RESUMEN
The two new ternary amalgams K1-xRbxHg11 [x = 0.472(7)] and Cs3-xCaxHg20 [x = 0.20(3)] represent two different examples of how to create ternary compounds from binaries by statistical atom substitution. K1-xRbxHg11 is a Vegard-type mixed crystal of the isostructural binaries KHg11 and RbHg11 [cubic, BaHg11 structure type, space group Pm3Ì m, a = 9.69143(3) Å, Rietveld refinement], whereas Cs3-xCaxHg20 is a substitution variant of the Rb3Hg20 structure type [cubic, space group Pm3Ì n, a = 10.89553(14) Å, Rietveld refinement] for which a fully substituted isostructural binary Ca phase is unknown. In K1-xRbxHg11, the valence electron concentration (VEC) is not changed by the substitution, whereas in Cs3-xCaxHg20, the VEC increases with the Ca content. Amalgams of electropositive metals form polar metal bonds and show "bad metal" properties. By thermal analysis, magnetic susceptibility and resistivity measurements, and density functional theory calculations of the electronic structures, we investigate the effect of the structural disorder introduced by creating mixed-atom occupation on the physical properties of the two new polar amalgam systems.
RESUMEN
The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.
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Proteínas Bacterianas/genética , ADN Helicasas/genética , Replicación del ADN/genética , Farmacorresistencia Bacteriana/genética , Roturas del ADN de Cadena Simple/efectos de los fármacos , Proteínas de Unión al ADN/genética , Geobacillus stearothermophilus/efectos de los fármacos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/patogenicidad , Plásmidos/efectos de los fármacos , Plásmidos/genética , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Tetraciclina/farmacología , Transactivadores/genéticaRESUMEN
Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5'-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate.
Asunto(s)
Roturas del ADN de Cadena Simple , ADN Helicasas/metabolismo , Replicación del ADN , Transactivadores/metabolismo , ADN Helicasas/química , Modelos Biológicos , Plásmidos/genética , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes , Transactivadores/químicaRESUMEN
Dengue is one of the most important arthropod-borne viral diseases. It is endemic in > 125 countries including Pakistan, with a global incidence of 50-200 million. We determined the frequency of different serotypes of dengue virus to highlight its hyperendemicity in Rawalpindi, Pakistan. Between May and October 2015 we analysed the serum samples of 140 patients with a suspicion of dengue, using ELISA and multiplex polymerase chain reaction. One hundred and eight were infected with serotype 2, 16 with serotype 3, 7 with serotype 4 and 3 with serotype 1. Three patients were infected with serotypes 1 and 2, and 1 each with serotypes 1 and 4 and serotypes 2 and 3. Incidence of dengue has increased many fold in the past 50 years and has expanded to areas that were previously free from the disease. Serotype 2 was predominant in our population followed by serotype 3. There is currently no specific treatment for dengue, and vector control and vaccination are the only effective methods to prevent future outbreaks.
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Virus del Dengue/clasificación , Dengue/epidemiología , Dengue/virología , Brotes de Enfermedades , Reacción en Cadena de la Polimerasa Multiplex , Adolescente , Adulto , Anciano de 80 o más Años , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Incidencia , Lactante , Masculino , Pakistán/epidemiología , SerotipificaciónRESUMEN
BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) remains a prevalent and devastating disease. Recently, there has been an increase in SCCHN cases that are associated with high-risk human papillomavirus (HPV) infection. The clinical characteristics of HPV-positive and HPV-negative SCCHN are known to be different but their molecular features are only recently beginning to emerge. MicroRNAs (miRNAs, miRs) are small, non-coding RNAs that are likely to play significant roles in cancer initiation and progression where they may act as oncogenes or tumor suppressors. Previous studies in our laboratory showed that miR-363 is overexpressed in HPV-positive compared to HPV-negative SCCHN cell lines, and the HPV type 16-E6 oncoprotein upregulates miR-363 in SCCHN cell lines. However, the functional role of miR-363 in SCCHN in the context of HPV infection remains to be elucidated. METHODS: We analyzed miR-363 levels in SCCHN tumors with known HPV-status from The Cancer Genome Atlas (TCGA) and an independent cohort from our institution. Cell migration studies were conducted following the overexpression of miR-363 in HPV-negative cell lines. Bioinformatic tools and a luciferase reporter assay were utilized to confirm that miR-363 targets the 3'-UTR of myosin 1B (MYO1B). MYO1B mRNA and protein expression levels were evaluated following miR-363 overexpression in HPV-negative SCCHN cell lines. Small interfering RNA (siRNA) knockdown of MYO1B was performed to assess the phenotypic implication of reduced MYO1B expression in SCCHN cell lines. RESULTS: MiR-363 was found to be overexpressed in HPV-16-positive compared to the HPV-negative SCCHN tumors. Luciferase reporter assays performed in HPV-negative JHU028 cells confirmed that miR-363 targets one of its two potential binding sites in the 3'UTR of MYO1B. MYO1B mRNA and protein levels were reduced upon miR-363 overexpression in four HPV-negative SCCHN cell lines. Increased miR-363 expression or siRNA knockdown of MYO1B expression reduced Transwell migration of SCCHN cell lines, indicating that the miR-363-induced migration attenuation of SCCHN cells may act through MYO1B downregulation. CONCLUSIONS: These findings demonstrate that the overexpression of miR-363 reduces cellular migration in head and neck cancer and reveal the biological relationship between miR-363, myosin 1b, and HPV-positive SCCHN.
