Biomedical subjects

Ping Wang

Publications and source records attributed to Ping Wang.

At least 325 records · Page 18Linked to original sources

PubMed2002

Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold.

Amino Acid Motifs↗

PubMed2002

Amino-terminal fragment of urokinase-type plasminogen activator inhibits its plasminogen activation.

The amino terminal fragment (ATF, Ser(1)-Lys(135)) of urokinase-type plasminogen activator (uPA) containing an epidermal growth factor-like (EGF) and kringle domain is critically involved in some important functions of uPA, such as receptor binding and chemotactic activity. In this report, the effect of ATF on single-chain uPA (sc-uPA) induced plasminogen activation was investigated. It was shown that sc-uPA-induced activation of Glu-plasminogen or Lys-plasminogen was significantly inhibited in the presence of ATF. In addition, sc-uPA activation to two-chain uPA (tc-uPA) by Lys-plasmin and plasminogen activation to plasmin by tc-uPA were both found to be inhibited by ATF. The inhibition of these activations was significantly attenuated but not diminished when ATF was pretreated with immobilized carboxypeptidase B (CPB), indicating that the C-terminal Lys(135) as well as internal Lys/Arg residue binding was involved in the mechanism. Kinetic analysis showed that sc-uPA activation by Lys-plasmin competitively inhibited by ATF and CPB pretreated ATF (CPB-ATF) with an inhibitory constant (K(i)) of 3.8+/-0.31 and 12.4 +/- 1.8 microM, respectively. In contrast to sc-uPA-induced Glu- or Lys-plasminogen activation, sc-uPA-induced mini-plasminogen activation, sc-uPA activation by mini-plasmin and mini-plasminogen activation by tc-uPA were not affected by ATF. These findings suggested that the inhibitory effects of ATF on sc-uPA activation by Lys-plasmin and Glu- or Lys-plasminogen activation by tc-uPA were related to the binding of ATF (by its C-terminal Lys(135) and internal Lys/Arg residue) with the kringle 1-4 of plasmin and plasminogen, respectively.

Amino Acid Substitution↗

PubMed2002

Association of tapasin and COPI provides a mechanism for the retrograde transport of major histocompatibility complex (MHC) class I molecules from the Golgi complex to the endoplasmic reticulum.

Tapasin is a subunit of the transporter associated with antigen processing (TAP). It associates with the major histocompatibility complex (MHC) class I. We show that tapasin interacts with beta- and gamma-subunits of COPI coatomer. COPI retrieves membrane proteins from the Golgi network back to the endoplasmic reticulum (ER). The COPI subunit-associated tapasin also interacts with MHC class I molecules suggesting that tapasin acts as the cargo receptor for packing MHC class I molecules as cargo proteins into COPI-coated vesicles. In tapasin mutant cells, neither TAP nor MHC class I are detected in association with the COPI coatomer. Interestingly, tapasin-associated MHC class I molecules are antigenic peptide-receptive and detected in both the ER and the Golgi. Our data suggest that tapasin is required for the COPI vesicle-mediated retrograde transport of immature MHC class I molecules from the Golgi network to the ER.

Antiporters↗

PubMed2002

C2A activates a cryptic Ca(2+)-triggered membrane penetration activity within the C2B domain of synaptotagmin I.

Synaptotagmin (syt) I, an integral membrane protein localized to secretory vesicles, is a putative Ca(2+) sensor for exocytosis. Its N terminus spans the membrane once, and its cytoplasmic domain contains two conserved C2 domains, designated C2A and C2B. The isolated C2A domain penetrates membranes in response to Ca(2+); isolated C2B does not. Here, we have addressed the function of each C2 domain, but in the context of the intact cytoplasmic domain (C2A-C2B), by using fluorescent reporters placed in the Ca(2+)-binding loops of either C2A or C2B. Surprisingly, these reporters revealed that, analogous to C2A, a Ca(2+)-binding loop in C2B directly penetrates into lipid bilayers. Penetration of each C2 domain was very rapid (k(on) approximately 10(10) M(-1) x s(-1)) and resulted in high affinity C2A-C2B-liposome complexes (K(d) approximately 13-14 nM). C2B-bilayer penetration strictly depended on the presence, but not the membrane binding activity, of an adjacent C2A domain, severing C2A from C2B after protein synthesis abolished the ability of C2B to dip into bilayers in response to Ca(2+). The activation of C2B by C2A was also displayed by the C2 domains of syt III but not the C2 domains of syt IV. A number of proteins contain more than one C2 domain; the findings reported here suggest these domains may harbor cryptic activities that are not detected when they are studied in isolation.

