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Biomedical subjects

G Bailey

Publications and source records attributed to G Bailey.

At least 37 records · Page 2Linked to original sources

Sending a copy of the letter to the general practitioner also to the parents in a child development centre: does it work?

Following concerns that a considerable extra workload would be involved in sending a copy of the GP outpatient letter to parents following a medical consultation, 100 consecutive letters from the CDC were analysed to see if they could be sent to parents. Changes would have been necessary in 11, of which 6 would need an extra letter to the GP conveying additional specific information. Letters were generally intelligible, but it was recommended that they could be written in a more simple way for parents. Following these recommendations, copies of letters to the GP were sent to parents and the practice reviewed 2 years later. GP letters from 100 consecutive patients who had just visited the Centre were analysed and found to have been sent in 94. Of the remaining six, five were not sent because they contained information specific to the GP. It was felt that this could be rectified fairly easily. An anonymous questionnaire, sent to those receiving a copy of the letter, indicated that it had been very well received. It was felt that this policy was most helpful to the parents and justified a small additional workload.

Child↗

Dry extracorporeal shock wave lithotripsy for treatment of ureterolithiasis and nephrolithiasis in a dog.

A second-generation lithotriptor was used to perform dry extracorporeal shock wave lithotripsy in a dog with ureterolithiasis, nephrolithiasis, and chronic renal failure. Previous studies on the use of lithotripsy in dogs have involved first-generation machines and have primarily concentrated on acute and chronic effects of lithotripsy in experimental models. Treatment in this dog resulted in resolution of ureteral obstruction, ureterolithiasis, and nephrolithiasis, and avoided complications associated with ureteral and renal surgery. The only complication was substantial hematuria of 12 hours' duration immediately after the procedure. Second-generation lithotripsy may offer an effective treatment for ureterolithiasis or nephrolithiasis in selected dogs.

Animals↗

Dietary chlorophyllin is a potent inhibitor of aflatoxin B1 hepatocarcinogenesis in rainbow trout.

Epidemiological and experimental evidence indicates a strong relationship between diet and cancer. The purpose of this study was to examine the potential of chlorophyllin (CHL), a food-grade derivative of the ubiquitous green plant pigment chlorophyll, to inhibit experimental carcinogenesis. We report that CHL is a potent, dose-responsive inhibitor of aflatoxin B1 DNA adduction and hepatocarcinogenesis in the rainbow trout model when fed with carcinogen. CHL neither promoted nor suppressed carcinogenesis with chronic postinitiation feeding. By molecular dosimetry analysis, reduced aflatoxin B1-DNA adduction accounted quantitatively for reduced tumor response up to 2000 ppm dietary CHL, but an additional protective mechanism was operative at 4000 ppm CHL. The finding of potent inhibition (up to 77%) at CHL levels well within the chlorophyll content of some green leafy vegetables may have important implications in intervention and dietary management of human cancer risks.

Aflatoxin B1↗

Evidence for ras gene mutation in 2-amino-3-methylimidazo[4,5-f]quinoline-induced colonic aberrant crypts in the rat.

Aberrant crypt foci (ACF) are putative preneoplastic lesions that develop after treatment of animals with colon carcinogens, including cooked-meat heterocyclic amines such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Male F344 rats given IQ by gavage on alternating days for 2 wk (130 mg/kg body weight) and killed 12 wk after the final carcinogen dose had an average of 4.4 ACF/colon and an average of 3.2 crypts/focus. The DNA from these ACF was amplified by the polymerase chain reaction and analyzed by 3'-primer mismatch and direct sequencing methods for mutations in the Ki-ras proto-oncogene. Of the 37 IQ-induced ACF screened, three contained a GGT-->GAT mutation in codon 12 and one contained a GGC-->GCC mutation in codon 13. The approximately 11% frequency of mutation in IQ-induced ACF is within the range of previous ACF studies of azoxymethane, which reported a 7-37% incidence of Ki-ras mutation. These findings suggest that for both compounds, ras mutations occur during early stages of colorectal tumorigenesis. However, while ras mutations can be detected with increasing frequency in azoxymethane-induced adenomas and carcinomas, they are reportedly absent in IQ-induced colon tumors. Thus, for IQ and related compounds additional factors (possibly increased cell proliferation) may be important in the later stages of colorectal tumorigenesis.

Animals↗

Mechanisms of chlorophyllin anticarcinogenesis against aflatoxin B1: complex formation with the carcinogen.

