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Biomedical subjects

M Plantavid

Publications and source records attributed to M Plantavid.

At least 55 records · Page 3Linked to original sources
PubMed1991

Luteinizing hormone pulse frequency and in vitro bioactivity in male idiopathic infertility.

We investigated 51 patients with idiopathic oligospermia and 10 control subjects. Blood samples were collected every 20 minutes from 10 P.M. to 10 A.M. and luteinizing hormone (LH) pulsatility was analyzed. A pool of all samples obtained from each subject was used to measure bioactive LH in an in vitro mouse Leydig cell bioassay and immunoactive LH in an immunoradiometric assay. Mean immunoactive LH pulse frequency was higher and mean bioactive to immunoactive LH ratio was lower in infertile men than in controls. There was a significant negative correlation between bioactive LH to immunoreactive LH ratio and LH pulse frequency. These data indicate that the defect in the gonadal axis in oligospermic men resides not in the hypothalamic-pituitary function but rather in the testis itself.

Adult↗
PubMed1991

Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor.

In this paper we demonstrate that cytoskeletons isolated from A431 cells have associated with them high activities of several kinases involved in inositol lipid metabolism, such as phosphatidylinositol kinase, phosphatidylinositol phosphate kinase, and diacylglycerol kinase. In addition also phospholipase C activity was detected on isolated cytoskeletons. Controlled extraction of the cytoskeletons followed by in vitro polymerization of actin demonstrated an association of the kinases to the actin filament system consisting of actin and a number of actin-binding proteins. The cytoskeleton-associated lipid kinase activities were significantly increased upon treatment of intact cells with EGF. These data suggest that the association of the phosphoinositide kinases, diacylglycerol kinase, phospholipase C, and also the EGF receptor to the cytoskeleton may play a role in the efficient signal transduction induced by EGF, by providing a matrix for the various components involved in signal transduction.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

LH pulsatility and in vitro bioactivity in women with anorexia nervosa-related hypothalamic amenorrhea.

LH nocturnal pulsatility and bioactivity to immunoreactivity (B/I) ratio were determined in 16 patients with anorexia nervosa-related hypothalamic amenorrhea and low sex steroid levels, and in 12 normal women in the midfollicular phase. The patients were subdivided into 2 groups: IA (N = 7) without, and IB (N = 9) with documented recent weight gain. Blood samples were taken from each subject at 10-min intervals from 00.00 to 06.00 h. Immunoreactive LH data were analysed with cluster analysis algorithm. A pool of aliquots from all the samples was used to evaluate bioactive LH, immunoreactive LH and LH B/I ratio in each subject. LH pulse frequency was lower in Group IA than in controls, whereas it did not differ significantly between Group IB and controls. LH pulse amplitude was lower in Group IA, and higher in Group IB than in controls. LH B/I ratio was below the control range in 3/16 patients. In conclusion, persistent hypothalamic amenorrhea does not require a permanent inhibition of the GnRH pulse generator; transient inhibition of pulsatility and qualitative abnormalities of gonadotropins could be involved in the mechanism, at least in some patients.

Adolescent↗
PubMed1990

Relationship between phosphoinositide kinase activities and protein tyrosine phosphorylation in plasma membranes from A431 cells.

Production of PtdIns(4)P and PtdIns(4,5)P2 by plasma-membrane preparations from A431 cells was selectively stimulated in a dose-dependent manner by epidermal growth factor (EGF) in the presence of Na3VO4. Na3VO4 itself mimicked this effect, which was overcome after treatment by a specific phosphotyrosyl phosphatase isolated from A431 cells. PtdIns and PtdIns(4)P kinase activities were present in phosphotyrosyl-proteins isolated from EGF- and/or Na3VO4-stimulated A431 cells by immunoaffinity using an anti-phosphotyrosine antibody. These data suggest for the first time an EGF-dependent regulation of PtdIns 4-kinase and PtdIns(4)P 5-kinase activities by a mechanism involving a tyrosine-phosphorylation process.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

The novel inositol lipid phosphatidylinositol 3,4-bisphosphate is produced by human blood platelets upon thrombin stimulation.

