Dyeing the cross-sections
2 - Phloroglucinol/HCl is used to dye lignified cell walls red
Phloroglucinol/HCl (figure 2) is used to dye lignified cell walls red. This dye works best for herb-chronology because it pronounces the structures you are interested in (the vessels and lignified parenchyma that often adds to the visualization of growth rings in the roots). To prepare the dye dissolve 1-2 points of a spattle of the phenol derivative phloroglucinol (also known as phloroglucin) in 20 ml of 75% alcohol. Use HCl conc. (≥32%) as the other reagent. Using dropper bottles you first apply one drop of phloroglucinol/EtOH to the cutting and, after 5-10 seconds or so, add one drop of HCl. In most cases this produces instantaneous reddish colouring of the lignified tissue. For thin cuttings where only the vessels are lignified colouring may take a little while. In this case do not take the photographs too quickly or you might end up with photos of low contrast. To help speed up colouring you might add a second drop of HCl. On the other hand, thicker cuttings or those that have a lignified parenchyma may colour quickly and deeply so that you should take the photo quickly to prevent dark photographs. In this case you should reduce the amounts of dye applied to the cuttings. If you do not produce satisfactory colouring try to add some more phloroglucinol to the alcoholic solution.
Monday, July 28, 2008
"The Fungal Kingdom: Diverse and Essential Roles in Earth's Ecosystem," June 2008
"The Fungal Kingdom: Diverse and Essential Roles in Earth's Ecosystem," (June 2008)
Prepared by Arturo Casadevall, Joe Heitman, and Merry Buckley
Fungi can cause a number of life-threatening diseases but they also are becoming increasingly useful to science and manufacturing every year. However, many people, scientists among them, are largely unaware of the roles fungi play in the world around us. Research on fungi and fungal diseases are seriously neglected as a result – a situation with grave negative repercussions for human health, agriculture, and the environment. The Fungal Kingdom explores the roles fungi play in the world around us.
http://www.asm.org/ASM/files/ccLibraryFiles/Filename/000000004018/Fungal_Kingdom.pdf
Prepared by Arturo Casadevall, Joe Heitman, and Merry Buckley
Fungi can cause a number of life-threatening diseases but they also are becoming increasingly useful to science and manufacturing every year. However, many people, scientists among them, are largely unaware of the roles fungi play in the world around us. Research on fungi and fungal diseases are seriously neglected as a result – a situation with grave negative repercussions for human health, agriculture, and the environment. The Fungal Kingdom explores the roles fungi play in the world around us.
http://www.asm.org/ASM/files/ccLibraryFiles/Filename/000000004018/Fungal_Kingdom.pdf
Wednesday, July 23, 2008
Prehistoric mystery organism verified as giant fungus
Prehistoric mystery organism verified as giant fungus
‘Humongous fungus’ towered over all life on land
April 23, 2007
Scientists at the University of Chicago and the National Museum of Natural History in Washington, D.C., have produced new evidence to finally resolve the mysterious identity of what they regard as one of the weirdest organisms that ever lived.
Their chemical analysis indicates that the organism was a fungus, the scientists report in the May issue of the journal of Geology, published by the Geological Society of America. Called Prototaxites (pronounced pro-toe-tax-eye-tees), the organism went extinct approximately 350 million years ago.
Prototaxites has generated controversy for more than a century. Originally classified as a conifer, scientists later argued that it was instead a lichen, various types of algae or a fungus. Whatever it was, it stood in tree-like trunks more than 20 feet tall, making it the largest-known organism on land in its day.
“No matter what argument you put forth, people say, well, that’s crazy. That doesn’t make any sense,” said C. Kevin Boyce, an Assistant Professor in Geophysical Sciences at Chicago. “A 20-foot-tall fungus doesn’t make any sense. Neither does a 20-foot-tall algae make any sense, but here’s the fossil.”
The Geology paper adds a new line of evidence indicating that the organism is a fungus. The fungus classification first emerged in 1919, with Francis Hueber of the National Museum of Natural History in Washington, D.C., reviving the idea in 2001. His detailed studies of internal structure have provided the strongest anatomical evidence that Prototaxites is not a plant, but a fungus.
“Fran Hueber has contributed more to our understanding of Prototaxites than anyone else, living or dead,” said Carol Hotton, also of the National Museum of Natural History. “He built up a convincing case based on the internal structure of the beast that it was a giant fungus, but agonized over the fact that he was never able to find a smoking gun in the form of reproductive structures that would convince the world that it was indeed a fungus,” Hotton said.
Co-authoring the Geology paper with Boyce, Hotton and Hueber himself were Marilyn Fogel, George Cody and Robert Hazen of the Carnegie Institution of Washington, and Andrew Knoll of Harvard University. Their work was funded by NASA’s Astrobiology Institute and by the American Chemical Society Petroleum Fund.
Prototaxites lived worldwide from approximately 420 million to 350 million years ago. During this period, which spans part of the Silurian and Devonian periods of geologic time, terrestrial Earth looked quite alien in comparison to the modern world.
Simple vascular plants, the ancestors of the familiar conifers, ferns and flowering plants of today, began to diversify on land during the Devonian Period. “Initially, they’re just stems. They don’t have roots. They don’t have leaves. They don’t have anything like that,” Boyce said.
Millipedes, wingless insects and worms were among the other organisms making a living on land by then, but no backboned animals had yet evolved out of the oceans. “That world was a very strange place,” Boyce said.
Although vascular plants had established themselves on land 40 million years before the appearance of Prototaxites, the tallest among them stood no more than a couple feet high. By the end of the Devonian, approximately 345 million years ago, large trees, ferns, seeds, leaves and roots had all evolved. “They’re all there. They just exploded over this one time period,” Boyce said.
Canadian paleontologist Charles Dawson published the first research on Prototaxites in 1859, based on specimens found along the shores of Gaspé Bay in Quebec, Canada. Hueber pored through Dawson’s field notebooks, written “in a completely illegible scrawl,” Hotton said.
“Fran spent months deciphering them for clues about the localities where specimens had been collected, how Dawson interpreted them and other information that helped understand this humongous fungus,” she said.
Hueber also traveled to Canada, Australia and Saudi Arabia to collect specimens. He tediously sliced them into hundreds of thin sections and made thousands of images taken through microscopes to determine the organism’s identity.
Now Boyce, Hotton and their colleagues have produced independent evidence that supports Hueber’s case. The team did so by analyzing two varieties—isotopes—of carbon contained in Prototaxites and the plants that lived in the same environment approximately 400 million years ago.
The metabolism of plants is limited by photosynthesis. Deriving their energy from the sun and their carbon from carbon dioxide in the air, any given type of plant will typically contain a similar ratio of carbon-12 to carbon-13 as another plant of the same type. “But if you’re an animal, you will look like whatever you eat,” Boyce said. And Prototaxites displayed a much wider variation in its ratio of carbon-12 to carbon-13 content than would be expected in any plant.
Geological processes can alter the isotopic composition of fossils, but Boyce and his colleagues conducted tests to verify that the carbon isotopic composition of the specimens they analyzed stemmed from organic rather than geologic factors.
As for why these bizarre organisms grew so large, “I’ve wondered whether it enabled Prototaxites to distribute its spores widely, allowing it to occupy suitable marshy habitats that may have been patchily distributed on the landscape,” Hotton said.
The relatively simple Devonian ecosystems certainly seemed to contain nothing to prevent them from growing slowly for a long time. Plant-eating animals had not yet evolved, Boyce said. But even if Prototaxites hadn’t been eaten by the dinosaurs and elephants that came much later, they probably grew too slowly to rebuild from regular disturbances of any kind, Boyce said.
“It’s hard to imagine these things surviving in the modern world,” he said.
from:
http://www-news.uchicago.edu/releases/07/070423.fungus.shtml
‘Humongous fungus’ towered over all life on land
April 23, 2007
Scientists at the University of Chicago and the National Museum of Natural History in Washington, D.C., have produced new evidence to finally resolve the mysterious identity of what they regard as one of the weirdest organisms that ever lived.
Their chemical analysis indicates that the organism was a fungus, the scientists report in the May issue of the journal of Geology, published by the Geological Society of America. Called Prototaxites (pronounced pro-toe-tax-eye-tees), the organism went extinct approximately 350 million years ago.
Prototaxites has generated controversy for more than a century. Originally classified as a conifer, scientists later argued that it was instead a lichen, various types of algae or a fungus. Whatever it was, it stood in tree-like trunks more than 20 feet tall, making it the largest-known organism on land in its day.
“No matter what argument you put forth, people say, well, that’s crazy. That doesn’t make any sense,” said C. Kevin Boyce, an Assistant Professor in Geophysical Sciences at Chicago. “A 20-foot-tall fungus doesn’t make any sense. Neither does a 20-foot-tall algae make any sense, but here’s the fossil.”
The Geology paper adds a new line of evidence indicating that the organism is a fungus. The fungus classification first emerged in 1919, with Francis Hueber of the National Museum of Natural History in Washington, D.C., reviving the idea in 2001. His detailed studies of internal structure have provided the strongest anatomical evidence that Prototaxites is not a plant, but a fungus.
