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Biomedical subjects

B J Del Tito

Publications and source records attributed to B J Del Tito.

12 recordsLinked to original sources
PubMed1998

Automated fluorescent analysis procedure for enzymatic mutation detection.

The Enzymatic Mutation Detection (EMD) assay detects mutations or polymorphisms in DNA. The assay procedure takes <1 h and is followed by electrophoretic detection. We report an automated procedure, using fluorescently labeled probe and quantitative analysis on the ABI Prism 377 DNA Sequencer, that improves on earlier methods (1, 2) by eliminating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio. The EMD assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves the heteroduplex molecules at the mismatch site, forming two shorter fragments that are resolved by gel electrophoresis. Unlike existing mutation techniques, the EMD method uses a single protocol to identify point mutations, deletions, and insertions for all DNA fragments. Test DNA samples are assayed directly from PCR reactions, and fragments up to 4 kb in size have been assayed successfully. A independent analysis on the p53 tumor suppressor gene from clinical samples has shown 100% sensitivity and 94% specificity. Because the fluorescent EMD assay has been optimized for high signal-to-noise ratios, mutations can be identified in mixed samples containing up to a 20-fold excess of normal DNA.

Animals↗
PubMed1995

Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli.

In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.

Bacterial Proteins↗
PubMed1991

The use of particle concentration fluorescence immunoassay technology for the analysis of rDNA products.

The electrophoretic and immunological techniques typically used to detect potentially useful biopharmaceutical proteins are sensitive with detection limits in the nanogram range. However, quantitation of a recombinant protein can be cumbersome, and involve large numbers of samples throughout process optimization schemes. Although electrophoretic methods (i.e., SDS-PAGE and Western blots) now avail themselves to quantitation by densitometry, these techniques are time consuming because of the lack of appropriate automated systems. Biological activity assays, when available, often require relatively pure material and are not suitable for analyzing and quantitating impure or semi-purified samples, typical of the fermentation milieu. The optimization of several rDNA-derived protein systems from both prokaryotic and eukaryotic hosts has been completed using PCFIA, a rapid, sensitive system with high throughput. The development of Particle Concentration Fluorescence Immunoassay (CFIA) procedures for several of these rDNA-derived proteins of interest as potential biopharmaceuticals (e.g., alpha-1-antitrypsin, tPA, soluble CD4, and a malaria vaccine candidate) are discussed.

Binding, Competitive↗
PubMed1990

Effects of mutations altering SOS regulation on a nalidixic acid-inducible system for the production of heterologous proteins in Escherichia coli.

The major leftward early promoter of phage lambda pL, has frequently been used to drive expression of heterologous genes in Escherichia coli. pL is typically maintained fully repressed by the lambda cl protein. When induction of heterologous protein synthesis is desired, one of several potential mechanisms of destroying cl function is employed and the expression of the foreign gene commences. One method of derepressing pL involves exposing cells to nalidixic acid, which results in the "activation" of RecA protein and the subsequent RecA-mediated proteolytic cleavage of cl. Activated RecA also mediates the cleavage of the E. coli LexA protein, resulting in induction of the SOS regulon (at least 15 E. coli genes, including recA). We have examined the effect of two chromosomal mutations on the productivity of nalidixic acid inductions. One of the tested mutations (recA o) increased the intracellular concentration of RecA prior to induction; the other (lexAind-) resulted in a mutated lexA protein insensitive to RecA-mediated cleavage. These mutations were introduced into a strain carrying a cl+ defective lysogen. Synthesis of two heterologous proteins, human alpha 1-antitrypsin and a fusion protein partially derived from the Plasmodium falciparum circumsporozoite surface antigen, was examined in the wild-type and mutant strains. The maximum alpha-1 antitrypsin concentration achieved was improved by 50% when the recA o strain was used rather than the wild-type; however, only smaller changes (20% or less) in the maximum concentration of the malaria fusion protein were observed. Use of the lexAind- strain resulted in a decrease in the maximum concentration attained for both heterologous products.

Animals↗
PubMed1988

Detection of alpha 1-antitrypsin from recombinant Escherichia coli lysates utilizing the particle concentration fluorescence immunoassay.

