The effect of basic fibroblast growth factor 2 on the bovine corpus luteum depends on the stage of the estrous cycle and modulates prostaglandin F2α action

The roles of fibroblast growth factor 2 (FGF2) in the corpus luteum (CL) function and its modulatory effect on prostaglandin (PG) F_2α during the bovine estrous cycle were studied using the following design of in vivo and in vitro experiments: (1) effects of FGF2 and FGF receptor 1 inhibitor (PD173074) on bovine CL function in the early (PGF_2α-resistant) and mid (PGF_2α-responsive) luteal stage in vivo, (2) the modulatory effect of FGF2 on PGF_2α action during the luteal phase in vivo and (3) effects of FGF2 and PD173074 on bovine CL secretory function in vitro. Cows were treated by injection into the CL with: (1) saline (control), (2) FGF2, (3) PD173074, (4) FGF2 followed by intramuscular (i.m.) PGF_2α, (5) PD173074 followed by i.m. PGF_2α and (6) i.m. PGF_2α as a positive control. For in vitro experiments, CL explants were treated with the aforementioned factors. Progesterone (P₄) concentrations of blood samples or culture media were determined by radioimmunoassay. Relative mRNA expressions of the genes involved in angiogenesis and steroidogenesis were determined by quantitative real-time PCR. Although FGF2 treatment on day 4 of the estrous cycle did not change the cycle length, FGF2 with PGF_2α decreased the P₄ concentrations observed during the estrous cycle compared to the control group (P < 0.001). Moreover, FGF2 treatment on day 10 prolonged CL function as indicated by a significantly greater concentration of P₄ on day 21 compared to the control group. In the in vitro study, FGF2 decreased cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase (HSD3B1) mRNA expression (P < 0.01) and decreased P₄ production in the early-stage CL (P < 0.001). However, FGF2 + PGF_2α or PGF_2α alone resulted in an elevation of steroidogenic acute regulatory protein and CYP11A1 mRNA expression and P₄ secretion in the early-stage CL (P < 0.01). In the mid-luteal phase, FGF2 upregulated CYP11A1 and HSD3B1 mRNA expression (P < 0.01), while FGF2 + PGF_2α increased only HSD3B1 mRNA expression (P < 0.001). In conclusion, FGF2 seems to play a modulatory role in CL development or luteolysis, differentially regulating steroidogenesis and angiogenic factors as well as PGF_2α actions.     

Effect of experimental infection with Listeria monocytogenes on the development of pregnancy and on concentrations of progesterone,oestrone sulphate and 15-ketodihydro-PGF2α in the goat

The effect of Listeria monocytogenes infection on hormone levels in pregnant goats was studied. Four goats (Group I) received an intravenous inoculation of a bacterial culture (Type 1) on Days 69–77 and another four goats (Group II) received a similar inoculation on Days 105–106 of gestation. Five non-inoculated goats were used as controls. Plasma was analysed for progesterone, oestrone sulphate and 15-ketodihydro-PGF_2α. The status of the foetus was followed using real-time ultrasonography.Three of the four goats in Group I aborted 8–10 days after inoculation. The fourth goat gave birth to a normal live kid at term. The three goats which aborted showed clinical signs of disease in connection with abortion. In Group II, all goats aborted after 9–11 days. All the goats showed clinical symptoms of disease from a few days after inoculation and the symptoms continued until abortion. The clinical symptoms of disease were more pronounced in Group II than in Group I. L. monocytogenes was isolated from all aborted foetuses. None of the control goats aborted.Ultrasound examination revealed foetal death either immediately before or up to 2 days before abortion. Mummification had begun in the foetus that had been dead for 2 days before expulsion.In comparison with pre-inoculation plasma levels in Group I, a significant decrease (P < 0.05) in progesterone levels and an increase in 15-ketodihydro-PGF_2α levels were observed from Days 4 and 6 after inoculation, respectively. In Group II, a significant decrease (P < 0.05) in progesterone levels and an increase in 15-ketodihydro-PGF_2α levels in plasma were observed from Days 8 and 6, respectively. The oestrone sulphate levels decreased slightly in the inoculated goats a few days before abortion.The pattern of changes in levels around abortion was similar to the pattern present in the control animals around parturition. However, oestrone sulphate levels did not increase in the inoculated groups before abortion in contrast to goats which delivered healthy kids. The changes in levels of 15-ketodihydro-PGF_2α in goats that aborted indicated that the endocrine foetal-placental function was disturbed, which was most likely due to the establishment and development of L. monocytogenes in the placenta and foetus.     

