Postpartum curettage in patients with HELLP-syndrome does not result in accelerated recovery.

OBJECTIVE: Our purpose was to examine the impact of immediate postpartum curettage on the recovery of patients with HELLP-syndrome (hemolysis, elevated liver enzymes, and low platelets). SUBJECTS: Between January 1994 and July 1997 all patients who presented with HELLP-syndrome in our institution underwent immediate postpartum curettage (n=24). Their outcome was compared with the recovery of women with HELLP-syndrome who were delivered without postpartum curettage between 1987 and 1993 (n=20). Clinical and laboratory data were analyzed. RESULTS: No significant difference could be found between both groups in terms of normalization of serum glutamic-oxaloacetic transaminase (GOT), serum glutamic-pyruvic transaminase (GPT), lactic dehydrogenase (LDH), and quantitative platelet count. Postpartum hospitalization time was identical in both groups. CONCLUSION: In our retrospective study no benefit is achieved by immediate postpartum curettage in patients with HELLP-syndrome. In order to eliminate the possible bias of retrospective analysis, we now plan a randomized study to further investigate the impact of immediate postpartum curettage.

Activation of glomerular mesangial cells by the terminal membrane attack complex of complement.

Treatment of cultured renal glomerular mesangial cells (MC) with nonlytic concentrations of the purified components (C5b-9) of the terminal membrane attack complex (MAC) of complement induced significant functional alterations characteristic of cellular activation. C5b-9-treated MC released large quantities of primarily vasodilatory prostaglandins. In addition, the secretion of an MC-derived auto-growth factor (MC interleukin 1) was greatly enhanced. Examination of the action of C5b-9 on MC phospholipid metabolism indicated that complement induced the activation of phospholipases, leading to quantitative changes in the fatty acid profile of MC membrane phospholipids. These findings demonstrate that cultured MC are highly responsive to nonlytic concentrations of the C5b-9 complex, and suggest that the mesangial deposition of the MAC in many forms of glomerular disease, with resultant cellular activation, may play a major role in the hemodynamic and cellular proliferative events characteristic of these disorders.

Lipid composition of functional domains of the lymphocyte plasma membrane.

Plasma membrane vesicles from calf T-lymphocytes were fractionated by affinity chromatography on Con A-Sepharose. One subfraction eluted freely from the affinity column (fraction 1), while a second one adhered specifically to the column (fraction 2). While both fractions were derived exclusively from the plasma membrane, fraction 2 carried the high-affinity receptor for the mitogen concanavalin A and was distinct from fraction 1 with respect to its polypeptide pattern and the content of some plasma membrane-associated enzymes, suggesting the existence of functional plasma membrane domains. These functionally distinct fractions showed different lipid composition. The adherent fraction was enriched in phosphatidylcholine, while the relative amount of phosphatidylethanolamine and phosphatidylserine was reduced. Furthermore, the relative amount of saturated fatty acids was enhanced in the phospholipids of the adherent plasma membrane fraction. This could be shown in total phospholipids, as well as in separated individual phospholipids. We could therefore demonstrate that lipid heterogeneity may exist in plasma membranes of cells without structural polarity. Similar results were obtained when T-lymphocytes were stimulated with the mitogen concanavalin A. The functional domain, consisting of the high-affinity concanavalin A receptor, several enzymes and distinct lipid compositional pattern, thus seems to constitute a relatively stable structural entity of the lymphocyte plasma membrane.

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose.

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.

Characterization of cellular and extracellular plasma membrane vesicles from a non-metastasizing lymphoma (Eb) and its metastasizing variant (ESb).

Plasma membrane fragments from two variants of a murine lymphoma, Eb and ESb, with different metastatic capacity were investigated. Plasma membranes were isolated from tumor cells recovered from the peritoneal cavity. They differed in their lipid composition, indicating a more fluid state of the plasma membranes derived from the highly metastatic tumor line ESb. Extracellular membrane vesicles could be isolated from the ascites of the tumor-bearing mice. The shedding capacity of ESb cells was much higher than that of Eb cells. The extracellular membranes by chemical analysis and the measurement of marker enzymes proved to be derived from the plasma membranes. However, they differed from the plasma membranes from which they were derived in several aspects: the lipid to protein ratio was diminished; the activities of some plasma membrane-associated enzymes were lower while others were identical in plasma membranes and extracellular membranes; the content of saturated fatty acids in phospholipids was enhanced in extracellular membranes. These effects were more pronounced in the highly metastasizing tumor line ESb. It is thus concluded that shedding of extracellular membranes is not a random process. The biochemical differences found in the plasma membranes and the extracellular membranes of the two tumor lines are discussed with respect to the different metastatic capacity of the tumors.

