Milk from dams fed an obesogenic diet combined with a high-fat/high-sugar diet induces long-term abnormal mammary gland development in the rabbit.

Alterations to the metabolic endocrine environment during early life are crucial to mammary gland development. Among these environmental parameters, the initial nutritional event after birth is the consumption of milk, which represents the first maternal support provided to mammalian newborns. Milk is a complex fluid that exerts effects far beyond its immediate nutritional value. The present study, therefore, aimed to determine the effect of the nutritional changes during the neonatal and prepubertal periods on the adult mammary phenotype. Newborn rabbits were suckled by dams fed a high-fat/high-sugar obesogenic (OD) or a control (CON) diet and then subsequently fed either the OD or CON diets from the onset of puberty and throughout early pregnancy. Mammary glands were collected during early pregnancy (Day 8 of pregnancy). Rabbits fed with OD milk and then subjected to an OD diet displayed an abnormal development of the mammary gland: the mammary ducts were markedly enlarged (P < 0.05) and filled with abundant secretory products. Moreover, the alveolar secretory structures were disorganized, with an abnormal aspect characterized by large lumina. Mammary epithelial cells contained numerous large lipid droplets and exhibited fingering of the apical membrane and abnormally enlarged intercellular spaces filled with casein micelles. Leptin has been shown to be involved in modulating several developmental processes. We therefore analyzed its expression in the mammary gland. Mammary leptin mRNA was strongly expressed in rabbits fed with OD milk and subjected to an OD diet by comparison with the CON rabbits. Leptin transcripts and protein were localized in the epithelial cells, indicating that the increase in leptin synthesis occurs in this compartment. Taken together, these findings suggest that early-life nutritional history, in particular through the milking period, can determine subsequent mammary gland development. Moreover, they highlight the potentially important regulatory role that leptin may play during critical early-life nutritional windows with respect to long-term growth and mammary function.

Real-time study of interactions between a composite DNA regulatory region (HIV-1 LTR NRE) and several transcription factors of nuclear extracts.

Here we describe the first real-time study of nuclear protein interaction with a composite DNA regulatory region. We studied the interplay between the three target sites of the negative regulatory element (NRE) of HIV-1 LTR, comprising a noncanonical GATA site overlapping two negative regulatory regions, USF and NFIL-6, and their corresponding transcription factors in nuclear extracts. By bandshift analysis, no GATA binding activity could be detected between LTR NRE and different nuclear extracts, although evidenced by in vitro footprinting. Additionally, the LTR NRE and a USF oligonucleotide showed identical retarded complexes. BIAcore study of these interactions revealed the binding of huGATA-3, as well as USF, to the immobilized LTR NRE oligonucleotide. Competition analyses, performed with GATA, USF, and NFIL-6 oligonucleotides, clearly showed that this regulatory region could bind both huGATA-3 and USF factors. Finally, the presence of USF and huGATA-3 proteins in the complexes formed with LTR NRE was ascertained using specific anti-huGATA-3 and anti-USF2 polyclonal antibodies.

ATP hydrolysis-dependent formation of a dynamic ternary nucleoprotein complex with MutS and MutL.

Functional interactions of Escherichia coli MutS and MutL in mismatch repair are dependent on ATP. In this study, we show that MutS and MutL associate with immobilised DNA in a manner dependent on ATP hydrolysis and with an ATP concentration near the solution K m of the ATPase of MutS. After removal of MutS, MutL and ATP, much of the protein in this ternary complex is not stably associated, with MutL leaving the complex more rapidly than MutS. The rapid dissociation reveals a dynamic interaction with concurrent rapid association and dissociation of proteins from the DNA. Analysis by surface plasmon resonance showed that the DNA interacting with dynamically bound protein was more resistant to nuclease digestion than the DNA in MutS-DNA complexes. Non-hydrolysable analogs of ATP inhibit the formation of this dynamic complex, but permit formation of a second type of ternary complex with MutS and MutL stably bound to the immobilised DNA.

Analysis of interactions between huGATA-3 transcription factor and three GATA regulatory elements of HIV-1 long terminal repeat, by surface plasmon resonance.

Relative affinities of transcriptional regulatory elements for their respective factor have been essentially studied by bandshift analysis. Here we report a real-time study of factor/DNA interactions using a surface plasmon resonance approach and further characterization of recovered proteins involved in this interaction. For this purpose, human GATA-3, either recombinant or in nuclear extracts, and three natural GATA elements of the HIV-1 long terminal repeat (sites 1, 2, and 3) were chosen, in which only site 2 is a noncanonical GATA site. Direct analysis of sensorgrams, with recombinant huGATA-3, allowed the comparison of association and dissociation profiles of the three DNA regions and their ranking according to their relative affinities. This result, confirmed by competitions with each GATA site, demonstrated the higher relative affinity (at least sevenfold) of site 3. Interactions between the canonical and unique GATA site 3 and nuclear extracts were also studied in real time and provided information on its association and dissociation rates for native huGATA-3. Finally, recovered protein was identified as genuine huGATA-3 by SDS-PAGE, Western blotting, and bandshift assays.