Localization of the binding site for transforming growth factor-beta in human alpha2-macroglobulin to a 20-kDa peptide that also contains the bait region

alpha2-Macroglobulin (alpha2M) functions as a major carrier of transforming growth factor-beta (TGF-beta) in vivo. The goal of this investigation was to characterize the TGF-beta-binding site in alpha2M. Human alpha2M, which was reduced and denatured to generate 180-kDa subunits, bound TGF-beta1, TGF-beta2, and NGF-beta in ligand blotting experiments. Cytokine binding was not detected with bovine serum albumin that had been reduced and alkylated, and only minimal binding was detected with purified murinoglobulin. To localize the TGF-beta-binding site in alpha2M, five cDNA fragments, collectively encoding amino acids 122-1302, were expressed as glutathione S-transferase (GST) fusion proteins. In ligand blotting experiments, TGF-beta2 bound only to the fusion protein (FP3) that includes amino acids 614-797. FP3 bound 125I-TGF-beta1 and 125I-TGF-beta2 in solution, preventing the binding of these growth factors to immobilized alpha2M-methylamine (alpha2M-MA). The IC50 values were 33 +/- 5 and 26 +/- 6 nM for TGF-beta1 and TGF-beta2, respectively; these values were comparable with or lower than those determined with native alpha2M or alpha2M-MA. A GST fusion protein that includes amino acids 798-1082 of alpha2M (FP4) and purified GST did not inhibit the binding of TGF-beta to immobilized alpha2M-MA. FP3 (0.2 microM) neutralized the activity of TGF-beta1 and TGF-beta2 in fetal bovine heart endothelial (FBHE) cell proliferation assays; FP4 was inactive in this assay. FP3 also increased NO synthesis by RAW 264.7 cells, mimicking an alpha2M activity that has been attributed to the neutralization of endogenously synthesized TGF-beta. Thus, we have isolated a peptide corresponding to 13% of the alpha2M sequence that binds TGF-beta and neutralizes the activity of TGF-beta in two separate biological assays.     

Binding of human plasma sialoglycoproteins by the B cell-specific lectin CD22. Selective recognition of immunoglobulin M and haptoglobin

CD22 is a cell-surface receptor of resting mature B cells that recognizes sialic acid (Sia) in the natural structure Sia alpha 2-6Gal beta 1-4GlcNAc (Powell, L. D., Jain, R. K., Matta, K. L., Sabesan, S., and Varki, A. (1995) J. Biol. Chem. 270, 7523-7532). Human umbilical vein endothelial cells (HEC) treated with inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) display increases in cell-surface CD22 ligands, caused by increased expression of the enzyme beta-galactoside alpha 2,6-sialyltransferase (Hanasaki, K., Varki, A., Stamenkovic, I., and Bevilacqua, M. P. (1994) J. Biol. Chem. 269, 10637-10643; Hanasaki, K., Varki, A., and Powell, L. D. (1995) J. Biol Chem. 270, 7533-7542). Thus, CD22 could direct potential interactions between mature B cells and endothelial cells during inflammatory states. However, this would have to occur in the presence of blood plasma, which contains many sialoglycoproteins known to carry alpha 2-6-linked sialic acids. We show here that human plasma can indeed inhibit Sia-dependent binding of a recombinant soluble chimeric form of human CD22 (CD22Rg) to TNF-alpha activated HEC. Affinity adsorption of individual human plasma samples with immobilized CD22Rg showed that, of the numerous alpha 2-6-sialic acid containing glycoproteins in plasma, only three polypeptides with apparent molecular mass (under reducing conditions) of 74, 44, and 25 kDa bound, and were specifically eluted with alpha 2-6-sialyllactose. NH2-terminal amino acid sequencing of these high affinity CD22 ligands revealed that they are subunits of immunoglobulin M (IgM) and haptoglobin. Purified human IgM from pooled human plasma can be quantitatively bound by CD22Rg, and binding is blocked by alpha 2-6-sialyllactose, but not by alpha 2-3-sialyllactose. Pretreatment by sialidase or by mild periodate oxidation of sialic acid side chains abolishes these interactions. IgM at physiological concentrations also inhibits CD22Rg binding to TNF-alpha-activated HEC in a manner dependent not only upon its sialylation but also requiring its intact multimeric structure. These data show that CD22 is capable of highly selective recognition of certain multimeric plasma sialoglycoproteins that carry alpha 2-6-linked sialic acids. Notably, the two proteins that are selectively recognized are known to be involved in immune and inflammatory responses. Haptoglobin synthesis by the liver is markedly increased during the "acute phase response" to systemic inflammation, while IgM is the major product resulting from activation of resting CD22-positive B cells.     

