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Kinds of DNA Terms modified by DNA Selected AbstractsDetection of bacterial DNA by PCR and reverse hybridization in the 16S rRNA gene with particular reference to neonatal septicemiaACTA PAEDIATRICA, Issue 2 2001S Shang Aim: The clinical diagnosis of sepsis is difficult, particularly in neonates. It is necessary to develop a rapid and reliable method for detecting bacteria in blood and cerebrospinal fluid (CSF) Polymerase chain reaction (PCR) and reverse hybridization of the 16S rRNA gene would permit fast and sensitive determination of the presence of bacteria and differentiate gram-positive bacteria from gram-negative ones in clinical specimens. Methods: We developed a pair of primers according to the gene encoding 16SrRNA found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with PCR, and with reverse hybridization using a universal bacterial probe, a gram-positive probe and a gram-negative probe. Results: A 371 bp DNA fragment was amplified from 20 different bacterial species. No signal was observed when human DNA and viruses were used as templates. The sensitivity could be improved to 10T -12 g. All 26 culture-positive clinical samples (22 blood samples and 4 CSF samples) were positive with PCR. The gram-negative and gram-positive probes hybridized to clinical samples and to known bacterial controls, as predicted by Gram's stain characteristics. Conclusions: Our results suggest that the method of PCR and reverse hybridization is rapid, sensitive and specific in detecting bacterial infections. This finding may be significant in the clinical diagnosis of sepsis in neonates. [source] Perinatal development of the rat kidney: Apoptosis and epidermal growth factorCONGENITAL ANOMALIES, Issue 3 2003Toshiya Okada ABSTRACT, Localization of apoptotic cells in the kidney of perinatal rats was examined by the terminal deoxynucleotidyl transferase,mediated d,UTP,biotin nick end labeling (TUNEL) method and electron microscopy. Perinatal changes in the percentage of kidney cells with DNA fragmentation were determined by flow cytometric analysis. Through observation of two successive sections, the relationship between the localization of the epidermal growth factor receptor (EGFR) positive cells and TUNEL positive cells in the kidney was determined. From fetal day 18 to neonatal day 5, TUNEL positive cells were noted in immature glomeruli, collecting ducts and interstitium. Electron microscopically, chromatin condensed nuclei and apoptotic bodies were seen in the same tissue component as the TUNEL positive cells. The percentage of DNA fragmented cells significantly increased from fetal days 18 to 20 and significantly decreased from fetal days 20 to 22, while they still remained low in the neonatal period. The TUNEL positive cells in immature glomeruli and collecting ducts were not reactive to the EGFR antibody. The TUNEL positive cells were not observed in the proximal tubular cells, which were positive to EGFR antibody. These results indicate that apoptotic cells are present in the kidney throughout the perinatal period in the rat and that EGF plays an important role in perinatal development of the rat kidney. [source] Amplifying Nuclear and Mitochondrial DNA from African Elephant Ivory: a Tool for Monitoring the Ivory TradeCONSERVATION BIOLOGY, Issue 6 2003KENINE E. COMSTOCK cacería furtiva; elefante africano; Loxodonta africana; marfil; microsatélites Abstract: The ability to extract DNA from ivory provides the basis for genetically tracking the origin of poached ivory and thus has important implications for elephant conservation and management. We describe a method to isolate and amplify both genomic and mitochondrial DNA from African elephant ivory that requires very small amounts of ivory taken from any location on the tusk. We pulverized ivory and isolated DNA with a modified QIAamp kit. Ivory as old as 10 to 20 years, stored at ambient conditions, was amenable to DNA isolation with this method. The isolated DNA was robustly amplified at 16 elephant microsatellite loci and two mitochondrial DNA loci. This method has important applications for the forensic analysis of poached African elephant ivory. It enables determination of where stronger antipoaching efforts are needed and provides the basis for monitoring the extent of the trade as well as the consequences of future international trade decisions. Resumen: La habilidad para extraer ADN del marfil proporciona la base para rastrear genéticamente el origen de marfil furtivo y por tanto tiene implicaciones importantes para la conservación y el manejo de elefantes. Describimos un método para aislar y amplificar ADN genómico y mitocondrial de marfil de elefante africano que requiere de cantidades muy pequeñas de marfil tomadas de cualquier parte del colmillo. Pulverizamos el marfil y aislamos el ADN con un equipo QIAamp modificado. Con este método, fue posible aislar el ADN de marfil de 10 a 20 años, conservado en condiciones ambientales. El ADN aislado fue amplificado robustamente en 16 loci microsatélite y dos loci de ADN mitocondrial. Este método tiene aplicaciones importantes para el análisis forense de marfil de elefantes africanos cazados furtivamente. Permite la identificación de sitios donde se requieren mayores esfuerzos para combatir la cacería furtiva y proporciona la base para monitorear la extensión del comercio así como las consecuencias de decisiones futuras de comercio internacional. [source] Genetic Diversity and Tests of the Hybrid Origin of the Endangered Yellow LarkspurCONSERVATION BIOLOGY, Issue 6 2001Jason A. Koontz The total number of individuals in these two populations is estimated to be <100. We used allozyme and random amplified polymorphic DNA ( RAPD) markers to (1) assess levels and patterns of genetic diversity in one wild population and two cultivated populations and (2) test the hypothesis that D. luteum is of hybrid origin between D. decorum and D. nudicaule. These data will be used to aid in developing a management plan to conserve the species. The wild population maintains high levels of genetic diversity. Genetic data indicate that both cultivated populations, especially