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Neoplasias de Cabeza y Cuello/genética , MicroARNs/genética , Miosina Tipo I/genética , Interferencia de ARN , Regiones no Traducidas 3' , Anciano , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , ARN Mensajero/genéticaRESUMEN
When the replication of a plasmid based on sucrose selection is deregulated via the inc1 and inc2 mutations, high copy numbers (7,000 or greater) are attained while the growth rate on minimal medium is negligibly affected. Adaptions were assumed to be required in order to sustain the growth rate. Proteomics indicated that indeed a number of adaptations occurred that included increased expression of ribosomal proteins and 2-oxoglutarate dehydrogenase. The operating space prescribed by a basic flux model that maintained phenotypic traits (e.g. growth, byproducts, etc.) within typical bounds of resolution was consistent with the flux implications of the proteomic changes.
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Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Plásmidos/metabolismo , Proteoma/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Dosificación de Gen , Glucosa/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Modelos Biológicos , Mutación , Fenotipo , Plásmidos/genética , Proteínas Ribosómicas/metabolismoRESUMEN
For small-copy-number pUC-type plasmids, the inc1 and inc2 mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due to inc mutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of the inc mutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ~1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C, inc2 mutants of pNTC8485 exhibited a copy number of ~7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use of inc mutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications.
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Replicación del ADN , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Mutación , Plásmidos , Medios de Cultivo/química , Escherichia coli/metabolismo , Sacarosa/metabolismo , TemperaturaRESUMEN
The essential DNA helicase, PcrA, regulates recombination by displacing the recombinase RecA from the DNA. The nucleotide-bound state of RecA determines the stability of its nucleoprotein filaments. Using single-molecule fluorescence approaches, we demonstrate that RecA displacement by a translocating PcrA requires the ATPase activity of the recombinase. We also show that in a 'head-on collision' between a polymerizing RecA filament and a translocating PcrA, the RecA K72R ATPase mutant, but not wild-type RecA, arrests helicase translocation. Our findings demonstrate that translocation of PcrA is not sufficient to displace RecA from the DNA and assigns an essential role for the ATPase activity of RecA in helicase-mediated disruption of its filaments.
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Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , Rec A Recombinasas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , ADN de Cadena Simple/metabolismo , Geobacillus stearothermophilus/enzimología , Transporte de ProteínasRESUMEN
The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.
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Antígenos Bacterianos/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Replicación del ADN , Plásmidos/genética , Replicón , Bacillus anthracis/metabolismo , Bacillus anthracis/patogenicidad , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Variaciones en el Número de Copia de ADN , Orden Génico , Mutagénesis , Sistemas de Lectura Abierta , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The RNase III endonuclease Dicer plays a key role in generation of microRNAs (miRs). We hypothesized that Dicer regulates cancer cell susceptibility to immune surveillance through miR processing. Indeed, Dicer disruption up-regulated intercellular cell adhesion molecule (ICAM)-1 and enhanced the susceptibility of tumor cells to antigen-specific lysis by cytotoxic T-lymphocytes (CTLs), while expression of other immunoregulatory proteins examined was not affected. Blockade of ICAM-1 inhibited the specific lysis of CTLs against Dicer-disrupted cells, indicating a pivotal role of ICAM-1 in the interaction between tumor cells and CTL. Both miR-222 and -339 are down-regulated in Dicer-disrupted cells and directly interacted with the 3' untranslated region (UTR) of ICAM-1 mRNA. Modulation of Dicer or these miRs inversely correlated with ICAM-1 protein expression and susceptibility of U87 glioma cells to CTL-mediated cytolysis while ICAM-1 mRNA levels remained stable. Immunohistochemical and in situ hybridization analyses of 30 primary glioblastoma tissues demonstrated that expression of Dicer, miR-222, or miR-339 was inversely associated with ICAM-1 expression. Taken together, Dicer is responsible for the generation of the mature miR-222 and -339, which suppress ICAM-1 expression on tumor cells, thereby down-regulating the susceptibility of tumor cells to CTL-mediated cytolysis. This study suggests development of novel miR-targeted therapy to promote cytolysis of tumor cells.
