RESUMEN
The organic polymer lignin is a component of plant cell walls, which like (hemi)-cellulose is highly abundant in nature and relatively resistant to degradation. However, extracellular enzymes released by natural microbial consortia can cleave the ß-aryl ether linkages in lignin, releasing monoaromatic phenylpropanoids that can be further catabolised by diverse species of bacteria. Biodegradation of lignin is therefore important in global carbon cycling, and its natural abundance also makes it an attractive biotechnological feedstock for the industrial production of commodity chemicals. Whilst the pathways for degradation of lignin-derived aromatics have been extensively characterised, much less is understood about how they are recognised and taken up from the environment. The purple phototrophic bacterium Rhodopseudomonas palustris can grow on a range of phenylpropanoid monomers and is a model organism for studying their uptake and breakdown. R. palustris encodes a tripartite ATP-independent periplasmic (TRAP) transporter (TarPQM) linked to genes encoding phenylpropanoid-degrading enzymes. The periplasmic solute-binding protein component of this transporter, TarP, has previously been shown to bind aromatic substrates. Here, we determine the high-resolution crystal structure of TarP from R. palustris as well as the structures of homologous proteins from the salt marsh bacterium Sagittula stellata and the halophile Chromohalobacter salexigens, which also grow on lignin-derived aromatics. In combination with tryptophan fluorescence ligand-binding assays, our ligand-bound co-crystal structures reveal the molecular basis for high-affinity recognition of phenylpropanoids by these TRAP transporters, which have potential for improving uptake of these compounds for biotechnological transformations of lignin.
Asunto(s)
Proteínas Bacterianas/genética , Biodegradación Ambiental , Lignina/genética , Proteínas de Unión al ARN/genética , Rhodopseudomonas/genética , Factores de Transcripción/genética , Transporte Biológico/genética , Regulación Bacteriana de la Expresión Génica/genética , Ligandos , Lignina/química , Lignina/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Oxidorreductasas/genética , Periplasma/genética , Periplasma/microbiología , Proteínas de Unión Periplasmáticas/genética , Proteobacteria/genética , Proteobacteria/crecimiento & desarrollo , Rhodopseudomonas/crecimiento & desarrolloRESUMEN
BACKGROUND/OBJECTIVES: Solar (actinic) keratosis (AK) is an emergent concern worldwide and is associated with an increased risk of development of non-melanoma skin cancer, especially squamous cell carcinoma. Daylight-mediated photodynamic therapy (DL-PDT) using methyl aminolaevulinate cream has proved to be an effective, nearly painless, and more convenient alternative to conventional PDT for the treatment of AK. In a phase III, randomised, controlled trial performed in Australia, the mean irradiance (light intensity) received by patients during DL-PDT treatment, assessed via a spectroradiometer, was 305 W/m(2) (min. 40 to max. 585 W/m(2) ) with similar efficacy irrespective of intensity or dose. The objective of the present meteorological study was to assess the suitability of natural daylight to perform DL-PDT for the treatment of face and scalp AK during different periods of the year and different geographical locations and latitudes across Australia. METHODS: To determine daylight irradiance during a complete year in eight different geographical locations throughout Australia, we used meteorological software (Meteonorm, Meteotest, Bern, Switzerland), and available solar radiation and weather data from 1986-2005. RESULTS: The average daily irradiance remained within the levels (40-585 W/m(2) ) measured during the clinical DL-PDT study in Australia, throughout the year and in all geographical locations investigated (yearly average from Darwin 548 W/m(2) to Hobart 366 W/m(2) ). CONCLUSIONS: DL-PDT for the treatment of face and scalp AK in Australia can be performed effectively throughout the entire year as long as weather conditions permit daylight exposure and allow participants to remain under direct light for 2 h.