“Fran Hueber has contributed more to our understanding of Prototaxites than anyone else, living or dead,” said Carol Hotton, also of the National Museum of Natural History. “He built up a convincing case based on the internal structure of the beast that it was a giant fungus, but agonized over the fact that he was never able to find a smoking gun in the form of reproductive structures that would convince the world that it was indeed a fungus,” Hotton said.
Co-authoring the Geology paper with Boyce, Hotton and Hueber himself were Marilyn Fogel, George Cody and Robert Hazen of the Carnegie Institution of Washington, and Andrew Knoll of Harvard University. Their work was funded by NASA’s Astrobiology Institute and by the American Chemical Society Petroleum Fund.
Prototaxites lived worldwide from approximately 420 million to 350 million years ago. During this period, which spans part of the Silurian and Devonian periods of geologic time, terrestrial Earth looked quite alien in comparison to the modern world.
Simple vascular plants, the ancestors of the familiar conifers, ferns and flowering plants of today, began to diversify on land during the Devonian Period. “Initially, they’re just stems. They don’t have roots. They don’t have leaves. They don’t have anything like that,” Boyce said.
Millipedes, wingless insects and worms were among the other organisms making a living on land by then, but no backboned animals had yet evolved out of the oceans. “That world was a very strange place,” Boyce said.
Although vascular plants had established themselves on land 40 million years before the appearance of Prototaxites, the tallest among them stood no more than a couple feet high. By the end of the Devonian, approximately 345 million years ago, large trees, ferns, seeds, leaves and roots had all evolved. “They’re all there. They just exploded over this one time period,” Boyce said.
Canadian paleontologist Charles Dawson published the first research on Prototaxites in 1859, based on specimens found along the shores of Gaspé Bay in Quebec, Canada. Hueber pored through Dawson’s field notebooks, written “in a completely illegible scrawl,” Hotton said.
“Fran spent months deciphering them for clues about the localities where specimens had been collected, how Dawson interpreted them and other information that helped understand this humongous fungus,” she said.
Hueber also traveled to Canada, Australia and Saudi Arabia to collect specimens. He tediously sliced them into hundreds of thin sections and made thousands of images taken through microscopes to determine the organism’s identity.
Now Boyce, Hotton and their colleagues have produced independent evidence that supports Hueber’s case. The team did so by analyzing two varieties—isotopes—of carbon contained in Prototaxites and the plants that lived in the same environment approximately 400 million years ago.
The metabolism of plants is limited by photosynthesis. Deriving their energy from the sun and their carbon from carbon dioxide in the air, any given type of plant will typically contain a similar ratio of carbon-12 to carbon-13 as another plant of the same type. “But if you’re an animal, you will look like whatever you eat,” Boyce said. And Prototaxites displayed a much wider variation in its ratio of carbon-12 to carbon-13 content than would be expected in any plant.
Geological processes can alter the isotopic composition of fossils, but Boyce and his colleagues conducted tests to verify that the carbon isotopic composition of the specimens they analyzed stemmed from organic rather than geologic factors.
As for why these bizarre organisms grew so large, “I’ve wondered whether it enabled Prototaxites to distribute its spores widely, allowing it to occupy suitable marshy habitats that may have been patchily distributed on the landscape,” Hotton said.
The relatively simple Devonian ecosystems certainly seemed to contain nothing to prevent them from growing slowly for a long time. Plant-eating animals had not yet evolved, Boyce said. But even if Prototaxites hadn’t been eaten by the dinosaurs and elephants that came much later, they probably grew too slowly to rebuild from regular disturbances of any kind, Boyce said.
“It’s hard to imagine these things surviving in the modern world,” he said.
from:
http://www-news.uchicago.edu/releases/07/070423.fungus.shtml
Circumventing the PDR pathway in fungi.
Fig. 1. Circumventing the PDR pathway in fungi. (A) In cells with clinically important resistance to azole drugs, high-level transcription of PDR efflux-pump genes involves the recruitment of RNA polymerase II, which depends on a drug-induced interaction between the ScPdr1p/Pdr3p and mediator complexes. The efflux pumps reduce the intracellular concentration of the drug below that required to inhibit the azole target Erg11p, allowing normal cell growth. (B) Binding of a multifunctional azole to the XBD domain of ScPdr1p/Pdr3p blocks expression of the drug pumps responsible for multidrug efflux and inhibits drug efflux by occupying a binding site in residual efflux pumps. The intracellular concentration of the drug is thus sufficient to block ergosterol biosynthesis in the endoplasmic reticulum. Reduced ergosterol content of membranes, production of toxic methylated sterols, and oxidative damage kill the fungal cell. Alternatively, other antifungals directly inhibit the electrogenic plasma membrane H+-ATPase Pma1p, preventing the uptake of nutrients driven by the plasma membrane electrochemical gradient. The cells die rapidly because of a limited cellular energy supply and a loss of ion balance. Partial inhibition of Pma1p activity compromises the activity of both MFS and ABC transporters and increases the potency of azole drugs. [View Larger Version of this Image (55K GIF file)]
Outwitting Multidrug Resistance to Antifungals
Science 18 July 2008:
Vol. 321. no. 5887, pp. 367 - 369
DOI: 10.1126/science.1159746
Prev | Table of Contents | Next
Perspective
Outwitting Multidrug Resistance to Antifungals
Brian C. Monk and Andre Goffeau*
The economic cost of fungal infection and its mortality associated with multidrug resistance remain unacceptably high. Recent understanding of the transcriptional regulation of plasma membrane efflux pumps of modest specificity provides new avenues for the development of broad-spectrum fungicides. Together with improved diagnosis and indirect intervention via inhibition of the energy supply for drug efflux, we envisage multifunctional azole analogs that inhibit not only ergosterol biosynthesis and drug efflux-pump activity but also activation of the transcriptional machinery that induces drug efflux-pump expression.
Department of Oral Sciences, Faculty of Dentistry, University of Otago, Post Office Box 647, Dunedin, New Zealand; and Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, 1348, Belgium.
* To whom correspondence should be addressed. E-mail: andre.goffeau@uclouvain.be
Eight hundred million years of evolution have generated 1.5 million fungal species that occupy many distinct ecological niches, yet only 300 fungi cause disease in humans (1). The identification of antifungals that act specifically against these pathogens is a particular challenge because of fungal diversity, individualized pathways for infection, and fungal use of multiple mechanisms that circumvent exogenous toxins. These highly regulated mechanisms include innate resistance to specific antifungal drugs, formation of biofilms, selection of spontaneous mutations that increase expression or decrease susceptibility of the drug target (2), stress-related tolerance that enhances short-term survival (3, 4), modification of chromosomal ploidy (5), and overexpression of multidrug efflux pumps (6). Fortunately, compared with infections caused by drug-resistant bacteria, those caused by resistant fungal pathogens and their spread to other patients occur relatively infrequently. However, the economic cost of fungal infection and its associated mortality, especially in debilitated and high-investment patients, remain unacceptably high.
A Clinical Perspective
The most prominent fungal pathogens affecting humans include Aspergillus fumigatus, Candida albicans, C. glabrata, C. parasilosis, C. tropicalis, C. krusei, and Cryptococcus neoformans (7). Although the skin, mucosal surfaces, and immune system usually provide robust defenses, weakened immunodefenses dramatically increase susceptibility to debilitating and life-threatening opportunistic fungal infections. Fungal infections are normally treated with a modest repertoire of drugs derived from five antifungal classes that target DNA and RNA synthesis, ergosterol, the ergosterol biosynthetic pathway, or the biosynthesis of the cell-wall component 1,3-β-D-glucan (Table 1). Unfortunately, the prophylactic use of fungistatic azoles such as fluconazole has been associated with an increased frequency of innate or acquired drug resistance in clinical isolates and the selection of non-albicans Candida, non-fumigatus Aspergillus, opportunistic yeastlike fungi, zygomycetes, and hyaline molds. Despite the fact that broader-spectrum third-generation azole drugs and the more expensive echinocandin class of antifungals prevent an increased proportion of life-threatening infections, Candida species remain the fourth most common cause of hospital-acquired bloodstream infection and kill 40% of those patients, whereas disseminated Aspergillus infections kill up to 80% of affected patients.
Mechanisms of Multidrug Resistance
Because of its economic and clinical impact, a focus on multidrug resistance rather than resistance to specific antifungals in pathogenic fungi is timely. Multidrug resistance, called pleiotropic drug resistance (PDR) in Saccharomyces cerevisiae, is an ancient phenomenon that preceded the modern use of antifungals (8). The adenosine triphosphate (ATP)–binding cassette (ABC) and major facilitator superfamily (MFS) transporter families responsible for multidrug resistance operate in all fungi. We distinguish among the transporters that belong to different species by using the prefix Sc for S. cerevisiae, Cg for C. glabrata, or Ca for C. albicans.
