cry1Ac toxin. Cry1Aa, Cry1Ab, and Cry1Ac exhibit essentially identical toxicities toward M. sexta larvae and show a high degree of sequence and presumed structural identities. These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity. Investigators in our laboratory previously demonstrated that the cloned 210-kDa glycoprotein BT-R1 binds all three Cry1A toxins (T. P. Keeton and L. A. Bulla, Jr., Appl. Environ. Microbiol. 63:3419-3425, 1997). This protein remains a common binding protein even after being subjected to various midgut membrane preparation and processing protocols. The method used to isolate proteins from the M. sexta larval midgut in no significant way affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1A toxin to Cry1Ac binding proteins other than BT-R1. Alterations in blot substrate and blocking, hybridization, and washing buffers support these conclusions. Collectively, these results indicate that in M. sexta the cadherin-like BT-R1 protein is a common high-affinity receptor protein for the Cry1A family of toxins.

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins.

Toxins isolated from the venom of the Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic type phospholipases A2 and postsynaptic neurotoxins. Toxicon 35, 1193-1203, 1997.-Two sets of proteins have been purified from the venom of the Brazilian coral snake, Micrurus frontalis frontalis. One set has mol. wts, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in the 8000-13,000 range and includes some proteins which are toxic to mice and others which are not. These proteins appear to be isoforms of postsynaptic toxins. The other set shows phospholipase A2 (PLA2) activity and the toxic members of this set promote hemorrhage in mice in a manner closely resembling that produced by PLA2s isolated from the venom of the Australian tiger snake (Notechis scutatus scutatus). These PLA2s migrate on SDS-PAGE with apparent mol. wts in the 18,000-22,000 range which is characteristic of PLA2s that have an alpha-helix D similar to pancreatic PLA2s. Elapid venom PLA2s of the type which typically migrate on SDS-PAGE with mol. wts in the 13,000-16,000 range and do not have alpha-helix D have not been detected in M. f. frontalis venom.

Further characterization of BT-R1, the cadherin-like receptor for Cry1Ab toxin in tobacco hornworm (Manduca sexta) midguts.

BT-R1, the Manduca sexta midgut receptor for the crystal toxin Cry1Ab produced by Bacillus thuringiensis ssp. berliner, was partly purified by gel filtration from M. sexta brush border membrane vesicles in the presence of the detergent CHAPS. Fractions containing BT-R1 were tested for their stability against degradation as indicated by retention of Cry1Ab binding on ligand blots. At 4 degrees C and pH 7.4 in the presence of Ca2+, BT-R1 was stable for up to 48 h but a 65% loss of binding was observed after 100 h. Under the same conditions, no loss of binding was observed in the presence of EGTA after 100 h. Cry1Ab binding decreased markedly as pH increased from 6 to 10 for incubations of 24 h at 4 degrees C. Increasing the temperature of incubation from 4 to 37 degrees C also decreased Cry1Ab binding. Neither metal ions nor free sulfhydryl groups are involved in Cry1Ab binding to BT-R1. A trypsin-like, metal-ion-dependent proteolytic activity co-eluted with BT-R1 during gel filtration. This endoproteolytic activity was unaltered by the addition of Cry1Ab. BT-R1 did not co-elute with peaks of aminopeptidase, alkaline phosphatase, alpha-glucosidase, beta-glucosidase and beta-galactosidase activities. When BT-R1 in the gel filtration fraction was further purified on a Mono Q anion exchange column, partial separation of the trypsin-like activity from BT-R1 was observed. BT-R1 could be removed from the appropriate Mono Q fraction by immunoprecipitation with only a slight decrease in this activity. These results demonstrate that there is no copurification of BT-R1 and these enzymes and that BT-R1 is unlikely to form complexes with them. Binding of Cry1Aa and Cry1Ac to BT-R1 in gel filtration fractions is similar to that of Cry1Ab, indicating that BT-R1 may be the high-affinity receptor for the Cry1A toxins. Binding of Cry1Ab to a 120 kDa protein has not been observed in this study.

Bovine granulocyte chemotactic protein-2 is secreted by the endometrium in response to interferon-tau (IFN-tau).

