RESUMO
Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.
Assuntos
Membrana Celular/metabolismo , Glicolipídeos/biossíntese , Mycobacterium marinum/metabolismo , Fenóis/metabolismo , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Regulação Bacteriana da Expressão Gênica/fisiologia , Glicolipídeos/química , Glicolipídeos/metabolismo , Estrutura Molecular , Mutação , Mycobacterium marinum/genética , Fenóis/química , Estrutura Terciária de Proteína , Especificidade da EspécieRESUMO
Juzen-taiho-to (JTT) is an immune-boosting formulation of ten medicinal herbs. It is used clinically in East Asia to boost the human immune functions. The active factors in JTT have not been clarified. But, existing evidence suggests that lipopolysaccharide (LPS)-like factors contribute to the activity. To examine this possibility, JTT was subjected to a series of analyses, including high resolution mass spectrometry, which suggested the presence of structural variants of LPS. This finding opened a possibility that JTT contains immune-boosting bacteria. As the first step to characterize the bacteria in JTT, 16S ribosomal RNA sequencing was carried out for Angelica sinensis (dried root), one of the most potent immunostimulatory herbs in JTT. The sequencing revealed a total of 519 bacteria genera in A. sinensis. The most abundant genus was Rahnella, which is widely distributed in water and plants. The abundance of Rahnella appeared to correlate with the immunostimulatory activity of A. sinensis. In conclusion, the current study provided new pieces of evidence supporting the emerging theory of bacterial contribution in immune-boosting herbs.
Assuntos
Medicamentos de Ervas Chinesas/química , Probióticos/química , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Angelica sinensis/metabolismo , Angelica sinensis/microbiologia , Linhagem Celular , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Metagenômica , Probióticos/farmacologia , RNA Ribossômico 16S/metabolismo , Rahnella/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections.
Assuntos
Parede Celular/enzimologia , Farmacorresistência Bacteriana/fisiologia , Ácido Graxo Sintases/metabolismo , Glicolipídeos/biossíntese , Lipídeos/biossíntese , Mycobacterium/enzimologia , Tioléster Hidrolases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Parede Celular/genética , Desenho de Fármacos , Ácido Graxo Sintases/genética , Deleção de Genes , Glicolipídeos/genética , Humanos , Lipídeos/genética , Mycobacterium/genética , Mycobacterium/patogenicidade , Infecções por Mycobacterium/tratamento farmacológico , Infecções por Mycobacterium/enzimologia , Infecções por Mycobacterium/genética , Tioléster Hidrolases/genéticaRESUMO
Phenolic glycolipids (PGLs) are non-covalently bound components of the outer membrane of many clinically relevant mycobacterial pathogens, and play important roles in pathogen biology. We report a mutational analysis that conclusively demonstrates that the conserved acyltransferase-encoding gene papA5 is essential for PGL production. In addition, we provide an in vitro acyltransferase activity analysis that establishes proof of principle for the competency of PapA5 to utilize diol-containing polyketide compounds of mycobacterial origin as acyl-acceptor substrates. Overall, the results reported herein are in line with a model in which PapA5 catalyses the acylation of diol-containing polyketides to form PGLs. These studies advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids and suggest that PapA5 might be an attractive target for exploring the development of antivirulence drugs.
