Javier Puente

Enhancement of human natural killer cell activity by opioid peptides : similar response to methionine-enkephalin and β-endorphin

We studied the effect of methionine-enkephalin (MET) and beta-endorphin upon human peripheral blood lymphocyte natural killer cell (NKC) activity in a group of healthy volunteers (n = 27; 17 male and 10 female, age +/- SD and range of 32 +/- 6, 25-43 years and 36 +/- 11, 22-65 years, respectively). Aliquots from some individual samples were preincubated separately with different concentrations of either peptide (n = 12), while others were tested with only one of these substances (MET, n = 6; beta-endorphin, n = 9). Using each individual as its own control, MET (10(-8) and 10(-6) M) and beta-endorphin (10(-10) and 10(-8) M) significantly increased NKC activity (NKCA) (at least 20% over base value, effector-to-target cell ratio, 40:1) in 7 out of 15 and 7 out of 19 subjects, respectively. Results obtained from the rest of the samples were mixed, e.g., changes observed in NKCA were not significant or showed significance with only one of the peptide concentrations studied. Cells from individuals showing a significant increase in NKC lytic function following preincubation with either MET or beta-endorphin responded similarly to the other peptide (in both cases 5 out of 6 subjects), suggesting that enhancement of NKCA by MET and beta-endorphin may work through a similar mechanism.

Studies of the in vitro human plasma degradation of methionine-enkephalin.

1. Incubation of [3H]tyrosine methionine-enkephalin (6 x 10(-9) M final concentration) with human platelet-poor plasma (1:9 ratio to Trizma Base buffer, pH 7.4) results mostly (greater than 95%) in hydrolysis of the tyrosyl-glycine peptide bond. This enzymatic reaction is essentially completed within 90 min, showing a half-life, Km and Vmax of 12.8 +/- 2.5 min, 0.70 +/- 0.01 mM and 17.90 +/- 1.05 mumol/L/min, respectively. These values are comparable to those previously reported for the human plasma degradation of leucine-enkephalin. 2. As expected hydrolysis of the methionine-enkephalin tyrosyl-glycine peptide bond was blocked by the known aminopeptidase inhibitors bestatin and puromycin (IC50 1.2 +/- 0.4 and 4.3 +/- 2.4 microM, respectively) but not by either thiorphan or captopril. 3. Neither the storing (up to 60 days) nor the freezing and thawing (up to ten times during a 60 days periods) significantly changed the above kinetic parameters, showing the stability of the plasma methionine-enkephalin degrading aminopeptidase.

γ-glutamyltranspeptidase activity and cyclic AMP levels in rat liver and mammary gland during the lactogenic cycle and in the oestradiol-progesterone pseudo-induced pregnancy

The mammary gland is a tissue subject to close hormonal control over the whole course of the lactation cycle with the most striking changes in hormonal balance occurring at parturition and the onset of lactation [ 11. Studies of enzyme changes in the tissue at this tra@tion have shown dramatic increases in the activity of those enzymes involved in the biosynthesis of the milk constituents and, in some cases, a hormonal regulation of their activity has been implied [2,3]. Although the membrane-bound enzyme 7-glutamyltranspeptidase has been found in most secretory tissues its activity has not yet been fully studied in mammary gland where, as the enzyme initiating the r-glutamyl cycle [4], it may be expected to fill an important role in regulating the entry of amino acids into the cell. The enzyme has been reported to be regulated by cyclic AMP in liver [5] while, in rat seminal vesicles, its activity appears to be testosteronedependent [6]. This investigation is a study of 7-glutamyltranspeptidase in the liver and mammary glands taken from pregnant and lactating rats and from rats in which mammary involution (caused by weaning of the pups) had been allowed to proceed for 5 days. Parallel studies were also carried out on the same tissues from hormonally induced pseudopregnant animals. The results show that the enzyme increases slowly in the pregnant mammary gland and then sharply in

Inhibition of cyclin-dependent kinase 5 but not of glycogen synthase kinase 3-β prevents neurite retraction and tau hyperphosphorylation caused by secretable products of human T-cell leukemia virus type I-infected lymphocytes

Human T‐cell leukemia virus type I (HTLV‐I)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T181 in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH‐SY5Y cells incubated with supernatant from MT‐2 cells (HTLV‐I‐infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T181 is attributable to glycogen synthase kinase 3‐β (GSK3‐β) and cyclin‐dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH‐SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3‐β and CDK5 in this process by using specific kinase inhibitors (LiCl, TDZD‐8, and roscovitine). Changes in both GSK3‐β active and inactive forms were followed by measuring the regulatory phosphorylable sites (S9 and Y216, inactivating and activating phosphorylation, respectively) together with changes in β‐catenin protein levels. Our results showed that LiCl and TDZD‐8 were unable to prevent MT‐2 supernatant‐mediated neurite retraction and also that neither Y216 nor S9 phosphorylations were changed in GSK3‐β. Thus, GSK3‐β seems not to play a role in T181 hyperphosphorylation. On the other hand, the CDK5 involvement in tau phosphorylation was confirmed by both the increase in its enzymatic activity and the absence of MT‐2 neurite retraction in the presence of roscovitine or CDK5 siRNA transfection.