Asunto(s)
Dermatosis Facial/tratamiento farmacológico , Queratosis Actínica/tratamiento farmacológico , Fotoquimioterapia , Dermatosis del Cuero Cabelludo/tratamiento farmacológico , Luz Solar , Ácido Aminolevulínico/uso terapéutico , Australia , Humanos , Meteorología/estadística & datos numéricos , Fármacos Fotosensibilizantes/uso terapéutico , Factores de TiempoRESUMEN
Australia has the highest prevalence of actinic keratoses (AK) worldwide. Because of the risk of transformation of AK to invasive squamous cell carcinomas, consensus guidelines recommend that AK are removed using appropriate therapies to prevent progression to invasive disease. Daylight photodynamic therapy (PDT) is emerging as an efficacious treatment for AK, particularly for patients who require treatment of large areas of chronic actinic damage that can be exposed easily to daylight. Daylight PDT with methyl aminolevulinate (MAL) cream is a simple treatment for AK, almost painless, well tolerated and convenient, requiring minimal time in the clinic. Randomised controlled studies from northern Europe and Australia support the use of daylight PDT as an effective therapy for grade I and II AK on the face and scalp. There is sufficient daylight to conduct daylight PDT in Australia at any time of the year and during most weather conditions. Hence, daylight PDT with MAL can be included as an effective and well-tolerated new treatment option for the treatment of AK in Australia. These consensus recommendations provide guidelines for Australian clinicians on the use of daylight PDT in the treatment of diagnosed AK.
Asunto(s)
Ácido Aminolevulínico/análogos & derivados , Queratosis Actínica/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/uso terapéutico , Guías de Práctica Clínica como Asunto , Luz Solar , Ácido Aminolevulínico/uso terapéutico , Australia , Consenso , Dermatosis Facial/diagnóstico , Dermatosis Facial/tratamiento farmacológico , Femenino , Humanos , Queratosis Actínica/diagnóstico , Masculino , Pronóstico , Ensayos Clínicos Controlados Aleatorios como Asunto , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
The food-borne zoonotic pathogen Campylobacter jejuni has complex electron transport chains required for growth in the host, many of which contain cofactored periplasmic enzymes localized by the twin-arginine translocase (TAT). We report here the identification of two paralogues of the TatA translocase component in C. jejuni strain NCTC 11168, encoded by cj1176c (tatA1) and cj0786 (tatA2). Deletion mutants constructed in either or both of the tatA1 and tatA2 genes displayed distinct growth and enzyme activity phenotypes. For sulphite oxidase (SorAB), the multi-copper oxidase (CueO) and alkaline phosphatase (PhoX), complete dependency on TatA1 for correct periplasmic activity was observed. However, the activities of nitrate reductase (NapA), formate dehydrogenase (FdhA) and trimethylamine N-oxide reductase (TorA) were significantly reduced in the tatA2 mutant. In contrast, the specific rate of fumarate reduction catalysed by the flavoprotein subunit of the methyl menaquinone fumarate reductase (MfrA) was similar in periplasmic fractions of both the tatA1 and the tatA2 mutants and only the deletion of both genes abolished activity. Nevertheless, unprocessed MfrA accumulated in the periplasm of the tatA1 (but not tatA2) mutant, indicating aberrant signal peptide cleavage. Surprisingly, TatA2 lacks two conserved residues (Gln8 and Phe39) known to be essential in Escherichia coli TatA and we suggest it is unable to function correctly in the absence of TatA1. Finally, only two TAT chaperones (FdhM and NapD) are encoded in strain NCTC 11168, which mutant studies confirmed are highly specific for formate dehydrogenase and nitrate reductase assembly, respectively. Thus, other TAT substrates must use general chaperones in their biogenesis.
