RESUMO
Buildup of volatile fatty acids (VFAs) in anaerobic digesters (ADs) often results in acidification and process failure. Understanding the dynamics of microbial communities involved in VFA degradation under stable and overload conditions may help optimize anaerobic digestion processes. In this study, five triplicate mesophilic completely mixed AD sets were operated at different organic loading rates (OLRs; 1-6 g chemical oxygen demand [COD] LR-1day-1), and changes in the composition and abundance of VFA-degrading microbial communities were monitored using amplicon sequencing and taxon-specific quantitative PCRs, respectively. AD sets operated at OLRs of 1-4 g COD LR-1day-1 were functionally stable throughout the operational period (120 days) whereas process instability (characterized by VFA buildup, pH decline, and decreased methane production rate) occurred in digesters operated at ≥ 5 g COD LR-1day-1. Though microbial taxa involved in propionate (Syntrophobacter and Pelotomaculum) and butyrate (Syntrophomonas) degradation were detected across all ADs, their abundance decreased with increasing OLR. The overload conditions also inhibited the proliferation of the acetoclastic methanogen, Methanosaeta, and caused a microbial community shift to acetate oxidizers (Tepidanaerobacter acetatoxydans) and hydrogenotrophic methanogens (Methanoculleus). This study's results highlight the importance of operating ADs with conditions that promote the maintenance of microbial communities involved in VFA degradation.
Assuntos
Bactérias Anaeróbias/metabolismo , Reatores Biológicos/microbiologia , Ácidos Graxos Voláteis/metabolismo , Microbiota , Anaerobiose , Firmicutes/metabolismo , Metano/metabolismo , Methanomicrobiaceae/metabolismo , EsgotosRESUMO
Phthalocyanine (Pc) 4, like many photosensitizers for photodynamic therapy (PDT), localizes to intracellular membranes, especially mitochondria. Pc 4-PDT photodamages Bcl-2 and Bcl-xL, antiapoptotic proteins interacting with the permeability transition pore complex that forms at contact sites between the inner and outer mitochondrial membranes. These complexes and the inner membrane are unique in containing the phospholipid cardiolipin. Nonyl-acridine orange (NAO) is a specific probe of cardiolipin. Here we show evidence for fluorescence resonance energy transfer from NAO to Pc 4, defining a binding site for the photosensitizer. This observation establishes an innovative tool for exploring the localization of other photosensitizers and additional fluorescent, mitochondrion-localizing drugs having appropriate spectral properties.
Assuntos
Laranja de Acridina/análogos & derivados , Cardiolipinas/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Indóis/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Neoplasias da Próstata/metabolismo , Laranja de Acridina/química , Sítios de Ligação , Cardiolipinas/química , Corantes/química , Humanos , Indóis/química , Masculino , Microscopia Confocal , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Neoplasias da Próstata/tratamento farmacológico , Espectrometria de Fluorescência , Frações Subcelulares/metabolismo , Células Tumorais CultivadasRESUMO
The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.
Assuntos
Indóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Receptores de GABA-A/fisiologia , Animais , Antineoplásicos/farmacocinética , Isoquinolinas/farmacocinética , Rim/efeitos dos fármacos , Rim/efeitos da radiação , Masculino , Mitocôndrias/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/efeitos dos fármacosRESUMO
Competition for molecular oxygen (O(2)) among respiratory microorganisms is intense because O(2) is a potent electron acceptor. This competition leads to the formation of microoxic environments wherever microorganisms congregate in aquatic, terrestrial and host-associated communities. Bacteria can harvest O(2) present at low, even nanomolar, concentrations using high-affinity terminal oxidases. Here, we report the results of surveys searching for high-affinity terminal oxidase genes in sequenced bacterial genomes and shotgun metagenomes. The results indicate that bacteria with the potential to respire under microoxic conditions are phylogenetically diverse and intriguingly widespread in nature. We explore the implications of these findings by highlighting the importance of microaerobic metabolism in host-associated bacteria related to health and disease.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Metabolismo Energético , Oxigênio/metabolismo , Aerobiose , Anaerobiose , Bactérias/enzimologia , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: As a potential therapy for malignant glioma, we tested the phthalocyanine photosensitizer Pc 4 for: (1) rapid clearance from the vasculature, (2) specificity for glioma, and (3) tumoricidal photosensitizing capability. STUDY DESIGN/MATERIALS AND METHODS: Parenchymal injection of U87 cells into athymic rat brains (N = 100) was followed after 12 days by tail vein injection of 0.5 mg/kg Pc 4. After 1 day, the tumor was illuminated with either 5 (N = 11) or 30 (N = 16) J/cm(2) red light at 672 nm. Sacrifice was 1 day later. The brains from these 27 animals underwent H&E (necrosis) and TUNEL assay (apoptosis) histology. Pc 4 concentration of explanted brains and tumors (N = 16), and all blood samples (N = 52) were determined by HPLC-MS 1 day post Pc 4 administration. RESULTS: Tumor-specific apoptosis was almost uniformly seen; however, necrosis was found mostly in the high-light-dose group. Pc 4 concentration in bulk tumor averaged 3.8 times greater than in normal brain. CONCLUSIONS: These results warrant expanding this pre-clinical study to seek effective baseline Pc 4 drug- and light-doses and infusion-to-photoirradiation timing that would be necessary for a Pc 4-mediated PDT clinical trial for glioma patients.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Indóis/uso terapêutico , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioma/metabolismo , Glioma/patologia , Humanos , Indóis/administração & dosagem , Indóis/farmacocinética , Injeções Intravenosas , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/farmacocinética , Ratos , Ratos NusRESUMO
Photodynamic therapy (PDT), a treatment for cancer and for certain benign conditions, utilizes a photosensitizer and light to produce reactive oxygen in cells. PDT is primarily employed to kill tumor and other abnormal cells, so it is important to ask how this occurs. Many of the photosensitizers currently in clinical or pre-clinical studies of PDT localize in or have a major influence on mitochondria, and PDT is a strong inducer of apoptosis in many situations. The purpose of this review is to critically evaluate all of the recently published research on PDT-induced apoptosis, with a focus on studies providing mechanistic insights. Components of the mechanism whereby PDT causes cells to undergo apoptosis are becoming understood, as are the influences of several signal transduction pathways on the response. Future research should be directed to elucidating the role(s) of the multiple steps in apoptosis in directing damaged cells to an apoptotic vs. necrotic pathway and for producing tumor ablation in conjunction with tissue-level mechanisms operating in vivo.