Differential Action Research Articles

Differential Cytotoxicity and Particle Action of Hydroxyapatite Nanoparticles in Human Cancer Cells

While hydroxyapatite nanoparticles (HAPNs) have been reported to exhibit anticancer effects on several types of human cancer cells, no investigation has been performed to compare their cytotoxicity with different types of cancer cells. The objective of the present study is to investigate the cytotoxic action of HAPNs in different types of human cancer cell and to explore the possible mechanisms involved. Rod-shaped HAPNs were prepared by the aqueous precipitation method and then labeled with fluorescein isothiocyanate to visualize the cellular uptake and distribution. Their cytotoxicity to three human carcinoma cell lines (gastric cancer cells [MGC80-3], cervical adenocarcinoma epithelial cells [HeLa] and hepatoma cells [HepG2], as well as to normal human hepatocyte cells [L-02]) was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was characterized by the changes in nuclear morphology with 4',6-diamidino-2-phenylindole staining and by flow cytometric analysis with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Furthermore, the activity of apoptotic proteins (caspase-3, -8 and -9), intracellular reactive oxygen species and glutathione levels were analyzed in HAPN-treated cells. The cellular uptake of HAPNs was studied using flow cytometry analysis, and changes in intracellular calcium levels were investigated using the Ca(2+)-sensitive fluorescent dye, fluo-3 AM. HAPNs significantly inhibited cell proliferation and induced apoptosis of cancer cells with an order of MGC80-3 > HepG2 > HeLa, but had no impact on normal hepatic cells (L-02). The increase in apoptosis was accompanied by the activation of caspase-3 and -9, but not activation of caspase-8. Moreover, HAPN treatment led to reactive oxygen species generation and decreased intracellular glutathione in cancer cells, with the most remarkable reactive oxygen species burst in HeLa cells. The degree of cytotoxicity did not correlate with the cellular uptake efficiency of HAPNs. However, more HAPNs were found inside the nucleus of MGC80-3 cells, and an increase in the intracellular calcium level was observed in all cancer cells, with the highest level also detected in MGC80-3. Varying cytotoxicity of HAPNs was observed in different cancer cell types. Our results suggest that possible mechanisms of cytotoxicity in various types of cancer cells could be different. The elevated calcium concentration and nuclear localization of the particles might be the main mechanism of growth inhibition by HAPNs in cancer cells.

Antiangiogenic therapy effects on age-associated matrix metalloproteinase-9 (MMP-9) and insulin-like growth factor receptor-1 (IGFR-1) responses: a comparative study of prostate disorders in aged and TRAMP mice

Senescence is associated with hormonal imbalance and prostatic disorders. Angiogenesis is fundamental for the progression of malignant lesions and is a promising target for prostate cancer treatment. The aim was to characterize matrix metalloproteinase-9 (MMP-9) and insulin-like growth factor receptor-1 (IGFR-1) responses in the prostate during senescence and following antiangiogenic and/or androgen ablation therapies, comparing them to cancer progression features in TRAMP mice. Aged male mice (52-week-old FVB) were submitted to antiangiogenic treatments with SU5416 (6 mg/kg; i.p.) and/or TNP-470 (15 mg/kg; s.c). Finasteride (20 mg/kg; s.c.) was administered alone or associated to both inhibitors. Dorsolateral prostate was collected for light microscopy, and immunohistochemistry and Western blotting collected for MMP-9 and IGFR-1. Senescence led to inflammation and different proliferative lesions in the prostate, as well as to increased MMP-9 and IGFR-1, resembling TRAMP mice prostatic microenvironment. Antiangiogenic therapies promoted recovery and/or interruption of age-associated alterations, presenting differential effects on the molecules studied. SU5416 acted mainly on MMP-9, whereas TNP-470 showed its best influence on IGFR-1 levels. Finasteride administration, alone or in combination with antiangiogenic agents, also resulted in regression of inflammation and neoplastic lesions, besides having a negative modulatory effect on both MMP-9 and IGFR-1. We concluded that stimulated tissue remodeling and proliferative processes during senescence predisposed the prostate to malignant disorders. The combination of different agents was more effective to minimize prostatic imbalance during this period, probably due to the differential action of each drug on factors involved in cell proliferation and extracellular matrix remodeling, resulting in a broader spectrum of effects following the combined treatment.