Radioactive PtdIns(3)P was detected in human platelets incubated with [32P]Pi, but remained unaffected by thrombin treatment. In contrast, [32P]PtdIns(3,4)P2 was absent from resting platelets, but was produced by thrombin-activated platelets in a dose- and time-dependent manner. [32P]PtdInsP3 was never found under these conditions. These changes are similar to those elicited in other cells by platelet-derived growth factor or the oncogene product pp60c-src.

Blood Platelets↗
PubMed1989

Association of phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities with the cytoskeleton in human platelets.

The inositol lipid kinases were investigated in the cytoskeletons of human platelets. In the absence of added lipids the kinases were only barely detectable in the Triton-soluble fractions and undetectable in cytoskeletons of resting cells. However at least 30% of the total phosphatidylinositol kinase was present in the cytoskeleton as revealed by saturation of the enzyme. Phosphatidylinositol 4-phosphate kinase was also found in significant amounts in the cytoskeletons. On the other hand, both enzymes being only recovered in the particulate fraction of the cells, we suggest that inositol lipid kinases may be present near the anchoring points of the cytoskeletons at the membranes.

1-Phosphatidylinositol 4-Kinase↗
PubMed1989

Increased levels of serum follicle-stimulating hormone and luteinizing hormone associated with intrinsic testicular hyperthermia in oligospermic infertile men.

A Negative correlation between spermatozoa output and serum gonadotropin levels, as well as between scrotal temperature and spermatozoa output, has been found in man. However, no studies have been done on the relationship between scrotal temperature and serum gonadotropin levels. This paper reports such data from 212 infertile men. The upper limit for normal scrotal temperature was defined as the 90th percentile value (35.3 C) of a control group of 64 fertile men whose mean serum FSH and LH levels were 6.0 +/- 0.8 (+/- SE) and 6.4 +/- 0.7 IU/L, respectively. This value for scrotal temperature (35.3 C) was used to classify infertile men into 3 groups: bilateral hyperthermia (n = 56), unilateral hyperthermia (n = 40), and bilateral normothermia (n = 116). In the unilateral and bilateral hyperthermic groups serum LH and FSH levels were significantly increased compared with those in the normothermic group. The mean serum testosterone values were similar in all groups. To study the relationships between serum gonadotropin levels or spermatozoa output and scrotal temperature, the infertile men also were divided into classes according to their spermatozoa output. These classes were subdivided into two groups, normothermic or hyperthermic, according to whether the left scrotal temperature was equal to or less than, or more than 35.3 C. For the infertile men whose spermatozoa output was more than 60 X 10(6) spermatozoa/ejaculate (normospermia), there was no significant difference between the serum gonadotropin levels of the normothermic (n = 42) and the hyperthermic (n = 20) groups. Among the oligospermic men (spermatozoa output, 0.1-60 X 10(6) spermatozoa/ejaculate), the hyperthermic group (n = 65) had significantly higher serum gonadotropin levels and significantly smaller testicular volumes than the normothermic group (n = 71). The two oligospermic groups also had significantly higher serum FSH values than the infertile normospermic groups. These results were not linked to the presence of a varicocele or a history of cryptorchidism, as the prevalence of varicocele and cryptorchidism was equally distributed within the groups studied. We conclude that the increase in serum gonadotropin levels in the case of a decrease in spermatozoa output is significantly greater in the presence of associated scrotal hyperthermia.

Adult↗
PubMed1988

Effects of two methylxanthines, pentoxifylline and propentofylline, on arachidonic acid metabolism in platelets stimulated by thrombin.