Asunto(s)
Campylobacter jejuni/enzimología , Campylobacter jejuni/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Transporte de Proteínas , Campylobacter jejuni/genética , Campylobacter jejuni/crecimiento & desarrollo , Enzimas/metabolismo , Eliminación de Gen , Proteínas de Transporte de Membrana/genética , Chaperonas Moleculares/genética , Proteínas Periplasmáticas/metabolismoRESUMEN
The biodegradation of lignin, one of the most abundant carbon compounds on Earth, has important biotechnological applications in the derivation of useful products from lignocellulosic wastes. The purple photosynthetic bacterium Rhodopseudomonas palustris is able to grow photoheterotrophically under anaerobic conditions on a range of phenylpropeneoid lignin monomers, including coumarate, ferulate, caffeate, and cinnamate. RPA1789 (CouP) is the periplasmic binding-protein component of an ABC system (CouPSTU; RPA1789, RPA1791-1793), which has previously been implicated in the active transport of this class of aromatic substrate. Here, we show using both intrinsic tryptophan fluorescence and isothermal titration calorimetry that CouP binds a range of phenylpropeneoid ligands with K d values in the nanomolar range. The crystal structure of CouP with ferulate as the bound ligand shows H-bond interactions between the 4-OH group of the aromatic ring with His309 and Gln305. H-bonds are also made between the carboxyl group on the ferulate side chain and Arg197, Ser222, and Thr102. An additional transport system (TarPQM; RPA1782-1784), a member of the tripartite ATP-independent periplasmic (TRAP) transporter family, is encoded at the same locus as rpa1789 and several other genes involved in coumarate metabolism. We show that the periplasmic binding-protein of this system (TarP; RPA1782) also binds coumarate, ferulate, caffeate, and cinnamate with nanomolar K d values. Thus, we conclude that R. palustris uses two redundant but energetically distinct primary and secondary transporters that both employ high-affinity periplasmic binding-proteins to maximise the uptake of lignin-derived aromatic substrates from the environment. Our data provide a detailed thermodynamic and structural basis for understanding the interaction of lignin-derived aromatic substrates with proteins and will be of use in the further exploitation of the flexible metabolism of R. palustris for anaerobic aromatic biotransformations.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Rhodopseudomonas/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Calorimetría , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Enlace de Hidrógeno , Ligandos , Lignina/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Diffuse field change with actinic keratoses (AK) is a ubiquitous skin disease in Australia, with potential for malignant transformation. We report on 35 consecutive patients who had field therapy with single session photodynamic therapy (PDT) using 1 h incubation time for 5-aminolevulinic acid (5-ALA) or 3 h for methyl-aminolevulinate (MAL). METHODS: We retrospectively telephone surveyed our patient cohort regarding their satisfaction and perceptions of the effectiveness, side-effect profile and benefits of PDT. We also reviewed all patients' notes for significant side-effects. RESULTS: Sixty-nine per cent (n = 24/35) of patients responded to the telephone survey; 66% (n = 16/24) of the respondents reported good clearance of AK and claimed a good cosmetic outcome. All respondents reported moderate or severe pain (42% and 58%, respectively) during the illumination phase. Twenty per cent of all patients treated had suffered from one or more of the following side-effects: pustulation; severe erythema; and skin erosions. CONCLUSIONS: Overall, our results compared favourably with previously published studies using 5-ALA or MAL PDT. However, our patient cohort experienced a greater side-effect profile. This may have been due to our patients having greater disease burden compared to other studies and possibly due to our use of topical retinoids prior to PDT in selected patients.
Asunto(s)
Dermatosis Facial/tratamiento farmacológico , Queratosis Actínica/tratamiento farmacológico , Satisfacción del Paciente , Fotoquimioterapia , Dermatosis del Cuero Cabelludo/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Ácido Aminolevulínico/análogos & derivados , Ácido Aminolevulínico/uso terapéutico , Humanos , Persona de Mediana Edad , Fotoquimioterapia/efectos adversos , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
Titanium dioxide and zinc oxide nanoparticles are being increasingly formulated in sunscreens. While the same compounds, in larger particle form, work by reflecting UV radiation, in nanoparticle form, they absorb UV radiation, resulting in photocatalysis, releasing reactive oxygen species. These reactive oxygen species are known to have the capability to alter DNA. Previous studies suggest that this photocatalytic process may not be significant, because the nanoparticles do not penetrate below the level of the stratum corneum. However, some recent studies suggest that nanoparticles may, under certain circumstances, breach that barrier. The majority of those studies have used animal skin models rather than human skin.