摘要:

The most primitive mechanism of cellular protection involves the expression of a polypeptide family named heat shock or stress proteins (hsps). Some of these hsps are present in unstressed cells and play an important role in the folding and translocation of polypeptides across membranes. Thus, they have been termed molecular chaperones. Hsps are expressed in response to an array of stresses, including hyperthermia, oxygen radicals, heavy metals, ethanol, and amino acid analogues. In addition, the heat shock response is induced during clinically relevant situations such as ischemia/reperfusion and circulatory and hemorrhagic shock. All of the above stresses have in common that they disturb the tertiary structure of proteins and have adverse effects on cellular metabolism. Pretreatment of cells with a mild stress, sufficient to induce the expression of hsps, results in protection to subsequent insults. This phenomenon has been coined “stress tolerance” and is apparently caused by the resolubilization of proteins that were denatured during the stress. In addition, cellular structures (microfilaments and centrosomes) and processes (transcription, splicing, and translation) are stabilized or repaired during a second stress in stress tolerant cells and organisms. There is a great body of evidence indicating a direct role of hsps in the stabilization of these events. The intrinsic capacity of hsps to protect cells has potential relevance as a natural mechanism of organ protection during harmful environmental conditions and operative procedures, and in the combat against pathogens.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Endothelial activation and damage are common endpoints of a complex process that may result in multiple organ dysfunction syndrome (MODS). The influence of continuous intravenous heparinization on plasma levels of circulating adhesion molecules was studied in 28 trauma patients (injury severity score between 15 and 25 points) and 28 sepsis patients secondary to abdominal surgery. According to a prospective, randomized sequence the patients received either unfractionated heparin (aim: activated partial thromboplastin time (aPTT) approximately 2 × normal) (trauma-heparin (n = 14); sepsis-heparin (n = 14)) or not (trauma (n = 14); sepsis (n = 14)). Plasma levels of circulating soluble endothelial leukocyte adhesion molecule-1 (sELAM-1), vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), and granule membrane protein-140 (sGMP-140) were serially measured from arterial blood samples for 5 days. Approximately 600 U/h of heparin were given to increase aPTT to approximately 60 s. Plasma levels of all adhesion molecules increased in all groups. This increase was significantly (p< .05) highest in both sepsis groups (sepsis: sELAM-1: from 50 ± 11 to 84 ± 19 ng/mL; sICAM-1: 410 ± 68 to 700 ± 95 ng/mL), but did not differ significantly between the treated and nontreated patients (sepsis-heparin: sELAM-1: from 60 ± 131 to 88 ± 20 ng/mL; sICAM-1: from 398 ± 99 to 686 ± 119 ng/mL). Trauma patients showed a less pronounced increase in all adhesion molecules without differences between the two subgroups. Only sGMP-140 increased significantly (p< .05) more in the trauma (from 102 ± 20 to 169 ± 16 ng/mL) than in the trauma-heparin group (from 109 ± 19 to 132 ± 17 ng/mL). It is summarized that continuous heparinization with approximately 600 U/h did not attenuate the rise in circulating adhesion molecules in sepsis and trauma patients. The study findings suggest that heparin in this dose regimen may be unlikely to influence endothelial inflammation or endothelial function in critically ill patients.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to “shed” the membrane-associated receptor for TNF-α (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in “cell-free” supernatant produced by stimulated macrophage populations applying125I-TNF-α and biotinylated TNF-α ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying125I-TNF-α and biotinylated TNF-α as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47–52 kDa with lesser bands also visible at approximately 26–32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47–52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The hypothesis that cardiac functional abnormalities that occur after major burn trauma are paralleled by an increased incidence of apoptosis in cardiac myocytes was examined. Adult Sprague-Dawley rats were given a full thickness scald burn comprising 43 ± 1% of the total body surface area or were manipulated identically but not exposed to burn injury (sham burn); burned rats were fluid resuscitated with lactated Ringer's solution. Tissues from burn and sham burn animals were then examined by the TUNEL (TdT-mediated dUTP nick end labeling) assay and light microscopy to determine the presence of apoptosis 24 and 48 h after burn trauma. In parallel, the mechanical function of the heart was assayed in separate groups of rats. Tissues harvested from the hearts of sham-treated animals showed essentially no apoptosis, whereas a small number of apoptotic cells were noted in the intestinal villi and liver of sham-treated animals. Twenty-four hours after burn trauma, there was a marked increase in apoptotic cells in the left ventricle (+916%), and the number of apoptotic cells remained increased by eightfold 48 h postburn. Apoptosis was noted predominately in the subendocardial tissue of the left ventricle. The appearance of apoptotic cells was paralleled by a decrease in cardiac mechanical function with significant decreases in left ventricular pressure and ±dP/dtmax. Burn injury also increased apoptosis in the small intestine significantly, whereas apoptosis in the liver did not increase with burn trauma. These data suggest that the apoptosis of the cardiac myocytes that occurs after burn trauma may contribute, in part, to postburn cardiac mechanical dysfunction.