In this study, we mapped differences in the promoter regions of two genes of p28-Omp locus, genes 14 and 19, whose expression is influenced by macrophage and tick cell environments. Primer extension and quantitative RT-PCR analysis were performed to map transcription start sites and to demonstrate that E. chaffeensis regulates transcription in a host cell-specific manner. Promoter regions of genes 14 and 19 were evaluated to map differences in gene expression and to locate RNA polymerase binding sites.
Primer extension (PE) analysis of p28-Omp genes 14 and 19. Panel A has a cartoon spanning all 22 genes [37]. This panel also has an expansion of cartoons for genes 14 and 19 with predicted transcripts, the primers used for the PE analysis and sequences of the primer extended products with transcription start sites identified with asterisks. PE analysis products resolved on a sequencing gel are shown in panel B. Blots on the left and right represent the data for transcripts of genes 14 and 19, respectively. A sequence ladder for the gene 14 analysis was prepared by using the same primer used for the PE analysis but with a DNA template spanning the gene 14 sequence. For gene 19, PE analysis was performed with RRG 44 primer, and the sequencing ladder was generated by using RRG20-PEXT primer with a gene 19 DNA template. (Lane 1, E. chaffeensis RNA from tick cells; lane 2, E. chaffeensis RNA from macrophages).
graphpad instat extension 14
The entire non-coding sequences upstream to genes 14 and 19 were evaluated to identify sequences similar to the consensus E. coli RNA polymerase binding site sequences, -10 and -35, and ribosome binding site sequences (RBS) (Figure 3). Consensus -10 and -35 elements were identified and are located few bases upstream to the transcription start sites mapped by primer extension analysis (Figure 3). Similarly, putative RBS sequences [22] were identified 7 and 4 nucleotides upstream to the initiation codon of genes 14 and 19, respectively. Genes 14 and 19 sequences upstream to the predicted -10 and -35 sequences differed considerably in their lengths and homology (Figure 3A and 3B). The gene 14 upstream sequence is 581 bp in length, which is 273 bp longer than the gene 19 upstream sequence (308 bp). The sequences included several gene-specific direct repeats and palindrome sequences. In addition, a unique 14 nucleotide-long 'G' rich sequence was detected in the gene 19 sequence. The consensus -35 sequence was identical for both the genes, whereas the -10 and RBS sequences differed by one nucleotide each (Figure 3C). Relative distances of the consensus -10 and -35 sequences from transcription start sites also remained the same for both the genes (Figure 3C).
P28-Omp genes 14 and 19 promoter region sequence analysis. Upstream sequences of genes 14 (panel A) and 19 (panel B) were evaluated for the presence of direct repeats (red text), palindromic sequences (pink text) and for the presence of unique sequences (G-rich region), consensus -35 and -10 regions (green text) and ribosome binding sites (blue text). Panel C shows the comparison of -10, -35 and ribosome binding sites of genes 14 and 19 with the E. coli consensus sequences. Transcription start sites for the genes mapped by primer extension analysis are identified with bold and grey color highlighted text or with an asterisk. Dashes were introduced in the p28-Omp gene 19 sequence to create alignment with the gene 14 sequence.
Table 3 shows histopathologic features of BRAFV600E mutation-positive and negative papillary thyroid microcarcinomas. Twenty-eight microcarcinomas (22%) were well circumscribed including 18 completely encapsulated tumors and the remaining 101 microcarcinomas (78%) displayed infiltrative tumor borders (Figures 1 and 2). Significantly, 57% of the circumscribed tumors (16 of 28) were BRAFV600E mutation negative in contrast to 23% of infiltrative (23 of 101) microcarcinoma (P=0.001). Cystic change but not back-to-back arrangement of tumor cells was significantly associated with the mutation, as noted in nearly 80% (43 of 54) of microcarcinoma with cystic change. Tumor-associated stromal reaction, eg, desmoplasia, fibrosis and/or sclerosis (Figures 1 and 2) was significantly more frequent in mutation-positive tumors (P=0.002), but stromal calcification, psammoma bodies, and lymphocytic infiltrate were not. Classic nuclear features of papillary thyroid carcinoma were significantly associated with the mutation. Eighteen microcarcinomas showed minimal microscopic extrathyroidal extension and all but one were positive for BRAFV600E mutation (Figure 2a). Mutated tumors were more frequently associated with tumor cells with moderate amount of homogeneous eosinophilic cytoplasm (plump pink cells), especially at the infiltrating front (Figure 2b), intratumoral osteoclast-like multinucleated giant cells (41 of 52, 79%; Figure 1e) and lymphovascular invasion (14 of 17, 82%) but these findings did not reach statistical significance.
