Targeted Synthesis of NIR Luminescent Rhenium Diimine cis,trans-[Re( NN )(CO)2 (L)2 ]n+ Complexes Containing N-Donor Axial Ligands: Photophysical, Electrochemical, and Theoretical Studies.

The combined action of ultraviolet irradiation and microwave heating onto acetonitrile solution of [Re( NN )(CO)3 (NCMe)]OTf ( NN =phenantroline and neocuproine) afforded cis,trans-Re( NN )(CO)2 (NCMe)2 ]+ acetonitrile derivatives. Substitution of relatively labile NCMe with a series of aromatic N-donor ligands (pyridine, pyrazine, 4,4'-bipyridine, N-methyl-4,4'-bipyridine) gave a novel family of the diimine cis,trans-[Re( NN )(CO)2 (L)2 ]+ complexes. Photophysical studies of the obtained compounds in solution revealed unusually high absorption across the visible region and NIR phosphorescence with emission band maxima ranging from 711 to 805 nm. The nature of emissive excited states was studied using DFT calculations to show dominant contribution of 3 MLCT (dπ(Re)→π*( NN )) character. Electrochemical (CV and DPV) studies of the monocationic diimine complexes revealed one reduction and one oxidation wave assigned to reduction of the diimine moiety and oxidation of the rhenium center, respectively.

Genome-wide association analysis for ß-hydroxybutyrate concentration in Milk in Holstein dairy cattle.

BACKGROUND: Ketosis in dairy cattle has been shown to cause a high morbidity in the farm and substantial financial losses to dairy farmers. Ketosis symptoms, however, are difficult to identify, therefore, the amount of ketone bodies (mainly ß-hydroxybutyric acid, BHB) is used as an indicator of subclinical ketosis in cows. It has also been shown that milk BHB concentrations have a strong correlation with ketosis in dairy cattle. Mid-infrared spectroscopy (MIR) has recently became a fast, cheap and high-throughput method for analyzing milk components. The aim of this study was to perform a genome-wide association study (GWAS) on the MIR-predicted milk BHB to identify genomic regions, genes and pathways potentially affecting subclinical ketosis in North American Holstein dairy cattle. RESULTS: Several significant regions were identified associated with MIR-predicted milk BHB concentrations (indicator of subclinical ketosis) in the first lactation (SCK1) and second and later lactations (SCK2) in Holstein dairy cows. The strongest association was located on BTA6 for SCK1 and BTA14 on SCK2. Several SNPs on BTA6 were identified in regions and variants reported previously to be associated with susceptibility to ketosis and clinical mastitis in Jersey and Holstein dairy cattle, respectively. One highly significant SNP on BTA14 was found within the DGAT1 gene with known functions on fat metabolism and inflammatory response in dairy cattle. A region on BTA6 and three SNPs on BTA20 were found to overlap between SCK1 and SCK2. However, a novel region on BTA20 (55-63 Mb) for SCK2 was also identified, which was not reported in previous association studies. Enrichment analysis of the list of candidate genes within the identified regions for MIR-predicted milk BHB concentrations yielded molecular functions and biological processes that may be involved in the inflammatory response and lipid metabolism in dairy cattle. CONCLUSIONS: The results of this study confirmed several SNPs and genes identified in previous studies as associated with ketosis susceptibility and immune response, and also found a novel region that can be used for further analysis to identify causal variations and key regulatory genes that affect clinical/ subclinical ketosis.

Genome-wide association study for lactation persistency, female fertility, longevity, and lifetime profit index traits in Holstein dairy cattle.

Female fertility in Holstein cattle can decline when intense genetic selection is placed on milk production. One approach to improving fertility is to identify the genomic regions and variants affecting fertility traits and then incorporate this knowledge into selection decisions. The objectives of this study were to identify or refine the positions of the genomic regions associated with lactation persistency, female fertility traits (age at first service, cow first service to conception, heifer and cow nonreturn rates), longevity traits (herd life, indirect herd life, and direct herd life), and lifetime profit index in the North American Holstein dairy cattle population. A genome-wide association study was performed for each trait, using a single SNP (single nucleotide polymorphism) regression mixed linear model and imputed high-density panel (777k) genotypes. No associations were identified for fertility traits. Several peak regions were detected for lifetime profit index, lactation persistency, and longevity. The results overlap with previous findings and identify some novel regions for lactation persistency. Previously proposed causative and candidate genes supported by this work include DGAT1, GRINA, and CPSF1, whereas new candidate genes are SLC2A4RG and THRB. Thus, the chromosomal regions identified in this study not only confirm several previous findings but also highlight new regions that may contribute to genetic variation in lactation persistency and longevity-associated traits in dairy cattle.