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ARN Helicasas DEAD-box/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , MicroARNs/genética , Ribonucleasa III/metabolismo , Linfocitos T Citotóxicos/inmunología , Western Blotting , Línea Celular Tumoral , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Citotoxicidad Inmunológica/inmunología , ARN Helicasas DEAD-box/genética , Regulación hacia Abajo , Citometría de Flujo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Células HCT116 , Humanos , Inmunohistoquímica , Hibridación in Situ , Molécula 1 de Adhesión Intercelular/genética , Luciferasas/genética , Luciferasas/metabolismo , Mutación , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasa III/genética , Linfocitos T Citotóxicos/citología , TransfecciónRESUMEN
OBJECTIVES: Complications related to coronavirus disease 2019 (COVID-19) may lead to disseminated intravascular coagulation (DIC), which has been reported to be among the known causes of mortality in such patients. This study aims to analyse the incidence of DIC in COVID-19 non-survivors and to assess the association between DIC and its comorbidities. METHODS: The medical records of 154 non-survivors of COVID-19, hospitalised between April 2020 and July 2020, were retrospectively analysed. The International Society on Thrombosis and Haemostasis (ISTH) criteria for DIC were applied to identify the occurrence of coagulopathy. The receiver-operating characteristic (ROC) analysis was used to assess the association between DIC and its comorbidities. RESULTS: Out of 154 non-survivors, non-overt DIC was observed in 94.8% of the patients, whereas only 5.2% fulfilled the overt criteria of DIC with a mean age 64.6 years. The mortality rate was 4.5 times higher among men than women. The D-dimer level was >250 ng/ml in 68.8% of the patients including 88.9% of the non-overt and 100% of the overt DIC patients. Prothrombin time (PT) in non-overt and overt DIC cases was 17.3 s and 24.4 s, respectively. Thrombotic event and chronic kidney disease were found to be the main predictors of DIC (p < 0.0001 and 0.03, respectively) followed by diabetes mellitus (DM) and hypertension (statistically insignificant). CONCLUSIONS: Our study concludes that the ISTH DIC score cannot predict mortality as the COVID-19 related DIC differs from the sepsis-induced DIC. Among the seriously ill, older patients with comorbidities, increased levels of D-dimer and prolonged PT are more reliable parameters among COVID-19 non-survivors.
RESUMEN
OBJECTIVE: Although cervical cancer could be prevented through medical screening, it remains one of the top causes of cancer-related morbidity and mortality all over the world. A number of factors may contribute to cervical cancer screening behaviour of women. The aim of this study was to investigate factors related to cervical cancer screening behaviour of women in Kazakhstan. METHODS: This was a cross-sectional survey-based study with a total of 1189 participants. Women attending gynaecological clinics aged between 18 and 70 years were administered paper-based questionnaires about their awareness of cervical cancer, the associated risk factors, and cervical cancer screening. Student t test or Wilcoxon rank-sum test and chi-square test or Fisher's exact test, where appropriate, were used to determine associations with categorical independent variables. RESULTS: The mean age of participants was 36.5 ± 10.1 years. Less than half (45.7%) of the participants had been screened for cervical cancer. The key factors related to the cervical cancer screening behaviour of women in this study included age, having a larger number of children, regular menstrual function, awareness of Pap smear test, and free screening programme for cervical cancer, and the causal association of human papillomavirus with cervical cancer. CONCLUSION: This study revealed several significant factors predicting screening behaviour in Kazakhstani women. To improve the rate of screening, there is a need to increase public knowledge and awareness of cervical cancer and opportunities for the free screening programme in the female population of Kazakhstan.