Nonenzymatic hydrolysis reactions of adenosine 5′-triphosphate and related compounds: II. Kinetic studies of the hydrolysis of ATP and related compounds with various polyamines

Various structural features of polyamines which are responsible for the acceleration of the hydrolysis of ATP to ADP and P_i at pH 3–4 were surveyed by means of kinetic studies, leading to the following conclusions: 1) The ethyleneimine chain of the polyamines should be as long as possible; 2) the number of methylene carbon atoms between the two adjacent nitrogen atoms of the polyamine has to be two; 3) the terminal groups of the ethyleneimine chain should be primary amino groups.The rate of ATP hydrolysis in the presence of pentaethylenehexamine (pentaen), which possesses the above properties, was found to be 15 times as high as that of hydrolysis at pH 3.5 in the absence of amines. The kinetic data support the previous assumption that there is formation of an ATP-pentaen complex in the hydrolysis reaction. The formation constant of the complex has been calculated to be K = 1.9 × 10⁴M⁻¹ at pH 3.5 and 50°C from the kinetic data. From the temperature dependence of the rates for pentaen or tetraethylenepentamine, the thermodynamic data of these reactions have been obtained.On the other hand, it has been found that pentaen enhances the hydrolysis of GTP and UTP as well as ATP. No phosphate ester bonds of AMP, p-nitrophenylphosphate and α-d-glucose-1-phosphate were hydrolyzed. Therefore, it may be concluded that hydrolysis of the phosphate ester bond with the polyamine is characteristic of ATP, GTP and UTP.     

Effect of the first GnRH and two doses of PGF2α in a 5-day progesterone-based CO-Synch protocol on heifer pregnancy

The objectives were (1) to determine the effects of gonadorelin hydrochloride (GnRH) injection at controlled internal drug release (CIDR) insertion on Day 0 and the number of PGF2α doses at CIDR removal on Day 5 in a 5-day CO-Synch + CIDR program on pregnancy rate (PR) to artificial insemination (AI) in heifers; (2) to examine how the effect of systemic concentration of progesterone and size of follicles influenced treatment outcome. Angus cross beef heifers (n = 1018) at eight locations and Holstein dairy heifers (n = 1137) at 15 locations were included in this study. On Day 0, heifers were body condition scored (BCS), and received a CIDR. Within farms, heifers were randomly divided into two groups: at the time of CIDR insertion, the GnRH group received 100 μg of GnRH and No-GnRH group received none. On Day 5, all heifers received 25 mg of PGF2α at the time of CIDR insert removal. The GnRH and No-GnRH groups were further divided into 1PGF and 2PGF groups. The heifers in 2PGF group received a second dose of PGF2α 6 hours after the administration of the first dose. Beef heifers underwent AI at 56 hours and dairy heifers at 72 hours after CIDR removal and received 100 μg of GnRH at the time of AI. Pregnancy was determined approximately at 35 and/or 70 days after AI. Controlling for herd effect (P < 0.06), the treatments had significant effect on AI pregnancy in beef heifers (P = 0.03). The AI-PRs were 50.3%, 50.2%, 59.7%, and 58.3% for No-GnRH + PGF + GnRH, No-GnRH + 2PGF + GnRH, GnRH + PGF + GnRH, and GnRH + 2PGF + GnRH groups, respectively. The AI-PRs were ranged from 50% to 62.4% between herds. Controlling for herd effects (P < 0.01) and for BCS (P < 0.05), the AI pregnancy was not different among the treatment groups in dairy heifers (P > 0.05). The AI-PRs were 51.2%, 51.9%, 53.9%, and 54.5% for No-GnRH + PGF + GnRH, No-GnRH + 2PGF + GnRH, GnRH + PGF + GnRH, and GnRH + 2PGF + GnRH groups, respectively. The AI-PR varied among locations from 48.3% to 75.0%. The AI-PR was 43.5%, 50.4%, and 64.2% for 2.5 or less, 2.75 to 3.5, and greater than 3.5 BCS categories. Numerically higher AI-PRs were observed in beef and dairy heifers that exhibited high progesterone concentrations at the time of CIDR insertion (>1 ng/mL, with a CL). In addition, numerically higher AI-PRs were also observed in heifers receiving CIDR + GnRH with both high and low progesterone concentration (<1 ng/mL) initially compared with heifers receiving a CIDR only with low progesterone. In dairy heifers, there were no differences in the pregnancy loss between 35 and 70 days post-AI among the treatment groups (P > 0.1). In conclusion, GnRH administration at the time of CIDR insertion is advantageous in beef heifers, but not in dairy heifers, to improve AI-PR in the 5-day CIDR + CO-Synch protocol. In addition, in this study, both dairy heifers that received either one or two PGF2α doses at CIDR removal resulted in similar AI-PR in this study regardless of whether they received GnRH at CIDR insertion.     