Saccharomyces cerevisiae. PDR in S. cerevisiae is the best-understood multidrug resistance mechanism in fungi. Point mutations conferring resistance to chemically diverse drugs (including azoles) have been mapped in genes encoding the zinc-finger transcription factors ScPdr1p or ScPdr3p (9, 10). These gain-of-function mutations activate over 20 target genes, the major ones being either ATP-driven (ABC transporter genes ScPDR5, ScSNQ2, and ScYOR1) orproton motive force–driven (MFS transporter genes ScTPO1 and ScFLR1) efflux pumps (11, 12). Resistance to a wide spectrum of drugs is conferred via the activation of efflux-pump gene expression, which involves the binding of Pdr1p/Pdr3p to the consensus binding element PDRE (13, 14). Mechanisms regulating PDR in S. cerevisiae include mutation of PDR1/3, plasma membrane sphingolipid homeostasis, ScPdr3p autoregulation, ScPdr3p-specific activation due to loss of mitochondrial respiration, chaperone-specific differential regulation of ScPdr1p and ScPdr3p (15), and ScPdr1p-dependent compensatory expression of efflux pumps (16). Yeast cells incubated with antifungals and other drugs transiently activate ScPdr1p/Pdr3p (16). Drugs such as itraconazole and progesterone bind to a 250–amino acid hydrophobic xenobiotic binding domain (XBD) of ScPdr1p/Pdr3p, enabling a specific association with the KIX domain of the Gal11p subunit of the mediator complex that recruits RNA polymerase II for expression of the ScPdr1p/Pdr3p-controlled genes (17) (Fig. 1A). Other transcription factors such as Yrr1p, Stb5p, Rdr1p, Yrm1p, and Yap1p also contribute to the expression of the various efflux transporter genes (17, 18).
Pathogenic fungi. The human pathogen C. glabrata uses the transcription factor CgPdr1p to control expression of the ABC multidrug efflux pumps CgCdr1p and CgCdr2p through mechanisms very similar to those of its close relative S. cerevisiae. The pumps are induced by treatment with diverse drugs and are highly expressed in respiration-defective mutants. Antifungal binding to the CgPdr1p XBD induces multidrug resistance via a KIX domain from the C. glabrata mediator complex (19). Mutants overexpressing CgPdr1p coordinately regulate 11 genes homologous to ScPdr1/ScPdr3p targets (20). These similarities support the use of S. cerevisiae in developing tools that are directly applicable to antifungal resistance in C. glabrata.
Multiple azole resistance stemming from longterm prophylaxis is frequently found in clinical isolates of the more distant pathogen C. albicans (21). The resistance mechanisms of this diploid species are complex and include mutations in single genes, loss of heterozygosity, chromosomal rearrangements, and selective segregation of chromosomal fragments (22). About 85% of fluconazole-resistant clinical isolates show multidrug resistance due to overexpression of the ABC transporters CaCdr1p and CaCdr2p (homologs of the S. cerevisiae ScPdr5p) and the major facilitator superfamily (MFS) pump CaMdr1p (homolog of ScFlr1p). The efflux functions of these transporters can be cloned in S. cerevisiae (23, 24). Expression of the CaCdr1p and CaCdr2p pumps is controlled by the transcription factor CaTac1p (25), which shares about 20% identity with ScPdr1/ScPdr3p. CaTac1p and ScPdr1p/ScPdr3p recognize substantially different PDREs (25), and CaTac1p causes more focused transcription than ScPdr1p/Pdr3p (26, 27). High doses of the female steroid hormone progesterone transiently upregulate, via steroid-specific PDREs, the same core of ABC transporters induced by antifungal intervention or gain-of-function mutations in the transcription factors (26, 28). Fluconazole-resistant clinical isolates often constitutively overexpress the MFS transporter CaMdr1p, either by itself or in combination with the azole target CaErg11p and/or the CaCdr1p and CaCdr2p ABC pumps. Although the MFS transporter CaMdr1p seems more efficient than its homolog ScFlr1p, it is overexpression of ABC transporters that confers clinically important, high-level azole resistance.
Preliminary data on non-albicans Candida species, Cryptococcus neoformans, and A. fumigatus (29) suggest that various resistance phenomena identified in C. albicans may operate in these pathogenic fungi and that PDR-related transcriptional mechanisms may contribute to their multidrug resistance.
Prospects
The long-awaited structural resolution of antifungal binding sites in the azole target ScErg11p as well as in the drug efflux pumps related to ScPdr5p would undoubtedly provide insight into multidrug resistance and guide strategies for impairing their activities. Meanwhile, and despite molecular mechanisms of differing complexity contributing to multidrug resistance in pathogenic fungi, a newly detected Achilles heel may be the transcriptional control of the antifungal efflux pumps. Of particular interest is the discovery that PDR transcriptional activators bind substrates of the efflux pumps they induce. We therefore may anticipate the development of novel multifunctional azole analogs. Erg11p would still be their primary target. Inclusion of a novel substituent would then enable inhibition of XBD-dependent coupling of Pdr1p/Pdr3p with its cognate mediator complex plus physical blockade of efflux via PDR transporters (Fig. 1B). The structures of itraconazole and fluconazole suggest that a fluconazole-like scaffold could be modified to antagonize not only Erg11p but also the transcriptional XBD and the active site from efflux pumps. Similarly, the dependence of the transient steroid response on interactions with the XBD domain of CgPdr1p, and possibly CaTac1p, indicates that a steroid hormone antagonist could increase the potency of azoles used against vaginal infections. Functional overexpression of Erg11p and both MFS and ABC drug efflux pumps from pathogenic fungi has been accomplished with an activated PDR5 promoter in a S. cerevisiae host whose major PDR genes had been deleted (24). This approach has allowed the assessment of innate and overexpression-related resistance to antifungals and the discovery of efflux-pump inhibitors. Similarly, ScPdr1p-regulated overexpression of functional ScPdr5p-related pump homologs is expected to provide screens for the identification of the novel broad-spectrum azoles or narrower-spectrum steroid antagonists hypothesized above (30). By minimizing pump expression, drug pump activity, and the opportunity for stress responses, these drugs should transform the fungistatic azoles into potent fungicides.
A complementary strategy is the identification of new targets whose dysfunction kills fungi rapidly, thus avoiding the emergence of both drug tolerance and efflux-mediated resistance. About 250 genes deemed essential in S. cerevisiae encode products that are at least 40% conserved across a broad range of fungi, including the fungal pathogens C. glabrata, C. albicans, C. neoformans, and A. fumigatus (31). Only about 50 of these gene products show less than 40% homology with human proteins. One of these is the plasma membrane proton pump (Pma1p), which generates the electrochemical gradient that fungi require for ion balance, nutrient uptake, and energy production. Pma1p inhibitors are fungicidal, indirectly block the activity of both ABC and MFS drug efflux pumps (32), and substantial resistance to them has yet to be detected.
Finally, diagnosis of disseminated fungal infections is too slow because conventional identification requires phenotypic examination of colonies grown for at least 48 hours on selective medium. The identification of drug resistance often requires a further step. Polymerase chain reaction amplification of ribosomal RNA intervening transcribed sequences followed by DNA pyrosequencing should halve the time needed for species-level fungal identification (33, 34). Translation of this technology into the clinic will allow the early identification of fungal species, including innately resistant species or those susceptible to the development of multidrug resistance. The application of appropriate prophylaxis with existing and novel antifungals and of ongoing surveillance will save many lives.
References and Notes
1. L. H. Taylor, S. M. Latham, M. E. Woolhouse, Philos. Trans. R. Soc. London Ser. B 356, 983 (2001). [ISI] [Medline]
2. D. Sanglard, J. Billie, in Candidia and Candidiasis, R. Calderone, Ed. (American Society for Microbiology Press, Washington, DC, 2002), pp. 349–383.