the north Sonoma population, have several allozymes and RAPD markers not found in the wild population and could be used to establish new populations of D. luteum or to enhance the diversity and size of the wild population. The allozyme data did not reveal any fixed differences between D. decorum and D. nudicaule, although allele frequencies of the putative parental populations differed. At these loci, D. luteum resembled D. nudicaule more than D. decorum . Many unique RAPD markers distinguish each of the three species. The diagnostic markers from populations of D. nudicaule and D. decorum were not additive in the putative hybrid, and these data indicate that D. luteum is not of recent hybrid origin. Conservation of the yellow larkspur should include strategies that use the cultivated populations of D. luteum, but hybridizing D. decorum and D. nudicaule to "recreate"D. luteum is not recommended. Resumen:Delphidium luteum ( Ranunculaceae), un delfinio en peligro de extinción, está restringido a dos poblaciones silvestres cerca de Bodega Bay, California. Se estima que el total de individuos en estas dos poblaciones es de <100. Utilizamos marcadores de alozimas y RAPD para (1) evaluar los niveles y patrones de diversidad genética en una población silvestre y dos poblaciones cultivadas y (2) probar la hipótesis de que D. luteum es de origen híbrido entre D. decorum y D. nudicaule. Estos datos serán utilizados para ayudar a desarrollar un plan de manejo para conservar la especie. La población silvestre mantiene altos niveles de diversidad genética. Los datos genéticos indican que ambas poblaciones cultivadas, especialmente en la población de Sonoma norte, tienen varias alozimas y marcadores RAPD que no se encuentran en poblaciones silvestres y podrían utilizarse para establecer nuevas poblaciones de D. luteum o reforzar la diversidad y tamaño de la población silvestre. Los datos de alozimas no revelaron diferencias fijas entre D. decorum y D. nudicaule, aunque las frecuencias alélicas de las poblaciones parentales putativas fueron diferentes. En estos loci, D. luteum fue más semejante a D. nudicaule que a D. decorum. Muchos marcadores RADP únicos distinguen a cada una de las tres especies. Los marcadores diagnóstico de poblaciones de D. decorum y D. nudicaule no fueron aditivos en el híbrido putativo, y estos datos indican que D. luteum no es de origen híbrido reciente. La conservación del delfinio amarillo debería incluir estrategias que usen las poblaciones cultivadas de D. luteum; sin embargo, no se recomienda la hibridación de D. decorum y D. nudicaule para "recrear" a D. luteum. [source] Phylogenetic Reanalysis of the Saudi Gazelle and Its Implications for ConservationCONSERVATION BIOLOGY, Issue 4 2001Robert L. Hammond The Saudi gazelle ( Gazella saudiya) was endemic to the Arabian peninsula but is now considered extinct in the wild and is potentially a candidate for captive breeding and reintroduction. Using 375 base pairs of mitochondrial DNA (mtDNA) cytochrome b gene derived from museum samples collected from the wild prior to the presumed extinction of this species, we show that G. saudiya is the sister taxon of the African dorcas gazelle ( G. dorcas). Reciprocal monophyly of G. saudiya mtDNA haplotypes with G. dorcas, coupled with morphological distinctiveness, suggests that it is an evolutionarily significant unit. These data indicate that captive populations identified previously as potential sources of G. saudiya for captive breeding appear incorrectly designated and are irrelevant to the conservation of G. saudiya. The polymerase chain reaction,restriction fragment length polymorphism ( PCR-RFLP) analysis of several private collections of living gazelles in Saudi Arabia provides no evidence for the survival of G. saudiya. We recommend that field surveys be undertaken to establish whether G. saudiya is indeed extinct in the wild and that other private collections within the Arabian peninsula be screened genetically. We urge caution when captive animals of unknown provenance are used to investigate the phylogenetics of cryptic species groups. Resumen: La identificación de poblaciones taxonómicamente apropiadas de especies en peligro para programas de reproducción en cautiverio y de reintroducción es fundamental para su éxito. La Gacela Saudi (Gazella saudiya) fue endémica a la península de Arabia pero ahora está considerada como extinta en su medio y es un candidato potencial para reproducción en cautiverio y reintroducción. Utilizando 375 pares de bases de ADN mitocondrial (ADNmt) del gene citocromo b derivados de muestras de museos colectadas en el medio silvestre antes de la extinción de la especie, mostramos que G. saudiya es el taxón hermano de la gacela dorcas africana (G. dorcas). La monofilia recíproca de haplotipos de ADNmt de G. saudiya con G. dorcas, aunado a diferencias morfológicas, sugiere que es una unidad evolutiva significativa. Estos datos indican que las poblaciones cautivas identificadas previamente como fuente potencial de G. saudiya para reproducción en cautiverio están incorrectamente identificadas y son irrelevantes para la conservación de G. saudiya. El análisis PCR-RFLP de varias colecciones privadas de gacelas vivas en Arabia Saudita no proporcionan evidencia para la supervivencia de G. saudiya. Recomendamos que se realicen muestreos en el campo para establecer si en efecto G. saudiya está extinta en su hábitat y que se examinen genéticamente las otras colecciones privadas en la península Arábiga. Recomendamos precaución cuando animales cautivos de origen desconocido son utilizados para investigar la filogenia de grupos de especies crípticas. [source] Cells meeting our immunophenotypic criteria of endothelial cells are large plateletsCYTOMETRY, Issue 2 2007Michiel H. Strijbos Abstract Background Circulating endothelial cells (CEC) are shed from damaged vasculature, making them a rational choice to serve as surrogate marker for vascular damage. Currently, various techniques and CEC definitions are in use, and their standardization and validation is needed. A flow cytometric single platform assay defining CEC as forward light scatter (FSC)low-to-intermedate, sideward