The Relationship between Substance Use and Depressive Symptoms among Colombian Adolescents / Relación entre el uso de sustancias y los síntomas depresivos entre adolescentes colombianos

ABSTRACT Objective: To explore the relationship between substance use and depressive symptomatology among adolescents in Colombia. Methods: Adolescents aged 13-19 years from a rural city in Colombia completed the Spanish version of the Alcohol, Smoking and Substance Involvement Screening Test and the Kutcher Adolescent Depression Scale-6 (KADS-6). Results: Regression analyses indicated that gender, having used cannabis, having used sedatives, and having a family member who used illegal drugs were predictive of higher symptom levels of depression according to KADS-6. Conclusion: The results of this study suggested that substance use was associated with depressive symptoms in this sample of Colombian adolescents.

RESUMEN Objetivo: Explorar la relación entre el uso de sustancias y la sintomatologia depresiva entre adolescentes en Colombia. Métodos: Los adolescentes de 13 a 19 años de una ciudad rural en Colombia completaron la versión en español de la Prueba de Detección de Alcohol, Tabaco y Sustancias (ASSIST, en inglés) y la Escala de Depresión pasa Adolescentes de Kutcher-6 (KADS-6, en inglés). Resultados: Los análisis de regresión indicaron que tanto el género, como el haber consumido cannabis, usado sedantes, y tener un miembro de la familia que usó drogas ilegales, fueron predictores de niveles más altos de sintomas depresivos de acuerdo con la escala KADS-6. Conclusión: Los resultados de este estudio sugirieron que el uso de sustancias estaba asociado con sintomas depresivos en esta muestra de adolescentes colombianos.

Development and Validation of a Measure of Attitudes toward Fluffy Women.

BACKGROUND: There is an absence of research on the newly evolved term "fluffy" which describes body image and personality features among women. Research on "fluffiness" among Caribbean peoples has been limited by the lack of valid and reliable measures of the concept. OBJECTIVE: This project addresses this problem by exploring the internal consistency reliability and the concurrent and discriminant validity of the Attitudes toward Fluffy Women Scale (ATFW) using a mixture of past and present students from The University of the West Indies (UWI), Mona, and the University of Technology (UTech), Kingston. METHOD: Past or present students from The UWI, Mona, and UTech, Kingston, were recruited for the study through the use of convenience sampling. A total of 80 students (38 males, 47.5%; 42 females, 52.5%) participated in the study. RESULTS: Overall, the ATFW was found to have an acceptable degree of internal consistency reliability (α = 0.90). The scale also had reasonably good concurrent validity as evidenced by moderate correlations with scores on the Attitudes Toward Obese Persons Scale (r = -0.42) and acceptable discriminant validity as demonstrated through low correlations with a Bogardus Social Distance Scale designed to assess prejudice toward people living with the human immunodeficiency virus [HIV] (r = 0.29). This pattern of scores suggests that the majority of the stable variance underlying the ATFW assesses the "fluffy" concept (17.6%) while a smaller degree of the variability (8%) measures a conceptually similar but distinct concept. CONCLUSION: The Attitudes toward Fluffy Women scale was found to be a reliable and valid scale for assessing the attitudes of young adults toward fluffy women.

Subjective well-being of adults with homozygous sickle cell disease in Jamaica.

OBJECTIVES: This study compared the subjective well-being of adults with homozygous sickle cell (SS) disease to a matched group of healthy adult peers. The differential influence of sociodemographic factors on the subjective well-being of Sickle Cell patients was also examined. METHODS: The Ferran and Powers Quality of Life Index and the Positive and Negative Affect Schedule were used to assess subjective well-being. Seventy-five homozygous sickle cell (SS) disease patients and sixty-seven matched controls (adults with normal haemoglobin: AA) from the Sickle Cell Disease Cohort Study in Jamaica were interviewed. Analysis of variance (ANOVA) was used to examine differences between the groups. RESULT: Patients with Sickle cell (SS) disease were less satisfied than matched controls with their lives overall, their health and functioning, social and economic situation and psychological functioning. Sickle cell disease patients reported lower levels of positive affect but similar levels of negative affect as controls. Unemployed sickle cell disease patients were less satisfied than all other adults with their lives overall, health and functioning, psychological functioning and social and economic situation. Sickle cell disease patients with lower occupational status were less satisfied with their family life than all other adults. CONCLUSIONS: These findings suggest that subjective well-being is compromised in patients with homozygous sickle cell disease. These patients may benefit from interventions designed to improve their subjective well-being.

