A regime shift in the Sun-Climate connection with the end of the Medieval Climate Anomaly.

Understanding the influence of changes in solar activity on Earth's climate and distinguishing it from other forcings, such as volcanic activity, remains a major challenge for palaeoclimatology. This problem is best approached by investigating how these variables influenced past climate conditions as recorded in high precision paleoclimate archives. In particular, determining if the climate system response to these forcings changes through time is critical. Here we use the Wiener-Granger causality approach along with well-established cross-correlation analysis to investigate the causal relationship between solar activity, volcanic forcing, and climate as reflected in well-established Intertropical Convergence Zone (ITCZ) rainfall proxy records from Yok Balum Cave, southern Belize. Our analysis reveals a consistent influence of volcanic activity on regional Central American climate over the last two millennia. However, the coupling between solar variability and local climate varied with time, with a regime shift around 1000-1300 CE after which the solar-climate coupling weakened considerably.

Antisense targeting of oxytocinergic hypothalamus neurons induces cytoplasmic triple helix-like immunoreactivity.

Intracerebroventricular injections of oligonucleotide probes complementary to oxytocin mRNA are known to decrease systemic oxytocin levels. In this study we show that immunoreactive oxytocin in the magnocellular hypothalamic perikarya and in their neurohypophysial projections remains unaffected by intracerebroventricular injections with an oxytocin antisense probe in rats. Hybridization signal for oxytocin mRNA was increased in the supraoptic and paraventricular nuclei in these animals. Immunocytochemistry with a monoclonal antibody, raised against triple helical DNA resulted in an accumulation of cytoplasmic reaction product in many of the magnocellular oxytocin immunoreactive neurons and in a fraction of the Herring bodies inthe posterior pituitary lobe in the antisense treated rats. Such immunostaining could be abolished by pretreating sections with RNase H. Animals injected with a mismatch probe instead of the antisense probe were devoid of cytoplasmic or axonal triple helix immunostaining. Our findings indicate that oxytocinergic transcripts in magnocellular hypothalamic neurons form triple helix-like aggregates upon specific antisense targeting rather than being degraded by endogenous RNases. While de novo transcription of oxytocin is probably stimulated, systemic release of the nonapeptide may be impaired.

Vitamin D receptor and retinoid X receptor interactions in motion.

Vitamin D receptor (VDR) and retinoid X receptor (RXR) are members of the nuclear receptor superfamily and they bind target DNA sequences as heterodimers to regulate transcription. This article surveys the latest findings regarding the roles of dimerizing RXR in VDR function and emphasizes potential areas for future developments. We first highlight the importance of dimerization with RXR for both the ligand-independent (hair growth) and ligand-dependent functions of VDR (calcium homeostasis, bone development and mineralization, control of cell growth and differentiation). Emerging information regarding the regulatory control of dimerization based on biochemical, structural, and genetic studies is then presented. Finally, the main focus of this article is a new dynamic perspective of dimerization functions, based on recent research with fluorescent protein chimeras in living cells by microscopy. These studies revealed that both VDR and RXR constantly shuttle between the cytoplasm and the nucleus and between subnuclear compartments, and showed the transient nature of receptor--DNA and receptor--coregulator interactions. Because RXR dimerizes with most of the nuclear receptors, regulation of receptor dynamics by RXR has a broad significance.

Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the limbic system of the rat.

We used immunocytochemistry to obtain a complete cellular and subcellular mapping of the 1,25-dihydroxyvitamin D3 receptor protein (VDR) in the rat limbic system. We observed specific VDR immunostaining in the nucleus as well as in the perinuclear cytoplasm of neuronal cells. The limbic system consists of a variety of neuronal structures, and is known to have influence on memory, behavior, emotions and reproduction. In the hippocampal formation, we found strong nuclear staining as well as less distinguished cytoplasmic VDR staining in CA1, CA3 and CA4. The CA2 area showed a unique cytoplasmic predominance of VDR. The amygdala was found to exhibit specific patterns of VDR distribution in the various regions of the nucleus. We observed distinct differences of VDR localization within the limbic preoptic areas of the hypothalamus. Further parts of the brain we analyzed included the mammillary bodies, the indusium griseum and the cingulate cortex. The subcellular distribution of VDR in regions of the limbic system suggests a specific functional role of the receptor protein and indicates a role for calcitriol as a neuroactive steroid.

