Evidence for a relationship between bovine erythrocyte lipid membrane peculiarities and immune pressure from ruminal ciliates.

Erythrocytes of bovines and other ruminants have a strikingly anomalous phospholipid composition, with low or absent phosphatidylcholine (PC) together with high sphingomyelin (SM) content. Here, we report the presence in normal bovine serum of high levels of anti-phospholipid antibodies of IgM isotype against, PC and the phosphono analogue of phosphatidylethanolamine, aminoethylphosphonolipid (AEPL), normally produced by rumen ciliates. In contrast, no antibodies were detected against SM or N-acyl-phosphatidylethanolamine (NAPE), the major components of bovine erythrocytes.

A bioreactor model system specifically designed for Tetrahymena growth and cholesterol removal from milk.

This work describes the configuration and operation of a bioreactor system especially designed for Tetrahymena cultivation and its use for milk improvement, particularly cholesterol elimination by the action of this cell. An advantage of the proposed method is the re-use of the growth medium; thus, the medium is used twice to provide two batches of Tetrahymena biomass without the need of further inoculation. This makes the procedure of producing the cell biomass faster and more economical.

The use of Tetrahymena thermophila mutant cell line for removal of cholesterol from milk.

The nonpathogenic ciliate Tetrahymena thermophila converts cholesterol from foodstuffs into provitamin D compounds in high yields. However, prolonged incubation with wild-type strain CU-399 at high densities results in a final deterioration of milk properties, possibly as a result of secreted hydrolases. Here we attempted to solve this problem using MS-1 Tetrahymena strain, a stable mutant with a low rate of hydrolase secretion.

Phospholipase A1 from Trypanosoma cruzi infective stages generates lipid messengers that activate host cell protein kinase c.

Here we have studied phospholipase A1 (Plase A1) from Trypanosoma cruzi infective stages and it's possible role regarding the interaction with mammalian host cells. Plase A1 was mainly detected as a membrane-bound activity in the infective amastigote and trypomastigote stages, being remarkably higher with respect to the non-infective epimastigotes. It is noteworthy that only the infective stages secreted Plase A1.

Free fatty acids induce cell differentiation to infective forms in Trypanosoma cruzi.

Intestinal extracts of Triatoma infestans induce cell differentiation of Trypanosoma cruzi epimastigotes into the infective metacyclic form. Part of this effect can be explained by the presence of haemoglobin fragments, which stimulate trypanosomal adenylate cyclase. In this work we examined the metacyclogenic activity of lipids present in this intestinal extract.

Phospholipase activity on N-acyl phosphatidylethanolamines is critically dependent on the N-acyl chain length.

We have recently shown that an endogenous phospholipase A2 from bovine erythrocytes does not hydrolyse NAPEs (N-acyl L-alpha-phosphatidylethanolamines), which accumulate remarkably in this system [Florin-Christensen, Suarez, Florin-Christensen, Wainszelbaum, Brown, McElwain and Palmer (2001) Proc. Natl. Acad.

The improvement of milk by cultivation with ciliates.

The exceptional nutritional values of milk have long been recognized. However, important drawbacks of milk and dairy products are their relatively high cholesterol contents and their low proportion of polyunsaturated fatty acids. Hypersensitivity to milk proteins can also draw the public away from fluid milk consumption.

A unique phospholipid organization in bovine erythrocyte membranes.

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A(2), an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE.

Lysosomal phospholipase A1 in Trypanosoma cruzi: an enzyme with a possible role in the pathogenesis of Chagas' disease.

We found that, as in African trypanosomes, endogenous phospholipase A(1) (Plase A(1)) activity can catalyse extensive deacylation of phospholipids upon cell death in all life stages of Trypanosoma cruzi. A major lysosomal Plase A(1) was purified and characterized. The enzyme products can explain the lesions surrounding degenerating T.

Babesia bovis-stimulated macrophages express interleukin-1beta, interleukin-12, tumor necrosis factor alpha, and nitric oxide and inhibit parasite replication in vitro.

The tick-transmitted hemoparasite Babesia bovis causes an acute infection that results in persistence and immunity against challenge infection in cattle that control the initial parasitemia. Resolution of acute infection with this protozoal pathogen is believed to be dependent on products of activated macrophages (Mphi), including inflammatory cytokines and nitric oxide (NO) and its derivatives. B.

Isolation of a delta7-cholesterol desaturase from Tetrahymena thermophila.

Cell-free preparations of Tetrahymena thermophila catalyze the direct desaturation of cholesterol to delta7-dehydrocholesterol (provitamin D3). The activity was isolated in the microsomal fraction from Tetrahymena homogenates. Delta7-desaturase activity was stimulated fivefold by the addition of 6 mM ATP.

Phosphatidylcholine formation is the predominant lipid biosynthetic event in the hemoparasite Babesia bovis.

This work examines the lipid composition and metabolism of bovine red blood cells infected by apicomplexan Babesia parasites, organisms closely related to Plasmodium sp. We found that erythrocytes infected with Babesia bovis (i-RBC) accumulate lipids and show striking increases in phosphatidylcholine, phosphatidic acid, diacylglycerol and cholesteryl esters as compared to uninfected erythrocytes cultured under the same conditions (n-RBC). A similar pattern was observed in cultures of erythrocytes infected with Babesia bigemina.

Mapping the catalytic pocket of phospholipases A2 and C using a novel set of phosphatidylcholines.