Patterns of depressive symptoms among patients with HIV infection / Patrones de síntomas depresivos entre pacientes con infección por VIH

OBJECTIVE: To identify the level of depressive symptoms among patients with HIV infection and to examine the reported patterns of depressive symptoms not confounded by the physical manifestations of HIV-infection. METHOD: A total of 191 patients with HIV infection (75 males (39%) and 116 females (61%), mean age 40.48 ± 10 years), from three HIV clinics were administered the Beck Depression Inventory - II as well as a demographic questionnaire as part of a larger study. RESULTS: Moderate to severe depressive symptoms were reported by 17.3% of the HIV-infected patients with females reporting significantly higher levels of depressive symptoms than males. A principal components analysis identified three clusters of depressive symptoms: cognitive-affective, negative cognitions and somatic symptoms. The HIV-infected patients were found to display mainly cognitive-affective symptoms of depression. CONCLUSION: HIV-infected patients, especially female patients, may be at an increased risk of experiencing high levels of depressive symptoms. It is recommended that HIV-infected patients be routinely screened for depression, particularly cognitive-affective symptoms of depression.

OBJETIVO: Identificar el nivel de sintomas depresivos entre pacientes con infección por VIHy examinar los patrones reportados sobre los sintomas de depresión, no confundidos por las manifestaciones físicas de la infección por VIH. MÉTODO: A un total de 191 pacientes con infección por VIH (75 varones (39%) y 116 hembras (61%), con edadpromedio 40.45 ± 10 anos), de tres clinicas de VIH, se les aplicó el Inventario de Depresión de Beck II, asi como una encuesta demográfica como parte de un estudio más grande. RESULTADOS: Un 17.3% de los pacientes infectados por VIH, informaron sintomas depresivos de moderados a severos, reportando las hembras niveles de sintomas de depresión significativamente más altos que los varones. Un análisis de los componentes principales identificó tres grupos de sintomas depresivos: cognitivos afectivos, cogniciones negativas y sintomas somáticos. Se halló que los pacientes infectados por VIHpresentaban principalmente sintomas afectivos cognitivos de depresión. CONCLUSIÓN: Los pacientes infectados con VIH, especialmente las hembras, pueden hallarse en un mayor riesgo de experimentar niveles altos de sintomas depresivos. Se recomienda que los pacientes infectados con VIHsean sometidos deforma rutinaria a tamizajes de depresión, particularmente de los sintomas de depresión cognitivos afectivos.

Validation of the Beck Depression Inventory II in HIV-positive patients.

OBJECTIVE: Research on depression among HIV-positive patients has been limited by the lack of a valid and reliable measure of depression. This project addresses this problem by exploring the internal consistency reliability and the concurrent and discriminant validity of the Beck Depression Inventory-II (BDI-II) using HIV-positive patients in Jamaica. METHOD: Patients from three HIV clinics in Jamaica (n = 191 patients; 61% female, 39% male, mean age 40.5-10 years) were administered the BDI-II along with the Centre for Epidemiological Studies -Depression Scale (CES-D) and the Social Provisions Scale. RESULTS: Overall, the BDI-II was found to have a high degree of reliability (alpha = 0.89). The scale also had good concurrent validity as evidenced by a high correlation with scores on the CES-D (r = 0.74) and acceptable discriminant validity as demonstrated through a moderate correlation with the Social Provisions Scale (r = -0.42). This pattern of scores suggests that the majority of the variance underlying the BDI-II assesses depression (55%) while a smaller degree of the variability (18%) measures a conceptually similar but distinct concept. CONCLUSION: The BDI-II is a sufficiently reliable and valid measure for assessing depression in HIV-positive patients.