light scatter (SSC)low, CD45,, CD31++ and CD146+ is a promising approach to enumerate CEC because of its simplicity (Mancuso et al., Blood 2001;97:3658,3661). Here, we set out to confirm the endothelial nature of these cells. Methods We isolated cells with a FSClow-to-intermediate, SSClow, CD31++, CD45dim immunophenotype (termed "cells meeting our immunophenotypic criteria for endothelial cells" [CMOIC]) from healthy donors to study the expression of endothelium-associated markers using several techniques. Special attention was paid to reagents identifying the endothelial cell-specific marker CD146. We compared antigen expression patterns of CMOIC with those of the HUVEC endothelial cell line and lymphocytes. Electron microscopy was used to detect the presence of endothelial cell-specific Weibel,Palade bodies in the sorted cells. Results CD146 expression was negative on CMOIC for all tested CD146 mAbs, but positive on HUVEC cells and a minor subset of T lymphocytes. Using flow cytometry, we found no expression of any endothelium-associated marker except for CD31 and CD34. HUVEC cells were positive for all endothelial markers except for CD34. Evaluation of CMOIC morphology showed a homogenous population of cells with a highly irregular nucleus-like structure and positive endothelial immunohistochemistry. CMOIC contained neither nuclei nor DNA. Electron microscopy revealed the absence of a nucleus, the absence of endothelial specific Weibel,Palade bodies, and revealed CMOIC to be large platelets. Conclusion The vast majority of cells with the immunophenotype FSClow-to-intermediate, SSClow, CD45,, CD31++ do not express CD146 and are large platelets rather than endothelial cells. © 2007 Clinical Cytometry Society. [source] Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipientsCYTOPATHOLOGY, Issue 6 2008M. Koukoulaki Objective:, BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients. Patients and methods:, We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood. Results:, Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR. Conclusions:, Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation. [source] Trials update in walesCYTOPATHOLOGY, Issue 2007A. Fiander Three ongoing studies will be presented and discussed. Prevalence of Human Papillomavirus Infection in a South Wales Screening population Methods: A total of 10 000 consecutive, anonymous liquid based cytology screening samples were collected over a five month period in 2004. Age, cytology result and social deprivation score was provided for each specimen. The methodology was chosen to ensure inclusion of all women attending routine cervical screening, avoiding potential constraints associated with obtaining individual informed consent. The liquid based cytology samples were processed and reported by the receiving cytology laboratory and the residual specimens sent to the HPV Research Laboratory, Wales College of Medicine, where they were processed and stored at -80°C until analysis. High risk and low risk HPV Typing was undertaken using PCR , EIA (Jacobs et al 1997). Full high risk typing was performed on HPV positive specimens. Results: The study population had a mean age of 38 years with 92% negative, 5% borderline and 3% dyskaryotic cytology. The average social deprivation score was 17.4 (based upon the Welsh Index of multiple deprivation). The following results will be presented: HPV prevalence by age. HPV prevalence by cytology result. Type specific HPV prevalence in single and multiple infection. Conclusion: This study represents the largest type specific HPV Prevalence Study in the UK to date. As such it will form a useful base line against which to access performance of marketed HPV tests and evaluating the impact following implementation of HPV vaccination. [Funded by Welsh Office for Research and Development] CRISP , 1 Study (Cervical Randomized Intervention Study Protocol -1) Background: Indole-3-carbinol (I3C) and Diindolylmethane (DIM) are found in cruciferous vegetables and have been identified as compounds that could potentially prevent or halt carcinogenesis. I3C spontaneously forms DIM in vivo during acid digestion. I3C has been shown to prevent the development of cervical cancer in HPV 16 transgenic mice and both I3C and DIM have been shown to promote cell death in cervical cancer cell models. DIM is the major active bi-product of I3C and preliminary data indicate that DIM is active in cervical dysplasia and may be better tolerated than I3C. Aim: To investigate chemoprevention of high grade cervical neoplasia using Diindolylmethane (DIM) supplementation in women with low grade cytological abnormalities on cervical cytology. Objectives: To observe any reduction in the prevalence of histological proven high-grade cervical intraepithelial neoplasia (CIN) after 6 months of supplementation. ,,To observe any reduction in the prevalence of cytological abnormalities. ,,To observe any changes in the clinical appearance of the cervix. To assess acceptability and monitor any side effects of DIM supplementation. ,,To assess whether any benefit is seen in relation to Human Papillomavirus (HPV) status including HPV Type, Viral load and integration. Methods: This is a double blind randomized placebo-controlled trial involving 600,700 women with low grade cytological abnormalities on a cervical smear. Randomization is in the ratio of 2 : 1 in favour of active medication. Women with first mildly dyskaryotic smear or second borderline smear are eligible. They are asked to take two capsules daily for 6 months. At the end of 6 months they undergo repeat cervical cytology, HPV testing and colposcopy. Results: A progress report will be given for this ongoing study. [Funded: - Cancer Research UK] Type Specific HPV Infection in Welsh Cervical Cancers Background: Whilst there have been numerous studies of HPV infection associated with cervical cancer and on prevalence of Human Papillomavirus in diverse populations there have been no studies of these variables in the same population. Against a background of prophylactic HPV vaccination it is important to assess potential protection against cervical cancer within a given population. The most