3. R. D. Cannon et al., Microbiology 153, 3211 (2007).[Abstract/Free Full Text]
4. L. E. Cowen, Nat. Rev. Microbiol. 6, 187 (2008). [CrossRef] [ISI] [Medline]
5. A. Selmecki, M. Gerami-Nejad, C. Paulson, A. Forche, J. Berman, Mol. Microbiol. 68, 624 (2008). [CrossRef] [ISI] [Medline]
6. E. Balzi, M. Wang, S. Leterme, L. Van Dyck, A. Goffeau, J. Biol. Chem. 269, 2206 (1994).[Abstract/Free Full Text]
7. M. A. Pfaller, D. J. Diekema, Clin. Microbiol. Rev. 20, 133 (2007).[Abstract/Free Full Text]
8. Y. Gbelska, J. J. Krijger, K. D. Breunig, FEMS Yeast Res. 6, 345 (2006). [CrossRef] [ISI] [Medline]
9. E. Carvajal, H. B. van den Hazel, A. Cybularz-Kolaczkowska, E. Balzi, A. Goffeau, Mol. Gen. Genet. 256, 406 (1997). [CrossRef] [ISI] [Medline]
10. A. Nourani, D. Papajova, A. Delahodde, C. Jacq, J. Subik, Mol. Gen. Genet. 256, 397 (1997). [CrossRef] [ISI] [Medline]
11. J. DeRisi et al., FEBS Lett. 470, 156 (2000). [CrossRef] [ISI] [Medline]
12. B. Rogers et al., J. Mol. Microbiol. Biotechnol. 3, 207 (2001). [CrossRef] [ISI] [Medline]
13. D. J. Katzmann, T. C. Hallstrom, Y. Mahe, W. S. Moye-Rowley, J. Biol. Chem. 271, 23049 (1996).[Abstract/Free Full Text]
14. Y. M. Mamnun, R. Pandjaitan, Y. Mahe, A. Delahodde, K. Kuchler, Mol. Microbiol. 46, 1429 (2002). [CrossRef] [ISI] [Medline]
15. K. Gulshan, W. S. Moye-Rowley, Eukaryot. Cell 6, 1933 (2007).[Free Full Text]
16. A. Kolaczkowska, M. Kolaczkowski, A. Goffeau, W. S. Moye-Rowley, FEBS Lett. 582, 977 (2008). [Medline]
17. B. Akache, S. MacPherson, M. A. Sylvain, B. Turcotte, J. Biol. Chem. 279, 27855 (2004).[Abstract/Free Full Text]
18. S. Le Crom et al., Mol. Cell. Biol. 22, 2642 (2002).[Abstract/Free Full Text]
19. J. K. Thakur et al., Nature 452, 604 (2008). [CrossRef] [ISI] [Medline]
20. J. P. Vermitsky et al., Mol. Microbiol. 61, 704 (2006). [CrossRef] [ISI] [Medline]
21. T. C. White, K. A. Marr, R. A. Bowden, Clin. Microbiol. Rev. 11, 382 (1998).[Abstract/Free Full Text]
22. A. Coste et al., Eukaryot. Cell 6, 1889 (2007).[Abstract/Free Full Text]
23. R. Prasad, P. De Wergifosse, A. Goffeau, E. Balzi, Curr. Genet. 27, 320 (1995). [CrossRef] [ISI] [Medline]
24. E. Lamping et al., Eukaryot. Cell 6, 1150 (2007).[Abstract/Free Full Text]
25. A. T. Coste, M. Karababa, F. Ischer, J. Bille, D. Sanglard, Eukaryot. Cell 3, 1639 (2004).[Abstract/Free Full Text]
26. D. Banerjee et al., Eukaryot. Cell 7, 68 (2008).[Abstract/Free Full Text]
27. T. T. Liu et al., Eukaryot. Cell 6, 2122 (2007).[Abstract/Free Full Text]
28. B. Larsen, S. Anderson, A. Brockman, M. Essmann, M. Schmidt, Yeast 23, 795 (2006). [CrossRef] [ISI] [Medline]
29. A. M. Nascimento et al., Antimicrob. Agents Chemother. 47, 1719 (2003).[Abstract/Free Full Text]
30. J. Cernicka et al., Int. J. Antimicrob. Agents 29, 170 (2007). [CrossRef] [ISI] [Medline]
31. M. Liu et al., Eukaryot. Cell 5, 638 (2006).[Abstract/Free Full Text]
32. B. C. Monk et al., Antimicrob. Agents Chemother. 49, 57 (2005).[Abstract/Free Full Text]
33. A. M. Borman, C. J. Linton, S. J. Miles, E. M. Johnson, J. Antimicrob. Chemother. 61 (suppl. 1), i7 (2008).[Abstract/Free Full Text]
34. B. L. Boyanton Jr., R. A. Luna, L. R. Fasciano, K. G. Menne, J. Versalovic, Arch. Pathol. Lab. Med. 132, 667 (2008). [ISI] [Medline]
35. Supported by NIH grant DE016885.
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Leslie Roberts and Stephen Simpson (18 July 2008)
Science 321 (5887), 355. [DOI: 10.1126/science.321.5887.355]
| Summary » | PDF »
Vol. 321. no. 5887, pp. 367 - 369
DOI: 10.1126/science.1159746
Prev | Table of Contents | Next
Perspective
Outwitting Multidrug Resistance to Antifungals
Brian C. Monk and Andre Goffeau*
The economic cost of fungal infection and its mortality associated with multidrug resistance remain unacceptably high. Recent understanding of the transcriptional regulation of plasma membrane efflux pumps of modest specificity provides new avenues for the development of broad-spectrum fungicides. Together with improved diagnosis and indirect intervention via inhibition of the energy supply for drug efflux, we envisage multifunctional azole analogs that inhibit not only ergosterol biosynthesis and drug efflux-pump activity but also activation of the transcriptional machinery that induces drug efflux-pump expression.
Department of Oral Sciences, Faculty of Dentistry, University of Otago, Post Office Box 647, Dunedin, New Zealand; and Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, 1348, Belgium.
* To whom correspondence should be addressed. E-mail: andre.goffeau@uclouvain.be
Eight hundred million years of evolution have generated 1.5 million fungal species that occupy many distinct ecological niches, yet only 300 fungi cause disease in humans (1). The identification of antifungals that act specifically against these pathogens is a particular challenge because of fungal diversity, individualized pathways for infection, and fungal use of multiple mechanisms that circumvent exogenous toxins. These highly regulated mechanisms include innate resistance to specific antifungal drugs, formation of biofilms, selection of spontaneous mutations that increase expression or decrease susceptibility of the drug target (2), stress-related tolerance that enhances short-term survival (3, 4), modification of chromosomal ploidy (5), and overexpression of multidrug efflux pumps (6). Fortunately, compared with infections caused by drug-resistant bacteria, those caused by resistant fungal pathogens and their spread to other patients occur relatively infrequently. However, the economic cost of fungal infection and its associated mortality, especially in debilitated and high-investment patients, remain unacceptably high.
A Clinical Perspective
The most prominent fungal pathogens affecting humans include Aspergillus fumigatus, Candida albicans, C. glabrata, C. parasilosis, C. tropicalis, C. krusei, and Cryptococcus neoformans (7). Although the skin, mucosal surfaces, and immune system usually provide robust defenses, weakened immunodefenses dramatically increase susceptibility to debilitating and life-threatening opportunistic fungal infections. Fungal infections are normally treated with a modest repertoire of drugs derived from five antifungal classes that target DNA and RNA synthesis, ergosterol, the ergosterol biosynthetic pathway, or the biosynthesis of the cell-wall component 1,3-β-D-glucan (Table 1). Unfortunately, the prophylactic use of fungistatic azoles such as fluconazole has been associated with an increased frequency of innate or acquired drug resistance in clinical isolates and the selection of non-albicans Candida, non-fumigatus Aspergillus, opportunistic yeastlike fungi, zygomycetes, and hyaline molds. Despite the fact that broader-spectrum third-generation azole drugs and the more expensive echinocandin class of antifungals prevent an increased proportion of life-threatening infections, Candida species remain the fourth most common cause of hospital-acquired bloodstream infection and kill 40% of those patients, whereas disseminated Aspergillus infections kill up to 80% of affected patients.
Mechanisms of Multidrug Resistance
Because of its economic and clinical impact, a focus on multidrug resistance rather than resistance to specific antifungals in pathogenic fungi is timely. Multidrug resistance, called pleiotropic drug resistance (PDR) in Saccharomyces cerevisiae, is an ancient phenomenon that preceded the modern use of antifungals (8). The adenosine triphosphate (ATP)–binding cassette (ABC) and major facilitator superfamily (MFS) transporter families responsible for multidrug resistance operate in all fungi. We distinguish among the transporters that belong to different species by using the prefix Sc for S. cerevisiae, Cg for C. glabrata, or Ca for C. albicans.
Saccharomyces cerevisiae. PDR in S. cerevisiae is the best-understood multidrug resistance mechanism in fungi. Point mutations conferring resistance to chemically diverse drugs (including azoles) have been mapped in genes encoding the zinc-finger transcription factors ScPdr1p or ScPdr3p (9, 10). These gain-of-function mutations activate over 20 target genes, the major ones being either ATP-driven (ABC transporter genes ScPDR5, ScSNQ2, and ScYOR1) orproton motive force–driven (MFS transporter genes ScTPO1 and ScFLR1) efflux pumps (11, 12). Resistance to a wide spectrum of drugs is conferred via the activation of efflux-pump gene expression, which involves the binding of Pdr1p/Pdr3p to the consensus binding element PDRE (13, 14). Mechanisms regulating PDR in S. cerevisiae include mutation of PDR1/3, plasma membrane sphingolipid homeostasis, ScPdr3p autoregulation, ScPdr3p-specific activation due to loss of mitochondrial respiration, chaperone-specific differential regulation of ScPdr1p and ScPdr3p (15), and ScPdr1p-dependent compensatory expression of efflux pumps (16). Yeast cells incubated with antifungals and other drugs transiently activate ScPdr1p/Pdr3p (16). Drugs such as itraconazole and progesterone bind to a 250–amino acid hydrophobic xenobiotic binding domain (XBD) of ScPdr1p/Pdr3p, enabling a specific association with the KIX domain of the Gal11p subunit of the mediator complex that recruits RNA polymerase II for expression of the ScPdr1p/Pdr3p-controlled genes (17) (Fig. 1A). Other transcription factors such as Yrr1p, Stb5p, Rdr1p, Yrm1p, and Yap1p also contribute to the expression of the various efflux transporter genes (17, 18).