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Tyrosine kinases mediate cellular signal transduction to endotoxin. A class of tyrosine kinase inhibitors, the tyrphostins, have been shown to protect mice from endotoxin-induced lethality. Neonatal rats and mice have been shown to be uniquely susceptible to lethal endotoxic shock. In our study, the effect of a lipophilic tyrphostin, AG 556, on endotoxin-induced neonatal and adult mortality andin vitroneonatal splenic cell thromboxane (TxB2), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) production were examined. Neonatal rats (<24 h old) were administered tyrphostin (100 μg subcutaneous) 2 h before an approximate LD50dose ofSalmonella enteritidisendotoxin (.024 mg/kg/intracardiac). There was a significant decrease in mortality in the animals pretreated with 100 μg of tyrphostin (29% mortality in the treated group, n = 41 versus 53% in the vehicle control group, n = 40;p< .05). Also in adult rats tyrphostin (5 mg/kg intraperitoneal) 2 h before endotoxin (10 mg/kg intravenous) significantly improved survival (50% drug treated versus 84% in control, n = 12/group;p< .05). Adherent neonatal splenic cell mediator production of TxB2, TNF-α, and NO (measured by nitrite) in tyrphostin pretreated splenic cells were compared with endotoxin-stimulated splenic cellsin vitro. The studies (n = 4) demonstrate an increase (p< .05) in the production of TxB2, TNF-α, and NO in the endotoxin- (10 μg/mL) stimulated adherent splenic cells compared with basal. Tyrphostin pretreatment (10, 20, 50 μM) produced a dose-dependent decrease (p< .05) in endotoxin-stimulated TxB2and TNF-α production. NO production was not significantly reduced. In conclusion, tryphostin appears to have a protective effect on mortality in both adult and neonatal rat endotoxic shock. Tyrphostin decreased specific mediator production in stimulated neonatal cells. Thus, inhibition of signal transduction pathways of endotoxin activation by tyrosine kinase inhibition may provide an effective approach to treat endotoxic shock in the neonate.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Changes in the protein level of various subunits of G-protein and the activity of adenylate cyclase in rat liver plasma membranes during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The results show that the protein levels of Gαi-2 and Gαi-3 were unchanged during the early hypermetabolic (hyperglycemic) phase (9 h after CLP), whereas Gαai-2 and Gαai-3 were increased by 32.4 and 59.1%, respectively, during the late hypometabolic (hypoglycemic) phase (18 h after CLP) of sepsis. The protein levels of Gαs and Gβ remained unaltered during both the early and the late phases of sepsis. The activity of adenylate cyclase remained unchanged during the early phase, whereas it was decreased by 26% (p< .05) during the late phase of sepsis. Since the G-protein/adenylate cyclase signaling system mediates hormonal control of hepatic glucose metabolism, the observed increases in the Gαi-2 and Gαi-3 protein levels coupled with a decrease in the activity of adenylate cyclase may contribute to the development of the hypoglycemia during the late stage of sepsis.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Background and Aims: Experimental peritonitis causes gut intramucosal acidosis indicating intramucosal ischemia. However, tissue acidosis may reflect other conditions than ischemia. An increased mucosal-arterial Pco2difference (Pco2-gap) is suggested to be a more adequate measure of tissue ischemia than intramucosal pH (pHi). This study was performed to elucidate whether keeping cardiac index (Cl) and splanchnic blood flow normal or supranormal by administration of colloids and an inotropic drug could prevent the acidosis as well as reduce the Pco2-gap. A secondary aim was to study to what degree the low pHiin peritonitis really reflects ischemia.Subjects: 24 anesthetized pigs (18–27 kg) divided into four groups.Models: A Swan-Ganz catheter, transonic flow meters and catheters for blood sampling were applied. pHiwas calculated using tonometry. Standardized fecal peritonitis was induced, except in controls. One peritonitis group was given dextran (Group PDEX) and another in addition dobutamine (Group PDOB) to keep Cl normal or supranormal, respectively. Results: After 4 h, a significant drop in pHiwas found in all peritonitis groups, most pronounced in untreated peritonitis (to 7.09 ± .02). Corresponding values in Group PDEXand Group PDOBwere 7.22 ± .03 and 7.22 ± .01, respectively, and in controls 7.30 ± .02. The Pco2-gap and the mucosal-arterial [H+] difference ([H+]-gap) increased significantly in untreated peritonitis but did not increase in groups given dextran and dextran + dobutamine.Conclusion: Maintaining Cl in peritonitis attenuated the reduction in pHiand prevented the increased Pco2- and [H+]-gap. It seems justified from these data to conclude that the somewhat reduced pHiin treated peritonitis groups did not reflect tissue ischemia.