RESUMO
Increased temperatures associated with urbanization (the "urban heat island" effect) have been shown to impact a wide range of traits across diverse taxa. At the same time, climatic conditions vary at fine spatial scales within habitats due to factors including shade from shrubs, trees, and built structures. Patches of shade may function as microclimate refugia that allow species to occur in habitats where high temperatures and/or exposure to ultraviolet radiation would otherwise be prohibitive. However, the importance of shaded microhabitats for interactions between species across urbanized landscapes remains poorly understood. Weedy plants and their foliar pathogens are a tractable system for studying how multiple scales of climatic variation influence infection prevalence. Powdery mildew pathogens are particularly well suited to this work, as these fungi can be visibly diagnosed on leaf surfaces. We studied the effects of shaded microclimates on rates of powdery mildew infection on Plantago host species in (1) "pandemic pivot" surveys in which undergraduate students recorded shade and infection status of thousands of plants along road verges in urban and suburban residential neighborhoods, (2) monthly surveys of plant populations in 22 parks along an urbanization gradient, and (3) a manipulative field experiment directly testing the effects of shade on the growth and transmission of powdery mildew. Together, our field survey results show strong positive effects of shade on mildew infection in wild Plantago populations across urban, suburban, and rural habitats. Our experiment suggests that this relationship is causal, where microclimate conditions associated with shade promote pathogen growth. Overall, infection prevalence increased with urbanization despite a negative association between urbanization and tree cover at the landscape scale. These findings highlight the importance of taking microclimate heterogeneity into account when establishing links between macroclimate or land use context and prevalence of disease.
Assuntos
Microclima , Doenças das Plantas , Urbanização , Doenças das Plantas/microbiologia , Ascomicetos/fisiologiaRESUMO
BACKGROUND: Impaired cerebral autoregulation and cerebral hypoperfusion may play a critical role in the high morbidity and mortality in patients with sepsis-associated encephalopathy (SAE). Bedside assessment of cerebral autoregulation may help individualize hemodynamic targets that optimize brain perfusion. We hypothesize that near-infrared spectroscopy (NIRS)-derived cerebral oximetry can identify blood pressure ranges that enhance autoregulation in patients with SAE and that disturbances in autoregulation are associated with severity of encephalopathy. METHODS: Adult patients with acute encephalopathy directly attributable to sepsis were followed using NIRS-based multimodal monitoring for 12 consecutive hours. We used the correlation in time between regional cerebral oxygen saturation and mean arterial pressure (MAP) to determine the cerebral oximetry index (COx) as a measure of cerebral autoregulation. Autoregulation curves were constructed for each patient with averaged COx values sorted by MAP in 3 sequential 4-hour periods; the optimal pressure (MAPOPT), defined as the MAP associated with most robust autoregulation (lowest COx), was identified in each period. Severity of encephalopathy was measured with Glasgow coma scale (GCS). RESULTS: Six patients with extracranial sepsis met the stringent criteria specified, including no pharmacological sedation or neurologic premorbidity. Optimal MAP was identified in all patients and ranged from 55 to 115 mmHg. Additionally, MAPOPT varied within individual patients over time during monitoring. Disturbed autoregulation, based on COx, was associated with worse neurologic status (GCS < 13) both with and without controlling for age and severity of sepsis (adjusted odds ratio [OR]: 2.11; 95% confidence interval [CI]: 1.77-2.52; P < .001; OR: 2.97; 95% CI: 1.63-5.43; P < .001). CONCLUSIONS: In this high-fidelity group of patients with SAE, continuous, NIRS-based monitoring can identify blood pressure ranges that improve autoregulation. This is important given the association between cerebral autoregulatory function and severity of encephalopathy. Individualizing blood pressure goals using bedside autoregulation monitoring may better preserve cerebral perfusion in SAE than current practice.
