The Role of Gene Therapy in the Treatment of Retinal Diseases: A Review.

BACKGROUND: Gene therapy represents the therapeutic delivery of nucleic acid polymers into patient cells with the aim of treating an underlying disease. Over the past 2 decades this new therapy has made substantial progress owing to better understanding of the pathobiologic basis of various diseases coupled with growth of gene transfer biotechnologies. The eye, in particular, represents a suitable target for such therapy due to the immune privilege provided by the blood-ocular barrier, the ability to directly visualize, access and locally treat the cells and the minimal amount of vector needed given the size of this organ. It is not surprising therefore that several clinical trials are now ongoing in this field. OBJECTIVE: The purpose of this review was to provide an update on gene therapy for retinal diseases, discussing differences in treatment strategies, vector designs and surgical techniques. METHOD: Research was performed on PubMed, ClinicalTrials.gov, and Home Genetic Reference. We additionally utilized the internet database for genetics of retinal diseases, the portal for rare diseases and orphan drugs and the NCBI database Online Mendelian Inheritance in Man. No restriction was applied on the language of publications. RESULTS: We present the available results of current active clinical trials for inherited retinal disease such as Leber's congenital amaurosis type 2, choroideremia, Stargardt disease, achromatopsia and juvenile X-linked retinoschisis. We also illustrate a new approach of this therapy for the treatment of much more common ocular diseases such as age-related macular degeneration and diabetic retinopathy. CONCLUSION: Gene therapy represents an emerging and promising therapeutic approach for the treatment not only of rare inherited retinal diseases but also much more common retinal pathologies.

A single-step method for RNA isolation from tropical crops in the field.

The RNAzol RT reagent was used to provide pure RNA from human cells. We develop a protocol using RNAzol RT reagent to extract pure RNA from plants tissues and demonstrate that this RNA extraction method works not only at room temperature but also at elevated temperatures and provides the simplest and most effective single-step method to extract pure and undegraded RNA directly from tropical plants in the field. RNA extraction directly in a complex field environment opens up the way for studying gene-environment interactions at transcriptome level to decipher the complex regulatory network involved in multiple-stress responses.

Efecto de Debaryomyces hansenii en la respuesta antioxidante de juveniles de camarón blanco Litopenaeus vannamei / Effect of Debaryomyces hansenii on the antioxidant response of juvenile white shrimp Litopenaeus vannamei

Objetivo. Determinar la respuesta antioxidante [actividad de superóxido dismutasa (SOD) y catalasa (CAT)] así como la cuenta total de hemocitos (CTH) y el contenido de proteínas (CP) en camarones (Litopenaeus vannamei) expuestos a diferentes dosis y cepas de la levadura Debaryomyces hansenii (DH5, DH6, LL1), y un inmunoestimulante comercial (LAM). Materiales y métodos. Las levaduras fueron cultivadas y suministradas diariamente en concentraciones diferentes (104 – 106 UFC/mL) directamente a los tanques de cultivo de los camarones (8 ± 0.2 g) mientras que LAM fue aplicado una vez a la semana (0.5 mg/L). Los organismos fueron mantenidos bajo condiciones de laboratorio (28°C, 35%, 80% de recambio diario de agua, dieta comercial para camarón ad libitum). Los tratamientos fueron distribuidos por duplicado y los resultados evaluados a los 15 días con un análisis de varianza y una prueba de Tukey. Resultados. Se registró un CTH significativo (p<0.05) en los tratamientos con DH6 y LL1 (106 UFC/mL) comparada con el control, mientras que las cepas DH5 y DH6 revelaron un incremento significativo (p<0.05) de CP con la dosis de 104 UFC/mL. Los camarones tratados con LAM incrementaron significativamente (p<0.05) los valores de SOD y CAT. Conclusiones. Los resultados obtenidos demuestran que D. hansenii incrementa la respuesta antioxidante y CTH en camarones.

