An essential role of phosphatidylinositol 3-kinase in myogenic differentiation

The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137), strongly enhances myogenic differentiation in cultures of chicken-embryo myoblasts. It increases the size of the myotubes and induces elevated levels of the muscle-specific proteins MyoD, myosin heavy chain, creatine kinase, and desmin. Inhibition of PI 3-kinase activity with LY294002 or with dominant-negative mutants of PI 3-kinase interferes with myogenic differentiation and with the induction of muscle-specific genes. PI 3-kinase is therefore an upstream mediator for the expression of the muscle-specific genes and is both necessary and rate-limiting for the process of myogenesis.     

STT4 is an essential phosphatidylinositol 4-kinase that is a target of wortmannin in Saccharomyces cerevisiae

Wortmannin is a natural product that inhibits signal transduction. One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase). We show that wortmannin is toxic to the yeast Saccharomyces cerevisiae and present genetic and biochemical evidence that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast. In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol. In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol. Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not. In vitro, the PI 4-kinase activity of STT4, but not of PIK1, was potently inhibited by wortmannin. Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 conferred wortmannin resistance, as did deletion of phospholipase C-1. These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate.     

skittles, a Drosophila phosphatidylinositol 4-phosphate 5-kinase, is required for cell viability, germline development and bristle morphology, but not for neurotransmitter release

The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. We have cloned and generated mutations in a Drosophila PIP5K type I (skittles). Our analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, we found no evidence for PIP5KI involvement in neural secretion.     

PIK1, an essential phosphatidylinositol 4-kinase associated with the yeast nucleus

Transmission of mitogenic and developmental signals to intracellular targets is often mediated by inositol derivatives. Here we present the cloning and characterization of a gene from Saccharomyces cerevisiae, PIK1, encoding the enzyme that catalyses the first committed step in the production of the second messenger inositol-1,4,5-trisphosphate. PIK1 encodes a phosphatidylinositol 4-kinase (PI 4-kinase) essential for growth. Cells carrying PIK1 on a multicopy vector overexpress PI 4-kinase activity exclusively in a nuclear fraction, suggesting that PIK1 is part of a nuclear phosphoinositide cycle. Temperature-sensitive mutations, but not a null mutation, can be suppressed by high osmolarity or an elevated concentration of Ca2+. Conditional mutants have a cytokinesis defect as indicated by a uniform terminal phenotype of cells with large buds and fully divided nuclei. We suggest that PIK1 controls cytokinesis through the actin cytoskeleton.     

An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling

Transmembrane signaling by the phospholipase C-beta (PLC-beta) pathway is known to require at least three components: the receptor, the G protein, and the PLC. Recent studies have indicated that if the cytosol is allowed to leak out of HL60 cells, then G protein-stimulated PLC activity is greatly diminished, indicating an essential role for a cytosolic component(s). We now report the complete purification of one component based on its ability to reconstitute GTP gamma S-mediated PLC activity and identify it as the phosphatidylinositol transfer protein (PI-TP). Based on the in vitro effects of PI-TP, we surmise that it is involved in transporting PI from intracellular compartments for conversion to PI bisphosphate (PIP2) prior to hydrolysis by PLC-beta 2/PLC-beta 3, the endogenous PLC isoforms present in these cells.     

An essential role for the phosphatidylinositol transfer protein in the scission of coatomer-coated vesicles from the trans-Golgi network