[3H]Pentoxifylline and [3H]propentofylline were taken up by human platelets in a dose-dependent manner probably involving a passive diffusion through the plasma membrane. In vitro, the two drugs were able to inhibit platelet activation induced by thrombin. serotonin secretion was reduced from 57% to 38% and 28% in the presence of 1 mM pentoxifylline and 1 mM propentofylline, respectively. Platelet aggregation was inhibited in the same way. Modifications of [14C]arachidonic acid metabolism in human platelets stimulated by thrombin were then measured in the presence of drugs. Preincubation of platelets with 1 mM pentoxifylline or propentofylline inhibited the production of [14C]arachidonic acid metabolites, without any accumulation of free arachidonic acid, suggesting an action at a step preceding its conversion. Phosphatidylinositol and phosphatidylcholine hydrolysis measured upon thrombin treatment as well as phosphatidic acid production were reduced or suppressed in the presence of the drugs. A dose-dependence study showed that phosphatidylcholine hydrolysis was totally inhibited at 5.10(-4) M propentofylline, while phosphatidic acid formation was reduced by only 40%. Propentofylline was in general more efficient than pentoxifylline in inhibiting events occurring upon thrombin stimulation. Our results suggest that the two methylxanthines inhibit both phospholipase A2 and phospholipase C, the former displaying a greater sensitivity to the two drugs.

Arachidonic Acid↗
PubMed1988

Characterization of plasma membranes from A431 cells, isolated by self-generating Percoll gradient: a rapid isolation procedure to obtain plasma membranes with functional epidermal growth factor receptors.

Plasma membranes have been isolated from the human epidermoid carcinoma cell line A431 by a rapid fractionation of lysate on Percoll density gradient at pH 9.6. Endoplasmic reticulum, lysosomes and mitochondria sedimented at the bottom of gradient whereas plasma membranes focused at low density, as shown with specific markers. Plasma membranes displayed a 4.5- and 4.4-fold enrichment in [3H]concanavalin A and 5'-nucleotidase, respectively. This proteic fraction was further characterized by its lipid composition and phospholipid analysis. The cholesterol/phospholipid molar ratio was 0.45 in plasma membranes against 0.19 in lysate. Sphingomyelin increased from 7.5% of total phospholipids in lysate to 16.2% in plasma membranes, as well as phosphatidylserine which displayed a 1.5-fold enrichment in the plasma membrane fraction. This was at the expense of phosphatidylcholine (45.2% in lysate, against 35% in plasma membranes). Electron microscopy of the isolated material showed vesicles essentially free from endoplasmic reticulum and organelles. These plasma membranes retained the ability to bind 125I-labelled epidermal growth factor (125I-EGF) with a Kd = 4.7 nM and Bmax = 63 pmol/mg protein. EGF binding resulted in a stimulation of the phosphorylation protein reaction in the presence of [gamma-32P]ATP and sodium dodecyl sulfate polyacrylamide gels of phosphorylated proteins indicated that the radioactivity of the major band of molecular weight 170,000 was clearly enhanced by EGF binding. These results indicate that the EGF receptor and its intrinsic protein kinase activity were preserved during our plasma membrane isolation procedure.

Carcinoma, Squamous Cell↗
PubMed1988

[Effects of pentoxifylline and propentofylline on the turnover of polyphosphoinositides of the erythrocyte membrane and on the metabolism of arachidonic acid in platelets].

[gamma-32p] ATP incorporation into polyphosphoinositides of the erythrocyte membrane has been studied in the presence of increasing concentrations of pentoxifylline and propentofylline. Our results show an inhibitory effect on the labelling of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, higher with propentofylline. We have then demonstrated that these two drugs block the metabolism of arachidonic acid in platelets stimulated by thrombin. The inhibition is located at the first biochemical steps of platelet activation, probably on phospholipase C. These results are discussed in relation with the modifications of platelet cyclic AMP content.

Blood Platelets↗
PubMed1987

Phospholipase C from human sperm specific for phosphoinositides.

Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization.

Humans↗
PubMed1986

Studies of endogenous polyphosphoinositide hydrolysis in human platelet membranes. Evidence that polyphosphoinositides remain inaccessible to phosphodiesterase in the native membrane.

