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Biomedical subjects

D W Roberts

Publications and source records attributed to D W Roberts.

At least 73 records · Page 4Linked to original sources
PubMed1996

Construction of an improved mycoinsecticide overexpressing a toxic protease.

Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. A key aim of much recent work has been to increase the speed of kill and so improve commercial efficacy of these biocontrol agents. This might he achieved by adding insecticidal genes to the fungus, an approach considered to have enormous potential for the improvement of biological pesticides. We report here the development of a genetically improved entomopathogenic fungus. Additional copies of the gene encoding a regulated cuticle-degrading protease (Pr1) from Metarhizium anisopliae were inserted into the genome of M. anisopliae such that Pr1 was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Pr1 and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wild-type fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Thus, environmental persistence of the genetically engineered fungus is reduced, thereby providing biological containment.

Amino Acid Sequence↗
PubMed1996

Fate of biological control introductions: monitoring an Australian fungal pathogen of grasshoppers in North America.

In North America there are two generally recognized pathotypes (pathotypes 1 and 2) of the fungus Entomophaga grylli which show host-preferential infection of grasshopper subfamilies. Pathotype 3, discovered in Australia, has a broader grasshopper host range and was considered to be a good biocontrol agent. Between 1989 and 1991 pathotype 3 was introduced at two field sites in North Dakota. Since resting spores are morphologically indistinguishable among pathotypes, we used pathotype-specific DNA probes to confirm pathotype identification in E. grylli-infected grasshoppers collected at the release sites in 1992, 1993, and 1994. In 1992, up to 23% of E. grylli-infected grasshoppers of the subfamilies Melanoplinae, Oedipodinae, and Gomphocerinae were infected by pathotype 3, with no infections > 1 km from the release sites. In 1993, pathotype 3 infections declined to 1.7%. In 1994 grasshopper populations were low and no pathotype 3 infections were found. The frequency of pathotype 3 infection has declined to levels where its long-term survival in North America is questionable. Analyses of biocontrol releases are critical to evaluating the environmental risks associated with these ecological manipulations, and molecular probes are powerful tools for monitoring biocontrol releases.

Animals↗
PubMed1996

Acetaminophen toxicity in children: diagnostic confirmation using a specific antigenic biomarker.

Chronic acetaminophen (APAP) toxicity poses a difficult diagnostic challenge to the clinician. Signs and symptoms are nonspecific and no currently available laboratory study can confirm APAP as the causative agent of hepatic injury. In this study an antigenic biomarker for APAP toxicity was used to confirm the diagnosis of APAP-induced hepatic failure in two children with chronic APAP toxicity. APAP that has been metabolized to N-acetyl-benzoquinone imine (NAPQI) reacts with cellular proteins to form 3-(cystein-S-yl)-APAP protein adducts (3-Cys-A). Serum from both patients was submitted for quantitation of 3-Cys-A by a competitive inhibition enzyme-linked immunosorbent assay (ELISA). Concentrations of 3-Cys-A in the two patients were 1.97 and 2.77 nmol/mg protein, which are similar to concentrations found in adults with hepatic injury secondary to an overdose of APAP. Individuals with no exposure to APAP have no detectable 3-Cys-A in serum. It was concluded that 3-Cys-A is a useful marker of APAP intoxication after long-term ingestion of APAP when total dose and time course of ingestion are uncertain, and may prove to be a useful clinical and investigative tool in the study of APAP intoxication.

Acetaminophen↗
PubMed1996

Preoperative ketorolac increases bleeding after tonsillectomy in children.

