Real-time changes of the local vasoactive peptide systems (angiotensin,endothelin) in the bovine corpus luteum after induced luteal regression

There is evidence that angiotensin II (Ang II) and endothelin-1 (ET-1) may interact in an additive or synergistic way during luteal regression. The aim of the study was to investigate real time changes in luteal tissue of angiotensin and endothelin system members in mRNA expression, tissue concentrations, tissue localization, and ACE (angiotensin converting enzyme) antagonist application after prostaglandin F(2alpha) (PG) induced (days 8-12) luteal regression in cow. Corpora lutea (CL) were collected by transvaginal ovaryectomy before and 2, 4, 12, 24, 48, and 64 hr (n = 5/time point) after PG injection. ACE mRNA expression (RT-PCR) increased continuously and peaked at 12, 24 hr; ECE-1 (endothelin converting enzyme) peaked at 12 hr, and both peptides in tissue (Ang II and ET-1) increased significantly and peaked at 24 hr. The expression of receptors for Ang II (AT1R and AT2R) did not change in contrast to ET receptors (ETR-A and ETR-B), which were up-regulated. Localization in tissue revealed very weak staining for Ang II and ET-1 before PG application followed by a clear increase of staining predominantly in large luteal cells, but also in endothelial cells. In two experiments, the attempt was made to block ACE by the antagonist captopril with two different doses. In both experiments with captopril, progesterone levels were not significantly different from controls. Ang II alone seems to be not essential for functional luteolysis in bovine system. In conclusion, the results suggest that both Ang II and ET-1 are in parallel up-regulated during luteal regression and may act as vasoconstrictors during functional luteolysis, but also as apoptosis inducer during functional/structural luteolysis.     

Initial determination of DNA polymorphism of some Primula veris L. populations from Kosovo and Austria

Primula veris L. (Primulaceae) is a long lived perennial and well known pharmaceutical plant, widely collected for these reasons in almost all SE Europe and particularly in Kosovo. The aim of the study is to determine molecular polymorphism of cowslip (P. veris L.) populations from Kosovo. DNA extracted from leaves were  investigated in details for presence of polymorphism. RAPD analyses were conducted using 20 different short primers. Genomic DNA amplification profiles were analyzed and processed using data labelling. Comparison between cowslip populations in genetic composition revealed that samples from Bogaj were too distinct on their own. Molecular variation was observed to be more within populations (73 %) as compared to among populations (27 %). On the other hand, genetic distance of populations revealed that the highest genetic distance is between Leqinat and Maja e Madhe. Mean values of expected heterozygosity were highest in Bogaj population, while lowest in Maja e Madhe population. The obtained results indicated that Bogaj population are more polymorphic. From the obtained data it can be concluded that RAPD markers provided a useful technique to study genetic diversity in P. veris L. populations. This technology allows identification and assessment of the genetic similarities and differences among plant populations.     

HDL from apoA1 transgenic mice expressing the 4WF isoform is resistant to oxidative loss of function

HDL functions are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes human apoA1. We previously found that the 4WF isoform of human apoA1, in which the four tryptophan residues are substituted with phenylalanine, is resistant to MPO-mediated loss of function. The purpose of this study was to generate 4WF apoA1 transgenic mice and compare functional properties of the 4WF and wild-type human apoA1 isoforms in vivo. Male mice had significantly higher plasma apoA1 levels than females for both isoforms of human apoA1, attributed to different production rates. With matched plasma apoA1 levels, 4WF transgenics had a trend for slightly less HDL-cholesterol versus human apoA1 transgenics. While 4WF transgenics had 31% less reverse cholesterol transport (RCT) to the plasma compartment, equivalent RCT to the liver and feces was observed. Plasma from both strains had similar ability to accept cholesterol and facilitate ex vivo cholesterol efflux from macrophages. Furthermore, we observed that 4WF transgenic HDL was partially (∼50%) protected from MPO-mediated loss of function while human apoA1 transgenic HDL lost all ABCA1-dependent cholesterol acceptor activity. In conclusion, the structure and function of HDL from 4WF transgenic mice was not different than HDL derived from human apoA1 transgenic mice.     

