RESUMO
Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.
Assuntos
Escherichia coli , Biologia de Sistemas , Escherichia coli/genética , Etilenos , Laboratórios , Engenharia Metabólica , Pseudomonas syringae/genéticaRESUMO
Genetic modification of Rhodococcus jostii RHA1 was carried out in order to optimise the production of pyridine-2,4-dicarboxylic acid and pyridine-2,5-dicarboxylic acid bioproducts from lignin or lignocellulose breakdown, via insertion of either the Sphingobium SYK-6 ligAB genes or Paenibacillus praA gene respectively. Insertion of inducible plasmid pTipQC2 expression vector containing either ligAB or praA genes into a ΔpcaHG R. jostii RHA1 gene deletion strain gave 2-threefold higher titres of PDCA production from lignocellulose (200-287 mg/L), compared to plasmid expression in wild-type R. jostii RHA1. The ligAB genes were inserted in place of the chromosomal pcaHG genes encoding protocatechuate 3,4-dioxygenase, under the control of inducible Picl or PnitA promoters, or a constitutive Ptpc5 promoter, producing 2,4-PDCA products using either wheat straw lignocellulose or commercial soda lignin as carbon source. Insertion of Amycolatopsis sp. 75iv2 dyp2 gene on a pTipQC2 expression plasmid led to enhanced titres of 2,4-PDCA products, due to enhanced rate of lignin degradation. Growth in minimal media containing wheat straw lignocellulose led to the production of 2,4-PDCA in 330 mg/L titre in 40 h, with > tenfold enhanced productivity, compared with plasmid-based expression of ligAB genes in wild-type R. jostii RHA1. Production of 2,4-PDCA was also observed using several different polymeric lignins as carbon sources, and a titre of 240 mg/L was observed using a commercially available soda lignin as feedstock.
Assuntos
Ácidos Dicarboxílicos/metabolismo , Lignina/metabolismo , Engenharia Metabólica/métodos , Piridinas/metabolismo , Rhodococcus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas/genética , Família Multigênica/genética , Regiões Promotoras Genéticas/genética , Protocatecoate-3,4-Dioxigenase/genética , Protocatecoate-3,4-Dioxigenase/metabolismo , Rhodococcus/genéticaRESUMO
Deletion of the pcaHG genes, encoding protocatechuate 3,4-dioxygenase in Rhodococcus jostii RHA1, gives a gene deletion strain still able to grow on protocatechuic acid as the sole carbon source, indicating a second degradation pathway for protocatechuic acid. Metabolite analysis of wild-type R. jostii RHA1 grown on medium containing vanillin or protocatechuic acid indicated the formation of hydroxyquinol (benzene-1,2,4-triol) as a downstream product. Gene cluster ro01857-ro01860 in Rhodococcus jostii RHA1 contains genes encoding hydroxyquinol 1,2-dioxygenase and maleylacetate reductase for degradation of hydroxyquinol but also putative mono-oxygenase (ro01860) and putative decarboxylase (ro01859) genes, and a similar gene cluster is found in the genome of lignin-degrading Agrobacterium species. Recombinant R. jostii mono-oxygenase and decarboxylase enzymes in combination were found to convert protocatechuic acid to hydroxyquinol. Hence, an alternative pathway for degradation of protocatechuic acid via oxidative decarboxylation to hydroxyquinol is proposed.IMPORTANCE There is a well-established paradigm for degradation of protocatechuic acid via the ß-ketoadipate pathway in a range of soil bacteria. In this study, we have found the existence of a second pathway for degradation of protocatechuic acid in Rhodococcus jostii RHA1, via hydroxyquinol (benzene-1,2,4-triol), which establishes a metabolic link between protocatechuic acid and hydroxyquinol. The presence of this pathway in a lignin-degrading Agrobacterium sp. strain suggests the involvement of the hydroxyquinol pathway in the metabolism of degraded lignin fragments.
Assuntos
Agrobacterium/metabolismo , Proteínas de Bactérias/genética , Hidroquinonas/metabolismo , Hidroxibenzoatos/metabolismo , Lignina/metabolismo , Rhodococcus/metabolismo , Proteínas de Bactérias/metabolismo , Deleção de Genes , Redes e Vias Metabólicas , Família MultigênicaRESUMO
Interpretation of variants of uncertain significance, especially chromosomal rearrangements in non-coding regions of the human genome, remains one of the biggest challenges in modern molecular diagnosis. To improve our understanding and interpretation of such variants, we used high-resolution three-dimensional chromosomal structural data and transcriptional regulatory information to predict position effects and their association with pathogenic phenotypes in 17 subjects with apparently balanced chromosomal abnormalities. We found that the rearrangements predict disruption of long-range chromatin interactions between several enhancers and genes whose annotated clinical features are strongly associated with the subjects' phenotypes. We confirm gene-expression changes for a couple of candidate genes to exemplify the utility of our analysis of position effect. These results highlight the important interplay between chromosomal structure and disease and demonstrate the need to utilize chromatin conformational data for the prediction of position effects in the clinical interpretation of non-coding chromosomal rearrangements.