Activated alpha 2-macroglobulin promotes mitogenesis in rat vascular smooth muscle cells by a mechanism that is independent of growth-factor-carrier activity

Vascular smooth muscle cell (vSMC) proliferation is important in atherosclerosis. We previously demonstrated that methylamine-activated alpha 2-macroglobulin (alpha 2M) and transforming growth factor beta 1 (TGF-beta 1) cause a synergistic proliferative response in quiescent rat aortic vSMCs [Stouffer, G. A., La-Marre, J., Gonias, S. L. & Owens, G. K. (1993) J. Biol. Chem. 268, 18,340-18,344]. The first goal of this study was to determine whether the synergy is due to the ability of alpha 2M-methylamine (alpha 2M-MeNH2) to bind TGF-beta 1 and target the growth factor to vSMCs that express the alpha 2M receptor. Receptor-recognized alpha 2M derivatives without TGF-beta 1-binding activity, including ternary alpha 2M-trypsin, an 18-kDa proteolytic fragment of the alpha 2M subunit, and the corresponding recombinant receptor-binding fragment (rRBF) increased vSMC [3H]thymidine incorporation and cell number in a manner similar to alpha 2M-MeNH2. In combination with TGF-beta 1, each alpha 2M derivative caused a synergistic vSMC proliferative response. vSMCs responded comparably when treated with alpha 2M-MeNH2 and TGF-beta 1 simultaneously or in sequence. Furthermore, alpha 2M-MeNH2-TGF-beta 1 complexes increased [3H]thymidine incorporation no more than alpha 2M-MeNH2 alone. These results indicate that TGF-beta 1 binding to alpha 2M is not responsible for the synergistic mitogenic activity. Additional studies were undertaken to determine whether activated alpha 2M independently induces a signal-transduction response in vSMCs. alpha 2M-MeNH2 and rRBF caused a rapid, transient increase in vSMC inositol 1,4,5-trisphosphate. This response was pertussis-toxin insensitive. Receptor-associated protein (RAP; 170 nmol/L) inhibited 91-95% of the specific binding of 125I-alpha 2M-MeNH2 and 125I-rRBF to vSMC; however, RAP did not affect the inositol 1,4,5-trisphosphate response or the mitogenic response. These studies suggest that vSMCs express a receptor, other than low-density-lipoprotein-receptor-related protein, that transduces a signal in response to activated alpha 2M. This receptor may mediate the mitogenic activity of alpha 2M in vSMC culture.     

Nitric oxide and the haemodynamic profile of endotoxin shock in the conscious mouse