A particle concentration fluorescence immunoassay for the quantitative determination of human alpha 1-antitrypsin in Escherichia coli is described. The principle advantages of this system are speed, automation and a high level of sensitivity compared with enzyme assays and Western blot analysis. Two monoclonal antibodies recognizing different epitopes on the protein are used for the detection and quantitation of alpha 1-antitrypsin. This method has a measured range of 30-2500 ng/ml with a mean accuracy of 5.5% and a precision of 5.1%.

Epitopes↗
PubMed1984

Protection against 3-methylcholanthrene-induced skin tumorigenesis in Balb/C mice by ellagic acid.

Topical application of ellagic acid, a naturally occurring dietary plant phenol, to Balb/C mice resulted in significant protection against 3-methylcholanthrene (MCA)-induced skin tumorigenesis. Ellagic acid was found to be an effective inhibitor of tumor formation whether the tumor data are considered as percent mice with tumors, cumulative number of tumors, tumors per mouse or tumors per tumor bearing animal as a function of the number of weeks on test. By 8, 10, 12, 14, and 16 weeks of testing, the number of tumors per mouse in the group receiving MCA alone was 2.0, 3.4, 4.0, 4.9 and 5.3, respectively, whereas the corresponding numbers in the group receiving MCA plus 2 mumol ellagic acid were 0, 0.3, 0.4, 0.6 and 1.2, respectively. At the termination of the experiment (16 weeks) aryl hydrocarbon hydroxylase (AHH) activity in skin and liver and the extent of 3H-BP-binding to skin, liver and lung DNA were determined and both of these parameters were found to be significantly inhibited in the animals treated with ellagic acid. These results indicate that ellagic acid can inhibit the metabolism of polyaromatic hydrocarbons and modulate skin carcinogenesis induced by these chemicals.

Animals↗
PubMed1984

Ellagic acid: a potent naturally occurring inhibitor of benzo[a]pyrene metabolism and its subsequent glucuronidation, sulfation and covalent binding to DNA in cultured BALB/C mouse keratinocytes.

The metabolism of [3H]benzo[a]pyrene (BP) by cultured primary keratinocytes prepared from BALB/C mouse epidermis was found to be largely inhibited by the dietary plant phenol, ellagic acid. Varying concentrations of ellagic acid added to the keratinocyte cultures resulted in a dose-dependent inhibition of the cytochrome P-450-dependent monooxygenases aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECD). The major organic solvent-extractable metabolites found intracellularly in the cultured cells were trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP-7,8-diol) and 3-hydroxybenzo[a]pyrene (3-OH-BP), although small amounts of 9-hydroxybenzo[a]pyrene, quinones and trans-9,10-dihydro-9,10-dihydroxybenzo[a]-pyrene (BP-9,10-diol) were also present. The major organic solvent-extractable metabolites found in the extracellular culture medium were BP-7,8-diol and BP-9,10-diol, with smaller quantities of unconjugated phenols and quinones. The major intracellular and extracellular water-soluble metabolites of BP were conjugated with glucuronide (primarily 3-OH-BP and several BP-quinones), and to a lesser extent with sulfate (primarily BP-7,8-diol). Both intracellular and extracellular metabolism of organic solvent-extractable and water-soluble conjugates was significantly inhibited by ellagic acid in a dose-dependent manner. The intracellular enzyme-mediated binding of BP to mouse keratinocyte DNA was also largely inhibited in a dose-dependent fashion by ellagic acid. Our results indicate that cultured primary mouse keratinocytes offer a useful model system for studying factors affecting the metabolic activation and detoxification of polycyclic aromatic hydrocarbon carcinogens in the epidermis, and that polyphenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer that results from exposure to these environmental chemicals.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1984

In vivo metabolism of topically applied benzo[a]pyrene-4,5-oxide in neonatal rat skin.

The metabolism of benzo[a]pyrene (BP)-4,5-oxide in the skin and liver of neonatal rats was studied after topical application of the arene oxide in vivo. The metabolism of BP-4,5-oxide was time-dependent and showed a 2-h maximum for BP-4,5-dihydrodiol formation in both skin and liver. Product formation was also dose-dependent. Inhibitors of epoxide hydrolase such as clotrimazole, 1,1,1,-trichloropropene oxide, and cyclohexene oxide largely abolished the formation of BP-4,5-dihydrodiol. The rapid biotransformation of arene oxides such as BP-4,5-oxide in the skin emphasizes the potential importance of epoxide hydrolase in the activation and inactivation of polycyclic aromatic hydrocarbons. Furthermore, the topically applied arene oxide also penetrated the skin and was rapidly metabolized in the liver as well.