Assuntos
Efeitos da Posição Cromossômica/genética , Mapeamento Cromossômico , Cromossomos Humanos/genética , Rearranjo Gênico/genética , Predisposição Genética para Doença/genética , Genoma Humano/genética , Pontos de Quebra do Cromossomo , Regulação da Expressão Gênica/genética , Variação Genética/genética , Humanos , Hibridização in Situ Fluorescente , Cariótipo , Fenótipo , Translocação Genética/genéticaRESUMO
In cyanobacteria and chloroplasts, exposure to HL damages the photosynthetic apparatus, especially the D1 subunit of Photosystem II. To avoid chronic photoinhibition, a PSII repair cycle operates to replace damaged PSII subunits with newly synthesised versions. To determine the sub-cellular location of this process, we examined the localisation of FtsH metalloproteases, some of which are directly involved in degrading damaged D1. We generated transformants of the cyanobacterium Synechocystis sp. PCC6803 expressing GFP-tagged versions of its four FtsH proteases. The ftsH2-gfp strain was functional for PSII repair under our conditions. Confocal microscopy shows that FtsH1 is mainly in the cytoplasmic membrane, while the remaining FtsH proteins are in patches either in the thylakoid or at the interface between the thylakoid and cytoplasmic membranes. HL exposure which increases the activity of the Photosystem II repair cycle led to no detectable changes in FtsH distribution, with the FtsH2 protease involved in D1 degradation retaining its patchy distribution in the thylakoid membrane. We discuss the possibility that the FtsH2-GFP patches represent Photosystem II 'repair zones' within the thylakoid membranes, and the possible advantages of such functionally specialised membrane zones. Anti-GFP affinity pull-downs provide the first indication of the composition of the putative repair zones.
Assuntos
Membrana Celular/química , Peptídeo Hidrolases/análise , Complexo de Proteína do Fotossistema II/metabolismo , Synechocystis/química , Tilacoides/química , Membrana Celular/enzimologia , Microscopia Confocal , Synechocystis/enzimologia , Tilacoides/enzimologiaRESUMO
The parent core structure of mycosporine-like amino acids (MAAs) is 4-deoxygadusol, which, in cyanobacteria, is derived from conversion of the pentose phosphate pathway intermediate sedoheptulose 7-phosphate by the enzymes 2-epi-5-epivaliolone synthase (EVS) and O-methyltransferase (OMT). Yet, deletion of the EVS gene from Anabaena variabilis ATCC 29413 was shown to have little effect on MAA production, thus suggesting that its biosynthesis is not exclusive to the pentose phosphate pathway. Herein, we report how, using pathway-specific inhibitors, we demonstrated unequivocally that MAA biosynthesis occurs also via the shikimate pathway. In addition, complete in-frame gene deletion of the OMT gene from A. variabilis ATCC 29413 reveals that, although biochemically distinct, the pentose phosphate and shikimate pathways are inextricably linked to MAA biosynthesis in this cyanobacterium. Furthermore, proteomic data reveal that the shikimate pathway is the predominate route for UV-induced MAA biosynthesis.
Assuntos
Aminoácidos/biossíntese , Anabaena variabilis/metabolismo , Metiltransferases/metabolismo , Via de Pentose Fosfato , Ácido Chiquímico/metabolismo , Anabaena variabilis/genética , Anabaena variabilis/efeitos da radiação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deleção de Genes , Glicina/análogos & derivados , Glicina/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Metiltransferases/genética , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Proteômica/métodos , Raios Ultravioleta , GlifosatoRESUMO
Cyanobacteria contain a bidirectional [NiFe] hydrogenase which transiently produces hydrogen upon exposure of anoxic cells to light, potentially acting as a "valve" releasing excess electrons from the electron transport chain. However, its interaction with the photosynthetic electron transport chain remains unclear. By GFP-tagging the HoxF diaphorase subunit we show that the hydrogenase is thylakoid associated, comprising a population dispersed uniformly through the thylakoids and a subpopulation localized to discrete puncta in the distal thylakoid. Thylakoid localisation of both the HoxH and HoxY hydrogenase subunits is confirmed by immunogold electron microscopy. The diaphorase HoxE subunit is essential for recruitment to the dispersed thylakoid population, potentially anchoring the hydrogenase to the membrane, but aggregation to puncta occurs through a distinct HoxE-independent mechanism. Membrane association does not require NDH-1. Localization is dynamic on a scale of minutes, with anoxia and high light inducing a significant redistribution between these populations in favour of puncta. Since HoxE is essential for access to its electron donor, electron supply to the hydrogenase depends on a physiologically controlled localization, potentially offering a new avenue to enhance photosynthetic hydrogen production by exploiting localization/aggregation signals.