Assuntos
Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Glicolipídeos/biossíntese , Mycobacterium marinum/enzimologia , Aciltransferases/genética , Proteínas de Bactérias/genética , Análise Mutacional de DNA , DNA Bacteriano/genética , Lipoilação , Mutação , Mycobacterium marinum/genética , Deleção de SequênciaRESUMO
The mycobactin siderophore system is present in many Mycobacterium species, including M. tuberculosis and other clinically relevant mycobacteria. This siderophore system is believed to be utilized by both pathogenic and nonpathogenic mycobacteria for iron acquisition in both in vivo and ex vivo iron-limiting environments, respectively. Several M. tuberculosis genes located in a so-called mbt gene cluster have been predicted to be required for the biosynthesis of the core scaffold of mycobactin based on sequence analysis. A systematic and controlled mutational analysis probing the hypothesized essential nature of each of these genes for mycobactin production has been lacking. The degree of conservation of mbt gene cluster orthologs remains to be investigated as well. In this study, we sought to conclusively establish whether each of nine mbt genes was required for mycobactin production and to examine the conservation of gene clusters orthologous to the M. tuberculosis mbt gene cluster in other bacteria. We report a systematic mutational analysis of the mbt gene cluster ortholog found in Mycobacterium smegmatis. This mutational analysis demonstrates that eight of the nine mbt genes investigated are essential for mycobactin production. Our genome mining and phylogenetic analyses reveal the presence of orthologous mbt gene clusters in several bacterial species. These gene clusters display significant organizational differences originating from an intricate evolutionary path that might have included horizontal gene transfers. Altogether, the findings reported herein advance our understanding of the genetic requirements for the biosynthesis of an important mycobacterial secondary metabolite with relevance to virulence.
Assuntos
Proteínas de Bactérias/genética , Vias Biossintéticas/genética , Família Multigênica , Mycobacterium smegmatis/genética , Oxazóis/metabolismo , Filogenia , Sequência Conservada , Análise Mutacional de DNA , Ordem dos GenesRESUMO
A new diterpene with a fused 5-8-5 ring system was isolated from the fermentation broth of a soil actinomycete. The stereochemistry at C-15 was determined in an unusual manner using a decomposition product.
Assuntos
Diterpenos/química , Diterpenos/isolamento & purificação , Streptomyces/química , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Many species use Fe+2 and H2O2 to oxidize a wide variety of compounds to simpler molecules. Both pathogen killing by leukocytes (neutrophils and lymphocytes) and degradation of cellulose by brown rot fungi rely on excretion of Fe+2 ions and H2O2, the Fenton reagent. To elucidate the mechanism of Fenton oxidation of carbohydrates, ß1,3 glucan (laminaran), a major fungal wall polysaccharide, was oxidized using a molar ratio of monomer/Fe+2/H2O2 of 10:1:1 (primarily). We labeled the reaction products and profiled them as fluorescent-labeled molecules in polyacrylamide gels and as hydrophobic-tagged molecules using reverse phase liquid chromatography/mass spectrometry (HPLC/MS). Sub-stoichiometric concentrations of Fe+2 and H2O2 fragmented laminaran into smaller molecules containing carbonyl and carboxylic acid groups visible on fluorescent-labeled carbohydrate polyacrylamide gel electrophoresis. HPLC/MS analysis of glucan fragments showed masses consistent with six classes of molecules: aldoses, dialdoses, uronic acids, hexosuloses, aldonic acids, and hexulosonic acids. The results were consistent with published mechanisms where hydrogen radical (Hâ¢) abstraction from a C-H or O-H bond begins a cascade of reactions leading to 1) C-C bond cleavage to produce aldose/dialdose pairs; 2) oxo-group (O = ) addition to produce uronic and aldonic acids; 3) hydroxyl group (HO-) addition to produce gluconolactone and hexosuloses; and 4) hexulosonic acids. Most products resulted from regioselective H⢠abstractions characteristic of oxidations by ferryl-oxo ion [(FeO)+2] or perferryl-oxo ion [(FeO)+3] in close contact with specific positions in the glycan. Therefore, oxidations initiated by regioselectively-bound Fe ions were the predominant initiators of polysaccharide degradations.
