Biomedical subjects
Publications and source records attributed to P A Cox.
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We tested the brain tissues of the Chamorro people of Guam who died of amyotrophic lateral sclerosis/Parkinsonism dimentia complex (ALS/PDC) for the neurotoxin beta-methylamino-l-alanine (BMAA). We used validated high-pressure liquid chromatography and liquid chromatography-mass spectrometry analyses to test well-characterized archival tissues of the superior frontal gyrus from eight Chamorros from Guam and a comparison group of 15 Canadians. BMAA was found as a free amino acid in 83% of Chamorro ALS/PDC patients (3-10 microg/g) as a protein-associated amino acid in 100% of the Chamorro individuals (149-1190 microg/g). Both forms of BMAA were also found at comparable levels in two Canadians who died of progressive neurodegenerative disease. BMAA, which is produced by cyanobacteria, may be associated with some cases of neurodegenerative disease.
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In our ongoing program to find new anti-inflammatory compounds, 58 extracts from 46 different medicinal plant species, used in treatment of inflammatory disorders-38 plants from the traditional medicine of Western Samoa and eight originating from the indigenous medicine of the Shipibo-Conibo tribe of Peruvian Amazonia-ere evaluated. The ability of all extracts to inhibit cyclooxygenase-1 catalysed prostaglandin biosynthesis in vitro was examined. Of the plant species tested 14 showed moderate to strong inhibition; including 11 Samoan and three Peruvian species. Further, 12 Samoan and all eight Peruvian species were investigated on their inhibitory activity of ethyl phenylpropiolate induced rat ear oedema in vivo. Significant activity was shown by 10 of the Samoan and by all eight Peruvian species. An additional evaluation of the most active species was provided through a compilation of existing literature documenting traditional medicinal uses, pharmacological activity and chemical constituents. Several known cyclooxygenase-1 inhibitors were reported to which the observed pharmacological activity can be attributed at least partly. The combination of chemical and pharmacological literature data and our experimental data may help to explain the anti-inflammatory use of these species in indigenous medicine.
Prior ultraviolet (UV) irradiation of the site of application of hapten on murine skin reduces contact sensitization, impairs the ability of dendritic cells in the draining lymph nodes (DLN) to present antigen, and leads to development of hapten-specific suppressor T lymphocytes. We tested the hypothesis that UV-induced DNA damage plays a role in the impaired antigen-presenting activity of DLN cells. First, we assessed the location and persistence of cells containing DNA damage. A monoclonal antibody specific for cyclobutyl pyrimidine dimers (CPD) was used to identify UV-damaged cells in the skin and DLN of C3H mice exposed to UV radiation. Cells containing CPD were present in the epidermis, dermis, and DLN and persisted, particularly in the dermis, for at least 4 d after UV irradiation. When fluorescein isothiocyanate (FITC) was applied to UV-exposed skin, the DLN contained cells that were Ia+, FITC+, and CPD+; such cells from mice sensitized 3 d after UV irradiation exhibited reduced antigen-presenting function in vivo. We then assessed the role of DNA damage in UV-induced modulation of antigen-presenting cell (APC) function by using a novel method of increasing DNA repair in mouse skin in vivo. Liposomes containing T4 endonuclease V (T4N5) were applied to the site of UV exposure immediately after irradiation. This treatment prevented the impairment in APC function and reduced the number of CPD+ cells in the DLN of UV-irradiated mice. Treatment of unirradiated skin with T4N5 in liposomes or treatment of UV-irradiated skin with liposomes containing heat-inactivated T4N5 did not restore immune function. These studies demonstrate that cutaneous immune cells sustain DNA damage in vivo that persists for several days, and that FITC sensitization causes the migration of these to the DLN, which exhibits impaired APC function. Further, they support the hypothesis that DNA damage is an essential initiator of one or more of the steps involved in impaired APC function after UV irradiation.