Functional role of lipid metabolism in activated T-lymphocytes.

Exogenous long-chain fatty acids are readily taken up by unstimulated lymphocytes derived from the thymus of calves or rabbits and esterified to complex lipids, primarily phospholipids and triacylglycerols. Compared to saturated fatty acids, unsaturated fatty acids are incorporated preferentially. Furthermore, unsaturated fatty acids are transferred from triacylglycerols to phospholipids. The transfer cannot be observed with palmitic acid. With regard to individual phospholipid species, oleic acid and linoleic acid are found primarily in phosphatidylcholine. Arachidonic acid, however, is transferred to phosphatidylethanolamine and phosphatidylinositol as well. This suggests an arachidonic-specific transfer between individual phospholipids. Stimulation of the cells with the mitogen concanavalin A results in an enhanced incorporation of the fatty acids and an enhanced transfer from triacylglycerols to phospholipids. Triacylglycerols may thus be regarded as a labile intracellular storage pool that may be activated upon mitogenic stimulation.

Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes.

The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.

Densitometric quantitation of individual phospholipids from natural sources separated by one-dimensional thin-layer chromatography.

A method for the quantitation of small amounts of phospholipids derived from biological sources is described. Total phospholipid is determined by mineralization followed by the estimation of liberated phosphate by means of malachite green. The main phospholipid species are separated by one-dimensional thin-layer chromatography. The individual phospholipids are detected by charring with CuSO4/H3PO4. They may be directly quantitated by scanning the thin-layer chromatography plates with a laser densitometer.

Interaction of myelin basic protein with mononuclear cells: the primary reaction for the MEM and EMT tests.

The primary reaction of the macrophage electrophoretic mobility test (MEM) and its modifications (viz. the interaction of myelin basic protein (MBP) and mononuclear cells) has been investigated. The binding of MBP to mononuclear cells is rather weak, and on incubation with mononuclear cells the MBP is proteolytically degraded. A fast process leads to fragments with mol. wts in the range 9000-14,000, followed by a slower process leading to peptides smaller than 5000. Both binding and proteolytic degradation are the same for mononuclear cells from cancer patients and from control individuals.

The effect of several nucleic acid binding drugs on the cleavage of d(GGAATTCC) and pBR 322 by the Eco RI restriction endonuclease.

The endonucleolytic action of the EcoRI restriction enzyme on the double-stranded oligonucleotide d(GGAATTCC) and the supercoiled plasmid DNA pBR 233 is inhibited by actinomycin D, ethidium bromide, proflavin, distamycin A and netropsin. Half-maximal inhibition is observed at around 100 microM concentrations for the intercalating drugs, and around 0.1 to 1 microM concentrations for netropsin and distamycin A. The inhibitory activity of these drugs can be correlated with their affinity to the oligonucleotide and the plasmid DNA. Since at high concentrations of the drugs a complete inhibition is observed, it is concluded that the effect of the drugs on the stereochemistry of the EcoRI site is such that recognition is excluded.

Inhibition of Eco RI action by polynucleotides. A characterization of the non-specific binding of the enzyme to DNA.

The cleavage of the plasmid pBR322 by the restriction endonuclease Eco RI has been studied in the presence of various polynucleotides and the double stranded octanucleotide d-(GGAATTCC) in order to clarify whether there is a preferential interaction of Eco RI with DNA sequences other than -GAATTC-. The steady state kinetic analysis shows that all polynucleotides investigated with the possible exception of poly-dG.poly-dC inhibit the cleavage competitively with Ki values in the range of 10(-4) to 10(-5) [M nucleotides]. The Ki of d-(GGAATTCC) is 1.5.10(-6) [M nucleotides], indicating that the specific binding is approx. 2 orders of magnitude stronger than non-specific binding.