1. The release of cytokines following administration of endotoxin and the contribution of nitric oxide (NO) to the subsequent haemodynamic profile were investigated in the conscious mouse. 2. Administration of endotoxin (E. Coli, 026:B6, 12.5 mg kg(-1), i.v.) elevated the concentration of tumour necrosis factor-alpha (TNF-alpha) in the plasma within 0.5 h, reaching a maximum at 2 h and returning to control concentrations by 4 h. In addition, the concentration of interleukin-6 (IL-6) in the plasma was also elevated within 1 h, reaching a maximum at 3 h and remaining elevated throughout the 12 h of study. 3. Endotoxin (12.5 mg kg(-1), i.v.) induced the expression of a Ca2+-independent (inducible) NO synthase in the mouse heart and elevated the concentrations of nitrite and nitrate in the plasma within 4 h, reaching a maximum at 12 h. This was accompanied by a progressive fall in blood pressure over the same period. 4. The vasopressor effect of noradrenaline (0.5-4 microg kg(-1) min(-1), i.v.) administered as a continuous infusion was significantly attenuated 7 h after endotoxin (12.5 mg kg(-1), i.v). 5. The NO synthase inhibitor NG-monomethyl-L-arginine HCl (L-NMMA; 1-10 mg kg(-1), i.v. bolus) reversed the fall in blood pressure when administered 7 h after endotoxin (12.5 mg kg(-1), i.v.). 6. In an attempt to maintain a constant blood concentration, L-NMMA was administered as a continuous infusion (10 mg kg(-1) h(-1), i.v.), beginning 4 h after a lower dose of endotoxin (6 mg kg(-1), i.v.). Such treatment prevented the fall in blood pressure and the elevation of nitrite and nitrate in the plasma throughout the 18 h of observation. 7. The fall in blood pressure following endotoxin (3 mg kg(-1), i.v.) was significantly reduced throughout the 18 h of observation in homozygous mutant mice lacking the inducible NO synthase. 8. In summary, we have developed a model of endotoxin shock in the conscious mouse in which an overproduction of NO by the inducible NO synthase is associated with the haemodynamic disturbances. This model, which exhibits many of the characteristics of septic shock in man, will enable the study of the pathology of this condition in more detail and aid the investigation of potential therapeutic agents both as prophylactics and, more importantly, as treatments.     

Role of nitric oxide in gonadotropin-releasing hormone-dependent prostaglandin F2 alpha synthesis by frog (Rana esculenta) interrenal gland during post-reproduction

The aim of this study was to clarify the possible involvement of nitric oxide (NO) on prostaglandin (PG) E2-9-ketoreductase activity in the gonadotropin-releasing hormone (GnRH)-dependent PGF2 alpha synthesis by the interrenal gland of the female water frog, Rana esculenta, during the post-reproduction. Interrenal glands were incubated in vitro with GnRH, NO donor (sodium nitroprusside, SNP), and inhibitors of phospholipase C (compound 48/80), inositol triphosphate (decavanadate), calmodulin (calmidazolium), NO synthase (L-NAME), and PGE2-9-ketoreductase (palmitic acid). Production of PGE2 and PGF2 alpha and NO synthase and PGE2-9-ketoreductase activities were determined. GnRH and SNP increased PGF2 alpha production and PGE2-9-ketoreductase activity, and decreased production of PGE2 and GnRH increased NO synthase activity. GnRH effects were blocked by all inhibitors, except for palmitic acid, which did not affect NO synthase activity, which is increased by GnRH. This study indicates that NO may be involved in regulation of the R. esculenta post-reproduction through stimulation of PGE2-9-ketoreductase activity in GnRH-dependent PGF2 alpha synthesis by the frog interrenal gland.     

Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism

The relationship between glucokinase (GK) gene copy number and glucose homeostasis was studied in transgenic mice with additional copies of the entire GK gene locus (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22564-22569). The plasma glucose concentration was reduced by 25 +/- 3% and 37 +/- 4% in mice with one or two extra copies of the gene locus, respectively. The basis for the hypoglycemic phenotype was determined using metabolic tracer techniques in chronically cannulated, conscious mice with one extra GK gene copy. Under basal conditions (6-h fasted) transgenic mice had a lower blood glucose concentration (-12 +/- 1%) and a slightly higher glucose turnover rate (+8 +/- 3%), resulting in a significantly higher glucose clearance rate (+21 +/- 2%). Plasma insulin levels were not different, suggesting that increased glucose clearance was due to augmented hepatic, not islet, GK gene expression. Under hyperglycemic clamp conditions the transgenic mice had glucose turnover and clearance rates similar to the controls, but showed a lower plasma insulin response (-48 +/- 5%). Net hepatic glycogen synthesis was markedly elevated (+360%), whereas skeletal muscle glycogen synthesis was decreased (-40%). These results indicate that increased GK gene dosage leads to increased hepatic glucose metabolism and, consequently, a lower plasma glucose concentration. Increased insulin secretion was not observed, even though the transgene is expressed in islets, because hypoglycemia causes a down-regulation in islet GK content (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, in press).     

