Effects of ageing on the energy balance of food-restricted rats.

AIMS: Age can alter energy balance by decreasing the resting metabolic rate. Food restriction can also change energy balance by decreasing energy expenditure as a mechanism of energy conservation. We investigated the influence of food restriction on the energy balance of rats at different ages. METHODS: Wistar EPM-1 female rats were used at ages of 3, 9, 15 and 21 months. At each age, two food intake schedules were provided: control (ad libitum) and food restriction (50%). Animals remained under these schedules for 30 days, and throughout this period body weight, food intake, and stool collection were controlled daily. On the 30th day, animals were killed, blood was collected and the carcasses and faeces were processed for analysis by pump calorimetry. Blood glucose, T(3), T(4) and rT(3) levels were determined. RESULTS: Food restriction reduced energy gain and gross food efficiency of animals at different ages, but more so in older animals. Food-restricted rats also had lower energy expenditure than controls. This reduction was about 40% of the energy expenditure of control animals irrespective of age. Water content increased and fat content decreased in the carcass of food-restricted animals. Serum T(3) and T(4) levels were lower in food-restricted animals pointing out to a major role of thyroid hormones in the mechanism of energy conservation exhibited by food-restricted animals. CONCLUSIONS: The mechanism of energy conservation takes place in all restricted animals and is very important for survival and for species preservation, mainly in aged animals in which food restriction is frequently aggravated by senescence-related organic disorders.

Comparación de los valores de cortisol en sangre de cordón umbilical en pacientes con trabajo de parto inducido y espontáneo / Comparison of cortisol levels in umbilical cord blood in patients in induced and spontaneous labor

Objetivo: Observar si existe alguna variación del cortisol fetal medido en sangre de cordón umbilical entre trabajos de partos desencadenados espontáneamente y trabajos de partos inducidos con prostaglandinas.Material y método: Se estudiaron 73 embarazadas que concurrieron al Sanatorio Allende desde el 1 de julio al 30 de octubre de 1999. La mayoría eran primigestas, primíparas de 39 ñ 1 semanas de gestación, y con una edad promedio de 27,3 ñ 5,2 años. Todas cursaron embarazos normales. Se tuvo en cuenta las horas de trabajo de parto, la medicación, la inducción (considerando la dosis de misoprostol administrada), la frecuencia y la duración de las contracciones, las complicaciones del parto, el sexo, el test de Apgar y la hora de nacimiento del recién nacido. El grupo control incluyó a embarazadas con trabajos de parto espontáneos, y el grupo experimental los inducidos, comparándose los valores de cortisol en sangre fetal.Resultados: Se observó, analizando ambos grupos, una mayor edad materna en los partos inducidos, una menor proporción de primigestas, un menor promedio de semanas de gestación, un mayor número de embarazos de más de 40 semanas, una mayor duración del trabajo de parto, una menor utilización de oxitocina, un mayor uso de bromuro de hioscina, una mayor cantidad de episodios de polisistolia, hiperdinamia y más circulares de cordón. Al ser estas variables distintas en ambos grupos, se realizó un proceso de ajuste, con lo cual se observó una ligera diferencia no significativa con respecto a las horas de trabajo de parto y las semanas de gestación. No hubo diferencias significativas con respecto a la variable inducción. Como único punto, cabe resaltar el valor de cortisol en referencia a la paridad (las primigestas más elevado) y el sexo del feto (el femenino más elevado) tomando a ambos grupos como uno, al realizar el proceso de ajuste de las diferencias.Conclusiones: No se evidencia diferencia significativa en trabajos de partos inducidos con prostaglandinas con respecto a los espontáneos. Al ajustar las diferencias entre ambos grupos, se observó un aumento del cortisol significativo en fetos de sexo femenino (atribuible al azar) y, de igual modo, en primigestas, pudiéndose explicar a causa de que el período expulsivo es más largo (AU)

Synthesis, cytotoxicity, antitumor activity and sequence selective binding of two pyrazole analogs structurally related to the antitumor agents U-71,184 and adozelesin.