RESUMO
The cyclase 2-epi-5-epi-valiolone synthase (EVS) is reported to be a key enzyme for biosynthesis of the mycosporine-like amino acid shinorine in the cyanobacterium Anabaena variabilis ATCC 29413. Subsequently, we demonstrated that an in-frame complete deletion of the EVS gene had little effect on in vivo production of shinorine. Complete segregation of the EVS gene deletion mutant proved difficult and was achieved only when the mutant was grown in the dark and in a medium supplemented with fructose. The segregated mutant showed a striking colour change from native blue-green to pale yellow-green, corresponding to substantial loss of the photosynthetic pigment phycocyanin, as evinced by combinations of absorbance and emission spectra. Transcriptional analysis of the mutant grown in the presence of fructose under dark or light conditions revealed downregulation of the cpcA gene that encodes the alpha subunit of phycocyanin, whereas the gene encoding nblA, a protease chaperone essential for phycobilisome degradation, was not expressed. We propose that the substrate of EVS (sedoheptulose 7-phosphate) or possibly lack of its EVS-downstream products, represses transcription of cpcA to exert a hitherto unknown control over photosynthesis in this cyanobacterium. The significance of this finding is enhanced by phylogenetic analyses revealing horizontal gene transfer of the EVS gene of cyanobacteria to fungi and dinoflagellates. It is also conceivable that the EVS gene has been transferred from dinoflagellates, as evident in the host genome of symbiotic corals. A role of EVS in regulating sedoheptulose 7-phosphate concentrations in the photophysiology of coral symbiosis is yet to be determined.
Assuntos
Anabaena variabilis/enzimologia , Anabaena variabilis/crescimento & desenvolvimento , Carbono/farmacologia , Inositol/análogos & derivados , Liases/metabolismo , Ficobilissomas/metabolismo , Absorção , Anabaena variabilis/efeitos dos fármacos , Anabaena variabilis/genética , Cromatografia Líquida , Inositol/metabolismo , Espectrometria de Massas , Mutação/genética , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Fluorescência , Fosfatos Açúcares/análise , Fosfatos Açúcares/química , Transcrição Gênica/efeitos dos fármacosRESUMO
Route of the sun block: according to empirical evidence, sun-screening mycosporine-like amino acids (MAAs) in Eukarya originate from the shikimic acid pathway, whereas in cyanobacteria, biosynthesis of the MAA shinorine reportedly occurs through the pentose phosphate pathway. However, gene deletion shows that the cyanobacterium Anabaena variabilis ATCC 29143 does not biosynthesise shinorine exclusively by this route.
