CDR1 CDR2 MDR1 ERG
9. Perspectivas Futuras
Para além dos genes de resistência, actualmente presta-se especial atenção aos mecanismos de regulação da expressão subjacente, nomeadamente os que promovem a sua sobre-expressão ou a sub-expressão.
É nossa intenção continuar a desenvolver estudos sobre o tema em questão no âmbito de uma das principais linhas investigacionais em curso no Serviço e Laboratório de Microbiologia da Faculdade de Medicina do Porto. Tais estudos como principais objectivos:
- a realização de um screening num vasto número de estirpes clinicas relativamente à sobre-expressão de genes de resistência para perceber quais os mecanismos mais frequentes a nível nacional;
- o estudo da expressão destes genes após indução de resistência e posterior bloqueio com moduladores
- a compreenção do efeito dos moduladores na reversão da resistência, através de estudos de interacção fármaco proteína
Complementar estudos de efluxo com marcadores mais específicos como a rodamina e correlacionar com sobre-expressão dos genes
- a avaliação da expressão de genes de resistência em outras espécies de Candida nomeadamente C. parapsilosis ou C. glabrata, espécies constitutivamente mais resistentes que C. albicans
10. Bibliografia
Ahmed-Belkacem, A., Pozza, A., Macalou, S., Perez-Victoria, J. M., Boumendjel, A. & Di Pietro, A. (2006). Inhibitors of cancer cell multidrug resistance mediated by breast
cancer resistance protein (BCRP/ABCG2). Anticancer Drugs 17, 239-243.
Akins, R. A. (2005). An update on antifungal targets and mechanisms of resistance in
Candida albicans. Med Mycol 43, 285-318.
Albertson, G. D., Niimi, M., Cannon, R. D. & Jenkinson, H. F. (1996). Multiple efflux
mechanisms are involved in Candida albicans fluconazole resistance. Antimicrob Agents
Chemother 40, 2835-2841.
Arendrup, M. C., Denning, D. W., Pfaller, M. A., Diekema, D. J. & Rex, J. H. (2007).
Does one voriconazole breakpoint suit all Candida species? J Clin Microbiol 45, 2093; author reply 2094.
Arnaud, M. B., Costanzo, M. C., Skrzypek, M. S., Binkley, G., Lane, C., Miyasato, S. R. & Sherlock, G. (2005). The Candida Genome Database (CGD), a community resource
for Candida albicans gene and protein information. Nucleic Acids Res 33, D358-363.
Barchiesi, F., Hollis, R. J., McGough, D. A., Scalise, G., Rinaldi, M. G. & Pfaller, M. A. (1995). DNA subtypes and fluconazole susceptibilities of Candida albicans isolates
from the oral cavities of patients with AIDS. Clin Infect Dis 20, 634-640.
Bard, M., Lees, N. D., Turi, T., Craft, D., Cofrin, L., Barbuch, R., Koegel, C. & Loper, J. C. (1993). Sterol synthesis and viability of erg11 (cytochrome P450 lanosterol
demethylase) mutations in Saccharomyces cerevisiae and Candida albicans. Lipids 28, 963-967.
Calderone, R. A. (2002). Candida and Candidiasis. Washington, DC: ASM Press.
Cameron, M. L., Schell, W. A., Bruch, S., Bartlett, J. A., Waskin, H. A. & Perfect, J. R. (1993). Correlation of in vitro fluconazole resistance of Candida isolates in relation to
therapy and symptoms of individuals seropositive for human immunodeficiency virus type 1. Antimicrob Agents Chemother 37, 2449-2453.
Cederlund, H. & Mardh, P. A. (1993). Antibacterial activities of non-antibiotic drugs. J
Antimicrob Chemother 32, 355-365.
Chau, A. S., Mendrick, C. A., Sabatelli, F. J., Loebenberg, D. & McNicholas, P. M. (2004). Application of real-time quantitative PCR to molecular analysis of Candida
albicans strains exhibiting reduced susceptibility to azoles. Antimicrob Agents Chemother
48, 2124-2131.