Assuntos
Circulação Cerebrovascular , Encefalopatia Associada a Sepse , Idoso , Pressão Sanguínea/fisiologia , Circulação Cerebrovascular/fisiologia , Feminino , Homeostase , Humanos , Masculino , Pessoa de Meia-Idade , Oximetria , Encefalopatia Associada a Sepse/fisiopatologia , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
PURPOSE: Early mobilization in the intensive care unit (ICU) can improve patient outcomes but has perceived barriers to implementation. As part of an ongoing structured quality improvement project to increase mobilization of medical ICU patients by nurses and clinical technicians, we adapted the existing, validated Patient Mobilization Attitudes & Beliefs Survey (PMABS) for the ICU setting and evaluated its performance characteristics and results. MATERIALS AND METHODS: The 26-item PMABS adapted for the ICU (PMABS-ICU) was administered as an online survey to 163 nurses, clinical technicians, respiratory therapists, attending and fellow physicians, nurse practitioners, and physician assistants in one medical ICU. We evaluated the overall and subscale (knowledge, attitude, and behavior) scores and compared these scores by respondent characteristics (clinical role and years of work experience). RESULTS: The survey response rate was 96% (155/163). The survey demonstrated acceptable discriminant validity and acceptable internal consistency for the overall scale (Cronbach α: 0.82, 95% confidence interval: 0.76-0.85), with weaker internal consistency for all subscales (Cronbach α: 0.62-0.69). Across all respondent groups, the overall barrier score (range: 1-100) was relatively low, with attending physicians perceiving the lowest barriers (median [interquartile range]: 30 [28-34]) and nurses perceiving the highest (37 [31-40]). Within the first 10 years of work experience, greater experience was associated with a lower overall barrier score (-0.8 for each additional year; P = 0.02). CONCLUSIONS: In our medical ICU, across 6 different clinical roles, there were relatively low perceived barriers to patient mobility, with greater work experience over the first 10 years being associated with lower perceived barriers. As part of a structured quality improvement project, the PMABS-ICU may be valuable in assisting to identify specific perceived barriers for consideration in designing mobility interventions for the ICU setting.
Assuntos
Atitude do Pessoal de Saúde , Cuidados Críticos/psicologia , Deambulação Precoce/psicologia , Unidades de Terapia Intensiva/estatística & dados numéricos , Corpo Clínico Hospitalar/psicologia , Adulto , Cuidados Críticos/normas , Deambulação Precoce/normas , Feminino , Pesquisas sobre Atenção à Saúde , Humanos , Unidades de Terapia Intensiva/normas , Masculino , Pessoa de Meia-Idade , Melhoria de QualidadeRESUMO
Recently, a hybrid approach combining solid-state NMR spectroscopy and cryo-electron microscopy showed that the baseplate in green sulfur bacterium Chlorobaculum tepidum is a 2D lattice of BChl a-CsmA dimers [Nielsen, J. T.; et al., Nat. Commun. 2016, 7, 12454-12465]. While the existence of the BChl a-CsmA subunit was previously known, the proposed orientations of the BChl a pigments had only been elucidated from spectral data up to this point. Regarding the electronic structure of the baseplate, two models have been proposed. 2D electronic spectroscopy data were interpreted as revealing that at least four excitonically coupled BChl a might be in close contact. Conversely, spectral hole burning data suggested that the lowest energy state was localized, yet additional states are sometimes observed because of the presence of the Fenna-Matthews-Olson (FMO) antenna protein. To solve this conundrum, this work studies the chlorosome-baseplate complex from Chloroflexus aurantiacus, which does not contain the FMO protein. The results confirm that in both C. tepidum and C. aurantiacus, excitation energy is transferred to a localized low-energy trap state near 818 nm with similar rates, most likely via exciton hopping.