Dimerization with retinoid X receptors promotes nuclear localization and subnuclear targeting of vitamin D receptors.

The vitamin D receptor (VDR) acts as heterodimer with the retinoid X receptor alpha (RXR) to control transcriptional activity of target genes. To explore the influence of heterodimerization on the subcellular distribution of these receptors in living cells, we developed a series of fluorescent-protein chimeras. The steady-state distribution of the yellow fluorescent protein-RXR was more nuclear than the unliganded green fluorescent protein (GFP)-VDR. Coexpression of RXR-blue fluorescent protein (BFP) promoted nuclear accumulation of GFP-VDR by influencing both nuclear import and retention. Fluorescence resonance energy transfer microscopy (FRET) demonstrated that the unliganded GFP-VDR and RXR-BFP form heterodimers. The increase in nuclear heterodimer content correlated with an increase in basal transcriptional activity. FRET also revealed that calcitriol induces formation of multiple nuclear foci of heterodimers. Mutational analysis showed a correlation between hormone-dependent nuclear VDR foci formation and DNA binding. RXR-BFP also promoted hormone-dependent nuclear accumulation and intranuclear foci formation of a nuclear localization signal mutant receptor (nlsGFP-VDR) and rescued its transcriptional activity. Heterodimerization mutant RXR failed to alter GFP-VDR and nlsGFP-VDR distribution or activity. These experiments suggest that RXR has a profound effect on VDR distribution. This effect of RXR to promote nuclear accumulation and intranuclear targeting contributes to the regulation of VDR activity and probably the activity of other heterodimerization partners.

Rat oligodendrocytes express the vitamin D(3) receptor and respond to 1,25-dihydroxyvitamin D(3).

The present study investigates the presence of vitamin D receptor (VDR) in cells of the rat oligodendrocyte (OL) lineage. VDR transcripts were detected by in situ hybridization in a fraction of rat OL in secondary cultures. The VDR protein was shown to be co-localized in cells that are also recognized by an anti-myelin basic protein (MBP) antibody. Likewise, in vivo, VDR-positive cells were found in the brain white matter, such as the internal capsule of the striatum or the corpus callosum but also in the spinal cord. At least part of these positive cells in vivo correspond to OL, since they were co-stained by an anti-carbonic anhydrase II antiserum. Northern blot analyses of the CG-4 OL cell line demonstrated that the VDR transcripts are already found in the O-2A precursors. There was a two-fold increase in the relative abundance of these transcripts in differentiated OL or in type-2 astrocytes. 1, 25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] increased the pool of transcripts encoding its own receptor, the VDR. The hormone also enhanced the abundance of the mRNA of the nerve growth factor (NGF) and of its low-affinity receptor, the p75(NTR) protein. By contrast, the hormone had no effect on the levels of MBP or proteolipid protein (PLP) mRNA. This finding suggests that unlike retinoic acid (RA) or thyroid hormone, 1,25-(OH)(2)D(3) has no regulatory action on the synthesis of myelin proteins.

Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat olfactory system.

1. The rat olfactory system contains numerous target sites for 1,25-dihydroxyvitamin D3, as determined by receptor protein (VDR) immunocytochemistry and in situ hybridization. 2. Nuclear and cytoplasmic VDR immunoreactivity as well as the corresponding hybridization signal was observed in neurons in the olfactory epithelium, the olfactory bulb, and throughout the limbic system in locations also known to be glucocorticoid targets. 3. The widespread distribution of VDR indicates the distinct functional importance of 1,25-dihydroxyvitamin D3 for olfactory perception.

Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord.

A complete mapping study on the 1,25-dihydroxyvitamin D3 receptor immunoreactivity within the rat central nervous system was performed with a monoclonal and a polyclonal antibody. Specific immunostaining was observed within both nuclear and cytoplasmic compartments of a variety of cells in the cerebellum, mesopontine area, diencephalon, cortex, spinal cord, and limbic system. Both monoclonal and polyclonal antibodies provided similar staining patterns. The monoclonal antibody stained distinct domains within the nuclei of all and the cytoplasm of specific neuronal cell types, like motor neurons, Purkinje cells, and pyramidal cells of the cortex more clearly than the polyclonal antibody. The expression of vitamin D3 receptor in the rat central nervous system was confirmed by in situ hybridisation. The widespread distribution of vitamin D3 receptor in distinct portions of the sensory, motor, and limbic brain systems suggests multiple functional properties of 1,25-dihydroxyvitamin D3 in the central nervous system.