Pathogenic fungi. The human pathogen C. glabrata uses the transcription factor CgPdr1p to control expression of the ABC multidrug efflux pumps CgCdr1p and CgCdr2p through mechanisms very similar to those of its close relative S. cerevisiae. The pumps are induced by treatment with diverse drugs and are highly expressed in respiration-defective mutants. Antifungal binding to the CgPdr1p XBD induces multidrug resistance via a KIX domain from the C. glabrata mediator complex (19). Mutants overexpressing CgPdr1p coordinately regulate 11 genes homologous to ScPdr1/ScPdr3p targets (20). These similarities support the use of S. cerevisiae in developing tools that are directly applicable to antifungal resistance in C. glabrata.
Multiple azole resistance stemming from longterm prophylaxis is frequently found in clinical isolates of the more distant pathogen C. albicans (21). The resistance mechanisms of this diploid species are complex and include mutations in single genes, loss of heterozygosity, chromosomal rearrangements, and selective segregation of chromosomal fragments (22). About 85% of fluconazole-resistant clinical isolates show multidrug resistance due to overexpression of the ABC transporters CaCdr1p and CaCdr2p (homologs of the S. cerevisiae ScPdr5p) and the major facilitator superfamily (MFS) pump CaMdr1p (homolog of ScFlr1p). The efflux functions of these transporters can be cloned in S. cerevisiae (23, 24). Expression of the CaCdr1p and CaCdr2p pumps is controlled by the transcription factor CaTac1p (25), which shares about 20% identity with ScPdr1/ScPdr3p. CaTac1p and ScPdr1p/ScPdr3p recognize substantially different PDREs (25), and CaTac1p causes more focused transcription than ScPdr1p/Pdr3p (26, 27). High doses of the female steroid hormone progesterone transiently upregulate, via steroid-specific PDREs, the same core of ABC transporters induced by antifungal intervention or gain-of-function mutations in the transcription factors (26, 28). Fluconazole-resistant clinical isolates often constitutively overexpress the MFS transporter CaMdr1p, either by itself or in combination with the azole target CaErg11p and/or the CaCdr1p and CaCdr2p ABC pumps. Although the MFS transporter CaMdr1p seems more efficient than its homolog ScFlr1p, it is overexpression of ABC transporters that confers clinically important, high-level azole resistance.
Preliminary data on non-albicans Candida species, Cryptococcus neoformans, and A. fumigatus (29) suggest that various resistance phenomena identified in C. albicans may operate in these pathogenic fungi and that PDR-related transcriptional mechanisms may contribute to their multidrug resistance.
Prospects
The long-awaited structural resolution of antifungal binding sites in the azole target ScErg11p as well as in the drug efflux pumps related to ScPdr5p would undoubtedly provide insight into multidrug resistance and guide strategies for impairing their activities. Meanwhile, and despite molecular mechanisms of differing complexity contributing to multidrug resistance in pathogenic fungi, a newly detected Achilles heel may be the transcriptional control of the antifungal efflux pumps. Of particular interest is the discovery that PDR transcriptional activators bind substrates of the efflux pumps they induce. We therefore may anticipate the development of novel multifunctional azole analogs. Erg11p would still be their primary target. Inclusion of a novel substituent would then enable inhibition of XBD-dependent coupling of Pdr1p/Pdr3p with its cognate mediator complex plus physical blockade of efflux via PDR transporters (Fig. 1B). The structures of itraconazole and fluconazole suggest that a fluconazole-like scaffold could be modified to antagonize not only Erg11p but also the transcriptional XBD and the active site from efflux pumps. Similarly, the dependence of the transient steroid response on interactions with the XBD domain of CgPdr1p, and possibly CaTac1p, indicates that a steroid hormone antagonist could increase the potency of azoles used against vaginal infections. Functional overexpression of Erg11p and both MFS and ABC drug efflux pumps from pathogenic fungi has been accomplished with an activated PDR5 promoter in a S. cerevisiae host whose major PDR genes had been deleted (24). This approach has allowed the assessment of innate and overexpression-related resistance to antifungals and the discovery of efflux-pump inhibitors. Similarly, ScPdr1p-regulated overexpression of functional ScPdr5p-related pump homologs is expected to provide screens for the identification of the novel broad-spectrum azoles or narrower-spectrum steroid antagonists hypothesized above (30). By minimizing pump expression, drug pump activity, and the opportunity for stress responses, these drugs should transform the fungistatic azoles into potent fungicides.
A complementary strategy is the identification of new targets whose dysfunction kills fungi rapidly, thus avoiding the emergence of both drug tolerance and efflux-mediated resistance. About 250 genes deemed essential in S. cerevisiae encode products that are at least 40% conserved across a broad range of fungi, including the fungal pathogens C. glabrata, C. albicans, C. neoformans, and A. fumigatus (31). Only about 50 of these gene products show less than 40% homology with human proteins. One of these is the plasma membrane proton pump (Pma1p), which generates the electrochemical gradient that fungi require for ion balance, nutrient uptake, and energy production. Pma1p inhibitors are fungicidal, indirectly block the activity of both ABC and MFS drug efflux pumps (32), and substantial resistance to them has yet to be detected.
Finally, diagnosis of disseminated fungal infections is too slow because conventional identification requires phenotypic examination of colonies grown for at least 48 hours on selective medium. The identification of drug resistance often requires a further step. Polymerase chain reaction amplification of ribosomal RNA intervening transcribed sequences followed by DNA pyrosequencing should halve the time needed for species-level fungal identification (33, 34). Translation of this technology into the clinic will allow the early identification of fungal species, including innately resistant species or those susceptible to the development of multidrug resistance. The application of appropriate prophylaxis with existing and novel antifungals and of ongoing surveillance will save many lives.
References and Notes
1. L. H. Taylor, S. M. Latham, M. E. Woolhouse, Philos. Trans. R. Soc. London Ser. B 356, 983 (2001). [ISI] [Medline]
2. D. Sanglard, J. Billie, in Candidia and Candidiasis, R. Calderone, Ed. (American Society for Microbiology Press, Washington, DC, 2002), pp. 349–383.
3. R. D. Cannon et al., Microbiology 153, 3211 (2007).[Abstract/Free Full Text]
4. L. E. Cowen, Nat. Rev. Microbiol. 6, 187 (2008). [CrossRef] [ISI] [Medline]
5. A. Selmecki, M. Gerami-Nejad, C. Paulson, A. Forche, J. Berman, Mol. Microbiol. 68, 624 (2008). [CrossRef] [ISI] [Medline]
6. E. Balzi, M. Wang, S. Leterme, L. Van Dyck, A. Goffeau, J. Biol. Chem. 269, 2206 (1994).[Abstract/Free Full Text]
7. M. A. Pfaller, D. J. Diekema, Clin. Microbiol. Rev. 20, 133 (2007).[Abstract/Free Full Text]
8. Y. Gbelska, J. J. Krijger, K. D. Breunig, FEMS Yeast Res. 6, 345 (2006). [CrossRef] [ISI] [Medline]
9. E. Carvajal, H. B. van den Hazel, A. Cybularz-Kolaczkowska, E. Balzi, A. Goffeau, Mol. Gen. Genet. 256, 406 (1997). [CrossRef] [ISI] [Medline]
10. A. Nourani, D. Papajova, A. Delahodde, C. Jacq, J. Subik, Mol. Gen. Genet. 256, 397 (1997). [CrossRef] [ISI] [Medline]
11. J. DeRisi et al., FEBS Lett. 470, 156 (2000). [CrossRef] [ISI] [Medline]
12. B. Rogers et al., J. Mol. Microbiol. Biotechnol. 3, 207 (2001). [CrossRef] [ISI] [Medline]
13. D. J. Katzmann, T. C. Hallstrom, Y. Mahe, W. S. Moye-Rowley, J. Biol. Chem. 271, 23049 (1996).[Abstract/Free Full Text]
14. Y. M. Mamnun, R. Pandjaitan, Y. Mahe, A. Delahodde, K. Kuchler, Mol. Microbiol. 46, 1429 (2002). [CrossRef] [ISI] [Medline]
15. K. Gulshan, W. S. Moye-Rowley, Eukaryot. Cell 6, 1933 (2007).[Free Full Text]
16. A. Kolaczkowska, M. Kolaczkowski, A. Goffeau, W. S. Moye-Rowley, FEBS Lett. 582, 977 (2008). [Medline]
17. B. Akache, S. MacPherson, M. A. Sylvain, B. Turcotte, J. Biol. Chem. 279, 27855 (2004).[Abstract/Free Full Text]
18. S. Le Crom et al., Mol. Cell. Biol. 22, 2642 (2002).[Abstract/Free Full Text]
19. J. K. Thakur et al., Nature 452, 604 (2008). [CrossRef] [ISI] [Medline]
20. J. P. Vermitsky et al., Mol. Microbiol. 61, 704 (2006). [CrossRef] [ISI] [Medline]
21. T. C. White, K. A. Marr, R. A. Bowden, Clin. Microbiol. Rev. 11, 382 (1998).[Abstract/Free Full Text]
22. A. Coste et al., Eukaryot. Cell 6, 1889 (2007).[Abstract/Free Full Text]
23. R. Prasad, P. De Wergifosse, A. Goffeau, E. Balzi, Curr. Genet. 27, 320 (1995). [CrossRef] [ISI] [Medline]
24. E. Lamping et al., Eukaryot. Cell 6, 1150 (2007).[Abstract/Free Full Text]
25. A. T. Coste, M. Karababa, F. Ischer, J. Bille, D. Sanglard, Eukaryot. Cell 3, 1639 (2004).[Abstract/Free Full Text]
26. D. Banerjee et al., Eukaryot. Cell 7, 68 (2008).[Abstract/Free Full Text]
27. T. T. Liu et al., Eukaryot. Cell 6, 2122 (2007).[Abstract/Free Full Text]
28. B. Larsen, S. Anderson, A. Brockman, M. Essmann, M. Schmidt, Yeast 23, 795 (2006). [CrossRef] [ISI] [Medline]
29. A. M. Nascimento et al., Antimicrob. Agents Chemother. 47, 1719 (2003).[Abstract/Free Full Text]
30. J. Cernicka et al., Int. J. Antimicrob. Agents 29, 170 (2007). [CrossRef] [ISI] [Medline]
31. M. Liu et al., Eukaryot. Cell 5, 638 (2006).[Abstract/Free Full Text]
32. B. C. Monk et al., Antimicrob. Agents Chemother. 49, 57 (2005).[Abstract/Free Full Text]
33. A. M. Borman, C. J. Linton, S. J. Miles, E. M. Johnson, J. Antimicrob. Chemother. 61 (suppl. 1), i7 (2008).[Abstract/Free Full Text]
34. B. L. Boyanton Jr., R. A. Luna, L. R. Fasciano, K. G. Menne, J. Versalovic, Arch. Pathol. Lab. Med. 132, 667 (2008). [ISI] [Medline]