Assuntos
Proteínas de Bactérias/química , Bacterioclorofilas/química , Chloroflexus/metabolismo , Transferência de Energia , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , TemperaturaRESUMO
A series of spectroscopic measurements were performed on membrane fractions and detergent-solubilized complexes from the green sulfur bacterium (GSB) Chlorobaculum (Cba.) tepidum. The excitation migration through the entire GSB photosynthetic apparatus cannot be observed upon excitation of membranes in the chlorosome region at 77â¯K. In order to observe energy transfer from the Fenna-Matthews-Olson (FMO) protein to the reaction center (RC), FMO was directly excited at ~800â¯nm in transient absorption experiments. However, interpretation of the results is complicated by the spectral overlap between FMO and the RC. The availability of the Y16F FMO mutant, whose absorption spectrum is drastically different from that of the WT, has enabled the selection of spectral regions where either only FMO or the RC contributes. The application of a directed kinetic modeling approach, or target analysis, revealed the various decay and energy transfer pathways within the pigment-protein complexes. The calculated FMO-to-RC excitation energy transfer efficiencies are approximately 25% and 48% for the Y16F and WT samples, respectively.
RESUMO
The reaction center (RC) from the phototrophic bacterium Blastochloris viridis was the first integral membrane protein complex to have its structure determined by X-ray crystallography and has been studied extensively since then. It is composed of four protein subunits, H, M, L, and C, as well as cofactors, including bacteriopheophytin (BPh), bacteriochlorophyll (BCh), menaquinone, ubiquinone, heme, carotenoid, and Fe. In this study, we utilized mass spectrometry-based proteomics to study this protein complex via bottom-up sequencing, intact protein mass analysis, and native MS ligand-binding analysis. Its primary structure shows a series of mutations, including an unusual alteration and extension on the C-terminus of the M-subunit. In terms of quaternary structure, proteins such as this containing many cofactors serve to test the ability to introduce native-state protein assemblies into the gas phase because the cofactors will not be retained if the quaternary structure is seriously perturbed. Furthermore, this specific RC, under native MS, exhibits a strong ability not only to bind the special pair but also to preserve the two peripheral BCh's.
Assuntos
Proteínas de Bactérias/química , Espectrometria de Massas/métodos , Complexo de Proteínas do Centro de Reação Fotossintética/química , Alphaproteobacteria/química , Bacterioclorofilas/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteômica/métodosRESUMO
Cerebral blood flow (CBF) autoregulation maintains consistent blood flow across a range of blood pressures (BPs). Sepsis is a common cause of systemic hypotension and cerebral dysfunction. Guidelines for BP management in sepsis are based on historical concepts of CBF autoregulation that have now evolved with the availability of more precise technology for its measurement. In this article, we provide a narrative review of methods of monitoring CBF autoregulation, the cerebral effects of sepsis, and the current knowledge of CBF autoregulation in sepsis. Current guidelines for BP management in sepsis are based on a goal of maintaining mean arterial pressure (MAP) above the lower limit of CBF autoregulation. Bedside tools are now available to monitor CBF autoregulation continuously. These data reveal that individual BP goals determined from CBF autoregulation monitoring are more variable than previously expected. In patients undergoing cardiac surgery with cardiopulmonary bypass, for example, the lower limit of autoregulation varied between a MAP of 40 to 90 mm Hg. Studies of CBF autoregulation in sepsis suggest patients frequently manifest impaired CBF autoregulation, possibly a result of BP below the lower limit of autoregulation, particularly in early sepsis or with sepsis-associated encephalopathy. This suggests that the present consensus guidelines for BP management in sepsis may expose some patients to both cerebral hypoperfusion and cerebral hyperperfusion, potentially resulting in damage to brain parenchyma. The future use of novel techniques to study and clinically monitor CBF autoregulation could provide insight into the cerebral pathophysiology of sepsis and offer more precise treatments that may improve functional and cognitive outcomes for survivors of sepsis.