Patterns of depressive symptoms among patients with HIV infection.

OBJECTIVE: To identify the level of depressive symptoms among patients with HIV infection and to examine the reported patterns of depressive symptoms not confounded by the physical manifestations of HIV-infection. METHOD: A total of 191 patients with HIV infection (75 males (39%) and 116 females (61%), mean age 40.48 +/- 10 years), from three HIV clinics were administered the Beck Depression Inventory-II as well as a demographic questionnaire as part of a larger study. RESULTS: Moderate to severe depressive symptoms were reported by 17.3% of the HIV-infected patients with females reporting significantly higher levels of depressive symptoms than males. A principal components analysis identified three clusters of depressive symptoms: cognitive-affective, negative cognitions and somatic symptoms. The HIV-infected patients were found to display mainly cognitive-affective symptoms of depression. CONCLUSION: HIV-infected patients, especially female patients, may be at an increased risk of experiencing high levels of depressive symptoms. It is recommended that HIV-infected patients be routinely screened for depression, particularly cognitive-affective symptoms of depression.

Factors associated with depression in students at The University of the West Indies, Mona, Jamaica.

OBJECTIVE: This project examines the factors associated with depression in students attending the University of the West Indies, Mona Campus. METHOD: Students enrolled in the Foundation courses during the first and second semesters of the 2005/2006 academic year were administered the Brief Screen for Depression as well as a demographic questionnaire as part of a larger study. RESULTS: A wide cross-section of the university population was sampled (n = 690; 252 from semester one, 438 from semester two; 77% females, 23% males; age 16-62 years, median = 20 years, mean = 23.4 years +/- 7.4). Nearly 40% of students scored in the clinically depressed range. Students in the December wave of data collection had higher depression scores than those in the January wave. Consistent with international research, females reported significantly higher levels of depressive symptoms. Married students reported significantly lower depression scores than students in visiting relationships. Students who were combining employment and school reported lower depression scores than those who were not employed. Maternal education significantly influenced students' levels of depression such that students whose mothers had university or other tertiary education had lower depression scores while those whose mothers had primary or lower education had the highest depression scores. Students with a chronic condition or a disability scored higher than those without such problems on all three measures of depression. CONCLUSION: Depression may be a significant problem in students at The University of the West Indies, Mona campus.

Component structure of the SF-36 in Jamaicans with sickle cell disease

OBJECTIVES: Sickle cell disease (SCD) is the commonest genetic disorder in Jamaica and greatly affects the quality of life (QOL) of those who are afflicted. The Short Form 36 survey (SF-36) questionnaire is one of the most commonly utilized measures of QOL. Physicians cannot interpret QOL measures until the instruments being used to make assessment are adequately established in their population. The Jamaican cultural and educational systems expose its people to many stresses which likely impact on their QOL. It is thus postulated that the QOL construct may exhibit a different structure for the population with sickle cell disease. SUBJECTS AND METHODS: The SF-36 v.2 was interviewer administered to the Jamaican Sickle Cell Disease Cohort Study participants ('Cohort' sample) and a random sample of adult sickle cell unit patients ('Main'sample). Demographic data were also collected on both groups. Both of the samples did not meet the five rule criteria for compliance with the original SF-36 component structure. Hence, principal components analysis was used to determine the component structure of the SF-36 in both groups. RESULTS: Three dimensions may underlie the SF-36 for both groups and these could be labelled 'Physical Health', 'Mental Health' and 'Role Limitations'. This solution accounted for 45.8% of the variability underlying the SF-36 in the 'Cohort'sample and 54.6% of the variability in the 'Main' sample. CONCLUSIONS: It concluded that within Jamaican samples of patients with sickle cell disease, the SF-36 has a component structure which is quite distinct from that initially proposed by its creators.