Administration, Topical↗
PubMed1984

Epidermal benzo[a]pyrene metabolism and DNA-binding in Balb/C mice: inhibition by ellagic acid.

Topical application of ellagic acid, a common plant phenol, to control or to 3-methylcholanthrene (3-MC) pretreated Balb/C mice, resulted in significant inhibition of hepatic and epidermal microsomal aryl hydrogen hydroxylase activity, and of benzo[a]pyrene (BP) binding to epidermal and hepatic DNA in vivo. In vitro addition of ellagic acid (0.25 mM) to epidermal microsomal incubation systems from either control or 3-MC-treated animals resulted in 62-75% inhibition of BP binding to calf thymus DNA. These studies suggest that ellagic acid could prove useful in understanding and/or modulating polyaromatic hydrocarbon carcinogenesis.

Animals↗
PubMed1984

Clotrimazole, an inhibitor of epidermal benzo(a)pyrene metabolism and DNA binding and carcinogenicity of the hydrocarbon.

Clotrimazole, a topically applied imidazole antifungal agent widely used in dermatological practice, was shown to be a potent inhibitor of the epidermal metabolism of benzo(a)pyrene (BP) and its microsomal enzyme-mediated binding both to neonatal rat epidermal DNA in vivo and to calf thymus DNA in vitro. Varying concentrations of clotrimazole added to in vitro incubation systems resulted in a dose-dependent inhibition of cytochrome P-450-dependent microsomal aryl hydrocarbon hydroxylase (AHH) in control animals as well as in animals pretreated with topical application of known inducers of the enzyme. Inhibition of epidermal AHH by topically applied clotrimazole was time and dose dependent. The 50% inhibition of clotrimazole for epidermal AHH ranged from 0.12 to 0.25 microM, which suggests that clotrimazole is among the most potent inhibitors of epidermal AHH yet identified. Clotrimazole was also found to be a potent inhibitor of epoxide hydrolase activity in vitro with a 50% inhibition at 0.1 mM. High-pressure liquid chromatographic analysis of the metabolism of BP in rat epidermal microsomes revealed substantial inhibition of metabolite formation by clotrimazole. This occurred in microsomes prepared from untreated as well as animals pretreated with inducers of the enzyme. Furthermore, a single topical application of clotrimazole resulted in 80 and 30% induction of epidermal and hepatic glutathione S-transferase activity, respectively. Topical application of clotrimazole to the skin of BALB/c mice substantially increased the latent period for the development of skin tumors by 3-methylcholanthrene. These studies indicate that clotrimazole is an extremely potent inhibitor of epidermal BP metabolism and of the DNA-binding of polycyclic aromatic hydrocarbon (PAH) carcinogens, and is an enhancer of enzymes necessary for detoxification of the PAH. Clotrimazole also reduces the formation of carcinogenic and mutagenic metabolites of BP in vitro and in vivo and inhibits induction of skin tumors by the PAH. These data indicate that the imidazole antifungal clotrimazole offers promise as an agent useful for the modulation of PAH cancer risk in the skin.

Animals↗
PubMed1983

Inhibition of epidermal metabolism and DNA-binding of benzo[a]pyrene by ellagic acid.

Ellagic acid, a common plant phenol, was shown to be a potent inhibitor of epidermal microsomal aryl hydrocarbon hydroxylase (AHH) activity in vitro, and of benzo[a]pyrene (BP)-binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. The in vitro addition of ellagic acid (0.25-2.0 microM) resulted in a dose-dependent inhibition of AHH activity in epidermal microsomes prepared from control or carcinogen-treated animals. The I50 of ellagic acid for epidermal AHH was 1.0 microM making it the most potent inhibitor of epidermal AHH yet identified. In vitro addition of ellagic acid to microsomal suspensions prepared from control or coal tar-treated animals resulted in 90% inhibition of BP-binding to calf thymus DNA. Application of ellagic acid to the skin (0.5-10.0 mumol/10 gm body wt) caused a dose-dependent inhibition of BP-binding to epidermal DNA. Our results suggest that phenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer from environmental chemicals.

Animals↗