Urinary isoflavone excretion as a compliance measure in a soy intervention among young girls: a pilot study.

OBJECTIVE: To investigate the compliance of young girls with a soy intervention. DESIGN: An 8-week dietary intervention and urine sample collection. SETTING: Free-living girls. SUBJECTS: A convenience sample of 8- to 14-y-old girls (20 started and 17 finished the study) recruited through flyers distributed to staff members and previous study participants. INTERVENTION: The girls consumed one daily serving of soymilk, soy nuts, or tofu, completed 3-day food records, kept daily soy intake logs, and collected weekly urine samples. MAIN OUTCOME MEASURES: Compliance with the intervention was evaluated by daily soy intake logs, 3-day food records analyzed by the center's Food Composition and Food Groups Servings Databases, and weekly urinary isoflavone excretion using high-pressure liquid chromatography. The statistical analysis included paired t-tests, analysis of variance, and Spearman's rank-order correlation coefficients. RESULTS: Daily soy intake logs indicated a mean intake of 6.28 servings out of a maximum of 7.0 servings per week. The food records revealed a six-fold increase in isoflavone intake during the study period (P<0.01) which was confirmed by an increase in urinary isoflavone excretion of similar magnitude (23.3-142.1 nmol/mg creatinine, P=0.02). CONCLUSIONS: This study demonstrated the ability of young girls to consume one daily soy serving and the usefulness of urinary isoflavones as a primary compliance measure. The high urinary isoflavone excretion levels detected in girls as compared to adult women suggest less intestinal degradation and/or greater absorption of isoflavones in nonadult populations. This finding requires further investigations into the pharmacokinetics of isoflavones.

Nutritional changes among premenopausal women undertaking a soya based dietary intervention study in Hawaii.

BACKGROUND: We conducted a 2-year nutritional intervention among premenopausal women. The goal was to incorporate two daily servings of soya into the regular diet. This report describes the dietary modifications and assesses their nutritional adequacy with regard to major nutrients. METHODS: In this analysis of 100 intervention and 106 control subjects, women completed a validated food-frequency questionnaire at baseline; throughout the study, they participated in at least three unannounced 24-h recalls. RESULTS: At randomization, both groups were similar in age and body weight, reported low soya intake, and did not differ by intake of major nutrients and foods. According to the 24-h recalls, women in the intervention group consumed nearly two servings of soya per day, while the control women remained at 0.2 servings. In comparison with the control group, the intervention group consumed fewer dairy products, primarily milk, but also less meat, nuts and seeds. As a result of the dietary modification, the intervention women consumed less-saturated fat and cholesterol and more protein, dietary fibre, calcium and vitamins than the control group. CONCLUSION: These results suggest that women in the intervention group improved the overall quality of their diet by adding two servings of soya per day.

Ocular angiostrongyliasis without meningitis symptoms in Okinawa, Japan.

A 62-year-old female farmer presented with retinal detachment in her left eye, and an Angiostrongylus cantonensis worm was recovered by vitreous surgery. The case did not show typical clinical symptoms indicating meningitis, although the patient complained of a mild headache, a low-grade fever, and slight ataxia. The symptoms were treated as influenza before the onset of the retinal detachment. The present case is the first confirmed of ocular angiostrongyliasis in Japan.

High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry.

We have used intramolecular cross-linking, MS, and sequence threading to rapidly identify the fold of a model protein, bovine basic fibroblast growth factor (FGF)-2. Its tertiary structure was probed with a lysine-specific cross-linking agent, bis(sulfosuccinimidyl) suberate (BS(3)). Sites of cross-linking were determined by tryptic peptide mapping by using time-of-flight MS. Eighteen unique intramolecular lysine (Lys-Lys) cross-links were identified. The assignments for eight cross-linked peptides were confirmed by using post source decay MS. The interatomic distance constraints were all consistent with the tertiary structure of FGF-2. These relatively few constraints, in conjunction with threading, correctly identified FGF-2 as a member of the beta-trefoil fold family. To further demonstrate utility, we used the top-scoring homolog, IL-1beta, to build an FGF-2 homology model with a backbone error of 4.8 A (rms deviation). This method is fast, is general, uses small amounts of material, and is amenable to automation.

Direct effects of thyroid hormones on rat coronary artery: nongenomic effects of triiodothyronine and thyroxine.

To determine whether thyroid hormones, triiodothyronine (T3) and thyroxine (T4), have any direct, nongenomic effects on vascular smooth muscle cells, we evaluated the effects of these hormones on rat coronary arteries. Bolus injection of T3 or T4 elicited a transient, dose-dependent decrease in coronary perfusion pressure (CPP), as well as an increase in arterial vasodilation. Vasodilation occurred immediately after injection, peaked at 15 seconds, and lasted 80 seconds. Reverse T3 had no effect on CPP or vasodilation. The rapidity of these effects suggests that they are not mediated by the T3-nuclear receptor, but are direct, nongenomic effects of thyroid hormones. Our results also suggest that thyroid hormones may play a role in preventing myocardial ischemia by inducing coronary artery vasodilation.

Rational design of novel antimicrobials: blocking purine salvage in a parasitic protozoan.