Objetivos: La enfermedad de células falciformes (ECF) es el trastorno genético más común en Jamaica y afecta grandemente la calidad de vida (CdV) de quienes la padecen. El cuestionario de salud SF-36 es una de las mediciones de la CdV más comúnmente usadas. Los médicos no pueden interpretar lasmediciones de la CdV hasta que los instrumentos usados para realizar la evaluación se establezcan de forma adecuada a su población. La cultura y los sistemas educacionales en Jamaica, exponen a su población a muchas formas de estrés que afectan probablemente su CdV. De este modo, se postula que el constructo CdV puede presentar una estructura diferente para esta población. Métodos: El cuestionario SF-36 v.2 fue aplicado por el entrevistador a los participantes en un estudio de cohorte de la enfermedad de células falciformes en Jamaica (muestra de “cohorte”) y a una muestra aleatoria de pacientes adultos de la unidad de anemia falciforme (muestra “principal”). Se recogierondatos demográficos de ambos grupos. Ninguna de las muestras satisfizo los cinco criterios normativos de conformidad con la estructura original de los componentes del SF-36. Por consiguiente, se recurrió al análisis de los componentes principales a fin de determinar la estructura de componentes del SF-36 en ambos grupos. Resultados: Tres dimensiones pueden subyacer en el SF-36 para ambos grupos. Estas pueden ser llamadas “salud física”, “salud mental”, y “limitaciones de roles”. Esta solución dio cuenta del 45.8% de la variabilidad subyacente en el SF-36 en el caso de la muestra de “cohorte” y el 54.6% dela variabilidad en la muestra “principal”.Conclusiones: Se concluyó que en las muestras de pacientes de Jamaica con la enfermad de células falciformes, el SF-36 posee una estructura de componentes que puede ser bien distinta de la que inicialmente propusieron sus creadores.

Validation of the beck depression inventory-II in a Jamaican university student cohort / Validación del inventario de depresión de beck II en una cohorte de estudiantes universitarios jamaicanos

OBJECTIVE: This project explores the internal consistency reliability and the concurrent and discriminant validity of the Beck Depression Inventory-II (BDI-II) using a wide cross-section of the student population attending the University of the West Indies, Kingston, Jamaica. SUBJECTS AND METHODS: Students enrolled in UWI Foundation courses during the first and second semesters of the 2005/2006 academic year (n = 690; 77% females, 23% males; mean age = 23.4 +/- 7.4 years) were administered the BDI-II along with the Brief Screen for Depression (BSD), the Centre for Epidemiological Studies--Depression Scale (CES-D) and the UCLA Loneliness Scale--Revised. RESULTS: Overall, the BDI-II was found to have an acceptable degree of reliability (alpha = 0.90). The scale also had reasonably good concurrent validity as evidenced by high correlations with scores on the BSD (r = 0.74) and the CES-D (r = 0.71) and acceptable discriminant validity as demonstrated through moderate correlations with the UCLA Loneliness Scale (r = 0.50). This pattern of scores suggests that the majority of the variance underlying the BDI-II assesses depression (50% to 55%) while a smaller degree of the variability (25%) measures a conceptually similar but distinct concept. CONCLUSION: The BDI-II is a reliable and valid measure for assessing depression within the Jamaican population.