Exposure of mice to UVB radiation down-regulates the induction of contact hypersensitivity (CHS) responses to haptens applied to the site of irradiation. Concomittantly, the activity of antigen-presenting cells (APC) in the draining lymph nodes is decreased, and T lymphocytes that suppress the induction of CHS are induced. We assessed the rôle of DNA damage in modulation of the CHS response by UV irradiation by applying liposomes containing T4 endonuclease V (T4N5) to the UV-irradiated skin. Liposomal T4N5, which increases the rate of repair of cyclobutyl pyrimidine dimers (CPD) in DNA, prevented the reduction in the CHS response, the impairement in APC function, and the induction of transferrable immune suppression. Liposomes containing heat-inactivated T4N5 did not restore immune responsiveness. In this model, hapten-bearing APC from unirradiated mice also fail to induce CHS upon injection into UV-irradiated recipients. This systemic effect of UV irradiation on APC function was also prevented by application of liposomes containing active, but not inactive, T4N5. These studies support the hypothesis that DNA damage is an essential initiator of one or more steps leading to impaired immune responsiveness after UV irradiation. They further imply that the release of cytokines that modulate APC function after UV irradiation is triggered by DNA damage.
Ultraviolet-B irradiation of murine skin before contact sensitization alters the antigen-presenting activity of cells in the skin and draining lymph nodes (DLN), decreases the contact hypersensitivity (CHS) response, and induces hapten-specific Ts cells. We determined whether hapten-bearing antigen-presenting cells (APC) from the DLN of UV-irradiated mice induce Ts cells. APC from UV-irradiated, FITC-sensitized mice were isolated, and FITC+ cells were FACS-purified and injected into the hind footpads of naive, syngeneic mice. Unseparated and FACS-purified bright FITC+ cells (FITChi) from UV-irradiated mice induced Ts. However, dull (FITClo) FITC+ cells, T-cell-depleted FITChi cells, or DLN cells from C3H SCID mice failed to induce Ts. Injection of FITC-bearing DLN cells from nonirradiated mice into UV-irradiated recipients failed to immunize and induced Ts instead. Treatment of the UV-irradiated recipients with anti-TNF-alpha blocked these effects. Thus, transferable suppression is due to Thy-1+ cells, does not require UV-altered APC for induction, and may depend on TNF-alpha.
The phospholipase D-inhibitory activity of a methanol extract from the leaves of a New Zealand plant, Myrsine australis, has been attributed to two new saponins 1 and 2. Compound 1 was assigned as 3-0-[-beta-D-xylopyranosyl-(1-->2)-0-beta-D-glucopyranosyl-(1-->4)- -[0-beta-D -glucopyranosyl-(1-->2)]-alpha-L-arabinosyl]-16alpha-hydroxy-+ ++13beta,28-epoxyoleanane and 2 as 3beta-0-[-beta-rhamnopyranosyl-(1-->2)-0-beta-D-glucopyranosyl-(1-->4)-[ 0-beta-D-glucopranosyl]-alpha-L-arabinopyranosyl]-16alpha -hydroxy-13beta, 28-epoxyleanane. Compounds 1 and 2 showed IC50 values of 3 and 2 microM, respectively, versus phorbol 12-myrisate-13-acetate-stimulated phosphlipase D in human promyelocytic leukemic (HL-60) cells. Compounds 1 and 2 also inhibited fMLP (formyl-Met-Leu-Phe) stimulated phospholipase D with IC50 values of 8 and 24 microM, respectively.