Chen, C. G., Yang, Y. L., Shih, H. I., Su, C. L. & Lo, H. J. (2004). CaNdt80 is involved
in drug resistance in Candida albicans by regulating CDR1. Antimicrob Agents Chemother
48, 4505-4512.
Chong, P. P., Abdul Hadi, S. R., Lee, Y. L., Phan, C. L., Tan, B. C., Ng, K. P. & Seow, H. F. (2007). Genotyping and drug resistance profile of Candida spp. in recurrent and one-
off vaginitis, and high association of non-albicans species with non-pregnant status. Infect
Genet Evol 7, 449-456.
CLSI (2002).Reference method for broth dilution antifungal susceptibility testing of
yeasts. Approved Standard. CLSI document M27-A2. Wayne PA. Edited by W. National Committee for Clinical Laboratory Standards, Pa.
Correia, A., Sampaio, P., Almeida, J. & Pais, C. (2004). Study of molecular
epidemiology of candidiasis in portugal by PCR fingerprinting of Candida clinical isolates.
J Clin Microbiol 42, 5899-5903.
Costa-de-Oliveira, S., Araújo, R., Ana, S. D., Pina-Vaz, C. & Rodrigues, A. G. (2007).
Propofol promotes resistance to antifungals by reducing drug input into the fungal cell.
BMC Microbiology.
Coste, A., Turner, V., Ischer, F., Morschhauser, J., Forche, A., Selmecki, A., Berman, J., Bille, J. & Sanglard, D. (2006). A mutation in Tac1p, a transcription factor regulating
CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans. Genetics 172, 2139-2156.
Coste, A. T., Karababa, M., Ischer, F., Bille, J. & Sanglard, D. (2004). TAC1,
transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2. Eukaryot Cell 3, 1639- 1652.
Cowen, L. E., Nantel, A., Whiteway, M. S., Thomas, D. Y., Tessier, D. C., Kohn, L. M. & Anderson, J. B. (2002). Population genomics of drug resistance in Candida albicans.
Proc Natl Acad Sci U S A 99, 9284-9289.
de Micheli, M., Bille, J., Schueller, C. & Sanglard, D. (2002). A common drug-
responsive element mediates the upregulation of the Candida albicans ABC transporters CDR1 and CDR2, two genes involved in antifungal drug resistance. Mol Microbiol 43, 1197-1214.
de Oliveira, J. M., Cruz, A. S., Fonseca, A. F., Vaz, C. P., Rodrigues, A., Aurea, F., Maia, J. & Sousa, J. A. (1993). Prevalence of Candida albicans in vaginal fluid of
asymptomatic Portuguese women. J Reprod Med 38, 41-42.
Dheda, K., Huggett, J. F., Bustin, S. A., Johnson, M. A., Rook, G. & Zumla, A. (2004).
Validation of housekeeping genes for normalizing RNA expression in real-time PCR.
Biotechniques 37, 112-114, 116, 118-119.
Douglas, C. M., D'Ippolito, J. A., Shei, G. J. & other authors (1997). Identification of
the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother 41, 2471-2479.
Fling, M. E., Kopf, J., Tamarkin, A., Gorman, J. A., Smith, H. A. & Koltin, Y. (1991).
Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate. Mol Gen Genet 227, 318-329.
Frade, J. P., Warnock, D. W. & Arthington-Skaggs, B. A. (2004). Rapid quantification
of drug resistance gene expression in Candida albicans by reverse transcriptase LightCycler PCR and fluorescent probe hybridization. J Clin Microbiol 42, 2085-2093.
Franz, R., Michel, S. & Morschhauser, J. (1998). A fourth gene from the Candida
albicans CDR family of ABC transporters. Gene 220, 91-98.
Galgiani, J. N., Reiser, J., Brass, C., Espinel-Ingroff, A., Gordon, M. A. & Kerkering, T. M. (1987). Comparison of relative susceptibilities of Candida species to three antifungal
agents as determined by unstandardized methods. Antimicrob Agents Chemother 31, 1343- 1347.