Assuntos
Pressão Arterial , Circulação Cerebrovascular , Homeostase , Hipotensão/fisiopatologia , Sepse/fisiopatologia , Procedimentos Cirúrgicos Cardíacos , Ponte Cardiopulmonar , Cuidados Críticos , Humanos , Hipotensão/etiologia , Hipotensão/terapia , Monitorização Fisiológica , Planejamento de Assistência ao Paciente , Sepse/complicações , Sepse/terapiaRESUMO
OBJECTIVE: To systematically review and analyze the efficacy and tolerability of different antidepressant pharmacologic treatments for depressive symptoms in Parkinson's disease (PD) METHODS: We searched PubMed, EMBASE, Cochrane database (CENTRAL), clinicaltrials.gov, and bibliographies for randomized controlled trials investigating the efficacy of antidepressant medications versus a non-treatment, placebo, or active treatment groups for depressive symptoms in PD. Twenty of 3191 retrieved studies (1893 patients) were included, but not all could be meta-analyzed. We used a random-effects model meta-analysis to compare depression scores between an active drug and placebo or control group then used a network meta-analysis to compare the effectiveness of different antidepressant classes. The primary outcome was the efficacy of different classes of antidepressant medications in PD patients with depressive symptoms, measured by standardized mean difference (SMD) in depression score from baseline compared with control. RESULTS: Pairwise meta-analysis suggested that type B-selective monoamine oxidase inhibitors (SMD = -1.28, CI = -1.68, -0.88), selective serotonin reuptake inhibitors (SMD = -0.49, CI = -0.93, -0.05), and tricyclics (SMD = -0.83, CI = -1.53, -0.13) are effective antidepressants in PD. Network meta-analysis showed that monoamine oxidase inhibitors had the largest effect on depression in PD (SMD (vs selective serotonin reuptake inhibitors) = -0.78, CI = -1.55, -0.01), but these might not be considered traditional antidepressants given their type B selectivity. CONCLUSIONS: Although limited by few data, this review suggests that multiple antidepressant classes are potentially efficacious in the treatment of depression in PD, but that further comparative efficacy and tolerability research is needed.
Assuntos
Antidepressivos/uso terapêutico , Transtorno Depressivo/tratamento farmacológico , Doença de Parkinson/tratamento farmacológico , Antidepressivos Tricíclicos/uso terapêutico , Benzilaminas/uso terapêutico , Testes Diagnósticos de Rotina , Humanos , Metanálise em Rede , Doença de Parkinson/psicologia , Inibidores Seletivos de Recaptação de Serotonina/uso terapêuticoRESUMO
Delirium is a common and serious psychiatric syndrome caused by an underlying medical condition. It is associated with significant mortality and increased healthcare resource utilization. There are few biological markers of delirium, perhaps related to the etiologic heterogeneity of the syndrome. Functional near-infrared spectroscopy (fNIRS) is an optical topography system to measure changes in the concentration of oxygenated hemoglobin ([oxy-Hb]) in the cerebral cortex. We examined whether altered cortical brain activity in delirious patients with end stage liver disease (ESLD) is detected by fNIRS. We found that the [oxy-Hb] change during the verbal fluency task (VFT) was reduced in patients with ESLD compared with healthy controls (HC) in the prefrontal and bi-temporal regions. The [oxy-Hb] change during the sustained attention task (SAT) was elevated in patients with ESLD compared to HC in the prefrontal and left temporal regions. Notably, [oxy-Hb] change in the left dorsolateral prefrontal cortex during SAT showed a positive correlation with the severity of delirium. Our results suggest that [oxy-Hb] change in the prefrontal cortex during the sustained attention task measured with fNIRS might serve as a biological marker associated with delirium in ESLD patients.