OBJETIVO: Este proyecto explora la fiabilidad de la consistencia interna y la validez discriminante y concurrente del inventario de depresión de Beck II (IDB - II) usando una amplia sección transversal de la población estudiantil que asiste a la Universidad de West Indies, Kingston, Jamaica. SUJETOS Y MÉTODOS: A los estudiantes matriculados en los cursos de Fundación de UWI durante el primer y segundo semestres del año académico 2005/2006 (n = 690; 77% hembras 23% varones; edad promedio = 23.4 años B1 7.4) se les aplicó el IDB - II junto con la Prueba Breve para la Detección de la Depresión (BSD), la Escala de Depresión del Centro de Estudios Epidemiológicos (CES-D), y la Escala Revisada de Soledad de la Universidad de los Ángeles de California. RESULTADOS: En general, se halló que el IDB - II tenía un grado aceptable de fiabilidad (a = 0.90). La escala también tenía una validez concurrente razonablemente tal como lo evidencian las altas correlaciones con las puntuaciones del BSD (r = 0.74) y el CES-D (r = 0.71), y una validez discriminante aceptable como quedó demostrado mediante las correlaciones moderadas con la Escala de Soledad de UCLA (r = 0.50). Este patrón de puntuaciones sugiere que la mayor parte de la varianza que subyace en el IDB - II evalúa la depresión (50% a 55%) mientras que un grado menor de variabilidad (25%) mide un concepto conceptualmente similar pero distinto. CONCLUSIÓN: El IDB - II constituye un instrumento de medición fiable y válido para evaluar la depresión en la población jamaicana.

Validation of the beck depression inventory-II in a Jamaican university student cohort.

OBJECTIVE: This project explores the internal consistency reliability and the concurrent and discriminant validity of the Beck Depression Inventory-II (BDI-II) using a wide cross-section of the student population attending the University of the West Indies, Kingston, Jamaica. SUBJECTS AND METHODS: Students enrolled in UWI Foundation courses during the first and second semesters of the 2005/2006 academic year (n = 690; 77% females, 23% males; mean age = 23.4 +/- 7.4 years) were administered the BDI-II along with the Brief Screen for Depression (BSD), the Centre for Epidemiological Studies--Depression Scale (CES-D) and the UCLA Loneliness Scale--Revised. RESULTS: Overall, the BDI-II was found to have an acceptable degree of reliability (alpha = 0.90). The scale also had reasonably good concurrent validity as evidenced by high correlations with scores on the BSD (r = 0.74) and the CES-D (r = 0.71) and acceptable discriminant validity as demonstrated through moderate correlations with the UCLA Loneliness Scale (r = 0.50). This pattern of scores suggests that the majority of the variance underlying the BDI-II assesses depression (50% to 55%) while a smaller degree of the variability (25%) measures a conceptually similar but distinct concept. CONCLUSION: The BDI-II is a reliable and valid measure for assessing depression within the Jamaican population.

Component structure of the SF-36 in Jamaicans with sickle cell disease.

OBJECTIVES: Sickle cell disease (SCD) is the commonest genetic disorder in Jamaica and greatly affects the quality of life (QOL) of those who are afflicted. The Short Form 36 survey (SF-36) questionnaire is one of the most commonly utilized measures of QOL. Physicians cannot interpret QOL measures until the instruments being used to make assessment are adequately established in their population. The Jamaican cultural and educational systems expose its people to many stresses which likely impact on their QOL. It is thus postulated that the QOL construct may exhibit a different structure for the population with sickle cell disease. SUBJECTS AND METHODS: The SF-36 v.2 was interviewer administered to the Jamaican Sickle Cell Disease Cohort Study participants ('Cohort' sample) and a random sample of adult sickle cell unit patients ('Main'sample). Demographic data were also collected on both groups. Both of the samples did not meet the five rule criteria for compliance with the original SF-36 component structure. Hence, principal components analysis was used to determine the component structure of the SF-36 in both groups. RESULTS: Three dimensions may underlie the SF-36 for both groups and these could be labelled 'Physical Health', 'Mental Health' and 'Role Limitations'. This solution accounted for 45.8% of the variability underlying the SF-36 in the 'Cohort'sample and 54.6% of the variability in the 'Main' sample. CONCLUSIONS: It concluded that within Jamaican samples of patients with sickle cell disease, the SF-36 has a component structure which is quite distinct from that initially proposed by its creators.