1,25-Dihydroxyvitamin D3 receptors in the central nervous system of the rat embryo.

We have mapped areas within the central nervous system (CNS) of the developing fetal rat which immunostain for the 1,25-dihydroxyvitamin D3 receptor (VDR). The VDR was detected from days 12 to 21 of gestation throughout the CNS; immunostaining was particularly intense in the neuroepithelium and within the differentiating fields of various areas of the brain. Cells within the spinal cord, dorsal root, and other ganglia exhibited positive staining for the VDR. The intensity of staining for the VDR diminished or disappeared in the neuroepithelium throughout the CNS during the later days of development, while in the differentiating fields single VDR immunoreactive cells were observed. The presence of the VDR in the CNS was confirmed by in situ hybridization and RNA-based polymerase chain reaction methods with di-deoxy sequencing of the resultant DNA product. These results support the hypothesis that 1, 25-dihydroxyvitamin D3, through interactions with the VDR, may play a role in the development of the CNS.

1.25-Dihydroxyvitamin D3 receptor is partly colocalized with oxytocin immunoreactivity in neurons of the male rat hypothalamus.

With receptor immunocytochemistry, neurons receptive for the steroidhormone 1.25-dihydroxyvitamin D3 have been observed in hypothalamic nuclei. In the present paper we report that a fraction of 1.25-dihydroxyvitamin D3 receptor (VDR) immunoreactive neurons in the hypothalamus of male rats are immunoreactive for oxytocin (OT), suggesting a direct genomic action of this steroid on OT expression. While only 10% of neurons with OT immunofluorescence in the periventricular nucleus contained nuclear VDR immunostaining, up to 50% of the OT neurons in the supraoptic nucleus and 30% in the magnocellular portion of the paraventricular nucleus were VDR positive. VDR immunostaining in the magnocellular nuclei was in many cases confined to the perinuclear cytoplasm. We assume that 1.25-dihydroxyvitamin D3 has effects on hypothalamic peptidergic systems similar to other steroid hormones.

Liposomal incorporation changes the effect of 1.25-dihydroxyvitamin D3 on the phospholipase C signal transduction pathway and the eicosanoid cascade on keratinocytes in vitro.

1.25-dihydroxyvitamin D3 is of clinical importance (e.g. in the treatment of psoriasis) given its ability to regulate the proliferation and differentiation of human keratinocytes. 1.25-Dihydroxyvitamin D3 mediates its action via genomic and nongenomic pathways. The nongenomic actions begin with the activation of phospholipase C and the subsequent rapid rise in calcium within the cells. We incorporated 1.25-dihydroxyvitamin D3 in liposomes of varying compositions in an attempt to improve their effect/negative side effect ratio. The influence of empty liposomes (1 mM) and free and liposomally incorporated 1.25-dihydroxyvitamin D3 (10 nM) on the rapid release of sulfidoleucotrien and inositole 1,4,5 triphosphate was examined in keratinocytes in vitro. Free 10 nM 1.25-dihydroxyvitamin D3 provoked a rapid rise in sulfidoleucotriens within 30 seconds, followed by a swift decrease in sulfidoleucotrien and inositole 1,4,5-triphosphate concentration after 10 minutes. Empty liposomes and liposomal-incorporated 1.25-dihydroxyvitamin D3 did not show such a strong effect. These results suggest the occurrence of specific binding sites for 1.25-dihydroxyvitamin D3 on the membrane level that are incapable of recognizing 1.25-dihydroxyvitamin D3 trapped within liposomal membrane.

Synthetic wound dressings--evaluation of interactions with epithelial and dermal cells in vitro.

