Isocaloric maternal low-protein diet alters IGF-I,IGFBPs, and hepatocyte proliferation in the fetal rat

We investigated the effect of an isocaloric maternal low-protein diet during pregnancy in rats on the proliferative capacity of cultured fetal hepatocytes. The potential roles of these changes on the IGF-IGF-binding protein (IGFBP) axis, and the role of insulin and glucocorticoids in liver growth retardation, were also evaluated. Pregnant Wistar rats were fed a control (C) diet (20% protein) or a low-protein (LP) diet (8%) throughout gestation. In primary culture, the DNA synthesis of hepatocytes derived from LP fetuses was decreased by approximately 30% compared with control hepatocytes (P < 0.05). In parallel, in vivo moderate protein restriction in the dam reduced the fetal liver weight and IGF-I level in fetal plasma (P < 0.01) and augmented the abundance of 29- to 32-kDa IGFBPs in fetal plasma (P < 0.01) and fetal liver (P < 0.01). By contrast, the abundance of IGF-II mRNA in liver of LP fetuses was unaffected by the LP diet. In vitro, the LP-derived hepatocytes produced less IGF-I (P < 0.01) and more 29- to 32-kDa IGFBPs (P < 0.01) than hepatocytes derived from control fetuses. These alterations still appeared after 3-4 days of culture, indicating some persistence in programming. Dexamethasone treatment of control-derived hepatocytes decreased cell proliferation (54 +/- 2.3%, P < 0.01) and stimulated 29- to 32-kDa IGFBPs, whereas insulin promoted fetal hepatocyte growth (127 +/- 5.5%, P < 0.01) and inhibited 29- to 32-kDa IGFBPs. These results show that liver growth and cell proliferation in association with IGF-I and IGFBP levels are affected in utero by fetal undernutrition. It also suggests that glucocorticoids and insulin may modulate these effects.     

Neuronal untranslated BC1 RNA: targeted gene elimination in mice

Despite the potentially important roles of untranslated RNAs in cellular form or function, genes encoding such RNAs have until now received surprisingly little attention. One such gene encodes BC1 RNA, a small non-mRNA that is delivered to dendritic microdomains in neurons. We have now eliminated the BC1 RNA gene in mice. Three independent founder lines were established from separate embryonic stem cells. The mutant mice appeared to be healthy and showed no anatomical or neurological abnormalities. The gross brain morphology was unaltered in such mice, as were the subcellular distributions of two prototypical dendritic mRNAs (encoding MAP2 and CaMKIIalpha). Due to the relatively recent evolutionary origin of the gene, we expected molecular and behavioral consequences to be subtle. Behavioral analyses, to be reported separately, indicate that the lack of BC1 RNA appears to reduce exploratory activity.     

L-nucleotides and 8-methylguanine of d(C1m8G2C3G4C5LG6LC7G8C9G10)2 act cooperatively to promote a left-handed helix under physiological salt conditions