Amino Acid Sequence↗

PubMed2002

Estradiol administration improves splanchnic perfusion following trauma-hemorrhage and sepsis.

HYPOTHESIS: The female sex steroid 17beta-estradiol improves immune functions following trauma-hemorrhage in rodent models. Therefore, we hypothesized that 17beta-estradiol administration following trauma-hemorrhage would also improve cardiac output, splanchnic perfusion, and oxygen utilization, even after the induction of subsequent sepsis. SETTING: A university laboratory. INTERVENTION: Male rats underwent midline laparotomy (ie, soft tissue injury). They were bled to a mean arterial pressure of 35 to 40 mm Hg for 90 minutes and resuscitated over 60 minutes with lactated Ringer solution. At the beginning of resuscitation, 17beta-estradiol (l mg/kg) or a vehicle was administered. At 20 hours after resuscitation, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). MAIN OUTCOME MEASURES: At 5 hours after CLP, cardiac performance (via a left ventricular catheter), cardiac output, and organ blood flow were determined using strontium 85 microspheres. Blood samples were collected from the femoral artery and jugular, portal, and renal veins to determine systemic and regional oxygen delivery and consumption. Moreover, circulating levels of 17beta-estradiol, its adrenal precursor dehydroepiandrosterone (DHEA), and corticosterone were assessed by enzyme-linked immunosorbent assay. RESULTS: Hemorrhage and subsequent sepsis significantly depressed cardiac performance, cardiac output, organ perfusion, and oxygen consumption. Estrogen did not restore cardiac output or systemic oxygen consumption; nonetheless, it restored the depressed intestinal perfusion. Rats treated with estrogen had significantly elevated levels of plasma 17beta-estradiol, but the levels of DHEA or corticosterone were not affected. CONCLUSIONS: The increase in gut perfusion could represent a potential mechanism for the salutary effects of 17beta-estradiol following trauma-hemorrhage. Because 17beta-estradiol improves systemic and intestinal perfusion after trauma-hemorrhage and induction of subsequent sepsis, this agent appears to be a promising adjunct for the treatment of trauma victims.

Animals↗

PubMed2002

Flutamide induces relaxation in large and small blood vessels.

HYPOTHESIS: Flutamide, a testosterone receptor antagonist, produces various beneficial effects in male rats following hemorrhagic shock, possibly as a result of a direct vasodilating effect on large and small vessels in the rat. DESIGN, INTERVENTIONS, AND MAIN OUTCOME MEASURES: The aorta and the small intestine were isolated from normal male and female Sprague-Dawley rats. Isolated aortic rings were placed in the organ bath and constricted by 2 x 10(-7)M norepinephrine bitartrate (Sigma, St Louis, Mo). Flutamide-induced and testosterone-induced vascular relaxation dose-response curves were then determined. The dose-response curves of flutamide were also determined in the small blood vessels of the isolated small intestine under conditions of constant flow following preconstriction induced by 5 x 10(-6 )M norepinephrine bitartrate. The effects of prior incubation with testosterone (8 x 10(-5)M) and sex differences on flutamide-induced vascular relaxation were also examined in aortic rings and in the small intestine. Moreover, flutamide-induced relaxation in endothelium-denuded aortic rings and in aortic rings from animals subjected to trauma and hemorrhagic shock was examined. RESULTS: Flutamide induced significant relaxation in aortic rings and small intestinal blood vessels in healthy males. The flutamide-induced relaxation in vessels from normal males was partially attenuated by prior incubation with the male sex steroid testosterone, and was significantly lower in females. Flutamide-induced vascular relaxation in the aorta was partially attenuated by endothelium removal, but it was not significantly affected by trauma and hemorrhagic shock in male rats. CONCLUSIONS: Flutamide has a direct vasodilating effect on large and small vessels in rats, which involves sex-dependent mechanisms. Thus, the beneficial effects of flutamide on cardiovascular responses in males following trauma and hemorrhagic shock may be due to the direct vascular relaxation induced by this agent.

Androgen Antagonists↗

PubMed2002

Prevention of fumonisin B1-induced neural tube defects by folic acid.