PURPOSE: To compare the incidence of vomiting following codeine or ketorolac for tonsillectomy in children. METHODS: We had planned to enrol 240 patients, aged 2-12 yr undergoing elective tonsillectomy into a randomized, single-blind study in University Children's Hospital. The study was terminated, after 64 patients because interim analysis of the data by a blinded non-study scientist concluded that the patients were at undue risk of excessive perioperative bleeding. After induction of anaesthesia by inhalation with N2O/halothane or with propofol 2.5-3.5 mg.kg-1 i.v., the children were administered 150 micrograms.kg-1 ondansetron and 50 micrograms.kg-1 midazolam. Maintenance of anaesthesia was with N2O and halothane in O2. Subjects were administered either 1.5 mg.kg-1 codeine im or 1 mg.kg-1 ketorolac i.v. before the commencement of surgery. Intraoperative blood loss was measured with a Baxter Medi-Vac Universal Critical Measurement Unit. Postoperative management of vomiting and pain was standardized. Vomiting was recorded for 24 hr after anaesthesia. Data were compared with ANOVA, Chi-Square analysis and Fisher Exact Test. RESULTS: Thirty-five subjects received ketorolac. Demographic data were similar. The incidence of vomiting during the postoperative period was 31% in the codeine-group and 40% in the ketorolac-group. Intraoperative blood losses was 1.3 +/- 0.8 ml.kg-1 after codeine and 2.2 +/- 1.9 ml.kg-1 after ketorolac (mean +/- SD) P < 0.05. Five ketorolac-treated patients had bleeding which led to unscheduled admission to hospital, P < 0.05, Exact Test. CONCLUSION: Preoperative ketorolac increases perioperative bleeding among children undergoing tonsillectomy without beneficial effects.

Analgesics, Non-Narcotic↗
PubMed1996

Characterization and Ultrastructural Localization of Chitinases from Metarhizium anisopliae, M. flavoviride, and Beauveria bassiana during Fungal Invasion of Host (Manduca sexta) Cuticle.

Extracellular chitinases have been suggested to be virulence factors in fungal entomopathogenicity. We employed isoelectric focusing and a set of three fluorescent substrates to investigate the numbers and types of chitinolytic enzymes produced by the entomopathogenic fungi Metarhizium anisopliae, Metarhizium flavoviride, and Beauveria bassiana. Each species produced a variety of N-acetyl-(beta)-d-glucosaminidases and endochitinases during growth in media containing insect cuticle. M. flavoviride also produced 1,4-(beta)-chitobiosidases. The endochitinases could be divided according to whether they had basic or acidic isoelectric points. In contrast to those of the other two species, the predominant endochitinases of M. anisopliae were acidic, with isoelectric points of about 4.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the acidic chitinases of M. anisopliae into two major bands (43.5 and 45 kDa) with identical N-terminal sequences (AGGYVNAVYFY TNGLYLSNYQPA) similar to an endochitinase from the mycoparasite Trichoderma harzianum. Use of polyclonal antibodies to the 45-kDa isoform and ultrastructural immunocytochemistry enabled us to visualize chitinase production during penetration of the host (Manduca sexta) cuticle. Chitinase was produced at very low levels by infection structures on the cuticle surface and during the initial penetration of the cuticle, but much greater levels of chitinase accumulated in zones of proteolytic degradation, which suggests that the release of the chitinase is dependent on the accessibility of its substrate.

Journal Article↗
PubMed1996

Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles.

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine proteinase which has a tryptic specificity for basic residues and which may be involved in entomopathogenicity. Analytical and preparative isoelectric focusing methods were used to separate two trypsin components, produced during growth on cockroach cuticle, with isoelectric points of 4.4 (molecular mass, 30 kDa) and 4.9 (27 kDa). The catalytic properties of the proteases were analyzed by their kinetic constants and by a combination of two-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme overlay membranes. Both Pr2 isoforms preferentially cleave at the carboxyl sides of positively charged amino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine. Compared with the pathogen's subtilisin-like enzyme (Pr1), the pI 4.4 Pr2 isoform shows low activity against insoluble proteins in a host (Manduca sexta) cuticle. However, it degrades most cuticle proteins when they are solubilized, with high-molecular-weight basic proteins being preferentially hydrolyzed. Polyclonal antibodies raised against each Pr2 isoform were isotype specific. This allowed us to use ultrastructural immunocytochemistry to independently visualize each isoform during penetration of the host (M. sexta) cuticle. Both isoforms were secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules. Intracellular labelling was sparse.