Nitric oxide inhibits alpha2-adrenoceptor-mediated endothelium-dependent vasodilation

This study was designed to investigate the interaction between the NO/L-arginine pathway and the alpha2-adrenoceptor-mediated endothelium-dependent vasorelaxation. Reactivity of isolated resistance mesenteric arterial segments from mice lacking the gene for constitutive endothelial NO synthase (eNOS- mice, n=14) and from their wild-type controls (WT mice, n=46) was studied in isometric conditions in the presence of indomethacin (blocker of cyclooxygenase). Oxymetazoline (OXY, 0.01 to 30 micromol/L; a selective alpha2-adrenoceptor agonist) induced an endothelium-dependent relaxation of eNOS- but not WT arteries preconstricted either with phenylephrine or serotonin. In the presence of Nomega-nitro-L-arginine (l-NNA, 100 micromol/L), an inhibitor of NOS, OXY induced an endothelium-dependent relaxation of WT mesenteric arteries. l-NNA had no effect on the relaxation caused by OXY in eNOS- arterial rings. Therefore, the relaxation caused by OXY was independent of NO formation. To demonstrate the inhibitory role of NO on the alpha2-adrenoceptor-mediated relaxation, subthreshold (0.1 nmol/L) to threshold (1 nmol/L) concentrations of sodium nitroprusside (donor of NO) were added to l-NNA-treated arteries before OXY challenges: in these conditions, the alpha2-adrenoceptor-mediated relaxation of eNOS- and WT arteries was inhibited. OXY-induced relaxation was restored on readdition of methylene blue (1 micromol/L, inhibitor of guanylate cyclase), suggesting that cGMP may be the mechanism of inhibition of the alpha2-adrenergic pathway in the presence of NO. Finally, OXY-mediated relaxation was blocked by tetraethylammonium (1 mmol/L) but not glibenclamide (1 micromol/L), suggesting the involvement of an endothelium-derived hyperpolarizing factor that activates Ca2+-activated K+ channels. In conclusion, alpha2-adrenoceptor activation caused relaxation of isolated murine mesenteric arteries that was functionally blocked by NO through a mechanism that may involve activation of the soluble guanylate cyclase and cGMP formation. The endothelium-dependent alpha2-adrenoceptor-mediated relaxation is likely to be due to an endothelium-derived hyperpolarizing factor, whose release and/or production is reduced by concurrent NO formation.     

Insulin down-regulates the inducible nitric oxide synthase pathway: nitric oxide as cause and effect of diabetes?

Evidence in this paper indicates that insulin can down-regulate the inducible nitric oxide synthase (iNOS) pathway in vivo. The iNOS pathway is up-regulated in diabetes-prone rats and mice and is associated with an autoimmune process. However, the results presented here indicate that macrophage nitric oxide (NO) production and iNOS mRNA expression are also elevated in rats or mice made diabetic by streptozotocin injection in which there is no primary autoimmune component. Insulin administration reduces NO production in autoimmune-prone and streptozotocin-induced diabetic rodents. Finally, insulin decreases macrophage NO production in normal hosts. These results indicate that the autoimmune paradigm is inadequate to explain increased NO in diabetes. As a potential mechanism to explain insulin-mediated regulation of NO production, TGF-1 may be involved because 1) macrophages from diabetic mice produce less TGF-beta1 than macrophages from normal hosts; 2) the circulating TGF-beta1 level is lower in diabetic mice; and 3) insulin administration increases circulating TGF-beta1 in normal mice. Together, these results provide evidence that increased NO in diabetes is not only a cause but also an effect of beta-cell destruction and results in part from a heretofore unrecognized immunomodulatory activity of insulin.     

Oxidized alpha2-macroglobulin (alpha2M) differentially regulates receptor binding by cytokines/growth factors: implications for tissue injury and repair mechanisms in inflammation

Alpha2M binds specifically to TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), beta-nerve growth factor (beta-NGF), platelet-derived growth factor (PDGF), and TGF-beta. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of alpha2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized alpha2M exhibits increased binding to TNF-alpha, IL-2, and IL-6 and decreased binding to beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Hypochlorite oxidation of methylamine-treated alpha2M (alpha2M*), an analogue of the proteinase/alpha2M complex, also results in decreased binding to bFGF, beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Concomitantly, we observed decreased ability to inhibit TGF-beta binding and regulation of cells by oxidized alpha2M and alpha2M*. We then isolated alpha2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-beta compared with alpha2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.     