Interferon-tau (IFN-tau) is secreted by the bovine conceptus and may regulate synthesis of uterine endometrial cytokines to provide an environment that is conductive to embryo development and implantation. Interferon-tau stimulates secretion of an 8-kDa uterine protein (P8) in the cow. P8 was purified, digested to yield internal peptides, and partially sequenced to determine identity. Two internal peptides had 100% (13-mer) and 92% (12-mer) amino acid sequence identity with bovine granulocyte chemotactic protein-2 (bGCP-2). Bovine GCP-2 is an alpha-chemokine that acts primarily as a potent chemoattractant for granulocyte cells of the immune system. A peptide was synthesized based on a region of bGCP-2 that overlapped with a P8 peptide amino acid sequence, coupled to keyhole limpet hemocyanin, and used to generate high titer polyclonal antiserum in sheep. Western blots revealed that bGCP-2 was not released by endometrium from day 14 nonpregnant cows, but was released in response to 25 nM IFN-tau (p<0.05). Uterine GCP-2 exhibited high affinity to heparin agarose, a characteristic shared by all alpha chemokines. This is the first report describing presence of GCP-2 in the uterine endometrium and regulation by IFN-tau. The regulation of bGCP-2 by IFN-tau may have important implications for cytokine networking in the uterus during pregnancy. Also, the regulation of inflammation and angiogenesis by bGCP-2 working together with other cytokines may be integral to establishing early pregnancy and implantation in the cow.

Bovine granulocyte chemotactic protein-2 is secreted by the endometrium in response to interferon-tau (IFN-τ).

Interferon-tau (IFN-τ) is secreted by the bovine conceptus and may regulate synthesis of uterine endometrial cytokines to provide an environment that is conducive to embryo development and implantation. Interferon-τ stimulates secretion of an 8-kDa uterine protein (P8) in the cow. P8 was purified, digested to yield internal peptides, and partially sequenced to determine identity. Two internal peptides had 100% (13-mer) and 92% (12-mer) amino acid sequence identity with bovine granulocyte chemotactic protein-2 (bGCP-2). Bovine GCP-2 is an α-chemokine that acts primarily as a potent chemoattractant for granulocyte cells of the immune system. A peptide was synthesized based on a region of bGCP-2 that overlapped with a P8 peptide amino acid sequence, coupled to keyhole limpet hemocyanin, and used to generate high titer polyclonal antiserum in sheep. Western blots revealed that bGCP-2 was not released by endometrium from day 14 nonpregnant cows, but was released in response to 25 nM IFN-τ (p<0.05). Uterine GCP-2 exhibited high affinity to heparin agarose, a characteristic shared by all α chemokines. This is the first report describing presence of GCP-2 in the uterine endometrium and regulation by IFN-τ. The regulation of bGCP-2 by IFN-τ may have important implications for cytokine networking in the uterus during pregnancy. Also, the regulation of inflammation and angiogenesis by bGCP-2 working together with other cytokines may be integral to establishing early pregnancy and implantation in the cow.

Use of BACTEC radiometric culture method and polymerase chain reaction for the rapid screening of faeces and tissues for Mycobacterium paratuberculosis.

The BACTEC radiometric culture method for detection of Mycobacterium paratuberculosis was evaluated on faeces from cattle on a farm in quarantine for Johne's disease. A multiplex polymerase chain reaction (PCR) based on the IS900 sequence specific for M paratuberculosis and a genus specific 16S rRNA region was developed and used to test cultures showing evidence of mycobacterial growth in the BACTEC liquid radiometric culture medium. Using the BACTEC-PCR combination, confirmation of M paratuberculosis from faeces and tissue of clinically affected animals was achieved within 2 to 4 weeks and 1 week, respectively, a substantial improvement on traditional culture and identification methods. The PCR provided rapid exclusion of M paratuberculosis when other Mycobacterium spp were grown. The radiometric culture medium proved to be very sensitive for culturing Mycobacterium spp.

Use of polymerase chain reaction for rapid diagnosis of tuberculosis.

A DNA amplification assay using the polymerase chain reaction technique designed for the rapid identification of Mycobacterium bovis organisms was used to test 211 human mycobacterial isolates and 177 clinical specimens previously submitted for routine mycobacterial culture. The procedures described could be used by routine or specialist laboratories for identification of M. tuberculosis complex organisms in 4 h and/or as a rapid screening method for the direct detection of M. tuberculosis complex organisms in specimens.

Field comparison of the interferon-gamma assay and the intradermal tuberculin test for the diagnosis of bovine tuberculosis.

An extensive field comparison of the gamma interferon (IFN-gamma) assay and the single intradermal tuberculin test for the diagnosis of bovine tuberculosis was conducted in Australia. The specificity of the IFN-gamma assay was determined by testing more than 6000 cattle from tuberculosis-free herds and varied from 96.2% to 98.1%, depending on the cut-off point chosen to define a positive reactor. For the sensitivity trial, cattle from herds being de-populated because of bovine tuberculosis were examined with both assays. The sensitivity of the IFN-gamma assay was shown to be significantly higher than the single intradermal tuberculin test and varied from 76.8% to 93.6% depending on the method of interpretation. A maximum overall sensitivity of 95.2% was obtained by testing with the IFN-gamma and the tuberculin test in parallel. The superior sensitivity of the IFN-gamma assay and the ability to adjust the sensitivity of the system depending on the task involved, will provide the Australian Tuberculosis Eradication Campaign with a valuable additional test to enable it to accomplish its goals.