Anti-VEGF compounds in the treatment of neovascular age related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among elderly patients in developed countries. Although the pathogenesis of AMD is still largely unknown, it is now well known that vascular endothelial growth factor (VEGF) plays a pivotal role in the growth of the abnormal blood vessels (i.e. choroidal neovascularization, CNV) which characterizes the "wet form" of this ocular disease. Therefore, inhibiting VEGF has turned out to be a good way of more effectively controlling neovascular AMD. VEGF is a heparin-binding glycoprotein with potent angiogenic, mitogenic and vascular permeability-enhancing activities specific for endothelial cells. Currently two anti-VEGF compounds have been approved by the US Food and Drug Administration (FDA) for the treatment of neovascular AMD: pegaptanib and ranibizumab. Off-label usage of bevacizumab, an anti-VEGF agent similar to ranibizumab, has also become fairly common. The substantial improvement of visual acuity noticed in patients treated with ranibizumab has made this drug the gold standard for AMD therapy. However, as with many new therapies, there are unresolved issues, including safety, cost, and dosing frequency. This review describes in details the properties and efficacy of the three anti-VEGF agents in use in clinical practice. Promising emerging anti-VEGF strategies (VEGF-trap, small interfering RNA, tyrosine kinase inhibitors) which aim to improve outcomes, safety and treatment burden through novel mechanisms of action are also discussed.

Effectiveness of ranibizumab for neovascular age-related macular degeneration using clinician-determined retreatment strategy.

AIMS: To report the effectiveness of intravitreal ranibizumab treatment for neovascular age-related macular degeneration in a tertiary centre. METHODS: 1 year prospective cohort study of patients with a diagnosis of neovascular age-related macular degeneration on fundus fluorescein angiography treated with ranibizumab. Patients received three consecutive monthly treatments, followed by a clinician-determined re-treatment strategy. Data collected included demographic details, baseline and subsequent follow-up visit measurements, refraction protocol best corrected visual acuity (BCVA), contrast sensitivity (CS) and central foveal thickness (CFT) on optical coherence tomography. RESULTS: 81 patients were included in the study. The mean age was 79.5 years with a male:female ratio 32:49. The mean number of treatments was 5.6 ± 2.3. Visual outcomes at 12 months showed 17.1% gained ≥ 15 letters BCVA, 97.4% lost <15 letters and 2.5% lost ≥ 15 letters. Mean changes at 12 months were: BCVA +3.7 ± 11.1 (p<0.01); CS +2.3 ± 5.1 letters (p<0.001); CFT -100.1 ± 111.9 µm (p<0.001). CONCLUSIONS: Clinician-determined re-treatment after a three-dose initiation phase appears to be less effective in improving BCVA than in randomised controlled trials.

Incidence of neovascularization in the fellow eye of patients with unilateral retinal angiomatous proliferation.

AIMS: The aim of this study is to describe the incidence and characteristics of neovascularization in the fellow eye of patients with retinal angiomatous proliferation (RAP). METHODS: This is a retrospective study conducted on all patients with a diagnosis of unilateral RAP commencing treatment in a single centre between November 2002 and January 2010. Clinical biomicroscopic examination, fluorescein angiography, and if required, indocyanine green angiography, and optical coherence tomography were used to evaluate all patients. RESULTS: In all, 37 patients had a follow-up of ≥1 year, 28 ≥2 years, and 11 ≥3. Patients who developed RAP in the fellow eye were: 2 of 37 (5.4%) within 1 year of follow-up, 4 of 28 (14.2%) within 2 years, and 4 of 11 (36.3%) within 3 years. CONCLUSION: In our case series, the risk of neovascularization in the fellow eye of patients with unilateral RAP increased with time. Approximately one-third of patients with a 3-year follow-up developed a bilateral disease. Our findings warrant further large-scale investigation.

Melatonin inhibits aromatase promoter expression by regulating cyclooxygenases expression and activity in breast cancer cells.