Validation of the Brief Screen for Depression in a Jamaican cohort / Validación de la prueba breve para la detección de la depresión en una Cohorte Jamaicana

Research on depression in Jamaicans has been limited by the absence of a psychometrically sound measure of depression. This project attempts to rectify this problem by exploring the concurrent and discriminant validity of the Brief Screen for Depression (BSD) using a sample of 244 students attending the University of the West Indies, Mona, Jamaica. Participants were administered the BSD along with the Beck Depression Inventory (BDI), the Center for Epidemiologic Studies Depression scale (CES-D) the University of California at Los Angeles (UCLA) Loneliness Scale - Revised, (UCLA-R) and the Responding Desirably on Attitudes and Opinions scale (RD16). Overall, the BSD was found to have an acceptable level of concurrent validity as evidenced by high correlations with scores on the BDI (0.64) and the CES-D (0.62), and an acceptable level of discriminant validity as demonstrated through moderate correlations with the UCLA Loneliness Scale (0.40). In addition, the BSD was found to possess a moderate degree of sensitivity in identifying individuals who may be experiencing clinically significant symptoms of depression.

Validation of the Brief Screen for Depression in a Jamaican cohort.

Research on depression in Jamaicans has been limited by the absence of a psychometrically sound measure of depression. This project attempts to rectify this problem by exploring the concurrent and discriminant validity of the Brief Screen for Depression (BSD) using a sample of 244 students attending the University of the West Indies, Mona, Jamaica. Participants were administered the BSD along with the Beck Depression Inventory (BDI), the Center for Epidemiologic Studies Depression scale (CES-D) the University of California at Los Angeles (UCLA) Loneliness Scale - Revised, (UCLA-R) and the Responding Desirably on Attitudes and Opinions scale (RD16). Overall, the BSD was found to have an acceptable level of concurrent validity as evidenced by high correlations with scores on the BDI (0.64) and the CES-D (0.62), and an acceptable level of discriminant validity as demonstrated through moderate correlations with the UCLA Loneliness Scale (0.40). In addition, the BSD was found to possess a moderate degree of sensitivity in identifying individuals who may be experiencing clinically significant symptoms of depression.

Site-specific binding of the 9.5 kilodalton DNA-binding protein ORF80 visualized by atomic force microscopy.

Atomic force microscopy (AFM) has been used to examine the binding properties of the DNA-binding protein ORF80 to DNA. ORF80 is a 9.5 kDa protein that binds site-specifically to double-stranded DNA of the sequence TTAA-N(7)-TTAA. Direct sizing of the protein complexes on DNA fragments from the plasmid pRN1 with AFM shows that the protein ORF80 binds preferentially to two positions. These positions agree well with the ORF80 binding sites determined by footprinting analysis. The measurements allow an estimate of the stoichiometry of the DNA-protein complexes. In contrast to previous results, the single-molecule experiments suggest that only a low number of ORF80 molecules bind to a DNA-binding site.

The replication protein of the Sulfolobus islandicus plasmid pRN1.

The thermoacidophile crenarchaeote Sulfolobus ssp. is one of the best-studied Archaea. Cryptic and conjugative plasmids as well as viruses have been described for this genus. For the majority of the genetic elements only the genome sequence and the basic characteristics were determined. In contrast the fusellovirus SSV1 and the cryptic plasmid pRN1, which is the smallest known genetic element of the crenarchaeota, have been studied in more detail. The three gene products of the plasmid pRN1 have been characterized biochemically. The replication protein of the plasmid, a multifunctional enzyme, has a novel domain, termed prim/pol domain. This domain constitutes the first member of the DNA polymerase family E. Based on the biochemical characterization of the gene products a model of how pRN1 is replicated in vivo is proposed.

Binding of XPA and RPA to damaged DNA investigated by fluorescence anisotropy.

