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Biomedical subjects

D W Roberts

Publications and source records attributed to D W Roberts.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and regulatory analysis of starvation-stress gene, ssgA, encoding a hydrophobin-like protein from the entomopathogenic fungus, Metarhizium anisopliae.

The nucleotide (nt) sequence of a starvation-stress gene (ssgA) of the entomopathogenic fungus, Metarhizium anisopliae, and its deduced amino acid (aa) sequence were determined. The primary structure of the SSGA (96 aa; deduced M(r) = 9925; pI = 4.1) protein shares extensive similarities with fungal wall proteins of the 'hydrophobin' class, and the eight Cys residues and putative signal sequences are conserved. Secondary structure predictions suggest an additional resemblance to low-M(r) toxins and agglutinins. Northern (RNA) blot analysis and nuclear run-on assays demonstrated transcriptional control of expression of ssgA during nutrient deprivation and during formation of infection structures. Hybridizations of M. anisopliae genomic DNA indicate that there is only one form of ssgA in the genome.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and regulatory analysis of the cuticle-degrading-protease structural gene from the entomopathogenic fungus Metarhizium anisopliae.

The proteinaceous insect cuticle is an effective barrier against most microbes, but entomopathogenic fungi can breach it using extracellular proteases. We report here the isolation and characterization of a cDNA clone of the cuticle-degrading protease (Pr1) of Metarhizium anisopliae. The cDNA sequence revealed that Pr1 is synthesized as a large precursor (40.3 kDa) containing a signal peptide, a propeptide and the mature protein predicted to have a molecular mass of 28.6 kDa. The primary structure of Pr1 has extensive similarity with enzymes of the subtilisin subclass of serine endopeptidases and the serine, histidine and aspartate components of the active site in subtilisins are preserved. Proteinase K demonstrated the closest sequence similarity to Pr1 (61%) but Pr1 was twofold more effective than proteinase K at degrading isolated cuticles of Manduca sexta and 33-fold more effective at degrading structural proteins bound to the cuticle by covalent bonds. We postulate that the additional positively charged residues on the surface of the Pr1 molecule, as determined using proteinase K, may facilitate electrostatic binding to cuticle proteins which is a prerequisite for activity. Northern-blot analysis of RNA and nuclear run-on assays demonstrated transcriptional control of the expression of Pr1 during nutrient deprivation and during the formation of infection structures. Southern-blot analysis demonstrated that genes with significant homologies to Metarhizium Pr1 were present in the entomopathogens Aspergillus flavus and Verticillium lecanii but not Zoophthora (= Erynia) radicans.

Amino Acid Sequence↗
PubMed1992

The value of the local lymph node assay in quantitative structure-activity investigations.

The development of quantitative correlations between the physicochemical properties of a compound and its ability to act as a skin sensitizer is complicated by the number of variables associated with the current sensitization test data, combined with the absence of a truly objective end point. Recently, however, a novel approach to the assessment of skin sensitization potential, the local lymph node assay (LLNA), has been described, which determines the skin sensitization by measuring lymphocyte proliferation in lymph nodes draining the site of chemical exposure. The assay offers several advantages over traditional methods in the context of quantitative structure-activity relationship studies. In the present work, a range of bromoalkanes has been employed which demonstrate the robustness and reproducibility of the LLNA. Sensitizing activity increased with chain length up to a maximum at C15/C16, whereafter the response declined. The data were modelled against hydrophobicity, expressed as Clog P and (ClogP)2 to fit the biphasic nature of the results. The results demonstrate the utility of LLNA data for interpretation in the context of quantitative structure-activity relationships, the limited number of variables, inter-test reproducibility and quantitative end point, lending themselves to mathematical interpretations.

Animals↗
PubMed1992

Further development and clinical application of the stereotactic operating microscope.

Implementation of a Vicom-VME image processing workstation for the frameless stereotactic operating microscope system has allowed an improved interpolation algorithm for more accurate reconstruction of three-dimensional data, the integration graphically of multimodality imaging information, an additional stereotactic graphics display outside of the microscope optics and optional three-dimensional displays through communication with a Convex mini-supercomputer. A precision-milled coupling for reproducible attachment of the spark gap bracket to the microscope has eliminated the need for that transformation calculation during each case. Clinical utility has been greatest in providing navigational guidance to small subcortical or deep lesions and in defining the extent of preoperatively planned resection of larger, infiltrating tumors. Mean accuracy in the past 17 cases has been 3.5-6.5 mm. The frameless methodology has also enabled extracranial stereotactic surgical procedures in 7 patients.

Brain Neoplasms↗
PubMed1991

Frequency of urination and its effects on metabolism, pharmacokinetics, blood hemoglobin adduct formation, and liver and urinary bladder DNA adduct levels in beagle dogs given the carcinogen 4-aminobiphenyl.