BACKGROUND: Melatonin reduces the development of breast cancer interfering with oestrogen-signalling pathways, and also inhibits aromatase activity and expression. Our objective was to study the promoters through which melatonin modifies aromatase expression, evaluate the ability of melatonin to regulate cyclooxygenases and assess whether the effects of melatonin are related to its effects on intracellular cAMP, in MCF-7 cells. METHODS: Total aromatase mRNA, aromatase mRNA promoter regions and cyclooxygenases mRNA expression were determined by real-time RT-PCR. PGE(2) and cAMP were measured by kits. RESULTS: Melatonin downregulated the gene expression of the two major specific aromatase promoter regions, pII and pI.3, and also that of the aromatase promoter region pI.4. Melatonin 1 nM was able to counteract the stimulatory effect of tetradecanoyl phorbol acetate on PGE(2) production and inhibit COX-2 and COX-1 mRNA expression. Melatonin 1 nM elicited a parallel time-dependent decrease in both cyclic AMP formation and aromatase mRNA expression. CONCLUSIONS: This study shows that melatonin inhibits aromatase activity and expression by regulating the gene expression of specific aromatase promoter regions. A possible mechanism for these effects would be the regulation by melatonin of intracellular cAMP levels, mediated by an inhibition of cyclooxygenase activity and expression.

Enfoques mucosales en vacunologia de Neisseria

Meningococcal B strains accounts for some 72 percent and 28 percent of meningococcal diseases in infants and toddlers in Europe and the USA, respectively. Nevertheless, meningococcal diseases are rare in Cuba owing to the wide spread program on antimeningococcal vaccination in the country. Finlay Institute is one of the pioneering organizations in Neisseria Vaccinology mainly by its contribution to N. meningitidis serogroup B outer membrane-based bivalent vaccine, VA-MENGOC-BC™. This vaccine was given intramuscularly in more than 60 million doses corresponding 10,7 millions of them to Cuban young adults, children, and infants. However, most dangerous or commensally Neisseria strains enter and establish in the mucosa, where the secretory (S) IgA is the main specific guardian and is mainly induced by mucosal routes. However, few mucosal vaccines exist principally due to the absent of mucosal adjuvants. We develop a Finlay Adjuvant (AF) platform based in outer membrane vesicles (Proteoliposome, PL) and its derivate Cochleate (Co). AFPL1 derived from serogroup B N meningitidis is a potent Th1/CTL driving parenteral adjuvant. AFCo1 is a potent mucosal adjuvant. Therefore, we sought to go deeper in the possible mucosal cross recognition between N. meningitidis serogroups and Neisseria species and explore a concurrent mucosal and parenteral immunization strategy (SinTimVaS) in order to develop suitable mucosal vaccines. Experiments were conducted in Balb/c or C57Bl6 mice with mucosal and systemic immunization using AFCo1 and AFPL1. Human sera and saliva were also analyzed for cross cognition. Mucosal cross recognition at SIgA level in human saliva between N. meningitidis serogroups B, A, C, Y, and W135 were observed. This SIgA cross recognition response was also observed between pathogenic (N meningitidis serogroup B, N gonorrhoeae) and non-pathogenic strains (N flava, N lactamica). The possible influence of meningococcal vaccination ...(AU)

Melatonin as a selective estrogen enzyme modulator.

Melatonin exerts oncostatic effects on different kinds of tumors, especially on hormone-dependent breast cancer. The general conclusion is that melatonin, in vivo, reduces the incidence and growth of chemically-induced mammary tumors in rodents, and, in vitro, inhibits the proliferation and invasiveness of human breast cancer cells. Both studies support the hypothesis that melatonin inhibits the growth of breast cancer by interacting with estrogen-signaling pathways through three different mechanisms: (a) the indirect neuroendocrine mechanism which includes the melatonin down-regulation of the hypothalamic-pituitary-reproductive axis and the consequent reduction of circulating levels of gonadal estrogens, (b) direct melatonin actions at tumor cell level by interacting with the activation of the estrogen receptor, thus behaving as a selective estrogen receptor modulator (SERM), and (c) the regulation of the enzymes involved in the biosynthesis of estrogens in peripheral tissues, thus behaving as a selective estrogen enzyme modulator (SEEM). As melatonin reduces the activity and expression of aromatase, sulfatase and 17beta-hydroxysteroid dehydrogenase and increases the activity and expression of estrogen sulfotransferase, it may protect mammary tissue from excessive estrogenic effects. Thus, a single molecule has both SERM and SEEM properties, one of the main objectives desired for the breast antitumoral drugs. Since the inhibition of enzymes involved in the biosynthesis of estrogens is currently one of the first therapeutic strategies used against the growth of breast cancer, melatonin modulation of different enzymes involved in the synthesis of steroid hormones makes, collectively, this indolamine an interesting anticancer drug in the prevention and treatment of estrogen-dependent mammary tumors.