BACKGROUND: The mycotoxin fumonisin B1 (FB1) inhibits sphingolipid synthesis, blocks folate transport, and has been associated with increased incidences of cancer and neural tube defects. Results from reproductive studies in animal models in vivo and in vitro have demonstrated toxicity in some cases, but no specific terata after fumonisin exposure. No information is available about folic acid's potential to protect against this toxicity. METHODS: Neurulating mouse embryos were exposed to fumonisin or folinic acid in whole embryo culture and assessed for effects on growth and development. RESULTS: Fumonisin exposure inhibited sphingolipid synthesis, reduced growth, and caused cranial neural tube defects in a dose dependent manner. Supplemental folinic acid ameliorated the effects on growth and development, but not inhibition of sphingolipid synthesis. CONCLUSION: Fumonisin has the potential to inhibit embryonic sphingolipid synthesis and to produce embryotoxicity and neural tube defects. Folic acid can reverse some of these effects, supporting results showing that fumonisin disrupts folate receptor function.

Animals↗

PubMed2002

A modified surgical technique for the emergent treatment of giant ulcers concomitant with hemorrhage in the posterior wall of the duodenal bulb.

BACKGROUND: One of the most challenging and lethal complications of the giant duodenal ulcer is massive bleeding, but management of the bleeding ulcer and the difficult duodenal stump remains controversial. METHODS: We developed a modified surgical technique involving a partial gastrectomy, duodenostomy, and a duodenostomy tube placed through the duodenal stump enveloped around an omental patch. The surface of the ulcer was closed with a fibrin sealant. RESULTS: Between January 1994 and December 1999, 12 patients underwent this surgical technique. All of the patients had massive giant bleeding ulcers in the posterior wall of the duodenal bulb. No deaths occurred. The duodenostomy tubes were removed between postoperative days 12 and 22. The other complications after operation were few. The mean hospital stay was 18 days. No rebleeding or duodenal stump leaks were found within the 12- to 60-month follow-up. CONCLUSIONS: This technique completely eradicated the ulcer as seen at the 12- to 60-month postoperative follow-up examination.

Adult↗

PubMed2002

Metformin therapy throughout pregnancy reduces the development of gestational diabetes in women with polycystic ovary syndrome.

OBJECTIVE: To assess whether metformin safely reduced development of gestational diabetes in women with the polycystic ovary syndrome (PCOS). DESIGN: Prospective and retrospective study. SETTING: Outpatient clinical research center. PATIENT(S): The prospective study included 33 nondiabetic women with PCOS who conceived while taking metformin and had live births; of these, 28 were taking metformin through delivery. The retrospective study included 39 nondiabetic women with PCOS who had live birth pregnancies without metformin therapy. INTERVENTION(S): Metformin, 2.55 g/d, throughout pregnancy in women with PCOS. MAIN OUTCOME MEASURE(S): Development of gestational diabetes in women with PCOS. RESULT(S): Before metformin therapy, after covariance adjustment for age, the two cohorts did not differ in height, weight, basal metabolic index, insulin, insulin resistance, or insulin secretion. Both cohorts had high fasting insulin, were insulin resistant, and had high insulin secretion. Among the 33 women who received metformin, gestational diabetes developed in 1 of 33 (3%) pregnancies versus 8 of 12 (67%) of their previous pregnancies without metformin. Among the 39 women who did not take metformin, gestational diabetes developed in 14 of 60 (23%) pregnancies. When all live births were combined, gestational diabetes occurred in 22 of 72 pregnancies (31%) in women who did not take metformin versus 1 of 33 pregnancies (3%) in those who took metformin. With gestational diabetes as the response variable and age at delivery and treatment group (metformin or no metformin) as explanatory variables, the odds ratio for gestational diabetes in women with metformin versus without metformin was 0.093 (95% CI: 0.011 to 0.795). With gestational diabetes in 93 pregnancies as the response variable and age at delivery and treatment group (metformin no metformin) as explanatory variables, the odds ratio of gestational diabetes in pregnancies in women taking metformin versus without metformin was 0.115 (95% CI: 0.014 to 0.938). CONCLUSION(S): In PCOS, use of metformin is associated with a 10-fold reduction in gestational diabetes (31% to 3%). It also reduces insulin resistance and insulin secretion, thus decreasing the secretory demands imposed on pancreatic beta-cells by insulin resistance and pregnancy.

Adult↗

PubMed2002

Transcriptional analysis of genes encoding enzymes of the folate pathway in the human malaria parasite Plasmodium falciparum.

