David Fernández

Peptidase activities in human semen.

Enkephalins are one of the opioids present in human semen and to date their function in this tissue remains unknown. The present work studies enkephalin-degrading enzyme activities, puromycin-sensitive alanyl aminopeptidase (AAP-S), puromycin-insensitive alanyl aminopeptidase N (Ap N) and neprilysin (NEP) in human seminal fractions. AAP-S activity was not detected in any fractions, whereas Ap N appeared in soluble and particulate sperm fractions in seminal fluid and in prostasome fraction. With regard to NEP activity, this was exclusively located in prostasome membranes. The high activity values observed in the prostasome fraction suggested that these peptidases and their substrates could be involved in seminal physiology.

Pyroglutamyl peptidase I and prolyl endopeptidase in human semen: increased activity in necrozoospermia

Thyrotropin-releasing hormone (TRH) and its analogues have been reported to have important functions in human semen. In the present paper, we have characterized the activity of the TRH-degrading enzymes pyroglutamyl peptidase I and prolyl endopeptidase in the fluid and prostasomes of human semen and in subcellular fractions of the corresponding sperm. Enzymatic activities were measured fluorimetrically using beta-naphthylamine derivatives as substrate. Activity associated with both enzymes was detected in seminal fluid and in the prostasome fraction, as well as in soluble and particulate sperm subcellular fractions. Pyroglutamyl-peptidase I activity presented highest levels in the particulate sperm fraction, whereas the activity of prolyl endopeptidase was maximal in the soluble sperm fraction. In addition, we compared the activity of both enzymes in different seminal fractions in normozoospermic, fertile men and in subfertile patients with different abnormalities revealed by spermiogram analysis (astenozoospermia, necrozoospermia and teratozoospermia). The activities of pyroglutamyl peptidase I and prolyl endopeptidase in necrozoospermia were found to be higher in the corresponding soluble and particulate sperm fractions, respectively, with respect to those measured in normozoospermic semen. The results of the present study indicate that these enzymes may participate in regulating the levels of seminal TRH analogues and in mediating sperm death associated with necrozoospermia.

Developmental changes of pyroglutamate-peptidase I activity in several regions of the female and the male rat brain

In this work, we have described decreases of pGlu-peptidase I activity, detected using pGlu-beta-naphtylamide as substrate, from 9 to the 20 days after birth in five brain areas. In addition, we have found sexual differences in the enzyme activity in the hypothalamic-pituitary axis in all ages investigated. pGlu-peptidase cleaves the pGlu-aminoacid bond of several neuropeptides such as thyroliberin, luliberin and neurotensin. The decrease in the activity of pGlu-peptidase coincidents with the increases observed in brain thyroliberin concentration and the decreases in His-Pro diketopiperazine. It is suggested that this enzyme could play a part in the normal development of the rat brain.

Effect of imipramine on enkephalin-degrading peptidases

In recent years, there has been increasing evidence of the involvement of the endogenous opioid system in mental depression and its treatment. In this work, we have measured the effect of imipramine on enkephalin-degrading peptidases in several rat brain areas. Aminopeptidase activities have been assayed using Tyr-beta-naphthylamide as substrate and puromycin as selective inhibitor. Dansyl-D-Ala-Gly-Phe(pNO2)-Gly has been the substrate for neutral endopeptidase 24.11. Imipramine in vitro inhibits puromycin-sensitive activities in all brain areas studied, without affecting the rest of the enzymes assayed. However, subacute imipramine treatment increases neutral endopeptidase activity in the hypothalamus and chronic treatment increases this activity in the hypothalamus and the striatum. These results suggest to us that enkephalin-degrading peptidases are involved in the acute and chronic action mechanism of imipramine and reinforce the idea that the central enkephalinergic activity is dynamically changed during the treatment of depressive illness.

