Animal models and analytical approaches for understanding the relationships between wine and cancer.

We used two approaches for studying the relationships between wine consumption, wine composition and cancer In the first approach, a transgenic mouse model of human neurofibromatosis, combined with the use of well-defined, chemically purified diets, showed that red wine contains nonalcoholic components that can delay tumor onset. In additional studies, catechin, the main monomeric polyphenol of red wine, delayed tumor onset in this mouse model in a positive, linear relationship when incorporated into the diet at levels of 0.5-4 mmol/kg diet. In the second approach, low doses of the chemical carcinogen 2-amino-1-methyl-6-phenylimidazo(4, 5-b)pyridine (PhlP) were administered to rats, and formation of DNA adducts was evaluated by accelerator mass spectrometry. Consumption of red wine solids (the residue from red wine remaining after removal of alcohol and water) and the wine polyphenol quercetin did not influence PhlP-DNA adduct levels or induce liver enzymes (glutathione-S-transferase and quinone reductase). However, quercetin did alter distribution of PhlP in the rat tissues compared to control animals and animals fed other potential dietary chemopreventive agents, including phenylethyl isothiocyanate and sulforaphane. These studies demonstrate the feasibility of these approaches for studying the chemopreventive potential of dietary components at physiologic levels in

Gram-negative bacteraemia (GNB) after 428 unrelated donor bone marrow transplants (UD-BMT): risk factors, prophylaxis, therapy and outcome.

Gram-negative infection is an important cause of morbidity and mortality after unrelated donor-bone marrow transplantation (UD-BMT). We performed a retrospective case-control study to examine the risk factors, prophylaxis, therapy and outcome of Gram-negative bacteraemia (GNB) in 428 patients undergoing UD-BMT. The incidence of GNB was 3.6% in children and 19% in adults. Of the adults, 11% developed GNB >60 days post UD-BMT. Predisposing risk factors for GNB included 'high-risk' disease status, chronic graft-versus-host disease and use of systemic steroids. Fever, a raised C-reactive protein (CRP) and hypotension were common findings at presentation. Patients were routinely given prophylactic ciprofloxacin: resistance to this antibiotic was seen in 33% of isolates. We identified an age-matched control group undergoing UD-BMT over the same time period as the study group. Gram-positive bacteraemia was significantly more common in cases than controls. Mortality from GNB was 17% in children and 24% in adults. We conclude that GNB is a common complication of UD-BMT with a high associated mortality. Patients should be educated further to present rapidly with symptoms suggestive of infection.

Visual improvement despite radiologically stable disease after treatment with carboplatin in children with progressive low-grade optic/thalamic gliomas.

BACKGROUND: The purpose of this study was to examine the clinical and radiologic response to carboplatin by children with progressive optic/thalamic gliomas. PATIENTS AND METHODS: Between July 1997 and July 1999, 12 consecutive children were treated with monthly carboplatin for progressive optic/thalamic gliomas. RESULTS: Five children have completed 12 cycles of carboplatin and five children are currently receiving treatment. Two children had progressive disease noted both clinically and radiologically. Nine children have stable radiologic disease and one child has had a partial radiologic response to chemotherapy. Eight children have had regular visual assessments. Four children (three with stable radiology and one with a partial radiologic response) have had improvement in their vision. Three children with radiologically stable disease have had no change in vision. One child has had deterioration in vision despite radiologically stable disease. CONCLUSIONS: The results suggest that the clinical response of optic/thalamic gliomas to carboplatin, as measured by visual acuity and visual fields, may be better than predicted by radiologic assessment. These data suggest that a prospective clinical study is warranted of the role of carboplatin in children with progressive optic/thalamic gliomas and visual impairment.

Determination of antioxidant properties of aroma extracts from various beans.

Aroma extracts from fresh soybeans, mung beans, kidney beans, and azuki beans were prepared using simultaneous steam distillation and solvent extraction (SDE) under mild conditions (55 degrees C and 95 mmHg). Extracts were examined for antioxidative activities in two different assays. The aroma extracts isolated from all beans inhibited the oxidation of hexanal for nearly one month at a level of 250 microL/mL. Mung bean and soybean extracts inhibited malonaldehyde (MA) formation from cod-liver oil by 86% and 88%, respectively, at the 250 microL/mL level. Azuki and kidney bean extracts inhibited MA formation from cod-liver oil by 76% and 53%, respectively, at the 250 microL/mL level. The antioxidative activities of mung bean and soybean extracts were comparable with that of the natural antioxidant, alpha-tocopherol (vitamin E).

