Effects of sub-chronic exposure to naturally occurring N-terminally truncated metabolites of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), GIP(3-42) and GLP-1(9-36)amide, on insulin secretion and glucose homeostasis in ob/ob mice.

Glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important enteroendocrine hormones that are rapidly degraded by an ubiquitous enzyme dipeptidyl peptidase IV to yield truncated metabolites GIP(3-42) and GLP-1(9-36)amide. In this study, we investigated the effects of sub-chronic exposure to these major circulating forms of GIP and GLP-1 on blood glucose control and endocrine pancreatic function in obese diabetic (ob/ob) mice. A once daily injection of either peptide for 14 days had no effect on body weight, food intake or pancreatic insulin content or islet morphology. GLP-1(9-36)amide also had no effect on plasma glucose homeostasis or insulin secretion. Mice receiving GIP(3-42) exhibited small but significant improvements in non-fasting plasma glucose, glucose tolerance and glycaemic response to feeding. Accordingly, plasma insulin responses were unchanged suggesting that the observed enhancement of insulin sensitivity was responsible for the improvement in glycaemic control. These data indicate that sub-chronic exposure to GIP and GLP-1 metabolites does not result in physiological impairment of insulin secretion or blood glucose control. GIP(3-42) might exert an overall beneficial effect by improving insulin sensitivity through extrapancreatic action.

Development of laminin receptor agonists: identification of important functional residues by alanine scanning.

An antagonist of cellular adhesion and motility, acetyl-C-[S-Acm]-VIGYSGDRC-[S-Acm]-NH(2) (mEGF(33-42)), shares homology with the agonist sequence CDPGYIGSR-NH(2). It has been proposed that the latter peptide binds to the high affinity 67 kDa laminin receptor. Both peptides have equal affinities for the receptor and similar conformations have been derived for both. We have examined the importance of individual non-homologous residues with respect to receptor binding and antagonistic properties of mEGF(33-42). Alanine scanning of non-conserved residues in the N-terminal half of mEGF(33-42) caused loss of biological activity with respect to cell attachment, receptor binding and migratory response. Substitution of alanine for serine (position 6) caused loss of laminin-specific cell attachment and receptor binding activities. However, the peptide did stimulate migration suggesting that this peptide may be a non-specific stimulator of migration. In contrast, alanine substitution for the C-terminal Cys-S-Acm had no apparent effect on the attachment or receptor binding activities of the peptide but generated an agonist from the antagonist parent. Comparison of the modelled folds of the alanine containing peptides revealed the presence of significant helical content in those peptides capable of stimulating migration and suggests that a reduction in bulk in the N-terminal residues is not conducive to adopting a productive binding conformation.

Identification of potential activators of proteinase-activated receptor-2.

In order to identify physiological activators of proteinase-activated receptor-2 (PAR-2), a peptide chloromethane inhibitor (biotinyl-Ser-Lys-Gly-Arg-CH2Cl) based on the cleavage site for activation of PAR-2 was synthesised and tested with 12 trypsin-like serine proteinases. The second-order rate constant (ki/Ki) for the formation of the covalent proteinase-inhibitor complex varied by 2 x 10(5)-fold between the proteinases. Biotinyl-Ser-Lys-Gly-Arg-CH2Cl reacted very rapidly with trypsin, acrosin from sperm and tryptase from mast cells: the ki/Ki values with these proteinases were greater than 10(5) M(-1) x s(-1). Thus, the specificity of these proteinases matched the sequence of the activation site of PAR-2 and it can be concluded that these proteinases are potential physiological activators of PAR-2.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo.

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala(8)-substituted analogues of GLP-1, (Abu(8))GLP-1 and (Val(8))GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu(8))GLP-1 and (Val(8))GLP-1 exhibited moderate affinities (IC(50): 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC(50): 0.37 nM). (Abu(8))GLP-1 and (Val(8))GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val(8))GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu(8))GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val(8))GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala(8) in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val(8))GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach.

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine (b) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylase (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

Evidence that the major degradation product of glucose-dependent insulinotropic polypeptide, GIP(3-42), is a GIP receptor antagonist in vivo.