Use of DNA amplification for the rapid identification of Mycobacterium bovis.

DNA amplification using the polymerase chain reaction technique was evaluated for rapid identification of Mycobacterium bovis. Two oligonucleotide primers of 20 bases in length were constructed to target a region of the gene encoding the M. bovis secretory protein, MPB70. The amplification reaction produced a single product 372 bp in size which was readily detected by agarose gel electrophoresis. All 84 strains of M. bovis tested produced a positive signal in the amplification reaction. In addition all isolates fro the M. tuberculosis complex tested, with the exception of M. microti, gave a single band at 372 bp. No amplified product was detected when 24 other species of mycobacteria and species from four other genera were tested. The sensitivity of the test was such that a single viable cell could be detected in the reaction. This technique provides a simple and extremely sensitive method of identifying isolates of M. bovis and other pathogenic M. tuberculosis complex organisms.

Tuberculosis in captive seals: bacteriological studies on an isolate belonging to the Mycobacterium tuberculosis complex.

Culture of tuberculous lesions from six of 14 captive seals yielded an organism which, on the basis of biochemical and drug sensitivity tests, was identified as Mycobacterium bovis, although the organism showed a weak cording pattern and was glycerol tolerant. It was pathogenic in rabbits and guinea pigs and after passage the organism exhibited strong cord formation and was glycerol intolerant. Restriction endonuclease analysis and sodium dodecyl-sulphate polyacrylamide gel electrophoresis indicated that the organism belonged to the Mycobacterium tuberculosis complex. Restriction patterns indicated that infection in the six seals was from a single source. Western blotting with monoclonal antibody to M bovis identified antigens at 23 and 27 kDa in M bovis which were absent from the seal isolates.

Photoaffinity labeling of the rabbit reticulocyte guanine nucleotide exchange factor and eukaryotic initiation factor 2 with 8-azidopurine nucleotides. Identification of GTP- and ATP-binding domains.

We have covalently modified rabbit reticulocyte polypeptide chain initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) with the 8-azido analogs of GTP (8-N3GTP) and ATP (8-N3ATP). Of the five subunits of GEF, the Mr 40,000 polypeptide binds 8-[gamma-32P]N3GTP, and the Mr 55,000 and 65,000 polypeptides bind 8-[gamma-32P]N3ATP. Both 8-N3GTP and 8-N3ATP specifically label the beta-subunit of eIF-2. Covalent binding of 8-azidopurine analogs to the eukaryotic initiation factors is dependent on UV irradiation. Binding of 8-N3GTP and 8-N3ATP is specific for the guanine- and adenine-binding sites on the protein, respectively. GDP and GTP, but not ATP, inhibit the photoinsertion of 8-N3GTP to the protein. Similarly, ATP, but not GTP, inhibits the photoinsertion of 8-N3ATP. The inclusion of NADP+ in the reaction mixtures also interferes with the binding of 8-N3ATP to GEF. Mg2+ inhibits the binding of the 8-azido analogs of GTP and ATP to both eIF-2 and GEF, whereas EDTA stimulates the photoinsertion of these nucleotides. Identical results are obtained when the binding of GTP and ATP to these proteins, in the presence of Mg2+ or EDTA, is estimated by nitrocellulose membranes. In enzymatic assays, 8-N3GTP supports the activity of eIF-2 and GEF, indicating that the interaction of 8-N3GTP is catalytically relevant.

Advantages of a new agar medium in the primary isolation of Mycobacterium bovis.

Suspect tuberculous lesions from 116 cattle were examined histologically and cultured for Mycobacterium bovis using 5 different media. The media used were: B83, an agar medium incorporating bovine blood and sodium pyruvate; Middlebrook's agar; 2 variations of Stonebrink's medium; Löwenstein-Jensen medium. The B83 medium and a modification of Stonebrink's medium which had a lowered concentration of malachite green were most successful, detecting 95.2% of tuberculous animals when used together. The B83 medium detected isolates approximately 1 week earlier and had more colonies than the Stonebrink's modification. A combination of 2 slopes of B83 and 2 slopes of modified Stonebrink's medium is recommended for routine culture of samples.