AIMTo explore the changes of rat platelet phospholipase A2 (PLA2) mRNA content in bacteria infected rat and study the cDNA and amino acid sequences of the PLA2 structure to lay a good foundation for the development of new antibiotics.

METHODSThe PLA2 mRNA level in blood was determined by RT-PCR. The DNA sequence was cloned and analyzed.

RESULTSAfter injection of bacteria in rats, the mRNA level of PLA2 in blood increased markedly. The cDNA and amino acid sequence were highly homologous to other PLA2 cDNA from different tissues of the rat.

CONCLUSIONPlatelet PLA2 in blood responded quickly to bacteria infection in gene level. Therefore, the PLA2 protein was produced increasingly which was shown to control the infection with bacteria. Although there are little difference between PLA2 cDNA cloned from blood and other sources in DNA and amino acid sequences, the catalytic site for enzymatic activity and basic structure are identical.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; metabolism ; Male ; Molecular Sequence Data ; Phospholipases A ; blood ; genetics ; metabolism ; Phospholipases A2 ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Staphylococcal Infections ; blood ; Staphylococcus aureus

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phos-pholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A2 (PLA2) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA2. Acanthamoeba exhibited optimal phospholipase activities at 37degrees C and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA2-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.
Acanthamoeba/*enzymology/genetics/*isolation & purification/physiology ; Cell Adhesion ; Cells, Cultured ; Endothelial Cells/parasitology ; Humans ; Keratitis/*parasitology ; Phospholipase D/genetics/*metabolism ; Phospholipases A2/genetics/*metabolism ; Protozoan Proteins/genetics/*metabolism ; Soil/*parasitology