Ligand-binding characterization of xanthophyll carotenoids to solubilized membrane proteins derived from human retina

The macula of the human retina contains extraordinarily high concentrations of lutein and zeaxanthin, xanthophyll carotenoids that appear to play an important role in protecting against age-related macular degeneration, the leading cause of blindness among the elderly. It is likely that the uptake and stabilization of these carotenoids is mediated by specific xanthophyll-binding proteins. In order to purify and characterize such a binding protein, a carotenoid-rich membrane fraction derived from human macula or peripheral retina was prepared by homogenization, differential centrifugation, and detergent solubilization. Further purification was carried out using ion-exchange chromatography and gel-filtration chromatography coupled with continuous photodiode-array monitoring for endogenously associated xanthophyll carotenoids. The most highly purified preparations contained two major protein bands at 25 and 55 kDa that consistently co-eluted with endogenous lutein and zeaxanthin. The visible absorbance spectrum of the binding protein preparation closely matches the spectral absorbance of the human macular pigment, and it is bathochromically shifted about 10 nm from the spectrum of lutein and zeaxanthin dissolved in organic solvents. Binding of exogenously added lutein and zeaxanthin is saturable and specific with an apparent Kd of approximately 1 microM. Canthaxanthin and beta-carotene exhibit no significant binding activity to solubilized retinal membrane proteins when assayed under identical conditions. Other potential mammalian xanthophyll-binding proteins such as albumin, tubulin, lactoglobulin and serum lipoproteins possess only weak non-specific binding affinity for carotenoids when assayed under the same stringent binding conditions. This investigation provides the first direct evidence for the existence of specific xanthophyll-binding protein(s) in the vertebrate retina and macula. The possible roles of xanthophyll-binding proteins in normal macular function and in the pathogenesis of age-related macular degeneration remain to be elucidated.     

Metabolism of equilenin in MCF-7 and MDA-MB-231 human breast cancer cells

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17beta-dihydroequilenin in extrahepatic, estrogen-responsive tissues.     

Cancer of the prostate: influence of nutritional factors. Vitamins, antioxidants and trace elements

CANCER OF THE PROSTATE AND VITAMINS: Four vitamins have been studied, vitamins A, E, D and C. the results of these studies have been contradictory. Vitamin A and vitamin E would have a protective effect. ANTIOXIDANTS: Carotenes have an activity similar to that of vitamin A. Beta-carotene was positively associated with risk of cancer of the prostate in one study while two others were unable to demonstrate any relationship. Lycopene, the red color in fruits and vegetables, particularly tomatoes, would contribute to a lower risk of prostate cancer. TRACE ELEMENTS: Cadmium would increase the risk of cancer while selenium would have a protective effect. However studies concerning selenium carry certain methodological biases.     

Vitamin A supplementation at birth delays pneumococcal colonization in South Indian infants

Nasopharyngeal colonization is a risk factor for pneumococcal disease, a leading cause of complications and death in infants. We assessed the impact of vitamin A supplementation in reducing pneumococcal colonization in infants from an area with endemic vitamin A deficiency. We recruited 464 2-mo-old infants from a rural area in South India. Infants were randomly assigned to receive two 7000-microg retinol equivalent doses of vitamin A (n = 239) or placebo (n = 225) orally at birth, and nasopharyngeal specimens were collected at ages 2, 4 and 6 mo. We studied the effect of vitamin A on culture-confirmed pneumococcal colonization and on the distribution of pneumococcal serotypes. Analyses were conducted by intention-to-treat. The risk of colonization among infants aged 4 mo who were not colonized by age 2 mo was significantly reduced in the vitamin A group compared with the placebo group [odds ratio 0.51 (0.28, 0.92), P = 0.02). The odds of colonization were 27% lower in the treatment group than in the placebo group [odds ratio 0.73 (0.48, 1.1), P = 0.13]. No differences were detected in the prevalence of invasive serotypes. The risk of colonization with penicillin-resistant isolates was 74% lower in the vitamin A group than in the placebo group at 2 mo of age. However, the prevalence of penicillin-resistant isolates was only 4%. Neonatal vitamin A supplementation may play a role in lowering morbidity rates associated with pneumococcal disease by delaying the age at which colonization occurs.     

A randomized trial of the long-term effects of digital ocular compression in the late postoperative period

PURPOSE: To determine whether digital ocular compression is a viable technique to lower intraocular pressure in patients at least 3 months after trabeculectomy. PATIENTS AND METHODS: A 6-month prospective, randomized, controlled, single-masked trial of 29 patients who underwent a trabeculectomy at the Glaucoma Service of Wills Eye Hospital. Patients were assigned to two groups: ocular compression or cheekbone compression (control group). The ocular compression group performed compression to the operated eye three times a day in the pattern of 10 seconds of pressure, 5 seconds of rest, and 10 seconds of pressure. Pressure was applied with the index finger through the closed lid to the center of the cornea. Pressure was steady and firm, but not painful. No massaging was performed. The cheekbone compression group applied pressure to the zygomatic arch with an identical style and frequency. RESULTS: At 6 months, the change in mean intraocular pressure for the ocular compression group was 0.25 mm Hg compared with -0.44 mm Hg for the control group (P = 0.7). A few patients in both groups experienced large swings in intraocular pressure and mild to moderate discomfort. CONCLUSION: Ocular compression had little to no success in the long-term management of increased intraocular pressure in the late postoperative period in this study.     