The human urinary bladder carcinogen, 4-aminobiphenyl (ABP), is known to undergo hepatic metabolism to an N-hydroxy arylamine and its corresponding N-glucuronide. It has been proposed that these metabolites are both transported through the blood via renal filtration to the urinary bladder lumen where acidic pH can facilitate the hydrolysis of the N-glucuronide and enhance the conversion of N-hydroxy-4-aminobiphenyl (N-OH-ABP) to a reactive electrophile that will form covalent adducts with urothelial DNA. Blood ABP-hemoglobin adducts, which have been used to monitor human exposure to ABP, are believed to be formed by reactions within the erythrocyte involving N-OH-ABP that has entered the circulation from the liver or from reabsorption across the urothelium. To test these hypotheses directly, experimental data were obtained from female beagles given [3H]ABP (p.o., i.v., or intraurethrally). [3H]N-OH-ABP (i.v. or intraurethrally), or [3H]N-OH-ABP N-glucuronide (i.v.). Analyses included determinations of total ABP in whole blood and plasma, ABP-hemoglobin adducts in blood erythrocytes, ABP and N-OH-ABP levels (free and N-glucuronide) in urine, urine pH, frequency of urination (controlled by urethral catheter), rates of reabsorption of ABP and N-OH-ABP across the urothelium, and apparent volumes of distribution in the blood/tissue compartment. The major ABP-DNA adduct, N-(guan-8-yl)-4-aminobiphenyl, was also measured in urothelial and liver DNA using a sensitive immunochemical method. An analog/digital hybrid computer was then utilized to construct a multicompartmental pharmacokinetic model for ABP and its metabolites that separates: (a) absorption; (b) hepatic metabolism and distribution in blood and tissues; (c) ABP-hemoglobin adduct formation; (d) hydrolysis and reabsorption in the urinary bladder lumen; and (e) excretion. Using this model, cumulative exposure of the urothelium to free N-OH-ABP was simulated from the experimental data and used to predict ABP-DNA adduct formation in the urothelium. The results indicated that exposure to N-OH-ABP and subsequent ABP-DNA adduct formation are directly dependent on voiding frequency and to a lesser extent on urine pH. This was primarily due to the finding that, after p.o. dosing of ABP to dogs, the major portion of the total N-OH-ABP entering the bladder lumen was free N-OH-ABP (0.7% of the dose), with much lower amounts as the acid-labile N-glucuronide (0.3% of the dose).(ABSTRACT TRUNCATED AT 400 WORDS)

Aminobiphenyl Compounds↗
PubMed1991

Corpus callosotomy for intractable seizures in the pediatric age group.

The results of corpus callosotomy in 18 patients 16 years old and younger are presented. Eighty-three percent of our patients have had a significant improvement from the surgery (a decrease in seizure frequency of greater than 80% or no longer having generalized atonic, tonic, or tonic-clonic seizures). The procedure seems to be well tolerated in young patients, and we have not noted a postoperative deterioration in behavior, memory, or language function in our patients. One of our patients died in status epilepticus 3 months after surgery. Nevertheless, we have not encountered any serious morbidity in our other patients. Corpus callosotomy can be considered for children with intractable seizures, especially when generalized atonic, tonic, or tonic-clonic (whether primary or secondary) seizures are the major seizure type.

Adolescent↗
PubMed1991

Refinement of the relative alkylation index (RAI) model for skin sensitization and application to mouse and guinea-pig test data for alkyl alkanesulphonates.

A derivation, more rigorous than hitherto, of the Relative Alkylation Index (RAI) as a quantifier of carrier protein haptenation in skin sensitization tests is presented. It is shown that the RAI, which is a composite parameter made up of dose, reactivity and lipophilicity terms, is likely to require a higher weighting for the reactivity term in the case of non-adjuvant tests than in the case of Freund's adjuvant-based tests. Methyl alkane-sulphonates, RSO3Me with R ranging from n-C6H13 to n-C16H33, were found to be skin sensitizers in a mouse ear swelling test, in agreement with published findings in a guinea-pig adjuvant model. A structure-activity relationship consistent with the published RAI model was observed whereby, in tests at fixed molar induction (0.1 mM) and challenge concentrations (0.025 mM), the level of sensitization response at first increased with increasing chain length of R, then showed a reversal of this trend at the highest chain length (R = n-C16H33). That this is a genuine 'over-load effect', as reported for several other series of compounds examined in guinea-pig adjuvant models, is indicated by the finding that on reducing the induction concentration for the R = n-C16H33 compound the sensitization response was increased. Alkyl and alkenyl methane-sulphonates, MeSO3R (R = n-C12H25, n-C18H37 and R = oleyl) did not give significant sensitization in the mouse ear test. Although they are chemically less reactive than methyl alkanesulphonates, these compounds are reported to be strong sensitizers in guinea-pig adjuvant tests and to fit a common quantitative sensitization-structure-dose relationship with the methyl alkanesulphonates.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkanesulfonates↗
PubMed1991

Application of QSAR to biodegradation of linear alkylbenzene sulphonate (LAS) isomers and homologues.