Effects of prostaglandins E1, E2 and F2α on the cardiac chronotropism by affecting the cardiac ganglionic transmission in spinal dogs

The effects of several prostaglandins (PGs) injected through the subclavian artery toward the cardiac sympathetic ganglia of spinal dogs were studied by utilizing changes of the heart rate as indicator of ganglionic function. PGF_2α (10–270 μg) administered intra-arterially in the presence or absence of preganglionic stimulation produced weak positive chronotropic effects, which were increased by physostigmine. This positive chronotropic effect of F_2α after physostigmine was inhibited by hexamethonium plus atropine, and depressed after hemicholinium-3 except for the response elicited by the first dose of F_2α. PGE₁ and E₂ injected during preganglionic stimulation did not affect the heart rate. Intra-arterially administered epinephrine and dopamine depressed dose-dependently transmission in the cardiac ganglia, the effect being inhibited by E₁ and E₂ but not by F_2α. These results suggest that F_2α facilitates the release of acetylcholine from preganglionic nerve ending, whereas E₁ and E₂ antagonize the inhibitory actions of catecholamine in the cardiac ganglia.     

Pharmacophore models based studies on the affinity and selectivity toward 5-HT1A with reference to α1-adrenergic receptors among arylpiperazine derivatives of phenytoin

The study is focused on (2-alkoxy)phenylpiperazine derivatives of 1-(2-hydroxy-3-(4-arylpiperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione with alkyl or ester substituents at N3 of hydantoin ring, as well as a new designed and synthesized series of compounds with a free N3H group or N3-acetic acid terminal fragment. The compounds were assessed on their affinity for 5-HT_1A and ??₁-adrenoceptors and evaluated in functional bioassays for antagonistic properties. Classical molecular mechanics (MMFFs force field, MCMM, MacroModel) and DFT methods (B3LYP functional, Gaussian 0.3) were used to investigate 3D structure of the compounds. SAR analysis was based on two pharmacophore models, the one described by Barbaro et al. for ??₁-adenoceptor antagonist and the model of Lepailleur et al. for 5-HT_1A receptor ligands. All compounds exhibited significant to moderate affinities for 5-HT_1A receptors in nanomolar range (7-610 nM). The highest activity (7 nM) and selectivity (17.38) for 5-HT_1A was observed for 1-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (13a). Among new synthesized compounds 1-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione hydrochloride (20a) displayed the highest affinity (16.6 nM) and selectivity (5.72) for ??₁-AR.     

Immunocytochemical and biochemical studies on the localization and changes of 17 α-hydroxylase/C17–C20 lyase activity in immature rat ovary treated with PMSG and hCG

Summary The localization of cytochrome P-450 of 17 -hydroxylase/C17–C20 lyase (P-450_{17 , lyase}) and the changes of the enzyme activity were studied immunocytochemically and biochemically in the ovaries of immature rats treated with PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin). Immunocytochemically, P-450_{17 , lyase} was localized in both the theca interna cells and interstitial gland cells of the ovaries of immature rats treated with PMSG for 48 h. After hCG administration, the immunoreactive cells rapidly decreased in number in the PMSG-pretreated rat ovary. Namely, 6 h after the hCG injection, positive staining for P-450_{17 , lyase} was recognized only in a few theca interna cells, while 12 h after the injection to immunostained cells were detected in the ovary. Forty-eight hours after the hGC treatment (96 h after the PMSG injection), most of the theca interna cells and the interstitial gland cells became immunopositive for P-450_{17 , lyase} again. The 17 -hydroxylating activity of P-450_{17 , lyase} was 0.5, 0.22 and 0.03 nmol/min/mg protein in the ovarian microsomes of PMSG-treated, PMSG+hCG(3 h)-treated and PMSG+hCG(6 h)-treated rats, respectively. Changes of the hydroxylase activities in all the experimental groups are almost parallel to those of P-450 contents in the microsomes. These immunocytochemical and biochemical findings suggest that 1) P-450_{17 , lyase} is localized in both the theca interna cell and interstitial gland cell, and these cells are the main site of the androstenedione production in the ovary, and that 2) the decreased production of estrogen occurring just before ovulation is not brought about by the decreased activity of P-450_{17 , lyase}, but done by the loss of the enzyme.Supported by grants from the Ministry of Education, Science and Culture, Japan     