Verteporfin photodynamic therapy for subfoveal choroidal neovascularization in pathologic myopia: a 12-month retrospective review.

PURPOSE: To evaluate the visual outcome of patients with subfoveal choroidal neovascularization (CNV) secondary to pathologic myopia treated with verteporfin photodynamic therapy (PDT-V) and to verify the predictive role of visual and angiographic parameters. METHODS: This is a retrospective, interventional, consecutive case series study of subjects with subfoveal CNV secondary to pathologic myopia. All patients received PDT-V according to VIP guidelines. A complete ophthalmologic evaluation was performed on all patients and included best-corrected visual acuity (BCVA), fundus examination, and fluorescein angiography (FA, IMAGEnet System, Topcon Corp., Japan). CNV size (mm2) was directly measured on the early phase of the angiogram using the software included with the IMAGEnet package. All checks were scheduled at 3-month intervals for a period of 1 year. A review of medical and angiographic records was performed and assessed throughout a 12-month follow-up period. RESULTS: A total of 62 patients (62 eyes) were examined. Best-corrected visual acuity (BCVA) moderately decreased without reaching a statistically noticeable level throughout the followup; reduction in lesion size reached a significant level at the second checkup. A significant correlation between higher baseline BCVA and better final visual outcome was detected. CONCLUSIONS: Standardized PDT-V minimizes central vision deterioration in patients with CNV secondary to pathologic myopia. Better BCVA at presentation represents a good predictive sign.

Selective estrogen enzyme modulator actions of melatonin in human breast cancer cells.

Melatonin exerts oncostatic effects on different kinds of neoplasias, especially on estrogen-dependent mammary tumors. Current knowledge about the mechanisms by which melatonin inhibits the growth of breast cancer cells point to an interaction of melatonin with estrogen-responsive pathways. The intratumoral production of estrogens in breast carcinoma tissue plays a pivotal role in the proliferation of mammary tumoral cells and its blockade is one of the main objectives of the treatment of breast cancer. The aim of the present work is centered on the study of the role of melatonin in the control of some enzymes involved in the formation and transformation of estrogens in human breast cancer cells. The present study demonstrates that melatonin, at physiologic concentrations, modulates the synthesis and transformation of biologically active estrogens in MCF-7 cells, through the inhibition of sulfatase (STS) and 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) activity and expression, enzymes involved in the estradiol formation in breast cancer cells. Physiologic concentrations of melatonin also stimulate the activity and expression of estrogen sulfotransferase (EST), the enzyme responsible for the formation of the biologically inactive estrogen sulfates. The level of EST mRNA steady-state of cells treated with melatonin was three times higher than that in control cells. These findings which document that melatonin has an inhibitory effect on STS and 17beta-HSD1 and a stimulatory effect on EST, in combination with its previously described antiaromatase effect, can open up new and interesting possibilities in clinical applications of melatonin in breast cancer.

Melatonin and estradiol effects on food intake, body weight, and leptin in ovariectomized rats.

