The psychometric properties of the Italian version of the Hospital Anxiety and Depression Scale and its utility as a screening instrument for anxiety and depression in a non-psychiatric setting were evaluated. The questionnaire was administered twice to 197 breast cancer patients randomised in a phase III adjuvant clinical trial: before the start of chemotherapy and at the first follow-up visit. The presence of psychiatric disorders was evaluated at the follow-up visit using the Structured Clinical Interview for DSM-III-R in 132 patients. Factor analyses identified two strictly correlated factors. Crohnbach's alpha for the anxiety and depression scales ranged between 0.80 and 0.85. At follow-up, 50 patients (38%) were assigned a current DSM-III-R diagnosis, in most cases adjustment disorders (24%) or major depressive disorder (10%). Receiver operating characteristics (ROC) analysis was used to test the discriminant validity for both anxiety and depressive disorders. The comparison of the areas under the curve (AUC) between the two scales did not show any difference in identifying either anxiety (P = 0.855) or depressive disorders (P = 0.357). The 14-item total scale showed a high internal consistency (alpha = 0.89 and 0.88) and a high discriminating power for all the psychiatric disorders (AUC = 0.89; 95% CI = 0.83-0.94). The cut-off point that maximised sensitivity (84%) and specificity (79%) was 10. These results suggest that the total score is a valid measure of emotional distress, so that the Italian version of HADS can be used as a screening questionnaire for psychiatric disorders. The use of the two subscales as a 'case identifier' or as an outcome measure should be considered with caution.
Due to the resurgence of tuberculosis and the emergence of multidrug-resistant strains, fluoroquinolones (FQ) are being used in selected tuberculosis patients, but FQ-resistant strains of Mycobacterium tuberculosis have rapidly begun to appear. The mechanisms involved in FQ resistance need to be elucidated if the effectiveness of this class of antibiotics is to be improved and prolonged. By using the rapid-growing Mycobacterium smegmatis as a model genetic system, a gene was selected that confers low-level FQ resistance when present on a multicopy plasmid. This gene, lfrA, encodes a putative membrane efflux pump of the major facilitator family, which appears to recognize the hydrophilic FQ, ethidium bromide, acridine, and some quaternary ammonium compounds. It is homologous to qacA from Staphylococcus aureus, tcmA, ofStreptomyces glaucescens, and actII and mmr, both from Streptomyces coelicoler. Increased expression of lfrA augments the appearance of subsequent mutations to higher-level FQ resistance.The worldwide reemergence of tuberculosis as a major public health problem has been accompanied by an ominous increase in multidrug resistant strains (1). This increase has stimulated an intense search for new antimycobacterial agents, but at present only one additional class of drugs, the fluoroquinolones (FQ), has been added to the traditional anti-tuberculosis armamentarium (2). Introduced into clinical practice in the 1980s, the FQ were initially active against many pathogens (3), but their use has been limited by the rapid appearance of resistance in a large percentage of clinical isolates, especially in Staphylococcus aureus and Pseudomonas aeruginosa (41,42). The therapeutic use of the FQ in tuberculosis began only within the past 3-4 years, and they are generally reserved for infections resistant to other agents. However, most FQ are only moderately active against the mycobacteria, and unfortunately, FQ-resistant (FQr) clinical isolates of Mycobacterium tuberculosis have already appeared (4,5). If more can be learned about what determines the effectiveness of a particular FQ against the mycobacteria and the mechanisms by which resistance develops, new agents or strategies may be designed that can prevent or circumvent this resistance.The principal targets of the FQ are bacterial type II topoisomerases, including both the bacterial DNA gyrase, an essential type II topoisomerase that introduces supercoils into the DNA chromosome (6), and the highly homologous topoisomerase IV, which deconcatenates the chromosome after DNA replication (7,8). Mutations in a particular region of gyrA, which encodes the gyrase A subunit, have been associated with moderate-to-high level [>5x minimal inhibitory concentration (MIC)] FQ resistance in many species of bacteria, including M. tuberculosis (5), and similar mutations have been found in the homologous region of topoisomerase IV (9, 10). Mutations conferring low-level resistance have also beenThe publication costs of this article were defrayed in part by page charge p...
Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34 + cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.
Currently, polymerase chain reaction is the most used technique in many laboratories for either diagnostic or molecular biology purposes. Despite the large number of DNA sequences that can be easily analyzed, some GC-rich sequences are refractory to amplification due to the formation of secondary intramolecular structures. To overcome this problem, several molecules have been described to improve polymerization. Here we show that a combination of three additives-betaine, dimethyl sulfoxide, and 7-deaza-dGTP-was essential to achieve amplification of DNA sequences of three disease genes showing a GC content ranging from 67 to 79%.
The formation of triple helical DNA has been evoked in several cellular processes including transcription, replication, and recombination. Using conventional and affinity chromatography, we purified from Saccharomyces cerevisiae whole-cell extract a 35-kDa protein that avidly and specifically bound a purine motif triplex (with a K d of 61 pM) but not a pyrimidine motif triplex or duplex DNA. Peptide microsequencing identified this protein as the product of the STM1 gene. Confirmation that Stm1p is a purine motif triplex-binding protein was obtained by electrophoretic mobility shift assays using either bacterially expressed, recombinant Stm1p or whole-cell extracts from stm1⌬ yeast. Stm1p has previously been identified as G4p2, a G-quartet nucleic acidbinding protein. This suggests that some proteins actually recognize features shared by G4 DNA and purine motif triplexes, e.g. Hoogsteen hydrogen-bonded guanines. Genetically, the STM1 gene has been identified as a multicopy suppressor of mutations in several genes involved in mitosis (e.g. TOM1, MPT5, and POP2). A possible role for multiplex DNA and its binding proteins in mitosis is discussed.It has long been recognized that, under the proper conditions, certain DNA sequences preferentially adopt a structure composed of three nucleic acid strands (1). Triple helical or triplex DNA is a thermodynamically favored structure characterized by a third pyrimidine-rich (Py triplex) 1 or purine-rich (Pu triplex) DNA strand located within the major groove of a homopurine/homopyrimidine stretch of duplex DNA (reviewed in Ref. 2). Both intermolecular triplexes, where the third stand originates from a separate DNA molecule, and intramolecular triplexes (H-DNA), where the third stand originates from a proximal site on the same DNA molecule as its duplex acceptor, have been described. In intermolecular and intramolecular triplexes, stable interaction of the third strand is achieved through either specific Hoogsteen (Py triplex) or reverse Hoogsteen (Pu triplex) hydrogen bonding to the homopurine strand of the duplex, with the third strand adopting either a parallel (Py triplex) or antiparallel (Pu triplex) orientation relative to the homopurine acceptor. Base triplets in the pyrimidine motif include T*AT and C ϩ
Lactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated. In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA-linked protein in nuclear extracts.
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