Serum estradiol-17 beta, progesterone and respective uterine cytosol receptor concentrations in bitches with spontaneous pyometra

The role of serum estradiol-17 beta (E(2)) and progesterone (P(4)) in relation to uterine estrogen (ER) and progesterone receptors (PR) was investigated in canine cystic endometrial hyperplasia-pyometra (CEH-P). Blood and uterine samples were collected pre- and post-ovariohysterectomy, respectively, from 54 bitches presenting spontaneous CEH-P and 25 healthy control bitches. Competitive enzyme immunoassays (EIA) and enzyme ligand immunoassays (ELIA) were applied to estimate serum hormones and uterine cytosol active receptors, respectively. Animals were classified in the stages of first half of diestrus, second half of diestrus and early anestrus on the basis of reproductive history, clinical signs, uterine and ovarian macro- and microscopic inspection and serum P(4) concentration. Bitches with CEH-P, compared to their respective stage controls, exhibited (a) similar P(4) fluctuations, (b) higher E(2) concentrations, (c) lower PR concentrations during diestrus first and second half and (d) lower ER concentrations during diestrus first half and early anestrus. Negative correlation was detected between P(4) and ER within both CEH-P and control groups. It was concluded that P(4) was the main uterine receptor regulator for both PR and ER during diestrus and early anestrus in healthy and affected uteri. However, in CEH-P bitches, high P(4) levels in diestrus appeared to over-activate uterine PRs, leading to stronger PR self-down regulation and ER suppression. These findings indicate an increased sensitivity of CEH-P uterus to P(4) action. During early anestrus, a complementary role of endogenous E(2) was considered, since reduction of P(4) action appeared to permit uterine ER replenishment and activation by relatively high E(2) levels.     

Murine diet/tissue and human brain tumorigenesis alter <Emphasis Type="Italic">Mthfr/MTHFR</Emphasis> 5′-end methylation

Polymorphisms and decreased activity of methylenetetrahydrofolate reductase (MTHFR) are linked to disease, including cancer. However, epigenetic regulation has not been thoroughly studied. Our goal was to generate DNA methylation profiles of murine/human MTHFR gene regions and examine methylation in brain and liver tumors. Pyrosequencing in four murine tissues revealed minimal DNA methylation in the CpG island. Higher methylation was seen in liver or intestine in the CpG island shore 5′ to the upstream translational start site or in another region 3′ to the downstream start site. In the latter region, there was negative correlation between expression and methylation. Three orthologous regions were investigated in human MTHFR, as well as a fourth region between the two translation start sites. We found significantly increased methylation in three regions (not the CpG island) in pediatric astrocytomas compared with control brain, with decreased expression in tumors. Methylation in hepatic carcinomas was also increased in the three regions compared with normal liver, but the difference was significant for only one CpG. This work, the first overview of the Mthfr/MTHFR epigenetic landscape, suggests regulation through methylation in some regions, demonstrates increased methylation/decreased expression in pediatric astrocytomas, and should serve as a resource for future epigenetic studies.     

The Mediterranean monk seal Monachus monachus: status,biology, threats,and conservation priorities

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Oximes short-acting CB1 receptor agonists

New oximes short-acting CB1 agonists were explored by the introduction of an internal oxime and polar groups at the C3 alkyl tail of Δ⁸-THC. The scope of the research was to drastically alter two important physicochemical properties hydrophobicity (log P) and topological surface area (tPSA) of the compound, which play a critical role in tissue distribution and sequestration (depot effect). Key synthesized analogs demonstrated sub-nanomolar affinity for CB1, marked reduction in hydrophobicity (ClogP～2.5–3.5 vs 9.09 of Δ⁸-THC-DMH), and found to function as either agonists (trans-oximes) or neutral antagonists (cis-oximes) in a cAMP functional assay. All oxime analogs showed comparable affinity at the CB2 receptor, but surprisingly they were found to function as inverse agonists for CB2. In behavioral studies (i.e. analgesia, hypothermia) trans-oxime 8a exhibited a predictable fast onset (～20?min) and short duration of pharmacological action (～180?min), in contrast to the very prolonged duration of Δ⁸-THC-DMH (>24?h), thus limiting the potential for severe psychotropic side-effects associated with persistent activation of the CB1 receptor. We have conducted 100?ns molecular dynamic (MD) simulations of CB1 complexes with AM11542 (CB1 agonist) and both trans-8a and cis-8b isomeric oximes. These studies revealed that the C3 alkyl tail of cis-8b orientated within the CB1 binding pocket in a manner that triggered a conformational change that stabilized the CB1 receptor at its inactive-state (antagonistic functional effect). In contrast, the trans-8a isomer’s conformation was coincided with that of the AM11542 CB1 agonist-bound structure, stabilizing the CB1 receptor at the active-state (agonistic functional effect). We have selected oxime trans-8a based on its potency for CB1, and favorable pharmacodynamic profile, such as fast onset and predictable duration of pharmacological action, for evaluation in pre-clinical models of anorexia nervosa.     