An approach of initially testing various structure descriptors on a trial and error basis, followed by mechanistic hypothesis and simple mathematical modelling to refine the choice of descriptor, is applied to develop a quantitative structure-activity relationship (QSAR) correlating initial biodegradation relative rate constants with carbon number and substitution position for (4-sulphophenyl)-substituted alkanes (linear alkyl benzene sulphonates, LAS). The relative rate constants are found to be linearly correlated with a composite descriptor, [(0.5 + L-1)-12 + (0.5 + S-1)-12]1/2 where L and S are the carbon numbers of the longer and shorter chains, respectively, counting from the (4-sulphophenyl) substitution position.

Benzenesulfonates↗
PubMed1991

QSAR issues in aquatic toxicity of surfactants.

Acute lethal toxicity data for a range of anionic and non-ionic surfactants are analysed with the aim of determining whether QSARs can be developed relating toxicity to calculated log P (octanol/water) values. Approaches have been developed for dealing with deficiencies in the fragment method for calculation of log P when applied to surfactants, and when these are applied it is found that log P-based QSARs for surfactants can be developed and are very similar to QSARs established for non-surfactant organic compounds with non-specific modes of action.

Animals↗
PubMed1991

Experimental brain hyperthermia: techniques for heat delivery and thermometry.

An experimental canine brain model was developed to assess the effects of hyperthermia for a range of time and temperature endpoints, delivered within a specified distance of an interstitial microwave antenna in normal brain. The target temperature location was defined radially at 5.0 or 7.5 mm from the microwave source at the longitudinal location of maximum heating along the antenna in the left cerebral cortex. Temperatures were measured with fiberoptic probes in a coronal plane at this location in an orthogonal catheter at 1.0 mm intervals. Six antennas were evaluated, including dipole, modified dipole, and four shorted helical antennas with coil lengths from 0.5 to 3.9 cm. Antenna performance evaluated in tissue equivalent phantom by adjusting frequency at a fixed insertion depth of 7.8 cm or adjusting insertion depth at 915 MHz showed dipoles to be much more sensitive to insertion depth and frequency change than helical antennas. Specific absorption rate (SAR) was measured in a brain/skull phantom and isoSAR contours were plotted. In vivo temperature studies were also used to evaluate antenna performance in large and small canine brain tissues. A helical antenna with a 2.0 cm coil length driven at 915 MHz was chosen for the beagle experiments because of tip heating characteristics, well-localized heating along the coil length, and heating pattern appropriate to the smaller beagle cranial vault. Verification of lesion dimensions in 3-D was obtained by orthogonal MRI scans and histology to document the desired heat effect, which was to obtain an imagable lesion with well-defined blood-brain-barrier breakdown and necrotic zones. The desired lesion size was between 1.5 to 2.5 cm diameter radially, in the coronal plane with the greatest diameter.

Animals↗
PubMed1991

Critical considerations in the immunochemical detection and quantitation of antigenic biomarkers.

The formation of covalent adducts as a result of the interaction of metabolically activated chemicals with host macromolecules is a common critical event in mutagenic, carcinogenic, and immunologic phenomena. Because of their antigenicity and their immunogenicity, covalent adducts may be detected using sensitive immunochemical techniques. The immunochemical approaches to biomonitoring and molecular dosimetry of DNA damage are particularly attractive because they allow sensitive quantitation of specific DNA adducts present in small samples and do not rely on the use of radiolabeled adducts. Two examples of biomarker immunoassay development are presented: an avidin/biotin-amplified ELISA for the major DNA adduct of the human bladder carcinogen 4-aminobiphenyl (ABP), and a particle concentration fluorescent immunoassay (PCFIA) for the major protein adduct associated with toxicity by the prototype hepatotoxin acetaminophen. The examples illustrate critical steps in the development of biomarker immunoassays which include selection of the relevant adduct, preparation of an appropriate immunogen, immunization, characterization of antisera, and development of application-specific sample processing techniques for biomarker quantitation. Immunochemical procedures may be combined with other analytical techniques to form hybrid systems which take advantage of both the antigenicity and the physical or chemical properties of a biomarker to achieve greater specificity and/or sensitivity. The future usefulness of these new tools of molecular epidemiology will depend on a compound-by-compound validation of methods and critical evaluation of the biologic importance of the particular antigenic biomarker as an indicator of exposure and as an indicator of risk.

