RESUMO
Carbonic anhydrase 8 (CA8) is a member of the α-carbonic anhydrase family but does not catalyze the reversible hydration of carbon dioxide. In the present study, we examined the effects of CA8 on two human colon cancer cell lines, SW480 and SW620, by suppressing CA8 expression through shRNA knockdown. Our results showed that knockdown of CA8 decreased cell growth and cell mobility in SW620 cells, but not in SW480 cells. In addition, downregulated CA8 resulted in a significant decrease of glucose uptake in both SW480 and SW620 cells. Interestingly, stable downregulation of CA8 decreased phosphofructokinase-1 expression but increased glucose transporter 3 (GLUT3) levels in SW620 cells. However, transient downregulation of CA8 fails to up-regulate GLUT3 expression, indicating that the increased GLUT3 observed in SW620-shCA8 cells is a compensatory effect. In addition, the interaction between CA8 and GLUT3 was evidenced by pull-down and IP assays. On the other hand, we showed that metformin, a first-line drug for type II diabetes patients, significantly inhibited cell migration of SW620 cells, depending on the expressions of CA8 and focal adhesion kinase. Taken together, our data demonstrate that when compared to primary colon cancer SW480 cells, metastatic colon cancer SW620 cells respond differently to downregulated CA8, indicating that CA8 in more aggressive cancer cells may play a more important role in controlling cell survival and metformin response. CA8 may affect glucose metabolism- and cell invasion-related molecules in colon cancer, suggesting that CA8 may be a potential target in future cancer therapy.
Assuntos
Anidrases Carbônicas , Neoplasias do Colo , Neoplasias Colorretais , Diabetes Mellitus Tipo 2 , Metformina , Humanos , Transportador de Glucose Tipo 3/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias do Colo/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Glucose , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Adenosine-to-inosine (A-to-I) RNA editing is the most common posttranscriptional editing to create somatic mutations and increase proteomic diversity. However, the functions of the edited mutations are largely underexplored. To identify novel targets in lung adenocarcinoma (LUAD), we conducted a genome-wide somatic A-to-I RNA editing analysis of 23 paired adjacent normal and LUAD transcriptomes and identified 26,280 events, including known nonsynonymous AZIN1-S367G and novel RHOAiso2 (RHOA isoform 2)-R176G, tubulin gamma complex associated protein 2 (TUBGCP2)-N211S, and RBMXL1-I40 M mutations. We validated the edited mutations in silico in multiple databases and in newly collected LUAD tissue pairs with the SEQUENOM MassARRAY® and TaqMan PCR Systems. We selected RHOAiso2-R176G due to its significant level, isoform-specificity, and being the most common somatic edited nonsynonymous mutation of RHOAiso2 to investigate its roles in LUAD tumorigenesis. RHOAiso2 is a ubiquitous but low-expression alternative spliced isoform received a unique Alu-rich exon at the 3' RHOA mRNA to become an editing RNA target, leading to somatic hypermutation and protein diversity. Interestingly, LUAD patients harboring the RHOAiso2-R176G mutation were associated with aberrant RHOA functions, cancer cell proliferation and migration, and poor clinical outcomes in transcriptome analysis. Mechanistically, RHOAiso2-R176G mutation-expressing LUAD cells potentiate RHOA-guanosine triphosphate (GTP) activity to phosphorylate ROCK1/2 effectors and enhance cell proliferation and migration in vitro and increase tumor growth in xenograft and systemic metastasis models in vivo. Taken together, the RHOAiso2-R176G mutation is a common somatic A-to-I edited mutation of the hypermutated RHOA isoform 2. It is an oncogenic and isoform-specific theranostic target that activates RHOA-GTP/p-ROCK1/2 signaling to promote tumor progression.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , RNA , Proteômica , Adenosina , Adenocarcinoma de Pulmão/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Neoplasias Pulmonares/genética , Guanosina Trifosfato , Inosina , Mutação , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The human health risks caused by heavy metal contamination (As, Cd, Cr, Cu, Hg, Pb, Ni, and Zn) in the surface water of the Houjing River, the most contaminated river in southern Taiwan, were assessed in this study. Firstly, heavy metal contamination was evaluated by the contamination factors (CF) and the metal indexes (MI). Secondly, the human health risks due to heavy metal contamination were simulated using the Adaptive Risk Assessments Modeling System (ARAMS) through three scenarios; fish ingestion, dermal water contact, and incidental water ingestion during swimming. The hazard quotient (HQ) and the hazard index (HI) were used to evaluate non-carcinogenic risks, while carcinogenic risks were estimated by the lifetime cancer incidence risk index (CR) and the cumulative cancer risk (CCR). The results showed that the synergistic contamination of heavy metals in the surface water was severe (MI = 12.4), with the highest contribution from Cu, Ni, and Pb. Copper had the highest non-carcinogenic risk at the "adverse effect" level, while Ni and Cr had the highest carcinogenic risk at an "unacceptable" level. In addition, the cumulative risks of fish ingestion (HIFI = 6.75 and CCRFI = 1.25E-03) were significantly higher than those of the swimming scenarios (HI(DC + WI) = 1.94E-03 and CCR(DC + WI) = 9.32E-08). The results from this study will be beneficial for immediate and future contamination control measures and human health management plans for this study area. This study has also demonstrated the effectiveness of using ARAMS in human health risk assessment.