comprehensive analysis of HPV type specific cervical cancer is a meta-analysis published by the IARC in 2003. This however included only three UK based studies, totalling 118 cases, 75 of which were only investigated by HPV type PCR for four high risk types. None of this data was presented with associated population based prevalence data. Therefore, the research objectives for this study in combination with the first study above, are as follows: To determine the frequency of specific HPV types in cervical cancers in Wales. To compare the distribution of specific HPV types amongst cervical cancers with their prevalence in the general population. This will allow accurate delineation of the relationship between prevalence of specific HPV types in the general population and their association with clinically relevant disease. This information is a pre-requisite to assess the potential impact of prophylactic vaccination against HPV infection in Wales. Methods: Welsh Cervical Cancer specimens from 2000,2005 will be identified from pathology departments within Wales. The pathology of each tumour will be reviewed by a single Gynaecological Pathologist. The age of the patient and pathological features of the tumour will be noted. DNA will be extracted from the paraffin sections and HPV typed by PCR-EIA. Results: A progress report will be given for this ongoing study. [Funded by Welsh Office for Research and Development] [source] Significance of high-risk human papillomavirus detection by polymerase chain reaction in primary cervical cancer screeningCYTOPATHOLOGY, Issue 2 2001Y. L. Oh Significance of high-risk human papillomavirus detection by polymerase chain reaction in primary cervical cancer screening The purposes of this study were to evaluate the incidence of high-risk human papillomavirus (HPV) infection by polymerase chain reaction (PCR) and to assess its diagnostic usefulness in primary cervical screening. PCR testing for HPV type 16, 18, 31 and 33 was performed on 1305 specimens obtained during routine cervical cancer screening. We analysed the concurrent cervical smears and biopsy, and correlated them with the HPV infection status. We also evaluated histologically-proven cases with ASCUS smears according to HPV infection. HPV DNA was identified in eight (0.7%) of 1144 cytologically normal patients; nine (10.5%) of 86 ASCUS; seven (25.0%) of 28 LSIL; 26 (78.8%) of 33 HSIL; and in all of three squamous cell carcinomas (SCC). HPV positivity was significantly associated with cytohistological diagnosis for HSIL of more. In addition, HPV-positive ASCUS cases were found to be associated with histological abnormality rather than HPV-negative. The results indicate that high-risk HPV testing by PCR could be a useful adjunct tool for Pap smear in primary cervical screening. The combination of Pap smear and high-risk HPV testing by PCR might reduce unnecessary colposcopy-guided biopsy of women with cytological diagnosis of ASCUS. [source] Actin on DNA,An ancient and dynamic relationship,CYTOSKELETON, Issue 8 2010Kari-Pekka Skarp Abstract In the cytoplasm of eukaryotic cells the coordinated assembly of actin filaments drives essential cell biological processes, such as cell migration. The discovery of prokaryotic actin homologues, as well as the appreciation of the existence of nuclear actin, have expanded the scope by which the actin family is utilized in different cell types. In bacteria, actin has been implicated in DNA movement tasks, while the connection with the RNA polymerase machinery appears to exist in both prokaryotes and eukaryotes. Within the nucleus, actin has further been shown to play a role in chromatin remodeling and RNA processing, possibly acting to link these to transcription, thereby facilitating the gene expression process. The molecular mechanism by which actin exerts these newly discovered functions is still unclear, because while polymer formation seems to be required in bacteria, these species lack conventional actin-binding proteins to regulate the process. Furthermore, although the nucleus contains a plethora of actin-regulating factors, the polymerization status of actin within this compartment still remains unclear. General theme, however, seems to be actin's ability to interact with numerous binding partners. A common feature to the novel modes of actin utilization is the connection between actin and DNA, and here we aim to review the recent literature to explore how this connection is exploited in different contexts. [source] Bacteria of asymptomatic periradicular endodontic lesions identified by DNA-DNA hybridizationDENTAL TRAUMATOLOGY, Issue 5 2000J. J. Gatti Abstract , Possible inclusion of contaminant bacteria during surgery has been problematic in studies of periradicular lesions of endodontic origin. Therefore, in this study, two different surgical techniques were compared. A second problem is that some difficult to cultivate species may not be detected using bacteriological methods. Molecular techniques may resolve this problem. DNA-DNA hybridization technology has the additional advantage that DNA is not amplified. The purpose of this investigation was to determine if bacteria from periradicular endodontic lesions could be identified using DNA-DNA hybridization. A full thickness intrasulcular mucoperiosteal (IS) flap (n=20) or a submarginal (SM) flap (n=16) was reflected in patients with asymptomatic apical periodontitis. DNA was extracted and incubated with 40 digoxigenin-labeled whole genomic probes. Bacterial DNA was detected in all 36 lesions. Seven probes were negative for all lesions. In patients with sinus tract communication, in teeth lacking intact full coverage crowns, and in patients with a history of trauma, 4,13 probes provided positive signals. Seven probes were positive in lesions obtained by the IS, but not the SM technique. Two probes were in samples obtained with the SM technique, but not the IS. Only Bacteroides forsythus and Actinomyces naeslundii genospecies 2 were present in large numbers using either the IS or the SM technique. The SM flap technique, in combination with DNA-DNA hybridization, appeared to provide excellent data pertaining to periradicular