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is rapidly degraded in the circulation by dipeptidyl peptidase IV forming the N-terminally truncated peptide GIP(3-42). The present study examined the biological activity of this abundant circulating fragment peptide to establish its possible role in GIP action. Human GIP and GIP(3-42) were synthesised by Fmoc solid-phase peptide synthesis, purified by HPLC and characterised by electrospray ionisation-mass spectrometry. In GIP receptor-transfected Chinese hamster lung fibroblasts, GIP(3-42) dose dependently inhibited GIP-stimulated (10(-7) M) cAMP production (up to 75.4+/-5.4%; P<0.001). In BRIN-BD11 cells, GIP(3-42) was significantly less potent at stimulating insulin secretion (1.9- to 3.2-fold; P<0.001), compared with native GIP and significantly inhibited GIP-stimulated (10(-7) M) insulin secretion with maximal inhibition (48.8+/-6.2%; P<0.001) observed at 10(-7) M. In (ob/ob) mice, administration of GIP(3-42) significantly inhibited GIP-stimulated insulin release (2.1-fold decrease; P<0.001) and exaggerated the glycaemic excursion (1.4-fold; P<0.001) induced by a conjoint glucose load. These data indicate that the N-terminally truncated GIP(3-42) fragment acts as a GIP receptor antagonist, moderating the insulin secreting and metabolic actions of GIP in vivo.

Improved biological activity of Gly2- and Ser2-substituted analogues of glucose-dependent insulinotrophic polypeptide.

The therapeutic potential of glucagon-like peptide-1 (GLP-1) in improving glycaemic control in diabetes has been widely studied, but the potential beneficial effects of glucose-dependent insulinotropic polypeptide (GIP) have until recently been almost overlooked. One of the major problems, however, in exploiting either GIP or GLP-1 as potential therapeutic agents is their short duration of action, due to enzymatic degradation in vivo by dipeptidylpeptidase IV (DPP IV). Therefore, this study examined the plasma stability, biological activity and antidiabetic potential of two novel NH2-terminal Ala2-substituted analogues of GIP, containing glycine (Gly) or serine (Ser). Following incubation in plasma, (Ser2)GIP had a reduced hydrolysis rate compared with native GIP, while (Gly2)GIP was completely stable. In Chinese hamster lung fibroblasts stably transfected with the human GIP receptor, GIP, (Gly2)GIP and (Ser2)GIP stimulated cAMP production with EC(50) values of 18.2, 14.9 and 15.0 nM respectively. In the pancreatic BRIN-BD11 beta-cell line, (Gly2)GIP and (Ser2)GIP (10(-8) M) evoked significant increases (1.2- and 1.5-fold respectively; P<0.01 to P<0.001) in insulinotropic activity compared with GIP. In obese diabetic ob/ob mice, both analogues significantly lowered (P<0.001) the glycaemic excursion in response to i.p. glucose. This enhanced glucose-lowering ability was coupled to a significantly raised (P<0.01) and more protracted insulin response compared with GIP. These data indicate that substitution of the penultimate Ala2 in GIP by Gly or Ser confers resistance to plasma DPP IV degradation, resulting in enhanced biological activity, therefore raising the possibility of their use in the treatment of type 2 diabetes.

N-terminal His(7)-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity.

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His(7)-modified analogue of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC(50) values 32.9 and 6.7 nM, respectively) compared with native GLP-1 (IC(50) 0.37 nM). Similarly, both analogues stimulated cAMP production with EC(50) values of 16.3 and 27 nM respectively compared with GLP-1 (EC(50) 4.7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5.6 mM glucose (P<0.05 to P<0.001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes.

Analysis of the post-translational processing of chromogranin A in rat neuroendocrine tissue employing an N-terminal site-specific antiserum.