Identification of 5′-deoxypyridoxine-3-sulfate as the major urinary metabolite of 5′-deoxypyridoxine in rats with comments on the inhibition of arylsulfatase activity and manganese dioxide oxidation by neighboring groups

The major urinary metabolite of 5′-deoxypyridoxine in rats was shown to be identical to 5′-deoxypyridoxine-3-sulfate but different from 5′-deoxypyridoxine-4′-sulfate in its ultraviolet and infrared spectra, its migration in thin-layer chromatography, and its behavior in acid and base. Previous identification of the metabolite as 5′-deoxypyridoxine-4′-sulfate by other workers was based on its failure to be hydrolyzed by arylsulfatase and to be oxidized by manganese dioxide. We have now demonstrated that ortho-methyl groups inhibit arylsulfatase and that ortho-sulfate groups inhibit oxidation by manganese dioxide. Therefore, we conclude that under our conditions the major metabolite of 5′-deoxypyridoxine in the rat was the 3-sulfate.     

Nucleosides LXXXIII. Synthetic studies on nucleoside antibiotics. 11. Synthesis of methyl 4-amino-3,4-dideoxy-β-D-ribo-hexopyranoside and -hexopyranosiduronic acid (derivatives related to the carbohydrate moiety of gougerotin)

Methyl 4-amino-3,4-dideoxy-β-D-ribo-hexopyranoside (17) and its uronic acid (19) were synthesized via a series of reactions starting from 1,2:5,6-di-O-isopropylidene-3-O-tosyl-α-D-glucofuranose. A method suitable for the large scale preparation of 3,4-dideoxy- 1,2:5,6-di-O-isopropylidene-α-D-erythro-hex-3-enofuranose(2) was devised.     

Gamma-secretase inhibitor suppressed Notch1 intracellular domain combination with p65 and resulted in the inhibition of the NF-κB signaling pathway induced by IL-1β and TNF-α in nucleus pulposus cells

In this experiment, the cross-talk betweenNotch and the NF-κB signaling pathway was examined to reveal the mechanism of slowing down the type II collagen (ColII) and aggrecan degeneration affected by inflammatory cytokines. The expression levels of ColII and aggrecan in the intervertebral disc were observed through immunohistochemistry and hematoxylin-eosin staining+alcian blue staining, respectively. The expression levels of ColII, aggrecan, Runx2, and NF-κB in the nuclei of human nucleus pulposus cells (hNPCs) in each group, as well as the phosphorylation and acetylation levels of p65, were examined through Western blot analysis. The 293T cells were transfected with a plasmid containing the overexpressed relative domain of Notch1 intracellular domain (NICD1), and immunoprecipitation (IP) was performed to observe the combination of NICD1 and p65. HNPCs were transfected with a lentiviral-contained overexpression lacking the ANK region of NICD1, and IP was performed to observe the combination of NICD1 and p65. The expression of ColII and aggrecan in the intervertebral disc culture increased when γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-Sphenylglycine t-butyl ester (DAPT) was added to the disc culture medium. Western blot revealed that DAPT inhibited p65 phosphorylation and acetylation, and the p65 and p50 levels in the nucleus decreased. NICD1 was found to be combined with p65 in contrast to the reverse consequences after ANK domain deletion in hNPCs. In nucleus pulposus cells, the combination of p65 and the ANK domain of NICD1 is a critical procedure for the degeneration related to the NF-κB signaling pathway activation induced by IL-1β and TNF-α.     