Chlorophyllin (CHL), a food-grade derivative of the green plant pigment chlorophyll, has recently been shown in this laboratory to be a potent inhibitor in vivo of hepatic aflatoxin B1 (AFB1)-DNA adduction and hepatocarcinogenesis (Breinholt et al. (1995) Cancer Res. 55, 57-62). We report here that CHL forms a strong noncovalent complex with AFB1 in vitro (dissociation constant (Kd) by Scatchard analysis = 1.4 (+/- 0.4) microM based on copper chlorin content), which may contribute to its anticarcinogenic activity. Kd values for the related porphyrins chlorine e6, protoporphyrin IX, and zinc protoporphyrin IX were also of the same order of magnitude as that of the commercial CHL. Mole ratio analysis provided evidence that all porphyrins examined associate with AFB1 at an approximate one to one stoichiometric ratio. Energy minimization computer modeling of the complex indicates a favorable association energy of -20 kcal/mol, independent of oxidation state of the 8,9-double bond of AFB1. AFB1 incubated in vitro with liver microsomes in the presence of added CHL showed comparable levels of inhibition in the production of several phase 1 metabolites, including the postulated procarcinogenic metabolite AFB1 8,9-epoxide. Kinetic analysis of microsome-catalyzed AFB1-DNA adduction suggested a CHL inhibition constant near 10 microM chlorin. In vivo, addition of CHL to concentrated AFB1 solutions followed by gavage administration resulted in dose-dependent inhibition of hepatic AFB1-DNA adduction, whereas the same dosages of AFB1 and CHL incorporated into a single bolus of trout diet for gavage provided less protection at all CHL doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin B1↗

Contact-dependent transfer of the galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture.

In a study to investigate early interactions of Entamoeba histolytica with epithelial cell monolayers, we found that a monoclonal antibody (MAb), CD6, against an ameba surface antigen recognized the lateral surface of epithelial cells after coculture with trophozoites. Display of the CD6 antigen on the epithelial cells necessitated contact with active trophozoites. It was found neither at 4 degrees C, nor with prefixed trophozoites, nor with trophozoite-conditioned media, nor when a filter prevented direct contact. Monolayers exposed to amebic sonicates or detergent lysates showed random immunostaining. Access to the antigenic site was limited, as immunostaining occurred predominantly with subconfluent monolayers. CD6 epithelial cell binding was first observed after 5 min of coculture; trophozoite-mediated target cell lysis was not detected until 15 min following coculture. Epithelial cell immunostaining occurred with some other ameba-specific antibodies but not with MAbs raised against the 170-kDa subunit of the galactose-N-acetylgalactosamine (Gal/GalNAc)-specific lectin. The CD6 MAb as well as some other ameba-specific antibodies immunoprecipitated from trophozoite lysates the same bands as the MAbs against the cysteine-rich domain of the 170-kDa subunit of the Gal/GalNAc-specific lectin. Why the latter MAbs failed to stain epithelial cells in the vicinity of attached trophozoites is presently unknown. We concluded that E. histolytica trophozoites transferred the intact amebic Gal/GalNAc-specific lectin or a portion of it to the lateral surface of epithelial cells. This juxtacrine transfer preceded killing of target cells.

Animals↗

Cloning and expression of an NADP(+)-dependent alcohol dehydrogenase gene of Entamoeba histolytica.

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entamoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoelectric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP(+)-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADH1 clone had an open reading frame that was 360 amino acids long and encoded a protein of approximately 39 kDa (calculated size). EhADH1 showed a 62% amino acid identity with the tetrameric NADP(+)-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADH1 contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADH1 lacked a 23-amino acid stretch present in other alcohol dehydrogenases that includes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-S-transferase fusion protein showed the expected NADP(+)-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADH1 fusion protein were inhibited by pyrazole and 4-methylpyrazole.

Alcohol Dehydrogenase↗

Anticarcinogenesis in fish.