Assuntos
Córtex Cerebral/fisiopatologia , Delírio/psicologia , Doença Hepática Terminal/fisiopatologia , Doença Hepática Terminal/psicologia , Espectroscopia de Luz Próxima ao Infravermelho , Idoso , Atenção , Estudos de Casos e Controles , Comorbidade , Delírio/etiologia , Doença Hepática Terminal/complicações , Doença Hepática Terminal/diagnóstico , Humanos , Pessoa de Meia-Idade , Testes Neuropsicológicos , Curva ROCRESUMO
The cyanobacterial orange carotenoid protein (OCP) protects photosynthetic cyanobacteria from photodamage by dissipating excess excitation energy collected by phycobilisomes (PBS) as heat. Dissociation of the PBS-OCP complex in vivo is facilitated by another protein known as the fluorescence recovery protein (FRP), which primarily exists as a dimeric complex. We used various mass spectrometry (MS)-based techniques to investigate the molecular mechanism of this FRP-mediated process. FRP in the dimeric state (dFRP) retains its high affinity for the C-terminal domain (CTD) of OCP in the red state (OCPr). Site-directed mutagenesis and native MS suggest the head region on FRP is a candidate to bind OCP. After attachment to the CTD, the conformational changes of dFRP allow it to bridge the two domains, facilitating the reversion of OCPr into the orange state (OCPo) accompanied by a structural rearrangement of dFRP. Interestingly, we found a mutual response between FRP and OCP; that is, FRP and OCPr destabilize each other, whereas FRP and OCPo stabilize each other. A detailed mechanism of FRP function is proposed on the basis of the experimental results.
Assuntos
Cianobactérias/metabolismo , Processos Fotoquímicos , Cromatografia Líquida , Espectrometria de MassasRESUMO
In this paper we report the steady-state optical properties of a series of site-directed mutants in the Fenna-Matthews-Olson (FMO) complex of Chlorobaculum tepidum, a photosynthetic green sulfur bacterium. The FMO antenna complex has historically been used as a model system for energy transfer due to the water-soluble nature of the protein, its stability at room temperature, as well as the availability of high-resolution structural data. Eight FMO mutants were constructed with changes in the environment of each of the bacteriochlorophyll a pigments found within each monomer of the homotrimeric FMO complex. Our results reveal multiple changes in low temperature absorption, as well as room temperature CD in each mutant compared to the wild-type FMO complex. These datasets were subsequently used to model the site energies of each pigment in the FMO complex by employing three different Hamiltonians from the literature. This enabled a basic approximation of the site energy shifts imparted on each pigment by the changed amino acid residue. These simulations suggest that, while the three Hamiltonians used in this work provide good fits to the wild-type FMO absorption spectrum, further efforts are required to obtain good fits to the mutant minus wild-type absorption difference spectra. This demonstrates that the use of FMO mutants can be a valuable tool to refine and iterate the current models of energy transfer in this system.
Assuntos
Proteínas de Bactérias/química , Complexos de Proteínas Captadores de Luz/química , Dicroísmo Circular , Mutagênese Sítio-DirigidaRESUMO
Carotenoid-to-bacteriochlorophyll energy transfer has been widely investigated in bacteriochlorophyll (BChl) a-containing light harvesting complexes. Blastochloris viridis utilizes BChl b, whose absorption spectrum is more red-shifted than that of BChl a. This has implications on the efficiency and pathways of carotenoid-to-BChl energy transfer in this organism. The carotenoids that comprise the light-harvesting reaction center core complex (LH1-RC) of B. viridis are 1,2-dihydroneurosporene and 1,2-dihydrolycopene, which are derivatives of carotenoids found in the light harvesting complexes of several BChl a-containing purple photosynthetic bacteria. Steady-state and ultrafast time-resolved optical spectroscopic measurements were performed on the LH1-RC complex of B. viridis at room and cryogenic temperatures. The overall efficiency of carotenoid-to-bacteriochlorophyll energy transfer obtained from steady-state absorption and fluorescence measurements were determined to be â¼27% and â¼36% for 1,2-dihydroneurosporene and 1,2-dihydrolycopene, respectively. These results were combined with global fitting and target analyses of the transient absorption data to elucidate the energetic pathways by which the carotenoids decay and transfer excitation energy to BChl b. 1,2-Dihydrolycopene transfers energy to BChl b via the S2 â Qx channel with kET2 = (500 fs)(-1) while 1,2-dihydroneurosporene transfers energy via S1â Qy (kET1 = (84 ps)(-1)) and S2 â Qx (kET2 = (2.2 ps)(-1)) channels.