Human platelet plasma membranes incubated in the presence of [gamma-32P]ATP and 15 mM MgCl2 incorporated radioactivity mostly into phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), which represented together over 90% of the total lipid radioactivity. After washing, reincubation of prelabelled membranes revealed some hydrolysis of the two compounds by phosphomonoesterase(s), as detected by the release of radioactive inorganic phosphate (Pi) from the two phospholipids. This degradation attained 40%/30 min for PIP in the presence of 2 mM calcium and cytosol. The effect of calcium was observed at concentrations equal to or greater than 10(-4) M. In no case did calcium alone facilitate the formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). In contrast, simultaneous addition of 2 mM calcium and 2 mg/ml sodium deoxycholate promoted the formation of IP3 and IP2, indicating phosphodiesteratic cleavage of PIP2 and PIP. Phospholipase C activity was detected at calcium concentrations as low as 10(-7) M, in which case PIP2 hydrolysis was slightly more pronounced compared to PIP. Addition of cytosol increased to some extent the phospholipase C activity, suggesting that the low amount of enzyme remaining in the membrane is sufficient to promote submaximal degradation of PIP2 and PIP. We conclude that platelet polyphosphoinositides are present in the plasma membrane in a state where they remain inaccessible to phospholipase C, which is still fully active even at basal calcium concentrations, i.e., 10(-7) M. These results support the view that phosphodiesteratic cleavage of PIP2 promotes and thus precedes calcium mobilization brought about by IP3. The in vitro model presented here may prove very useful in future studies dealing with the mechanism rendering polyphosphoinositides accessible to phospholipase C attack upon agonist-receptor binding.

Adenosine Triphosphate↗
PubMed1985

Inhibition of transmembrane movement and metabolism of platelet activating factor (PAF-acether) by a specific antagonist, BN 52021.

Incorporation of 1-[3H]-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H] PAF-acether) into rabbit platelet phosphatidylcholine (PC) was inhibited by a specific antagonist, BN 52021 (IC50 5.6 X 10(-6) M, maximal effect, i.e 70% inhibition, at 10(-4) M). Under the same conditions, [3H] lyso-PAF-acether incorporation remained 9 fold lower, compared to PAF-acether, without any effect of BN 52021. Upon cell lysis, both phospholipids attained the same rate of metabolic conversion, corresponding to a 1.15-fold and a 12-fold increase for PAF-acether and lyso-PAF-acether, respectively. In none of these cases was BN 52021 effective. It is concluded that transmembrane movement of the two phospholipids represents the limiting step of their metabolism. The higher rate of PAF-acether conversion by intact platelets could involve its binding to a membrane receptor, as suggested by the inhibitory effect of BN 52021, the significance of which is discussed.

Animals↗
PubMed1985

Relationship between phospholipid metabolism and intracellular calcium mobilization during platelet activation.

This paper reviews some of our most recent studies concerning the relationship between phospholipid metabolism and calcium mobilization in thrombin-stimulated platelets. Evidence is provided that phospholipase C activation does not require any increase in cytoplasmic calcium but is subsequent to a yet unknown membrane modification rendering the substrate accessible to the enzyme upon thrombin-receptor binding. In contrast, the much higher calcium dependence of phospholipase A2 led us to conclude that cytoplasmic free calcium might increase to in excess of 1.6 microM even in the absence of external calcium. This is supported by our estimation that calcium is present in dense tubular system in rather large excess, as measured on purified vesicles derived from this organelle. We thus suggest that the level of inositol-1,4,5-tris-phosphate is rate limiting in the process of calcium mobilization which implies that calcium influx through the plasma membrane might become a critical event with weak agonist poorly activating phospholipase C.

Aminoquinolines↗
PubMed1983

Are polyphosphoinositides involved in platelet activation?

In human platelets, the amounts of triphosphoinositides (TPI) and diphosphoinositides (DPI) increase after 30 sec and level off after 120 sec of thrombin stimulation. After 180 sec of thrombin challenge, TPI and DPI increase accounts for 66 and 80%, respectively. Polyphosphoinositide changes roughly parallel the release of N-acetyl-beta-D-glucosaminidase and appear as a later event compared to aggregation and serotonin secretion. It is concluded that an increased phosphorylation of polyphosphoinositides might participate in platelets to the process of stimulus-activation coupling and might be linked to thrombin receptor occupancy. A role of DPI in platelet activation is suggested by the observation that DPI promote platelet aggregation, the mechanism of which is discussed.

Humans↗