The proteins XPA and RPA are assumed to be involved in primary damage recognition of global genome nucleotide excision repair. XPA as well as RPA have been each reported to specifically bind DNA lesions, and ternary complex formation with damaged DNA has also been shown. We employed fluorescence anisotropy measurements to study the DNA-binding properties of XPA and RPA under true equilibrium conditions using damaged DNA probes carrying a terminal fluorescein modification as a reporter. XPA binds with low affinity and in a strongly salt-dependent manner to DNA containing a 1,3-d(GTG) intrastrand adduct of the anticancer drug cisplatin or a 6-nt mismatch (K(D) = 400 nM) with 3-fold preference for damaged vs undamaged DNA. At near physiological salt conditions binding is very weak (K(D) > 2 microM). RPA binds to damaged DNA probes with dissociation constants in the range of 20 nM and a nearly 15-fold preference over undamaged DNA. The presence of a cisplatin modification weakens the affinity of RPA for single-stranded DNA by more than 1 order of magnitude indicating that binding to the lesion itself is not a driving force in damage recognition. Our fluorescence anisotropy assays also show that the presence of XPA does not enhance the affinity of RPA for damaged DNA although both proteins interact. In contrast, cooperative binding of XPA and RPA is observed in EMSA. Our results point to a damage-sensing function of the XPA-RPA complex with RPA mediating the important DNA contacts.

Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein.

There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of -6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85 degrees C.

The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein.

The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetrical binding sites separated by approximately 60 bp were found upstream of the orf80 gene. Both binding sites contain two TTAA motifs as well as other conserved bases. Fluorescence measurements show that short duplex DNAs derived from a single binding site sequence are bound with submicromolar affinity and moderate cooperativity by ORF80. On DNA fragments carrying both binding sites, a rather large protein-DNA complex is formed in a highly cooperative manner. ORF80 contains an N-terminal leucine zipper motif and a highly basic domain at its C-terminus. Compared to all known basic leucine zipper proteins the order of the domains is reversed in ORF80. ORF80 may therefore constitute a new subclass of basic leucine zipper DNA-binding proteins.

Photocrosslinking locates a binding site for the large subunit of human replication protein A to the damaged strand of cisplatin-modified DNA.

The repair proteins XPA, XPC and replication protein A (RPA) have been implicated in the primary recognition of damaged DNA sites during nucleotide excision repair. Detailed structural information on the binding of these proteins to DNA lesions is however lacking. We have studied the binding of human RPA (hRPA) and hRPA-XPA-complexes to model oligonucleo-tides containing a single 1, 3-d(GTG)-cisplatin-modification by photocrosslinking and electrophoretic mobility shift experiments. The 70 kDa subunit of hRPA can be crosslinked with high efficiency to cisplatin-modified DNA probes carrying 5-iodo-2"-deoxyuridin (5-IdU) as crosslinking chromophore. High efficiency crosslinking is dependent on the presence of the DNA lesion and occurs preferentially at its 5"-side. Examination of the crosslinking efficiency in dependence on the position of the 5-IdU chromophore indicates a specific positioning of hRPA with respect to the platination site. When hRPA and XPA are both present mainly hRPA is crosslinked to the DNA. Our mobility shift experiments directly show the formation of a stable ternary complex of hRPA, XPA and the damaged DNA. The affinity of the XPA-hRPA complex to the damaged DNA is increased by more than one order of magnitude as compared to hRPA alone.

Adenylosuccinate synthase from Saccharomyces cerevisiae: homologous overexpression, purification and characterization of the recombinant protein.

Adenylosuccinate synthase (EC 6.3.4.4) catalyses the first committed step in the synthesis of adenosine. We have overexpressed the cloned gene of Saccharomyces cerevisiae (ADE12) in S. cerevisiae. The recombinant enzyme exhibits similar kinetic behaviour to that of the native enzyme purified from S. cerevisiae. This ter-reactant dimeric enzyme shows Michaelis-Menten kinetics only with IMP. l-Aspartate and GTP display a weak negative co-operativity (Hill coefficient 0. 8-0.9). This negative co-operativity has not yet been reported for adenylosuccinate synthases from other organisms. Another unusual feature of the enzyme from S. cerevisiae is its negligible inhibition by adenine nucleotides and its pronounced inhibition by Cl(-) ions.