Assuntos
Proteínas de Bactérias/química , Bacterioclorofilas/química , Carotenoides/química , Hyphomicrobiaceae/metabolismo , Complexos de Proteínas Captadores de Luz/química , Proteínas de Bactérias/metabolismo , Transferência de Energia , Complexos de Proteínas Captadores de Luz/metabolismo , Fotossíntese , Espectrometria de FluorescênciaRESUMO
BACKGROUND: Severity of alcohol withdrawal syndrome (AWS) is associated with hospital mortality and length of stay. However, as there is no consensus regarding how to predict the development of severe alcohol withdrawal syndrome (SAWS), we sought to determine independent predictors of SAWS. METHODS: We conducted a systematic review and meta-analysis of studies evaluating hospitalized patients with AWS versus SAWS-delirium tremens (DT) and/or seizures. Random-effects meta-analysis [PRISMA guidelines] was performed on common baseline variables and predictive effects for development of SAWS were calculated using RevMan v5.2. Funnel plots were constructed, and tests of heterogeneity were performed. RESULTS: Of 226 studies screened, 17 met criteria and 15 were included in the meta-analysis. The primary findings were that an incident occurrence of DT or alcohol withdrawal seizures was significantly predicted by history of a similar event (OR 2.58 for DT vs. no-DT, 95% CI 1.41, 4.7; OR 2.8 for seizure vs. no-seizure, 95% CI 1.09, 7.19). Both a lower initial platelet count and serum potassium level were predictive of an incident occurrence of DT (platelet count mean difference [MD] -45.64/mm(3) vs. no-DT, 95% CI -75.95, -15.33; potassium level MD -0.26 mEq/l vs. no-DT, 95% CI -0.45, -0.08), seizures, and SAWS. Higher initial alanine aminotransferase was seen in patients with SAWS (MD 20.97 U/l vs. no-SAWS, 95% CI 0.89, 41.05). Higher initial serum gamma-glutamyl transpeptidase was seen in patients with incident alcohol withdrawal seizures (MD 202.56 U/l vs. no-seizure, 95% CI 3.62, 401.5). Significant heterogeneity was observed, and there was evidence of publication bias. Notably, neither gender nor comorbid liver disease was predictive. CONCLUSIONS: The course of prior episodes of AWS is the most reliable predictor of subsequent episodes. Thrombocytopenia and hypokalemia also correlate with SAWS. We propose further research into drinking patterns, gender, and medical comorbidities.
Assuntos
Etanol/efeitos adversos , Pacientes Internados , Índice de Gravidade de Doença , Síndrome de Abstinência a Substâncias/sangue , Síndrome de Abstinência a Substâncias/diagnóstico , Alanina Transaminase/sangue , Delirium por Abstinência Alcoólica/sangue , Delirium por Abstinência Alcoólica/diagnóstico , Delirium por Abstinência Alcoólica/epidemiologia , Convulsões por Abstinência de Álcool/sangue , Convulsões por Abstinência de Álcool/diagnóstico , Convulsões por Abstinência de Álcool/epidemiologia , Biomarcadores/sangue , Feminino , Humanos , Incidência , Masculino , Contagem de Plaquetas , Potássio/sangue , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Síndrome de Abstinência a Substâncias/epidemiologiaRESUMO
When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then "boosted" after 2 days with additional acetate, the cells become "obese" after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, â¼425 genes are upregulated ≥2-fold and â¼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes-encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)-are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.