Basic models for differential inhibition of enzymes

The possible preferential action exerted by an inhibitor on the transformation of one of two agonist substrates catalyzed by the same enzyme has recently been reported in studies on aldose reductase inhibition. This event was defined as “intra-site differential inhibition” and the molecules able to exert this action as “differential inhibitors”. This work presents some basic kinetic models describing differential inhibition. Using a simple analytic approach, the results show that differential inhibition can occur through either competitive or mixed type inhibition in which the inhibitor prevalently targets the free enzyme. The results may help in selecting molecules whose differential inhibitory action could be advantageous in controlling the activity of enzymes acting on more than one substrate.

Differential ERα-mediated rapid estrogenic actions of ginsenoside Rg1 and estren in human breast cancer MCF-7 cells

Recent studies indicated that both estren and Rg1 appear to be able to activate mitogen-activated protein kinase (MAPK) pathway in estrogen responsive cells. Rg1 could lead to MAPK activation through ligand-independent activation of estrogen receptor (ER), while estren could activate the Src-MAPK pathway in an ERE-independent manner. Thus, it is important to understand the mechanistic insights on the difference in transcriptional activation between estren and Rg1. The present study also addressed the differential abilities of Rg1 and estren in terms of the ability to activate ER and the ability to induce ER translocation in MCF-7 cells. Our data indicated that Rg1 could increase pS2 gene expression, and could recruit the co-activator steroid receptor co-activator-1 (SRC-1) to the pS2 promoter. Rg1 could also induce ERα nuclear translocation as well as ERα phosphorylation at Ser118 principally in the cytoplasm in MCF-7 cells. We deduced that estren induced ERE-dependent transcriptional activity and activated ERα at Ser118 occurred in the nucleus of MCF-7 cells. However, it was found to decrease pS2 gene expression and failed to induce the recruitment of SRC-1 to the pS2 promoter in MCF-7 cells. Our results suggest that the abilities of Rg1 and estren to regulate pS2 gene expression, to recruit co-activators as well as to induce sub-cellular distribution of ERα are dramatically different.

Structural Features Determining Flower-Promoting Activity of Arabidopsis FLOWERING LOCUS T

In Arabidopsis thaliana, the genes FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) have antagonistic roles in regulating the onset of flowering: FT activates and TFL1 represses flowering. Both encode small, closely related transcription cofactors of ∼175 amino acids. Previous studies identified a potential ligand binding residue as well as a divergent external loop as critical for the differences in activity of FT and TFL1, but the mechanisms for the differential action of FT and TFL1 remain unclear. Here, we took an unbiased approach to probe the importance of residues throughout FT protein, testing the effects of hundreds of mutations in vivo. FT is surprisingly robust to a wide range of mutations, even in highly conserved residues. However, specific mutations in at least four different residues are sufficient to convert FT into a complete TFL1 mimic, even when expressed from TFL1 regulatory sequences. Modeling the effects of these mutations on the surface charge of FT protein suggests that the affected residues regulate the docking of an unknown ligand. These residues do not seem to alter the interaction with FD or 14-3-3 proteins, known FT interactors. Potential candidates for differential mediators of FT and TFL1 activities belong to the TCP (for TEOSINTE BRANCHED1, CYCLOIDEA, PCF) family of transcription factors.

M‐Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure: Differential Actions of 2 cGMP Activating Therapeutics

BackgroundSystemic hypertension is a common characteristic in acute heart failure (HF). This increasingly recognized phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M‐atrial natriuretic peptide (M‐ANP), a novel particulate guanylyl cyclase (pGC) activator, and nitroglycerin, a soluble guanylyl cyclase (sGC) activator.Methods and ResultsHF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and following intravenous M‐ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Mean arterial pressure (MAP) was reduced by M‐ANP (139±4 to 118±3 mm Hg, P<0.05) and nitroglycerin (137±3 to 116±4 mm Hg, P<0.05); similar findings were recorded for pulmonary wedge pressure (PCWP) with M‐ANP (12±2 to 6±2 mm Hg, P<0.05) and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05). M‐ANP enhanced renal function with significant increases (P<0.05) in glomerular filtration rate (38±4 to 53±5 mL/min), renal blood flow (132±18 to 236±23 mL/min), and natriuresis (11±4 to 689±37 mEq/min) and also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation.ConclusionsOur results advance the differential cardiorenal actions of pGC (M‐ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M‐ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal.