Amino Acid Sequence↗
PubMed1996

Acetaminophen-induced hepatotoxicity. Analysis of total covalent binding vs. specific binding to cysteine.

Acetaminophen-induced hepatotoxicity is believed to be mediated by covalent binding of the reactive metabolite N-acetyl-p-benzoquinone imine to essential proteins in liver. It has been shown that the primary reaction of this metabolite with hepatic proteins is the formation of 3-(cysteine-S-yl)-acetaminophen adducts. The importance of covalent binding to other amino acids that may be formed by reaction of N-acetyl-p-benzoquinone imine with protein is unclear. Previously, we developed immunochemical assays for the acetaminophen cysteine adducts by immunizing animals with the conjugate 3-(N-acetylcystein-S-yl)acetaminophen-keyhole limpet hemocyanin, wherein the carboxyl group of the N-acetyl-cysteine moiety was coupled to amino groups on the protein. A very sensitive and specific immunochemical assay was developed for acetaminophen specifically bound to cysteine groups on protein [3-(cystein-S-yl)acetaminophen protein adducts]. Analysis of protein adducts indicated that after toxic doses, acetaminophen covalently bound at high levels to cysteine residues on a relatively small number of hepatic proteins. In the present work, a new antiacetaminophen antiserum was prepared by immunizing mice with 4-acetamidobenzoic acid coupled to keyhole limpet hemocyanin. Competitive ELISA data indicate that the resulting antiserum has excellent recognition of acetaminophen and related arylacetamide derivatives. Using this new antiserum, Western blot analyses of liver proteins from acetaminophen-intoxicated mouse livers were performed and compared with similar assays using the anti-3-(cystein-S-yl)acetaminophen antiserum. Visual and densitometric analyses of the Western blots indicate that the two antisera detect the same primary acetaminophen protein adducts; however, minor differences in the intensity of certain bands were observed. These differences may represent either differences in antibody accessibility to 3-(cystein-S-yl)acetaminophen adducts or differences in the proportion of acetaminophen bound to cysteine vs. binding to other amino acids.

Acetaminophen↗
PubMed1995

Ondansetron is a better prophylactic antiemetic than droperidol for tonsillectomy in children.

Both intravenous ondansetron (OND) and droperidol (DROP) have been observed to reduce vomiting after tonsillectomy in children. This randomized, double-blind investigation compared the effect of OND and DROP on vomiting after outpatient tonsillectomy in 276 healthy children age 2-12 yr. All subjects received a standardized anaesthetic, which consisted of induction with either propofol or halothane/N2O, vecuronium 0.1 mg x kg(-1) on an as needed basis, maintenance with halothane/N2O, midazolam and codeine, and reversal of neuromuscular blockade with neostigmine and atropine on an as needed basis. Subjects were given either OND 150 micrograms x kg(-1) or DROP 50 micrograms x kg(-1)iv after induction of anaesthesia. Rescue antiemetics in the hospital were administered to patients who vomited X 2 and X 4, respectively. Postoperative pain was treated with morphine, codeine and/or acetaminophen. For 24 hr following surgery, emesis was recorded by nursing staff while subjects were in the hospital, and by parents following discharge from hospital. The two groups were similar with respect to demographic data, induction technique and anaesthesia time. The frequency of in-hospital emesis was 16% in the OND-patients and 30% in the DROP-group, P <0.05. The OND-subjects required fewer rescue antiemetics, 5% vs 13%, P <0.05. The overall incidence of emesis was 45% in the OND-group and 57% in the DROP-group, P <0.05. In conclusion, ondansetron was a superior prophylactic antiemetic for tonsillectomy in children when compared to droperidol.