35. Supported by NIH grant DE016885.
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INTRODUCTION TO SPECIAL ISSUE
Deadly Defiance
Leslie Roberts and Stephen Simpson (18 July 2008)
Science 321 (5887), 355. [DOI: 10.1126/science.321.5887.355]
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Monday, July 14, 2008
Effects of psilocybin on time perception and temporal control of behaviour in humans.
Effects of psilocybin on time perception and temporal control of behaviour in humans.
J. Psychopharmacol. 2007 Jan;21(1):50-64. Epub 2006 May 19.
Wittmann M, Carter O, Hasler F, Cahn BR, Grimberg U, Spring P, Hell D, Flohr H, Vollenweider FX.
Generation Research Programme, Human Science Centre, Ludwig-Maximilian University Munich, Bad Tölz, Germany, and Heffter Research Centre, University Hospital of Psychiatry, Zürich, Switzerland.
Hallucinogenic psilocybin is known to alter the subjective experience of time. However, there is no study that systematically investigated objective measures of time perception under psilocybin. Therefore, we studied dose-dependent effects of the serotonin (5-HT)2A/1A receptor agonist psilocybin (4-phosphoryloxy-N, N-dimethyltryptamine) on temporal processing, employing tasks of temporal reproduction, sensorimotor synchronization and tapping tempo. To control for cognitive and subjective changes, we assessed spatial working memory and conscious experience. Twelve healthy human volunteers were tested under placebo, medium (115 microg/kg), and high (250 microg/kg) dose conditions, in a double-blind experimental design. Psilocybin was found to significantly impair subjects' ability to (1) reproduce interval durations longer than 2.5 sec, (2) to synchronize to inter-beat intervals longer than 2 sec and (3) caused subjects to be slower in their preferred tapping rate. These objective effects on timing performance were accompanied by working-memory deficits and subjective changes in conscious state, namely increased reports of 'depersonalization' and 'derealization' phenomena including disturbances in subjective 'time sense.' Our study is the first to systematically assess the impact of psilocybin on timing performance on standardized measures of temporal processing. Results indicate that the serotonin system is selectively involved in duration processing of intervals longer than 2 to 3 seconds and in the voluntary control of the speed of movement. We speculate that psilocybin's selective disruption of longer intervals is likely to be a product of interactions with cognitive dimensions of temporal processing -presumably via 5-HT2A receptor stimulation.
PMID: 16714323 [PubMed - indexed for MEDLINE]
J. Psychopharmacol. 2007 Jan;21(1):50-64. Epub 2006 May 19.
Wittmann M, Carter O, Hasler F, Cahn BR, Grimberg U, Spring P, Hell D, Flohr H, Vollenweider FX.
Generation Research Programme, Human Science Centre, Ludwig-Maximilian University Munich, Bad Tölz, Germany, and Heffter Research Centre, University Hospital of Psychiatry, Zürich, Switzerland.
Hallucinogenic psilocybin is known to alter the subjective experience of time. However, there is no study that systematically investigated objective measures of time perception under psilocybin. Therefore, we studied dose-dependent effects of the serotonin (5-HT)2A/1A receptor agonist psilocybin (4-phosphoryloxy-N, N-dimethyltryptamine) on temporal processing, employing tasks of temporal reproduction, sensorimotor synchronization and tapping tempo. To control for cognitive and subjective changes, we assessed spatial working memory and conscious experience. Twelve healthy human volunteers were tested under placebo, medium (115 microg/kg), and high (250 microg/kg) dose conditions, in a double-blind experimental design. Psilocybin was found to significantly impair subjects' ability to (1) reproduce interval durations longer than 2.5 sec, (2) to synchronize to inter-beat intervals longer than 2 sec and (3) caused subjects to be slower in their preferred tapping rate. These objective effects on timing performance were accompanied by working-memory deficits and subjective changes in conscious state, namely increased reports of 'depersonalization' and 'derealization' phenomena including disturbances in subjective 'time sense.' Our study is the first to systematically assess the impact of psilocybin on timing performance on standardized measures of temporal processing. Results indicate that the serotonin system is selectively involved in duration processing of intervals longer than 2 to 3 seconds and in the voluntary control of the speed of movement. We speculate that psilocybin's selective disruption of longer intervals is likely to be a product of interactions with cognitive dimensions of temporal processing -presumably via 5-HT2A receptor stimulation.
PMID: 16714323 [PubMed - indexed for MEDLINE]
Psilocybin can occasion mystical-type experiences having substantial and sustained personal meaning and spiritual significance.
Psilocybin can occasion mystical-type experiences having substantial and sustained personal meaning and spiritual significance.
Psychopharmacology (Berl). 2006 Aug;187(3):268-83; discussion 284-92. Epub 2006 Jul 7.
Griffiths RR, Richards WA, McCann U, Jesse R.
Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 5510, Nathan Shock Drive, Baltimore, MD 21224-6823, USA. rgriff@jhmi.edu
RATIONALE: Although psilocybin has been used for centuries for religious purposes, little is known scientifically about its acute and persisting effects. OBJECTIVES: This double-blind study evaluated the acute and longer-term psychological effects of a high dose of psilocybin relative to a comparison compound administered under comfortable, supportive conditions. MATERIALS AND METHODS: The participants were hallucinogen-naïve adults reporting regular participation in religious or spiritual activities. Two or three sessions were conducted at 2-month intervals. Thirty volunteers received orally administered psilocybin (30 mg/70 kg) and methylphenidate hydrochloride (40 mg/70 kg) in counterbalanced order. To obscure the study design, six additional volunteers received methylphenidate in the first two sessions and unblinded psilocybin in a third session. The 8-h sessions were conducted individually. Volunteers were encouraged to close their eyes and direct their attention inward. Study monitors rated volunteers' behavior during sessions. Volunteers completed questionnaires assessing drug effects and mystical experience immediately after and 2 months after sessions. Community observers rated changes in the volunteer's attitudes and behavior. RESULTS: Psilocybin produced a range of acute perceptual changes, subjective experiences, and labile moods including anxiety. Psilocybin also increased measures of mystical experience. At 2 months, the volunteers rated the psilocybin experience as having substantial personal meaning and spiritual significance and attributed to the experience sustained positive changes in attitudes and behavior consistent with changes rated by community observers. CONCLUSIONS: When administered under supportive conditions, psilocybin occasioned experiences similar to spontaneously occurring mystical experiences. The ability to occasion such experiences prospectively will allow rigorous scientific investigations of their causes and consequences.
PMID: 16826400 [PubMed - indexed for MEDLINE
Psychopharmacology (Berl). 2006 Aug;187(3):268-83; discussion 284-92. Epub 2006 Jul 7.
Griffiths RR, Richards WA, McCann U, Jesse R.
Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 5510, Nathan Shock Drive, Baltimore, MD 21224-6823, USA. rgriff@jhmi.edu
RATIONALE: Although psilocybin has been used for centuries for religious purposes, little is known scientifically about its acute and persisting effects. OBJECTIVES: This double-blind study evaluated the acute and longer-term psychological effects of a high dose of psilocybin relative to a comparison compound administered under comfortable, supportive conditions. MATERIALS AND METHODS: The participants were hallucinogen-naïve adults reporting regular participation in religious or spiritual activities. Two or three sessions were conducted at 2-month intervals. Thirty volunteers received orally administered psilocybin (30 mg/70 kg) and methylphenidate hydrochloride (40 mg/70 kg) in counterbalanced order. To obscure the study design, six additional volunteers received methylphenidate in the first two sessions and unblinded psilocybin in a third session. The 8-h sessions were conducted individually. Volunteers were encouraged to close their eyes and direct their attention inward. Study monitors rated volunteers' behavior during sessions. Volunteers completed questionnaires assessing drug effects and mystical experience immediately after and 2 months after sessions. Community observers rated changes in the volunteer's attitudes and behavior. RESULTS: Psilocybin produced a range of acute perceptual changes, subjective experiences, and labile moods including anxiety. Psilocybin also increased measures of mystical experience. At 2 months, the volunteers rated the psilocybin experience as having substantial personal meaning and spiritual significance and attributed to the experience sustained positive changes in attitudes and behavior consistent with changes rated by community observers. CONCLUSIONS: When administered under supportive conditions, psilocybin occasioned experiences similar to spontaneously occurring mystical experiences. The ability to occasion such experiences prospectively will allow rigorous scientific investigations of their causes and consequences.