Assessing RNA interactions with proteins by DRaCALA.

Discovery of RNA elements, including riboswitches and regulatory RNAs, has revealed additional regulatory mechanisms for transcript stability, transcript termination, and translational initiation. These regulatory RNA molecules act through direct binding to cellular targets including other RNA molecules, proteins, and low molecular weight metabolites. RNA-RNA interactions based on complementarity can be identified through bioinformatic analysis. However, identification of novel interactions between these regulatory RNA molecules and their partners other than complementary sequences is more challenging. We have developed a technique called Differential Radial Capillary Action of Ligand Assay (DRaCALA) to facilitate the detection of direct binding between RNA elements to proteins or low molecular weight ligands. Previously, we have described the adaptation of this technique to detect the binding interaction between Vc2 riboswitch to a signaling cyclic dinucleotide called cyclic-di-GMP. Here, we describe the adaptation of DRaCALA for identifying sequence-specific RNA-binding proteins directly from E. coli cell lysates expressing the recombinant binding protein. DRaCALA can be used to qualitatively and quantitatively assess RNA-protein interaction in whole cell lysate, determine the kinetics of the binding, and test for competitors. Using DRaCALA in a high-throughput format has the potential to rapidly identify sequence-specific RNA-binding proteins.

220 kV旁代主变操作引起差动保护动作分析

大兴变旁代主变过程导致差动保护动作，主变三侧开关跳闸，经短接CT电流端子模拟试验，确认为五防钥匙误碰CT电流端子，导致AB相短路，出现差流启动差动保护。本文提出加强现场防误碰措施、规范二次操作，以提高运行人员技术水平。 The process of bypassing transformer of Daxing leads to differential protection action and transformer’s three side of switch trip. According to the simulation experiment of short circuit CT terminals, it is sure that the thing that five-prevention key falsely touches the current terminals leads to short circuit phase A and B, the appearance of differential current and the start of differential protection. The paper proposes to enhance the measure of anti false touch and specify secondary operation, in order to improve the technology level of operators.

Imaging of corticosteroid receptors in live cells.

Adrenal corticosteroids (cortisol in humans or corticosterone in rodents) exert numerous effects on the central nervous system that regulate the stress response, mood, learning, and memory, and various neuroendocrine functions. The actions of corticosterone (CORT) in the brain are mediated via two receptor systems: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). It has been shown that the MR and GR are highly co-localized in the hippocampus. These receptors are mainly localized in the cytoplasm, and are translocated into the nucleus, after binding with hormones, to act as transcriptional factors. Thus, the subcellular dynamics of both receptors constitute one of the most important issues to be clarified. Given the differential actions of MR and GR in the central nervous system, it is of great consequence to elucidate how MR and GR are trafficked between the cytoplasm and nucleus and how their interactions are regulated by hormones and/or other molecules to exert their functional activities. In this chapter, we describe our recent studies of corticosteroid receptor dynamics in living cells, focusing on three points: (1) time-lapse imaging of green fluorescent protein (GFP)-labeled corticosteroid receptors; (2) intranuclear dynamics of GFP-labeled corticosteroid receptors using the technique of fluorescence recovery after photobleaching; and (3) the possibility of heterodimer formation using the fluorescence resonance energy transfer technique. We discuss various factors affecting the dynamics of these receptors. Furthermore, we present future directions in the in vivo molecular imaging of corticosteroid receptors at the whole brain level.

Reply to D’Amato et al. and Zeldis et al.: Screening of thalidomide derivatives in chicken and zebrafish embryos

We thank D’Amato et al. (1) and Zeldis et al. (2) for their interest and comments on our article in PNAS (3). We carried out a screen of lenalidomide and pomalidomide primarily in chicken and zebrafish embryos, but also in several in vitro assays. Our goal was to determine any differential actions or responses and to visualize effects live and in vivo (3). Identifying such differences, we reasoned, could help understand the underlying or differential mechanisms of actions of each of these compounds. In addition, this screen provides proof-of-principle to potentially use the chicken and zebrafish embryos to initially screen modified analogs of the compounds to identify those with cell/tissue-specific actions and identify potential adverse reactions (3, 4). This initial screen would require validation in higher species/clinical trials.