Reduced Calmodulin Expression Accelerates Transient Outward Potassium Current Inactivation in Diabetic Rat Heart

Background/Aims: In myocytes from diabetic hearts, the reduction in the amplitude of the transient outward potassium current (Ito) and the acceleration of its inactivation contribute to the action potential duration lengthening. Whereas the reduced amplitude is attributable to a reduced support of trophic factors, the mechanism underlying the acceleration of inactivation remains unknown. Ca2+/Calmodulin-dependent protein kinase II (CaMKII) modifies the inactivation kinetics of Ito. In this work we explored the role of CaMKII in the acceleration of Ito current inactivation observed in diabetic myocytes. Methods: We used patch-clamp and immunoblotting techniques in enzymatically-isolated myocytes from healthy and streptozotocin-induced diabetic rat hearts, and in blood samples from diabetic patients. Results: In control myocytes, inhibition of either calmodulin or CaMKII accelerated Ito current inactivation. However, in diabetic myocytes Ito inactivation was already accelerated, and did not respond to calmodulin or CaMKII inhibition. Calmodulin protein abundance was significantly reduced in diabetic myocytes. Incubation of diabetic myocytes with insulin recovered calmodulin expression to normal values. A similar pattern of calmodulin expression appears in the blood of diabetic patients. Insulin treatment also restored Ito current inactivation kinetics as well as the responsiveness to regulation by calmodulin. Conclusion: Diabetes-induced acceleration of Ito current inactivation is due to a reduced effect of CaMKII on Ito channels as a result of a diabetes-induced reduction in calmodulin protein expression. A correct follow up of the insulin treatment could prevent this alteration.

Ontogeny of puromycin-sensitive and insensitive aminopeptidase activities in several subcellular fractions of the rat brain

Puromycin-sensitive and insensitive aminopeptidase (aminopeptidase M) activities are measured in several subcellular fractions of the rat brain cortex and subcortex during the first postnatal month. Tyr-beta-naphthylamide has been used as substrate and 20 microM puromycin as selective inhibitor. We have found that puromycin-sensitive aminopeptidase activity increases twofold in the synaptosomal and mitochondrial fractions in the first 6-9 postnatal days, just during the period of axonal and dendritic growth. This enzyme also has significant age-related changes in the nuclear fraction. The developmental pattern is different, depending on the subcellular fraction analyzed. Significant developmental changes of puromycin-insensitive aminopeptidase (aminopeptidase M) are only found in the myelinic and microsomal fractions and they are less significant than those found in the puromycin-sensitive aminopeptidase. It has been suggested that these enzyme activities could be involved in processes of cell proliferation, differentiation, and maturation.

Membrane-bound tyrosine aminopeptidase activities in the rat brain throughout the estrous cycle

Puromycin sensitive and insensitive membrane-bound aminopeptidase activity levels during the estrous cycle in several brain areas have been described in this research. We have found the highest aminopeptidase M activity levels during the proetrous stage in the hypothalamus, the amygdala and the pituitary gland. Since this enzyme has been involved in opioid peptide metabolism, it is suggested that aminopeptidase M could play a part in the decrease in the inhibitory influence of the endogenous opioids peptides that participate in the LH surge.

A TCN Gateway Emulator

TCN standard makes use of the WTB bus and MVB vehicle buses to provide necessary services which a train may require. This architecture takes advantage of complex multiprocessor gateways to link a vehicle bus to the wire train bus. To provide a robust development and verification environment, a TCN gateway emulation application has been created. This program develops a virtual network to test the TCN protocol stack and applications built on top of it.

Effect of lidocaine administration on the enkephalin-degrading aminopeptidase activities in discrete areas of the rat brain

1. The levels of three aminopeptidase activities involved in the degradation of enkephalins (soluble, M and MII) in several rat brain areas were studied after lidocaine administration. 2. Soluble aminopeptidase activity showed decreases in the frontal cortex and in the pituitary gland after treatment. 3. No significant changes in the complete membrane-bound aminopeptidase activity were appreciated in any other of the brain areas. 4. However, decreases of the membrane-bound puromycin-insensitive aminopeptidase activity in the frontal cortex, the hippocampus and the thalamus, after lidocaine administration, were observed.