In vivo quantification of endotoxin-induced nitric oxide production in pigs from Na15NO3-infusion

1. In this investigation the NO production rate is quantified in the pig during normotensive endotoxin-induced shock with increased cardiac output and during subsequent treatment with the NO synthase inhibitor N omega-monomethy-L-arginine (L-NMMA). NO production rate was derived from the plasma isotope-enrichment of 15N-labelled nitrate (15NO3-). 2. Three groups of animals (control, n = 5; endotoxin, n = 6; endotoxin + L-NMMA, n = 6) were anaesthetized and instrumented for the measurement of systemic and pulmonary haemodynamics. Each animal received a primed-continuous infusion of stable, non-radioactively labelled Na15 NO3 (bolus 30 mg, infusion rate 2.1 mg h-1). Arterial blood samples were taken 5, 10, 15, 30, 60 and 90 min later and every 90 minutes until the end of the experiment. 3. Continuous i.v. infusion of endotoxin was incrementally adjusted until mean pulmonary artery pressure (PAP) reached 50 mmHg and subsequently titrated to keep mean PAP approximately 35 mmHg. Hydroxyethylstarch was administered as required to maintain mean arterial pressure (MAP) > 60 mmHg. Six hours after the start of the endotoxin continuous i.v. L-NMMA (1 mg kg-1 h-1) was administered to the endotoxin + L-NMMA group. Haemodynamic data were measured before as well as 9 h after the start of the endotoxin. 4. After conversion of NO3- to nitro-trimethoxybenzene and gas chromatography-mass spectrometry analysis the total NO3- pool, basal NO3- production rate and the increase per unit time in NO3- production rate were calculated from the time-course of the 15NO3- plasma isotope-enrichment. A two compartment model was assumed for the NO3- kinetics, one being an active pool in which newly generated NO3- appears and from which it is eliminated, the other being an inactive volume of distribution in which only passive exchange takes place with the active compartment. 5. Although MAP did not change during endotoxin infusion alone, cardiac output (CO) increased by 42 +/- 40% (P < 0.05 versus baseline) by the end of the experiment due to a significant (P < 0.05 versus baseline) fall in systemic vascular resistance (SVR) to 65 +/- 25% of the baseline value. L-NMMA given with endotoxin did not change MAP, and both CO and SVR were maintained close to the pre-shock levels. 6. Baseline plasma NO3- concentrations were 43 +/- 13 and 40 +/- 10 mumol l-1 in the control and endotoxin animals, respectively, and did not differ at the end of the experiment (39 +/- 8 and 44 +/- 15 mumol l-1, respectively). The mean NO3- pool and basal NO3- production rate were 1155 +/- 294 mumol and 140 +/- 32 mumol h-1, respectively, without any intergroup difference. Endotoxin significantly increased NO3- production rate (23 +/- 10 mumol h-2, P < 0.05 versus control (6 +/- 7 mumol h-2) and endotoxin + L-NMMA groups). L-NMMA given with endotoxin (-1 +/- 2 mumol h-2, P < 0.05 versus control and endotoxin groups) had no effect. 7. Analysis of the time course of the 15NO3- plasma isotope enrichment during primed-continuous infusion of Na15NO3 allowed us to quantify the endotoxin-induced increase in NO3- production rate independently of total NO3- plasma concentrations. Low-dose L-NMMA blunted the increase in NO3- production rate while maintaining basal NO3- formation.     

Transforming growth factor beta as a virulence mechanism for Leishmania braziliensis

Transforming growth factor beta (TGF-beta) has potent down-regulating effects on macrophages and is thus capable of influencing the fate of intramacrophage parasites, including leishmanias. We report the development of a mouse model for the study of the human pathogen Leishmania braziliensis and demonstrate, both in vitro and in vivo, a key regulatory role for TGF-beta in the pathogenesis of infection with this parasite. Recombinant TGF-beta added to cultures of murine peritoneal macrophages led to increased intracellular L. braziliensis replication, whereas addition of neutralizing anti-TGF-beta monoclonal antibody decreased levels of infection. Macrophages infected with L. braziliensis produced biologically active TGF-beta, with a direct correlation between amounts of TGF-beta induced by two parasite isolates and their relative virulence. In vivo, treatment with recombinant TGF-beta rendered avirulent parasites virulent and activated latent L. braziliensis infection. Activation of parasite replication was observed in mice which had been infected with L. braziliensis 15 weeks previously but had not developed lesions or had healed lesions, depending on the parasite isolate used to infect the mice. The exacerbation of L. braziliensis infection in vivo was associated with an increase of interleukin 10 mRNA in the draining lymph node. These results demonstrate that TGF-beta is able to alter the course of in vitro and in vivo infections with L. braziliensis, the latter being characterized by an increase in interleukin 10, an important Th2 helper-T-cell cytokine.     

Insulinomimetic effect on glucose transport by epidermal growth factor when combined with a major histocompatibility complex class I-derived peptide

Peptides derived from the alpha 1-region of the murine H-2Dk molecule enhance glucose uptake in rat adipose cells above the maximum obtained with insulin stimulation alone (Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now describe that epidermal growth factor (EGF) in combination with the same peptides, Dk-(61-85) and Dk-(62-85), stimulates cellular glucose uptake 5-7 times over the basal level, i.e. to 30-50% of the maximal insulin effect. EGF alone increased glucose uptake by only approximately 50% above basal and the peptide alone by 100% above basal. Maximal effect of EGF and peptide was reached in 10-20 min with 30 microM peptide (EC50 10-15 microM) and 50 nM EGF (EC50 1-2 nM). The effect of EGF and peptide on glucose uptake was additive to that of insulin and peptide until the maximal level attained with insulin and peptide was reached. The combined effect of EGF plus peptide on glucose transport was associated with a recruitment of GLUT4 molecules to the plasma membrane. However, the phosphatidylinositol (PI) kinase which is activated by insulin was not activated by EGF plus peptide. Thus, the effect of EGF plus peptide on glucose uptake seems independent of the activity status of the insulin receptor. 125I-Labeled EGF bound specifically to rat adipose cells with an apparent affinity of approximately 2 nM and Bmax approximately 5 x 10(3). However, the major histocompatibility complex (MHC) peptides did not affect EGF-stimulated internalization of EGF receptor, in contrast to their effect on the insulin receptors. Transforming growth factor alpha had an effect similar to EGF on glucose uptake. Three other peptides derived from other parts of murine MHC class I had no effect on glucose uptake in combination with EGF. Thus, EGF in combination with certain MHC class I-derived peptides is insulinomimetic concerning glucose transport and this effect is independent of the insulin receptor activity.     

Induction of alpha/beta interferon and dependent nitric oxide synthesis during Chlamydia trachomatis infection of McCoy cells in the absence of exogenous cytokine

The propensity of two Chlamydia trachomatis strains (L2/434/Bu [biovar LGV] and E/DK20/ON [biovar trachoma]) to induce putative host defense responses upon infection of McCoy (mouse) cell cultures was examined. Both strains induced production of alpha/beta interferon and nitric oxide (NO) by McCoy cells. NO synthesis was mediated by the inducible isoform of NO synthase as indicated by the ability of cycloheximide or the arginine analog NG-monomethyl-L-arginine to abolish NO production; the extent of the response was dependent upon the dose of chlamydiae applied. Incubation of McCoy cells with chloramphenicol prior to infection reduced NO production by strain 434 but not by DK20, suggesting that initial chlamydial metabolism was essential to induction by the LGV strain. Antibody inhibition studies indicated that NO synthesis was dependent upon production of alpha/beta interferon and induction via lipopolysaccharide. Overall, our findings show that chlamydiae are capable of the induction of interferon and NO in murine fibroblasts in the absence of exogenous cytokines. However, the role of NO as an antichlamydial effector could not be clearly demonstrated since treatment with an arginine analog, while suppressing NO production, gave no consistent enhancement of infected cell numbers.     

Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1)

The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.     

Utility of a linear array ultrasound endoscope in the evaluation of suspected pancreatic disease

Diethylcarbamazine (DEC) was discovered in 1947 as a potent therapeutic agent in lymphatic filariasis and has been a mainstay of antifilarial therapy over the past five decades (R. I. Hewitt, et al., 1947, Journal of Laboratory and Clinical Medicine 32, 1304-1313). Several hundred million doses of this drug have been administered to people. Despite its widespread and successful use over this prolonged time scale, its mechanism of action remains obscure (R. M. Maizels and D. A. Denham, 1992, Parasitology 105 Suppl. 549-560). Numerous studies suggest that DEC has no direct effect on the parasite (F. Hawking and W. Laurie, 1949, Lancet 2, 146-147) and that it exerts its action by stimulating host immune defense mechanisms (F. Hawking et al., 1948, Lancet 2, 730-731), or by activating host platelets to become microfilaricidal (J. Y. Cesbron et al., 1987, Nature 325(6104) 533-536). Recent data from two different laboratories suggest that NO may be involved in host defense against filarial parasites (T. V. Rajan et al., 1996, Infection and Immunity 64(8), 3351-3353; M. J. Taylor et al., 1996, Parasitology 112, 315-322). We investigated whether DEC stimulates the production of NO from murine macrophages or rat endothelial cells. DEC did not stimulate the synthesis or secretion of NO from either, nor did it synergize with interferon-gamma or tumor necrosis factor-alpha in the induction of inducible NO synthase (iNOS). In addition, there was no consistent increase in the output of inorganic nitrate, the end product of NO metabolism, in the urines of rats treated with DEC. These data suggest that DEC does not achieve its therapeutic efficacy through the induction of host iNOS.     

Building synthetic antibodies as adhesive ligands for integrins

An antibody engineering strategy was employed to build high affinity ligands and antagonists of integrins alpha v beta 3 and alpha IIb beta 3. Previously, we inserted the integrin recognition motif, RGD, into the antigen binding site of a human antibody and selected the optimal flanking sequences from a phage-display library (Barbas, C. F., Languino, L. R., and Smith, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10003-10007). The resulting antibody, Fab-9, blocked the function of integrin alpha v beta 3 but also bound to the ligand binding site of platelet integrin alpha IIb beta 3. In this report, the antibody engineering effort has been extended by 1) redesigning Fab-9 to achieve specificity for platelet integrin alpha IIb beta 3, 2) building non-RGD-containing antibodies that bind the ligand binding site of both beta 3-integrins, and 3) testing the hypothesis that peptides derived from complementarity determining regions (CDR) can be used to emulate the activity of the parent synthetic antibody. These goals were accomplished by subjecting the original antibody, Fab-9, to a "motif optimization" (MTF). A phage library was constructed in which the residues flanking the RGD motif in Fab-9 were maintained, but the RGDX sequence was randomized. This library was panned on purified alpha IIb beta 3 to identify high affinity binders. Four function-blocking antibodies lacking RGD, but with specificity for alpha IIb beta 3, were characterized. The antibody with the highest preference for alpha IIb beta 3, MTF-10, had an adhesion sequence of KGDN. This sequence is similar in primary structure to the active sequence within the disintegrin barbourin, which also antagonizes alpha IIb beta 3 (Scarborough, R. M., Rose, J. W., Hsu, M. A., Phillips, D. R., Fried, V. A., Campbell, A. M., Nannizzi, L., and Charo, I. F. (1991) J. Biol. Chem. 266, 9359-9362). MTF-10 had a 70-fold higher affinity for alpha IIb beta 3 than alpha v beta 3. Through our selection strategy, we also identified several antibodies that lack RGD but still blocked ligand binding to both integrins with high affinity. Therefore, the RGD sequence is not necessary for a high affinity interaction with the ligand binding site of beta 3-integrins. Further investigation showed that the activity of inhibitory antibodies could be emulated by synthetic peptides derived from the protein sequences of the antibody's HCDR3. CDR-derived peptides blocked ligand binding to integrins and maintained essentially the same specificity as the parent antibody.     

Association of apolipoprotein E with alpha2-macroglobulin in human plasma

Apolipoprotein (apo) E plays a central role in the transport of lipids among different organs and cell types, whereas alpha2-macroglobulin (alpha2M) is responsible for the binding and inactivation of plasma proteases, as well as the transport of various cytokines, growth factors, and hormones. In the present study, evidence is presented for direct binding of apoE with alpha2M in human plasma, based on the observation that two-dimensional non-denaturing gradient gel electrophoretic separation of plasma resulted in co-migration of apoE with alpha2M in a complex intermediate in size (18.5 nm in diameter) between low (LDL) and high density lipoproteins (HDL). ApoE associated with alpha2M could be immunoprecipitated from plasma with anti-human alpha2M antiserum. Purified apoE, labeled with 125I, bound to native and methylamine-activated alpha2M (alpha2M-MA) in vitro in a time- and concentration-dependent manner. ApoE bound to alpha2M-MA with greater affinity than alpha2M. The binding of apoE to both alpha2M and alpha2M-MA did not depend on the presence of lipid. Ingestion of an oral fat load resulted in a reduction in the amount of apoE associated with alpha2M. Sphingomyelin vesicles and very low density lipoproteins (VLDL), but not phosphatidylcholine vesicles or HDL3, inhibited the in vitro binding of 125I-labeled apoE3 to alpha2M and alpha2M-MA. Binding of 125I-labeled apoE3 was also partially inhibited by an excess of platelet-derived growth factor and beta-amyloid protein, but not interferon-gamma. Subjects with an apoE 4/4 phenotype had less apoE associated with alpha2M in plasma than subjects with an apoE 3/3 or 2/2 phenotype, corresponding to reduced in vitro binding of apoE4 with alpha2M or alpha2M-MA. Although the functional significance of apoE binding to alpha2M remains to be determined, the present results demonstrate that: 1) apoE is non-covalently bound to alpha2M in human plasma, 2) alpha2M-MA has a greater capacity to bind apoE than alpha2M, 3) various proteins or lipoproteins known to bind apoE or alpha2M can potentially affect the interaction of apoE with alpha2M, and 4) association of apoE with alpha2M or alpha2M-MA is dependent on apoE phenotype.     

Preschool children at risk for repeat injuries

BACKGROUND: Nitric oxide (NO) is cytostatic for proliferating cells, inhibits microbial growth, and down-regulates the synthesis of specific proteins. Studies were undertaken to determine the mechanism by which NO inhibits total protein synthesis and whether the inhibition correlates with established cytostatic activities of NO. MATERIALS AND METHODS: In in vitro experiments, various cell types were exposed to NO using either donors or expression of inducible NO synthase (iNOS). The capacity of NO to suppress total protein synthesis, measured by incorporation of 35S-methionine into protein, was correlated with the capacity of NO to suppress cell proliferation, viral replication, or iNOS expression. Phosphorylation of eIF-2 alpha was examined as a possible mechanism for the suppressed protein synthesis by NO. RESULTS: Both NO donors and expression of the iNOS suppressed total protein synthesis in L929 cells and A2008 human ovarian tumor cells in parallel with decreased cell proliferation. Suppressed protein synthesis was also shown to correlate with decreased vaccinia virus proliferation in murine peritoneal macrophages in an iNOS-dependent manner. Furthermore, iNOS expression in pancreatic islets or RAW264.7 cells almost completely inhibited total protein synthesis, suggesting that nonspecific inhibition of protein synthesis may be the mechanism by which NO inhibited the synthesis of specific proteins such as insulin or iNOS itself. This possibility was confirmed in RAW264.7 cells where the inhibition of total protein synthesis correlated with the decreased iNOS protein. The decrease in protein levels occurred without changes in iNOS mRNA levels, implicating an inhibition of translation. Mechanistic studies revealed that iNOS expression in RAW264.7 cells resulted in the phosphorylation of eIF-2 alpha and inhibition of the 80S ribosomal complex formation. CONCLUSIONS: These results suggest that NO suppresses protein synthesis by stimulating the phosphorylation of eIF-2 alpha. Furthermore, our observations indicate that nonspecific inhibition of protein synthesis may be a generalized response of cells exposed to high levels of NO and that inhibition of protein synthesis may contribute to many of the described cytostatic actions of NO.