Profiles of cytokines secreted by isolated human endometrial cells under the influence of chorionic gonadotropin during the window of embryo implantation

BackgroundSeveral studies have indicated that human pre-implantation embryo-derived chorionic gonadotropin (hCG) may influence the implantation process by its action on human endometrial epithelial and stromal cells. Despite reports indicating that hCG acts on these cells to affect the production of several cytokines and growth factors (e.g., MIF, IGF-I, VEGF, LIF, IL-11, GMCSF, CXL10 and FGF2), our understanding of the integral influence of hCG on paracrine interactions between endometrial stromal and epithelial cells during implantation is very limited.MethodsIn the present study, we examined the profile of 48 cytokines in the conditioned media of primary cell cultures of human implantation stage endometrium. Endometrial epithelial cells (group 1; n = 20), stromal cells (group 2; n = 20), and epithelial plus stromal cells (group 3; n = 20) obtained from mid-secretory stage endometrial samples (n = 60) were grown on collagen and exposed to different doses (0, 1, 10 and 100 IU/ml) of rhCG for 24 h in vitro. Immunochemical and qRT-PCR methods were used to determine cytokine profiles. Enrichment and process networks analyses were implemented using a list of cytokines showing differential secretion in response to hCG.ResultsUnder basal conditions, endometrial epithelial and stromal cells exhibited cell type-specific profiles of secreted cytokines. Administration of hCG (100 IU) resulted in significantly (P < 0.05) different cytokine secretion profiles indicative of macropinocytic transport (HGF, MCSF) in epithelial cells, signal transduction (CCL4, FGF2, IL-1b, IL-6, IL-17, VEGF) in stromal cells, and epithelial-mesenchymal transition (FGF2, HGF, IL-1b, TNF) in mixed cells. Overall, the administration of hCG affected cytokines involved in the immune response, chemotaxis, inflammatory changes, proliferation, cell adhesion and apoptosis.ConclusionsCG can influence the function of the endometrium during blastocyst implantation via its differential action on endometrial epithelial and stromal cells. CG may also affect complex paracrine processes in the different endometrial cell types.

Immunolocalization of cell cycle proteins (p57, p27, cyclin D3, PCNA and Ki67) in intrauterine growth retardation (IUGR) and normal human term placentas

Placental development involves a series of events that depend on the coordinated action of proliferation, differentiation and invasion of trophoblasts. Studies on cell cycle related proteins controlling these events are fairly limited. It is still not fully determined how placental tissue proliferation is affected by intrauterine growth retardation (IUGR). Information on cell cycle related proteins that control these events is limited and how they are affected in IUGR is not fully understood. The aim of this study was to understand the role of cell cycle regulators in IUGR placentas and to determine the spatio-temporal immunolocalization of these cell cycle regulators in human IUGR and normal term placentas. Placental samples were stained immunohistochemically with PCNA, Ki67, cyclin D3, p27 and p57 antibodies and were examined by light microscopy. In all regions of IUGR placentas, PCNA, Ki67 and cyclin D3 staining intensities were statistically significantly decreased compared to normal controls. p27 staining intensity of the IUGR group was statistically significantly increased in villous parts and chorionic plates in comparison with the normal term placentas. Moreover, p57 staining intensity was statistically significantly increased in all parts of the IUGR group compared to controls. The observed placental abnormalities in IUGR placentas may be associated with arrest mechanisms affecting cell proliferation and cell cycle alterations in IUGR.

Correction: Gene Expression Profiles in Human and Mouse Primary Cells Provide New Insights into the Differential Actions of Vitamin D3Metabolites

Correction: Gene Expression Profiles in Human and Mouse Primary Cells Provide New Insights into the Differential Actions of Vitamin D₃Metabolites

Differential Pharmacological Activity of JN403 between α7 and Muscle Nicotinic Acetylcholine Receptors

