Influence of the uterine inflammatory response after insemination with frozen-thawed semen on serum concentrations of acute phase proteins in mares.

The aim of this study was to investigate the clinical relevance of measuring blood concentrations of serum amyloid A (SAA), haptoglobin (Hp) and fibrinogen (Fib) in horse reproductive management, and changes in response to artificial insemination (AI) with frozen-thawed semen. Standardbred mares (n=18) with different reproductive status (eight healthy mares in first postpartum oestrus, five healthy barren mares and five mares with endometritis) were inseminated with frozen-thawed semen. Endometritis was evaluated during oestrus by bacteriological culture, cytology and presence of ultrasonically visible intrauterine fluid during oestrus. Concentrations of SAA, Hp and Fib were analysed in the blood in every 48h during oestrus and until 5, 6 or 7 days after AI. The day of sampling and number of blood samples varied between mares because of length of the oestrus and time of AI. Changes in concentrations of SAA, Hp and Fib were evaluated based on the day of sampling regard to AI and classification of the mares. There were no differences in SAA, Hp and Fib concentrations over time before or after AI or between the groups of mares. The insemination of mares with frozen-thawed semen did not increase the plasma concentrations of SAA, Hp and Fib above clinical threshold concentration and there were no differences between susceptible or healthy mares.

Higher than expected estradiol levels in aromatase inhibitor-treated, postmenopausal breast cancer patients.

OBJECTIVE: Vaginal estradiol is considered contraindicated in aromatase inhibitor (AI)-treated patients because of the risk of elevated estrogen levels. This leaves limited treatment options for patients experiencing gynecological symptoms. However, in clinical practice, no precise estimation has been performed of circulating estrogens and aromatase index in postmenopausal breast cancer patients on long-lasting AI or tamoxifen treatment. METHODS: Steroid hormones were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) and extraction radioimmunoassay (RIA). Postmenopausal AI-treated patients (n =33) were compared with tamoxifen-treated patients (n =34) and controls without vaginal treatment (n =56), with vaginal estradiol (n =25), or with estriol (n =11) treatment. RESULTS: By use of LC-MS/MS, median (range) estradiol plasma concentrations were 16.7 (2.4-162.6), 31.0 (13.4-77.1), 27.2 (7.8-115.8) and 33.3 (20.3-340.1) pmol/l in AI-treated breast cancer patients, tamoxifen-treated breast cancer patients, postmenopausal controls and postmenopausal controls on vaginal estradiol, respectively. The AI-treated group and subgroups had significantly lower estradiol and estrone concentrations than all other groups (p <0.05). There was extensive interindividual variation in estradiol concentration within the AI-treated group, measured using both LC-MS/MS (2.3-182.0 pmol/l) and extraction RIA (2.4-162.6 pmol/l). The AI-treated group had lower aromatase index compared to all other groups (p <0.05-0.001). CONCLUSION: Circulating estrogen levels may have been underestimated in previous longitudinal studies of AI-treated breast cancer patients. Additional studies are required to further evaluate the role of circulating estrogens in breast cancer patients suffering from gynecological symptoms.

The effects of presentation methods and semantic information on multi-ethnicity face recognition

Studies have shown that own-race faces are more accurately recognised than other-race faces. The present study examined the effects of own- and other-race face recognition when different ethnicity targets are presented to the participants together. Also the effect of semantic information on the recognition of different race faces was examined. The participants (N = 234) were presented with photos of own-race and other-race faces. For some participants the faces were presented with stereotypical names and for some not. As hypothesized, own-race faces were better recognised in target-present lineup and more correctly rejected in target-absent lineup than other-race faces. Concerning presentation method, both own-race and other-race faces were more correctly identified in target-present simultaneous than in target-present sequential lineups. No effects of stereotypical names on face recognition were found. The findings suggest that identifying multi-ethnicity perpetrators is a problematic and difficult task(AU)

La literatura ha demostrado que caras de la raza propia son reconocidas con mayor exactitud que las de otras razas. El presente estudio examinó los efectos del reconocimiento de caras en condiciones de presentación conjunta de personas de diferentes etnias. Asimismo, también se estudió el efecto de la información semántica en el reconocimiento de caras de razas distintas. Para ello, se le presentaron a los participantes (N=234) fotografías de caras de su propia y de otras razas. Para unos participantes, las fotografías de las caras fueron presentadas con nombres estereotipados y para otros no, Como se había hipotetizado, las caras de la raza propia, en comparación con caras de otras razas, fueron reconocidas mejor en ruedas de identificación con el sospechoso presente a la vez que se registraron mayores rechazos correctos en ruedas con el sospechoso ausente. Caras de otras razas fueron(AU)

Assessment of early postpartum reproductive performance in two high producing Estonian dairy herds.