Functional conformations of the nuclear 1alpha,25-dihydroxyvitamin D3 receptor.

The nuclear hormone 1alpha,25-dihydroxyvitamin D3 (VD) has important cell regulatory functions. Various synthetic VD analogues are under investigation to identify candidates with an improved therapeutic profile against hyperproliferative diseases. VD directly activates the transcription factor VD receptor (VDR), which in turn stimulates the expression of a cascade of primary and secondary VD-responsive genes. The activation of the VDR through binding of its natural and synthetic ligands is linked to a conformational change presenting the interface with co-activator proteins, referred to as the (trans)activation function 2 (AF-2) domain. Multiple conformations of the VDR might be the key to understanding a selective action of VD analogues. The method of limited protease digestion was used here to characterize up to three different functional VDR conformations stabilized individually by VD and its analogues. The relative potency of VDR ligands can be quantified in the interaction with these VDR conformations by determination of a functional dissociation constant, where a two-concentration-point comparison has already provided important information. In this way seven amino acid residues in the AF-2 domain have been analysed as potential ligand contact points. Interestingly, residues Phe-422 and Val-418 seem to interact with all tested VDR ligands, whereas VD analogues such as the anti-psoriatic drug MC903 displayed additional contact points within the AF-2 domain. Taken together, limited protease digestion is a powerful method for studying functional VDR conformations and seems to be very appropriate for screening VD analogues.

Sensitive induction of apoptosis in breast cancer cells by a novel 1,25-dihydroxyvitamin D3 analogue shows relation to promoter selectivity.

The biologically active form of vitamin D3, the nuclear hormone 1 alpha,25-dihydroxyvitamin D3 (VD), is an important regulator of cellular growth, differentiation, and death. The hormone mediates its action through the activation of the transcription factor VDR, which is a member of the superfamily of nuclear receptors. In most cases the ligand-activated VDR is found in complex with the retinoid X receptor (RXR) and stimulates gene transcription mainly from VD response elements (VDREs) that are formed by two hexameric core binding motifs and are arranged either as a direct repeat spaced by three nucleotides (DR3) or as an inverted palindrome spaced by nine nucleotides (1P9). The two VD analogues CB1093 and EB1089 are both very potent inhibitors of the proliferation of MCF-7 cultured breast cancer cells displaying approximately 100-fold lower IC50 values (0.1 nM) than the natural hormone. In addition, CB1093 is even more potent in vivo than EB1089 in producing regression of experimental mammary tumors. Moreover, both VD analogues induce apoptosis in MCF-7 cells, but CB1093 is effective at concentrations approximately 10-fold lower than EB1089. In accordance, the reduction of Bcl-2 protein expression showed CB1093 to be more potent than EB1089. This suggests that the antiproliferative effect of CB1093 may be related mainly to its apoptosis inducing effect, whereas EB1089 may preferentially have effects on growth arrest. EB1089 is known to result in a selectivity for the activation of IP9-type VDREs, whereas CB1093 shows a preference for the activation of DR3-type VDREs. This promoter selectivity suggests that the effects of VD and its analogues on growth arrest and the induction of apoptosis may be mediated by different primary VD responding genes. In conclusion, CB1093 was found to be a potent inhibitor of rat mammary tumor growth in vivo. CB1093 also displayed a high potency in vitro in the induction of apoptosis, a process that may be linked to a promoter selectivity for DR3-type VDREs.

Potent gene regulatory and antiproliferative activities of 20-methyl analogues of 1,25 dihydroxyvitamin D3.

The biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues.

The high affinity ligand binding conformation of the nuclear 1,25-dihydroxyvitamin D3 receptor is functionally linked to the transactivation domain 2 (AF-2).

The nuclear receptor for 1,25-dihydroxyvitamin D3 (VD), VDR, is a transcription factor that mediates all genomic actions of the hormone. The activation of VDR by ligand induces a conformational change within its ligand binding domain (LBD). Due to the lack of a crystal structure analysis, biochemical methods have to be applied in order to investigate the details of this receptor-ligand interaction. The limited protease digestion assay can be used as a tool for the determination of a functional dissociation constant (K(df)) of VDR with any potential ligand. This method provided with the natural hormone VD two protease-resistant fragments of the VDR LBD and with the 20-epi conformation of VD, known as MC1288, even an additional fragment of intermediate size. These fragments were interpreted as different receptor conformations and their decreasing size was found to be associated with decreasing ligand binding affinity. A critical amino acid for VDR's high ligand binding conformation has been identified by C-terminal receptor truncations and point mutations as phenylalanine 422. This amino acid appears to directly contact the ligand and belongs to the ligand-inducible activation function-2 (AF-2) domain. Moreover, functional assays supported the observation that high affinity ligand binding is directly linked to transactivation function.

