Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients.

Two kinds of membranes (plasma membranes and intracellular membranes) have been separated from human platelets by fractionation on Percoll gradients (successively at pH 7.4 and pH 9.6). On alkaline Percoll gradient, plasma membranes floated at low density, as shown with specific markers such as [3H]concanavalin A and monoacylglycerol lipase, whereas intracellular membranes sedimented in the higher densities and displayed a 5.6-12.4-fold enrichment in NADH diaphorase, antimycin insensitive NADH-cytochrome-c oxidoreductase and Ca2+-ATPase. Another criterion allowing differentiation of two membrane populations of human platelets was their lipid composition, which showed a cholesterol/phospholipid molar ratio of 0.5 in plasma membranes against 0.2 in intracellular membranes. Phospholipid analysis of the two kinds of membranes displayed also quite different profiles, since phosphatidylcholine increased from 30-32% in the plasma membrane to 52-66% in the intracellular membranes. This was at the expense of sphingomyelin (20-23% in plasma membrane, against 6.8-7.7% in intracellular membranes) and of phosphatidylserine (12-13% in plasma membrane, against 2-6% in intracellular membranes). Other striking differences between plasma membranes and intracellular membranes were obtained by SDS-polyacrylamide gel electrophoresis, which revealed the absence of actin and myosin in the intracellular membrane, whereas both proteins were present in significant amounts in plasma membranes. Finally, intracellular membranes but not plasma membranes were able to incorporate calcium. These results suggest that intracellular membrane fractions are derived from the dense tubular system and plasma membranes should correspond to the whole surface membrane of human platelets.

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. I. Studies on neutral glycerides.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various neutral glycerides. Triolein hydrolysis proceeded in the absence of di- and monoolein accumulation. Optimal conditions for di- and monoolein hydrolysis included an alkaline pH (9-10), a substrate concentration of 10 mM, and the presence of sodium deoxycholate (12 and 24 mM, respectively). Pancreatic colipase (bovine) had no effect on the activity of the two lipases. The comparison between the rates of hydrolysis of various substrates revealed the following order of decreasing enzyme activity: diolein greater than 1(3)-monoolein greater than tributyrin = triacetin greater than or equal to triolein = 2-monoolein. No hydrolysis of p-nitrophenylacetate and cholesteryloleate could be detected. Using 1-[3H]palmitoyl-2-[14C]linoleoyl-sn-glycerol, both enzymes displayed a strong preference for the 1-position, leading to the accumulation of 2-[14C]linoleoyl-sn-glycerol. Identical activities were found for the two lipases. It is concluded that the two cationic lipases from guinea pig pancreas represent a unique group of lipolytic enzymes different from other previously described enzymes, including classical pancreatic lipase, gastric and lingual enzymes, mold lipases and carboxylesterhydrolase.

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. II. Studies on glycerophospholipids.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various natural and synthetic phospholipids. Natural glycerophospholipids carrying a 1-acyl-bond were degraded in the following order of decreasing activity: phosphatidylcholine = phosphatidylinositol greater than 1-acyl-sn-glycero-3-phosphocholine greater than phosphatidylethanolamine greater than phosphatidylglycerol. Sodium deoxycholate was an activator with all the phospholipids tested, each one requiring its own optimal concentration of detergent. Whereas 1-alkyl-2-acyl-sn-glycero-3-phosphocholine remained fully insensitive to enzyme degradation, 2-acyl-sn-glycero-3-phosphocholine was hydrolysed to some extent. However, additional experiments involving time-course hydrolysis revealed that this was entirely due to the migration of the 2-acyl-chain to the sn-1 position. From studies using racemic or enantiomeric phosphatidylcholines, it was concluded that the enzymes are not stereospecific. Activity against 1-acylpropanediolphosphocholine was much lower than with 1-acyl-sn-glycero-3-phosphocholine, indicating that the 2-hydroxyl group (or the 2-acyl-ester group) participates in the substrate reactivity through a strong inductive effect. Some activity could be detected against 1,3-diacylglycero-2-phosphocholine (beta-phosphatidylcholine) and 1-acylglycol-2-phosphocholine. It is thus concluded that the failure of the lipases to hydrolyse the 2-acyl-bond in a natural phospholipid is due to the steric hindrance brought about by the acyl, alkyl or hydroxyl group present in the sn-1 position. The lipases might also be unable to hydrolyse acyl-ester bonds involving a secondary alcohol.