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Neoplasias del Cuello Uterino , Adolescente , Adulto , Anciano , Niño , Estudios Transversales , Detección Precoz del Cáncer , Femenino , Conocimientos, Actitudes y Práctica en Salud , Humanos , Kazajstán/epidemiología , Tamizaje Masivo , Persona de Mediana Edad , Prueba de Papanicolaou , Encuestas y Cuestionarios , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/epidemiología , Frotis Vaginal , Adulto JovenRESUMEN
Cervical cancer remains one of the top causes of cancer-related morbidity and mortality all over the world. Currently, however, there are no published studies to assess the knowledge of HPV and cervical cancer in Kazakhstan. This study aimed to assess the awareness of HPV, the knowledge of HPV as a cause of cervical cancer, and the awareness of HPV vaccination among Kazakhstani women visiting gynecological clinics across the country. In addition, the study aimed to identify the factors associated with the awareness of HPV and the HPV vaccine and knowledge of HPV as a major cause of cervical cancer. This was a cross-sectional survey-based study with 2,272 women aged between 18-70 years attending gynecological clinics, who were administered paper-based questionnaires. Data analysis included descriptive statistics consisting of mean values, standard deviations, and frequencies, where applicable. Differences in categorical variables between groups were analyzed using the Chi-square test with a significance value of <0.005. Crude odds ratio (OR) and adjusted odds ratio (AOR) with 95% corresponding confidence intervals were calculated in regression analysis using univariate and multivariable logistic regression models. The mean age of participants was 36.33±10.09 years. More than half (53%) of the participants had been screened for cervical cancer. Among those who were aware of HPV, 46% knew that HPV causes cervical cancer and 52% were aware of the HPV vaccine. The key factors related to outcome variables were age, ethnicity, education, family, number of deliveries, and menarche. From a subgroup analysis, results from the HPV test and Pap smear test were factors related to dependent variables such as awareness of HPV and awareness of HPV vaccination.
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Conocimientos, Actitudes y Práctica en Salud/etnología , Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus/uso terapéutico , Adulto , Anciano , Alphapapillomavirus/patogenicidad , Estudios Transversales , Detección Precoz del Cáncer/métodos , Femenino , Ginecología/educación , Ginecología/métodos , Humanos , Kazajstán , Conocimiento , Persona de Mediana Edad , Prueba de Papanicolaou/métodos , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/virología , Vacunas contra Papillomavirus/administración & dosificación , Vacunas contra Papillomavirus/provisión & distribución , Encuestas y Cuestionarios , Neoplasias del Cuello Uterino/prevención & control , Neoplasias del Cuello Uterino/virología , Frotis Vaginal/métodosRESUMEN
OBJECTIVES: To conduct a nationwide high-risk human papillomavirus (HR-HPV) infection genotyping analysis of women attending gynecological clinics and identify factors associated with HR-HPV infection. METHODS: A cross-sectional survey-based study with 759 participants. Demographics, lifestyle, and medical history data were collected by questionnaire completed by gynecologists during patients' visits. Cervical swabs were used for HPV genotyping using AmpliSens kit. Data analysis included descriptive statistics consisting of mean values, standard deviations, and frequencies, where applicable. Ordinal logistic regression was performed to identify factors associated with HPV infection status. RESULTS: The mean age of participants was 36.51 ± 10.09 years. The majority of participants were aged 26-35 years. Less than half of the women (39%) were HPV positive; 26% had single HR-HPV, and 13% had multiple HR-HPV infection. The most prevalent HR-HPV genotypes were HPV-16 (54%), HPV-51 (7%), HPV-68 (7%), and HPV-18 (6%). Ordinal logistic regression demonstrated that older age, not being single, and having a history of sexually transmitted infections, decrease the odds of HPV infection. CONCLUSION: This study identified high prevalence of HR-HPV among Kazakhstani women. Our results showed that adding HPV testing to compulsory cervical cancer screening in Kazakhstan could improve the screening program and decrease cervical cancer rates.