In conclusion, our findings suggest that BRAFV600E mutation is an early and phenotypically defining molecular event in papillary thyroid carcinoma. The mutation is associated with features predictive of a high risk of local recurrence, eg, extrathyroidal extension and nodal, specifically lateral cervical lymph node metastasis, even in microcarcinomas. Despite their small size, the phenotype of microcarcinomas with regard to the BRAFV600E mutation is distinctive, and the incidence of mutation remains similar to larger papillary thyroid carcinoma.
Ethosuximide is a medication used to treat seizure disorders in humans, and we previously demonstrated that ethosuximide can delay age-related changes and extend the lifespan of the nematode Caenorhabditis elegans. The mechanism of action of ethosuximide in lifespan extension is unknown, and elucidating how ethosuximide functions is important for defining endogenous processes that influence lifespan and for exploring the potential of ethosuximide as a therapeutic for age-related diseases. To identify genes that mediate the activity of ethosuximide, we conducted a genetic screen and identified mutations in two genes, che-3 and osm-3, that cause resistance to ethosuximide-mediated toxicity. Mutations in che-3 and osm-3 cause defects in overlapping sets of chemosensory neurons, resulting in defective chemosensation and an extended lifespan. These findings suggest that ethosuximide extends lifespan by inhibiting the function of specific chemosensory neurons. This model is supported by the observation that ethosuximide-treated animals displayed numerous phenotypic similarities with mutants that have chemosensory defects, indicating that ethosuximide inhibits chemosensory function. Furthermore, ethosuximide extends lifespan by inhibiting chemosensation, since the long-lived osm-3 mutants were resistant to the lifespan extension caused by ethosuximide. These studies demonstrate a novel mechanism of action for a lifespan-extending drug and indicate that sensory perception has a critical role in controlling lifespan. Sensory perception also influences the lifespan of Drosophila, suggesting that sensory perception has an evolutionarily conserved role in lifespan control. These studies highlight the potential of ethosuximide and related drugs that modulate sensory perception to extend lifespan in diverse animals.
Aging is a major factor that contributes to disease and disability in humans, but no medicines have been demonstrated to delay human aging. We previously conducted a screen for FDA-approved drugs that can extend the lifespan of the nematode worm C. elegans, resulting in the identification of ethosuximide, a medicine used to treat epilepsy. To elucidate the mechanism of action of ethosuximide in lifespan extension, we conducted a genetic screen for C. elegans mutations that cause resistance to ethosuximide. Here, we describe the identification of genes that are critical for ethosuximide sensitivity. These genes are necessary for the function of neurons that mediate sensory perception. Furthermore, ethosuximide treatment caused defects in sensory perception. These results indicate that ethosuximide affects lifespan by inhibiting neurons that function in the perception of sensory cues. These studies highlight the importance of sensory neurons in lifespan determination and demonstrate that a drug can act on specific cells within the nervous system to extend lifespan. Sensory perception also modulates Drosophila lifespan, suggesting this is an evolutionarily conserved relationship. Our results indicate that sensory perception may be a promising target for pharmacological extension of lifespan in a variety of animals.
Studies using vertebrate cultured cells have led to the proposal that T-type calcium channels may be the molecular target of ethosuximide in controlling seizures [20],[22],[35]. To determine if ethosuximide inhibits T-type calcium channels to extend lifespan, we analyzed a null mutation in the gene encoding the C. elegans orthologue of the mammalian T-type calcium channel, cca-1(gk30) [36],[37]. If ethosuximide inhibits T-type calcium channels to extend lifespan, then we predict that (1) a cca-1 loss-of-function mutant will be long-lived and (2) a cca-1 loss-of-function mutant will not respond to the lifespan extension caused by ethosuximide. cca-1(gk30) mutants displayed a mean adult lifespan of 15.2 days, which was not significantly different from the 16.1 day mean adult lifespan of wild-type animals (Figure 1A). cca-1(gk30) hermaphrodites treated with ethosuximide displayed a robust lifespan extension, indicating that ethosuximide does not require T-type calcium channels to extend C. elegans lifespan (Figure 1A).
Our previous studies of the related compound trimethadione demonstrated that osm-3 mutants are partially resistant to the mean lifespan extension caused by trimethadione and are fully resistant to the maximum lifespan extension cause by trimethadione [14]. These results are consistent with the conclusion that the lifespan extension caused by trimethadione is partly caused by inhibiting the function of chemosensory neurons. The finding that part of the lifespan extending activity of trimethadione was not suppressed by an osm-3 mutation raises the possibility that trimethadione functions by additional mechanisms to extend lifespan. 2ff7e9595c
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