Antiemetics↗
PubMed1995

Linear free energy relationships for reactions of electrophilic halo- and pseudohalobenzenes, and their application in prediction of skin sensitization potential for SNAr electrophiles.

Published kinetic data and linear free energy relationships for nucleophilic aromatic substitution reactions (SNAr reactions) are analyzed so as to derive a reactivity parameter (RP), defined as sigma sigma-(o,m,p) + 0.45 sigma*(ipso), quantifying the relative reactivities of SNAr electrophiles toward aniline in ethanol. It is shown that the dataset of Landsteiner and Jacobs, in which 20 SNAr electrophiles were classified on the basis of experimental evidence as either skin sensitizing and reactive to aniline or nonsensitizers and unreactive to aniline, can be equivalently classified on the basis of RP values and that predictive criteria can be defined as follows: sensitizing/aniline reactive: RP > 3.80; nonsensitizing/unreactive to aniline: RP < 3.65. These predictive criteria based on calculated RP values are applied to seven further SNAr electrophiles to predict which are sensitizers and which are not. There is close agreement between the predictions and the biological data. This validates the applicability of the reactivity parameter RP, derived from linear free energy relationships, to prediction of skin sensitization potential for SNAr electrophiles. More generally, the applicability of physical organic chemistry principles to structure-activity relationships in contact allergy is further demonstrated.

Animals↗
PubMed1995

Curvature-based nonfiducial registration for the Frameless Stereotactic Operating Microscope.

The Frameless Stereotactic Operating Microscope permits information from CT and MRI scans to be displayed in the operating microscope in the proper scale and perspective without using a mechanical frame in the process, when the microscope is positioned over the surgical field. This registration is currently done using fiducials that must be present when the imaging scans are taken. A new technique, based on the theory of curvatures, has been developed as an alternative to the use of fiducials for the registration of a patient's head position during surgery with diagnostic images from CT or MRI. Surface curvatures are estimated from a trace of points of the surface of the skin, obtained from both the diagnostic images and an intraoperative nonimaging ultrasonic rangefinder. The point traces need not be identical or evenly spaced. Plots of the resulting curvature fields are compared visually and the alignment determined. Phantom testing, using a human skull, has resulted in a median alignment error of 1.95 mm. Testing using a human subject with clinically obtained data has resulted in alignment errors on the order of 7 mm.

Computers↗
PubMed1995

Cytochrome P4502E1 inhibition by propylene glycol prevents acetaminophen (paracetamol) hepatotoxicity in mice without cytochrome P4501A2 inhibition.

Acetaminophen hepatotoxicity is associated with its biotransformation to the reactive metabolite N-acetyl-p-benzoquinone imine that binds to protein. Two forms of cytochrome P450, CYP2E1 and CYP1A2, have been implicated as primarily responsible for the bioactivation. To determine the relative contributions of these P450's, overnight fasted male NMRI mice were pretreated with 10 ml of 50% v/w propylene glycol/kg or fluvoxamine (10 mg/kg) at -80 and -20 min. relative to acetaminophen dosing to inhibit CYP2E1 and CYP1A2, respectively. Mice were sacrificed at 0.5 or 4 hr after a hepatotoxic dose of acetaminophen (300 mg/kg). Propylene glycol or propylene glycol plus fluvoxamine, but not fluvoxamine alone protected against acetaminophen hepatotoxicity as indicated by abolished increase in serum alanine aminotransferase activity, less depletion of hepatic glutathione and lower liver:body weight ratios. Propylene glycol inhibited the activity of CYP2E1 as indicated by 84% reduction in the clearance of 3 mg/kg dose of chlorzoxazone, whereas fluvoxamine inhibited the activity of CYP1A2 as indicated by 40% reduction in the clearance of a 10 mg/kg dose of caffeine. For this animal model, the data are consistent with the notion that hepatoxicity is associated with bioactivation of acetaminophen by CYP2E1 but not by CYP1A2.