Acetaminophen↗
PubMed1991

Immunohistochemical localization and quantification of the 3-(cystein-S-yl)-acetaminophen protein adduct in acetaminophen hepatotoxicity.

Acetaminophen overdose causes severe hepatotoxicity in humans and laboratory animals, presumably by metabolism to N-acetyl-p-benzoquinone imine: and binding to cysteine groups as 3-(cystein-S-yl)acetaminophen-protein adduct. Antiserum specific for the adduct was used immunohistochemically to demonstrate the formation, distribution, and concentration of this specific adduct in livers of treated mice and was correlated with cell injury as a function of dose and time. Within the liver lobule, immunohistochemically demonstrable adduct occurred in a temporally progressive, central-to-peripheral pattern. There was concordance between immunohistochemical staining and quantification of the adduct in hepatic 10,000g supernate, using a quantitative particle concentration fluorescence immunoassay. Findings include: 1) immunochemically detectable adduct before the appearance of centrilobular necrosis, 2) distinctive lobular zones of adduct localization with subsequent depletion during the progression of toxicity, 3) drug-protein binding in hepatocytes at subhepatotoxic doses and before depletion of total hepatic glutathione, 4) immunohistochemical evidence of drug binding in the nucleus, and 5) adduct in metabolically active and dividing hepatocytes and in macrophagelike cells in the regenerating liver.

Acetaminophen↗
PubMed1990

Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen protein adducts in subcellular liver fractions following a hepatotoxic dose of acetaminophen.

The hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein-S-yl)acetaminophen protein adducts. Using a sensitive and specific immunochemical assay, we quantitated the formation of these protein adducts in liver fractions and serum after administration of a hepatotoxic dose of acetaminophen (400 mg/kg) to B6C3F1 mice. Adducts in the cytosolic fraction increased to 3.6 nmol/mg protein at 2 hr and then decreased to 1.1 nmol/mg protein by 8 hr. Concomitant with the decrease in adducts in the cytosol, 3-(cystein-S-yl)acetaminophen protein adducts appeared in serum and their levels paralleled increases in serum alanine aminotransferase. Microsomal protein adducts peaked at 1 hr (0.7 nmol/mg protein) and subsequently decreased to 0.2 nmol/mg at 8 hr. The 4000 g pellet (nuclei, plasma membranes, and cell debris) had the highest level of adducts (3.5 nmol/mg protein), which remained constant from 1 to 8 hr. Evaluation of fractions purified from a 960 g pellet indicated that the highest concentration of 3-(cystein-S-yl)acetaminophen protein adducts was located in plasma membranes and mitochondria; peak levels were 10.3 and 5.1 nmol/mg respectively. 3-(Cystein-S-yl)acetaminophen protein adducts were detected in nuclei only after enzymatic hydrolysis of the proteins. The localization of high levels of 3-(cystein-S-yl)acetaminophen protein adducts in plasma membranes and mitochondria may play a critical role in acetaminophen toxicity.

Acetaminophen↗
PubMed1990

Acetaminophen-induced alterations in pancreatic beta cells and serum insulin concentrations in B6C3F1 mice.

Administration of acetaminophen (500 mg/kg) to male B6C3F1 mice resulted in alterations of pancreatic beta cell ultrastructure. These alterations were characterized by pronounced intercellular spaces, cytoplasmic vacuolization, damaged membranes of cytoplasm, secretory granules, and other organelles, and pyknotic nuclei with disrupted membranes. Concomitant with these changes, acetaminophen also caused increaes in serum insulin concentrations from 24 microU/ml at 0 time to 160 microU/ml at 8 hr and increases in serum alanine aminotransferase (ALT) concentrations from 42 to 13,279 U/liter, which indicated hepatic damage. Quantitation of 3-(cystein-S-yl)acetaminophen adducts in hepatic 10,000g supernatant protein using a particle concentration fluorescence immunochemical assay indicated a positive correlation between binding and the occurrence of the hepatotoxicity consistent with what has been previously reported; however, 3-(cystein-S-yl)acetaminophen protein adducts were not detected in pancreatic 10,000g supernatant. Immunohistochemical analysis of the liver and pancreas from acetaminophen-treated mice revealed acetaminophen-protein adducts in the centrilobular regions of the liver but not in the pancreatic islets. Doses of 100 and 200 mg/kg produced no evidence of hepatotoxicity and no increase in serum insulin; 300 mg/kg and higher doses produced both hepatotoxicity and increased serum insulin concentrations. A comparison of the time course for the increase in serum levels of ALT and insulin following a toxic dose of acetaminophen indicated that the increase in ALT preceded the increase in insulin. Thus the hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein-S-yl)acetaminophen protein adducts in liver, which supports the concept that this toxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinone imine; however, the toxicity of acetaminophen to beta cells in the pancreas is apparently not mediated by this mechanism.

Acetaminophen↗