PMID: 16826400 [PubMed - indexed for MEDLINE
Mystical-type experiences occasioned by psilocybin mediate the attribution of personal meaning and spiritual significance 14 months later
Mystical-type experiences occasioned by psilocybin mediate the attribution of personal meaning and spiritual significance 14 months later
R R Griffiths1*, W A Richards2, M W Johnson3, U D McCann3, and R Jesse4
1 Department of Psychiatry and Behavioral Sciences and Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
2 Johns Hopkins Bayview Medical Center, Baltimore, Maryland, USA
3 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
4 Council on Spiritual Practices, San Francisco, California, USA
* To whom correspondence should be addressed.
Abstract
Psilocybin has been used for centuries for religious purposes; however, little is known scientifically about its long-term effects. We previously reported the effects of a double-blind study evaluating the psychological effects of a high psilocybin dose. This report presents the 14-month follow-up and examines the relationship of the follow-up results to data obtained at screening and on drug session days. Participants were 36hallucinogen-naïve adults reporting regular participation in religious/spiritual activities. Oral psilocybin (30 mg/70 kg) was administered on one of two or three sessions, with methylphenidate (40 mg/70 kg) administered on the other session(s). During sessions, volunteers were encouraged to close their eyes and direct their attention inward. At the 14-month follow-up, 58% and 67%, respectively, of volunteers rated the psilocybin-occasioned experience as being among the five most personally meaningful and among the five most spiritually significant experiences of their lives; 64% indicated that the experience increased well-being or life satisfaction; 58% met criteria for having had a ‘complete’ mystical experience. Correlation and regression analyses indicated a central role of the mystical experience assessed on the session day in the high ratings of personal meaning and spiritual significance at follow-up. Of the measures of personality, affect, quality of life and spirituality assessed across the study, only a scale measuring mystical experience showed a difference from screening. When administered under supportive conditions, psilocybin occasioned experiences similar to spontaneously occurring mystical experiences that, at 14-month follow-up, were considered by volunteers to be among the most personally meaningful and spiritually significant of their lives.
Key Words: entheogen, hallucinogen, humans, mystical experience, psilocybin, psychedelic, religion, spiritual
First published on July 1, 2008
Journal of Psychopharmacology 2008, doi:10.1177/0269881108094300
© 2008 British Association for Psychopharmacolo
R R Griffiths1*, W A Richards2, M W Johnson3, U D McCann3, and R Jesse4
1 Department of Psychiatry and Behavioral Sciences and Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
2 Johns Hopkins Bayview Medical Center, Baltimore, Maryland, USA
3 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
4 Council on Spiritual Practices, San Francisco, California, USA
* To whom correspondence should be addressed.
Abstract
Psilocybin has been used for centuries for religious purposes; however, little is known scientifically about its long-term effects. We previously reported the effects of a double-blind study evaluating the psychological effects of a high psilocybin dose. This report presents the 14-month follow-up and examines the relationship of the follow-up results to data obtained at screening and on drug session days. Participants were 36hallucinogen-naïve adults reporting regular participation in religious/spiritual activities. Oral psilocybin (30 mg/70 kg) was administered on one of two or three sessions, with methylphenidate (40 mg/70 kg) administered on the other session(s). During sessions, volunteers were encouraged to close their eyes and direct their attention inward. At the 14-month follow-up, 58% and 67%, respectively, of volunteers rated the psilocybin-occasioned experience as being among the five most personally meaningful and among the five most spiritually significant experiences of their lives; 64% indicated that the experience increased well-being or life satisfaction; 58% met criteria for having had a ‘complete’ mystical experience. Correlation and regression analyses indicated a central role of the mystical experience assessed on the session day in the high ratings of personal meaning and spiritual significance at follow-up. Of the measures of personality, affect, quality of life and spirituality assessed across the study, only a scale measuring mystical experience showed a difference from screening. When administered under supportive conditions, psilocybin occasioned experiences similar to spontaneously occurring mystical experiences that, at 14-month follow-up, were considered by volunteers to be among the most personally meaningful and spiritually significant of their lives.
Key Words: entheogen, hallucinogen, humans, mystical experience, psilocybin, psychedelic, religion, spiritual
First published on July 1, 2008
Journal of Psychopharmacology 2008, doi:10.1177/0269881108094300
© 2008 British Association for Psychopharmacolo
Human hallucinogen research: guidelines for safety
Human hallucinogen research: guidelines for safety
M W Johnson1, W A Richards2, and R R Griffiths3*
1 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
2 Johns Hopkins Bayview Medical Center, Baltimore, Maryland, USA
3 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
* To whom correspondence should be addressed.
Abstract
There has recently been a renewal of human research with classical hallucinogens (psychedelics). This paper first briefly discusses the unique history of human hallucinogen research, and then reviews the risks of hallucinogen administration and safeguards for minimizing these risks. Although hallucinogens are relatively safe physiologically and are not considered drugs of dependence, their administration involves unique psychological risks. The most likely risk is overwhelming distress during drug action (‘bad trip’), which could lead to potentially dangerous behaviour such as leaving the study site. Less common are prolonged psychoses triggered by hallucinogens. Safeguards against these risks include the exclusion of volunteers with personal or family history of psychotic disorders or other severe psychiatric disorders, establishing trust and rapport between session monitors and volunteer before the session, careful volunteer preparation, a safe physical session environment and interpersonal support from at least two study monitors during the session. Investigators should probe for the relatively rare hallucinogen persisting perception disorder in follow-up contact. Persisting adverse reactions are rare when research is conducted along these guidelines. Incautious research may jeopardize participant safety and future research. However, carefully conducted research may inform the treatment of psychiatric disorders, and may lead to advances in basic science.
Key Words: 5-HT2A agonists, adverse reactions, DMT, entheogens, hallucinogens, human research, LSD, mescaline, psilocybin, psychedelics, safety guidelines
First published on July 1, 2008
Journal of Psychopharmacology 2008, doi:10.1177/0269881108093587
© 2008 British Association for Psychopharmacology
M W Johnson1, W A Richards2, and R R Griffiths3*
1 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
2 Johns Hopkins Bayview Medical Center, Baltimore, Maryland, USA
3 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
* To whom correspondence should be addressed.
Abstract
There has recently been a renewal of human research with classical hallucinogens (psychedelics). This paper first briefly discusses the unique history of human hallucinogen research, and then reviews the risks of hallucinogen administration and safeguards for minimizing these risks. Although hallucinogens are relatively safe physiologically and are not considered drugs of dependence, their administration involves unique psychological risks. The most likely risk is overwhelming distress during drug action (‘bad trip’), which could lead to potentially dangerous behaviour such as leaving the study site. Less common are prolonged psychoses triggered by hallucinogens. Safeguards against these risks include the exclusion of volunteers with personal or family history of psychotic disorders or other severe psychiatric disorders, establishing trust and rapport between session monitors and volunteer before the session, careful volunteer preparation, a safe physical session environment and interpersonal support from at least two study monitors during the session. Investigators should probe for the relatively rare hallucinogen persisting perception disorder in follow-up contact. Persisting adverse reactions are rare when research is conducted along these guidelines. Incautious research may jeopardize participant safety and future research. However, carefully conducted research may inform the treatment of psychiatric disorders, and may lead to advances in basic science.
Key Words: 5-HT2A agonists, adverse reactions, DMT, entheogens, hallucinogens, human research, LSD, mescaline, psilocybin, psychedelics, safety guidelines
First published on July 1, 2008
Journal of Psychopharmacology 2008, doi:10.1177/0269881108093587
© 2008 British Association for Psychopharmacology
A Very Memorable Trip
A Very Memorable Trip
By Greg Miller
ScienceNOW Daily News
1 July 2008
More than a year after taking a hallucinogenic drug in a carefully controlled experiment, most people rate the experience among the most personally meaningful and spiritually significant of their lives, researchers report online today in the Journal of Psychopharmacology. Such findings are helping to renew interest in research with hallucinogens, a field whose reputation long suffered from the psychedelic excesses of the 1960s.