Assuntos
Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/metabolismo , Glucose-1-Fosfato Adenililtransferase/genética , Triglicerídeos/metabolismo , Acetatos/metabolismo , Tamanho Celular , Chlamydomonas reinhardtii/enzimologia , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Lipase/genética , Lipase/metabolismo , Nitrogênio/metabolismo , Deleção de Sequência , Amido/metabolismoRESUMO
Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous ß-1, 4- and/or ß-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form "drapes" between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.
Assuntos
Clorófitas/ultraestrutura , Matriz Extracelular/química , Arabinose/análise , Comunicação Celular , Membrana Celular , Parede Celular/química , Parede Celular/ultraestrutura , Clorófitas/química , Cloroplastos , Microscopia Crioeletrônica , Retículo Endoplasmático , Galactose/análise , Complexo de Golgi , Hidrocarbonetos/análise , Lipídeos/análise , beta-Glucanas/análiseRESUMO
Light microscopy and deep-etch electron microscopy were used to visualize triacylglyceride (TAG)-filled lipid bodies (LBs) of the green eukaryotic soil alga Chlamydomonas reinhardtii, a model organism for biodiesel production. Cells growing in nitrogen-replete media contain small cytoplasmic lipid bodies (α-cyto-LBs) and small chloroplast plastoglobules. When starved for N, ß-cyto-LB formation is massively stimulated. ß-Cyto-LBs are intimately associated with both the endoplasmic reticulum membrane and the outer membrane of the chloroplast envelope, suggesting a model for the active participation of both organelles in ß-cyto-LB biosynthesis and packaging. When sta6 mutant cells, blocked in starch biosynthesis, are N starved, they produce ß-cyto-LBs and also chloroplast LBs (cpst-LBs) that are at least 10 times larger than plastoglobules and eventually engorge the chloroplast stroma. Production of ß-cyto-LBs and cpst-LBs under the conditions we used is dependent on exogenous 20 mM acetate. We propose that the greater TAG yields reported for N-starved sta6 cells can be attributed to the strain's ability to produce cpst-LBs, a capacity that is lost when the mutant is complemented by a STA6 transgene. Provision of a 20 mM acetate "boost" during N starvation generates sta6 cells that become so engorged with LBs-at the expense of cytoplasm and most organelles-that they float on water even when centrifuged. This property could be a desirable feature for algal harvesting during biodiesel production.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Meios de Cultura/metabolismo , Citoplasma/metabolismo , Metabolismo dos Lipídeos , Acetato de Potássio/metabolismo , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Chlamydomonas reinhardtii/ultraestrutura , Cloroplastos/ultraestrutura , Citoplasma/ultraestrutura , Nitrogênio/metabolismo , Tamanho das Organelas , Organelas/metabolismo , Triglicerídeos/metabolismoRESUMO
The human pathogen Campylobacter jejuni utilizes oxidative phosphorylation to meet all of its energy demands. The genome sequence of this bacterium encodes a number of respiratory enzymes in a branched electron transport chain that predicts the utilization of a number of electron transport chain donor and acceptor molecules. Three of these electron donor enzymes: hydrogenase, formate dehydrogenase, and 2-oxoglutarate:acceptor oxidoreductase (OOR), oxidize hydrogen, formate and alpha-ketoglutarate as electron donors, respectively. Mutations were created in these donor enzymes to isolate mutants in hydrogenase (HydB::CM), formate dehydrogenase (Fdh::CM), and OOR (OorB::CM), as well as a strain with insertions in both hydrogenase and formate dehydrogenase (Hyd::Fdh). These mutants are deficient in their respective enzyme activities and do not reduce the components of the electron transport chain when provided with their respective substrates. The presence of either hydrogen or formate in the media stimulated the growth of wild type (WT) C. jejuni (but not the associated mutant strains) and at least one of these alternative substrates is required for growth of the OOR mutant strain OorB::CM. Finally, the importance of hydrogenase, formate dehydrogenase and OOR as well as the complex I of C. jejuni are elucidated by chicken colonization assays, where the double mutant Hyd::Fdh, OorB::CM and nuo mutants are severely impaired in host colonization.