OBJECTIVE: The study in ovariectomized (Ovx) rats, as a model of menopausal status, of the effects of melatonin (M) and/or estradiol (E), associated or not with food restriction, on body weight (BW) and serum leptin levels. METHODS: Female SD rats (200-250 g) were Ovx and treated with E, M, E+M or its diluents. Control sham-Ovx rats were treated with E-M diluents. After 7 weeks being fed ad libitum, the animals were exposed for 7 more weeks to a 30% food restriction. We measured: food intake, BW, nocturnal and diurnal urinary excretion of sulphatoxymelatonin (aMT6s), leptin in midday and midnight blood samples, glucose, total cholesterol, LDL, HDL and triglycerides. RESULTS: Day/night rhythm of aMT6s excretion was preserved in all cases. The increase of aMT6s excretion in M-treated animals basically affected the nocturnal period. In animals fed ad libitum, E fully prevented Ovx-induced increase of BW, leptin and cholesterol. Melatonin reduced food intake and partially prevented the increase of BW and cholesterol, without changing leptin levels. Under food restriction, M was the most effective treatment in reducing BW and cholesterol. Leptin levels were similar in M, E or E+M treated rats, and lower than in untreated Ovx rats. CONCLUSIONS: Our result gives a preliminary experimental basis for a post-menopausal co-treatment with estradiol and melatonin. It could combine the effectiveness of estradiol (not modified by melatonin) with the positive effects of melatonin (improvement of sleep quality, prevention of breast cancer, etc.). The possible beneficial effects of melatonin which could justify its use, need to be demonstrated in clinical trials.

Inhibitory effects of pharmacological doses of melatonin on aromatase activity and expression in rat glioma cells.

Melatonin exerts oncostatic effects on different kinds of neoplasias, especially on oestrogen-dependent tumours. Recently, it has been described that melatonin, on the basis of its antioxidant properties, inhibits the growth of glioma cells. Glioma cells express oestrogen receptors and have the ability to synthesise oestrogens from androgens. In the present study, we demonstrate that pharmacological concentrations of melatonin decreases the growth of C6 glioma cells and reduces the local biosynthesis of oestrogens, through the inhibition of aromatase, the enzyme that catalyses the conversion of androgens into oestrogens. These results are supported by three types of evidence. Firstly, melatonin counteracts the growth stimulatory effects of testosterone on glioma cells, which is dependent on the local synthesis of oestrogens from testosterone. Secondly, we found that melatonin reduces the aromatase activity of C6 cells, measured by the tritiated water release assay. Finally, by (RT)-PCR, we found that melatonin downregulates aromatase mRNA steady-state levels in these glioma cells. We conclude that melatonin inhibits the local production of oestrogens decreasing aromatase activity and expression. By analogy to the implications of aromatase in other forms of oestrogen-sensitive tumours, it is conceivable that the modulation of the aromatase by pharmacological melatonin may play a role in the growth of glioblastomas.

New vaccines require potent adjuvants like AFPL1 and AFCo1.

Neisseria meningitidis B proteoliposome (AFPL1 when used as adjuvant) and its derivative-Cochleate (AFCo1) contain immunopotentiating and immunomodulating properties and delivery system capacities required for a good adjuvant. Additionally, they contain meningococcal protective antigens and permit packaging of other antigens and pathogen-associated molecular patterns (PAMP). Consequently, we hypothesized that they would function as good vaccine adjuvants for their own antigens and also for non-related antigens. AFPL1 is a detergent-extracted outer membrane vesicle of N. meningitidis B transformed into AFCo1 in calcium environment. Both are produced at Finlay Institute under good manufacture practices (GMP) conditions. We show their exceptional characteristics: combining in the same structure, the potentiator activity, polarizing agents and delivery system capacities; presenting multimeric protein copies; containing multiprotein composition and multi and synergistic PAMP components; acting with incorporated or co-administrated antigens; inducing type I IFN-gamma and IL-12 cytokines suggesting the stimulation of human plasmocytoid precursor and conventional dendritic cells, respectively, inducing a preferential Th1 immune response with TCD4(+), TCD8(+), cross-presentation and cytotoxic T-lymphocyte (CTL) in vivo responses; and functioning by parenteral and mucosal routes. AFPL1-AFCo1 protective protein constitutions permit per se their function as a vaccine. In addition to Phase IV Men BC vaccine, AFPL1 has ended the preclinical stage in an allergy vaccine and is concluding the preclinical stage of a nasal meningococcal vaccine. In conclusion, AFPL1 and AFCo1 induced signal 1, 2 and 3 polarizing to a Th1 (including CTL) response when they acted directly as vaccines or were used as adjuvants with incorporated or co-administered antigens by parenteral or mucosal routes. Both are very promising adjuvants.