Early postpartum (6 weeks) ovarian activity, hormonal profiles, uterine involution, uterine infections, serum electrolytes, glucose, milk acetoacetate and blood urea nitrogen (BUN) levels were studied in 2 Estonian high producing dairy herd with annual milk production of 7688 (Farm A) and 9425 (Farm B). From each farm 10 cows, with normal calving performance were used. Blood samples for the hormonal (PGF2alpha-metabolite, progesterone) analyses were withdrawn. On day 25 PP blood serum samples were taken for the evaluation of metabolic/electrolyte status. On the same day estimation of milk acetoacetate values was done. The ultrasound (US) was started on day 7 PP and was performed every 3rd day until the end of experiment. Uterine content, follicular activity and sizes of the largest follicle and corpus luteum were monitored and measured. Vaginal discharge and uterine tone were recorded during the rectal palpation. Each animal in the study was sampled for bacteriological examination using endometrial biopsies once a week. Two types of PGF2alpha-metabolite patterns were detected: elevated levels during 14 days PP, then decline to the basal level and then a second small elevation at the time of final elimination of the bacteria from the uterus: or elevated levels during first 7 days PP, then decline to the basal level and a second small elevation before the final elimination of bacteria. Endometritis was diagnosed in 5 cows in farm A and in 3 cows in farm B respectively. In farm A, 5 cows out of 10 ovulated during experimental period and in 1 cow cystic ovaries were found. In farm B, 3 cows out of 10 ovulated. In 3 cows cystic ovaries were found. Altogether 40% of cows had their first ovulation during the experimental period. Three cows in farm A and 5 cows in farm B were totally bacteria negative during the experimental period. The most frequent bacteria found were A. pyogenes, Streptococcus spp., E. coli., F. necrophorum and Bacteroides spp. The highest incidence of bacteriological species was found during the first 3 weeks in both farms. All animals were free from bacteria after 5th week PP in farm A and after 4th week in farm B respectively. Serum electrolytes and glucose levels were found to be within the reference limits for the cows in both farms. No significant difference was found between farms (p > 0.05). Low phosphorus levels were found in both farms. Significant difference (p < 0.05) was found in BUN levels between farms. In both farms milk acetoacetate values were staying within the reference range given for the used test (< 100 micromol/l). The uterine involution and bacterial elimination in the investigated cows could consider as normal but more profound metabolic studies could be needed to find reasons for later resumption of ovarian activity. Some recommendations to changing feeding regimes and strategies should also be given.

15-ketodihydro-PGF2 alpha, progesterone and cortisol profiles in heifers after induction of parturition by injection of dexamethasone.

In order to study rapid changes in 15-ketodihydro-PGF2 alpha, cortisol and progesterone in the period preceding parturition in cattle, pre-term parturition was induced in 4 late pregnant heifers. Parturitions were induced by 2 intramuscular injections of 20 mg dexamethasone with a 24-h interval. The first injection was made on days 254, 258, 264 and 265 in gestation, respectively. Twenty-four h before the first injection an intravenous polyurethane cannula was inserted. Blood samples were collected at least every hour until 12 h after parturition and during the second stage of labour at least 6 times per hour. Plasma was analysed for 15-ketodihydro-PGF2 alpha and progesterone by radioimmunoassays, and for cortisol by an ELISA. The average time from injection to parturition was 7.7 (6.6-8.9) days (mean (range)). Two of the heifers had retained foetal membranes (RFM). At the start of the experiment the levels of PGF2 alpha metabolite were low (< 300 pmol/L) and increased slowly to levels between 1000 and 2000 pmol/L at one day before parturition. During the last day, however, the levels increased rapidly and the highest levels (> 10,000 pmol/L) were reached at the time of delivery. No pulsatile release was seen. Immediately after foetal expulsion the PG-metabolite levels decreased rapidly in all animals. In the 2 animals with RFM, however, this decline ceased within a few h. The PG-metabolite levels in these animals then started to increase and reached levels as high as during parturition. Luteolysis occurred between 1.6 and 0.4 days before parturition in all animals. The cortisol profile showed a distinct peak at the time of parturition in the RFM heifers. This peak was absent in the non-RFM heifers. This study shows that the PGF2 alpha release at prepartal luteolysis and parturition is not pulsatile in cattle and that cortisol profiles in heifers with retained foetal membranes might differ from the profiles in non-RFM heifers at the time of parturition.