Gaur, N. A., Puri, N., Karnani, N., Mukhopadhyay, G., Goswami, S. K. & Prasad, R. (2004). Identification of a negative regulatory element which regulates basal transcription
Ghannoum, M. A. & Rice, L. B. (1999). Antifungal agents: mode of action, mechanisms
of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol
Rev 12, 501-517.
Gupta, A. K., Katz, H. I. & Shear, N. H. (1999). Drug interactions with itraconazole,
fluconazole, and terbinafine and their management. J Am Acad Dermatol 41, 237-249.
Gupta, V., Kohli, A., Krishnamurthy, S., Puri, N., Aalamgeer, S. A., Panwar, S. & Prasad, R. (1998). Identification of polymorphic mutant alleles of CaMDR1, a major
facilitator of Candida albicans which confers multidrug resistance, and its in vitro transcriptional activation. Curr Genet 34, 192-199.
Haugland, R. P. (1996). Handbook of fluorescence probes. Eugene Molecular Probes
Inc. .
Henry, K. W., Nickels, J. T. & Edlind, T. D. (2000). Upregulation of ERG genes in
Candida species by azoles and other sterol biosynthesis inhibitors. Antimicrob Agents
Chemother 44, 2693-2700.
Higuchi, R., Dollinger, G., Walsh, P. S. & Griffith, R. (1992). Simultaneous
amplification and detection of specific DNA sequences. Biotechnology (N Y) 10, 413-417.
Hitchcock, C. A. (1991). Cytochrome P-450-dependent 14 alpha-sterol demethylase of
Candida albicans and its interaction with azole antifungals. Biochem Soc Trans 19, 782- 787.
Hossain, M. A. & Ghannoum, M. A. (2000). New investigational antifungal agents for
treating invasive fungal infections. Expert Opin Investig Drugs 9, 1797-1813.
Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J. (1990). PCR Protocols. A
guide to methods and aplications. San Diego.
Jensen, J., Munoz, P., Guinea, J., Rodriguez-Creixems, M., Pelaez, T. & Bouza, E. (2007). Mixed fungemia: incidence, risk factors, and mortality in a general hospital. Clin
Infect Dis 44, e109-114.
Jones, T., Federspiel, N. A., Chibana, H. & other authors (2004). The diploid genome
sequence of Candida albicans. Proc Natl Acad Sci U S A 101, 7329-7334.
Karababa, M., Coste, A. T., Rognon, B., Bille, J. & Sanglard, D. (2004). Comparison of
gene expression profiles of Candida albicans azole-resistant clinical isolates and laboratory strains exposed to drugs inducing multidrug transporters. Antimicrob Agents Chemother
48, 3064-3079.
Karnani, N., Gaur, N. A., Jha, S., Puri, N., Krishnamurthy, S., Goswami, S. K., Mukhopadhyay, G. & Prasad, R. (2004). SRE1 and SRE2 are two specific steroid-
responsive modules of Candida drug resistance gene 1 (CDR1) promoter. Yeast 21, 219- 239.
Kerridge, D. (1986). Mode of action of clinically important antifungal drugs. Adv Microb
Physiol 27, 1-72.
Kohrer, K. & Domdey, H. (1991). Preparation of high molecular weight RNA. Methods
Enzymol 194, 398-405.
Kubista, M., Andrade, J. M., Bengtsson, M. & other authors (2006). The real-time
polymerase chain reaction. Mol Aspects Med 27, 95-125.
Loeffler, J. & Stevens, D. A. (2003). Antifungal drug resistance. Clin Infect Dis 36, S31-
41.
Lopes, M. (2001).Infecções nosocomiais fungicas em pediatria. In Faculdade de
Farmacia Lisboa: Universidade de Lisboa.
