Profiling circulating microRNAs in the serum of pregnant and non-pregnant pigs reveals a plethora of reproductive status-dependent microRNAs.

Circulating, non-coding RNAs, such as microRNAs (miRNAs) have been proposed to be powerful pathophysiological indicators of pregnancy in animals and humans. Since their discovery, it is known that miRNAs can take part in numerous biological processes, including cell proliferation and differentiation during early embryonic development and establishment of pregnancy. Our recent studies have indicated that maternal blood can carry miRNAs reported previously at the embryo-maternal interface in pigs. To expand the scope of our research, we tested the hypothesis that miRNAs previously identified in conceptuses, trophoblasts, endometrium and uterine lumen-derived extracellular vesicles (EVs) collected before Day 20 of pregnancy can show reproductive status-dependent profiles in the serum of cyclic and pregnant crossbred pigs. Custom-designed TaqMan arrays, multiplex real-time reverse transcription (RT)-PCR and real-time RT-PCR allowed us to identify a number of reproductive status-dependent miRNAs in serum samples collected from pigs during the estrous cycle or pregnancy (Days 16 and 20). We found that serum samples were enriched with miRNAs involved in processes important during the estrous cycle and early pregnancy, e.g. cell sensitivity and viability, angiogenesis, embryonic cell proliferation and differentiation. Further validation revealed different abundance of ssc-miR-143-3p and ssc-miR-125b in pregnant and non-pregnant animals and correlation of ssc-miR-125b levels with litter size. In addition, analyzed serum samples contained both EVs and Argonaute2 proteins, which are known to be involved in miRNA transportation and intercellular communication. In summary, we identified several circulating miRNAs that differ in abundance between cyclic and pregnant animals and could serve as potential indicators of reproductive status in pigs during breeding management.

Aminoalkynyldithianes. A new class of calcium channel blockers.

Several dithiane derivatives, prepared as intermediates for compounds structurally related to the therapeutically useful antimuscarinic agent oxybutynin, were effective inhibitors of calcium ion induced contraction of guinea pig ileal strips and of KCl-induced calcium entry into neuronal cells. Although the first member of this series, 2-[5-(diethylamino)-3-pentynyl]-1,3-dithiane (2a), was only marginally effective, its condensation product with diphenyl ketone, i.e. 2-[5-(diethylamino)-3-pentynyl]-2-(a,a-diphenyl-a- hydroxymethyl)-1,3-dithiane (3a), demonstrated weak, but significant, calcium channel antagonist activity. As part of a structure-activity relationship (SAR) study, various structural analogues of 2a and 3a were prepared and examined for calcium antagonist properties. In addition to these structural types, ring bridged (tricyclic) congeners of 3, i.e. 4, related bicyclic compounds 5, dehydroxylated derivatives 6, some homologous 2-[[[(N,N-disubstituted-amino)methyl]2- phenyl-1,3-dithianes (7), and a series of 2-[6-[N,N-disubstituted-amino)methyl]-1-hydroxy-1-phenyl- 4-hexynyl]-1,3-dithianes (8) were prepared and studied for calcium channel blocking activity. In general, greatest potency was noted in the tricyclic series 4; however, a definitive SAR could not be established. A structural similarity between several potent calcium antagonists having the structures 7c, 8b, and 8d and the well-known calcium channel blockers verapamil and tiapamil suggests these compounds may act at the same site. Compounds in the other classes (2-6) failed to show clearly defined SAR and their potency differed markedly in two tests for calcium channel antagonist activity. These results may indicate that the dithiane derivatives 2-6 produce their effects in a manner differing from that of the calcium channel antagonists diltiazem, verapamil, and nitrendepine.

Pharmacological profile of NPC 12626, a novel, competitive N-methyl-D-aspartate receptor antagonist.

The novel compound 2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid (NPC 12626) was evaluated for activity in a variety of tests associated with receptors for excitatory amino acids. NPC 12626 failed to inhibit the specific binding of RS-[3H] amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid or [3H] kainic acid to brain membranes in vitro but displaced both agonist and antagonist binding to N-methyl-D-aspartic acid (NMDA) receptors. Like cis-(+/-)-3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid, NPC 12626 competitively blocked NMDA-induced enhancement of [3H]-1-thienylcyclohexyl)piperidine binding. In the voltage-clamped frog oocyte expression system, NPC 12626 was a competitive inhibitor of NMDA-evoked inward current with a pA2 of 6.24. After both i.c.v. or i.p. administration, NPC 12626 was a potent anticonvulsant in the pentylenetetrazol, maximal electroshock and NMDA seizure models. Furthermore, low doses (25 mg/kg) of NPC 12626 given i.v. were effective in preventing damage to the CA1 region of hippocampus in the gerbil model of global ischemia. Unlike the noncompetitive NMDA antagonist, phencyclidine, but like cis-(+/-)-3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid and pentobarbital, NPC 12626 only partially substituted for phencyclidine in a drug discrimination study. The results of the current study indicate that NPC 12626 is a novel, systemically active and competitive NMDA receptor antagonist.