The differential action of the novel agonist JN403 at neuronal α7 and muscle nicotinic receptors (AChRs) was explored by using a combination of functional and structural approaches. Single-channel recordings reveal that JN403 is a potent agonist of α7 but a very low-efficacy agonist of muscle AChRs. JN403 elicits detectable openings of α7 and muscle AChRs at concentrations ~1000-fold lower and ~20-fold higher, respectively, than that for ACh. Single-channel activity elicited by JN403 is very similar to that elicited by ACh in α7 but profoundly different in muscle AChRs, where openings are brief and infrequent and do not appear in clusters at any concentration. JN403 elicits single-channel activity of muscle AChRs lacking the ε subunit, with opening events being more frequent and prolonged than those of wild-type AChRs. This finding is in line with the molecular docking studies predicting that JN403 may form a hydrogen bond required for potent activation at the α-δ but not at the α-ε binding site. JN403 does not elicit detectable Ca²⁺ influx in muscle AChRs but inhibits (±)-epibatidine-elicited influx mainly by a noncompetitive mechanism. Such inhibition is compatible with single-channel recordings revealing that JN403 produces open-channel blockade and early termination of ACh-elicited clusters, and it is therefore also a potent desensitizing enhancer of muscle AChRs. The latter mechanism is supported by the JN403-induced increase in the level of binding of [³H]cytisine and [³H]TCP to resting AChRs. Elucidation of the differences in activity of JN403 between neuronal α7 and muscle AChRs provides further insights into mechanisms underlying selectivity for α7 AChRs.

High-Throughput Screening Using the Differential Radial Capillary Action of Ligand Assay Identifies Ebselen As an Inhibitor of Diguanylate Cyclases

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

Analysis on Commutation Failure and DC Differential Protection Unwanted Action of Yunnan-Guangdong UHVDC

In order to reveal the commutation failure process and the reason for DC differential protection (87DCM) unwanted action of Yunnan-Guangdong UHVDC in 9.12th 2012, and to lessen the unnecessary outage and guarantee a safe and stable operation, the 9.12 outage is deeply studied, including commutation failure phenomena and equivalent circuit analysis, high voltage DC current, low voltage DC current and their differential current analysis and reason for 87DCM unwanted action, and the final conclusions come out that: (1) 9.12 commutation failure includes V1 failing to communicate to V3 of Y bridge and V2 failing to communicate to V4 of D bridge; (2) during commutation failure process, high voltage DC current and low voltage DC current all raise rapidly and both contain DC component, AC component and no-circle rising component, and theoretically high voltage DC current equals to low voltage DC current; (3) failure in low pass filter circuit of pole 1 low voltage CT leads to measuring differential current between high voltage and low voltage DC current, and finally leads to unwanted action of 87DCM.

Antioxidant/Oxidant Status and Cardiac Function in Bradykinin B1- and B2-Receptor Null Mice

Kinin-vasoactive peptides activate two G-protein-coupled receptors (R), B(1)R (inducible) and B(2)R (constitutive). Their complex role in cardiovascular diseases could be related to differential actions on oxidative stress. This study investigated impacts of B(1)R or B(2)R gene deletion in mice on the cardiac function and plasma antioxidant and oxidant status. Echocardiography-Doppler was performed in B(1)R (B(1)R(-/-)) and B(2)R (B(2)R(-/-)) deficient and wild type (WT) adult male mice. No functional alteration was observed in B(2)R(-/-) hearts. B(1)R(-/-) mice had significantly lowered fractional shortening and increased isovolumetric contraction time. The diastolic E and A waves velocity ratio was similar in all mice groups. Thus B(1)R(-/-) mice provide a model of moderate systolic dysfunction, whereas B(2)R(-/-) mice displayed a normal cardiac phenotype. Plasma antioxidant capacity (ORAC) was significantly decreased in both B(1)R(-/-) and B(2)R(-/-) mice whereas the vitamin C levels were decreased in B(2)R(-/-) mice only. Plasma ascorbyl free radical was significantly higher in B(1)R(-/-) compared to WT and B(2)R(-/-) mice. Therefore, the oxidative stress index, ascorbyl free radical to vitamin C ratio, was increased in both B(1)R(-/-) and B(2)R(-/-) mice. Hence, B(1)R and B(2)R deficiency are associated with increased oxidative stress, but there is a differential imbalance between free radical production and antioxidant defense. The interrelationship between the differential B(1)R and B(2)R roles in oxidative stress and cardiovascular diseases remain to be investigated.