Exposure of murine skin to suberythemal doses of ultraviolet-B (UVB) radiation before contact sensitization alters the activity of antigen-presenting cells (APC) in the draining lymph nodes (DLN), decreases the contact hypersensitivity (CHS) response, and induces hapten-specific Ts cells. We determined whether in vivo UVB irradiation alters the ability of hapten-bearing APC from the DLN to form clusters with hapten-specific T lymphocytes. When APC from UV-irradiated, fluorescein isothiocyanate (FITC)-sensitized mice were mixed with a FITC-specific T-cell line, significantly fewer clusters formed compared with FITC+ APC from unirradiated mice. A higher percentage of clusters formed with APC from UV-irradiated mice were Mac-1+ and bound both CD4+ and CD8+ T cells, whereas FITC+ APC from nonirradiated mice bound CD4+ cells. These results suggest that UVB irradiation interferes with the induction of CHS by altering the functional interaction between APC and T cells, perhaps by altering the population of APC in the skin.
A water soluble extract from the bark of the Samoan medicinal plant Alphitonia zizyphoides A. Gray (Rhamnaceae), enhances the plating efficiency in vitro of lymphoid cell lines as well as the survival of bone marrow cells and normal T and B lymphocytes. Furthermore, the inclusion of bark-extract into culture media enhances the cloning efficiency of a T-hybridoma cell line by more than 30 times at otherwise unsuitably low serum concentrations, but does not completely substitute for serum. The enhanced growth of a B-cell hybridoma is also paralleled by an increased production of monoclonal antibodies in cultures containing low cell densities.
Nutmeg, endemic to the Maluku Province of Indonesia (formerly known as the Spice Islands), has long been of importance both as a spice and as a commodity that was once of geopolitical significance. Although its botany, cultivation, and history have been studied, ethnobotanical studies of nutmeg in its place of origin are noticeably lacking. The ethnobotany of nutmeg, Myristica fragrans Houtt. (Myristicaceae), was studied in the Indonesian provinces of Maluku and Central and East Java. Historical and current indigenous uses of the fruit and seed are described. Information regarding medicinal aspects was gathered from vendors of herbal remedies, healers, and midwives. It was found that M.fragrans is still used for both culinary and medicinal purposes in its area of origin. M.fragrans is used for diarrhea, mouth sores, and insomnia. In addition, no evidence was found to support previously published reports of nutmeg's hallucinogenic or other psychoactive properties other than a mild sedative action. A renewed suggestion is made that anecdotal reports of nutmeg use as a hallucinogen be revaluated more critically.
High-field nmr experiments have been used to determine the structures of three new saponins, zizyphoisides C, D, and E [1-3], isolated from Alphitonia zizyphoides, a medicinal plant found in the Samoan rain forest. The saponins consist of the aglycone, jujubogenin, to which are attached three sugar units and an unsaturated side-chain. The three compounds are similar except for the location of an acetyl group on one of the sugar units.
For pharmaceuticals ranging from digitalis to vincristine the ethnobotanical approach to drug discovery has proven successful. The advent of high-throughput, mechanism-based in vitro bioassays coupled with candidate plants derived from pain-staking ethnopharmacological research has resulted in the discovery of new pharmaceuticals such as prostratin, a drug candidate for treatment of human immunodeficiency virus, as well as a variety of novel antiinflammatory compounds. Not all Western diseases are equally likely to be recognized by indigenous peoples. Gastrointestinal maladies, inflammation, skin infections and certain viral diseases are likely to be of high saliency to indigenous healers, whereas diseases such as cancer and cardiovascular illness are unlikely to be easily diagnosed by indigenous peoples. Yet indigenous remedies may indicate pharmacological activity for maladies such as schizophrenia, for which the biochemical mechanisms have yet to be discovered. Ethnopharmacological information can be used to provide three levels of resolution in the search for new drugs: (1) as a general indicator of non-specific bioactivity suitable for a panel of broad screens; (2) as an indicator of specific bioactivity suitable for particular high-resolution bioassays; (3) as an indicator of pharmacological activity for which mechanism-based bioassays have yet to be developed.