Melatonin prevents the estrogenic effects of sub-chronic administration of cadmium on mice mammary glands and uterus.

Cadmium (Cd) is a heavy metal classified as a human carcinogen. Occupational exposure, dietary consumption and cigarette smoking are sources of Cd contamination. Cd-induced carcinogenicity depends on its oxidative and estrogenic actions. A possible role of Cd in breast cancer etiology has been recently suggested. Melatonin, because of its antioxidant and antiestrogenic properties could counteract the toxic effects of this metalloestrogen. Our aim was both to determine the effects of relevant doses of Cd on mice mammary glands and uterus and to test whether melatonin would counteract its effects. Female mice of different ages and estrogenic status (prepuberal, adult intact, adult ovariectomized) were treated with CdCl(2) (2-3 mg/kg, i.p.), melatonin (10 microg/mL in drinking water), CdCl(2) + melatonin, or diluents. Whereas in prepuberal animals Cd disturbs mammary ductal growth and reduces the number of terminal end buds, in adults, regardless of the steroidal milieu, Cd exerts estrogenic effects on mammary glands, increasing lobuloalveolar development and ductal branching. Uterine weight also increased as a result of Cd treatment. The effects of Cd are partially inhibited by melatonin. In adult ovariectomized mice, Cd concentration in blood of animals treated with CdCl(2) + melatonin was lower than in mice receiving only Cd; the opposite effects were found in non-castrated animals. As Cd mimics the effect of estrogens, the high incidence of breast cancer in tobacco smokers and women working in industries related with Cd could be explained because of the properties of this metal. The effects of melatonin point to a possible role of this indoleamine as a preventive agent for environmental or occupational Cd contamination.

Effects of MT1 melatonin receptor overexpression on the aromatase-suppressive effect of melatonin in MCF-7 human breast cancer cells.

A major mechanism through which melatonin reduces the development of breast cancer is based on its anti-estrogenic actions by interfering at different levels with the estrogen-signalling pathways. Melatonin inhibits both aromatase activity and expression in vitro (MCF-7 cells) as well as in vivo, thus behaving as a selective estrogen enzyme modulator. The objective of this study was to study the effect of MT1 melatonin receptor overexpression in MCF-7 breast cancer cells on the aromatase-suppressive effects of melatonin. Transfection of the MT1 melatonin receptor in MCF-7 cells significantly decreased aromatase activity of the cells and MT1-transfected cells showed a level of aromatase activity that was 50% of vector-transfected MCF-7 cells. The proliferation of estrogen-sensitive MCF-7 cells in an estradiol-free media but in the presence of testosterone (an indirect measure of aromatase activity) was strongly inhibited by melatonin in those cells overexpressing the MT1 receptor. This inhibitory effect of melatonin on cell growth was higher on MT1 transfected cells than in vector transfected ones. In MT1-transfected cells, aromatase activity (measured by the tritiated water release assay) was inhibited by melatonin (20% at 1 nM; 40% at 10 microM concentrations). The same concentrations of melatonin did not significantly influence the aromatase activity of vector-transfected cells. MT1 melatonin receptor transfection also induced a significant 55% inhibition of aromatase steady-state mRNA expression in comparison to vector-transfected MCF-7 cells (p<0.001). In addition, in MT1-transfected cells melatonin treatment inhibited aromatase mRNA expression and 1 nM melatonin induced a higher and significant down-regulation of aromatase mRNA expression (p<0.05) than in vector-transfected cells. The findings presented herein point to the importance of MT1 melatonin receptor in mediating the oncostatic action of melatonin in MCF-7 human breast cancer cells and confirm MT1 melatonin receptor as a major mediator in the melatonin signalling pathway in breast cancer.