Assuntos
Anabaena variabilis/metabolismo , Glicina/análogos & derivados , Raios Ultravioleta , Cromatografia Líquida , Cicloexilaminas , Glicina/biossíntese , Espectrofotometria Ultravioleta , Espectrometria de Massas em TandemRESUMO
BACKGROUND: In cyanobacteria the photosystems are localised to, and maintained in, specialist membranes called the thylakoids. The mechanism driving the biogenesis of the thylakoid membranes is still an open question, with only two potential biogenesis factors, Vipp1 and Alb3 currently identified. METHODOLOGY/PRINCIPAL FINDINGS: We generated a slr1768 knockout using the pGEM T-easy vector and REDIRECT. By comparing growth and pigment content (chlorophyll a fluoresence) of the Δslr1768 mutant with the wild-type, we found that Δslr1768 has a conditional phenotype; specifically under high light conditions (130 µmol m(-2) s(-1)) thylakoid biogenesis is disrupted leading to cell death on a scale of days. The thylakoids show considerable disruption, with loss of both structure and density, while chlorophyll a density decreases with the loss of thylakoids, although photosynthetic efficiency is unaffected. Under low light (30 µmol m(-2) s(-1)) the phenotype is significantly reduced, with a growth rate similar to the wild-type and only a low frequency of cells with evident thylakoid disruption. CONCLUSIONS/SIGNIFICANCE: This is the first example of a gene that affects the maintenance of the thylakoid membranes specifically under high light, and which displays a phenotype dependent on light intensity. Our results demonstrate that Slr1768 has a leading role in acclimatisation, linking light damage with maintenance of the thylakoids.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/fisiologia , Clorofila/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fotossíntese/fisiologia , Synechocystis/metabolismo , Tilacoides/fisiologia , Aclimatação , Clorofila A , Luz , Synechocystis/genéticaRESUMO
The title refers to the question addressed in this paper, namely, to what degree if any technology, including nanotechnologies, in the form of products and processes, is capable of contributing to a good life. To answer that question, the paper will develop a meta-normative model whose primary purpose is to determine the essential conditions that any normative theory of the Good Life and Technology (T-GLAT) must adequately address in order to be able to account for, explain and evaluate the Contributive Capability of Technology for a Good Life (CCT-GL). By CCT-GL understand the capability of any technological product or process in its design and/or its use to contribute in some way, if any, to the good life of individuals and society at large. In this paper, the all-embracing term "technology" will be used to refer to both the products and processes of different technologies.
RESUMO
The COMT gene is thought to contribute to the cognitive/psychiatric phenotypes in 22q11.2 deletion syndrome. We measured these manifestations against the Val/Met alleles of the COMT gene, in 40 nonpsychotic 22q11DS children. The Val allele was associated with poor IQ, processing speed, executive function and a higher frequency of anxiety disorders, underscoring the importance of the COMT gene in the childhood psychopathology in 22q11DS.
Assuntos
Ansiedade/genética , Catecol O-Metiltransferase/genética , Transtornos Cognitivos/genética , Síndrome de DiGeorge/genética , Predisposição Genética para Doença , Adolescente , Criança , Função Executiva/fisiologia , Feminino , Genótipo , Humanos , Masculino , Metionina/genética , Testes Neuropsicológicos , Polimorfismo de Nucleotídeo Único , Escalas de Graduação Psiquiátrica , Valina/genéticaRESUMO
Chromosome 22q11.2 deletion syndrome (22q11DS) is associated with cognitive deficits and morphometric brain abnormalities in childhood and a markedly elevated risk of schizophrenia in adolescence/early adulthood. Determining the relationship between neurocognition and neuroimaging findings would yield crucial information about childhood neurodevelopment and provide a basis for the study of the trajectory that occurs on the pathway to psychosis. We compared morphometric brain findings between non-psychotic children with 22q11DS (n = 22) and healthy controls (n = 16), and examined the association between neurocognitive functioning and morphometric brain findings. Volumetric regional gray matter differences between the 22q11DS and control subjects were measured, and correlations of the regional gray matter volumes and neurocognition were performed. Children with 22q11DS demonstrated reductions in gray matter in several brain regions, chiefly the frontal cortices, the cingulate gyrus and the cerebellum. The volumetric reductions in these salient areas were associated with poor performance in sustained attention, executive function and verbal memory; however, the relation of brain volume with cognitive performance did not differ between the patient and control groups. Thus, children with 22q11DS demonstrate gray matter reductions in multiple brain regions that are thought to be relevant to schizophrenia. The correlation of these volumetric reductions with poor neurocognition indicates that these brain regions may mediate higher neurocognitive functions implicated in schizophrenia.
Assuntos
Encéfalo/patologia , Transtornos Cognitivos/patologia , Síndrome de DiGeorge/patologia , Fibras Nervosas Amielínicas/patologia , Adolescente , Atenção , Cerebelo/patologia , Criança , Função Executiva , Feminino , Lobo Frontal/patologia , Giro do Cíngulo/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Memória , Testes Neuropsicológicos , Tamanho do ÓrgãoRESUMO
Recurrent deletions of 2q32q33 have recently been reported as a new microdeletion syndrome. Clinical features of this syndrome include severe mental retardation, growth retardation, dysmorphic features, thin and sparse hair, feeding difficulties and cleft or high palate. The commonly deleted region contains at least seven genes. Haploinsufficiency of one of these genes, SATB2, a DNA-binding protein that regulates gene expression, has been implicated as causative in the cleft or high palate of individuals with 2q32q33 microdeletion syndrome. In this study we describe three individuals with smaller microdeletions of this region, within 2q33.1. The deletions ranged in size from 173.1 kb to 185.2 kb and spanned part of SATB2. Review of clinical records showed similar clinical features among these individuals, including severe developmental delay and tooth abnormalities. Two of the individuals had behavioral problems. Only one of the subjects presented here had a cleft palate, suggesting reduced penetrance for this feature. Our results suggest that deletion of SATB2 is responsible for several of the clinical features associated with 2q32q33 microdeletion syndrome.