Developmental profile of kainate receptor subunit KA1 revealed by Cre expression in YAC transgenic mice.

To determine the spatio-temporal expression in brain of the high-affinity kainate receptor subunit KA1, we generated transgenic mice expressing Cre recombinase from the KA1 gene on a chromosomally integrated 550 kb yeast artificial chromosome (YAC). Activity of the KA1 gene promoter during brain development was visualized by Cre immunohistochemistry, and by X-gal staining of beta-galactosidase induced by Cre recombinase in double transgenic KA1-Cre/lacZ indicator mice. During early brain development, expression from the YAC-carried KA1-Cre transgene was observed in all major brain areas, predicting a function for KA1 in the developing central nervous system. In the adult brain, KA1-Cre transgene expression was restricted mainly to hippocampal CA3 pyramidal and dentate gyrus granule cells, an adult expression pattern characteristic for the endogenous KA1 alleles. KA1-Cre transgenic mice may help in elucidating the role of floxed genes ablated in vivo in KA1 expressing neurons.

Induction of parturition with prostaglandin f2 alpha as a possible model to study impaired reproductive performance in the dairy cow.

Parturitions were induced in five cows, 2 weeks before term using prostaglandin (PG) F(2alpha). Two i.m. injections were performed with an interval of 24 h. All cows calved within 5 days (average 2.7 days) after the first injection of PGF(2alpha). Out of five cows, four had retained fetal membranes (RFM). Each animal was sampled for bacteriological examination using uterine biopsies twice a week during 42 days postpartum (PP). Jugular vein blood samples were withdrawn for PGF(2alpha)-metabolite and progesterone analyses five times per day during the first week PP and eight times per 24 h during the 2nd and 3rd weeks PP. From the 4th week, the sampling interval was reduced back to five times per day. From the 5th week PP, the sampling was reduced to two times per day and sampling was terminated after day 46 PP. Only morning samples were used for progesterone analyses. From day 10 PP, ultrasonography (US) was performed every 3rd day until day 39 PP for detection of ovarian activity and follicular dynamics. The highest incidence of bacteriological species was found during the first 3 weeks PP. After the 5th week of collection, all animals were free from bacteria. The species of bacteria found were Arcanobacterium (Actinomyces) pyogenes, Escherichia coli, alpha-hemolytic streptococcae and Pasteurella multocida. Immediately after parturition, very high levels of the PG-metabolite were seen in all animals, with a sharp decrease to line of significance around days 9-12 PP. Small increases above the line of significance were detected up to day 27 PP in cows with RFM, and after that time the levels were considered to be at baseline. Low levels of progesterone were seen in four animals during the whole experimental time. In one animal, an increase was seen on day 43 PP, which was maintained until the end of the experimental period on day 46 PP. Based on US, follicular waves were detected in all animals during the experimental period. In three animals, three non-ovulatory follicular waves were detected and in two animals, four non-ovulatory follicular waves were detected during 39 days of ultrasound sessions. Based on progesterone levels, only one animal was considered to have ovulated around day 40 PP. Results from the present study indicate that reproductive performance of cows after PG-induced parturitions differs from those of spontaneous cases of RFM. Differences regarding the resumption of ovarian activity were also observed between previous studies of dexamethasone-induced parturitions and the present study.

Studies on conditional gene expression in the brain.

This manuscript summarizes our recent attempts to regulate in vitro and in vivo the expression of genes encoding components and regulators of the postsynaptic machinery along with marker genes such as lacZ and GFP. In particular, we studied tTA-dependent regulation and utilized Cre in combination with reversible silencing by intron engineering of dominant negative alleles. We further present a "knockin" approach for on-site artificial regulation of chromosomal genes.

Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B.

We generated mouse mutants with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines had increased AMPAR calcium permeabilities in pyramidal neurons, and one showed elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induced NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants had mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlated with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons.

Uterine bacteriology, histology, resumption of ovarian activity and granulocyte function of the postpartum cow in different milking frequencies.

