Lipocortin represents a family of similar Ca++ depentent phospholipid-binding proteins capable of blocking the activity of phospholipase A2 (PLA2) in vitro. Generally, these proteins are believed to inhibit the release of arachidonic acid from photopholipids and the formation of lipid mediators such as prostaglandin, leukotriene, and platelet activating factor. Lipocortin 1, initially identified as a glucocorticoid- responsive protein in macrophages and neutrophils has been implicated in transmembrane signal transduction during growth factor-mediated cell proliferation and transformation. To define the synthesis and its regulation, we investigated the expression of lipocortin 1 in both the mRNA and protein level in U937 cell line in the presence of several differentiation factors. The results were as follows. 1. The expression of lipocortin 1 and its mRNA was increased during TPA-induced differentiation of U937 cells to maximum of 2-fold and 5-fold respectively. Both the protein and mRNA levels decreased after 48 hours. 2. With the treatment with IFN-gamma, the expression of CD16 was increased. However, the protein and mRNA levels of lipocortin 1 were, not changed significantly. 3. Neither the dexamethasone or hydrocortisone have any effects on the expression of lipocortin 1 in both TPA-differentiated and undifferentiated U937 cells. The results from this study would give a light on defining the functional role of lipocortin 1 in macro-moncycle cell lineage and possibly some informative clues for the pathogenic mechanisms of the inflammatory diseases.
Annexin A1* ; Annexins* ; Arachidonic Acid ; Cell Lineage ; Cell Proliferation ; Dexamethasone ; Humans ; Hydrocortisone ; Macrophages ; Neutrophils ; Phospholipases A2 ; Platelet Activating Factor ; RNA, Messenger ; Signal Transduction ; U937 Cells*

To produce recombinant phospholipase A(1) (PLA(1)) by Escherichian coli, the pla gene encoding PLA(1) was amplified from Serratia liquefaciens by PCR and cloned into two vectors pET20-b(+) and pET28-a(+). The two recombinant plasmids were then transformed into E. coli BL21 (DE3) individually to express PLA(1). E. coli BL21(DE3)/pET28a-pla yielded extracellular PLA(1) with an activity of 40.8 U/mL in batch cultivations of shaken flasks by auto-induction, which was accounted for 91% of total enzyme activity. On the basis of primal auto-induction medium, the optimized fermentation medium of PLA(1) contained tryptone 10 g/L, yeast extract 5 g/L, glucose 0.8 g/L, lactose 5 g/L, Na2HPO4 25 mmol/L, KH2PO4 25 mmol/L and 1 mmol/L MgSO4 (final concentration). Glycine (7.5 g/L) was added 6 h after inoculated. After incubated at 37 degrees C for 24 h, extracellular enzyme activity reached 128.7 U/mL.
Cloning, Molecular ; Culture Media ; Escherichia coli ; genetics ; growth & development ; metabolism ; Fermentation ; Lactose ; pharmacology ; Phospholipases A1 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Serratia liquefaciens ; enzymology

OBJECTIVETo test the changing expression level of Annexin I, cPLA(2) and PCNA in the palatine process of cleft-palate mice A/J and C57B/6j induced by dexamethasone. To discuss the developing mechanism of cleft-palate and the corresponding preventive methods.

METHODs Pregnant mice A/J and C57BL/6j were randomly divided into normal group as blank control, group with deformity induced by dexamethasone, group given VitB(12) as antagonist to deforming factor, group given only VitB(12). The relative quantity of Annexin I, Cpla2 and PCNA were detected by immunoblotting.

RESULTSAmong mice A/J, with the development of palatine process, Annexin I's expression level was increased in the normal group, and other groups showed the similar changes. Annexin I's expression level was significantly higher in group DEX and group DEX + VitB(12) than in normal group and VitB(12) group of, while there was no significant difference between normal group and VitB(12) group. But the changes of cPLA(2) and PCNA expression level was in an opposite direction, with development it decreased in the normal mice's palatine process. In mice C57B/6j there was no significant difference between normal group and group DEX on the measured quantity of Annexin I, cPLA(2) and

CONCLUSIONSAnnexin I and cPLA(2) introduce glucocorticoid to induce cleft-palate. VitB(12) can not inhibit DEX's enhancing effect on the expression level of Annexin I, but it can antagonize DEX's inhibiting effect on expression level of cPLA(2), which is probably one of the mechanisms why VitB(12) antagonize glucocorticoid's deforming effect.

Abnormalities, Drug-Induced ; etiology ; Animals ; Annexin A1 ; biosynthesis ; Cleft Palate ; chemically induced ; Dexamethasone ; toxicity ; Female ; Male ; Mice ; Mice, Inbred C57BL ; Palate ; embryology ; metabolism ; Phospholipases A ; biosynthesis ; Pregnancy ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Random Allocation

Most diabetic patients experience diabetic mellitus (DM) urinary bladder dysfunction. A number of studies evaluate bladder smooth muscle contraction in DM. In this study, we evaluated the change of bladder smooth muscle contraction between normal rats and DM rats. Furthermore, we used pharmacological inhibitors to determine the differences in the signaling pathways between normal and DM rats. Rats in the DM group received an intraperitoneal injection of 65 mg/kg streptozotocin and measured blood glucose level after 14 days to confirm DM. Bladder smooth muscle contraction was induced using acetylcholine (ACh, 10⁻⁴ M). The materials such as, atropine (a muscarinic receptor antagonist), U73122 (a phospholipase C inhibitor), DPCPX (an adenosine A1 receptor antagonist), udenafil (a PDE5 inhibitor), prazosin (an α₁-receptor antagonist), papaverine (a smooth muscle relaxant), verapamil (a calcium channel blocker), and chelerythrine (a protein kinase C inhibitor) were pre-treated in bladder smooth muscle. We found that the DM rats had lower bladder smooth muscle contractility than normal rats. When prazosin, udenafil, verapamil, and U73122 were pre-treated, there were significant differences between normal and DM rats. Taken together, it was concluded that the change of intracellular Ca²⁺ release mediated by PLC/IP3 and PDE5 activity were responsible for decreased bladder smooth muscle contractility in DM rats.
Acetylcholine ; Animals ; Atropine ; Blood Glucose ; Calcium Channels ; Humans ; Injections, Intraperitoneal ; Muscle, Smooth* ; Papaverine ; Prazosin ; Protein Kinase C ; Rats* ; Receptor, Adenosine A1 ; Receptors, Muscarinic ; Streptozocin ; Type C Phospholipases ; Urinary Bladder* ; Verapamil