Assuntos
Anormalidades Múltiplas/genética , Deleção Cromossômica , Cromossomos Humanos Par 2 , Deficiência Intelectual/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Fatores de Transcrição/genética , Criança , Pré-Escolar , Cromossomos Artificiais Bacterianos , Feminino , Humanos , Hibridização in Situ Fluorescente , MasculinoRESUMO
Cyanobacteria possess a differentiated membrane system and transport proteins into both the periplasm and thylakoid lumen. We have used green fluorescent protein (GFP)-tagged constructs to study the Tat protein transporter and Rieske Tat substrates in Synechocystis PCC6803. The Tat system has been shown to operate in the plasma membrane; we show here that it is also relatively abundant in the thylakoid membrane network, indicating that newly synthesized Tat substrates are targeted to both membrane systems. Synechocystis contains three Rieske iron-sulphur proteins, all of which contain typical twin-arginine signal-like sequences at their N-termini. We show that two of these proteins (PetC1 and PetC2) are obligate Tat substrates when expressed in Escherichia coli. The Rieske proteins exhibit differential localization in Synechocystis 6803; PetC1 and PetC2 are located in the thylakoid membrane, while PetC3 is primarily targeted to the plasma membrane. The combined data show that Tat substrates are directed with high precision to both membrane systems in this cyanobacterium, raising the question of how, and when, intracellular sorting to the correct membrane is achieved.
Assuntos
Membrana Celular/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Synechocystis/metabolismo , Tilacoides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Escherichia coli/genética , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana Transportadoras/genética , Microscopia Confocal , Dados de Sequência Molecular , Plasmídeos , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Synechocystis/genéticaRESUMO
BACKGROUND: S-PM2 is a phage capable of infecting strains of unicellular cyanobacteria belonging to the genus Synechococcus. S-PM2, like other myoviruses infecting marine cyanobacteria, encodes a number of bacterial-like genes. Amongst these genes is one encoding a MazG homologue that is hypothesized to be involved in the adaption of the infected host for production of progeny phage. METHODOLOGY/PRINCIPAL FINDINGS: This study focuses on establishing the occurrence of mazG homologues in other cyanophages isolated from different oceanic locations. Degenerate PCR primers were designed using the mazG gene of S-PM2. The mazG gene was found to be widely distributed and highly conserved among Synechococcus myoviruses and podoviruses from diverse oceanic provinces. CONCLUSIONS/SIGNIFICANCE: This study provides evidence of a globally connected cyanophage gene pool, the cyanophage mazG gene having a small effective population size indicative of rapid lateral gene transfer despite being present in a substantial fraction of cyanophage. The Prochlorococcus and Synechococcus phage mazG genes do not cluster with the host mazG gene, suggesting that their primary hosts are not the source of the mazG gene.
Assuntos
Bacteriófagos/genética , Cianobactérias/virologia , Transferência Genética Horizontal , Água do Mar , Proteínas Virais/genética , Bacteriófagos/isolamento & purificação , Sequência de Bases , Cianobactérias/genética , Genes Virais , Geografia , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
BACKGROUND: Mid-dilution haemodiafiltration (HDF) results in an improved middle molecule removal compared with standard HDF. The OLpur MD 190 haemodiafilter represents a new dialyser design exclusively for mid-dilution on-line HDF. Compared with standard haemodialysers, structural changes in the headers allow the infusion of high replacement fluid volumes after a first post-dilution and before a second pre-dilution stage. METHODS: We compared in vitro the new device [blood flow (QB) 400 ml/min, substitution flow (QS) 100 and 200 ml/min, dialysate flow (QD) 800 ml/min] with a conventional high-flux dialyser of the same surface area in haemodialysis (HD) (QD 500 ml/min) and post-dilution HDF (at QS 60, QD = 500 ml/min and at QS 100, QD = 800 ml/min) modes. Subsequently, we performed an initial clinical application of the new device in six mid-dilution HDF treatments of five end-stage renal disease patients (QB 400 ml/min, QS 200 ml/min, QD 800 ml/min, treatment duration 205+/-23 min). RESULTS: In vitro urea and beta2-microglobulin clearances in mid-dilution HDF were, respectively, 309.2+/-5.5 and 144.4+/-15.2 ml/min (QS 100) and 321.6+/-4.1 and 204.9+/-4.1 ml/min (QS 200), compared with 278.6+/- 17.2 and 94.0+/-7.6 ml/min in HD, and 310.8+/-10.2 and 123.0+/-6.5 ml/min (QS 60) and 323.6+/-11.2 and 158.0+/-10.3 ml/min (QS 100) in post-dilution HDF. The in vivo trials showed the clinical utility of the device and confirmed the in vitro data: urea and beta2-microglobulin clearances were, respectively, 324.6+/- 10.9 and 207.9+/-29.3 ml/min, while reduction ratios were 75.0+/-5.5 and 83.6+/-4.7%. CONCLUSION: Our preliminary results need confirmation in a prospective cross-over study. However, the Nephros MD 190 haemodiafilter promises to be a true technological step ahead in terms of improved beta2-microglobulin removal.