Chromogranin A (CgA) is a complex prohormone expressed as a constituent of the regulated secretory pathway of numerous neuroendocrine cells. Recent investigations have demonstrated that CgA is selectively cleaved to generate distinct peptides in different neuroendocrine tissues. This investigation employed a site-specific antiserum that detects residues 98-106 rat CgA to examine the amino-terminal processing of CgA to generate beta-granin and related peptides in rat neuroendocrine tissues. Immunohistochemistry revealed moderate to intense beta-granin-like immunostaining in cells scattered throughout the anterior pituitary, thyroid, in the islets of Langerhans and in the mucosa of the gastrointestinal tract. Variable intensities of immunostaining were observed in distinct clusters of chromaffin cells. Quantitatively, the highest concentration of beta-granin-like immunoreactivity was detected in pituitary extracts. Significantly lower concentrations were detected in adrenal and thyroid glands, brain, ventral and dorsal pancreatic lobes and gastrointestinal tissue extracts. Chromatography resolved three distinct beta-granin-like immunoreactants; a large CgA-like form, an intermediate molecular form presumably corresponding to beta-granin (rat CgA1-128) and small immunoreactants that coeluted with the synthetic peptide. Two beta-granin-like immunoreactants, 21 and 22 kDa, were detected following immunoblot analysis of pituitary extracts. This study has demonstrated that chromogranin A is subject to distinct amino-terminal patterns of tissue-and cell-specific processing to generate a beta-granin-like immunoreactant which is additionally cleaved in pancreatic, fundic and colonic tissue to generate previously unidentified peptides.

Lys9 for Glu9 substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39).

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLP-1's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His(7) and Ala(8), can greatly improve resistance to this enzyme. Little research has assessed what effect Glu(9)-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu(9) of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue, (Lys(9))GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1(9-36)amide. We investigated the in vivo antagonistic actions of (Lys(9))GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor.

Liver fluke (Fasciola hepatica)-derived peptides activate rat peritoneal mast cells.

Short peptides with sequences derived from those found in the tegumental antigen of Fasciola hepatica have been synthesised. Incubation of some of these peptides with rat peritoneal mast cells resulted in the degranulation of the cells as measured by a histamine release assay. This activity was shown to be associated with the proline-lysine-proline motif, which is responsible for the induction of mast cell degranulation by the mammalian bioactive peptide substance P. Studies on the mode of action of the fluke-derived peptide indicated that it was operating through the same biochemical pathways as substance P. The implications of these findings for the development of immune responses during parasite infections are discussed.

Cyclic AMP production and insulin releasing activity of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide.

Synthetic fragment peptides of glucose-dependent insulinotropic polypeptide (GIP) were evaluated for their ability to elevate cellular cAMP production and stimulate insulin secretion. In GIP receptor transfected CHL cells, GIP(4-42) and GIP(17-30) dose-dependently inhibited GIP-stimulated cAMP production (40 +/- 8%; p<0.01 and 15 +/- 6%; p<0.05, respectively), while GIP(1-16) exerted very weak agonist effects on cAMP production. In the clonal pancreatic beta-cell line, BRIN-BD11, GIP(1-16) demonstrated weak insulin releasing activity compared with native GIP. In contrast, GIP(4-42) and GIP (17-30) weakly antagonized the insulin releasing activity of the native peptide (23 +/- 6%; p<0.05 and 11 +/- 3%, respectively). These data demonstrate the critical role of the N-terminus and the involvement of regions of the C-terminal domain in generating full biological potency of GIP.

Early administration of the glucose-dependent insulinotropic polypeptide receptor antagonist (Pro3)GIP prevents the development of diabetes and related metabolic abnormalities associated with genetically inherited obesity in ob/ob mice.

AIMS/HYPOTHESIS: Ablation of gastric inhibitory polypeptide (GIP) receptor action is reported to protect against obesity and associated metabolic abnormalities. The aim of this study was to use prediabetic ob/ob mice to examine whether 60 days of chemical GIP receptor ablation with (Pro(3))GIP is able to counter the development of genetic obesity-related diabetes. MATERIALS AND METHODS: Young (5-7 weeks) ob/ob mice received once daily i.p. injections of either saline vehicle or (Pro(3))GIP (25 nmol kg(-1) day(-1)) over a 60 day period. Food intake, body weight and circulating glucose and insulin were measured at frequent intervals. At 60 days, glucose tolerance, response to native GIP, postprandial responses, insulin sensitivity, HbA(1c), circulating hormones and plasma lipids were assessed. RESULTS: Body weight and food intake in (Pro(3))GIP-treated mice did not differ from ob/ob controls. GIP receptor blockade significantly improved non-fasting glucose (p < 0.001), HbA(1c) (p < 0.05), glucose tolerance (p < 0.001), meal tolerance (p < 0.001) and insulin sensitivity (p < 0.05). Remarkably, (Pro(3))GIP treatment prevented the age-related development of diabetes, as none of these parameters differed significantly between treated ob/ob mice and normal age-matched lean controls. Circulating levels of glucagon, corticosterone, adiponectin and total cholesterol were unchanged by (Pro(3))GIP, while levels of triacylglycerol, LDL-cholesterol and resistin were decreased (p < 0.05) compared with those in control ob/ob mice. Plasma and pancreatic insulin concentrations were generally lower after (Pro(3))GIP treatment than in control ob/ob mice (p < 0.01), but plasma insulin levels remained substantially raised (p < 0.001) compared with those observed in lean controls. CONCLUSIONS/INTERPRETATION: These data indicate that sustained GIP receptor antagonism provides an effective means of preventing the development of many of the metabolic abnormalities of obesity-driven diabetes.