Lysyl oxidase and P-ATPase-7A expression during embryonic development in the rat.

Lysyl oxidase activity is critical for the assembly and cross-linking of extracellular matrix proteins, such as collagen and elastin. Moreover, lysyl oxidase activity is sensitive to changes in copper status and genetic perturbations in copper transport, e.g., mutations in the P-type ATPase gene, ATP7A, associated with cellular copper transport. Lysyl oxidase may also serve as a vehicle for copper transport from extracellular matrix cells. Herein, we demonstrate that sufficient lysyl oxidase functional activity is present in the rat embryo at gestation day (GD) 9 to be detected in conventional enzyme assays. Estimation of embryonic lysyl oxidase functional activity, however, required partial purification in order to remove inhibitors. From GD 9 to GD 15, lysyl oxidase activity was relatively constant when expressed per unit of protein or DNA. In contrast, the steady-state levels of lysyl oxidase and ATP7A mRNA, measured by RT-PCR and expressed relative to total RNA and cyclophilin mRNA, increased approximately fourfold from GD 9 to 15. The pattern of temporal expression for ATP7A was consistent with its possible role in copper delivery to lysyl oxidase.

Physiological importance of quinoenzymes and the O-quinone family of cofactors.

O-quinone cofactors derived from tyrosine and tryptophan are involved in novel biological reactions that range from oxidative deaminations to free-radical redox reactions. The formation of each of these cofactors appears to involve post-translational modifications of either tyrosine or tryptophan residues. The modifications result in cofactors, such as topaquinone (TPQ), tryptophan tryptophylquinone (TTQ), lysine tyrosylquinone (LTQ) or the copper-complexed cysteinyl-tyrosyl radical from metal-catalyzed reactions. Pyrroloquinoline quinone (PQQ) appears to be formed from the annulation of peptidyl glutamic acid and tyrosine residues stemming from their modification as components of a precursor peptide substrate. PQQ, a primary focus of this review, has invoked considerable interest because of its presence in foods, antioxidant properties and role as a growth-promoting factor. Although no enzymes in animals have been identified that exclusively utilize PQQ, oral supplementation of PQQ in nanomolar amounts increases the responsiveness of B- and T-cells to mitogens and improves neurologic function and reproductive outcome in rodents. Regarding TPQ and LTQ, a case may be made that the formation of TPQ and LTQ is also influenced by nutritional status, specifically dietary copper. For at least one of the amine oxidases, lysyl oxidase, enzymatic activity correlates directly with copper intake. TPQ and LTQ are generated following the incorporation of copper by a process that involves the two-step oxidation of a specified tyrosyl residue to first peptidyl dopa and then peptidyl topaquinone to generate active enzymes, generally classed as "quinoenzymes." Limited attention is also paid to TTQ and the copper-complexed cysteinyl-tyrosyl radical, cofactors important to fungal and bacterial redox processes.

HPLC method for the quantification of procyanidins in cocoa and chocolate samples and correlation to total antioxidant capacity.

Monomeric and oligomeric procyanidins present in cocoa liquors and chocolates were separated and quantified in four different laboratories using a normal-phase high-performance liquid chromatography (HPLC) method with fluorescence detection. Procyanidin standards through decamers were obtained by extraction from cocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography, and final purification by preparative normal-phase HPLC. The purity of each oligomeric fraction was assessed using HPLC coupled to mass spectrometry. A composite standard was then prepared, and calibration curves were generated for each oligomeric class using a quadratic fit of area sum versus concentration. Results obtained by each of the laboratories were in close agreement, which suggests this method is reliable and reproducible for quantification of procyanidins. Furthermore, the procyanidin content of the samples was correlated to the antioxidant capacity measured using the ORAC assay as an indicator for potential biological activity.

Structural and functional consequences of haloenol lactone inactivation of murine and human glutathione S-transferase.