High-affinity nuclear receptor binding of 20-epi analogues of 1,25-dihydroxyvitamin D3 correlates well with gene activation.

The hormone 1,25-dihydroxyvitamin D3 (VD) has the potential for clinical use in several diseases, such as cancer, osteoporosis, and psoriasis. The action of VD is mediated by primary responding genes that contain in their promoter region a binding site for the transcription factor VDR. Most of the known VD response elements are formed by a direct repeat of two hexameric core binding motifs spaced by three nucleotides (DR3) bound by a heterodimer of VDR and the retinoid X receptor (RXR). Various VD analogues have been developed in order to optimize the therapeutic profile of VD. This report presents a novel experimental system that may help in the understanding of the structural basis for the high potency of a VD analogue like KH1060, which is a 20-epi-22-oxa-derivative of VD. In human breast cancer cells, MCF-7, the half-maximal gene activation values for KH1060 and seven of its structural precursors were determined on a DR3-type VD response element. These eight analogues cover conservative structural changes from 20-epi-VD (MC1288) to KH1060. With a modified version of the limited protease digestion assay the functional affinity of the analogues to VDR was measured. The functional receptor affinity of the eight analogues was found to be directly proportional to their potency in VDR-RXR-mediated gene activity.

Thiazolidine diones, specific ligands of the nuclear receptor retinoid Z receptor/retinoid acid receptor-related orphan receptor alpha with potent antiarthritic activity.

Rat adjuvant arthritis is a chronic T cell-dependent autoimmune disease with many similarities to rheumatoid arthritis. We have identified a class of thiazolidine diones with high potency in suppressing chronic inflammation and joint destruction in this experimental model. The lead compound CGP 52608 (1-(3-allyl-4-oxothiazolidine-2-ylidene)-4-methylthiosemicarbazone) exhibits antiarthritic activity at daily oral doses between 0.01 and 1 mg/kg and was shown to specifically activate the retinoid Z receptor/retinoid acid receptor-related orphan receptor alpha (RZR/RORalpha) in low nanomolar concentrations. This receptor is a novel member of the superfamily of ligand-inducible transcription factors, and we have recently identified the pineal gland hormone melatonin as a natural ligand. Structure-activity relationship studies with 13 closely related analogues of CGP 52608 revealed a striking correlation between RZR/RORalpha activation and antiarthritic activity. We therefore suggest that nuclear signaling via RZR/RORalpha is a key mechanism in mediating the antiarthritic effects of these thiazolidine diones and may open a novel therapeutic approach for the treatment of rheumatoid arthritis and other autoimmune diseases. The existence of a nuclear melatonin receptor may lead to a better understanding of the immunomodulatory actions of melatonin.

The anti-proliferative effect of vitamin D3 analogues is not mediated by inhibition of the AP-1 pathway, but may be related to promoter selectivity.

The hormone 1,25-dihydroxyvitamin D3 (VD) is able to induce cellular differentiation and to inhibit cellular proliferation, which provides it with an interesting therapeutic potential in cancer. However, side effects of VD on homeostasis (eg hypercalcemia) had made the need for the development of VD analogues with low calcemic effect. On the human breast cancer cell line MCF-7 we obtained with the VD analogue EB1089 an about 100-fold higher anti-proliferative effect than with VD. We found that this difference in biological activity is neither related to increased functional affinity to the VD receptor nor to repression of AP-1 activity. The physiologically most prominent complex of the VD receptor is a heterodimer with the retinoid X receptor that binds VD response elements formed two hexameric core binding motifs being arranged either as direct repeats spaced by 3 nucleotides (DR3s) or as inverted palindromes spaced by 9 nucleotides (IP9s). We observed that EB1089 stimulates transcriptional activation from IP9-type elements at clearly lower concentrations than from DR3-type elements. It is possible that IP9-type response elements play an important role in or contribute to the control of cell proliferation, so that promoter-selectivity may explain the high anti-proliferative effect of EB1089.