Lipocortin-like anti-phospholipase A2 activity of endonexin.

Endonexin (protein II, 32.5 kDa) has been purified to homogeneity from bovine liver in the following steps: selective extraction by EGTA from membranes precipitated with Triton X-100/calcium; chromatography on DEAF-TSK 545 at pH 7.0, endonexin being eluted at 0.1 M NaCl; affinity chromatography on polyacrylamide-immobilized phosphatidylserine; gel filtration on TSK 3000. The amino acid composition was essentially similar to that previously reported. Using [3H]oleic acid-labelled Escherichia coli membranes as substrate, endonexin inhibited phospholipase A2 from pig pancreas. Maximal inhibition was 55 and 70%, whereas 50% inhibition occurred at 480 and 120 nM endonexin and lipocortin II, respectively. These data could be related to common features shared by both lipocortins/calpactins and endonexin, i.e. the presence of a consensus sequence and the ability to bind to anionic phospholipids in a calcium-dependent manner.

Isolation of two 67 kDa calcium-binding proteins from pig lung differing in affinity for phospholipids and in anti-phospholipase A2 activity.

Two 67 kDa proteins adsorbed to membranes in the presence of Ca2+ have been purified to homogeneity from pig lung using conventional procedures, followed by calcium-dependent affinity chromatography on polyacrylamide-immobilized phosphatidylserine. The two proteins were, respectively, excluded (67E) and retained (67R) on the column in the presence of Ca2+. On the basis of amino acid composition and isoelectric point, 67R was identified as 67 kDa calelectrin/calcimedin, whereas 67E could be differentiated from albumin, calregulin, 67 kDa fragment of protein kinase C and surfactant-associated proteins. Only 67R was slightly phosphorylated by protein kinase C, reacted with an antibody raised against 32.5 kDa endonexin and inhibited pig pancreas phospholipase A2 in a way similar to that of lipocortin or endonexin. These data bring further support to the view that inhibition of phospholipase A2 by lipocortin or other related proteins involves interaction with the lipid/water interface. They also provide evidence for a new kind of Ca2+-binding protein (67E), whose role still remains to be determined.

Rehospitalization because of respiratory syncytial virus infection in premature infants younger than 33 weeks of gestation: a prospective study. IRIS Study Group.

OBJECTIVE: To collect data on hospitalization for respiratory syncytial virus (RSV) infections and presumptive risk factors for rehospitalization among premature infants in Spain. DESIGN: Observational, prospective, longitudinal, multicenter study. SETTING: Fourteen Spanish neonatal units with an annual birth cohort of 57,000 infants. PATIENTS: All children (n = 680) born < or =32 weeks of gestational age between April 1, 1998, and March 31, 1999, and discharged from the hospital before March 31, 1999, were included in the study. A total of 96 were excluded because of administration of prophylactic treatment (n = 55) or were lost to follow-up (n = 41). Five children died during the study period, but death was related to RSV in only 1 case. METHODS AND MAIN OUTCOME MEASURES: Neonatal and demographic data were recorded at the initial visit. Infants were prospectively followed at monthly intervals up to March 31, 1999. In patients rehospitalized for respiratory disorders, further data about RSV status and morbidity were collected. A comparison was made between children rehospitalized for RSV infection and those who were not. The influence of factors on the probability of rehospitalization for RSV infection was assessed by logistic regression analysis. RESULTS: Of the 584 evaluable patients 118 (20.2%) were rehospitalized for respiratory disease during the study period. The causative pathogen was identified in 89 (75.4%) hospital admissions. Of these 59 (66.3%) were a result of RSV infection in 53 children; 6 were reinfections. In a logistic regression model significant independent prognostic variables included: lower risk of RSV hospitalization with increase gestational age [odds ratio (OR), 0.85; 95% confidence interval (CI), 0.72 to 0.99; P < 0.047]; higher risk with chronic lung disease (OR = 3.1; 95% CI 1.22 to 7.91; P < 0.016); and living with school age siblings (OR = 1.86; 95% CI 1.01 to 3.4; P < 0.048). CONCLUSION: This large descriptive study has enabled us to define the influence of specific risk factors that increase the risk of rehospitalization for RSV infection in preterm infants. Such studies help to define the appropriate role of available prophylactic interventions and establish treatment guidelines.