In vitro Human Plasma Leucine5-Enkephalin Degradation Is Inhibited by a Select Number of Drugs with the Phenothiazine Molecule in Their Chemical Structure

A number of drugs with the phenothiazine molecule in their chemical structure inhibit in a dose-dependent manner human plasmatic aminopeptidase leucine5-enkephalin (LEU) metabolism. Half-life peptide degradation was significantly increased by thioridazine > fluphenazine > As-1397 [10-(α-diethylaminopropionyl)phenothiazine] ≧ promethazine ≧ chlorpromazine (final drug conc. 10–4M); t1/2 (± SD) 21.2 ± 1.1, 19.6 ± 1.0, 17.2 ± 0.9, 17.1 ± 1.0, and 17.1 ± 1.1 min, respectively. Control and bacitracin (known aminopeptidase inhibitor) values were 11.8 ± 1.0 and 31.3 ± 1.7 min, respectively. These drugs significantly decreased (listed in the same order) LEU degradation initial velocity; Iv (± SD) 0.77 ± 0.2, 0.82 ± 0.2, 0.92 ± 0.3, 0.93 ± 0.2, 0.94 ± 0.3 pg LEU/min, respectively. Control and bacitracin 1.10 ± 0.3 and 0.20 ± 0.1 pg LEU/min, respectively. Values represent results from 5 samples, each obtained by pooling 6 individual plasmas (4 male and 2 female; n = 30 healthy, drug-free volunteers). However, neither the phenothiazines ethopropazine, methotrimeprazine, prochlorperazine and trifluoperazine nor the various commonly used heterocyclic antipsychotics tested, e.g., molindone, loxapine, clozapine, haloperidol, sulpiride and thiothixene inhibited plasma LEU degradation kinetics. Our results failed to show correlations between chemical structure, antipsychotic properties and ability to inhibit plasmatic aminopeptidase LEU degradation. Whereas, presence of the phenothiazine molecule appears to be necessary for enzyme inhibition, only five out of nine substituted phenothiazines tested exhibited this effect. Furthermore, there was a lack of correlation between phenothiazines antipsychotic properties and their capacity to inhibit aminopeptidase activity, a property shown by promethazine (antihistaminic) and As-1397 (selective butyrylcholinesterase inhibitor) but lacking in prochlorperazine and trifluoperazine. Our results provide information which could lead to the rational design of agents capable to modulate the bioavailability of enkephalin and other endogenous aminopeptidase-degraded peptides believed to be involved in the etiology and/or pathophysiology associated with various disease conditions. Whether their development could find useful pharmacological applications remains to be explored.

Endogenous Opioid‐Like Peptides in Headache. An Overview.

La large distribution des peptides opioides et de leurs recepteurs au niveau du systeme nerveux central et peripherique suggere que ces substances ont un role biologique varie dans la modulation de la douleur

Levels of interleukin-21 in patients with untreated chronic periodontitis.

BACKGROUND A growing body of evidence suggested that interleukin (IL)-21 enhances the effector phase during T-cell responses. The aim of our study is to determine the levels of IL-21 in periodontal sites from patients with chronic periodontitis and controls. METHODS The population studied consisted of 34 patients (15 with chronic periodontitis and 19 healthy patients). Twenty samples (10 gingival crevicular fluid [GCF] and 10 gingival biopsies) were collected from each group before the patients with periodontitis received periodontal treatment. Total protein concentrations were measured in all samples; the presence of IL-21 was confirmed by immunohistochemistry and Western blot, and IL-21 levels were quantified through an enzyme-linked immunosorbent assay. Statistical analyses were performed using statistical software. Data were expressed as patient means ± SDs or medians (interquartile ranges) by using the χ(2), Student t, and Mann-Whitney U tests. RESULTS GCF IL-21 was mainly detected in patients with chronic periodontitis (P <0.05). Levels of IL-21 in gingival tissues were significantly higher in patients with chronic periodontitis compared to healthy individuals (P <0.05). The Western blot and immunohistochemical staining confirmed the presence of IL-21 in periodontal tissues and GCF. CONCLUSION IL-21 was highly expressed in patients with chronic periodontitis, especially in gingival biopsies; therefore, IL-21 might play a role in the T-cell response.