We identified the phosphatidylinositol transfer protein (PITP) as being responsible for a powerful latent, nucleotide-independent, Golgi-vesiculating activity that is present in the cytosol but is only manifested as an uncontrolled activity in a cytosolic protein subfraction, in which it is separated from regulatory components that appear to normally limit its action to the scission of COPI-coated buds from trans-Golgi network membranes. A specific anti-PITP antibody that recognizes the two mammalian PITP isoforms fully inhibited the capacity of the cytosol to support normal vesicle generation as well as the uncontrolled vesiculating activity manifested by the cytosolic protein subfraction. The phosphatidylinositol- (PI) loaded form of the yeast PITP, Sec14p, but not the phosphatidylcholine- (PC) loaded form of the protein, was capable of substituting for the cytosolic subfraction in promoting the scission of coated buds from the trans-Golgi network. At higher concentration, however, Sec14p, when loaded with PI, but not with PC or phosphatidylglycerol, caused by itself an indiscriminate vesiculation of uncoated Golgi membranes that could be suppressed by PC-Sec14p, which also suppresses the uncontrolled vesiculation caused by the cytosolic subfraction. We propose that, by delivering PI to specific sites in the Golgi membrane near the necks of coated buds, PITP induces local changes in the organization of the lipid bilayer, possibly involving PI metabolites, that triggers the fusion of the ectoplasmic faces of the Golgi membrane necessary for the scission of COPI-coated vesicles.     

A requirement for phosphatidylinositol 3-kinase in pseudopod extension

Phagocytosis requires actin assembly and pseudopod extension, two cellular events that coincide spatially and temporally. The signal transduction events underlying both processes may be distinct. We tested whether phagocytic signaling resembles that of growth factor receptors, which induce actin polymerization via activation of phosphatidylinositol 3-kinase (PI 3-kinase). Fcgamma receptor-mediated phagocytosis was accompanied by a rapid increase in the accumulation of phosphatidylinositol 3,4,5-trisphosphate in vivo, and addition of wortmannin (WM) or LY294002, two inhibitors of PI 3-kinase(s), inhibited phagocytosis but not Fcgamma receptor-directed actin polymerization. However, both compounds prevented maximal pseudopod extension, suggesting that PI 3-kinase inhibition produced a limitation in membrane required for pseudopod extension. Availability of plasma membrane was not limiting for phagocytosis, because blockade of ingestion in the presence of WM was not overcome by reducing the number of particles adhering to macrophages. However, decreasing bead size, and hence the magnitude of pseudopod extension required for particle engulfment, relieved the inhibition of phagocytosis in the presence of WM or LY294002 by up to 80%. The block in phagocytosis of large particles occurred before phagosomal closure, because both compounds inhibited spreading of macrophages on substrate-bound IgG. Macrophage spreading on IgG was accompanied by exocytic insertion of membrane from an intracellular source, as measured by the dye FM1-43. These results indicate that one or more isoforms of PI 3 kinase are required for maximal pseudopod extension but not phagocytosis per se. We suggest that PI 3-kinase is required for coordinating exocytic membrane insertion and pseudopod extension.     

A role for phosphatidylinositol 3-kinase in generating T cell help for B cell growth and differentiation

Evidence supporting the importance of the 3-phosphoinositide signaling pathway in lymphocyte activation is rapidly accumulating. In our study, we assessed the effects of two PI 3-kinase inhibitors, wortmannin and LY294002, on T cells as a means to analyze the role of the PI 3-kinase-signaling pathway in the generation of T cell help for B cell growth and differentiation. For these studies, B cells were cocultured with CD3-activated mitomycin C-treated T cells to induce B cell responsiveness. Of interest, wortmannin or LY294002 pretreatment of the T cell population significantly inhibited T cell-dependent induction of B cell proliferation and differentiation. The failure of wortmannin-treated CD3-activated mitomycin C-treated T cells to provide help in driving the differentiation of B cells to Ig-secreting cells could not be corrected by the addition of exogenous IL-2. Further studies designed to elucidate the mechanism by which wortmannin-treated T cells failed to provide B cell help indicated that wortmannin and LY294002 significantly inhibited the induction of CD40 ligand and, to a lesser extent, intercellular adhesion molecule-1 expression. These results suggest that the PI 3-kinase-signaling pathway, or other wortmannin- and LY294002-sensitive pathways, may be important for the induction of expression of crucial interaction molecules, such as CD40 ligand, on T cells and thus indicates that D-3 phosphoinositides play a pivotal role in regulating T cell-dependent B cell activation.     