Natural vitamin D3 response elements formed by inverted palindromes: polarity-directed ligand sensitivity of vitamin D3 receptor-retinoid X receptor heterodimer-mediated transactivation.

VDR, the nuclear receptor for 1,25-dihydroxyvitamin D3 (VD), is a member of the superfamily of nuclear hormone receptors and controls multiple aspects of homeostasis, cell growth, and differentiation. VDR can function as a homodimer, but heterodimerization with the retinoid X receptor (RXR), retinoic acid receptor, or thyroid hormone receptor increases its affinity for response elements in the promoter of target genes. All natural VD response elements identified so far consist of direct repeats of a variety of hexameric core binding motifs with a preferential spacing of three nucleotides (DR3s). However, all four VD signalling pathways function also on response elements formed by inverted palindromes, although these sequences were not of natural origin. Here, we report the identification of two VD response elements consisting of inverted palindromes spaced by nine nucleotides (IP9s) in the promoters of the human calbindin D9k gene and the rat osteocalcin gene. Like most DR3-type VD response elements, both IP9s are preferentially bound by VDR-RXR heterodimers with a 5'-RXR-VDR-3' polarity, whose transcriptional activity can be enhanced by costimulation with 9-cis retinoic acid. We demonstrate that changing the response element orientation relatively to the basal promoter decreases the sensitivity of transcriptional activation by VD by about 10-fold. Our findings indicate that inverted palindromes are as functional as direct repeats. Furthermore, we suggest that the orientation of a nuclear receptor complex in relation to the basic transcriptional machinery, which is directed by heterodimer polarity and response element orientation, influences the ligand sensitivity of the respective target gene expression.

The nuclear receptor for melatonin represses 5-lipoxygenase gene expression in human B lymphocytes.

The two subtypes of retinoid Z receptor (RZR alpha and beta) and the three splicing variants of retinoid orphan receptor (ROR alpha 1, alpha 2, and alpha 3) form a subfamily within the superfamily of nuclear hormone receptors. Very recently we found that the pineal gland hormone melatonin is a natural ligand of RZR alpha and RZR beta. Ligand-induced transcriptional control is therefore proposed to mediate physiological functions of melatonin in the brain where RZR beta is expressed, but also in peripheral tissues, where RZR alpha was found. However, no natural RZR responding genes have been identified yet. Here, we report that a response element in the promoter of 5-lipoxygenase binds specifically RZR alpha and ROR alpha 1, but not ROR alpha 2 and alpha 3. 5-Lipoxygenase is a key enzyme in the biosynthesis of leukotrienes, which are known to be allergic and inflammatory mediators. We could show that the activity of the whole 5-lipoxygenase promoter as well as of the RZR response element fused to the heterologous thymidine kinase promoter could be repressed by melatonin. The hormone down-regulated the expression of 5-lipoxygenase about 5-fold in B lymphocytes, which express RZR alpha. In contrast, 5-lipoxygenase mRNA levels were not affected in differentiated monocytic and granulocytic cell lines, which do not express RZR alpha. This indicates that 5-lipoxygenase is the first natural RZR alpha responding gene. Furthermore, our results open up a new perspective in understanding the involvement of melatonin in inflammatory and immunological reactions.

Vitamin D3-thyroid hormone receptor heterodimer polarity directs ligand sensitivity of transactivation.

The nuclear receptors for 1,25-dihydroxyvitamin D3 (VD) and 3,5,3'-triiodothyronine (T3), that is, VDRs and T3Rs respectively, control aspects of homeostasis, cell growth and differentiation. They activate transcription from response elements consisting of direct repeats, palindromes and inverted palindromes of a variety of hexameric core-binding motifs. VDRs bind preferentially to direct repeats spaced by three nucleotides, whereas T3Rs bind to direct repeats spaced by four nucleotides. VDRs and T3Rs can function as homodimers but heterodimerization with retinoid X or retinoic acid receptors increases their affinity for DNA in vitro and resulting transcriptional activity in vivo. We recently observed the formation of VDR-T3R heterodimers. Here we show that the polarity of the binding of such heterodimers to the VD response element of the rat 9K (relative molecular mass 9,000) calbindin gene promoter was 5'-T3R-VDR-3', whereas on the mouse 28K calbindin VD response element this polarity was reversed to 5'-VDR-T3R-3'. We also show that the ligand for the downstream receptor controls the transcriptional activity of the heterodimeric complex. Thus, polarity seems to be an important regulatory property of heterodimeric nuclear receptor complexes.