Assuntos
Hemodiafiltração/instrumentação , Falência Renal Crônica/terapia , Membranas Artificiais , Diálise Renal/instrumentação , Adulto , Idoso , Estudos de Coortes , Desenho de Equipamento , Segurança de Equipamentos , Feminino , Hemodiafiltração/métodos , Humanos , Técnicas In Vitro , Falência Renal Crônica/diagnóstico , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Avaliação da Tecnologia BiomédicaRESUMO
YidC/OxaI play essential roles in the insertion of a wide range of membrane proteins in Eschericha coli and mitochondria, respectively. In contrast, the chloroplast thylakoid homolog Albino3 (Alb3) facilitates the insertion of only a specialized subset of proteins, and the vast majority insert into thylakoids by a pathway that is so far unique to chloroplasts. In this study, we have analyzed the role of Alb3 in the cyanobacterium Synechocystis sp. PCC6803, which contains internal thylakoids that are similar in some respects to those of chloroplasts. The single alb3 gene (slr1471) was disrupted by the introduction of an antibiotic cassette, and photoautotrophic growth resulted in the generation of a merodiploid species (but not full segregation), indicating an essential role for Alb3 in maintaining the photosynthetic apparatus. Thylakoid organization is lost under these conditions, and the levels of photosynthetic pigments fall to approximately 40% of wild-type levels. Photosynthetic electron transport and oxygen evolution are reduced by a similar extent. Growth on glucose relieves the selective pressure to maintain photosynthetic competence, and under these conditions, the cells become completely bleached, again indicating that Alb3 is essential for thylakoid biogenesis. Full segregation could not be achieved under any growth regime, strongly suggesting that the slr1471 open reading frame is essential for cell viability.
Assuntos
Proteínas de Arabidopsis/fisiologia , Cianobactérias/metabolismo , Synechocystis/metabolismo , Proteínas de Arabidopsis/metabolismo , Southern Blotting , Membrana Celular/metabolismo , Clorofila/química , Cloroplastos/metabolismo , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Glucose/metabolismo , Luz , Microscopia Confocal , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Modelos Genéticos , Mutação , Fases de Leitura Aberta , Oxigênio/metabolismo , Fotossíntese , Plasmídeos/metabolismo , Pressão , Tilacoides/metabolismo , Fatores de TempoRESUMO
The transport and sorting of extracytoplasmic proteins in cyanobacteria is made complex by the presence of a highly differentiated membrane system. Proteins destined for the periplasm and thylakoid lumen are initially transported by Sec- and Tat-type pathways but little is known of the mechanisms that ultimately direct them to the correct destinations. We have generated a Synechocystis PCC6803 transformant that expresses a fusion protein comprising the Tat-specific targeting signal of Escherichia coli TorA linked to green fluorescent protein (GFP). Immunoblotting indicates the presence of mature-size GFP but no precursor form, demonstrating that efficient translocation has taken place. Confocal microscopy and immunogold electron microscopy reveal GFP to be almost exclusively located in the periplasm, with almost no protein evident in the thylakoid network. These data point to the operation of highly effective sorting pathways for soluble proteins in this cyanobacterium. The observed sorting of the GFP suggests that either (a) the Tat apparatus is located only in the plasma membrane or (b) the TorA-GFP is targeted across either membrane but the GFP is subsequently directed to the periplasm, perhaps by a default sorting pathway to this compartment.