Protein tyrosine phosphorylation activates rat splenic type II phosphatidylinositol 4-kinase in vitro

Cystatin A (acid cysteine proteinase inhibitor; ACPI) is a natural inhibitor of cysteine proteinases. It has been suggested that an inverse correlation exists between cystatin A and malignant progression. We wanted to assess the biological and clinical significance of cystatin A in infiltrative breast carcinoma by immunohistochemical staining. Formalin-fixed paraffin-embedded material from 440 cases treated during the years 1988-1991 was used in the study. After exclusion of patients with disseminated disease at diagnosis, previous contralateral breast carcinoma, and absence of follow-up data, 384 patients could be included in the survival analysis. For immunohistochemical analysis of cystatin A, we used monoclonal cystatin A antibody WR-23/2/3/3, the binding of which was detected by the avidin-biotin-peroxidase method. Immunohistochemical analysis of Bcl-2 and p53 was also done, and mitotic activity was evaluated. Positive staining for cystatin A was found in 52 of 440 cases. The staining was irregular but showed irrefutably positive areas within neoplastic tissue. Most of the positive tumors were of the ductal infiltrative type, but two were mucinous carcinomas, one medullary and one squamous cell carcinoma. No lobular carcinomas showed positive staining. Focal cystatin A positivity was seen in myoepithelial cells of benign ducts. Occasional apoptotic bodies within the neoplasm showed strong positivity for cystatin A. Tumors positive for cystatin A were of larger size and had higher mitotic activity than cystatin A-negative tumors. Cystatin A was associated with negative Bcl-2 staining, but there was no statistically significant association between axillary lymph node status or p53 immunostaining. The risk for breast cancer-related death was significantly higher in patients with cystatin A-positive tumors than in those with cystatin A-negative ones. The risk increase was significant also in lymph node-negative patients. After adjusting for the effect of tumor size, histological grade, and lymph node status, cystatin A-positive patients still had a higher risk of death. Patients with cystatin A and p53 coexpression had a higher risk of death than the other patients. The findings reveal a new variant of aggressive breast cancer. This type of carcinoma may develop during tumor progression through genetic instability that allows cystatin A expression and gives growth advantage to a clone of tumor cells.     

NSF function in neurotransmitter release involves rearrangement of the SNARE complex downstream of synaptic vesicle docking

The SNARE hypothesis has been proposed to explain both constitutive and regulated vesicular transport in eukaryotic cells, including release of neurotransmitter at synapses. According to this model, a vesicle targeting/docking complex consisting primarily of vesicle- and target-membrane proteins, known as SNAREs, serves as a receptor for the cytosolic N-ethylmaleimide-sensitive fusion protein (NSF). NSF-dependent hydrolysis of ATP disassembles the SNARE complex in a step postulated to initiate membrane fusion. While features of this model remain tenable, recent studies have challenged fundamental aspects of the SNARE hypothesis, indicating that further analysis of these components is needed to fully understand their roles in neurotransmitter release. We have addressed this issue by using the temperature-sensitive Drosophila NSF mutant comatose (comt) to study the function of NSF in neurotransmitter release in vivo. Synaptic electrophysiology and ultrastructure in comt mutants have recently defined a role for NSF after docking in the priming of synaptic vesicles for fast calcium-triggered fusion. Here we report that an SDS-resistant neural SNARE complex, composed of the SNARE polypeptides syntaxin, n-synaptobrevin, and SNAP-25, accumulates in comt mutants at restrictive temperature. Subcellular fractionation experiments indicate that these SNARE complexes are distributed predominantly in fractions containing plasma membrane and docked synaptic vesicles. Together with the electrophysiological and ultrastructural analyses of comt mutants, these results indicate that NSF functions to disassemble or otherwise rearrange a SNARE complex after vesicle docking and that this rearrangement is required to maintain the readily releasable pool of synaptic vesicles.     

Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels

Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their alpha1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein-protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.     

An essential role for ectodomain shedding in mammalian development

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.     