The outcome of the functioning filter after subsequent cataract extraction

OBJECTIVE: To evaluate and compare the outcome of functioning filtration surgery followed by cataract surgery with posterior intraocular lens implantation by both phacoemulsification and extracapsular cataract extraction (ECCE) techniques in glaucomatous eyes. PATIENTS AND METHODS: We retrospectively evaluated the clinical course of 77 eyes (68 patients) that after successful trabeculectomy, underwent cataract surgery by either phacoemulsification or ECCE techniques. We determined the frequency of partial and absolute failure following cataract surgery by either phacoemulsification or ECCE in eyes with functioning trabeculectomies. Partial failure of intraocular pressure (IOP), control after cataract extraction was defined as the need for an increased number of antiglaucoma medications or argon laser trabeculoplasty to maintain IOP < or =21mm Hg. Complete failure of IOP control after cataract surgery was defined as an IOP >21 mm Hg on at least two consecutive measurements one or more weeks apart or the performance of additional filtration surgery. Failure rates were calculated using the Kaplan-Meier actuarial method. Failure rates between phacoemulsification and ECCE subgroups were compared using the log rank test. RESULTS: The probability of partial failure by the third postoperative year after cataract surgery was 39.5% in the phacoemulsification subgroup and 37.3% in the ECCE subgroup. This small difference is not statistically significant (P = 0.48). The probability of complete failure by the fourth postoperative year after cataract surgery was 12.0% in the phacoemulsification subgroup and 12.5% in the ECCE subgroup. This difference is also not statistically significant (P = 0.77). At the 6-month follow-up visit, visual acuity of both groups improved one or more lines in 87.0% of patients, and worsened one or more lines in 3.9% of patients. Sixty-one percent achieved visual acuity of 20/40 or better. The most frequent complication was posterior capsular opacification requiring laser capsulotomy that occurred in 31.2% of patients. CONCLUSION: Cataract extraction by either phacoemulsification or ECCE following trabeculectomy surgery may be associated with a partial loss of the previously functioning filter and the need for more antiglaucoma medications to control IOP.     

Dopamine transporter binding without cocaine-like behavioral effects: synthesis and evaluation of benztropine analogs alone and in combination with cocaine in rodents

Rationale: Previous SAR studies demonstrated that small halogen substitutions on the diphenylether system of benztropine (BZT), such as a para-Cl group, retained high affinity at the cocaine binding site on the dopamine transporter. Despite this high affinity, the compounds generally had behavioral effects different from those of cocaine. However, compounds with meta-Cl substitutions had effects more similar to those of cocaine. Objectives: A series of phenyl-ring analogs of benztropine (BZT) substituted with 3′-, 4′-, 3′,4′′- and 4′,4′′-position Cl-groups were synthesized and their pharmacology was evaluated in order to assess more fully the contributions to pharmacological activity of substituents in these positions. Methods: Compounds were synthesized and their pharmacological activity was assessed by examining radioligand binding and behavioral techniques. Results: All of the compounds displaced [³H]WIN 35,428 binding with affinities ranging from 20 to 32.5 nM. Affinities at norepinephrine ([³H]nisoxetine) and serotonin ([³H]citalopram) transporters, respectively, ranged from 259 to 5120 and 451 to 2980 nM. Each of the compounds also inhibited [³H]pirenzepine binding to muscarinic M₁ receptors, with affinities ranging from 0.98 to 47.9 nM. Cocaine and the BZT analogs produced dose-related increases in locomotor activity in mice. However, maximal effects of the BZT analogs were uniformly less than those produced by cocaine, and were obtained 2–3 h after injection compared to the relatively rapid onset (within 30 min) of cocaine effects. In rats trained to discriminate IP saline from 29 μmol/kg cocaine (10 mg/kg), cocaine produced a dose-related increase in responding on the cocaine lever, reaching 100% at the training dose; however, none of the BZT analogs fully substituted for cocaine, with maximum cocaine responding from 20 to 69%. Despite their reduced efficacy compared to cocaine in cocaine discrimination, none of the analogs antagonized the effects of cocaine. As has been reported previously for 4′-Cl-BZT, the cocaine discriminative-stimulus effects were shifted leftward by co-administration of the present BZT analogs. Conclusions: The present results indicate that although the BZT analogs bind with relatively high affinity and selectivity at the dopamine transporter, their behavioral profile is distinct from that of cocaine. The present results suggest that analogs of BZT may be useful as treatments for cocaine abuse in situations in which an agonist treatment is indicated. These compounds possess features such as reduced efficacy compared to cocaine and a long duration of action that may render them particularly useful leads for the development of therapeutics for cocaine abusers. Electronic Publication