Synthesis and cytotoxic evaluation of novel pyrimidine deoxyapiothionucleosides

The synthesis of novel pyrimidine deoxyapiothionucleosides of d- and l-series was realized following application of a versatile and high-yielding scheme, which utilized inexpensive l- and d-arabinose as starting materials, respectively, and which makes use of a regio- and stereo-selective Pummerer rearrangement reaction for the coupling of the nucleobase with the thiosugar moiety. Some of the targeted compounds have shown selective cytotoxic effects (with IC₅₀ <10 μM) against specific cancer cell lines. All of the tested compounds had no cytotoxic effect on the normal cell line tested.     

An in silico erythropoiesis model rationalizing synergism between stem cell factor and erythropoietin

Stem cell factor (SCF) and erythropoietin (EPO) are two most recognized growth factors that play in concert to control in vitro erythropoiesis. However, exact mechanisms underlying the interplay of these growth factors in vitro remain unclear. We developed a mathematical model to study co-signaling effects of SCF and EPO utilizing the ERK1/2 and GATA-1 pathways (activated by SCF and EPO) that drive the proliferation and differentiation of erythroid progenitors. The model was simplified and formulated based on three key features: synergistic contribution of SCF and EPO on ERK1/2 activation, positive feedback effects on proliferation and differentiation, and cross-inhibition effects of activated ERK1/2 and GATA-1. The model characteristics were developed to correspond with biological observations made known thus far. Our simulation suggested that activated GATA-1 has a more dominant cross-inhibition effect and stronger positive feedback response on differentiation than the proliferation pathway, while SCF contributed more to the activation of ERK1/2 than EPO. A sensitivity analysis performed to gauge the dynamics of the system was able to identify the most sensitive model parameters and illustrated a contribution of transient activity in EPO ligand to growth factor synergism. Based on theoretical arguments, we have successfully developed a model that can simulate growth factor synergism observed in vitro for erythropoiesis. This hypothesized model can be applied to further computational studies in biological systems where synergistic effects of two ligands are seen.     

RNA-Mediated Feedback Control of Transcriptional Condensates

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Paraoxonase 1 R/Q alleles are associated with differential accumulation of saturated versus 20:5n3 fatty acid in human adipose tissue

Serum paraoxonase 1 (PON1) function has been associated with human cardiovascular disease. The projected mechanism postulates interaction of PON1 with lipoproteins and insulin signaling resulting in alterations in lipid homeostasis. Recently, PON2 was shown to directly regulate triglyceride accumulation in macrophages and PON1 was detected in the interstitial space of adipocytes. The aims of the present study were a) to examine the relationship of the PON1 function with serum parameters related to lipid homeostasis, and b) to examine a possible role of PON1 in the regulation of lipid composition in the human adipose tissue. Two important genetic variations with functional impact on PON1 activity in humans are the Q192R and the L55M. The present study evaluated the impact of the Q192R and the L55M polymorphisms in a cross-section of the population on the island of Crete, as regards to PON1 activity, plasma lipids/lipoproteins, parameters of the metabolic syndrome, and the fatty acid composition of the adipose tissue. We detected a significant association of the polymorphisms with blood pressure, fasting blood glucose, triglycerides, apolipoprotein B, serum iron, and homocysteine. Furthermore, a novel function is suggested for PON1 on the fatty acid composition in the adipose tissue through the positive association of the R allele with saturated fatty acid and of the Q allele with 20:5n3 fatty acid deposition.     

Insight in the transport behavior of copper glycinate complexes through the porcine gastrointestinal membrane using an Ussing chamber assisted by mass spectrometry analysis

An Ussing chamber study was conducted in order to investigate the transport behavior of copper glycinate complexes through a porcine gastrointestinal membrane. Organic copper complexes such as copper tri- and tetraglycinates (GGG–Cu(II) and GGGG–Cu(II)) were used as model system. In a novel analytical approach the Ussing chamber was combined with mass spectrometry. Therefore, relevant analytical methods based on MALDI-MS and a coupling of capillary electrophoresis to ICP-MS and ESI-MS were developed for the determination of copper complexes in the mucosal and serosal half-chambers. It was found that 86.1±8.5% of copper triglycinate but only 20.8±9.9% of copper tetraglycinate penetrated the digestive membrane without modification. Furthermore, inorganic copper species were not detected but a new copper complex (m/z 442) was found to be formed in both compartments of the Ussing chamber.