All parasitic protozoa obtain purine nucleotides solely by salvaging purine bases and/or nucleosides from their host. This observation suggests that inhibiting purine salvage may be a good way of killing these organisms. To explore this idea, we attempted to block the purine salvage pathway of the parasitic protozoan Tritrichomonas foetus. T. foetus is a good organism to study because its purine salvage depends primarily on a single enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), and could provide a good model for rational drug design through specific enzyme inhibition. Guided by the crystal structure of T. foetus HGXPRTase, we used structure-based drug design to identify several non-purine compounds that inhibited this enzyme without any detectable effect on human HGPRTase. One of these compounds, 4-[N-(3, 4-dichlorophenyl)carbamoyl]phthalic anhydride (referred to as TF1), was selected for further characterization. TF1 was shown to be a competitive inhibitor of T. foetus HGXPRTase with respect to both guanine (in the forward reaction; Ki = 13 microM) and GMP (in the reverse reaction; Ki = 10 microM), but showed no effect on the homologous human enzyme at concentrations of up to 1 mM. TF1 inhibited the in vitro growth of T. foetus with an EC50 of approximately 40 microM. This inhibitory effect was associated with a decrease in the incorporation of exogenous guanine into nucleic acids, and could be reversed by supplementing the growth medium with excess exogenous hypoxanthine or guanine. Thus, rationally targeting an essential enzyme in a parasitic organism has yielded specific enzyme inhibitors capable of suppressing that parasite's growth.

Characterization of receptors with a new negative image: use in molecular docking and lead optimization.

The characterization of receptor binding sites is an important aspect of molecular docking, molecular recognition, and the structure-based design process. This characterization can take several forms: the receptor surface itself can be delineated or described, the space adjacent to the surface can be chemically mapped, or a negative image of the protein binding region can be generated. In this report, we describe a new method of constructing a negative image through generation of a set of spheres. These spheres lie along the receptor surface, and their centers represent possible ligand atom positions. By the method in which they are constructed, these spheres carry a limited amount of energetic and chemical information in addition to their primary geometric information. We test the accuracy of the image by comparing sphere positions to the positions of bound ligand atoms and propose a figure of merit for such tests. Then, we use the spheres to orient ligands in enzyme active sites and show how they can be used to generate low scoring configurations more efficiently than other approaches that search orientation space. In addition, two novel applications of these spheres are described: they are used to help identify structural differences among families of enzymes and to suggest points for ligand modification in analog design.

Thyroid-stimulating antibody and TSH-binding inhibitor immunoglobulin in 277 Graves' patients and in 686 normal subjects.

TSH receptor antibodies (TRAb) are believed to cause hyperthyroidism of Graves' disease. Thyroid-stimulating antibody (TSAb) and TSH-binding inhibitor immunoglobulin (TBII) have been measured as TRAb to diagnose Graves' disease and to follow Graves' patients. We intended to evaluate the clinical value of TRAb (TSAb and TBII) assay in establishing the diagnosis of Graves' disease and in predicting its clinical course. TSAb and TBII were studied in 686 normal subjects and in 277 Graves' patients before antithyroid drug therapy. We followed serial changes of TSAb and TBII in 30 Graves' patients before, during and after antithyroid drug therapy over 3.5-9 yr. We measured TSAb as a stimulator assay and TBII as a receptor assay. Both TSAb and TBII were distributed normally in 686 normal subjects. ROC curves demonstrated that both TSAb and TBII had high sensitivity and specificity for the diagnosis of Graves' disease, and were equally sensitive and specific; 150% was chosen as cut-off value for TSAb and 10% for TBII. Of the 277 untreated Graves' patients, 254 (92%) had positive TSAb and positive TBII. All of the 277 untreated Graves' patients had positive TRAb (TSAb and/or TBII). We followed the serial changes of TSAb and TBII in 30 Graves' patients over 3.5-9 yr. During antithyroid drug therapy, TSAb and TBII activities decreased and disappeared in 27 patients (Group A), but continued to be high in the other 3 (Group B). The former 27 Group A patients achieved remission, but the latter 3 Group B patients continued to have hyperthyroidism. Of the 27 Group A patients, 16 (59%) had parallel decreases of TSAb and TBII activities; in 6, the changes were predominantly observed in either TSAb or TBII, and in 4, complex changes in TSAb and TBII activities were observed. Disappearance of TSAb and appearance of TSBAb was seen in one. The other 3 Group B patients continued to have high TSAb and TBII activities and to have hyperthyroidism. In conclusion, TSAb and TBII are of clinical value in establishing the diagnosis of Graves' disease and in predicting its clinical course. We clearly demonstrated its diagnostic usefulness. Both TSAb and TBII have high sensitivity and specificity. All of the 277 untreated Graves' patients had TRAb (TSAb and/or TBII). Serial changes of TSAb and TBII during therapy differ from one patient to another, and can be classified into several groups. Changes in TSAb and TBII activities reflect the clinical courses of Graves' patients. The simultaneous measurement of both TSAb and TBII is clinically useful, since TSAb and TBII reflect two different aspects of TRAb. TSAb and TBII are different.