Rainbow trout respond to a range of natural and synthetic dietary tumor modulators. Observed modulations of final tumor incidence include inhibition, promotion and cocarcinogenesis, depending on modulator, carcinogen, target organ, and relative order of carcinogen and modulator exposure. Despite several obvious limitations (e.g. lung, colon, mammary gland, bladder are not available as target organs), the trout model possesses several important features that have made it valuable for tumor modulation studies. (1) The comparative advantage. Since rodents are not perfect human surrogates, studies with alternative vertebrates such as trout have provided important comparative mechanism information for confident extrapolation of animal studies to humans. For example, beta-naphthoflavone appears to inhibit aflatoxin B1 hepatocarcinogenicity by species-independent mechanisms that readily extrapolate to humans. By contrast other modulators, including butylated hydroxyanisole, inhibit aflatoxin B1 hepatocarcinogenesis in rats by an enabling mechanism shown to be absent in trout, namely the induction of an aflatoxin B1-specific glutathione S-transferase isozyme. Interestingly, evidence is presently lacking that this determinant mechanism would be operative in humans. (2) The sensitivity advantage. Trout sensitivity and small body size at exposure have permitted tumor studies with carcinogens and HPLC-purified anticarcinogen intermediates too scarce to study in rodents. (3) The low cost advantage. The very low cost of trout tumor studies has enabled statistically challenging issues in molecular dosimetry, dose-response, and risk-benefit analysis to be addressed using as many as 9600 animals per tumor study at modest budget. In particular, these designs provide modulator-mediated alterations in precisely determined carcinogen TD50 values, rather than changes in simple tumor incidence, to quantify more rigorously modulator potencies for tumor inhibition or promotion as a function of dietary concentrations.

Animals↗

Primary structures of alcohol and aldehyde dehydrogenase genes of Entamoeba histolytica.

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite E. histolytica under the anaerobic conditions found in the lumen of the colon. With the goal of finding new targets for anti-amebic drugs, the E. histolytica NADP(+)-dependent alcohol dehydrogenase gene (EhADH1; EC 1.1.1.2) and an aldehyde dehydrogenase gene (EhALDH1; EC 1.3.2) were cloned. The EhADH1 alcohol dehydrogenase gene encoded -39 kDa protein with 62 and 60% amino acid identities, respectively, with NADP(+)-dependent alcohol dehydrogenases of anaerobic bacteria Thermoanaerobium brockii and Clostridia beijerinckii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. An EhADH1-glutathione-S-transferase fusion protein showed the expected NADP(+)-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADH1 fusion protein were inhibited by pyrazole and 4-methyl pyrazole. The E. histolytica aldehyde dehydrogenase EhALDH1 gene encoded a 60 kDa protein, which showed a 36% amino acid identity over a 451 amino acid overlap with the human stomach aldehyde dehydrogenase (ALDH3).

Alcohol Oxidoreductases↗

Expression of ras genes in rainbow trout liver.

Members of the ras gene family have been studied in a variety of species. Two ras genes expressed in the normal liver of rainbow trout, ras-1 and ras-2, as well as a portion of a genomic ras-1 allele, are described for the first time in this report. Over 500 bp of trout ras-1 and at least 300 bp of trout ras-2 genes expressed in normal liver have been sequenced; DNA homology to the human ras genes ranges from 76.8% to 87.1%. The base changes resulting from over 400 million years of evolutionary divergence between the species were primarily silent, with few changes in protein sequence. The partial DNA sequence of the genomic ras-1 allele has 86.8% homology to the first two exons of human c-Ha-ras, and its intron has several conserved sequences characteristic of vertebrate intron-exon junctions. However, the predicted amino acid sequence of trout ras-1 differs at only one of the first 172 amino acid residues from human c-Ki-ras with the alternate exon 4b. Since trout ras-1 differs at 17 and 18 residues from the human c-N-ras and c-Ha-ras proteins, respectively, over this region, we conclude that trout ras-1 is a c-Ki-ras gene. The highly conserved nature of this gene suggests that the ras p21 protein has identical functions in normal and neoplastic cells among higher and lower vertebrates.

Animals↗

Analysis of ras gene mutations in rainbow trout liver tumors initiated by aflatoxin B1.

The suspect human hepatocarcinogen aflatoxin B1 (AFB1) is a well-known potent initiator of hepatic tumors in rainbow trout (Oncorhynchus mykiss). Both hepatocellular carcinomas and mixed hepatocellular/cholangiocellular carcinomas are induced by AFB1 in trout, with the mixed form predominating. We previously isolated two c-ras genes from trout liver cDNA, and in the present study we analyzed DNA from 14 AFB1-induced trout liver tumors for point mutations in exon 1 of both genes. Using the polymerase chain reaction (PCR) and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated tumor DNAs showed evidence of activating point mutations in the trout c-Ki-ras gene. Of the 10 mutant ras genotypes, seven were codon 12 GGA----GTA transversions, two were codon 13 GGT----GTT transversions, and one was codon 12 GGA----AGA transition. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive evidence for two codon 12 GGA----GTA mutations, one codon 12 GGA----AGA mutation, and one codon 13 GGT----GTT mutation, in complete agreement with the oligonucleotide hybridization results. No mutations were detected in exon 1 of a second trout ras gene also expressed in liver, nor in DNA from control livers. This is the first report of experimentally induced ras gene point mutations in a lower vertebrate fish model. The results indicate that the hepatocarcinogen AFB1 induces c-Ki-ras gene mutations in trout similar to those in rat liver tumors.