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Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Adulto , Anciano , Instituciones de Atención Ambulatoria , Estudios Transversales , Detección Precoz del Cáncer , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Papillomaviridae/genética , Infecciones por Papillomavirus/epidemiología , PrevalenciaRESUMEN
Bacillus anthracis contains two megaplasmids, pXO1 and pXO2, that are critical for its pathogenesis. Stable inheritance of pXO1 in B. anthracis is dependent upon the tubulin/FtsZ-like RepX protein encoded by this plasmid. Previously, we have shown that RepX undergoes GTP-dependent polymerization in vitro. However, the polymerization properties and localization pattern of RepX in vivo are not known. Here, we utilize a RepX-green fluorescent protein (GFP) fusion to show that RepX forms foci and three distinct forms of polymeric structures in B. anthracis in vivo, namely straight, curved, and helical filaments. Polymerization of RepX-GFP as well as the nature of polymers formed were dependent upon concentration of the protein inside the B. anthracis cells. RepX predominantly localized as polymers that were parallel to the length of the cell. RepX also formed polymers in Escherichia coli in the absence of other pXO1-encoded products, showing that in vivo polymerization is an inherent property of the protein and does not require either the pXO1 plasmid or proteins unique to B. anthracis. Overexpression of RepX did not affect the cell morphology of B. anthracis cells, whereas it drastically distorted the cell morphology of E. coli host cells. We discuss the significance of our observations in view of the plasmid-specific functions that have been proposed for RepX and related proteins encoded by several megaplasmids found in members of the Bacillus cereus group of bacteria.
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Bacillus anthracis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Plásmidos , Multimerización de Proteína , Factores de Virulencia/metabolismo , Bacillus anthracis/genética , Proteínas Bacterianas/genética , Proteínas del Citoesqueleto/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Virulencia/genéticaRESUMEN
Human papillomaviruses (HPVs) have been implicated in the pathogenesis of a subset of squamous cell carcinoma of the head and neck (SCCHN). The goal of this study was to compare the cellular gene expression profiles of HPV-positive and HPV-negative oropharyngeal carcinomas with those of the normal oral epithelium. Using Affymetrix Human U133A GeneChip, our results showed that 397 genes were differentially expressed in HPV-positive SCCHN compared to the normal oral epithelium. The upregulated genes included those involved in cell cycle regulation (CDKN2A), cell differentiation (SFRP4) and DNA repair (RAD51AP1), while the downregulated genes included those involved in proteolysis (PRSS3). We also found 162 differentially expressed genes in HPV-negative SCCHN compared to the normal oral mucosa. The upregulated genes included those involved in cell proliferation (AKR1C3) and transcription regulation (SNAPC1), while downregulated genes included those involved in apoptosis (CLU) and RNA processing (RBM3). Our studies also identified a subgroup of 59 differentially expressed genes in HPV-positive SCCHN as compared to both HPV-negative SCCHN and normal oral tissues. Such upregulated genes included those involved in nuclear structure and meiosis (SYCP2), DNA repair (RFC5), and transcription regulation (ZNF238). Genes involved in proteolysis (KLK8) and signal transduction (CRABP2) were found to be downregulated in HPV-positive SCCHN. The results of GeneChip experiments were validated by quantitative real-time RT-PCR analysis of a few representative genes. Our results reveal specific gene expression patterns in HPV-positive and HPV-negative oropharyngeal squamous carcinomas that may serve as potential biomarkers for the development of SCCHN.
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Carcinoma de Células Escamosas/genética , Papillomavirus Humano 16/genética , Neoplasias Orofaríngeas/genética , Infecciones por Papillomavirus/genética , Infecciones Tumorales por Virus/genética , Carcinoma de Células Escamosas/virología , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Análisis por Micromatrices , Neoplasias Orofaríngeas/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Infecciones Tumorales por Virus/virologíaRESUMEN
PcrA is an essential helicase in Gram-positive bacteria, but its precise role in cellular DNA metabolism is currently unknown. The Staphylococcus aureus PcrA helicase has both 5'-->3' and 3'-->5' helicase activities. In this work, we have studied the binding of S.aureus PcrA to a variety of DNA substrates that represent intermediates in DNA replication, repair, recombination and transcription. PcrA bound poorly or not at all to single-stranded DNA, double-stranded DNA with blunt ends, partially double-stranded DNA containing fork and bubble structures, and duplex DNA substrates containing either 5' or 3' single-stranded oligo dT tails. Interestingly, PcrA bound with high affinity to partially duplex DNA containing hairpin structures adjacent to a 6 nt long 5' single-stranded region and one unpaired nucleotide (flap) at the 3' end. However, PcrA did not detectably bind to partial duplexes with folded regions adjacent to a 6 nt long 3' single-stranded tail (with or without a 1 nt flap at the 5' end). PcrA showed moderate helicase activity with partially double-stranded DNAs containing 3' or 5' single-stranded oligo dT tails, the 3'-->5' helicase activity being more efficient than its 5'-->3' helicase activity. Interestingly, PcrA showed maximal helicase activity with substrates containing folded structures and 5' single-stranded tails, suggesting that its 5'-->3' helicase activity is greatly stimulated in the presence of specific structures. However, the 3'-->5' helicase activity of PcrA did not appear to be affected by the presence of folded substrates containing 3' single-stranded tails. Our data indicate that PcrA may recognize DNA substrates with specific structures in vivo and its 5'-->3' and 3'-->5' helicase activities may be involved in distinct cellular processes.