Two pyrazole analogs structurally related to the antitumor agents adozelesin and U-71,184 respectively were synthesized. By using a polymerase chain reaction approach, both compounds show selective binding to A + T rich sequences exactly as reference compound U-71,184. In in vitro assays, against L1210 cell lines, both derivatives showed cytotoxicity in the pM range, values comparable with the natural target compound (+)-CC-1065. The most active compound showed very high antitumor activity in mice implanted with L1210 cells (ILS% 363).

Targeting of the HIV-1 long terminal repeat with chromomycin potentiates the inhibitory effects of a triplex-forming oligonucleotide on Sp1-DNA interactions and in vitro transcription.

We have studied the effects of chromomycin and of a triple-helix-forming oligonucleotide (TFO) that recognizes Sp1 binding sites on protein-DNA interactions and HIV-1 transcription. Molecular interactions between chromomycin, the Sp1 TFO and target DNA sequences were studied by gel retardation, triplex affinity capture using streptavidin-coated magnetic beads and biosensor technology. We also determined whether chromomycin and a TFO recognizing the Sp1 binding sites of the HIV-1 long terminal repeat (LTR) inhibit the activity of restriction enzyme HaeIII, which recognizes a sequence (5'-GGCC-3') located within these Sp1 binding sites. The effects of chromomycin and the TFO on the interaction between nuclear proteins or purified Sp1 and a double-stranded oligonucleotide containing the Sp1 binding sites of the HIV-1 LTR were studied by gel retardation. The effects of both chromomycin and TFO on transcription were studied by using an HIV-1 LTR-directed in vitro transcription system. Our results indicate that low concentrations of chromomycin potentiate the effects of the Sp1 TFO in inhibiting protein-DNA interactions and HIV-1-LTR-directed transcription. In addition, low concentrations of chromomycin do not affect binding of the TFO to target DNA molecules. The results presented here support the hypothesis that both DNA binding drugs and TFOs can be considered as sequence-selective modifiers of DNA-protein interactions, possibly leading to specific alterations of biological functions. In particular, the combined use of chromomycin and TFOs recognizing Sp1 binding sites could be employed in order to abolish the biological functions of promoters (such as the HIV-1 LTR) whose activity is potentiated by interactions with the promoter-specific transcription factor Sp1.

In vitro and in vivo binding of a CC-1065 analogue to human gene sequences: a polymerase-chain reaction study.

In this paper we analyse the in vitro sequence selectivity of the CC-1065 analogue 2-[[5-[(1H-indol-2-yl]carbonyl)-1H-indol-2-yl] carbonyl]-7-methyl-1,2,8,8a-tetrahydrocyclopropa [c]-pyrrolo-[3,2-e]-indol-4-one (U-71184) employing the polymerase-chain reaction (PCR). In addition, we determined whether alteration of PCR by U-71184 is detected when DNA is isolated from cells cultured in the presence of this drug. As molecular model systems we employed the human estrogen receptor gene, the Ha-ras oncogene and the chromosome X-linked, (CGG)-rich fragile X mental retardation-1 gene. The first conclusion that can be drawn from the experiments reported in our paper is that U-71184 inhibits PCR in a sequence-dependent manner. A second conclusion of our experiments is that PCR performed on DNA from U-71184-treated cells is inhibited when the primers amplifying the estrogen receptor gene region are used. This approach might bring important information on both in vivo uptake of the drug by target cells and binding to DNA.

Targeting of the Sp1 binding sites of HIV-1 long terminal repeat with chromomycin. Disruption of nuclear factor.DNA complexes and inhibition of in vitro transcription.

Sequence selectivity of DNA-binding drugs has recently been reported in a number of studies employing footprinting and gel retardation approaches. In this paper, we studied the biochemical effects of the sequence-selective binding of chromomycin to the long terminal repeat of the human immunodeficiency type I virus. Deoxyribonuclease I (E.C.3.1.21.1) footprinting, arrested polymerase chain reaction, gel retardation and in vitro transcription experiments have demonstrated that chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1. Accordingly, interactions between nuclear proteins and Sp1 binding sites are inhibited by chromomycin, and this effect leads to a sharp inhibition of in vitro transcription.

Alteration of the expression of human estrogen receptor gene by distamycin.