Concanavalin A modulates tyrosine phosphorylation and activation of a type II phosphatidylinositol 4-kinase in rat splenic lymphocytes

Activation of rat splenic lymphocytes by concanavalin A resulted in two-fold increase in Ptdlns 4-kinase activity and rapid tyrosine phosphorylation of the enzyme. The activation kinetics showed a strong correlation with tyrosine phosphorylation state of the enzyme. Characterization of the enzyme activity suggests that it is a type II PtdIns 4-kinase. Kinetic analysis of the enzyme reaction showed three-fold decrease in Km for PtdIns and two-fold increase in Vmax in Con A stimulated cells. These results suggest that a type II PtdIns 4-kinase is an integral component of the early signal transduction machinery during T-cell activation by concanavalin A and is actively regulated by protein tyrosine phosphorylation-dephosphorylation.     

Synaptic physiology and ultrastructure in comatose mutants define an in vivo role for NSF in neurotransmitter release

N-Ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic protein thought to play a key role in vesicular transport in all eukaryotic cells. Although NSF was proposed to function in the trafficking of synaptic vesicles responsible for neurotransmitter release, only recently have in vivo experiments begun to reveal a specific function for NSF in this process. Our previous work showed that mutations in a Drosophila NSF gene, dNSF1, are responsible for the temperature-sensitive paralytic phenotype in comatose (comt) mutants. In this study, we perform electrophysiological and ultrastructural analyses in three different comt alleles to investigate the function of dNSF1 at native synapses in vivo. Electrophysiological analysis of postsynaptic potentials and currents at adult neuromuscular synapses revealed that in the absence of repetitive stimulation, comt synapses exhibit wild-type neurotransmitter release at restrictive (paralytic) temperatures. In contrast, repetitive stimulation at restrictive temperatures revealed a progressive, activity-dependent reduction in neurotransmitter release in comt but not in wild type. These results indicate that dNSF1 does not participate directly in the fusion of vesicles with the target membrane but rather functions in maintaining the pool of readily releasable vesicles competent for fast calcium-triggered fusion. To define dNSF1 function further, we used transmission electron microscopy to examine the distribution of vesicles within synaptic terminals, and observed a marked accumulation of docked vesicles at restrictive temperatures in comt. Together, the results reported here define a role for dNSF1 in the priming of docked synaptic vesicles for calcium-triggered fusion.     

Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes

Insulin stimulates glucose transport in its target cells by recruiting the glucose transporter Glut 4 from an intracellular compartment to the cell surface. Previous studies have indicated that phosphatidylinositol 3-kinase (PI 3-kinase) is a necessary step in this insulin action. We have investigated whether PI 3-kinase activation is sufficient to promote Glut 4 translocation in transiently transfected adipocytes. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged Glut 4 and a constitutively active form of PI 3-kinase (p110*). The expression of p110* induced the appearance of epitope-tagged Glut 4 at the cell surface at a level similar to that obtained after insulin treatment, whereas a kinase-dead version of p110* had no effect. The p110* effect was observed over a wide range of the transfected cDNA. When subcellular fractionation of adipocytes was performed, p110* was found, similar to the endogenous PI 3-kinase, enriched in the low density microsomal compartment, which also contains the Glut 4 vesicles. This could suggest that a specific localization of PI 3-kinase in this compartment is required for the action on Glut 4. The observations made with PI 3-kinase are in contrast with those seen with the MAP kinase cascade. Indeed, a constitutively active form of MAP kinase kinase had no effect on Glut 4 translocation in basal conditions. At the highest degree of expression, the constitutively active form of MAP kinase kinase slightly inhibited the insulin stimulation of Glut 4 translocation. Taken together, our results indicate that Glut 4 translocation can be efficiently promoted by an active form of PI 3-kinase but not by the activation of the MAP kinase pathway.     