The structure and thermal stability of a hetero chiral decaoligodeoxyribonucleotide duplex d(C1m8 G2C3G4C5LG6LC7G8C9G10)d(C11m8G12C13G14C15LG16LC17G18C19G20) (O1) with two contiguous pairs of enantiomeric 2'-deoxy-L-ribonucleotides (C5LG6L/C15LG16L) at its centre and an 8-methylguanine at position 2/12 was analysed by circular dichroism, NMR and molecular modelling. O1 resolves in a left-handed helical structure already at low salt concentration (0.1 M NaCl). The central L2-sugar portion assumes a B* left-handed conformation (mirror-image of right-handed B-DNA) while its flanking D4-sugar portions adopt the known Z left-handed conformation. The resulting Z4-B2*-Z4 structure (left-handed helix) is the reverse of that of B4-Z2*-B4 (right-handed helix) displayed by the nearly related decaoligodeoxyribonucleotide d(mC1G2mC3G4C5L G6LmC7G8mC9G10)2, at the same low salt concentration (0.1 M NaCl). In the same experimental conditions, d(C1m8G2C3G4C5G6C7G8C9G10)2 (O2), the stereoregular version of O1, resolves into a right-handed B-DNA helix. Thus, both the 8-methylguanine and the enantiomeric step CLpGL at the centre of the molecule are needed to induce left-handed helicity. Remarkably, in the various heterochiral decaoligodeoxyribonucleotides so far analysed by us, when the central CLpGL adopts the B* (respectively Z*) conformation, then the adjacent steps automatically resolves in the Z (respectively B) conformation. This allows a good optimisation of the base-base stackings and base-sugar van der Waals interactions at the ZB*/B*Z (respectively BZ*/Z*B) junctions so that the Z4-B2*-Z4 (respectively B4-Z2*-B4) helix displays a Tm (approximately 65 degrees C) that is only 5 degrees C lower than the one of its homochiral counterpart. Here we anticipate that a large variety of DNA helices can be generated at low salt concentration by manipulating internal factors such as sugar configuration, duplex length, nucleotide composition and base methylation. These helices can constitute powerful tools for structural and biological investigations, especially as they can be used in physiological conditions.     

The N-terminal adenosine triphosphate binding domain of Hsp90 is necessary and sufficient for interaction with estrogen receptor

To understand how the molecular chaperone Hsp90 participates in conformational maturation of the estrogen receptor (ER), we analyzed the interaction of immobilized purified avian Hsp90 with mammalian cytosolic ER. Hsp90 was either immunoadsorbed to BF4 antibody-Sepharose or GST-Hsp90 fusion protein (GST.90) was adsorbed to glutathione-Sepharose. GST.90 was able to retain specifically ER, similarly to immunoadsorbed Hsp90. When cells were treated with estradiol and the hormone treatment was maintained during cell homogenization, binding, and washing steps, GST.90 still interacted efficiently with ER, suggesting that ER may form complexes with Hsp90 even after its activation by hormone and salt extraction from nuclei. The GST.90-ER interaction was consistently reduced in the presence of increasing concentrations of potassium chloride or when cytosolic ER-Hsp90 complexes were previously stabilized by molybdate, indicating that GST.90-ER complexes behave like cytosolic Hsp90-ER complexes. A purified thioredoxin-ER fusion protein was also able to form complexes with GST.90, suggesting that the presence of other chaperones is not required. ER was retained only by GST.90 deletion mutants bearing an intact Hsp90 N-terminal region (1-224), the interaction being more efficient when the charged region A was present in the mutant (1-334). The N-terminal fragment 1-334, devoid of the dimeric GST moiety, was also able to interact with ER, pointing to the monomeric N-terminal adenosine triphosphate binding region of Hsp90 (1-224) as the region necessary and sufficient for interaction. These results contribute to understand the Hsp90-dependent process responsible for conformational competence of ER.     

Spatial codes in dendritic BC1 RNA

BC1 RNA is a dendritic untranslated RNA that has been implicated in local translational control mechanisms in neurons. Prerequisite for a functional role of the RNA in synaptodendritic domains is its targeted delivery along the dendritic extent. We report here that the targeting-competent 5' BC1 domain carries two dendritic targeting codes. One code, specifying somatic export, is located in the medial-basal region of the 5' BC1 stem-loop structure. It is defined by an export-determinant stem-bulge motif. The second code, specifying long-range dendritic delivery, is located in the apical part of the 5' stem-loop domain. This element features a GA kink-turn (KT) motif that is indispensable for distal targeting. It specifically interacts with heterogeneous nuclear ribonucleoprotein A2, a trans-acting targeting factor that has previously been implicated in the transport of MBP mRNA in oligodendrocytes and neurons. Our work suggests that a BC1 KT motif encodes distal targeting via the A2 pathway and that architectural RNA elements, such as KT motifs, may function as spatial codes in neural cells.