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Biomedical subjects

A Ahumada

Publications and source records attributed to A Ahumada.

10 recordsLinked to original sources
PubMed1998

The Zn(II) binding motifs of E. coli DNA topoisomerase I is part of a high-affinity DNA binding domain.

Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs. Three subclones containing these tetracysteine motifs were expressed and purified. Subclone ZD1 contained the minimal tetracysteine motifs sequence. A larger subclone ZD2 corresponded to a region bordered by two protease sensitive sites. Subclone ZD3 also included the 14-kDa C-terminal domain that has been shown to bind DNA. Subclones ZD1 and ZD2 were found to bind one and two Zn(II), respectively, and neither had detectable DNA binding activity. ZD3 could bind three Zn(II) and had higher DNA binding affinity than the 14-kDa C-terminal domain. The complex formed between ZD3 and a single-stranded 31mer could be detected by the gel shift assay while the complex formed by the 14-kDa C-terminal domain was not stable under gel electrophoresis conditions. The three Zn(II) binding motifs appeared to be part of a high-affinity DNA binding domain.

Amino Acid Sequence↗
PubMed1995

Expression and DNA-binding properties of the 14K carboxyl terminal fragment of Escherichia coli DNA topoisomerase I.

DNA sequence coding for the last 121 amino acids of Escherichia coli topoisomerase I was synthesized by PCR and cloned into a plasmid under the control of the T7 promoter. Induction of T7 RNA polymerase in E. coli carrying the plasmid clone resulted in over-expression of this C-terminal domain fragment previously shown to confer higher DNA binding affinity to the enzyme. Purification to homogeneity was achieved by phosphocellulose and single-stranded DNA agaraose chromatography. Direct interaction between this 14K domain and poly(dA) was demonstrated by UV spectroscopy. Noncovalent complexes formed between this fragment and oligo(dT) 8 and oligo(dT) 16 can also be trapped by photo-crosslinking.

Bacteriophage T7↗
PubMed1994

A new microassay for the determination of alkaline phosphatase activity in early mouse concepti.

A new enzymatic assay for the determination of alkaline phosphatase (APase) activity in preimplantation mouse concepti is described. This method allows estimation of APase activity of concepti extracts using the fluorogenic substrate, 3,6-fluorescein diphosphate (FDP). For measuring APase activity, 0.1% Nonidet P-40 was used to solubilize the enzyme. Control assays showed that this procedure does not modify the enzyme activity. According to the Km obtained for APase from mouse concepti (between 1-2 microM), the initial concentration of FDP was 20 microM, which is 10 fold the Km. The assay sensitivity allows continuous recording of the product generated and a reliable determination in less than 20 min. Results show that APase activity in mouse concepti may be detected from the 2-cell stage, increasing exponentially towards the blastocyst stage.

Alkaline Phosphatase↗
PubMed1992

Sealed reticulocyte ghosts. An experimental model for the study of Fe2+ transport.

Sealed right-side-out reticulocyte ghosts transported and accumulated iron offered as 59Fe(2+)-ascorbate (Km = 1.1 microM). The uptake of iron by ghosts presented the characteristics of a transporter-mediated process: it responded to osmotic challenge, the rate of transport increased when iron was present in the opposing side, and the transport rate showed the temperature dependence typical of membrane-mediated processes. The transport of iron was dependent on an associated influx of Cl- in order to keep electroneutrality. Other transition metals, such as Cu2+, Zn2+, and Co2+, inhibited the transport of Fe2+. The overall characteristics of the system make reticulocyte sealed ghosts a very useful model in determining the basic mechanisms of membrane iron transport.

Animals↗
PubMed1992

In vitro effect of hCG on steroidogenesis in the testicular tissue from a patient with complete androgen resistance.

The present study was performed to determine the in vitro steroidogenic capacity of a gonadal sample from a patient suffering from a complete androgen resistance syndrome. Testosterone and estradiol production by the testicular tissue from this patient as well as gonadotropin binding to a membrane fraction prepared from this tissue were measured. hCG bound with high affinity but with a very low capacity and the gonadotropin induced a clear dose response for both testosterone and estradiol production. The ED50 of hCG on testosterone and estradiol production were 2.5 and 5.0 nM, respectively. We conclude that estradiol originates from Leydig cell activity, since estradiol synthesis does not depend on testosterone availability and it shows a clear hCG dose response.

Adolescent↗