Temporal Lobe Cortical Thickness Correlations Differentiate the Migraine Brain from the Healthy Brain

BackgroundInterregional cortical thickness correlations reflect underlying brain structural connectivity and functional connectivity. A few prior studies have shown that migraine is associated with atypical cortical brain structure and atypical functional connectivity amongst cortical regions that participate in sensory processing. However, the specific brain regions that most accurately differentiate the migraine brain from the healthy brain have yet to be determined. The aim of this study was to identify the brain regions that comprised interregional cortical thickness correlations that most differed between migraineurs and healthy controls.MethodsThis was a cross-sectional brain magnetic resonance imaging (MRI) investigation of 64 adults with migraine and 39 healthy control subjects recruited from tertiary-care medical centers and their surrounding communities. All subjects underwent structural brain MRI imaging on a 3T scanner. Cortical thickness was determined for 70 brain regions that cover the cerebral cortex and cortical thickness correlations amongst these regions were calculated. Cortical thickness correlations that best differentiated groups of six migraineurs from controls and vice versa were identified.ResultsA model containing 15 interregional cortical thickness correlations differentiated groups of migraineurs from healthy controls with high accuracy. The right temporal pole was involved in 13 of the 15 interregional correlations while the right middle temporal cortex was involved in the other two.ConclusionsA model consisting of 15 interregional cortical thickness correlations accurately differentiates the brains of small groups of migraineurs from those of healthy controls. Correlations with the right temporal pole were highly represented in this classifier, suggesting that this region plays an important role in migraine pathophysiology.     

FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells

Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d.     

Comparison of diagnostic performances of ten different immunoassays detecting anti-CCHFV IgM and IgG antibodies from acute to subsided phases of Crimean-Congo hemorrhagic fever

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a geographically widespread tick-borne arbovirus that has been recognized by the WHO as an emerging pathogen needing urgent attention to ensure preparedness for potential outbreaks. Therefore, availability of accurate diagnostic tools for identification of acute cases is necessary.A panel comprising 121 sequential serum samples collected during acute, convalescent and subsided phase of PCR-proven CCHFV infection from 16 Kosovar patients was used to assess sensitivity. Serum samples from 60 healthy Kosovar blood donors were used to assess specificity. All samples were tested with two IgM/IgG immunofluorescence assays (IFA) from BNITM, the CCHFV Mosaic 2 IgG and IgM indirect immunofluorescence tests (IIFT) from EUROIMMUN, two BlackBox ELISAs for the detection of CCHFV-specific IgM and IgG antibodies (BNITM), two Anti-CCHFV ELISAs IgM and IgG from EUROIMMUN using recombinant structural proteins of CCHFV antigens, and two ELISAs from Vector-Best (IgM: μ-capture ELISA, IgG: indirect ELISA using immobilized CCHFV antigen). Diagnostic performances were compared between methods using sensitivity, specificity, concordance and degree of agreement with particular focus on the phase of the infection.In early and convalescent phases of infection, the sensitivities for detecting specific IgG antibodies differed for the ELISA test. The BlackBox IgG ELISA yielded the highest, followed by the EUROIMMUN IgG ELISA and finally the VectorBest IgG ELISA with the lowest sensitivities. In the subsided phase, the VectorBest IgM ELISA detected a high rate of samples that were positive for anti-CCHFV IgM antibodies. Both test systems based on immunofluorescence showed an identical sensitivity for detection of anti-CCHFV IgM antibodies in acute and convalescent phases of infection.Available serological test systems detect anti-CCHFV IgM and IgG antibodies accurately, but their diagnostic performances vary with respect to the phase of the infection.     

Growth hormone, but not luteinizing hormone, acts with luteal peptides on prostaglandin F2alpha and progesterone secretion by bovine corpora lutea in vitro

Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-I (IGF-I) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.