Assuntos
Peróxido de Hidrogênio/química , Ferro/química , beta-Glucanas/química , Estereoisomerismo , TemperaturaRESUMO
Several Mycobacterium tuberculosis strains, Mycobacterium leprae, and other mycobacterial pathogens produce a group of small-molecule virulence factors called phenolic glycolipids (PGLs). PGLs play key roles in pathogenicity and host-pathogen interaction. Thus, elucidation of the PGL biosynthetic pathway will not only expand our understanding of natural product biosynthesis, but may also illuminate routes to novel therapeutics to afford alternative lines of defense against mycobacterial infections. In this study, we report an investigation of the enzymatic requirements for the production of long-chain p-hydroxyphenylalkanoate intermediates of PGL biosynthesis. We demonstrate a functional cooperation between a coenzyme A-independent stand-alone didomain initiation module (FadD22) and a 6-domain reducing iterative type I polyketide synthase (Pks15/1) for production of p-hydroxyphenylalkanoate intermediates in in vitro and in vivo FadD22-Pks15/1 reconstituted systems. Our results suggest that Pks15/1 is an iterative type I polyketide synthase with a relaxed control of catalytic cycle iterations, a mechanistic property that explains the origin of a characteristic alkyl chain length variability seen in mycobacterial PGLs. The FadD22-Pks15/1 reconstituted systems lay an initial foundation for future efforts to unveil the mechanism of iterative catalysis control by which the structures of the final products of Pks15/1 are defined, and to scrutinize the functional partnerships of the FadD22-Pks15/1 system with downstream enzymes of the PGL biosynthetic pathway.
Assuntos
Coenzima A/metabolismo , Ácidos Graxos/biossíntese , Glicolipídeos/biossíntese , Mycobacterium marinum/enzimologia , Fenóis/metabolismo , Policetídeo Sintases/metabolismo , Glicolipídeos/química , Fenóis/química , Policetídeo Sintases/química , Policetídeo Sintases/genética , Estrutura Terciária de ProteínaRESUMO
The catalytic oxidation of alkenes by most iron porphyrins using a variety of oxygen sources, but generally not dioxygen, yields the epoxide with minor quantities of other products. The turnover numbers for these catalysts are modest, ranging from a few hundred to a few thousand depending on the porphyrin structure, axial ligands, and other reaction conditions. Halogenation of substituents increases the activity of the metalloporphyrin catalyst and/or makes it more robust to oxidative degradation. Oxidation of cyclohexene by 5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)porphyrinato iron(III), ([Fe(III)(tppf(20))]) and H(2)O(2) is typical of the latter: the epoxide is 99 % of the product and turnover numbers are about 350.1-4 Herein, we report that dynamic organic nanoparticles (ONPs) of [Fe(III)(tppf(20))] with a diameter of 10 nm, formed by host-guest solvent methods, catalytically oxidize cyclohexene with O(2) to yield only 2-cyclohexene-1-one and 2-cyclohexene-1-ol with approximately 10-fold greater turnover numbers compared to the non-aggregated metalloporphyrin in acetonitrile/methanol. These ONPs facilitate a greener reaction because the reaction solvent is 89 % water and O(2) is the oxidant in place of synthetic oxygen sources. This reactivity is unexpected because the metalloporphyrins are in close proximity and oxidative degradation of the catalyst should be enhanced, thus causing a significant decrease in catalytic turnovers. The allylic products suggest a different oxidative mechanism compared to that of the solvated metalloporphyrins. These results illustrate the unique properties of some ONPs relative to the component molecules or those attached to supports.
Assuntos
Cicloexenos/química , Compostos Férricos/química , Metaloporfirinas/química , Nanopartículas/química , Oxigênio/química , Água/química , Catálise , Isótopos , Cinética , OxirreduçãoRESUMO
Phenolic glycolipids (PGLs) are polyketide-derived virulence factors produced by Mycobacterium tuberculosis, M. leprae, and other mycobacterial pathogens. We have combined bioinformatic, genetic, biochemical, and chemical biology approaches to illuminate the mechanism of chain initiation required for assembly of the p-hydroxyphenyl-polyketide moiety of PGLs. Our studies have led to the identification of a stand-alone, didomain initiation module, FadD22, comprised of a p-hydroxybenzoic acid adenylation domain and an aroyl carrier protein domain. FadD22 forms an acyl-S-enzyme covalent intermediate in the p-hydroxyphenyl-polyketide chain assembly line. We also used this information to develop a small-molecule inhibitor of PGL biosynthesis. Overall, these studies provide insights into the biosynthesis of an important group of small-molecule mycobacterial virulence factors and support the feasibility of targeting PGL biosynthesis to develop new drugs to treat mycobacterial infections.