Newly synthesized phosphatidylinositol phosphates are required for synaptic norepinephrine but not glutamate or gamma-aminobutyric acid (GABA) release

Newly synthesized phosphatidylinositol phosphates have been implicated in many membrane-trafficking reactions. They are essential for exocytosis of norepinephrine in PC12 cells and chromaffin cells, suggesting a function in membrane fusion. We have now studied the role of phosphatidylinositol phosphates in synaptic vesicle exocytosis using synaptosomes. Under conditions where phosphorylation of phosphatidylinositols is blocked, norepinephrine secretion was nearly abolished whereas glutamate and GABA release was still elicited. Thus phosphatidylinositides are essential only for some membrane fusion reactions, and exocytotic release mechanisms differ between neurotransmitters.     

Protein kinase C-zeta as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose transport

Insulin provoked rapid increases in enzyme activity of immunoprecipitable protein kinase C-zeta (PKC-zeta) in rat adipocytes. Concomitantly, insulin provoked increases in 32P labeling of PKC-zeta both in intact adipocytes and during in vitro assay of immunoprecipitated PKC-zeta; the latter probably reflected autophosphorylation, as it was inhibited by the PKC-zeta pseudosubstrate. Insulin-induced activation of immunoprecipitable PKC-zeta was inhibited by LY294002 and wortmannin; this suggested dependence upon phosphatidylinositol (PI) 3-kinase. Accordingly, activation of PI 3-kinase by a pYXXM-containing peptide in vitro resulted in a wortmannin-inhibitable increase in immunoprecipitable PKC-zeta enzyme activity. Also, PI-3,4-(PO4)2, PI-3,4,5-(PO4)3, and PI-4,5-(PO4)2 directly stimulated enzyme activity and autophosphoralytion in control PKC-zeta immunoprecipitates to levels observed in insulin-treated PKC-zeta immunoprecipitates. In studies of glucose transport, inhibition of immunoprecipitated PKC-zeta enzyme activity in vitro by both the PKC-zeta pseudosubstrate and RO 31-8220 correlated well with inhibition of insulin-stimulated glucose transport in intact adipocytes. Also, in adipocytes transiently expressing hemagglutinin antigen-tagged GLUT4, co-transfection of wild-type or constitutive PKC-zeta stimulated hemagglutinin antigen-GLUT4 translocation, whereas dominant-negative PKC-zeta partially inhibited it. Our findings suggest that insulin activates PKC-zeta through PI 3-kinase, and PKC-zeta may act as a downstream effector of PI 3-kinase and contribute to the activation of GLUT4 translocation.     

Phosphatidylinositol-4-phosphate 5-kinase localized on the plasma membrane is essential for yeast cell morphogenesis

Phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2), an important element in eukaryotic signal transduction, is synthesized either by phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P 5K) from phosphatidylinositol 4-phosphate (PtdIns(4)P) or by phosphatidylinositol-5-phosphate 4-kinase (PtdIns(5)P 4K) from phosphatidylinositol 5-phosphate (PtdIns(5)P). Two Saccharomyces cerevisiae genes, MSS4 and FAB1, are homologous to mammalian PtdIns(4)P 5Ks and PtdIns(5)P 4Ks. We show here that MSS4 is a functional homolog of mammalian PtdIns(4)P 5K but not of PtdIns(5)P 4K in vivo. We constructed a hemagglutinin epitope-tagged form of Mss4p and found that Mss4p has PtdIns(4)P 5K activity. Immunofluorescent and fractionation studies of the epitope-tagged Mss4p suggest that Mss4p is localized on the plasma membrane, whereas Fab1p is reportedly localized on the vacuolar membrane. A temperature-sensitive mss4-1 mutant was isolated, and its phenotypes at restrictive temperatures were found to include increased cell size, round shape, random distribution of actin patches, and delocalized staining of cell wall chitin. Thus, biochemical and genetic analyses on Mss4p indicated that yeast PtdIns(4)P 5K localized on the plasma membrane is required for actin organization.     