Association of the 5′ HS4 sequence of the chicken β‐globin locus control region with human EF1α gene promoter induces ubiquitous and high expression of human CD55 and CD59 cDNAs in transgenic rabbits

Whatever its field of application, animal transgenesis aims at a high level of reproducible and stable transgene expression. In the case of xenotransplantation, prevention of hyperacute rejection of grafts of animal origin requires the use of organs expressing human inhibitors of complement activation such as CD55 (DAF) and CD59. Pigs transgenic for these molecules have been produced, but with low and variable levels of expression. In order to improve cDNA expression, a vector containing the 5HS4 region from the LCR of the chicken globin locus and the promoter and the first intron from the human EF1 gene, was used to coexpress human CD55 and CD59 cDNAs in transgenic rabbits. The transgenic lines with the 5HS4 region displayed dramatically enhanced CD55 and CD59 mRNA concentrations in brain, heart, kidney, liver, lung, muscle, spleen and aortic endothelial cells in comparison with the transgenic lines without the 5HS4 region. In the absence of the 5HS4 region, only some of the transgenic lines displayed specific mRNAs and at low levels. Human CD55 and CD59 proteins were detectable in mononuclear cells from transgenic rabbits although at a lower level than in human mononuclear cells. On the other hand, primary aortic endothelial cells from a bitransgenic line were very efficiently protected in vitro against human complementdependent lysis. Transgenic rabbits harbouring the two human inhibitors of complement activation, CD55 and CD59, can therefore be used as new models in xenotransplantation. Moreover, the vector containing the 5HS4 region from the LCR of the chicken globin locus seems appropriate not only for xenotransplantation but also for any other studies involving transgenic animals in which cDNAs have to be expressed at a high level in all cell types.     

Cyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type‐Iα (PKG‐Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG‐Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti‐apoptotic Bcl‐2‐related proteins in NCI‐H460 and A549 non‐small cell lung cancer (NSCLC) cells. 1H‐(1,2,4)oxadiazolo(4,3‐a)quinoxalin‐1‐one (ODQ), which blocks endogenous NO‐induced activation of cGMP/PKG‐Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c‐IAP1, livin, and survivin. DT‐2 (inhibitor of PKG‐Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c‐IAP1, livin, and survivin expression. Gene knockdown of PKG‐Iα expression using small‐interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c‐IAP1, livin, survivin, and Mcl‐1 expression. Inhibition of PKG‐Iα kinase activity with DT‐2 dramatically enhanced pro‐apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT‐2 and cisplatin increased apoptosis compared with cisplatin or DT‐2 alone, showing a synergistic effect. The data suggest that the PKG‐Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c‐IAP1, livin, survivin, and Mcl‐1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin. J. Cell. Biochem. 113: 3587–3598, 2012. © 2012 Wiley Periodicals, Inc.     

Single Nucleotide Polymorphisms in the 3′UTR of VPAC-1 Cooperate in Modulating Gene Expression and Impact Differently on the Interaction with miR525-5p

Complex immune and neurodegenerative disorders are the result of multiple interactions between common genetic variations having, individually, a weak effect on the disease susceptibility or resistance. Interestingly, some genes have been found to be associated with more than one disease although not necessarily the same SNPs are involved. In this context, single nucleotide polymorphisms in the 3′UTR region of type 1 receptor (VPAC-1) for vasoactive intestinal peptide (VIP) have been reported to be associated with some immune-mediated as well as with neurodegenerative diseases such as Alzheimer''s Disease (AD). Here, we demonstrate that variations at the 3′UTR of the VPAC-1 gene act synergistically to affect the expression of the luciferase as well as of the GFP reporter genes expressed in HEK293T cells. Moreover, the miRNA 525-5p, previously shown by us to target the 3′UTR of VPAC-1, is more efficient in decreasing GFP expression when co-expressed with constructs carrying the allele C at rs896 (p<10^-3) suggesting that this miRNA regulates VPAC-1 expression at different levels depending on rs896 polymorphism and thus adding complexity to the network of disease susceptibility.     

The Effect of NeuroAid (MLC901) on Cholestasis-Induced Spatial Memory Impairment with Respect to the Expression of BAX,BCL-2, BAD,PGC-1α and TFAM Genes in the Hippocampus of Male Wistar Rats

Neurochemical Research - Cholestasis is a bile flow reduction that is induced following Bile Duct Ligation (BDL). Cholestasis impairs memory and induces apoptosis. Apoptosis consists of two...     

Structure–activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps

Cytosolic phospholipase A₂α (cPLA₂α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA₂α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA₂α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA₂α and FAAH, respectively.