Mass spectrometric analysis of proteolysis products of haloenol lactone-modified glutathione S-transferase isozyme mGSTP1 indicates that the haloenol lactone 3-cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone is covalently attached to the protein at Cys-47. Comparisons of the extent of adduct formation with losses in enzymatic activity indicate that mGSTP1 exhibits greatest reactivity toward the haloenol lactone, followed by mGSTM1 and mGSTA3. Activities of mGSTP1 and mGSTM1 decrease in inverse proportion to haloenol lactone concentration, whereas modification had no apparent effect on catalytic activity of mGSTA3. Decreases in activity agree with the extent of protein modification observed in ESI mass spectra for mGSTP1 and mGSTM1 but not for mGSTA3. Kinetic studies employing recombinant human proteins with replacement of cysteine by serine at Cys-47 and Cys-101 indicate that rapid inactivation (t1/2 = 2 min) occurs only when residue 47 is cysteine. Mass spectra of C47S-hGSTP1 incubated with haloenol lactone demonstrate covalent attachment of a haloenol lactone-glutathione conjugate and suggest that an ester forms between the lactone and Ser-47. Therefore, we propose that initial opening of the lactone ring is promoted by Cys-47 through thioester formation between the lactone carbonyl and the Cys-47 sulfhydryl. Enol-keto tautomerization and enzyme-mediated hydrolytic cleavage of the thioester produces a reactive alpha-bromoketone which reacts a second time with Cys-47 and inactivates the enzyme. These results suggest that Pi class GSTs have thioesterase activity and that haloenol lactone inactivation occurs through an enzyme-mediated process.

Haloenol lactone is a new isozyme-selective and active site-directed inactivator of glutathione S-transferase.

A haloenol lactone derivative has been synthesized and found to be an isozyme-selective and active site-directed inactivator of glutathione S-transferase (GST). Preincubation of the haloenol lactone (100 microM) with murine Alpha, Mu, or Pi GST isozyme (1.0 microM) at pH 6.5, 37 degrees C resulted in time-dependent loss of enzyme activity with highly selective inhibition of the Pi isozyme (t1/2, approximately 2 min). In a separate experiment, a 10-fold excess of the lactone was incubated with GST-Pi isozyme at 37 degrees C for 3 h, followed by dialysis against Nanopure water. GST activity lost upon incubation with the lactone could not be restored by exhaustive dialysis, and only 8% of enzyme activity for the modified GST remained relative to the control that was treated identically except the lactone was omitted from the incubation. Both control and modified GST were characterized using electrospray ionization mass spectrometry. No native GST (23,478 Da) was observed in the spectrum of modified GST. Instead, protein incubated with the lactone exhibited an increase in molecular mass of 230 Da relative to control GST. The lactone (100 microM) was incubated with GST Pi isozyme (1.0 microM) in the presence of the competitive inhibitor S-hexylglutathione (10 microM), which suppressed time-dependent inhibition of GST by the lactone. The results suggest that this haloenol lactone is an irreversible and active site-directed inhibitor of GST that appears to inhibit the enzyme through two consecutive steps of nucleophilic attack.

Purification, mass spectrometric characterization, and covalent modification of murine glutathione S-transferases.

Three cytosolic glutathione S-transferase (GST, EC 2.5.1.18) isozymes were purified from livers of male CD-1 mice to homogeneity using affinity chromatography and gradient elution. Isozyme molecular masses and purities were determined using electrospray ionization mass spectrometry (ESI/MS), HPLC, and electrophoretic methods. Isozymes were assigned to alpha, pi, and mu classes based on kinetic and electrophoretic properties. Molecular masses determined by mass spectrometry were 25 271.4 +/- 2, 23 478.8 +/- 2, and 25 838.4 +/- 2 Da for alpha, pi, and mu isozymes, respectively. Molecular masses for pi and mu class isozymes agreed with molecular masses derived from nucleic acid sequences, but the molecular mass of the alpha class isozyme was 42 Da greater than calculated for a reported sequence. Analysis of tryptic digests of GST-alpha using tandem mass spectrometry determined that the molecular weight discrepancy could be attributed to N-terminal acetylation. Effects of covalent attachment of 4-hydroxy-2-nonenal (HNE) on catalytic activities of each GST isozyme were investigated by incubating GSTs with HNE. The extent of HNE-protein adduct formation was determined using ESI/MS. Catalytic activity of HNE-modified GSTs toward CDNB was measured. ESI mass spectra of modified enzymes show that the extent of HNE modification to the isozymes (pi > alpha > mu) coincides with the loss of activity seen for each protein. ESI mass spectra also indicated that glutathione-protein adducts form in isozymes during incubations with HNE even though purified proteins had been dialyzed against water before incubations. Glutathionylation of isozymes increased as the concentration of HNE in the incubations was raised. Dialysis of GSTs against 2 mM S-hexylglutathione (to displace GSH) before incubations eliminated S-glutathionylation. Reactivities of these GSTs toward HNE differed and is related to the number of cysteine residues (alpha < mu < pi) within the amino acid sequence.