Folate metabolism in Plasmodium falciparum is essential for cell growth and replication, and the target of important antimalarial agents. The pathway comprises a series of enzymes that convert GTP to derivatives of tetrahydrofolate, which are cofactors in one-carbon transfer reactions. We investigated the expression of five of the genes encoding these enzymes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using a threshold detection technique. We followed changes in mRNA levels as parasites progress through the erythrocytic cell cycle and examined this process in two cloned lines of diverse origins, as well as under stress conditions, induced by either removal of important metabolites or challenge by folate enzyme inhibitors. Although conventionally regarded as performing housekeeping functions, these genes show disparate levels of and changes in expression through the cell cycle, but respond quite uniformly to folate pathway-specific stress factors, with no evidence of feedback at the transcriptional level. Overall, the two genes involved in the thymidylate cycle (encoding dihy-drofolate reductase-thymidylate synthase, dhfr-ts, and serine hydroxymethyltransferase, shmt) gave the most abundant transcripts. However, only the latter showed major variation across the cell cycle, with a peak around the time of onset of DNA replication, possibly indicative of a regulatory function.

Animals↗

PubMed2002

Estrogen replacement therapy, thrombophilia, and atherothrombosis.

In a consecutive case series, cross-sectional study of 401 women referred for hyperlipidemia therapy, (110 [27%] on estrogen replacement therapy [ERT]), we assessed whether ERT-mediated thrombophilia and heritable thrombophilia (20210 G-->A prothrombin gene [PTG], Factor V Leiden gene mutation [FV]) interacted as risk factors for atherothrombotic cardiovascular disease (ATCVD). Thirty-eight percent of women (152/401) had > or = 1 ATCVD event, 57 (14%) had > or = 2 ATCVD events. Fifteen women (3.7%) were PTG heterozygotes, 24 (6.0%) were FV heterozygotes, (there was 1 double heterozygote [0.25%]); 363 (91%) were wild-type normal for both genes. Of the 152 women with > or = 1 ATCVD event, 21 (14%) had > or = 1 thrombophilic gene mutation, versus 17/249 (7%) without events (X(2) = 5.4, P =.02). In women on ERT and with both genes wild-type normal, 23 of 96 (24%) had > or = 1 ATCVD event versus 8 of 14 (57%) on ERT and with > or = 1 thrombophilic mutation, X(2) = 6.6, P =.01. By stepwise logistic regression, in 401 women (152 with > or = 1 ATCVD event, 249 no events), positive explanatory variables for ATCVD included FV and/or PTG (risk odds ratio, 2.59, 95% confidence interval [CI] 1.26 to 5.36, P =.01) and a PTG*ERT interaction term (risk odds ratio, 2.27, 95% CI 1.36 to 3.79, P =.0017). After deleting 23 FV heterozygotes and 14 PTG heterozygotes and 1 double heterozygote from the 401 women, ERT was protective against ATCVD events, with a risk odds ratio of 0.50 and 95% CI of 0.29 to 0.87 P =.014. PTG and FV may increase risk for ATCVD, particularly in the presence of ERT, whereas ERT may be protective against ATCVD when PTG and FV are absent.

Cardiovascular Diseases↗

PubMed2002

Pregnancy outcomes among women with polycystic ovary syndrome treated with metformin.