Assuntos
Coenzima A Ligases , Inibidores Enzimáticos/farmacologia , Glicolipídeos , Macrolídeos/farmacologia , Mycobacterium tuberculosis/enzimologia , Fenóis , Fatores de Virulência , Adenosina/química , Adenosina/metabolismo , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/metabolismo , Glicolipídeos/antagonistas & inibidores , Glicolipídeos/biossíntese , Glicolipídeos/química , Humanos , Macrolídeos/química , Modelos Químicos , Mycobacterium tuberculosis/genética , Parabenos/química , Parabenos/metabolismo , Fenóis/antagonistas & inibidores , Fenóis/química , Fenóis/metabolismo , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/biossíntese , Fatores de Virulência/químicaRESUMO
From Toki-shakuyaku-san, an herbal formulation for 'cleansing stagnated blood,' a key gene regulatory compound was purified and identified through a screening based on DNA microarray and real-time PCR.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Medicina Herbária , Poli-Inos/química , Poli-Inos/farmacologia , Trombose/metabolismo , Células Cultivadas , Humanos , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Poli-Inos/isolamento & purificaçãoRESUMO
We report an efficient and rapid means for the synthesis of tetrakis(pentafluorophenyl)porphyrin (TPPF(20)) derivatives by microwave irradiation in an environmentally acceptable solvent. The selective displacement of the para-fluorine groups in TPPF(20) by primary amines occurs in yields between 70 and 95%. This method demonstrates that TPPF(20) is an ideal platform for the rapid formation of porphyrin conjugates for therapeutic, catalytic, and other applications. [reaction: see text]
Assuntos
Aminas/química , Micro-Ondas , Porfirinas/síntese química , Catálise , Cristalografia por Raios X , Conformação Molecular , Estrutura Molecular , Porfirinas/química , EstereoisomerismoRESUMO
Melanoma is a malignancy with increasing incidence. Although primary tumors that are localized to the skin can be successfully treated by surgical removal, there is no satisfactory treatment for metastatic melanoma, a condition that has currently an estimated 5-year survival of just 6%. During the last decade, ß- or α-emitter-radiolabeled peptides that bind to different receptors on a variety of tumors have been investigated as potential therapeutic agents in both the preclinical and clinical settings with encouraging results. A recent study demonstrated that 188-Rhenium ((188)Re)-labeled, via HYNIC ligand, fungal melanin-binding decapeptide 4B4 was effective against experimental MNT1 human melanoma and was safe to normal melanized tissues. The availability of radiolanthanides with diverse nuclear emission schemes and half-lives provides an opportunity to expand the repertoire of peptides for radionuclide therapy of melanoma. The melanin-binding decapeptide 4B4 was radiolabeled with (177)Lu, (166)Ho, and (153)Sm via a DO3A chelate. The stability studies of Ln*-DO3A-4B4 in phosphate-buffered saline, serum, and a hydroxyapatite assay demonstrated that (177)Lu-labeled peptide was more stable than (166)Ho- and (153)Sm-labeled peptides, most likely because of the smallest ionic radius of the former allowing for better complexation with DO3A. Binding of Ln*-DO3A-4B4 to the lysed highly melanized MNT1 melanoma cells demonstrated the specificity of peptides binding to melanin. In vivo biodistribution data for (177)Lu-DO3A-4B4 given by intraperitoneal administration to lightly pigmented human metastatic A2058 melanoma-bearing mice demonstrated very high uptake in the kidneys and low tumor uptake. Intravenous administration did not improve the tumor uptake. The plausible explanation of low tumor uptake of (177)Lu-DO3A-4B4 could be its decreased ability to bind to melanin during in vitro binding studies in comparison with (188)Re-HYNIC-4B4, exacerbated by the very fast clearance from the blood and the kidneys "sink" effect.