A set of radioiodinatable phosphatidylcholines (PCs) derivatized with the Bolton-Hunter reagent (BHPCs) was synthesized to probe the substrate recognition and activity of phospholipases. A common feature of this series is the presence of a bulky 4-hydroxyphenyl group at the end of the fatty acyl chain attached to position sn-2. The distance between the end group and the glycerol backbone was varied by changing the length of the intervening fatty acyl chain (3-25 atoms).

A method for distinguishing 1-acyl from 2-acyl lysophosphatidylcholines generated in biological systems.

Phospholipases A(1) and A(2) frequently coexist in biological systems. Generation of lysophosphatidylcholine (LPC) in such systems cannot be assigned to any of these types of enzymes unless the position of the fatty acid in the lysocompound can be unambiguously determined. We here present a simple method to achieve this purpose.

Positional specificity of a phospholipase A activity induced by wounding, systemin, and oligosaccharide elicitors in tomato leaves.

Phospholipase A (PLA) activity, as measured by the accumulation of (14)C-lysophosphatidylcholine in leaves of tomato plants, increased rapidly and systemically in response to wounding. The increase in PLA activity in the systemic unwounded leaves was biphasic in wild-type tomato plants, peaking at 15 min and again at 60 min, but the second peak of activity was absent in transgenic prosystemin antisense plants. Supplying young excised tomato plants with the polypeptide hormone systemin also caused (14)C-lysophosphatidylcholine to increase to levels similar to those induced by wounding, but the increase in activity persisted for >2 hr.

Lysosomal glycerophosphocholine phosphodiesterase in Tetrahymena.

The purification and characterization of a novel phosphodiesterase (PDE) is presented. The activity was detected in the extracellular medium of Tetrahymena thermophila cultures, by the release of p-nitrophenol from p-nitrophenylphosphocholine (PNPPC) with an acidic pH optimum. In cell homogenates, it is sedimentable, shows a latency similar to that of acid phosphatase and is co-secreted with this enzyme, indicating that it is a lysosomal hydrolase.

A method for the preparation of Tetrahymena thermophila phospholipase A1 suitable for large-scale production.

A rapid and economical method for the purification of phospholipase A1 (PLA1) from the extracellular medium of the ciliate Tetrahymena thermophila is presented. Essentially, the procedure, here designated as purification by selective interaction (PSI), entails the incubation of media containing PLA1 with liposomes made of soy bean phospholipids. The PLA1-lipid complexes are precipitated by the addition of CaCl2 and collected by centrifugation.

Micellar lipoproteins as the possible storage and translocation form of intracellular diacylglycerol.

Previous work indicated that diacylglycerol (DG) molecules translocate across the cytoplasm of mammalian cells, a process relevant to the signalling role of this lipid as protein kinase C activator. Here we investigated the possible mechanism underlying DG translocation. We examined the interaction between 1,2-di-[1-14C]oleoyl-sn-glycerol and rat liver cytosol (rlc) using assays based on Lipidex-1000 and on coelution on Sepharose CL 6B.

Temperature acclimation of Trypanosoma cruzi epimastigote and metacyclic trypomastigote lipids.

This study examines the changes in cellular lipids that take place when Trypanosoma cruzi epimastigotes and metacyclic trypomastigotes are transferred from 28 to 37 degrees C. We found a rise in the sterol to phospholipid ratio, as well as in the triacylglycerol and steryl ester cellular content in T. cruzi epimastigotes.

Diversity of roles of protein kinase C alpha in the proliferation of Swiss 3T3 cells.

We examined the role of protein kinase C alpha (PKC alpha ) in the stimulation of DNA synthesis of Swiss 3T3 cells induced by bombesin, platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA). We found that cells in which this kinase had been down-regulated showed a partially abrogated mitogenic response to bombesin. The response to PDGF was unaltered; however, the response to PMA was completely suppressed.

The molecular basis of the self/nonself selectivity of a coelenterate toxin.

Coelenterates produce potent hemolysins inhibited by sphingomyelin (SM). Remarkably, instead of this lipid, their membranes contain a phosphono analogue of it. Using coelenterolysin (CL), a toxin produced by the sea anemone Phymactis clematis, we have examined a possible connection between these two peculiar traits.

Uptake and metabolism of L-serine by Pneumocystis carinii carinii.

Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids.

Differential hydrolysis of immobilized phosphatidylcholines by phospholipases A2 and C.

A novel phospholipid, 1-fatty acyl-2-(12-aminododecyl) phosphatidylcholine (APC), was synthesized and reacted with two different activated agarose matrices, differing in the spacer arm length: N-hydroxysuccinimidylester agarose (1-atom spacer arm) and N-hydroxysuccinimidylester-6-aminohexanoic acid agarose (8-atom spacer arm). Both immobilized phosphatidylcholines were readily degraded by Bacillus cereus phospholipase C at similar rates. By contrast, Crotalus adamanteus phospholipase A2 hydrolyzed long-spacer arm phosphatidylcholine, but had less than one tenth of the activity towards the short-spacer arm one.

Occurrence of specific sterols in Pneumocystis carinii.

We have examined the sterol composition and biosynthesis of rat Pneumocystis carinii. We found a number of lipid components among the nonsaponifiable fraction that appear unique to P. carinii.

Counteracting effects of E1a transformation on cAMP growth inhibition.

Several signaling molecules have been identified which act as inhibitors of epithelial cell growth. The mechanisms for this negative growth regulation are still poorly understood. In the case of TGF-beta, inhibition of keratinocyte cell growth can be totally prevented by transformation with an intact early region 1a (E1a) oncogene.