BACKGROUND: We sought to determine whether metformin, which had facilitated conception in 72 oligoamenorrhoeic women with polycystic ovary syndrome (PCOS), would safely reduce the rate of first trimester spontaneous abortion (SAB) and increase the number of live births without teratogenicity. METHODS: Seventy-two oligoamenorrheic women with PCOS conceived on metformin (2.55 g/day). They were prospectively assessed in an outpatient clinical research centre. Outcome measures included number of first trimester SAB, live births, normal ongoing pregnancies >or=13 weeks, gestational diabetes (GD), congenital defects (CD), birthweight and height, as well as weight, height, and motor and social development during the first 6 months of life. RESULTS: Of the 84 fetuses, to date there have been 63 normal live births without CD (75%), 14 first trimester SAB (17%), and seven ongoing pregnancies >or=13 weeks with normal sonograms without CD (8%). Previously, without metformin, 40 of the 72 women had 100 pregnancies (100 fetuses) with 34 (34%) live births and 62 (62%) first trimester SAB. In current pregnancies on metformin in these 40 women (46 pregnancies, 47 fetuses), there have been 33 live births (70%), two pregnancies ongoing >/=13 weeks (4%), and 12 SAB (26%) (P < 0.0001). There was no maternal lactic acidosis, and no maternal or neonatal hypoglycaemia. Fasting entry serum insulin was a significant explanatory variable for total (previous and current) first trimester SAB, odds ratio 1.32 (for each 5 micro U/ml rise in insulin), 95% CI 1.09-1.60 (P = 0.005). On metformin, GD developed in 4% of pregnancies versus 26% of previous pregnancies without metformin, P = 0.025. There have been no major CD in the 63 live births or CD by sonography in the seven fetuses <or=13 weeks. In the 63 live births, neither weight nor height differed from the normal neonatal population. At 6 month follow-up, height was greater (P = 0.008) and weight did not differ from the normal paediatric population; motor and social development were normal. CONCLUSIONS: Metformin therapy during pregnancy in women with PCOS was safely associated with reduction in SAB and in GD, was not teratogenic, and did not adversely affect birthweight or height, or height, weight, and motor and social development at 3 and 6 months of life.

Abortion, Spontaneous↗

PubMed2002

Novel approach to prevent the transition from the hyperdynamic phase to the hypodynamic phase of sepsis: role of adrenomedullin and adrenomedullin binding protein-1.

OBJECTIVE: To determine whether the combined administration of adrenomedullin and adrenomedullin binding protein-1 (AM/AMBP-1) has any modulatory effects on the cardiovascular response during the progression of sepsis. SUMMARY BACKGROUND DATA: Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Recent studies have shown that AM, a newly reported potent vasodilator peptide, plays a major role in initiating the hyperdynamic response. Moreover, the reduced vascular responsiveness to AM appears to be responsible for the transition from the hyperdynamic phase to the hypodynamic phase of sepsis. Although the novel AMBP-1 augments AM-mediated action in vitro, it remains unknown whether AM/AMBP-1 maintains vascular responsiveness to AM at the late stage of sepsis. METHODS: Sepsis was induced by cecal ligation and puncture (CLP) in adult male rats. Human AMBP-1 (40 microg/kg body weight) was infused intravenously at the beginning of sepsis for 20 minutes and synthetic AM (12 microg/kg body weight) was continuously administrated for the entire study period using an Alzert micro-osmotic pump, beginning 3 hours before the induction of sepsis. At 20 hours after the onset of sepsis (i.e., the late stage), cardiac output, systemic oxygen delivery, stroke volume, total peripheral resistance, and organ blood flow in the liver, gut, kidneys, and heart were determined using radioactive microspheres. Plasma levels of transaminases (ALT, AST) and lactate were also measured. Additional studies were conducted to determine whether administration of AM alone or AMBP-1 alone alters the cardiovascular response at 20 hours after CLP. In additional rats, the necrotic cecum was excised at 20 hours after CLP following AM/AMBP-1 treatment, the peritoneal cavity irrigated with saline, and the midline incision closed in layers. Survival was then examined for a period of 10 days thereafter. RESULTS: Administration of AM/AMBP-1 prevented the decrease in the measured systemic and regional hemodynamic parameters at 20 hours after the onset of sepsis. Moreover, AM/AMBP-1 significantly attenuated hepatic damage and the elevation of plasma lactate, and prevented hemoconcentration. Treatment with AM/AMBP-1 reduced the overall 10-day mortality rate from 57% to 7%. Neither AM nor AMBP-1 alone was sufficient to maintain cardiovascular stability at 20 hours after CLP. CONCLUSIONS: Since AM/AMBP-1 delays or even prevents the transition from the hyperdynamic phase to the hypodynamic phase of sepsis, attenuates tissue injury, and decreases sepsis-induced morality, these agents should provide a novel approach for maintaining cardiovascular stability and preventing cell and organ damage during the progression of polymicrobial sepsis.

Adrenomedullin↗

PubMed2002

Administration of human inter-alpha-inhibitors maintains hemodynamic stability and improves survival during sepsis.

