ABSTRACT
Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp. OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 10(4) cells. Fusobacterium nucleatum ss. polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples.
Subject(s)
Aptamers, Nucleotide , Biofilms/classification , Dental Plaque/microbiology , Gingiva/microbiology , Gram-Negative Bacteria/classification , Gram-Positive Bacteria/classification , Actinomyces/classification , Bacterial Load , Bacteroidaceae/classification , Campylobacter/classification , DNA Probes , DNA, Bacterial/genetics , Deltaproteobacteria/classification , Digoxigenin , Fusobacterium nucleatum/classification , Gram-Negative Bacteria/genetics , Gram-Positive Bacteria/genetics , Haemophilus/classification , Humans , Lactobacillus/classification , Luminescence , Nucleic Acid Hybridization , Periodontitis/microbiology , Pilot Projects , Porphyromonas gingivalis/classification , Prevotella/classification , RNA, Ribosomal, 16S/genetics , Species Specificity , Staphylococcus/classification , Streptococcus/classificationABSTRACT
Embelin has been reported to exhibit therapeutic activity in cancer. In this study glioblastoma cells and human astrocytes were treated with Embelin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of Embelin broadly sensitized malignant glioma cells to TRAIL-mediated apoptosis. Notably, human astrocytes were not significantly affected by the combined treatment consisting of Embelin and TRAIL. Combined treatment with Embelin and TRAIL augmented the activation of initiator caspases-8/-9 and effector caspases-3/-7, respectively. Furthermore, Embelin down-regulated the expression of the long- and short-isoform of c-FLIP. In addition, forced expression of the short isoform of c-FLIP (S) attenuated apoptosis induced by the combination of Embelin and TRAIL. Embelin did not modulate the mRNA levels of c-FLIP (S), suggesting that Embelin modulates the expression of c-FLIP in a posttranscriptional manner. In summary, the short isoform of c-FLIP is a key regulator in TRAIL-Embelin-mediated apoptosis in malignant glioma.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzoquinones/pharmacology , Brain Neoplasms/drug therapy , CASP8 and FADD-Like Apoptosis Regulating Protein/antagonists & inhibitors , Glioma/drug therapy , TNF-Related Apoptosis-Inducing Ligand/agonists , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/physiology , Apoptosis Regulatory Proteins/drug effects , Apoptosis Regulatory Proteins/metabolism , Brain Neoplasms/metabolism , Brain Neoplasms/physiopathology , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Cell Line , Cell Line, Tumor , Down-Regulation/drug effects , Down-Regulation/physiology , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , Glioma/metabolism , Glioma/physiopathology , Humans , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/drug effects , RNA, Messenger/metabolism , X-Linked Inhibitor of Apoptosis Protein/antagonists & inhibitors , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
The soy isoflavone Daidzein has been reported to exhibit therapeutic activity in cancer. In this study glioblastoma cells and human astrocytes were treated with Daidzein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of Daidzein in combination with TRAIL induces rapid apoptosis in glioma cells. Notably, human astrocytes were not affected by the combined treatment consisting of Daidzein and TRAIL. Combined treatment with Daidzein and TRAIL augmented the activation of caspase-9, suggesting that Daidzein modulated the intrinsic apoptotic pathway. Daidzein did not modulate the expression of death receptors, c-FLIP, XIAP and survivin. However, Daidzein down-regulated bcl-2 and over-expression of bcl-2 attenuated apoptosis induced by the combination of Daidzein and TRAIL. In summary, bcl-2 is a key regulator in TRAIL-Daidzein mediated cell death in malignant glioma.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Glioma/pathology , Isoflavones/pharmacology , Proto-Oncogene Proteins c-bcl-2/biosynthesis , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Astrocytes/drug effects , Blotting, Western , Cell Line, Tumor , Flow Cytometry , Humans , Receptors, TNF-Related Apoptosis-Inducing Ligand/biosynthesisABSTRACT
The flavonoid Myricetin has been reported to exhibit therapeutic activity in cancer. In this study glioblastoma cells and human astrocytes were treated with Myricetin, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of Myricetin in combination with TRAIL induces rapid apoptosis in glioma cells. Notably, human astrocytes were not affected by the combined treatment consisting of Myricetin and TRAIL. Combined treatment with Myricetin and TRAIL augmented the activation of initiator caspases-8/-9 and effector caspases-3/-7. Furthermore, Myricetin down regulated the expression of the long and short isoform of c-FLIP and bcl-2 and over-expression of the short isoform of c-FLIP (S) and bcl-2 attenuated apoptosis induced by the combination of Myricetin and TRAIL. Furthermore, Myricetin did not modulate the mRNA levels of c-FLIP, suggesting that Myricetin modulates the expression of c-FLIP in a posttranscriptional manner. In summary, the short isoform of c-FLIP and bcl-2 are key regulators in TRAIL-Myricetin mediated cell death in malignant glioma.