Assuntos
Elementos da Série dos Lantanídeos/farmacologia , Melaninas/metabolismo , Melanoma/diagnóstico por imagem , Melanoma/metabolismo , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Elementos da Série dos Lantanídeos/química , Elementos da Série dos Lantanídeos/farmacocinética , Melanoma/radioterapia , Camundongos , Camundongos Nus , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ensuring biosecurity for pathogen-free rodents generally requires processing all materials that come in direct contact with the animals, including feed, to reduce or eliminate unwanted adventitious agents. A common method of processing animal feed is gamma irradiation. Irradiation is performed offsite and requires transport of feed from the irradiator to the point of use, potentially resulting in surface contamination of the packaging. We tested whether an autoclave could be used to provide a flash disinfection cycle to decontaminate the outer feed packaging while having a limited effect on nutritional feed quality. We developed a standardized and repeatable method, which involved attaching sterile glass vials containing Pseudomonas aeruginosa- and Staphylococcus aureus-laden culture broth onto the bag's surface, to validate effectiveness of the process. Nutritional analyses verified that the flash process had minimal effect on feed quality. Gas chromatography-mass spectrometry confirmed that subjecting feed packaging to the elevated cycle temperatures and pressures did not result in feed contamination by the packaging materials. The lowest autoclave setting that produced consistent surface disinfection, as determined by 3 consecutive negative cultures, was exposure of the bag surface to a chamber temperature of at least 82 degrees C for a minimum of 2 min. This flash disinfection process has been implemented successfully in 5 vivaria supporting more than 35,000 rodent cages daily.
Assuntos
Ração Animal/microbiologia , Desinfecção/métodos , Contaminação de Alimentos/prevenção & controle , Embalagem de Alimentos , Raios gama , Ração Animal/análise , Ração Animal/efeitos da radiação , Desinfecção/normas , Contaminação de Alimentos/análise , Cromatografia Gasosa-Espectrometria de MassasRESUMO
A screening methodology called 'genomic screening' was established to identify natural products that can regulate cellular gene expression. Application of genomic screening to Keishi-bukuryo-gan (KBG), a Japanese herbal medicine formulation, identified a previously unnoticed transcriptional effect by linoleic acid, a known KBG component. The approach opens up a possibility to develop cell-permeable molecular tools for functional genomics research and sets a stage to evaluate the potential of natural products for transcription therapies.
Assuntos
Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Genoma/genética , Produtos Biológicos/química , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/metabolismoRESUMO
Diacyl phthiocerol esters and their congeners are mycobacterial virulence factors. The biosynthesis of these complex lipids remains poorly understood. Insight into their biosynthesis will aid the development of rationally designed drugs that inhibit their production. In this study, we investigate a biosynthetic step required for diacyl (phenol)phthiocerol ester production, i.e., the reduction of the keto group of (phenol)phthiodiolones. We utilized comparative genomics to identify phthiodiolone ketoreductase gene candidates and provide a genetic analysis demonstrating gene function for two of these candidates. Moreover, we present data confirming the existence of a diacyl phthiotriol intermediate in diacyl phthiocerol biosynthesis. We also elucidate the mechanism underlying diacyl phthiocerol deficiency in some mycobacteria, such as Mycobacterium ulcerans and Mycobacterium kansasii. Overall, our findings shed additional light on the biosynthesis of an important group of mycobacterial lipids involved in virulence.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium/enzimologia , Mycobacterium/patogenicidade , Fatores de Virulência/biossíntese , Virulência , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Mycobacterium/genética , PlasmídeosRESUMO
This commentary discusses the recent reports in RNA by Yan and colleagues and Westman and colleagues of the apparent failure of ribavirin to bind to recombinant eIF4E and inhibit 7-methyl guanosine cap-dependent exogenous mRNA translation of cell extracts in vitro. Measuring binding by using affinity chromatography of matrix-immobilized proteins and by using protein emission fluorescence spectroscopy in the presence of nucleotide ligands, as well as limitations of using cell extracts for the assessment of mechanisms of mRNA translation are discussed. Possible reasons for the discordant findings of Yan and colleagues and Westman and colleagues are suggested, and direct observation of the specific binding of ribavirin to eIF4E by using mass spectrometry is presented.