OBJECTIVES: The major forms of human inter-alpha-inhibitor proteins circulating in the plasma are inter-alpha-inhibitor (IalphaI, containing one light peptide chain called bikunin and two heavy chains) and pre-alpha-inhibitor (PalphaI, containing one light and one heavy chain). Although it has been reported that a decrease in IalphaI/PalphaI is correlated with an increased mortality rate in septic patients, it remains unknown whether administration of IalphaI/PalphaI early after the onset of sepsis has any beneficial effects on the cardiovascular response and outcome of the septic animal. The aim of this study, therefore, was to determine whether IalphaI and PalphaI have any salutary effects on the depressed cardiovascular function, liver damage, and mortality rate after polymicrobial sepsis. DESIGN: Prospective, controlled, randomized animal study. SETTING: A university research laboratory. SUBJECTS: Male adult rats were subjected to polymicrobial sepsis by cecal ligation and puncture or sham operation followed by the administration of normal saline (i.e., resuscitation). MEASUREMENTS AND MAIN RESULTS: At 1 hr after cecal ligation and puncture, human IalphaI/PalphaI at a dose of 30 mg/kg body weight or vehicle (normal saline, 1 mL/rat) were infused intravenously over a period of 30 mins. At 20 hrs after cecal ligation and puncture (i.e., the late, hypodynamic stage of sepsis), cardiac output was measured by using a dye dilution technique, and blood samples were collected for assessing oxygen content. Oxygen delivery, consumption, and extraction ratio were determined. Plasma concentrations of liver enzymes alanine aminotransferase and aspartate aminotransferase as well as lactate and tumor necrosis factor-alpha also were measured. In additional animals, the necrotic cecum was excised at 20 hrs after cecal ligation and puncture with or without IalphaI/PalphaI treatment, and survival was monitored for 10 days thereafter. The results indicate that administration of human IalphaI/PalphaI early after the onset of sepsis maintained cardiac output and systemic oxygen delivery, whereas it increased oxygen consumption and extraction at 20 hrs after cecal ligation and puncture. The elevated concentrations of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-alpha, and lactate were attenuated by IalphaI/PalphaI treatment. In addition, administration of human IalphaI/PalphaI improved the survival rate from 30% to 89% in septic animals at day 10 after cecal ligation and puncture and cecal excision. CONCLUSION: Human IalphaI/PalphaI appears to be a useful agent for maintaining hemodynamic stability and improving survival during the progression of polymicrobial sepsis.

Alpha-Globulins↗

PubMed2002

Mechanisms of the beneficial effect of adrenomedullin and adrenomedullin-binding protein-1 in sepsis: down-regulation of proinflammatory cytokines.

OBJECTIVE: Our recent study indicates that administration of adrenomedullin (AM) in combination with AM-binding protein-1 (AMBP-1) before sepsis (i.e., pretreatment) maintains cardiovascular stability and reduces the mortality rate. The aim of the present study was to determine whether administration of AM/AMBP-1 after the onset of sepsis (posttreatment) has any salutary effects on the septic host, and if so, whether AM/AMBP-1 down-regulates proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6. DESIGN: Prospective, controlled, randomized animal study. SETTING: A university research laboratory. SUBJECTS: Male adult Sprague-Dawley rats. INTERVENTIONS: Rats were subjected either to polymicrobial sepsis by cecal ligation and puncture or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). MEASUREMENTS AND MAIN RESULTS: At 5 hrs after cecal ligation and puncture, AM (12 microg/kg body weight) and AMBP-1 (40 microg/kg body weight) were administered intravenously over 1 hr. At 20 hrs after cecal ligation and puncture (i.e., the late, hypodynamic stage of sepsis), cardiac output, stroke volume, total peripheral resistance, systemic oxygen delivery, and organ blood flow were determined by radioactive microspheres, and circulating concentrations of proinflammatory cytokines were measured using enzyme-linked immunosorbent assay kits. Moreover, plasma concentrations of transaminases and lactate were measured. The results indicated that administration of AM/AMBP-1 at 5 hrs after cecal ligation and puncture prevented the decrease in measured systemic and regional hemodynamic variables and reduced plasma concentrations of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 at 20 hrs after the onset of sepsis. Moreover, administration of AM/AMBP-1 attenuated hepatic damage and the increase in plasma lactate and prevented hemoconcentration. CONCLUSION: Administration of AM/AMBP-1 may provide a novel approach to the treatment of sepsis. Moreover, because AM/AMBP-1 significantly reduced circulating concentrations of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6, down-regulation of those proinflammatory cytokines by AM/AMBP-1 appears to play an important role for the beneficial effects of these agents in polymicrobial sepsis.