Adenoma, Bile Duct↗

Replication blocks and sequence interaction specificities in the codon 12 region of the c-Ha-ras proto-oncogene induced by four carcinogens in vitro.

We have examined the region surrounding codon 12 in the human c-Ha-ras proto-oncogene in vitro to determine the reaction intensities at the guanine nucleotides after exposure to the mycotoxin aflatoxin B1, to one of its human metabolites aflatoxin M1, to dimethyl sulfate, and to the major ultimate carcinogen of benzo(a)pyrene, benzo(a)pyrene diol-epoxide. Among the adducts produced, those at N-7-guanyl sites are alkali-labile and can be identified using a variation of the Maxam-Gilbert sequencing procedure. Data indicate that the guanine nucleotides of codon 12 have above average potential for adduct formation by the genotoxins when compared to other guanine sites, but were not the strongest sites. This codon 12 region has been inserted into single-stranded M13 phage, exposed to several of the genotoxins, and used as a template for DNA synthesis in vitro. There is a sequence-specific variation in polymerase inhibition at various adducted nucleotide sites, but replication blocks are not preferentially seen at the carcinogen-adducted guanines of codon 12. These results indicate that the predominance of point mutations which are detected in vivo at codon 12 do not reflect sequence-mediated preferential susceptibility of these sites to initial DNA adduction or to replication errors. The mechanisms controlling these sequence effects are not currently understood, and attempts to predict relative alkylation or termination frequencies based solely on local DNA sequence are not reliable.

Aflatoxin B1↗

Site-specific aflatoxin B1 adduction of sequence-positioned nucleosome core particles.

The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles. We have circumvented this problem by using nucleosomes that are homogenous in DNA sequence and hence in DNA-histone contact points. A cloned DNA fragment containing a sea urchin 5 S gene which precisely positions a histone octamer was employed. By using 32P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels. The small methylating agent dimethyl sulfate and the bulky alkylating agent aflatoxin B1-dichloride (AFB1-Cl2) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in the sequence-positioned core particle. We find dimethyl sulfate to bind with equal preference to naked or nucleosomal DNA. In contrast, AFB1-Cl2 binding is suppressed an average of 2.4-fold at guanyl sites within nucleosomes compared with AFB1-Cl2 affinity at the corresponding site in naked DNA. The DNA is more accessible in regions near the particle boundary. We observe no other histone-imposed localized changes in AFB1-Cl2 sequence specificity. Further, sites of DNase I cleavage or proposed DNA bending show neither enhanced nor reduced AFB1-Cl2 adduction to N7-guanine. Since AFB1-Cl2 binding sites lie in the major groove, nucleosomal DNA appears accessible to AFB1-Cl2 at all points of analysis but with an access which is uniformly restricted in the central 100 nucleotides of the core particle. The data available do not indicate further localized or site-specific perturbations in DNA interactions with the two carcinogens studied.

Aflatoxin B1↗

Gene-specific differences in the aflatoxin B1 adduction of chicken erythrocyte chromatin.

Mature and immature chicken erythrocyte nuclei were treated with activated aflatoxin B1 (2,3-dichloroaflatoxin B1), producing covalently bound DNA adducts. This reaction produces alkali-labile sites in the DNA which can be identified by using a variation of the Maxam-Gilbert sequencing procedure. We determined the aflatoxin B1 accessibility of defined regions of the erythroid genome by using different specific probes and monitoring the disappearance of similar-sized fragments generated by restriction enzyme digestion. The genes studied were the erythroid-specific beta-globin and histone H5 genes, which are potentially active in mature erythroid nuclei and transcriptionally active in immature erythrocytes, and the vitellogenin and ovalbumin genes, which are both transcriptionally inactive in these cells. The beta-globin and histone H5 genes were more accessible than the repressed vitellogenin and ovalbumin genes to aflatoxin B1 modification in mature and immature erythroid chromatin. Micrococcal nuclease was used to probe the nucleosomal organization of active (beta-globin and histone H5) and repressed (vitellogenin and ovalbumin) genes in chicken erythrocytes. The vitellogenin and ovalbumin genes show a canonical nucleosome repeat pattern in mature and immature chicken erythrocyte nuclei. In contrast, the beta-globin and histone H5 genes lack a distinct nucleosomal repeat pattern in these cells. These results support the hypothesis that transcriptionally active genes are preferentially accessible to carcinogen modification because of their disrupted chromatin structure.

Aflatoxin B1↗