[Multidrug resistance in lymphoproliferative syndromes].
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biomedical subjects
M Giacca
Publications and source records attributed to M Giacca.
A protein target site in an early replicated human DNA sequence: a highly conserved binding motif.
We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.
Base Composition↗
Presence of transcription signals in two putative DNA replication origins of human cells.
We describe the purification and cloning of human DNA replicated at the onset of S phase in HL60 cells synchronized with aphidicolin. A survey of the overall structural properties of these sequences did not show any distinctive features except for an enrichment in Cot0 DNA. The two longer fragments were completely sequenced and studied in more detail. Both were shown to contain transcriptional signals associated with promoters and/or enhancers, such as the binding sites of Sp1, T antigen and nuclear factor III. In one instance, a binding site for a known cellular transcription factor (USF/MLTF) was located inside the sequence by footprinting. Accordingly, by CAT assay and Northern blot, the same sequence was shown to contain an active promoter. The significance of these findings with respect to the role of transcription in initiation of DNA replication at the origin is discussed. None of the tested fragments exhibited autonomously replicating sequence (ARS) activity in transfected cells. The problems connected with the detection of ARS activity in human cells are critically examined.
Aphidicolin↗
Sequence-specific DNA binding protein(s) that bind(s) to a putative human DNA replication origin.
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Cloning, Molecular↗
Characterization of human DNA sequences synthesized at the onset of S-phase.
We developed a method of enrichment for DNA replicated at the onset of S-phase in synchronized human HL60 cells. About 200 such sequences were cloned. The analysis of this selected DNA sample showed that: 1) the cloned DNA fragments derive from a limited number (750-1500) of replicons; 2) there is no extensive homology between different DNA fragments; 3) they are not significantly enriched in highly repeated sequences; 4) they are enriched in snap-back (Cot = o) DNA. The sequence of the longest fragment revealed the presence of numerous signals collected in a few hundred nucleotides: 1) homology with the origin of replication of human Papovaviruses usually associated with potential stem-loop structures; 2) binding sites for known transcription factors and for another nuclear factor; 3) potential binding sites for the chromosome "scaffold".
Base Sequence↗
Cluster analysis of antibiotic susceptibility patterns of clinical isolates as a tool in nosocomial infection surveillance.
Hospital infections represent a major epidemiological problem. The first step in the detection of nosocomial infections consists in assessing the probability that two or more isolates from different patients are similar or different. Many methods are available for typing purposes. Among these, antibiotic susceptibility patterns do not need extra cost or extra work and are available "on line" every moment they are needed. A mathematical technique of elaboration is proposed for disk zone sizes, in order to assess the probability of two or more clinical isolates to be the same strain. Antibiograms performed according to Kirby-Bauer are evaluated detecting zone sizes by a computer controlled device and then submitted to cluster analysis. Similarity of strains is reported in a dendrogram, in which strains are successively fused. Strains that share a common susceptibility pattern are considered a "cluster". At last, epidemiological maps are constructed for each group of strains, in which all the isolates are reported, ordered for patients, plotted on the day the specimen was collected, drawn in a different shape according to the source of specimen, and shadowed by the pattern of its cluster. This method of reporting data directly allows to detect cross infections among patients and can be used as a first typing step before other more expensive procedures.
Anti-Bacterial Agents↗
Multivariate analysis of antibiograms for typing Pseudomonas aeruginosa.
A method for typing Pseudomonas aeruginosa using antibiotic susceptibility patterns is presented, which allows recognition of clusters of the same strain among clinical isolates from different patients, thus indicating whether cross infection has occurred. An index of similarity (the euclidean or the oblique distance), which includes all the differences of disk zone sizes among isolates, is computed and then elaborated by a clustering algorithm that successively groups all the isolates in larger clusters. The results of clustering are presented as dendrograms, whose terminal branches are pruned down to a level below which differences are casual; isolates that still appear on a common branch are considered identical. The reliability of this technique for detecting nosocomial cross infections was assessed by comparing its results with that of serotyping and pyocin typing. Only 2 of 31 (6.4%) clusters detected by multivariate analysis were not confirmed, while 4 of 33 (12.1%) clusters were recognized by serotyping and pyocin typing, but not by multivariate analysis. In at least two instances the differences in susceptibility patterns were due to cytoplasmic R factors. The routine use of antibiogram data for typing purposes should be considered an essential part of nosocomial infection control.
Anti-Bacterial Agents↗
[A year of blood cultures at the Associated Hospitals of Trieste: a critical evaluation of the clinico-laboratory approach to the patient with suspected bacteremia].
The authors evaluated the 2956 blood cultures performed in a multispecialist hospital in Trieste (Italy) during a whole year. A computer assisted analysis of the data pointed out that a single blood culture performed in a bacteremic patient could reveal 93% of positivities, two blood cultures in the same day at least 96.6% and three at least 98.3%. Furthermore, in suspected bacteremic patients who received several blood cultures in subsequent days, the chance for a second day culture to reveal a bacteremia not pointed out in the first day was less than 4%, while it was 0% for the following days. These results assess the importance of a correct approach to the suspected bacteremic patient, and point out the usefulness of performing at most three blood cultures in the first day of clinical suspicion of bacteremia.
Bacteriological Techniques↗
Image analysis in quantitative PCR. An application for the measurement of c-erbB-2 oncogene amplification in DNA from human tumours.
We present an application of image analysis for the direct quantification of PCR products after gel electrophoresis and ethidium bromide staining of DNA. This procedure has been applied to the development of an assay based on competitive PCR for the measurement of the degree of amplification of c-erbB-2 oncogene in DNA from human tumours. In this method two DNA species (genomic and competitor) compete for PCR amplification. Since results are calculated from the final competitor/genomic ratio any variable affecting the rate of PCR amplification has no effect on the accuracy of the ratio measurement. Results are reported which show that even large variations in the experimental conditions (number of PCR cycles, sample volumes and extracted DNA quality) did not interfere with the precision of the measurement of the competitor/genomic ratio.
Binding, Competitive↗
Expression of multidrug resistance gene (MDR-1) in human normal leukocytes.
BACKGROUND: The mdr-1 gene, which codes for a 170-kd transmembrane glycoprotein (P170), is frequently overexpressed in multidrug resistant (MDR) tumor cell lines and in spontaneous tumors, including leukemia and lymphoma. However, it is also constitutively expressed as a normal gene in normal tissues. METHODS: We used human mdr-1 cDNA and three anti-P170 monoclonal antibodies (MoAbs: MRK-16, C-219 and JSB-1) to investigate the normal peripheral blood leukocyte content of mdr-1 specific mRNA and of P170 through immunocytochemistry and flow cytometry. RESULTS: We did not find any increase in mdr-1-specific mRNA, while small amounts of P170 were easily detectable in about two thirds of the lymphocytes and monocytes and in about one third of the granulocytes. The level of P170 expression in leukocytes was similar to that found in non-MDR tumor cell lines. CONCLUSIONS: mdr-1 is constitutively expressed in human normal leukocytes at levels that cannot significantly affect drug resistance. Accordingly, low-level mdr-1 expression in leukemic cells should not be considered a label of pleiotropic drug resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