Natural Killer Cell Activity in Patients With Septic Shock

ATURAL KILLER CELLS (NKC), a small subpopulation (approximately 8%) of lymphoid cells,’ which in vitro present a spontaneous ability to lyse transformed, virally infected, and some normal cells in a nonrestricted fashion, have been shown to play an important role in the immune surveillance against primary tumors and metastases.1-4 The high incidence of malignancy and the special susceptibility to viral infections shown by individuals with a selective and marked deficit in NKC function further stresses the importance of these cells as an in vivo defense system against disease.4,5 A number of endogenous mediators have been suggested to play a role in the etiology of septic shock.‘-* This heterogeneous condition, arising as a complication of septicemia, is accompanied by numerous pathophysiologic changes.’ Reports of a bactericidal response of NKC to direct contact with certain bacteria, either through a direct action or via the Iymphokineactivation of other immune cells,‘o91’ led us to examine NKC activity (NKCA) in a population of septic shock patients in critical conditions and compare it with values obtained from a group of normal volunteers.

Flow cytometric analysis of lymphocyte subsets in migraine patients during and outside of an acute headache attack

We have conducted flow cytometric studies of two subsets of lymphocyte markers in groups of migraineurs during (n = 12; group B) and outside (n = 10; group C) of a migraine without aura attack (total n = 22; group A), including a group of patients tested in both of these phases (n = 5; group D), and compared these results with those obtained from a population of age-comparable, sex- and race-matched healthy volunteers (n = 12; group E). Comparison of the first set of lymphocytes (CD3+CD16+56+, CD3-CD16+56+, CD3-CD19+, CD3+CD19+, and CD3+HLA-DR+) between the patients in group A and the controls (group E) showed differences, reflecting greater group A percentages of CD3+CD16+CD56+ and CD3-CD19+ lymphocytes. Furthermore, these differences reached statistical significance only for the CD3+CD16+CD56+ lymphocytes, and then solely for the patients in group C (Scheffes test, p< 0.05). Paired analysis of the above lymphocyte markers for subjects in group D failed to show significant differences between patients when they were having and not having a migraine attack, raising the possibility that results from a larger study could show meaningful increases in percentages of CD3+CD16+CD56+ lymphocytes as one of the immune parameters useful for differentiating migraineurs from controls. Comparison of a second set of lymphocyte markers (CD19+CD5+, CD20+CD72-, CD20-CD72+, CD20+CD72+) among either the different groups of patients or between the patients and controls failed, however, to show statistically significant differences, emphasizing the apparent specificity of the findings described above for CD3+CD16+CD56+ lymphocytes. Our results, albeit of a preliminary nature, suggest the occurrence of significant, differential changes in lymphocyte subset immunophenotyping between groups of pain-free migraineurs and patients during an acute migraine episode or controls. Corroboration of these findings may prove useful in clinical laboratory practice to identify changes in immunological parameters specifically associated with migraineurs, and help towards a better understanding of the etiology and pathophysiology of this condition.

Calmodulin and cyclic nucleotide-phosphodiesterase activities in rat mammary gland during the lactogenic cycle

Numerous studies [l-4] have documented that cyclic AMP plays a central role in the hormonal regulation of cell growth and function and that its content in the mammary gland increases progressively during the growth stage of pregnancy and then, at parturition, decreases rapidly to the low levels seen during the lactational period. These findings suggest that cyclic AMP is a negative controlling factor for lactogenesis. It has also been demonstrated that 3’,5’nucleotide phosphodiesterase, the enzyme which hydrolyses cyclic nucleotides, is activated by calmodulin, a thermostable low M, protein already studied in several tissues, but not in mammary gland [S]. Calmodulin is an intracellular calcium receptor protein that binds to Ca2+ when their concentration increases in response to stimulus. This binding induces a distinct change in the shape of the cahnodulin molecule which in turn is capable of binding to any of several enzymes, activating them and so setting in motion the biochemical changes that produce directly the response to the stimulus. Calmodulin can also indirectly modify cell activation by affecting the concentration of calcium itself and that of other important cellular regulators, including cyclic AMP. The finding that Ca*+, in cooperation with calmodulin, can alter cyclic nucleotides concentration indicates one way by which the actions of the 2 regulatory agents may be integrated in regulating the lactogenic cycle in mammary gland. and its activating capacity for the enzyme, both from bovine brain and mammary gland, and analyse its kinetic properties. Our results support the view that calmodulin plays a regulatory role during lactogenesis.