The postpartum uterine bacteriology, histology, resumption of ovarian activity and polymorphonuclear granulocyte (PMN) number and function in 18 Swedish dairy cows were studied. Cows were milked either 2x (n = 9) or 3x per day (n = 9). Endometrial biopsy samples for bacteriological and histological investigations were collected during 8 weeks postpartum, starting within one week after calving. Milk samples for progesterone determination were collected twice a week until the cows had shown normal reproductive cyclicity. Blood samples for granulocyte function (phagocytic capacity and total number) were collected from each animal on the same days as when the biopsies were obtained. All animals in both groups were free from bacteria at the latest after 6 weeks post-partum and there was no difference regarding bacterial elimination and bacterial species between milking groups. No difference regarding uterine histology between milking groups was seen. In both groups, 8 cows had normal to slight infiltration of leukocytes in the endometrium at the end of sample collection. No changes in granulocyte function could be seen in the 2 milking groups. Resumption of ovarian activity was detected on day 45.6 +/- 9.3 (mean +/- SD) postpartum in the 2x milking group and 36.6 +/- 9.0 (mean +/- SD) post-partum in the 3x milking group (p = 0.05). Based on our findings, an increased milking frequency from 2 to 3 times a day did not influence the uterine function postpartum.

The AMPA receptor subunit GluR-B in its Q/R site-unedited form is not essential for brain development and function.

Calcium permeability of L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in excitatory neurons of the mammalian brain is prevented by coassembly of the GluR-B subunit, which carries an arginine (R) residue at a critical site of the channel pore. The codon for this arginine is created by site-selective adenosine deamination of an exonic glutamine (Q) codon at the pre-mRNA level. Thus, central neurons can potentially control the calcium permeability of AMPARs by the level of GluR-B gene expression as well as by the extent of Q/R-site editing, which in postnatal brain, positions the R codon into >99% of GluR-B mRNA. To study whether the small amount of unedited GluR-B is of functional relevance, we have generated mice carrying GluR-B alleles with an exonic arginine codon. We report that these mutants manifest no obvious deficiencies, indicating that AMPAR-mediated calcium influx into central neurons can be solely regulated by the levels of Q/R site-edited GluR-B relative to other AMPAR subunits. Notably, a targeted GluR-B gene mutant with 30% reduced GluR-B levels had 2-fold higher AMPAR-mediated calcium permeability in hippocampal pyramidal cells with no sign of cytotoxicity. This constitutes proof in vivo that elevated calcium influx through AMPARs need not generate pathophysiological consequences.

Novel galanin receptor ligands.

Galanin is a neuroendocrine peptide which is 29/30 amino acids in length and is recognised by G-protein-coupled central nervous system receptors via its N-terminus. We synthesised several galanin receptor ligands and fragments around C-terminal extensions of galanin(1-13) to yield chimeric peptides with C-terminals corresponding to bioactive peptides like bradykinin(2-9), mastoparan, neuropeptide Y(25-36) or substance P(5-11), respectively. We also synthesised short galanin analogs in which galanin(1-13) was C-terminally elongated with Lys14; different pharmacologically active small molecules were then attached to the epsilon-amino group of Lys14. Several cysteine-substituted linear and ring closed analogs of galanin(1-9) and galanin(1-16) were also synthesised. The equilibrium binding constants for these peptides at hypothalamic galanin receptors were determined and found in the subnanomolar to micromolar range. The large number of peptides and their binding affinities presented here permit structure-activity relationship analysis of peptide-type ligands to galanin receptors.

Bacteriological and histological investigation of the postpartum bovine uterus in two Estonian dairy herds.

Postpartum uterine infections, endometrial histology and resumption of ovarian activity in cows were studied in 2 Estonian dairy herds with different herd sizes, milk yields and management systems. Ten cows at Farm A and 5 cows at Farm B were studied in the experiment. All cows in the study had normal calving performance. Endometrial biopsies for bacteriological and histological examinations were collected once a week starting on the second week postpartum and continuing for 7 weeks postpartum. Milk progesterone samples were collected twice a week during the whole study period. In both herds, the uterine flora contained mainly facultative anaerobic bacteria (Streptococcus spp., E. coli, Staphylococcus spp., Proteus vulgaris). Among obligate anaerobic bacteria only Bacteroides spp. were found. After 7 weeks of collection at farm A, a bacterial uterine flora still persisted in 2 of the cows. At farm B, on the other hand, bacterial elimination was complete after 6 weeks. Presence of inflammatory cells in uterine histology specimens remained higher at the end of collection and resumption of ovarian activity was delayed at farm A. After 7 weeks postpartum, only 6 of the 10 cows at farm A had resumed ovarian cyclicity, while at farm B the first oestrous cycle had occurred in all cows. The study showed that differences regarding uterine infections and their clearance occurred between farms and, despite these differences, cows with normal calving performance will effectively recover without any treatment.

Mutagenesis study on human galanin receptor GalR1 reveals domains involved in ligand binding.