Predicted prostate specific antigen results using transrectal ultrasound gland volume. Differentiation of benign prostatic hyperplasia and prostate cancer.

METHODS: The diagnostic performance of transrectal ultrasound (TRUS) gland volume and prostate specific antigen (PSA) results were evaluated in 204 men consecutively scheduled to undergo transurethral prostatic resection (TUR). RESULTS: Nonpalpable prostate cancer was detected by TRUS alone in 18% (29 of 161) and by TUR alone in 9% (14/161), for an overall cancer incidence of 27%. A predicted PSA value (TRUS gland volume x 0.20 ng/ml/g = polyclonal PSA) was used for comparison with serum PSA for each patient. TRUS positive predictive value improved from 52% to 86% when serum PSA exceeded the predicted value. The specificity and positive predictive value of PSA at 2.5 ng/ml were 23% and 37%, respectively, which increased to 88% and 72%, respectively, when serum PSA exceeded the predicted value. CONCLUSIONS: Predicted PSA values produce decision levels near the 95th percentile for each patient and assist individual biopsy decisions better than grouped gland volume ranges. Wider application of TRUS and PSA in any clinical setting or early detection program is now possible.

Prostate cancer: transrectal ultrasound and pathology comparison. A preliminary study of outer gland (peripheral and central zones) and inner gland (transition zone) cancer.

A study was conducted to compare results of transrectal ultrasound with pathologic findings on 116 patients who underwent radical prostatectomy for treatment of prostate cancer. In 96% (111 of 116), transrectal ultrasound guided biopsies of a hypoechoic lesion proved cancer; seven patients had known Stage A cancer; one patient had cancer detected by palpation and not detected by ultrasound. Cancers in the outer gland (peripheral and central zones) were compared with cancers in the inner gland (transition zone) by both ultrasound and pathology. Forty-eight percent (52 of 108) of cancers originating in the outer gland showed extraprostatic extension (Stage C disease). The primary sites of tumor escape from the outer gland were the prostatic capsule (38%), anterior fibromuscular stroma (5%), seminal vesicle (18%), the base of the gland at the neurovascular bundle (21%), and the apex (31%). Twenty-two percent (17 of 54) of cancers originating in the inner gland (transition zone) showed extraprostatic extension (Stage C disease). The primary sites of tumor escape from the inner gland were the anterior fibromuscular stroma (6%) and apex (11%). Both histologic and biologic differences between outer and inner gland cancers were found when tumor size was controlled. Gleason scores were significantly different for inner and outer gland cancers, with mean scores of 6.2 +/- 1.6 and 7.4 +/- 0.9, respectively. An odds ratio of 8.6 confirmed the increased risk of extraprostatic extension for outer gland cancer. Outer gland cancers showed increased aggressive behavior of both histologic and biologic nature. The difference in biologic aggressiveness of outer and inner gland cancers has definite implications for treatment options. Use of other diagnostic parameters, such as DNA ploidy, may help to determine which cancers to treat and when to treat them; this may have more relevance for cancers originating in the inner gland. Strategic transrectal ultrasound guided biopsy affords accurate tumor mapping and staging when modes of internal spread and escape of cancer from both outer and inner gland are known. Thus, transrectal ultrasound may be our "window of observation" through which additional research may explain the histologic and biologic discrepancies between outer and inner gland cancers.

Use of transrectal ultrasound and prostate-specific antigen in diagnosis of prostatic intraepithelial neoplasia.

1. PIN can present as a hypoechoic lesion on ultrasound. 2. Biopsy results prove a close relationship between PIN and cancer. 3. Measurements of age, lesion size, and PSA for diagnoses of PIN were intermediate values between non-cancer and cancer. 4. Sequential, precise transrectal ultrasound-guided biopsies of hypoechoic lesions are now possible, and close follow-up of patients with diagnoses of PIN is therefore possible.