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Nitric oxide (NO) has been implicated in the pathophysiology of hemorrhagic shock. We investigated the influence of l-arginine (the precursor of NO synthesis),NG-nitro-l-arginine methyl ester (l-NAME) and aminoguanidine (AG) (inhibitors of NO synthase, with selectivity toward the constitutive and inducible isoforms, respectively) on the survival rate in a rat model of hemorrhagic shock. Anesthetized, male Sprague-Dawley rats (300–350 g) were subjected to hemorrhagic shock for 30 min followed by intravenous injection (1 mL/kg) with normal saline, l-arginine (30 mg/kg), l-NAME (10 mg/kg), l-NAME+l-arginine, AG (1,10,100 mg/kg) or AG (100 mg/kg)+l-arginine (n = 5 per group). Hemorrhagic shocked rats treated with saline died within 90 min. In contrast, l-NAME increased the survival time to >72 h in shocked rats. AG (1, 10, and 100 mg/kg) increased the survival time of shocked animals to 150 min, 230 min, and >72 h, respectively. Shocked rats treated with l-arginine died within 80 min, and those that received l-NAME+l-arginine and AG+l-arginine died within 120 min and 110 min, respectively. l-NAME and AG (dose dependently) reduced macroscopic and microscopic injuries, nitrate/nitrite, PGE2and creatinine production, and inhibited GOT activity in shocked animals. l-arginine reversed the beneficial effects of AG and l-NAME, suggesting the involvement of NO in the pathophysiology of hemorrhagic shock.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Altered endotoxin (LPS) signal transduction in macrophages (Mø) may mediate development of organ dysfunction in sepsis. C3H/HeJ Mø have a specific genetic defect that renders them “tolerant” toin vitroLPS activation. LPS tolerance can be induced in normal C3H/HeN Mø followingin vitroLPS pretreatment. In these experiments,in vitroLPS-stimulated activation of Mø mitogen-activated protein (MAP) kinases were compared in C3H/HeJ and C3H/HeN mice. C3H/HeJ and C3H/HeN Moslash; were cultured ± 10 ng/mL LPS pretreatment for 24 h, then stimulated with 0–1,000 ng/mL LPS for 6 h. Western blots were performed on lysates with monoclonal antibody to active ERK1,2 (p42/44), stress-activated protein kinase (SAPK, p54/46), and p38 kinase. Supernatant TNF or IL-1 was determined by bioassay. High dose LPS stimulation activated ERK, SAPK, and p38 kinases in both C3H/HeN and C3H/HeJ Mø. ERK activation, p46 SAPK, and p38 activation were inhibited in C3H/HeN Mø after LPS pretreatment, whereas they were unchanged or increased in HeJ Mø. TNF secretion was significantly decreased in C3H/HeN Mø following LPS pretreatment, but absent in C3H/HeJ Mø at all times. Mø from normal C3H/HeN mice rendered endotoxin tolerant byin vitro, low dose LPS pretreatment have specific signal transduction defects that are not present in genetically LPS hyporesponsive C3H/HeJ mice.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Endotoxin-induced vascular hyporesponsiveness could potentially involve alterations of vascular smooth muscle (VSM) myoplasmic free calcium (Cam) mobilization mechanisms. Contractile function and Cam(fura-2 microfluorometry) regulation were evaluatedin vitrousing coronary (COR) and mesenteric (MES) artery preparations (100–250 μm inner diameter) isolated from guinea pigs 16 h after intraperitoneal (i.p.) injection of either saline (control; CON) orEscherichia coliendotoxin lipopolysaccharide (LPS; 4 mg/kg). Concentration-response relationships to K+(5–100 mM) were significantly enhanced in both COR and MES arteries isolated from LPS-treated animals. In contrast, contractile responses to prostaglandin F2α(PGF2α; 1–100 μM) were markedly impaired in COR and MES arteries from LPS-treated animals, while endothelin-1 (ET; 1–100 nM)-mediated contractile responses of these arteries were enhanced at the maximal dose (100 nM). In COR arteries, PGF2α(1–100 μM) and ET (1–100 nM) produced biphasic increases in Camin both CON and LPS groups. No significant differences were observed in either the initial transient peak or secondary sustained Camresponses between groups, suggesting a lack of effect of LPS upon intracellular Ca2+release or Ca2+influx mechanisms in COR arteries. Exposure of MES arteries to PGF2αand ET produced concentration-dependent increases in Camin both groups. However, Camresponses of MES arteries lacked initial peak responses, suggesting potential differences in Cammobilization between COR and MES arteries. Camresponses to K+(80 mM) and PGF2α(1–100 μM) were similar in MES arteries from both groups; however, ET-mediated increases in Camwere significantly blunted in LPS compared with CON MES arteries. Thus, endotoxemia produced differential effects upon depolarization (K+) and receptor (PGF2α, ET)-mediated contractile responses in both COR and MES arteries. Reductions in VSM Cammobilization appear unlikely as a mechanism for LPS-induced impairment of contractile function of COR and MES arteries; other mechanisms (i.e., decreased Ca2+sensitivity of contractile proteins) may be involved in. effects of LPS upon VSM function of COR and MES arteries.

ISSN:1073-2322    

出版商:OVID    

年代:1999

数据来源: OVID