Adrenomedullin↗

PubMed2002

Differential alterations in cardiovascular responses during the progression of polymicrobial sepsis in the mouse.

Although the mouse has been extensively used to study immune consequences of sepsis and other genetic anomalies, the changes in various cardiovascular parameters such as cardiac output, organ perfusion, as well as oxygen utilization have not been characterized in this species during sepsis. To determine this, polymicrobial sepsis was induced in male adult C3H/NeN mice by cecal ligation and puncture (CLP, two punctures with a 22-gauge needle). The animals were then resuscitated with normal saline subcutaneously. At 5 or 24 h after CLP (time points previously shown to be within the hyperdynamic and hypodynamic stage of sepsis, respectively, in the rat), cardiac output and blood flow in major organs were determined using a well-established radioactive microsphere method, and stroke volume and total peripheral resistance were calculated. In addition, oxygen delivery and consumption were determined. The results indicate that cardiac output, stroke volume, oxygen delivery and consumption, and blood flow in the liver, small intestine, spleen, and kidneys increased significantly at 5 h after CLP. This was associated with significantly decreased total peripheral resistance. In contrast, total peripheral resistance increased and the other above-mentioned parameters, as well as mean arterial pressure, decreased significantly at 24 h after the onset of sepsis. Thus, the cardiovascular response to polymicrobial sepsis in the mouse is characterized by an early hyperdynamic phase (i.e., 5 h after CLP) followed by a late hypodynamic phase (24 h post-CLP). Since the radioactive microsphere technique provides a reliable method for determining various hemodynamic parameters in the mouse, the correlation between the cardiovascular response and immune or potentially genetic alterations can be examined in this species during the progression of sepsis.

Animals↗

PubMed2002

Endotoxic shock alters the pharmacokinetics of lidocaine and monoethylglycinexylidide.

Significant hepatic dysfunction occurs following endotoxin administration. Although the metabolism of lidocaine to one of the primary metabolites of lidocaine, monoethylglycinexylidide (MEGX), has been used as a marker of hepatic function under various conditions, it remains unknown whether these compounds can be used in vivo to evaluate hepatic function in a rat model of endotoxic shock. To study this, cytochrome P450-3A4 (CYP3A4) was determined after harvesting hepatic microsomes, hepatic blood flow was determined using radioactive microspheres, and the pharmacokinetics of lidocaine and MEGX were evaluated. Adult male Sprague-Dawley rats were divided into endotoxin (45 mg/kg, intraperitoneally; n = 28) or control (n = 32) groups. The CYP3A4 was significantly reduced after endotoxic shock. Carboxylesterase (hydrolase S) content, which was used as a control for microsomal protein, was not significantly different between groups. Total hepatic blood flow was significantly decreased (36.2 +/- 8.4 mL/min/100 g tissue vs. 120.4 +/- 10.6 mL/min/100 g tissue), which was due to the decreased portal blood flow. For the lidocaine and MEGX experiment, lidocaine (2 mg/kg) was administered followed by serial blood samples collected up to 2 h for determination of serum lidocaine and MEGX concentrations. Mean arterial pressure (MAP) was recorded throughout the experiment. The MAP was significantly lower in the endotoxin treated rats vs. control 7.5 to 8 h following endotoxin administration. Serum concentrations of lidocaine were higher in endotoxic shock versus control animals at 2 h following lidocaine administration (1.5 +/- 0.13 mg/L vs. 0.11 +/- 0.03 mg/L). Similarly, MEGX concentrations were significantly higher in endotoxic shock versus control animals (0.55 +/- 0.04 mg/L vs. 0.16 +/- 0.02, respectively) under such conditions. These data demonstrate that the elimination of lidocaine and MEGX is impaired during endotoxic shock. The elevated lidocaine and MEGX concentrations are likely to be the result of primarily reduced hepatic blood flow and secondarily due to impaired CYP450, one of which was CYP3A4. The reduced elimination of MEGX concentrations is not due to decreased hepatic metabolism of the compound via carboxylesterase. The ratio of MEGX to lidocaine concentrations, which decreased significantly following endotoxic shock, appears to be a useful measure of hepatic function during endotoxic shock where profound reductions of hepatic blood flow are observed in addition to significant reductions in CYP450. The use of only MEGX concentrations in this endotoxic shock model is not useful in evaluating liver function.

Animals↗

PubMed2002

A PCR-based strategy to generate integrative targeting alleles with large regions of homology.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

Alleles↗