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , ADN/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , ADN/química , ADN Helicasas/aislamiento & purificación , Conformación de Ácido Nucleico , Especificidad por SustratoRESUMEN
Initiation of DNA replication requires the generation of a primer at the origin of replication that can be utilized by a DNA polymerase for DNA synthesis. This can be accomplished by several means, including the synthesis of an RNA primer by a DNA primase or RNA polymerase, by nicking of one strand of the DNA to generate a free 3'-OH end that can be used as a primer, and by the utilization of the OH group present in an amino acid such as serine within an initiation protein as a primer. Furthermore, some single-stranded DNA genomes can utilize a snap-back 3'-OH end generated due to self-complementarity as a primer for DNA replication. The different modes of initiation require the generation of highly organized DNA-protein complexes at the origin that trigger the initiation of replication. A large majority of small, multicopy plasmids of Gram-positive bacteria and some of Gram-negative bacteria replicate by a rolling-circle (RC) mechanism (for previous reviews, see Refs.). More than 200 rolling-circle replicating (RCR) plasmids have so far been identified and, based on sequence homologies in their replication regions, can be grouped into approximately seven families (Refs., and http://www.essex.ac.uk/bs/staff/osborn/DPR-home.htm). This review will focus on plasmids of the pT181 family that replicate by an RC mechanism. So far, approximately 25 plasmids have been identified as belonging to this family based on the sequence homology in their double-strand origins (dsos) and the genes encoding the initiator (Rep) proteins. This review will highlight our current understanding of the structural features of the origins of replication, and the DNA-protein and protein-protein interactions that result in the generation of a replication-initiation complex that triggers replication. It will discuss the molecular events that result in the precise termination of replication once the leading-strand DNA synthesis has been completed. This review will also discuss the various biochemical activities of the initiator proteins encoded by the plasmids of the pT181 family and the mechanism of inactivation of the Rep activity after supporting one round of leading-strand replication. Finally, the review will outline the mechanism of replication of the lagging strand of the pT181 plasmid as well as the limited information that is available on the role of host proteins in pT181 leading- and lagging-strand replication.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Plásmidos/biosíntesis , Plásmidos/metabolismo , Origen de Réplica/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión al ADN/química , Origen de Réplica/genéticaRESUMEN
In the United States, penicillin-resistant variants of the Tennessee (Tenn) (23 F)-4 clone account for a substantial proportion of the very-high-level penicillin-resistant (MIC 8 microg/ml) infections in the 7-valent pneumococcal protein conjugate vaccine (PCV 7) era. Serotype 19 A strains account for an increasing proportion of penicillin-nonsusceptible Streptococcus pneumoniae infections. Sequential transformations of the Tenn (23 F)-4 clone (penicillin MIC 0.1 microg/ml) were performed with four penicillin-nonsusceptible serotype 19 A international clones (penicillin MIC): S. Africa (19 A)-7 (0.5 microg/ml), Hungary (19 A)-6 (2 microg/ml), Slovakia (19 A)-11 (8 microg/ml), and South Africa (19 A)-13 (8 microg/ml). Fifty-two transformants were characterized by MICs, serogroup-specific PCR, pbp PCR restriction profile and sequence, psp A PCR restriction profile, and erm/mef PCR. A subset was analyzed with multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. Serotype 23 F transformants with penicillin MIC >or= 8 microg/ml were detected through a single transformation with the Hungary (19 A)-6 clone or serial transformations using two to three different clones. Forty-four percent (14/32) of the transformants incorporated >or=1 new MLST allele. Using encapsulated donors, very-high-level penicillin resistant variants of the Tenn (23 F)-4 clone were detected. In addition to detecting stepwise increases in penicillin MIC, a 12-fold increase in penicillin MIC was achieved through a single transformation. This large increase in MIC may explain why this clone is commonly associated with very-high-level resistance in natural populations. Recombination within the MLST housekeeping genes was commonly detected in the transformants that had acquired penicillin resistance.