The effects of distamycin on the expression of the estrogen receptor gene were determined in the MCF7 human breast cancer cell line. Estrogen receptor (ER) RNA transcripts were analyzed by Northern blotting and RT-PCR using specific oligonucleotides for the 5' upstream region and for ER cDNA. After ex vivo distamycin treatment of the cells the expression of the canonical ER mRNA isoform of 6.3 kb is strongly inhibited, without appreciable alteration of the accumulation of 5' upstream ER mRNA isoforms. These results suggest that distamycin alters the transcriptional activity of the ER gene causing a change in the ratio between the canonical transcript and other isoforms containing 5' upstream regions.

Binding of distamycin and chromomycin to human immunodeficiency type 1 virus DNA: a non-radioactive automated footprinting study.

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we studied sequence-selectivity of the binding of chromomycin and distamycin to DNA by performing DNase I footprinting and analysis of the cleaved fragments by the Pharmacia ALF DNA Sequencing System. As a model system we employed the long terminal repeat of the human immunodeficiency type 1 virus. The main conclusion of our experiments is that automated analysis of DNase I footprinting is a fast and reliable technique to study drugs-DNA interactions. The results obtained suggest that distamycin and chromomycin differentially interact with the long terminal repeat of the human immunodeficiency type 1 virus; this differential binding depends upon the DNA sequences recognized. The data presented are consistent with a preferential binding of distamycin to DNA sequences of the binding sites of nuclear factor kappa B and transcription factor IID. By contrast, distamycin exhibits only weak binding to DNA sequences recognized by the promoter-specific transcription factor Sp1. Unlike distamycin, chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1.

Differential effects of distamycin analogues on amplification of human gene sequences by polymerase-chain reaction.

In this report we analyse the effects of distamycin and five distamycin analogues on amplification by polymerase-chain reaction (PCR) of two gene sequences displaying a different A+T/G+C content. The first was a 5' region of the human oestrogen receptor (ER) gene, containing a (TA)26 stretch; the second was a CG-rich sequence of the human Ha-ras oncogene. The results obtained unequivocally demonstrate that the addition of one pyrrole ring significantly improves the ability of distamycin derivatives to interfere with PCR-mediated amplification of the human ER genomic region carrying a (TA)26 stretch. The distamycin analogues analysed differ in the number of pyrrole rings and in the presence of an N-formyl, an N-formimidoyl or a retroamide group at position X1. Among compounds carrying the same number of pyrrole rings, those carrying an N-formyl or an N-formimidoyl group retain a similar inhibitory activity. The retroamide analogues, on the contrary, are much less efficient in inhibiting PCR-mediated amplification of the 5'ER region. With respect to sequence selectivity both distamycin and distamycin analogues exhibit a sequence preference, since they do not inhibit PCR amplification of Ha-ras CG-rich gene regions, with the exception of a distamycin analogue carrying four pyrrole rings.

Polymerase-chain reaction as a tool for investigations on sequence-selectivity of DNA-drugs interactions.

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we performed polymerase-chain reaction (PCR) experiments to study the in vitro effects of distamycin, daunomycin, chromomycin and mithramycin. As model systems we employed the human estrogen receptor (ER) gene and the Harvey-ras (Ha-ras) oncogene, in order to obtain PCR products significantly differing for the A + T/G + C frequency ratio. Distamycin, daunomycin, chromomycin and mithramycin are indeed known to differentially bind to different DNA regions depending upon the DNA sequences recognized. The main conclusion of our experiments is that distamycin, daunomycin, chromomycin and mithramycin inhibit polymerase-chain reaction in a sequence-dependent manner. Distamycin inhibits indeed PCR mediated amplification of AT-rich regions of the human estrogen receptor gene, displaying no inhibitory effects on PCR-mediated amplification of GC-rich sequences of Ha-ras oncogene. By contrast daunomycin, chromomycin and mithramycin were found to inhibit PCR-mediated amplification of the Ha-ras GC-rich oncogene sequences. We propose that polymerase-chain reaction technique could be applied to study the in vivo interactions of DNA-binding drugs to specific genes in intact cells.