bacteria. These results supported other studies that provide evidence of a bacterial presence and persistence in periradicular lesions. [source] Genetics of anxiety disorders: the complex road from DSM to DNA,DEPRESSION AND ANXIETY, Issue 11 2009Jordan W. Smoller M.D. Sc.D. Abstract Anxiety disorders are among the most common psychiatric disorders, affecting one in four individuals over a lifetime. Although our understanding of the etiology of these disorders is incomplete, familial and genetic factors are established risk factors. However, identifying the specific casual genes has been difficult. Within the past several years, advances in molecular and statistical genetic methods have made the genetic dissection of complex disorders a feasible project. Here we provide an overview of these developments, with a focus on their implications for genetic studies of anxiety disorders. Although the genetic and phenotypic complexity of the anxiety disorders present formidable challenges, advances in neuroimaging and experimental animal models of anxiety and fear offer important opportunities for discovery. Real progress in identifying the genetic basis of anxiety disorders will require integrative approaches that make use of these biologic tools as well as larger-scale genomic studies. If successful, such efforts may yield novel and more effective approaches for the prevention and treatment of these common and costly disorders. Depression and Anxiety, 2009. © 2009 Wiley-Liss, Inc. [source] Genetic variation in D7S1875 repeat polymorphism of leptin gene is associated with increased risk for depression: a case-control study from IndiaDEPRESSION AND ANXIETY, Issue 9 2009Manav Kapoor M.Sc. Abstract Background: Epidemiologic data suggest an association between obesity and depression, however findings vary considerably across different studies. Both depression and obesity are disabling disorders associated with loss over appetite control, influenced by genetic and environmental factors and are risk factors for diseases like hypertension, cardiovascular disorders, etc. This study attempts to establish a link between the symptoms of depression, metabolic disorders, and obesity, to unravel the underlying association/s. Methods: This exploratory case,control study comprises 133 clinically diagnosed depressed individuals and 136 age matched controls. DNA from all 269 subjects was genotyped for D7S1875 repeat polymorphism in the promoter region of Leptin (LEP) gene using polymerase chain reaction. Results: Frequency of the shorter allele of D7S1875 (<208,bp) was 0.73 in the depressive group versus 0.67 in the control group (P=.01). Cases homozygous for D7S1875,208,bp alleles had significantly higher value of systolic (130 versus 122; P<.009) and diastolic (85.4 versus 81; P=.01) blood pressure (SBP and DBP) than the individuals homozygous for<208,bp allele. A similar trend was observed for SBP (127.8 versus 123.6; P=.03) among controls homozygous for the longer or the shorter allele. Thus, the LEP gene appears to be an important genetic determinant for susceptibility to depression in the Indian population (OR=1.4913, 95% CI=1.0334,2.1522; P=.04). Conclusions: Our findings suggest that LEP gene variants could be related to depression and associated co-morbidities such as hypertension. Depression and Anxiety, 2009. © 2009 Wiley-Liss, Inc. [source] Tanning and Cutaneous MalignancyDERMATOLOGIC SURGERY, Issue 4 2008SHERRIF F. IBRAHIM MD BACKGROUND Exposure to ultraviolet radiation (UVR) results in a darkening of the skin known as tanning. Recently, it has been shown that tanning is a response to UVR-induced DNA damage and represents the skin's efforts to protect itself against further injury. Despite the link between UVR and cutaneous malignancy, people continue to pursue tanning from natural and artificial sources. This trend is reflected in the exponential rise in skin cancer incidence. OBJECTIVE The objective of this study was to review our current understanding of the factors controlling the tanning response and the relationship to cutaneous carcinogenesis, as well as the impact that the multibillion dollar tanning industry has had on the practice of dermatology. MATERIALS AND METHODS Extensive literature review was conducted in subjects related to tanning and the relationship to cutaneous malignancy. RESULTS Our knowledge of tanning and its effects on the skin has increased tremendously. It is clear that tanning contributes to the development of skin cancer. Despite this information, the incidence of skin cancer continues to increase exponentially. CONCLUSIONS Skin cancer poses a major public health concern and tanning remains the most modifiable risk factor in its etiology. Social, economic, and legislative issues have become tightly intertwined with the complex nature of human behavior in the continued pursuit of an activity that clearly has detrimental effects on one's health. [source] Human Papillomavirus and Overexpression of P16INK4a in Nonmelanoma Skin CancerDERMATOLOGIC SURGERY, Issue 3 2004Ingo Nindl PhD Background. P16INK4a overexpression has been identified as a specific biomarker in high-risk human papillomavirus (HPV),infected cervical (pre)cancer lesions. Objective. To evaluate the overexpression of this cyclin-dependent kinase inhibitor in skin tumors depending on HPV infections, we analyzed normal skin, benign skin disease, and skin cancer specimens. Methods. Biopsies of 23 patients with normal histology (3), psoriasis (2), verrucae vulgaris (2), actinic keratoses (5), squamous cell carcinoma (SCC) in situ (3), Bowen's carcinoma (1), and SCC (7) were analyzed. Specimens of 23 patients were immunostained using the monoclonal antibody E6H4 specific for p16INK4a. HPV status was assessed by a polymerase chain reaction (PCR) system to detect all currently known HPV types. MY (MY09/MY11 and MYN9/MYN10)-, CP (CP65/CP70 and CP66/CP69)-nested PCR, and three single PCR methods CN1, CN3, and CN4 were used in a first step, and HPV typing was performed by restriction fragment length polymorphism analysis. Only ,-globin,positive patients were included in this study. Results. HPV DNA was detected in all actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC, in 50% (one of two) of verrucae vulgaris, in 66% (two of three) of normal skin, and in none of two psoriasis. P16INK4a expression was not detected in normal skin, psoriasis, and verrucae vulgares. Overexpression of p16INK4a was detected in a subset of dysplastic cells (10% to 80%) of all skin (pre)cancer lesions such as actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC infected with HPV independent of sun exposure. Conclusion. P16INK4a appears to be overexpressed in a portion of dysplastic cells from actinic keratoses and SCC. Further studies to examine the association of HPV infection and the overexpression of p16INK4a are warranted. [source] Keratoacanthoma: A Clinico-Pathologic EnigmaDERMATOLOGIC SURGERY, Issue 2004Robert A. Schwartz MD Background. Keratoacanthoma (KA) is an extraordinary entity. Once considered a benign neoplasm that resembled a highly malignant one (pseudomalignancy), it is now viewed in an opposite light as a cancer that resembles a benign neoplasm (pseudobenignity). Objective. The goal was to delineate the malignant potential of this neoplasm based on the author's experience and a review of recent data and research and to emphasize the KA as a possible part of an autosomal dominant familial cancer syndrome, the Muir,Torre syndrome. Methods. This is a review of the literature. Results. In this work, the KA is reviewed with recent advances emphasized. Conclusion. KA is an abortive malignancy that rarely progresses into an invasive SCC. The KA may serve as a marker for the important autosomal dominant familial cancer syndrome, the Muir,Torre syndrome, as a result of a defective DNA mismatch repair gene. [source] Mechanism of DNA replication-dependent transcriptional activation of the acetylcholinesterase gene in the Ciona intestinalis embryoDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2009Yumiko Kataoka The acetylcholinesterase-encoding gene in the ascidian Ciona intestinalis (Ci-AChE) is expressed in tail muscle cells from the gastrula stage. When the embryo was continuously treated with aphidicolin from the 32-cell stage, Ci-AChE was not expressed even when control embryos reached the tailbud stage. This result suggests that Ci-AChE acquires the competence to be transcribed after passing through a certain number of DNA replication cycles. A lacZ reporter gene containing the 5, flanking region of Ci-AChE was expressed in the tail muscle cells. Aphidicolin treatment from the 32-cell stage affected, but did not completely suppress, the expression of lacZ. A bisulfite sequencing analysis was carried out to examine the methylation status of four regions within the 5, flanking sequence and the first exon. However, all of these regions remained unmethylated from the 16-cell to 110-cell stages. The results suggested that the DNA of the Ci-AChE locus is not responsible for counting the rounds of replication. We examined the expression of the C. intestinalis MyoD (Ci-MyoD), a transcription factor that activates Ci-AChE. Aphidicolin treatment from the 32-cell stage suppressed the expression of Ci-MyoD, even when control embryos reached the gastrula stage. These results suggest that a lack of Ci-MyoD is critical to the suppression of Ci-AChE in aphidicolin-treated embryos. [source] Epigenetic regulation of the imprinted U2af1-rs1 gene during retinoic acid-induced differentiation of embryonic stem cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2006Noelia Andollo Epigenetic modifications such as DNA methylation and changes in chromatin structure are changes in the chemical composition or structure of DNA that work by regulating gene expression. Their mechanisms of action have been generally studied in imprinted genes. The present work analyzes the involvement of these mechanisms in the expression of the U2af1-rs1 imprinted gene during the differentiation process of embryonic stem (ES) cells induced by retinoic acid. By DNA digestion with methylation-dependent or independent restriction enzymes and consecutive Southern blot, we have found that methylation of the U2af1-rs1 gene increases in differentiated ES cells and in embryoid bodies. However, northern blot and real-time reverse transcription,polymerase chain reaction analysis showed a higher expression of the U2af1-rs1 gene in differentiated ES cells and in embryoid bodies than in undifferentiated ones. On the other hand, the sensitivity to DNase-I assay demonstrated an open chromatin conformation for differentiated cells with regard to undifferentiated ES cells. Our results suggest that the expression of the U2af1-rs1 gene would be regulated by changes in chromatin structure rather than by DNA methylation during the RA-induced process of differentiation of ES cells. [source] Bioenergetics and the epigenome: Interface between the environment and genes in common diseasesDEVELOPMENTAL DISABILITIES RESEARCH REVIEW, Issue 2 2010Douglas C. Wallace Abstract Extensive efforts have been directed at using genome-wide association studies (GWAS) to identify the genes responsible for common metabolic and degenerative diseases, cancer, and aging, but with limited success. While environmental factors have been evoked to explain this conundrum, the nature of these environmental factors remains unexplained. The availability of and demands for energy constitute one of the most important aspects of the environment. The flow of energy through the cell is primarily mediated by the mitochondrion, which oxidizes reducing equivalents from hydrocarbons via acetyl-CoA, NADH + H+, and FADH2 to generate ATP through oxidative phosphorylation (OXPHOS). The mitochondrial genome encompasses hundreds of nuclear DNA (nDNA)-encoded genes plus 37 mitochondrial DNA (mtDNA)-encoded genes. Although the mtDNA has a high mutation rate, only milder, potentially adaptive mutations are introduced into the population through female oocytes. In contrast, nDNA-encoded bioenergetic genes have a low mutation rate. However, their expression is modulated by histone phosphorylation and acetylation using mitochondrially-generated ATP and acetyl-CoA, which permits increased gene expression, growth, and reproduction when calories