Many receptor mutants were generated and several NH2-terminally modified galanin analogs synthesized to define the regions of hGalR1 involved in galanin binding. Ligand binding properties and functionality of mutant receptors were evaluated. The His264Ala and Phe282Ala receptor mutants, although deficient in binding in the concentration range of galanin used, remained functional albeit at least 20-fold less efficient than the wild-type receptor in the inhibition of stimulated cAMP production. Hence, His264 and Phe282 of hGalR1 are directly involved in galanin binding. NH2-terminal carboxylic acid analogs of galanin (1-16) have a very low affinity for the wild-type receptor, but substantially increased affinity for the Glu271Lys-hGalR1, suggesting that the NH2-terminus of galanin binds to the receptor near the transmembrane (TM) VI. Based on these findings and computer-aided molecular modeling, we propose a binding site model for the hGalR1 receptor (possibly also for other galanin receptor subtypes): galanin binds with its NH2-terminus to the pocket between TM III and TM VI, Trp2 of galanin interacts with His264 of the receptor, and Tyr9 is involved in an aromatic-aromatic type of interaction with Phe282 of ECIII of GalR1.

Mutagenesis and ligand modification studies on galanin binding to its GTP-binding-protein-coupled receptor GalR1.

In this study, a large number of receptor mutants were generated and several N-terminally modified galanin analogues synthesized to refine the previously proposed binding site model for galanin to its GTP-binding-protein-coupled receptor GalR1. In addition to ligand-binding studies, the functionality of mutant receptors was evaluated by assessing their ability to mediate galaninergic inhibition of isoproterenol-stimulated adenylyl cyclase activity. The His264Ala and Phe282Ala receptor mutants, although deficient in binding in the concentration range of galanin used, remain functional albeit 20-fold less efficient than the wild-type receptor in mediating inhibition of stimulated cAMP production by galanin. The His267Ala mutant is, apart from being deficient in galanin binding, also severely impaired in functional coupling. While His264 and Phe282 seem to be important in forming the binding pocket for galanin, His267 might play a role in forming or stabilizing the active conformation of the GalR1 receptor rather than directly participating in the formation of the binding pocket for galanin. N-terminal carboxylic acid analogues of galanin have low affinity to wild-type GalR1, but substantially increased affinity to the Glu271Lys receptor mutant. This, together with the finding that an alanine substitution of Phe115 in TM III results in a tenfold decrease in affinity for galanin, suggests that the N-terminus of galanin interacts with Phe115. In contrast to the Phe282Ala mutation in TM VII, a conservative mutation of Phe282 to tyrosine did not alter the affinity for galanin. Thus, the interaction between Tyr9 of galanin and Phe282 is likely to be of an aromatic-aromatic nature.

Prostaglandin F2alpha release associated with an embryo transfer procedure in the mare.

The pattern of the main metabolite of prostaglandin (PG) F2alpha was recorded following a nonsurgical embryo transfer technique in 9 mares under field conditions in Estonia. Three patterns were observed. Two of them were characterised by PG release, thereas the third was not. A tendency towards a shortened cycle was seen in 3 mares. Observations were made regarding the manipulation of the uterus as being normal or difficult to perform. In general, mares where the procedure was considered difficult were also found to have a PG release.

Galanin receptors: involvement in feeding, pain, depression and Alzheimer's disease.

Galanin, a neuroendocrine peptide with a multitude of functions, binds to and acts on specific G-protein coupled receptors. Only one galanin receptor subtype, GalRI, has been cloned so far, although pharmacological evidence suggests the presence of more than one galanin receptor subtype. These receptors mediate via different Gi/Go-proteins the inhibition of adenylyl cyclase, opening of K+-channels and closure of Ca2+-channels. Galanin inhibits secretion of insulin, acetylcholine, serotonin and noradrenaline, while it stimulates prolactin and growth hormone release. Determination of structural components of galanin receptors required for binding of the peptide ligand as carried out recently will facilitate the screening and design of molecules specifically acting on galaninergic systems with therapeutic potential in Alzheimer's disease, feeding disorders, pain and depression.

Delineation of the peptide binding site of the human galanin receptor.

Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th

Galanin--a neuropeptide with inhibitory actions.

1. Galanin is a 29 (in humans 30) amino acids long neuropeptide with mostly inhibitory, hyperpolarizing actions. 2. Differential structural requirements of truncated forms of galanin and differential agonist/antagonist behaviour of chimeric peptides, high affinity galanin receptor ligands suggest the presence of pharmacologically distinct galanin receptor subtypes. 3. The galanin receptor from human Bowes melanoma cell line--a member of G-protein coupled receptor superfamily--has been cloned. 4. Galanin acts via Gi/G(o) proteins inhibiting cAMP production, inositol phosphate turnover, opening K+ channels or closing Ca2+ channels.