Melatonin inhibits both ER alpha activation and breast cancer cell proliferation induced by a metalloestrogen, cadmium.

Cadmium (Cd) is a heavy metal affecting human health both through environmental and occupational exposure. There is evidence that Cd accumulates in several organs and is carcinogenic to humans. In vivo, Cd mimics the effect of estrogens in the uterus and mammary gland. In estrogen-responsive breast cancer cell lines, Cd stimulates proliferation and can also activate the estrogen receptor independent of estradiol. The ability of this metalloestrogen to increase gene expression in MCF7 cells is blocked by anti-estrogens suggesting that the activity of these compounds is mediated by ER alpha. The aims of this work were to test whether melatonin inhibits Cd-induced proliferation in MCF7 cells, and also to study whether melatonin specifically inhibits Cd-induced ER alpha transactivation. We show that melatonin prevents the Cd-induced growth of synchronized MCF7 breast cancer cells. In transient transfection experiments, we prove that both ER alpha- and ER beta-mediated transcription are stimulated by Cd. Melatonin is a specific inhibitor of Cd-induced ER alpha-mediated transcription in both estrogen response elements (ERE)- and AP1-containing promoters, whereas ER beta-mediated transcription is not inhibited by the pineal indole. Moreover, the mutant ER alpha-(K302G, K303G), unable to bind calmodulin, is activated by Cd but becomes insensitive to melatonin treatment. These results proved that melatonin inhibits MCF7 cell growth induced by Cd and abolishes the stimulatory effect of the heavy metal in cells expressing ER alpha at both ERE-luc and AP1-luc sites. We can infer from these experiments that melatonin regulates Cd-induced transcription in both ERE- and AP1 pathways. These results also reinforce the hypothesis of the anti-estrogenic properties of melatonin as a valuable tool in breast cancer therapies.

Genetic mapping of a caffeoyl-coenzyme A 3-O-methyltransferase gene in coffee trees. Impact on chlorogenic acid content.

Chlorogenic acids (CGA) are involved in the bitterness of coffee due to their decomposition in phenolic compounds during roasting. CGA mainly include caffeoyl-quinic acids (CQA), dicaffeoyl-quinic acids (diCQA) and feruloyl-quinic acids (FQA), while CQA and diCQA constitute CGA sensu stricto (CGA s.s.). In the two cultivated species Coffea canephora and Coffea arabica, CGA s.s. represents 88% and 95% of total CGA, respectively. Among all enzymes involved in CGA biosynthesis, caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is not directly involved in the CGA s.s. pathway, but rather in an upstream branch leading to FQA through feruloyl-CoA. We describe how a partial cDNA corresponding to a CCoAOMT encoding gene was obtained and sequenced. Specific primers were designed and used for studying polymorphism and locating the corresponding gene on a genetic map obtained from an interspecific backcross between Coffea liberica var. Dewevrei and Coffea pseudozanguebariae. Offspring of this backcross were also evaluated for the chlorogenic acid content in their green beans. A 10% decrease was observed in backcross progenies that possess one C. pseudozanguebariae allele of the CCoAOMT gene. This suggests that CGA s.s. accumulation is dependent on the CCoAMT allele present and consequently on the activity of the encoded isoform, whereby CGA accumulation increases as the isoform activity decreases. Possible implications in coffee breeding are discussed.

Regiospecific transglycolytic synthesis and structural characterization of 6-O-alpha-glucopyranosyl-glucopyranose (isomaltose).

The enzymatic synthesis of 6-O-alpha-glucopyranosyl-glucopyranose (isomaltose) was achieved. The regiospecific transglycosylation reaction was catalyzed by a crude preparation of alpha-D-glucosidase from Aspergillus niger, using p-nitrophenyl alpha-D-glucopyranose as the donor and glucopyranose as the acceptor. The yield of the reaction was 59% on a molar basis with respect to the donor. The structural identity of the product was fully determined by HPLC, HPAEC-PAD, ionspray mass spectrometry and (13)C NMR.