Lopez-Ribot, J. L., McAtee, R. K., Lee, L. N., Kirkpatrick, W. R., White, T. C., Sanglard, D. & Patterson, T. F. (1998). Distinct patterns of gene expression associated
with development of fluconazole resistance in serial candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob
Agents Chemother 42, 2932-2937.
Losberger, C. & Ernst, J. F. (1989). Sequence of the Candida albicans gene encoding
actin. Nucleic Acids Res 17, 9488.
Lyons, C. N. & White, T. C. (2000). Transcriptional analyses of antifungal drug
resistance in Candida albicans. Antimicrob Agents Chemother 44, 2296-2303.
Maenza, J. R., Merz, W. G., Romagnoli, M. J., Keruly, J. C., Moore, R. D. & Gallant, J. E. (1997). Infection due to fluconazole-resistant Candida in patients with AIDS:
Magill, S. S., Shields, C., Sears, C. L., Choti, M. & Merz, W. G. (2006). Triazole cross-
resistance among Candida spp.: case report, occurrence among bloodstream isolates, and implications for antifungal therapy. J Clin Microbiol 44, 529-535.
Marr, K. A., Rustad, T. R., Rex, J. H. & White, T. C. (1999). The trailing end point
phenotype in antifungal susceptibility testing is pH dependent. Antimicrob Agents
Chemother 43, 1383-1386.
Meletiadis, J., Chanock, S. & Walsh, T. J. (2006). Human pharmacogenomic variations
and their implications for antifungal efficacy. Clin Microbiol Rev 19, 763-787.
Millard, P. J., Roth, B. L., Thi, H. P., Yue, S. T. & Haugland, R. P. (1997).
Development of the FUN-1 family of fluorescent probes for vacuole labeling and viability testing of yeasts. Appl Environ Microbiol 63, 2897-2905.
Muller, F. M., Staudigel, A., Salvenmoser, S., Tredup, A., Miltenberger, R. & Herrmann, J. V. (2007). Cross-resistance to medical and agricultural azole drugs in yeasts
from the oropharynx of human immunodeficiency virus patients and from environmental Bavarian vine grapes. Antimicrob Agents Chemother 51, 3014-3016.
Odds, F. C., Brown, A. J. & Gow, N. A. (2003). Antifungal agents: mechanisms of
action. Trends Microbiol 11, 272-279.
Pfaffl, M. W., Tichopad, A., Prgomet, C. & Neuvians, T. P. (2004). Determination of
stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett 26, 509-515.
Pfaller, M. A., Rex, J. H. & Rinaldi, M. G. (1997). Antifungal susceptibility testing:
technical advances and potential clinical applications. Clin Infect Dis 24, 776-784.
Pfaller, M. A., Diekema, D. J., Rex, J. H. & other authors (2006). Correlation of MIC
with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints. J Clin Microbiol 44, 819-826.
Pierson, C. A., Eckstein, J., Barbuch, R. & Bard, M. (2004). Ergosterol gene expression
in wild-type and ergosterol-deficient mutants of Candida albicans. Med Mycol 42, 385-389.
Pina-Vaz, C., Rodrigues, A. G., Sansonetty, F., Martinez-De-Oliveira, J., Fonseca, A. F. & Mardh, P. A. (2000). Antifungal activity of local anesthetics against Candida
Pina-Vaz, C., Sansonetty, F., Rodrigues, A. G., Costa-de-Oliveira, S., Martinez-de- Oliveira, J. & Fonseca, A. F. (2001a). Susceptibility to fluconazole of Candida clinical
isolates determined by FUN-1 staining with flow cytometry and epifluorescence microscopy. J Med Microbiol 50, 375-382.
Pina-Vaz, C., Sansonetty, F., Rodrigues, A. G., Costa-Oliveira, S., Tavares, C. & Martinez-de-Oliveira, J. (2001b). Cytometric approach for a rapid evaluation of
susceptibility of Candida strains to antifungals. Clin Microbiol Infect 7, 609-618.