The new study follows up with 36 volunteers who participated in earlier experiments led by psychopharmacologist Roland Griffiths of Johns Hopkins University in Baltimore, Maryland. The researchers monitored the mostly middle-aged subjects while they took a strong dose of psilocybin, the active ingredient in hallucinogenic mushrooms. All of the volunteers had indicated at least some participation in religious or spiritual activities--such as meditating or going to church--and the researchers instructed them to direct their attention inward while under the drug's sway. None had previous experience with hallucinogens. On questionnaires completed after the drug had worn off, and again 2 months later, they rated the experience as highly significant, the researchers reported in a 2006 paper in Psychopharmacology. Volunteers frequently described a sense of greater truth or a sense of the unity of all things while on the drug, for example.
The experience remained highly significant to most of the volunteers 14 months later, the researchers now report: 58% rated it among the five most personally meaningful experiences of their lives and 67% rated it among the five most spiritually significant. And 64% said the experience had improved their sense of well-being or life satisfaction. It's remarkable, Griffiths says, that people continued to rate their 8-hour experience in the lab as similar in significance to life events such as the birth of a first child.
The findings suggest to Griffiths that hallucinogenic drugs may provide a way to investigate the neurobiology of religious experiences by evoking in the lab the kinds of mystical experiences traditionally achieved by prayer, meditation, or fasting. Would the drug have the same effect on a group of atheist or agnostic subjects? "We're dying to do that study," he says.
In the meantime, Griffiths's team is recruiting volunteers for a clinical trial to test whether similar psilocybin experiences can reduce anxiety and depression in cancer patients. A few studies in the late 1960s and early 1970s suggested that the hallucinogen LSD might ease suffering in terminal cancer patients, but that line of investigation was dropped and largely forgotten, says David Nichols, a psychopharmacologist at Purdue University in West Lafayette, Indiana. Although such patients often receive heavy doses of pain drugs along with antidepressants and anxiety drugs, Nichols says hallucinogens might provide a better alternative. "If you could change their perception of death and reduce their stress in that way, it would improve their quality of life because their consciousness wouldn't be dulled by sedatives or narcotics," he says.
By Greg Miller
ScienceNOW Daily News
1 July 2008
More than a year after taking a hallucinogenic drug in a carefully controlled experiment, most people rate the experience among the most personally meaningful and spiritually significant of their lives, researchers report online today in the Journal of Psychopharmacology. Such findings are helping to renew interest in research with hallucinogens, a field whose reputation long suffered from the psychedelic excesses of the 1960s.
The new study follows up with 36 volunteers who participated in earlier experiments led by psychopharmacologist Roland Griffiths of Johns Hopkins University in Baltimore, Maryland. The researchers monitored the mostly middle-aged subjects while they took a strong dose of psilocybin, the active ingredient in hallucinogenic mushrooms. All of the volunteers had indicated at least some participation in religious or spiritual activities--such as meditating or going to church--and the researchers instructed them to direct their attention inward while under the drug's sway. None had previous experience with hallucinogens. On questionnaires completed after the drug had worn off, and again 2 months later, they rated the experience as highly significant, the researchers reported in a 2006 paper in Psychopharmacology. Volunteers frequently described a sense of greater truth or a sense of the unity of all things while on the drug, for example.
The experience remained highly significant to most of the volunteers 14 months later, the researchers now report: 58% rated it among the five most personally meaningful experiences of their lives and 67% rated it among the five most spiritually significant. And 64% said the experience had improved their sense of well-being or life satisfaction. It's remarkable, Griffiths says, that people continued to rate their 8-hour experience in the lab as similar in significance to life events such as the birth of a first child.
The findings suggest to Griffiths that hallucinogenic drugs may provide a way to investigate the neurobiology of religious experiences by evoking in the lab the kinds of mystical experiences traditionally achieved by prayer, meditation, or fasting. Would the drug have the same effect on a group of atheist or agnostic subjects? "We're dying to do that study," he says.
In the meantime, Griffiths's team is recruiting volunteers for a clinical trial to test whether similar psilocybin experiences can reduce anxiety and depression in cancer patients. A few studies in the late 1960s and early 1970s suggested that the hallucinogen LSD might ease suffering in terminal cancer patients, but that line of investigation was dropped and largely forgotten, says David Nichols, a psychopharmacologist at Purdue University in West Lafayette, Indiana. Although such patients often receive heavy doses of pain drugs along with antidepressants and anxiety drugs, Nichols says hallucinogens might provide a better alternative. "If you could change their perception of death and reduce their stress in that way, it would improve their quality of life because their consciousness wouldn't be dulled by sedatives or narcotics," he says.
Saturday, July 5, 2008
Identification of a serotonin/glutamate receptor complex implicated in psychosis
Identification of a serotonin/glutamate receptor complex implicated in psychosis
The psychosis associated with schizophrenia is characterized by alterations in sensory processing and perception1, 2. Some antipsychotic drugs were identified by their high affinity for serotonin 5-HT2A receptors (2AR)3, 4. Drugs that interact with metabotropic glutamate receptors (mGluR) also have potential for the treatment of schizophrenia5, 6, 7. The effects of hallucinogenic drugs, such as psilocybin and lysergic acid diethylamide, require the 2AR8, 9, 10 and resemble some of the core symptoms of schizophrenia10, 11, 12. Here we show that the mGluR2 interacts through specific transmembrane helix domains with the 2AR, a member of an unrelated G-protein-coupled receptor family, to form functional complexes in brain cortex. The 2AR–mGluR2 complex triggers unique cellular responses when targeted by hallucinogenic drugs, and activation of mGluR2 abolishes hallucinogen-specific signalling and behavioural responses. In post-mortem human brain from untreated schizophrenic subjects, the 2AR is upregulated and the mGluR2 is downregulated, a pattern that could predispose to psychosis. These regulatory changes indicate that the 2AR–mGluR2 complex may be involved in the altered cortical processes of schizophrenia, and this complex is therefore a promising new target for the treatment of psychosis.
Letter
Nature 452, 93-97 (6 March 2008) | doi:10.1038/nature06612; Received 2 November 2007; Accepted 20 December 2007; Published online 24 February 2008
The psychosis associated with schizophrenia is characterized by alterations in sensory processing and perception1, 2. Some antipsychotic drugs were identified by their high affinity for serotonin 5-HT2A receptors (2AR)3, 4. Drugs that interact with metabotropic glutamate receptors (mGluR) also have potential for the treatment of schizophrenia5, 6, 7. The effects of hallucinogenic drugs, such as psilocybin and lysergic acid diethylamide, require the 2AR8, 9, 10 and resemble some of the core symptoms of schizophrenia10, 11, 12. Here we show that the mGluR2 interacts through specific transmembrane helix domains with the 2AR, a member of an unrelated G-protein-coupled receptor family, to form functional complexes in brain cortex. The 2AR–mGluR2 complex triggers unique cellular responses when targeted by hallucinogenic drugs, and activation of mGluR2 abolishes hallucinogen-specific signalling and behavioural responses. In post-mortem human brain from untreated schizophrenic subjects, the 2AR is upregulated and the mGluR2 is downregulated, a pattern that could predispose to psychosis. These regulatory changes indicate that the 2AR–mGluR2 complex may be involved in the altered cortical processes of schizophrenia, and this complex is therefore a promising new target for the treatment of psychosis.
Letter
Nature 452, 93-97 (6 March 2008) | doi:10.1038/nature06612; Received 2 November 2007; Accepted 20 December 2007; Published online 24 February 2008
FIGURE 1. Receptor interaction.
González-Maeso et al.2 find that the metabotropic glutamate receptor 2 (mGluR2) and serotonin 5-HT2A receptor (2AR) physiologically bind each other, leading to reciprocal regulation of their functions. Agonists that stimulate mGluR2 are antipsychotic, whereas 2AR agonists, such as hallucinogens, have the opposite effect. It is conceivable that the clinically significant anti-schizophrenic effects of LY2140023, an mGluR2 agonist1, derive from reducing the excessive — and hence hallucinogen-like — activity of 2AR. DOI, 2,5-dimethoxy-4-iodoamphetamine.
From the following article:
Neuroscience: A complex in psychosis
Solomon H. Snyder
Nature 452, 38-39(6 March 2008)
doi:10.1038/452038a
Neuroscience: A complex in psychosis
Neuroscience: A complex in psychosis
Solomon H. Snyder1
Abstract
The molecular basis of psychoses such as schizophrenia remains largely mysterious. The interaction between two of the brain receptors involved adds to evidence that will help in the search for explanations.
This is a story that involves three types of receptor in the brain that influence human perception and behaviour (those for the neurotransmitters dopamine, serotonin and glutamate), and the drugs that block or enhance their activity. Such drugs are used by researchers to investigate the causes of psychotic disorders such as schizophrenia, and by clinicians to treat patients.
Nature 452, 38-39 (6 March 2008) | doi:10.1038/452038a; Published online 5 March 2008
Solomon H. Snyder1
Abstract
The molecular basis of psychoses such as schizophrenia remains largely mysterious. The interaction between two of the brain receptors involved adds to evidence that will help in the search for explanations.
This is a story that involves three types of receptor in the brain that influence human perception and behaviour (those for the neurotransmitters dopamine, serotonin and glutamate), and the drugs that block or enhance their activity. Such drugs are used by researchers to investigate the causes of psychotic disorders such as schizophrenia, and by clinicians to treat patients.
Nature 452, 38-39 (6 March 2008) | doi:10.1038/452038a; Published online 5 March 2008
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