Differential action of glycoprotein hormones: significance in cancer progression.

Growth of multicellular organisms depends on maintenance of proper balance between proliferation and differentiation. Any disturbance in this balance in animal cells can lead to cancer. Experimental evidence is provided to conclude with special reference to the action of follicle-stimulating hormone (FSH) on Sertoli cells, and luteinizing hormone (LH) on Leydig cells that these hormones exert a differential action on their target cells, i.e., stimulate proliferation when the cells are in an undifferentiated state which is the situation with cancer cells and promote only functional parameters when the cell are fully differentiated. Hormones and growth factors play a key role in cell proliferation, differentiation, and apoptosis. There is a growing body of evidence that various tumors express some hormones at high levels as well as their cognate receptors indicating the possibility of a role in progression of cancer. Hormones such as LH, FSH, and thyroid-stimulating hormone have been reported to stimulate cell proliferation and act as tumor promoter in a variety of hormone-dependent cancers including gonads, lung, thyroid, uterus, breast, prostate, etc. This review summarizes evidence to conclude that these hormones are produced by some cancer tissues to promote their own growth. Also an attempt is made to explain the significance of the differential action of hormones in progression of cancer with special reference to prostate cancer.

Inhibitory Effect of Submaximal Doses of Ghrelin on Gonadotropin Secretion in Women

The effect of ghrelin on gonadotropin secretion has been equivocal. Recent data have shown an inhibitory effect of repeated injections of ghrelin on nocturnal LH and FSH secretion in women. The aim of this study was to investigate the effect of submaximal doses of ghrelin on the diurnal secretion of gonadotropins. Ten normally cycling women received 2 consecutive dosages of ghrelin (0.15 μg/kg and 0.30 μg/kg) intravenously in the early and late follicular phases of the cycle. Saline was injected in the preceding cycle. Blood samples in relation to ghrelin or saline administration (time 0 and 90 min) were taken at -15, 0, 30, 90, 120, 150, and 180 min. Serum estradiol concentrations were significantly higher in the late than in the early follicular phase. Following ghrelin administration, serum LH and FSH levels decreased significantly, in relation to the saline injection, in the late (p<0.01), although FSH values showed a within the group decrease also in the early follicular phase (p<0.05). The study suggests a differential action of ghrelin on diurnal gonadotropin secretion throughout the follicular phase of the cycle.

Gene Expression Profiles in Human and Mouse Primary Cells Provide New Insights into the Differential Actions of Vitamin D3 Metabolites

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) had earlier been regarded as the only active hormone. The newly identified actions of 25-hydroxyvitamin D3 (25(OH)D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) broadened the vitamin D3 endocrine system, however, the current data are fragmented and a systematic understanding is lacking. Here we performed the first systematic study of global gene expression to clarify their similarities and differences. Three metabolites at physiologically comparable levels were utilized to treat human and mouse fibroblasts prior to DNA microarray analyses. Human primary prostate stromal P29SN cells (hP29SN), which convert 25(OH)D3 into 1α,25(OH)2D3 by 1α-hydroxylase (encoded by the gene CYP27B1), displayed regulation of 164, 171, and 175 genes by treatment with 1α,25(OH)2D3, 25(OH)D3, and 24R,25(OH)2D3, respectively. Mouse primary Cyp27b1 knockout fibroblasts (mCyp27b1 −/−), which lack 1α-hydroxylation, displayed regulation of 619, 469, and 66 genes using the same respective treatments. The number of shared genes regulated by two metabolites is much lower in hP29SN than in mCyp27b1 −/−. By using DAVID Functional Annotation Bioinformatics Microarray Analysis tools and Ingenuity Pathways Analysis, we identified the agonistic regulation of calcium homeostasis and bone remodeling between 1α,25(OH)2D3 and 25(OH)D3 and unique non-classical actions of each metabolite in physiological and pathological processes, including cell cycle, keratinocyte differentiation, amyotrophic lateral sclerosis signaling, gene transcription, immunomodulation, epigenetics, cell differentiation, and membrane protein expression. In conclusion, there are three distinct vitamin D3 hormones with clearly different biological activities. This study presents a new conceptual insight into the vitamin D3 endocrine system, which may guide the strategic use of vitamin D3 in disease prevention and treatment.