Assuntos
Metais Pesados , Rios , Animais , China , Monitoramento Ambiental , Humanos , Indústrias , Metais Pesados/análise , Medição de RiscoRESUMO
Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide due to metastasis. Paraspeckle component 1 (PSPC1) upregulation has been identified as an HCC pro-metastatic activator associated with poor patient prognosis, but with a lack of targeting strategy. Here, we report that PSPC1, a nuclear substrate of PTK6, sequesters PTK6 in the nucleus and loses its metastasis driving capability. Conversely, PSPC1 upregulation or PSPC1-Y523F mutation promotes epithelial-mesenchymal transition, stemness, and metastasis via cytoplasmic translocation of active PTK6 and nuclear translocation of ß-catenin, which interacts with PSPC1 to augment Wnt3a autocrine signaling. The aberrant nucleocytoplasmic shuttling of active PTK6/ß-catenin is reversed by expressing the PSPC1 C-terminal interacting domain (PSPC1-CT131), thereby suppressing PSPC1/PTK6/ß-catenin-activated metastasis to prolong the survival of HCC orthotopic mice. Thus, PSPC1 is the contextual determinant of the oncogenic switch of PTK6/ß-catenin subcellular localizations, and PSPC1-CT131 functions as a dual inhibitor of PSPC1 and PTK6 with potential for improving cancer therapy.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , beta Catenina/metabolismo , Adulto , Idoso , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Estimativa de Kaplan-Meier , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Regulação para Cima , Proteína Wnt3A/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the "mucosal escape response," this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.
Assuntos
Proteínas de Bactérias/fisiologia , Cólera/microbiologia , Mucosa Intestinal/microbiologia , Fator sigma/fisiologia , Vibrio cholerae/fisiologia , Animais , Cólera/patologia , Modelos Animais de Doenças , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Íleo/microbiologia , Íleo/fisiopatologia , Mucosa Intestinal/fisiopatologia , Microscopia Confocal , Movimento/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/análise , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator sigma/deficiência , Vibrio cholerae/ultraestruturaRESUMO
Microporous activated carbon fibers (ACFs) were developed for CO2 capture based on potassium hydroxide (KOH) activation and tetraethylenepentamine (TEPA) amination. The material properties of the modified ACFs were characterized using several techniques. The adsorption breakthrough curves of CO2 were measured and the effect of relative humidity in the carrier gas was determined. The KOH activation at high temperature generated additional pore networks and the intercalation of metallic K into the carbon matrix, leading to the production of mesopore and micropore volumes and providing access to the active sites in the micropores. However, this treatment also resulted in the loss of nitrogen functionalities. The TEPA amination has successfully introduced nitrogen functionalities onto the fiber surface, but its long-chain structure blocked parts of the micropores and, thus, made the available surface area and pore volume limited. Introduction of the power of time into the Wheeler equation was required to fit the data well. The relative humidity within the studied range had almost no effects on the breakthrough curves. It was expected that the concentration of CO2 was high enough so that the impact on CO2 adsorption capacity lessened due to increased relative humidity.
