Tobacco budworm dihydrofolate reductase is a promising target for insecticide discovery.

Structural differences in dihydrofolate reductases from different species have been exploited to develop specific inhibitory molecules, such as chemotherapeutic agents, antibiotics or antihelminthics, that show species specificity or selectivity. As dihydrofolate reductase (DHFR) is a crucial enzyme for the synthesis of purines, pyrimidines and some amino acids, and also because developing insects show a remarkably rapid rate of cell division, DHFR is a potentially promising target for the discovery of novel insecticides. We have thus isolated and characterized the enzyme from a serious agricultural pest, Heliothis (Helicoverpa) virescens, the tobacco budworm. Sequencing tryptic peptides of the 35 000-fold purified DHFR allowed the subsequent isolation of a partial cDNA, with the full Dhfr gene sequence obtained from a genomic library. The H. virescens Dhfr spans 4 kb, with three introns, and encodes 185 amino acids. The enzyme shows an overall similarity of approximately 68% with DHFR from other metazoans, which has facilitated the molecular modeling of the protein. DHFRs from insects appear to have strikingly reduced sensitivity to inhibition by methotrexate, compared with the vertebrate enzymes, and this reduction was also reflected in the total binding energy seen after modeling experiments. Four residues that may be characteristic of insect DHFR, as well as a unique cysteine in the H. virescens DHFR active site, offer insight into the nature of inhibitor selectivity and provide suitable target sites for insecticide discovery.

Expression and purification of the hormone binding domain of the Drosophila ecdysone and ultraspiracle receptors.

Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists. USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein. Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM. Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate. In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer. The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer.

Photodynamic therapy using a protoporphyrinogen oxidase inhibitor.

The use of endogenously created porphyrins as an alternative to photosensitizer injection for photodynamic therapy is a rapidly evolving area of study. One common method to induce porphyrin synthesis and accumulation in cells is the topical, oral, or parenteral administration of 5-aminolevulinic acid, a precursor for heme biosynthesis. Porphyrin accumulation may also be elicited by the use of enzyme inhibitors of the heme biosynthetic pathway. Groups of DBA/2 mice bearing SMT-F mammary tumors were placed on a diet containing 0-4000 ppm of a protoporphyrinogen oxidase inhibitor, FP-846. This agent blocks a critical step in porphyrin metabolism and results in elevated intracellular levels of protoporphyrin IX. Light treatment of tumors produced both initial and long-term regression that was dependent on the amount of inhibitor, the duration of inhibitor exposure to animals, and the amount of light used in PDT. Tumor regression occurred without significant destruction of normal tissues in the treatment field and without initial vascular constriction or blood flow stasis. Tumor cure in animals given 4000 ppm FP-846 in feed for 3 days and 300 J/cm2 602-670 nm light (23% cure) was similar to the response in animals given 10 mg/kg Photofrin and the same light dose (20%).

Simultaneous determination of vitamins B2 (riboflavin) and B6 (pyridoxine) in infant formula products by reverse phase liquid chromatography.

An ion pair liquid chromatographic (LC) method is described for the simultaneous determination of vitamins B2 and B6 in infant formula products. The method eliminates many of the UV absorbing ingredients present in the infant formulations. The method involves extraction of the vitamins with distilled water followed by a filtration step and the subsequent analysis of the filtrate by LC. Vitamins B2 and B6 and their vitamers are separated from each other as well as from ingredients in the sample matrix on a 5 microns Spherisorb ODS column. The mobile phase consists of 85% 0.04 M triethylammonium phosphate (pH 3.0), 10% methanol, 5% acetonitrile, and the counter ion, octane sulfonic acid sodium salt. The method is reproducible for both vitamins with relative standard deviations (RSD) ranging from +/- 0.3 to +/- 2.6% depending on the infant formula product. The recoveries of the vitamins from various infant formula products are greater than 97%. The analysis for vitamins B2 and B6 in various lots of infant formula compared well with the official AOAC methods.

Liquid chromatographic determination of thiamine in infant formula products by using ultraviolet detection.

A liquid chromatographic (LC) method has been developed for determination of thiamine in infant formula products. The method involves the following steps: (a) dissolution of the formula with water, (b) pH adjustment to induce protein precipitation, (c) filtration, (d) concentration of thiamine by using a cation exchange column and extraction system, (e) cleanup of adsorbed thiamine and other contaminants on the ion exchange column by washing with water and then methanol, (f) elution of thiamine with a mixture of methanol-2M potassium chloride buffer, (g) analysis for thiamine by liquid chromatography. Thiamine is separated from its phosphate esters, the mono-, di-, and triphosphates, as well as its antagonists oxythiamine and pyrithiamine on a 6 micron particle size column and a mobile phase of 40mM triethyl-ammonium phosphate buffer-methanol (pH 7.7) (90 + 10). The method is reproducible, with relative standard deviations ranging from +/- 0.76 to +/- 1.2%, depending on the infant formula product tested. Recovery of thiamine from various infant formula products is greater than 99%. Analysis for thiamine of several commercially available infant formulas at different levels of fortification gave results that ranged from 122 to 216% of the declared levels. These results agree well with those obtained using the AOAC fluorometric method.