Comparative testing of seven wound dressings (WD) has been performed with human HaCaT keratinocyte and mouse 3T3 fibroblast cultures. To assess biocompatibility, morphologic examinations were combined with cell counting. Supernatants were subjected to measurements of tissue peptide antigen (TPS), soluble intercellular adhesion molecule 1 (ICAM-1), and interleukins (IL-1 alpha, -1 beta, -6). Furthermore, monoxygenation, the reduced glutathione/oxidized gluthathione (GSH/GSSG) ratio and lipid peroxides were determined. Initial morphologic events were noted within the first day of exposure to WD. After 72 h, inhibition of cell growth was observed in the presence of hydrocolloids and hydrogels. The cytochrome-P-450-dependent ethoxyresorufin 0-deethylation rate and the GSH/GSSG ratio were not altered by WD in HaCaT cells. Lipid peroxide generation, IL-1 and ICAM-1 were scarcely detectable. TPS and IL-6 release indicate the presence of an 'activated stage' of keratinocytes and fibroblasts exposed to WD. Peptide release in vivo may contribute to the beneficial effects of modern dressings in the treatment of superficial cutaneous wounds.

Sense- and antisense-targeting of oxytocinergic systems in the rat hypothalamus.

3' phosphorothioate modified sense or antisense oligonucleotides to oxytocin transcripts were used for in vivo targeting of oxytocin (OT) neurons in the rat hypothalamus. Intracerebroventricular injections of antisense probe resulted in a loss of systemic OT. However, abundant immunoreactive OT as well as oxytocin mRNA hybridization signal was visualized in the hypothalamo neurohypophysial system (HNS) of these animals. RT-PCR of hypothalamic homogenates revealed clearly detectable amounts of cytoplasmic OT mRNA in spite of sense or antisense treatment. Immunostaining with an antibody to DNA-RNA triple helix resulted in cytoplasmic reaction product in the HNS in the antisense group, which was not found when tissue sections had been pretreated with RNase. Animals injected with the sense probe showed a less pronounced but significant loss of systemic OT while immunoreactivity for this peptide in the posterior lobe seemed to be unaffected. RT-PCR of OT encoding mRNA extracted from sense injected rats indicated that these transcripts were of smaller size than samples from antisense treated animals or controls. Immunostaining with the triple helix antibody revealed distinct immunoreactive dots in cellular nuclei throughout the brain in the sense group. Our findings suggest that sense and antisense probes may not readily be employed as "functional antagonists" since peptidergic neurons are probably capable of responding in various ways to the treatment. RNase H may be less important in hypothalamic neurons as commonly suggested. Targeted transcripts are likely to form complexes which may somehow interact with secretion. Triple helix formation in the nucleus may not be able to induce an efficient transcriptional arrest. Although endocrine and behavioral changes observed in antisense treated animals seem to confirm the hypothesis that a selective translational "knock out" can be achieved with in vivo hybridization strategies, the actual underlying molecular events are far from being understood. On the other hand, sense or antisense strategies may provide valuable insights into molecular and cellular events associated with neurosecretion.

Interaction of liposomal incorporated vitamin D3-analogues and human keratinocytes.

The influence of different liposomal qualities, loaded with a variety of vitamin D3-analogues, on the proliferation and interleucine 1 alpha-release (IL-1 alpha) of human keratinocytes was examined by fluorimetric and colorimetric measurements to optimize their use for psoriasis treatment. In comparison, the effects of the free drugs, as 25-hydroxyvitamin D3, calcipotriol, and calcitriol, as well as of empty liposomes have been studied. At the interaction between empty liposomes (< 200 nm) and HaCaT-cells has been looked by electron microscopy. Empty liposomes, made of DMPC as well as of egg-PC, can be used as drug carrier without any inhibiting effect on the proliferation of human keratinocytes at lipid concentrations of < 10(-4) M. Under the influence of the free drugs investigated an inhibition of cell growth as well as of the IL 1 alpha-release was measured at drug concentrations of > or = 10(-8) M. In comparison the related liposomal drug formulations didn't show any diminishing in the proliferation effects caused by the free drugs. A significant improvement, however, was only found in the action of DMPC-incorporated 25-hydroxyvitamin D3 at drug concentration of 10(-7) M. These results suggest that there is no remarkable improvement in the action of liposomal incorporated vitamin D3-analogues neither related to their proliferation nor their IL1 alpha-releasing effects. The influence of liposomal incorporated vitamin D3-analogues in keeping small their negative side effects has to be investigated at a more relevant model.