RESUMO
The mosaic-structured Vibrio cholerae genome points to the importance of horizontal gene transfer (HGT) in the evolution of this human pathogen. We showed that V. cholerae can acquire new genetic material by natural transformation during growth on chitin, a biopolymer that is abundant in aquatic habitats (e.g., from crustacean exoskeletons), where it lives as an autochthonous microbe. Transformation competence was found to require a type IV pilus assembly complex, a putative DNA binding protein, and three convergent regulatory cascades, which are activated by chitin, increasing cell density, and nutrient limitation, a decline in growth rate, or stress.
Assuntos
Quitina/fisiologia , Transformação Bacteriana , Vibrio cholerae O1/genética , Vibrio cholerae/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Braquiúros/microbiologia , Quitina/metabolismo , Meios de Cultura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fímbrias/biossíntese , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/metabolismo , Mutação da Fase de Leitura , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Modelos Biológicos , Mutação , Fenótipo , Regulon , Fator sigma/metabolismo , Vibrio cholerae/crescimento & desenvolvimento , Vibrio cholerae/metabolismo , Vibrio cholerae/fisiologia , Vibrio cholerae O1/crescimento & desenvolvimento , Vibrio cholerae O1/metabolismo , Vibrio cholerae O1/fisiologiaRESUMO
The type IV bundle-forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for virulence in orally challenged human volunteers and for the localized adherence and autoaggregation in vitro phenotypes. BFP filament biogenesis and function are encoded by the 14-gene bfp operon. The BFP assembly complex, containing a BfpB-His6 fusion protein, was chemically cross-linked in situ, and the complex was then purified from BFP-expressing EPEC by a combination of nickel- and BfpB antibody-based affinity chromatography. Characterization of the isolated complex by immunoblotting using BFP protein-specific antibodies showed that at least 10 of the 14 proteins specified by the bfp operon physically interact to form an oligomeric complex. Proteins localized to the outer membrane, inner membrane, and periplasm are within this complex, thus demonstrating that the complex spans the periplasmic space. A combination of immunofluorescence and immuno-gold thin-section transmission electron microscopy studies localized this complex to one pole of the cell.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/ultraestrutura , Proteínas de Fímbrias/química , Fímbrias Bacterianas/metabolismo , Óperon , Polaridade Celular , Cromatografia de Afinidade , Reagentes de Ligações Cruzadas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Immunoblotting , Microscopia Eletrônica , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Chitin, an insoluble polymer of GlcNAc, is an abundant source of carbon, nitrogen, and energy for marine microorganisms. Microarray expression profiling and mutational studies of Vibrio cholerae growing on a natural chitin surface, or with the soluble chitin oligosaccharides (GlcNAc)(2-6), GlcNAc, or the glucosamine dimer (GlcN)2 identified three sets of differentially regulated genes. We show that (i) ChiS, a sensor histidine kinase, regulates expression of the (GlcNAc)(2-6) gene set, including a (GlcNAc)2 catabolic operon, two extracellular chitinases, a chitoporin, and a PilA-containing type IV pilus, designated ChiRP (chitin-regulated pilus) that confers a significant growth advantage to V. cholerae on a chitin surface; (ii) GlcNAc causes the coordinate expression of genes involved with chitin chemotaxis and adherence and with the transport and assimilation of GlcNAc; (iii) (GlcN)2 induces genes required for the transport and catabolism of nonacetylated chitin residues; and (iv) the constitutively expressed MSHA pilus facilitates adhesion to the chitin surface independent of surface chemistry. Collectively, these results provide a global portrait of a complex, multistage V. cholerae program for the efficient utilization of chitin.
Assuntos
Quitina/metabolismo , Vibrio cholerae/metabolismo , Adesão Celular/fisiologia , Quitina/genética , Regulação Bacteriana da Expressão Gênica , Cinética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Regulon/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vibrio cholerae/genética , Vibrio cholerae/fisiologiaRESUMO
Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon. Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins. This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex. These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts. The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU. Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments.