Recombinant bactericidal/permeability-increasing protein (rBPI21) in combination with sulfadiazine is active against Toxoplasma gondii. (33/7831)

The activity of recombinant bactericidal/permeability-increasing protein (rBPI21), alone or in combination with sulfadiazine, on the intracellular replication of Toxoplasma gondii was assessed in vitro and in mice with acute toxoplasmosis. rBPI21 markedly inhibited the intracellular growth of T. gondii in human foreskin fibroblasts (HFFs). Following 72 h of exposure, the 50% inhibitory concentration of rBPI21 for T. gondii was 2.6 micrograms/ml, whereas only slight cytotoxicity for HFF cells was observed at the concentrations tested. Subsequent mathematical analyses revealed that the combination of rBPI21 with sulfadiazine yielded slight to moderate synergistic effects against T. gondii in vitro. Infection of mice orally with C56 cysts or intraperitoneally (i.p.) with RH tachyzoites resulted in 100% mortality, whereas prolongation of the time to death or significant survival (P = 0.002) was noted for those animals treated with 5 to 20 mg of rBPI21 per kg of body weight per day. Treatment with rBPI21 in combination with sulfadiazine resulted in significant (P = 0.0001) survival of mice infected i.p. with tachyzoites but not of mice infected orally with T. gondii cysts. These results indicate that rBPI21 is active in vitro and in vivo against T. gondii and that its activity is significantly enhanced when it is used in combination with sulfadiazine. To our knowledge, this is the first report of the activity of rBPI21 against a protozoan parasite.  (+info)

SH2-B is required for nerve growth factor-induced neuronal differentiation. (34/7831)

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced neuronal differentiation.  (+info)

Modeling the effects of proteins on pH in plasma. (35/7831)

Stewart's model of plasma acid-base balance (Can. J. Physiol. Pharmacol. 61: 1444-1461, 1983) has three weaknesses in the treatment of weak acids: 1) the combination of all weak acids into one entity, 2) inappropriate chemistry for the protein combination with H+, and 3) undocumented values for the dissociation parameters. The present study models serum albumin acid-base properties by fixed negative charges and the association of H+ with the imidazole side chain of histidine. This model has three parameters: 1) the net negative fixed charge (21 eq/mol), 2) the number of histidine residues (16/mol), and 3) the association constant for the imidazole side chain (1.77 x 10(-7) eq/l), all determined from published values. The model was compared with that of Figge, Mydosh, and Fencl (J. Lab. Clin. Med. 120: 713-719, 1992) and with the pH data of Figge, Rossing, and Fencl (J. Lab. Clin. Med. 117: 453-467, 1991). The predictions of pH were excellent, comparable to those found by Figge, Mydosh, and Fencl. The model has the advantages that its structure and parameter values are supported by the literature and that the acid-base effects of factors modifying protein can be investigated.  (+info)

Inflammatory response after inhalation of bacterial endotoxin assessed by the induced sputum technique. (36/7831)

BACKGROUND: Organic dusts may cause inflammation in the airways. This study was performed to assess the usefulness of the induced sputum technique for evaluating the presence of airways inflammation using inhaled endotoxin (lipopolysaccharide) as the inducer of inflammation. METHODS: To characterise the inflammatory response after inhalation of endotoxin, 21 healthy subjects inhaled 40 micrograms lipopolysaccharide and were examined before and 24 hours after exposure. Examinations consisted of a questionnaire for symptoms, spirometric testing, blood sampling, and collection of induced sputum using hypertonic saline. Eleven of the subjects inhaled hypertonic saline without endotoxin exposure as controls. Cell counts, eosinophilic cationic protein (ECP), and myeloperoxidase (MPO) were determined in blood and sputum. RESULTS: A significantly higher proportion of subjects reported respiratory and general symptoms after endotoxin inhalation. MPO and the number of neutrophils in the blood were higher and spirometric values were decreased after the lipopolysaccharide challenge. In the sputum MPO, ECP, and the numbers of neutrophils and lymphocytes were higher after the lipopolysaccharide challenge. No significant differences were found after the inhalation of hypertonic saline compared with before, except for a significantly lower number of lymphocytes in the sputum. CONCLUSIONS: The results support previous studies that inhaled endotoxin causes an inflammation at the exposure site itself, as well as general effects. Sampling of sputum seems to be a useful tool for assessing the presence of airways inflammation, and the inhalation of hypertonic saline used to induce sputum did not significantly interfere with the results found after inhalation of lipopolysaccharide.  (+info)

Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells. (37/7831)

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors caused a rapid (<3 min) translocation of the cytosolic fluorescence to ruffle-like membrane structures. This response was not observed in cells expressing GFP only and was completely inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004. Membrane-targeted PI 3-kinase also caused membrane localization of BtkPH-GFP that was slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain, which causes X-linked agammaglobulinemia, was introduced into the fluorescent construct, no translocation was observed after stimulation. In contrast, the E41K mutation, which confers transforming activity to native Btk, caused significant membrane localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type BtkPH-GFP, interfered with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of changes in this phospholipid in living cells.  (+info)

Betamethasone-mediated vascular dysfunction and changes in hematological profile in the ovine fetus. (38/7831)

Glucocorticoid administration to fetal sheep induces a sustained systemic blood pressure rise and an associated increase in femoral vascular resistance. We utilized a small vessel myograph to compare isometric vascular responses of small femoral arterial branches from fetal sheep infused intravenously with either betamethasone or vehicle in vivo from 128 days gestation. Changes in hematological parameters were also determined. Betamethasone was infused for 48 h to produce fetal plasma betamethasone concentrations similar to those observed in human fetuses after maternal treatment with betamethasone to accelerate fetal lung maturation. When compared with vessels removed from vehicle-infused fetuses, vessels obtained from betamethasone-treated fetuses exhibited 1) enhanced sensitivity to depolarizing potassium solutions; 2) no differences in response to the thromboxane mimetic U-46619 or norepinephrine; and 3) differential responses to vasodilators, enhanced sensitivity to ACh, but decreased response to bradykinin and forskolin. In addition, erythrocyte and leukocyte counts were increased in betamethasone-infused fetuses. These observations indicate that multiple mechanisms operate to increase fetal vascular resistance during antenatal betamethasone exposure.  (+info)

Lipoprotein(a) stimulates growth of human mesangial cells and induces activation of phospholipase C via pertussis toxin-sensitive G proteins. (39/7831)

BACKGROUND: Renal disease is commonly associated with hyperlipidemia and correlates with glomerular accumulation of atherogenic lipoproteins, for example, lipoprotein(a) [Lp(a)], and mesangial hypercellularity. Specific binding of Lp(a) to mesangial cells and induction of c-myc and c-fos expression has been demonstrated. Therefore, in this study, we investigated a possible growth stimulatory effect and mode of action of Lp(a) in human mesangial cells. METHODS: Lp(a) was purified from the regenerate fluid of a dextran sulfate column-based low-density lipoprotein apheresis system. Human mesangial cells were isolated by a sequential sieving technique from patients undergoing tumor nephrectomy. DNA synthesis was measured by [3H]-thymidine incorporation. The intracellular calcium concentration ([Ca2+]i) was determined by Fura 2-fluorescence, and inositol 1,4,5-trisphosphate (1,4,5-IP3) concentration was measured by a radioreceptor assay. RESULTS: The data show that Lp(a) bound to the cells with a Kd of 17.0 micrograms/ml and increased DNA synthesis and cell proliferation. Lp(a) caused a rapid increase in 1,4,5-IP3 and [Ca2+]i via a pertussis toxin-sensitive mechanism. The phospholipase C (PLC) inhibitor U73122 abolished Lp(a)-induced cell proliferation. In contrast, vasopressin-induced increase in 1,4,5-IP3 and [Ca2+]i was pertussis toxin insensitive. CONCLUSION: This study revealed that Lp(a) stimulates growth of human mesangial cells. Lp(a)-induced signaling involves binding to a receptor and stimulation of PLC via Gi proteins. Stimulation of PLC appears to be essential for the growth stimulatory effect of Lp(a). Whether these effects of Lp(a) contribute to the pathophysiology of renal disease needs to be determined.  (+info)

Tranexamic acid increases peritoneal ultrafiltration volume in patients on CAPD. (40/7831)

OBJECTIVE: The preservation of ultrafiltration (UF) capacity is crucial to maintaining long-term continuous ambulatory peritoneal dialysis (CAPD).The aim of the present study was to investigate whether the antiplasmin agent tranexamic acid (TNA) increases UF volume in CAPD patients. PATIENTS AND METHODS: Fifteen patients on CAPD, 5 with UF loss and 10 without UF loss, were recruited for the study. The effect of TNA was evaluated with respect to changes in UF volume, peritoneal permeability, peritoneal clearance, bradykinin (BK), and tissue plasminogen activator (tPA) concentration. SETTING: Dialysis unit of the Saiseikai Central Hospital. RESULTS: In patients with UF loss, 2 weeks of treatment with oral TNA produced a significant increase in UF volume in all subjects (5/5).TNA also produced a significant increase in peritoneal clearances of urea and creatinine (Cr). However, the peritoneal equilibration test (PET) revealed that TNA had no effect on dialysate/plasma (D/P) Cr, Kt/V, or the protein catabolic rate (PCR).TNA also had no effect on net glucose reabsorption. In contrast, significant decreases in BK and blood tPA concentrations in response to TNA treatment were noted. BK concentration in drainage fluid was also reduced. In the case of patients without UF loss,TNA produced an increase in UF volume in 70% (7/10). However, no differences were found in blood and drainage BK and tPA concentrations between theTNA treatment and nontreatment periods in these patients. A comparison of basal BK and tPA concentration showed that there were no differences in these parameters between patients with UF loss and those without loss of UF. Furthermore,TNA given intraperitoneally to a patient also produced a marked increase in UF volume. CONCLUSION: The present study suggests thatTNA enhances UF volume in patients both with and without UF loss. SinceTNA did not affect peritoneal permeability and glucose reabsorption, the mechanism by which TNA exerts an enhancing action on UF is largely unknown. We speculate that it may be associated with suppression of the BK and/or tPA system, at least in patients with UF loss.  (+info)