are abundant. Phosphorylation, acetylaton, and cellular redox state also regulate most signal transduction pathways and activities of multiple transcription factors. Thus, mtDNA mutations provide heritable and stable adaptation to regional differences while mitochondrially-mediated changes in the epigenome permit reversible modulation of gene expression in response to fluctuations in the energy environment. The most common genomic changes that interface with the environment and cause complex disease must, therefore, be mitochondrial and epigenomic in origin. © 2010 Wiley-Liss, Inc. Dev Disabil Res Rev 2010;16:114,119. [source] Genetic basis of rett syndromeDEVELOPMENTAL DISABILITIES RESEARCH REVIEW, Issue 2 2002Ignatia B. Van den Veyver Abstract The origin of Rett syndrome has long been debated, but several observations have suggested an X-linked dominant inheritance pattern. We and others have pursued an exclusion-mapping strategy using DNA from a small number of familial Rett syndrome cases. This work resulted in the narrowing of the region likely to harbor the mutated gene to Xq27.3-Xqter. After systematic exclusion of several candidate genes, we discovered mutations in MECP2, the gene that encodes the transcriptional repressor, methyl-CpG-binding protein 2. Since then, nonsense, missense, or frameshift mutations have been found in at least 80% of girls affected with classic Rett syndrome. Sixty-four percent of mutations are recurrent C > T transitions at eight CpG dinucleotides mutation hotspots, while the C-terminal region of the gene is prone to recurrent multinucleotide deletions (11%). Most mutations are predicted to result in total or partial loss of function of MeCP2. There is no clear correlation between the type and position of the mutation and the phenotypic features of classic and variant Rett syndrome patients, and XCI appears to be a major determinant of phenotypic severity. Further research focuses on the pathogenic consequences of these mutations along the hypothesis of loss of transcriptional repression of a small number of genes that are essential for neuronal function in the maturing brain. MRDD Research Reviews 2002;8:82,86. © 2002 Wiley-Liss, Inc. [source] Cytoplasmic localization of oocyte-specific variant of porcine DNA methyltransferase-1 during early developmentDEVELOPMENTAL DYNAMICS, Issue 7 2009Young Sun Jeong Abstract DNA methyltransferase-1 (Dnmt1) is involved in the maintenance of genomic methylation patterns. Rather than full-length Dnmt1, mouse oocytes have a truncated variant called Dnmt1o. Immunofluorescence data showed that Dnmt1o localized to the cytoplasm, but this has not been confirmed using more direct methods. The cytoplasmic localization of Dnmt1o has been assigned to the main cause of global DNA demethylation in early mouse embryos. We studied localization of Dnmt1o in mouse and pig embryos. We identified pig Dnmt1o protein and its transcript with unique 5,-end sequence. Physically separating mouse and pig 2-cell embryos into their nuclear and cytoplasmic components demonstrated that Dnmt1o of both species localized to the cytoplasm. Cloned pig embryos had Dnmt1o as the main form, with no indication of somatic Dnmt1. These findings indicate that Dnmt1o is cytoplasmic during early development; its presence in both pig and mouse embryos further suggests that Dnmt1o is conserved in mammals. Developmental Dynamics 238:1666,1673, 2009. © 2009 Wiley-Liss, Inc. [source] A challenge for regenerative medicine: Proper genetic programming, not cellular mimicryDEVELOPMENTAL DYNAMICS, Issue 12 2007Angie Rizzino Abstract Recent progress in stem cell biology and the reprogramming of somatic cells to a pluripotent phenotype has generated a new wave of excitement in regenerative medicine. Nonetheless, efforts aimed at understanding transdifferentiation, dedifferentiation, and the plasticity of cells, as well as the ability of somatic cells to be reprogrammed, has raised as many questions as those that have been answered. This review proffers the argument that many reports of transdifferentiation, dedifferentiation, and unexpected stem cell plasticity may be due to aberrant processes that lead to cellular look-alikes (cellular mimicry). In most cases, cellular look-alikes can now be identified readily by monitoring gene expression profiles, as well as epigenetic modifications of DNA and histone proteins of the cells involved. This review further argues that progress in regenerative medicine will be significantly hampered by failing to address the issue of cellular look-alikes. Developmental Dynamics 236:3199,3207, 2007. © 2007 Wiley-Liss, Inc. [source] An olig2 reporter gene marks oligodendrocyte precursors in the postembryonic spinal cord of zebrafishDEVELOPMENTAL DYNAMICS, Issue 12 2007Hae-Chul Park Abstract Continuous production of new neurons and glia in adult mammals occurs within specialized proliferation zones of the forebrain. Neural cell proliferation and neurogenesis is more widespread in adult amphibians, reptiles, and fish but the identity of neural stem cell populations in these organisms has not been fully described. We investigated expression of a reporter gene driven by olig2 regulatory DNA at postembryonic stages in zebrafish. We show that olig2 expression marks a discrete population of spinal cord radial glia in larvae and adults that divide continuously. olig2+ radial glia have hallmarks of stem cells and their divisions appear to be asymmetric, producing new oligodendrocytes but not neurons or astrocytes. Developmental Dynamics 236:3402,3407, 2007. © 2007 Wiley-Liss, Inc. [source] Nucleolar colocalization of TAF1 and testis-specific TAFs during Drosophila spermatogenesisDEVELOPMENTAL DYNAMICS, Issue 10 2007Chad E. Metcalf Abstract In Drosophila, testis-specific TBP-associated factors (tTAFs) predominantly localize to spermatocyte nucleoli and regulate the transcription of genes necessary for spermatocyte entry into meiosis. tTAFs are paralogs of generally expressed TAF subunits of transcription factor IID (TFIID). Our recent observation that the generally expressed TAF1 isoform TAF1-2 is greatly enriched in testes prompted us to explore the functional