Ana María Riverón

Entamoeba histolytica TATA‐box binding protein binds to different TATA variants in vitro

The ability of Entamoeba histolytica TATA binding protein (EhTBP) to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study the in vitro EhTBP DNA‐binding activity and to determine the TATA‐EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts (NE) and the TATTTAAA oligonucleotide, which corresponds to the canonical TATA box for E. histolytica, was demonstrated by gel‐shift assays. In these experiments a single NE‐TATTTAAA oligonucleotide complex was detected. Complex was retarded by anti‐EhTBP Igs in supershift experiments and antibodies also recognized the cross‐linked complex in Western blot assays. Recombinant EhTBP formed specific complexes with TATA variants found in E. histolytica gene promoters and other TATA variants generated by mutation of TATTTAAA sequence. The dissociation constants of recombinant EhTBP for TATA variants ranged between 1.04 (±0.39) × 10−11 and 1.60 (±0.37) × 10−10 m. TATTTAAA and TAT_ _AAA motifs presented the lowest KD values. Intriguingly, the recombinant EhTBP affinity for TATA variants is stronger than other TBPs reported. In addition, EhTBP is more promiscuous than human and yeast TBPs, probably due to modifications in amino acids involved in TBP‐DNA binding.

Comparative Study of Three Methods for Non-radioactive In Vivo DNA Labeling in Escherichia coli Using Nucleoside Analogs

Abstract In the present work, a comparative study of 5-FdUrd, thy -, and metabolic in vivo labeling methods for plasmid and chromosomal DNA in E. coli DH5α cells was performed in order to achieve the best thymidine substitution method by 5-BrdUrd. According to the colorimetric immunoenzymatic results, we found that the minimal detectable labelled DNA (MDLD) was 312 pg with the 5-FdUrd and thy - methods for 5-BrdUrd labelled plasmid DNA. 5-BrdUrd replaced about 96% of the total thymidine by 5-FdUrd methods; for the thy - and metabolic labeling methods, the MDLD value was 1,25 ng for denatured 5-BrdUrd chromosomal DNA. Pyrimidine nucleoside analogues were also evaluated as immunochemical markers for their in vivo introduction into DNA.

Apolipoprotein E alleles in Cuban patients with mild cognitive impairment.

Background: Apolipoprotein E (ApoE) ∊4 genotype is the most clearly documented risk factor for Alzheimer’s disease (AD). Epidemiological studies demonstrate an accelerated rate of progression to dementia and AD in patients with mild cognitive impairment (MCI). We assessed the ApoE allele and genotypes frequencies in Cuban patients with MCI. Methods: We performed ApoE genotyping of 74 Cuban patients more than 65 years old. Cognitive assessments included the Mini-Mental State Examination (MMSE) and a cognitive battery for evaluating memory, attention, perception, and executive function. Results: Cognitive impairments were characterized by amnesia and executive deficits in patients with MCI. The Apo ∊4 allele frequency was 0.196 in patients with MCI, 10-fold higher than that in the controls. Patients carrying the ∊4 allele exhibited poorer performance in MMSE and tests assessing executive function and short-term memory than noncarriers. Conclusions: The patients exhibited amnestic MCI multiple domains. Cognitive performance was worse in patients who carried the ApoE ∊4 allele.

PCR of Immobilized DNA Molecules Isolated from Human Blood Cells by a Non‐Enzymatic Method

Abstract DNA molecules suitable for amplification by Polymerase Chain Reaction were obtained by immobilizing whole blood or isolated leukocytes and incubating the immobilized cells for one hour with the known non‐enzymatic solution described for preparing intact DNA molecules for PFGE. Cell immobilization was done in agarose gels and punches of 1.2 mm of diameter had the amount of DNA needed for amplifying chromosomal and mitochondrial sequences, many times. The approach was successfully used in preparing DNA molecules from multiple samples in flat‐bottom 96‐well ELISA plates. The procedure is simple and does not demand special conditions for sample transportation or conservation; thus, it should be useful to collect and process samples under field conditions in epidemiological studies.

Algorithm to Predict MiniCHEF Electrophoresis Patterns of Entamoeba histolytica DNA

*Departamento de Biologia Molecular, Division de Neurociencias, Centro Nacional de Investigacion Cientifica, Havana, Cuba **Programa de Biomedicina Molecular, Centro de Investigacion en Ciencia Aplicada y Tecnologia Avanzada del I.P.N. (CICATA-IPN), Mexico City, Mexico ***Departamento de Patologia Experimental, ****Programa Multidisciplinario en Biomedicina Molecular, Centro de Investigacion y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico

Identification of small nuclear DNA molecules of the cuban sugarcane cultivar Ja60-5 via pulsed field gel electrophoresis

We present evidence of the existence of at least 7 small nuclear DNA molecules in the Ja60-5 sugarcane cultivar genome. These small molecules were separated by means of transversal alternating field electrophoresis (TAFE); their sizes ranged from about 230–3000 kbp, and they hybridized with telomeric, subcentromeric, and various nuclear DNA probes. Here, we also report the use and the modifications of DNA from microorganisms, to immobilize nuclear DNA from sugarcane suitable for analysis via pulsed field gel electrophoresis. The protocol is simple, inexpensive, and easy to perform.