Molecular docking to ensembles of protein structures.

Until recently, applications of molecular docking assumed that the macromolecular receptor exists in a single, rigid conformation. However, structural studies involving different ligands bound to the same target biomolecule frequently reveal modest but significant conformational changes in the target. In this paper, two related methods for molecular docking are described that utilize information on conformational variability from ensembles of experimental receptor structures. One method combines the information into an "energy-weighted average" of the interaction energy between a ligand and each receptor structure. The other method performs the averaging on a structural level, producing a "geometry-weighted average" of the inter-molecular force field score used in DOCK 3.5. Both methods have been applied in docking small molecules to ensembles of crystal and solution structures, and we show that experimentally determined binding orientations and computed energies of known ligands can be reproduced accurately. The use of composite grids, when conformationally different protein structures are available, yields an improvement in computational speed for database searches in proportion to the number of structures.

Seventeen alpha-hydroxylase deficiency with one base pair deletion of the cytochrome P450c17 (CYP17) gene.

Mutation of the cytochrome P450c17 (CYP17) gene causes 17 alpha-hydroxylase deficiency (17OHD). Recently, several researchers have elucidated the molecular basis of 17OHD by gene analysis. We experienced a case of 17OHD and intended to reveal the abnormality of the CYP17 gene in this Japanese female with 17OHD. Leukocytes were obtained from the patient, her mother and sister, and normal control subjects. We amplified the CYP17 gene using polymerase chain reaction and performed the sequence analysis using the dideoxy terminator method and restriction enzyme analysis. We found that the patient had one base-pair deletion at the position of amino acid 438. An identical result was obtained with restriction enzyme analysis. This G deletion altered the reading frame and resulted in a premature stop codon at position 443; the ligand of heme iron (Cys: cystine 442) was absent. This small mutation may account for the patient's clinical manifestations of 17OHD. This is the first case of 17OHD with only one base pair deletion of the CYP17 gene.

Flexible ligand docking using a genetic algorithm.

Two computational techniques have been developed to explore the orientational and conformational space of a flexible ligand within an enzyme. Both methods use the Genetic Algorithm (GA) to generate conformationally flexible ligands in conjunction with algorithms from the DOCK suite of programs to characterize the receptor site. The methods are applied to three enzyme-ligand complexes: dihydrofolate reductase-methotrexate, thymidylate synthase-phenolpthalein and HIV protease-thioketal haloperidol. Conformations and orientations close to the crystallographically determined structures are obtained, as well as alternative structures with low energy. The potential for the GA method to screen a database of compounds is also examined. A collection of ligands is evaluated simultaneously, rather than docking the ligands individually into the enzyme.

Application of distance geometry to the proton assignment problem.

Assignment of the hydrogen spectrum is the first step in the conventional procedure for the determination of molecular structure by 1H-nmr. In this paper, we explore the possibility of directly exploiting the distances derived from nuclear Overhauser effect experiments to generate a three-dimensional structure that is then assigned based on knowledge of the connectivity or primary sequence. This effort is analogous to that of the protein crystallographers in tracing electron density of the peptide chain. In particular, we compare structures produced by distance geometry to known peptide secondary structures to see what level of information is required to "trace" the backbone alpha-carbon and amide hydrogens and the beta-carbon hydrogens. We conclude that this approach is only useful with excellent quality stereo-resolved data.

Effects of limited input distance constraints upon the distance geometry algorithm.

In this paper we examine the distance geometry (DG) algorithm in the form used to determine the structure of proteins. We focus on three aspects of the algorithm: bound smoothing with the triangle inequality, the random selection of distances within the bounds, and the number of distances needed to specify a structure. Computational experiments are performed using simulated and real data for basic pancreatic trypsin inhibitor (BPTI) from nmr and crystallographic measurements. We find that the upper bounds determined by bound smoothing to be a linear function of the true crystal distance. A simple model that describes the results obtained with randomly selected trial distances is proposed. Using this representation of the trial distances, we show that BPTI DG structures are more compact than the true crystal structure. We also show that the DG-generated structures no longer resemble test structures when the number of these interresidue distance constraints is less than the number of degrees of freedom of the protein backbone. While the actual model will be sensitive the way distances are chosen, our conclusions are likely to apply to other versions of the DG algorithm.

Ocular esterase composition in albino and pigmented rabbits: possible implications in ocular prodrug design and evaluation.

A methodology was developed to determine the proportion of acetyl- (AChE) and butyrylcholinesterase (BuChE) in the albino and pigmented rabbit eye. It was found that BuChE contributed over 75% of the cholinesterase activity in all the ocular tissues but the corneal epithelium of the albino rabbit. This esterase was principally responsible for the parabolic chain length dependence of ocular hydrolysis of model naphthyl ester prodrugs reported previously. In contrast, when incubated with AChE, the rate of hydrolysis of these esters decreased monotonically with increasing ester chain length. Together these findings suggest that esters whose chain length exceeds 4 carbons will be hydrolyzed primarily by BuChE. It is suggested that the dominance of BuChE in ocular tissues is another factor which merits consideration in the design and evaluation of ocular ester prodrugs.