[Secondary recurrent hepatitis in ventricular arrhythmia]. / Hépatite récurrente secondaire à une arythmie ventriculaire.

INTRODUCTION: A major episode of hepatic cytolysis, rapidly regressive and occasionally recurrent, evokes a bilary or toxic pathology. We report an exceptional case in which several episodes of cytolysis were secondary to a paroxystic cardiac rhythm disorder. OBSERVATION: A 44 year-old woman was hospitalized 3 times during 4 months for episodes of asthenia and rapidly regressive cytolysis. Lithiasic, viral, metabolic, autoimmune and toxic causes were eliminated. A cardiac rhythm disorder was revealed during the last two episodes and diagnosis was made of intermittent and asymptomatic ventricular tachycardia, secondary to arryhthmogenic right ventricular dysplasia, and was confirmed on the electrocardiogram, cardiac scan and magnetic resonance imaging. Treatment of the cardiac rhythm led to the absence of further relapse. COMMENTS: In our patient, the clinical and chronological imputability appeared highly probable. The responsibility of perturbed cardiac rhythm in the genesis of an ischemic hepatopathy is classical, but little documented. Our observation confirms that severe unexplained cytolysis requires systematic search for a cardiac rhythm disorder, even in the absence of a known or symptomatic cardiopathy.

[Collagen gastroenterocolitis]. / Une gastroentérocolite collagène.

INTRODUCTION: Collagenous gastroenterocolitis is a recently known rare cause of chronic diarrhoea, that raises numerous nosological and diagnostic problems. OBSERVATION: A 41 year-old woman was hospitalised for severe diarrhoea, diagnosed as collagenous gastroenterocolitis. Gastroscopy and ileocolonoscopy were macroscopically normal, but a 20 to 40 microns thick sub-epithelial collagenous band was revealed in the gastric, duodenal and colic biopsies. Parenteral nutrition and treatment with salazopyrine and prednisolone progressively normalised the transit. Three months later, only a 30 microns colic mucosa collagenous band persisted. All the biopsies taken during control gastro-colonoscopy 2 years later were histologically normal. After 5 years follow-up and absence of treatment, the patient no longer presented diarrhoea or biological abnormality. COMMENTS: This exceptional observation is a reminder that sub-epithelial collagen deposits are not always limited to the colon and therefore justify, in patients with collagenous colitis, systematic gastro-duodenal and ileum biopsies.

[Acute hepatitis due to ecstasy]. / Hépatite aiguë à l'ecstasy.

BACKGROUND: Ecstasy is a synthetic amphetamine which causes a wide variety of adverse effects. Hepatic toxicity was only recently demonstrated but can be quite severe. CASE REPORT: A 27-year-old male with no past medical or surgical history developed jaundice without fever. He was a regular user of ecstasy and had recently increased the number of doses consumed. No evidence of a viral, alcoholic, metabolic or autoimmune mechanism was found which could explain the hepatitis. Complete cure was obtained by discontinuing ecstasy. DISCUSSION: Few cases of ecstasy hepatic toxicity have been reported. Ecstasy was undoubtedly the causal agent in this case since other known causes of acute hepatitis were excluded, confirming the hepatotoxicity of ecstasy reported in the literature. The liver disease has been reported to range form acute regressive hepatitis to fatal liver failure. Iterative exposure can lead to fibrosis. The pathophysiological mechanism of this toxic effect is not well elucidated. Ischemia alone cannot explain all the clinical forms described, particularly cases without hyperpyrexia. Ecstasy must be added to the list of potential causes of acute hepatitis. Exposure must always be searched for in cases of acute hepatitis in young subjects.