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Early European visitors to Samoa tended to denigrate the authenticity and efficacy of Samoan herbal medicine, yet bioassays indicate pharmacological activity in over 86% of Samoan medicinal plants. Novel anti-inflammatory compounds have been isolated from Alphitonia zyzyphoides and Erythrina variegata, and the anti-HIV compound prostratin has been isolated from Homalanthus nutans. Unfortunately, both Samoan ethnopharmacology and Samoan rain forests are threatened. In order to prevent logging, funds were raised to build a needed village school in exchange for a village covenant to protect the 30,000 acre Falealupo forest. Subsequently, four additional rainforest reserves have been established. Hopefully such conservation measures can save the ethnopharmacological knowledge of Samoa.
Exposing the skin of mice to UV radiation interferes with the induction of delayed and contact hypersensitivity immune responses initiated at nonirradiated sites. The identity of the molecular target in the skin for these immunosuppressive effects of UV radiation remains controversial. To test the hypothesis that DNA is the target for UV-induced systemic immunosuppression, we exposed C3H mice to UV radiation and then used liposomes to deliver a dimer-specific excision repair enzyme into the epidermis in situ. The application of T4 endonuclease V encapsulated in liposomes to UV-irradiated mouse skin decreased the number of cyclobutane pyrimidine dimers in the epidermis and prevented suppression of both delayed and contact hypersensitivity responses. Moreover, the formation of suppressor lymphoid cells was inhibited. Control, heat-inactivated endonuclease encapsulated in liposomes had no effect. These studies demonstrate that DNA is the major target of UV radiation in the generation of systemic immunosuppression and suggest that the primary molecular event mediating these types of immunosuppression by UV radiation is the formation of pyrimidine dimers. Furthermore, they illustrate that the delivery of lesion-specific DNA repair enzymes to living skin after UV irradiation is an effective tool for restoring immune function and suggest that this approach may be broadly applicable to preventing other alterations caused by DNA damage.
The unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor were expressed in bacterial and used to produce subunit-specific polyclonal antisera. Antibodies immobilized on protein A-Sepharose were used to isolate naturally occurring alpha-specific populations of GABAA receptors from rat brain that retained the ability to bind [3H]muscimol, [3H]flunitrazepam, [3H]Ro15-1788, and [125I]iodo-clonazepam with high affinity. Pharmacological characterization of these subtypes revealed marked differences between the isolated receptor populations and was generally in agreement with the reported pharmacological profiles of GABAA receptors in cells transiently transfected with alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 combinations of subunits. Additional subtypes were also identified that bind [3H]muscimol but do not bind benzodiazepines with high affinity. The majority of GABAA receptor oligomers contains only a single type of alpha subunit, and we conclude that alpha 1, alpha 2, alpha 3, and alpha 5 subunits exist in vivo in combination with the beta subunit and gamma 2 subunit.
Bioactive molecules occur in plants as secondary metabolites and as defence mechanisms against predation, herbivores, fungal attack, microbial invasion and viral infection. Throughout the world indigenous peoples have discovered plants with pharmacological activity; many useful drugs such as vincristine, reserpine, quinine, and even aspirin, have their origins in indigenous ethnopharmacologies. Three features characterize indigenous cultures that are most likely to have discovered useful natural products: (1) possession of a conservative ethnomedical tradition with established cultural mechanisms for accurate transmission of ethnopharmacological knowledge; (2) residence in an area with a diverse flora; and (3) continuity of residence in the area over many generations. Ethnopharmacological data derived from such cultures can be considered as analogues of human bioassay data. Yet indigenous ethnomedical traditions are rapidly disappearing. To preserve indigenous discoveries and make them accessible and useful to Western medicine, collaboration is required between linguistically sophisticated ethnobotanists, pharmacognosists, natural product chemists and pharmacologists. Ethnopharmacologists must be aware of all three components of indigenous medical traditions (a cosmological foundation, a repertoire of crude natural products, and a health care delivery system) to facilitate transfer of useful compounds and information to Western medicine, as well as to protect the interests and cultural integrity of indigenous peoples.