Pina-Vaz, C., Rodrigues, A. G., Costa-de-Oliveira, S., Ricardo, E. & Mardh, P. A. (2005). Potent synergic effect between ibuprofen and azoles on Candida resulting from
blockade of efflux pumps as determined by FUN-1 staining and flow cytometry. J
Antimicrob Chemother 56, 678-685.
Polak, A. & Scholer, H. J. (1975). Mode of action of 5-fluorocytosine and mechanisms of
resistance. Chemotherapy 21, 113-130.
Poulain, D., Tronchin, G., Dubremetz, J. F. & Biguet, J. (1978). Ultrastructure of the
cell wall of Candida albicans blastospores: study of its constitutive layers by the use of a cytochemical technique revealing polysaccharides. Ann Microbiol (Paris) 129, 141-153.
Prasad, R., De Wergifosse, P., Goffeau, A. & Balzi, E. (1995). Molecular cloning and
characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals. Curr Genet 27, 320-329.
Rex, J. H., Cooper, C. R., Jr., Merz, W. G., Galgiani, J. N. & Anaissie, E. J. (1995).
Detection of amphotericin B-resistant Candida isolates in a broth-based system.
Antimicrob Agents Chemother 39, 906-909.
Rex, J. H., Nelson, P. W., Paetznick, V. L., Lozano-Chiu, M., Espinel-Ingroff, A. & Anaissie, E. J. (1998). Optimizing the correlation between results of testing in vitro and
therapeutic outcome in vivo for fluconazole by testing critical isolates in a murine model of invasive candidiasis. Antimicrob Agents Chemother 42, 129-134.
Rex, J. H., Pfaller, M. A., Walsh, T. J. & other authors (2001). Antifungal susceptibility
testing: practical aspects and current challenges. Clin Microbiol Rev 14, 643-658, table of contents.
Ririe, K. M., Rasmussen, R. P. & Wittwer, C. T. (1997). Product differentiation by
analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245, 154-160.
Roemer, T., Jiang, B., Davison, J. & other authors (2003). Large-scale essential gene
identification in Candida albicans and applications to antifungal drug discovery. Mol
Microbiol 50, 167-181.
Rogers, P. D., Kramer, R. E., Crews, J. K. & Lewis, R. E. (2003). The activity of
amphotericin B against Candida albicans is not directly associated with extracellular calcium concentration. J Antimicrob Chemother 51, 305-312.
Rutledge, R. G. & Cote, C. (2003). Mathematics of quantitative kinetic PCR and the
application of standard curves. Nucleic Acids Res 31, e93.
Saini, P., Prasad, T., Gaur, N. A., Shukla, S., Jha, S., Komath, S. S., Khan, L. A., Haq, Q. M. & Prasad, R. (2005). Alanine scanning of transmembrane helix 11 of Cdr1p ABC
antifungal efflux pump of Candida albicans: identification of amino acid residues critical for drug efflux. J Antimicrob Chemother 56, 77-86.
Sandven, P. (2000). Epidemiology of candidemia. Rev Iberoam Micol 17, 73-81.
Sanglard, D., Kuchler, K., Ischer, F., Pagani, J. L., Monod, M. & Bille, J. (1995).
Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 39, 2378-2386.
Sanglard, D., Ischer, F., Monod, M. & Bille, J. (1997). Cloning of Candida albicans
genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene. Microbiology 143 ( Pt 2), 405-416.
Sanglard, D. & Odds, F. C. (2002). Resistance of Candida species to antifungal agents:
molecular mechanisms and clinical consequences. Lancet Infect Dis 2, 73-85.
Sanglard, D., Ischer, F., Parkinson, T., Falconer, D. & Bille, J. (2003). Candida
albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob Agents Chemother 47, 2404-2412.
Schuetzer-Muehlbauer, M., Willinger, B., Egner, R., Ecker, G. & Kuchler, K. (2003).
Shimokawa, O. & Nakayama, H. (1989). A Candida albicans mutant conditionally
defective in sterol 14 alpha-demethylation. J Med Vet Mycol 27, 121-125.