relationship between general TAFs and tTAFs during spermatogenesis. Analysis by immunofluorescence microscopy revealed that among the general TFIID subunits examined (TATA-box binding protein [TBP], TAF1, TAF4, TAF5, and TAF9), only TAF1 colocalized with the tTAF Mia in spermatocyte nucleoli. Nucleolar localization of TAF1, but not Mia, was disrupted in tTAF mutant flies, and TAF1 dissociated from DNA prior to Mia as spermatocytes entered meiosis. Taken together, our results suggest stepwise assembly of a testis-specific TFIID complex (tTFIID) whereby a TAF1 isoform, presumably TAF1-2, is recruited to a core subassembly of tTAFs in spermatocyte nucleoli. Developmental Dynamics 236:2836,2843, 2007. © 2007 Wiley-Liss, Inc. [source] Transient expression of thyroid hormone nuclear receptor TR,2 sets S opsin patterning during cone photoreceptor genesisDEVELOPMENTAL DYNAMICS, Issue 5 2007M.L. Applebury Abstract Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR,2, the nuclear thyroid hormone receptor , isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR,2 acts, we compared the spatiotemporal expression of TR,2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR,2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR,2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR,2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR,2. The mechanism by which TR,2 functions was probed in transgenic animals with TR,2 ablated, TR,2 that is DNA binding defective, and TR,2 that is ligand binding defective. These studies show that TR,2 is necessary for dorsal repression, but not ventral activation of S opsin. TR,2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR,2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors. Developmental Dynamics 236:1203,1212, 2007. © 2007 Wiley-Liss, Inc. [source] Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 3 2005Teun P. De Boer Abstract Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (,62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals. Developmental Dynamics 233:864,871, 2005. © 2005 Wiley-Liss, Inc. [source] Prep2: Cloning and expression of a new prep family memberDEVELOPMENTAL DYNAMICS, Issue 3 2002Klaus Haller Abstract We describe Prep2, a new murine homeobox-containing gene closely related to Prep1. The PREP2 protein belongs to the three amino acid loop extension (TALE) superclass of homeodomain-containing proteins and encodes a polypeptide of 462 residues. As for PREP1, PREP2 binds an appropriate site on DNA as a heterodimer with PBX1A. Northern analysis, immunoblotting, immunohistochemistry, and in situ hybridization show widespread Prep2 expression during organogenesis and in the adult. The data suggest that Prep2 functions to varying degrees in a broad array of tissues and developmental processes. © 2002 Wiley-Liss, Inc. [source] Abnormal myelin formation in rhizomelic chondrodysplasia punctata type 2 (DHAPAT-deficiency)DEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 7 2000László Sztriha MD PhD The case of a Yemeni girl with isolated peroxisomal acyl-CoA:dihydroxyacetonephosphateacyltransferase (DHAPAT) deficiency is reported. She had rhizomelic chondrodysplasia punctata, microcephaly, failure to thrive, delayed motor and mental development, and spastic quadriplegia. Deficient de novo plasmalogen synthesis in her fibroblasts as a result of low DHAPAT activity was found, while her very-long-chain fatty acid profile, phytanic acid concentration, alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) activity, and peroxisomal 3-ketoacyl-CoA thiolase protein were normal. A mutation in her DHAPAT complementary DNA resulted in the substitution of an arginine residue in the protein at position 211 by a histidine (R211H). Magnetic resonance imaging showed abnormal white matter signal in the centrum semiovale involving the arcuate fibers, while the corpus callosum was normal. DHAPAT and alkyl-DHAP synthase initiate the synthesis of plasmalogens, which are major constituents of myelin phospholipids. The reported girl's abnormal formation of myelin is probably related to the inadequacy of plasmalogen biosynthesis, which is likely to be due to deficient DHAPAT activity. [source] Liposome-mediated transfection of mature taste cellsDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2005Ana Marie Landin Abstract The introduction and expression of exogenous DNA in neurons is valuable for analyzing a range of cellular and molecular processes in the periphery, e.g., the roles of transduction-related proteins, the impact of growth factors on development and differentiation, and the function of promoters specific to cell type. However, sensory receptor cells, particularly chemosensory cells, have been difficult to transfect. We have successfully introduced plasmids expressing green and Discosoma Red fluorescent proteins (GFP and DsRed) into rat taste buds in primary culture. Transfection efficiency increased when delaminated taste epithelium was redigested with fresh protease, suggesting that a protective barrier of extracellular matrix surrounding taste cells may normally be present. Because taste buds are heterogeneous aggregates of cells, we used ,-gustducin, neuronal cell adhesion molecule (NCAM), and neuronal ubiquitin carboxyl terminal hydrolase (PGP9.5), markers for defined subsets of mature taste cells, to demonstrate that liposome-mediated transfection targets multiple taste cell types. After testing eight commercially available lipids, we identified one, Transfast, that is most effective on taste cells. We also demonstrate the effectiveness of two common "promiscuous" promoters and one promoter that taste cells use endogenously. These studies should permit ex vivo strategies for studying development and cellular function in taste cells. © 2005 Wiley Periodicals, Inc. J. Neurobiol, 2005 [source] In vivo analysis reveals different apoptotic pathways in pre- and postmigratory cerebellar granule cells of rabbitDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2004Laura Lossi Abstract Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly-ADP-ribose polymerase-1 (PARP-1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 437,452, 2004 [source] |