Lipid products of phosphoinositide 3-kinase and phosphatidylinositol 4',5'-bisphosphate are both required for ADP-dependent platelet spreading

We have shown previously that ADP released upon platelet adhesion mediated by alphaIIb beta3 integrin triggers accumulation of phosphatidylinositol 3',4'-bisphosphate (PtdIns-3,4-P2) (Gironcel, D. , Racaud-Sultan, C., Payrastre, B., Haricot, M., Borchert, G., Kieffer, N., Breton, M., and Chap, H. (1996) FEBS Lett. 389, 253-256). ADP has also been involved in platelet spreading. Therefore, in order to study a possible role of phosphoinositide 3-kinase in platelet morphological changes following adhesion, human platelets were pretreated with specific phosphoinositide 3-kinase inhibitors LY294002 and wortmannin. Under conditions where PtdIns-3, 4-P2 synthesis was totally inhibited (25 microM LY294002 or 100 nM wortmannin), platelets adhered to the fibrinogen matrix, extended pseudopodia, but did not spread. Moreover, addition of ADP to the medium did not reverse the inhibitory effects of phosphoinositide 3-kinase inhibitors on platelet spreading. Although synthetic dipalmitoyl PtdIns-3,4-P2 and dipalmitoyl phosphatidylinositol 3',4', 5'-trisphosphate restored only partially platelet spreading, phosphatidylinositol 4',5'-bisphosphate (PtdIns-4,5-P2) was able to trigger full spreading of wortmannin-treated adherent platelets. Following 32P labeling of intact platelets, the recovery of [32P]PtdIns-4,5-P2 in anti-talin immunoprecipitates from adherent platelets was found to be decreased upon treatment by wortmannin. These results suggest that the lipid products of phosphoinositide 3-kinase are required but not sufficient for ADP-induced spreading of adherent platelets and that PtdIns-4,5-P2 could be a downstream messenger of this signaling pathway.     

An essential role for free radicals and derived species in signal transduction

OBJECTIVE: To examine whether there is generation of oxygen free radicals (OFR) and lipid peroxidation of cell membrane after volume replacement for burn shock, and to study the relationship between OFR injury and enterogenous endotoxemia. METHODS: Forty-seven burn patients were involved in this study. Among them, 18 had delayed fluid resuscitation (DR) and the others had early fluid resuscitation (ER) within 6 hours postburn. Sixty-six gnotobiotic rats were used in a collaborating experiment as burn models. They were divided into 4 groups: sham injury (n = 6), early resuscitation (n = 24), late resuscitation (n = 24) and vitamins E and C treatment group (n = 12). All the rats, except those in the sham injury group, were inflicted with 40% total body surface area (TBSA) third-degree burns. OFR was determined in the blood of patients with electron spin resonance (ESR). S/W ratio and tau c values of patients' erythrocytes were measured with ESR spectrometer. Blood superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities, malondialdehyde contents and plasma endotoxin levels were assayed. Rats were sacrificed at the 12th, 24th, 48th and 72nd hour after injury. Plasma endotoxin levels, mucosal SOD, GSHPx and malondialdehyde (MDA), as well as diamine oxidase activity of ileum were determined. Cultures of mesenteric lymph nodes (MLN), liver, spleen, heart, lung, kidney and blood were done. RESULTS: A significant increase in blood OFR contents and plasma MDA, and a significant decline in blood SOD and GSHPx were found after resuscitation in DR group as compared with those in ER group. Both strong to weak spectra component (S/W) ratio and tau c value were higher in DR group in contrast with those in ER group. Higher elevation in plasma endotoxin level in DR group was seen. In DR group, plasma MDA content was correlated with S/W ratio, tau c value and plasma endotoxin level. In rats, the level of mucosal MDA, plasma endotoxin and incidence of bacterial translocation (BT) were significantly higher. Mucosal SOD, GSHPx and diamine oxidase (DAO) activity were significantly lower in DR group as compared with those in ER group. In DR group, mucosal MDA content was negatively correlated with mucosal DAO activity, while the latter was negatively correlated with BT. After treatment with vitamins E and C, mucosal MDA content decreased, plasma endotoxin and BT significantly declined and mucosal DAO heightened. CONCLUSIONS: Tissue reperfusion might induce the production of OFR, resulting in lipid peroxidation injury, especially to intestinal mucosa, and resulting in disruption of mucosal barrier function followed by endotoxemia and BT.