Shimokawa, O. & Nakayama, H. (1992). Increased sensitivity of Candida albicans cells
accumulating 14 alpha-methylated sterols to active oxygen: possible relevance to in vivo efficacies of azole antifungal agents. Antimicrob Agents Chemother 36, 1626-1629.
Silver, P. M., Oliver, B. G. & White, T. C. (2004). Role of Candida albicans transcription
factor Upc2p in drug resistance and sterol metabolism. Eukaryot Cell 3, 1391-1397.
Tortorano, A. M., Peman, J., Bernhardt, H. & other authors (2004). Epidemiology of
candidaemia in Europe: results of 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance study. Eur J Clin Microbiol Infect Dis 23, 317-322.
Tortorano, A. M., Kibbler, C., Peman, J., Bernhardt, H., Klingspor, L. & Grillot, R. (2006). Candidaemia in Europe: epidemiology and resistance. Int J Antimicrob Agents 27,
359-366.
Vanden Bossche, H., Willemsens, G. & Marichal, P. (1987). Anti-Candida drugs--the
biochemical basis for their activity. Crit Rev Microbiol 15, 57-72.
Vazquez, J. A., Arganoza, M. T., Vaishampayan, J. K. & Akins, R. A. (1996). In vitro
interaction between amphotericin B and azoles in Candida albicans. Antimicrob Agents
Chemother 40, 2511-2516.
Wada, S., Niimi, M., Niimi, K., Holmes, A. R., Monk, B. C., Cannon, R. D. & Uehara, Y. (2002). Candida glabrata ATP-binding cassette transporters Cdr1p and Pdh1p expressed
in a Saccharomyces cerevisiae strain deficient in membrane transporters show phosphorylation-dependent pumping properties. J Biol Chem 277, 46809-46821.
Wada, S., Tanabe, K., Yamazaki, A., Niimi, M., Uehara, Y., Niimi, K., Lamping, E., Cannon, R. D. & Monk, B. C. (2005). Phosphorylation of candida glabrata ATP-binding
cassette transporter Cdr1p regulates drug efflux activity and ATPase stability. J Biol Chem
280, 94-103.
Walsh, T. J., Pappas, P., Winston, D. J. & other authors (2002). Voriconazole
compared with liposomal amphotericin B for empirical antifungal therapy in patients with neutropenia and persistent fever. N Engl J Med 346, 225-234.
White, T. C. (1997a). Increased mRNA levels of ERG16, CDR, and MDR1 correlate with
increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 41, 1482-1487.
White, T. C. (1997b). The presence of an R467K amino acid substitution and loss of
allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans. Antimicrob Agents Chemother 41, 1488-1494.
White, T. C., Marr, K. A. & Bowden, R. A. (1998). Clinical, cellular, and molecular
factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11, 382-402.
White, T. C., Holleman, S., Dy, F., Mirels, L. F. & Stevens, D. A. (2002). Resistance
mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother 46, 1704-1713.
Wiederhold, N. P. & Lewis, R. E. (2003). The echinocandin antifungals: an overview of
the pharmacology, spectrum and clinical efficacy. Expert Opin Investig Drugs 12, 1313- 1333.
Wirsching, S., Michel, S., Kohler, G. & Morschhauser, J. (2000a). Activation of the
multiple drug resistance gene MDR1 in fluconazole-resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor. J Bacteriol 182, 400-404.
Wirsching, S., Michel, S. & Morschhauser, J. (2000b). Targeted gene disruption in
Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates. Mol Microbiol 36, 856-865.
Xu, Z., Zhang, L. X., Zhang, J. D., Cao, Y. B., Yu, Y. Y., Wang, D. J., Ying, K., Chen, W. S. & Jiang, Y. Y. (2006). cDNA microarray analysis of differential gene expression
and regulation in clinically drug-resistant isolates of Candida albicans from bone marrow transplanted patients. Int J Med Microbiol 296, 421-434.