Acetaminophen↗
PubMed1995

Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes.

The zygomycetous fungus Entomophaga grylli is a pathogen that shows host-specific variance to grasshopper subfamilies. Three pathotypes of the E. grylli species complex were differentiated by three molecular techniques. In the first method, the three pathotypes showed different fragment patterns generated by random amplification of polymorphic DNA (RAPD). There was little or no interisolate variability in RAPD fragment patterns within each pathotype. Passage of an isolate of pathotype 3, originally from an Australian grasshopper (Praxibulus sp.), through a North America grasshopper resulted in no differences in the resultant RAPD fragment patterns. In the second method, polymorphic RAPD fragments were used to probe the genomic DNA from the three pathotypes, and pathotype-specific fragments were found. In the third method, restriction fragments from genomic DNA of the three pathotypes were cloned and screened for pathotype specificity. A genomic probe specific for each pathotype was isolated. These probes did not hybridize to DNA from Entomophaga aulicae or from grasshoppers. To facilitate the use of RAPD analysis and other molecular tools to identify pathotypes, a method for extracting DNA from resting spores from infected grasshoppers was developed. The DNA from the fractured resting spores was of sufficient integrity to be blotted and probed with the pathotype-specific DNA probes, thus validating the use of these probes for pathotype identification in field-collected grasshoppers.

Animals↗
PubMed1995

Direct stereotactic targeting of the globus pallidus: an MRI-based anatomic variability study.

An anatomic variability study of the spatial relationship between the imaged globus pallidus, the optic tract, and the anterior and posterior commissures was performed using T1-weighted gradient echo three-dimensional volume acquisition MRI of 20 patients. Imaging data were resized, reformatted, and manually segmented for the above structures, and mean values and standard deviations for their medial, superior, lateral, and inferior limits determined. Findings demonstrate that the globus pallidus can be consistently and reliably imaged by MRI, that direct stereotactic targeting of this structure can account for normal anatomic variability which may be as much as several millimeters, and that the risk of injury to the optic tract may be reduced with this technique.

Adult↗
PubMed1994

Characterization of a novel carboxypeptidase produced by the entomopathogenic fungus Metarhizium anisopliae.

Preparative isoelectric focusing and gel filtration chromatography were used to purify a carboxypeptidase produced by the entomopathogenic fungus Metarhizium anisopliae during growth on cockroach cuticle. The enzyme was inhibited by diisopropyl fluorophosphate, implying involvement of a serine residue in catalysis. However, the M. anisopliae enzyme differed from most serine carboxypeptidases in also being inhibited by the metal chelator 1,10-phenanthroline and in being a small (30 kDa), basic (pI 9.97) protein with a neutral pH optima (pH 6.8). These properties resemble those exhibited by some metalloproteases but the enzyme is not inhibited by Cd2+; nor do Zn2+ or Co2+ restore activity in enzyme inhibited with phenanthroline. The amino-terminal sequence (22 residues) showed no similarity to other protein sequences. Unlike previously reported fungal carboxypeptidases, the M. anisopliae enzyme is powerfully inhibited by potato carboxypeptidase inhibitor. The carboxypeptidase shows a broad primary specificity toward amino acids with hydrophobic side groups in a series of N-blocked dipeptides, with substrates with phenylalanine being the most rapidly hydrolyzed. The S1 subsite also accommodated Glu, confirming its low selectivity. Proline at P1 or P1 resulted in a very poor substrate. The specificity of the carboxypeptidase complements that of the subtilisin-like protease (Pr1) of M. anisopliae. Both Pr1 and the carboxypeptidase are produced during carbon and nitrogen deprivation, which indicates that the exopeptidase functions with Pr1 to degrade peptides to supply amino acids during starvation and pathogenicity.

Amino Acid Sequence↗