Peptides containing acylated C-terminal gem diamines: novel irreversible inactivators of the cysteine and serine proteinases.

This study reports on the synthesis of peptides containing C-terminal acylated gem-diamines and their utilization for the preparation of irreversible inactivators of the serine and cysteine proteinases. We have succeeded in obtaining an inhibitor Acetyl-Val-Pro-g-Val-CO-O-C(6)H(4)-NO(2) of neutrophil and pancreatic elastases that functions in a time-dependent manner, indicative of the action of an irreversible inactivator, functioning, most probably, through the formation of a long-lived acyl enzyme intermediate. In addition, we have demonstrated the irreversible inhibition of the cysteine proteinase bovine cathepsin B, by chloroacetyl and bromoacetyl derivatives of a dipeptide gem-diamine, Cbz-Phe-g-Ala-CO-CH(2)Hal (Hal = Br, Cl).

The inactivation of bovine cathepsin B by novel N-chloro-acetyl-dipeptides: application of the Houghten 'tea bag' methodology to inhibitor synthesis.

In this study, a series of N-chloro-acetylated dipeptides were synthesised by the application of Houghten's methodology of multiple analog peptide syntheses (MAPS). The peptides, all of which contain a C-terminal free acid, were tested as inactivators of bovine cathepsin B, in an attempt at exploiting the known and, amongst the cysteine proteinases, unique carboxy dipeptidyl peptidase activity of the protease. We have succeeded in obtaining a number of effective inactivators, the most potent of which-chloroacetyl-Leu-Leu-OH, inactivates the enzyme with an apparent second-order rate constant of 3.8 x 10(4)M(-1)min(-1). In contrast, the esterified analog, chloroacetyl-Leu-Leu-OMe, inactivates the enzyme some three orders of magnitude less efficiently, lending credence to our thesis that a free carboxylic acid moiety is an important determinant for inhibitor effectiveness. This preliminary study has highlighted a number of interesting features about the specificity requirements of the bovine proteinase and we believe that our approach has great potential for the rapid delineation of the subsite specificities of cathepsin B-like proteases from various species.

A peptide corresponding to the neuropilin-1-binding site on VEGF(165) induces apoptosis of neuropilin-1-expressing breast tumour cells.

There is increasing evidence that vascular endothelial growth factor (VEGF) has autocrine as well as paracrine functions in tumour biology. Vascular endothelial growth factor-mediated cell survival signalling occurs via the classical tyrosine kinase receptors Flt-1, KDR/Flk-1 and the more novel neuropilin (NP) receptors, NP-1 and NP-2. A 24-mer peptide, which binds to neuropilin-1, induced apoptosis of murine and human breast carcinoma cells, whereas a peptide directed against KDR had no effect. Both anti-NP1 and anti-KDR peptides induced endothelial cell apoptosis. Confocal microscopy using 5-(6)-carboxyfluorescein-labelled peptides showed that anti-NP1 bound to both tumour and endothelial cells, whereas anti-KDR bound endothelial cells only. This study demonstrates that NP-1 plays an essential role in autocrine antiapoptotic signalling by VEGF in tumour cells and that NP1-blockade induces tumour cell and endothelial cell apoptosis. Specific peptides can therefore be used to target both autocrine (tumour cells) and paracrine (endothelial cells) signalling by VEGF.

Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping reading frames.

The gene encoding the phosphoprotein of the pneumovirus pneumonia virus of mice (PVM) has been cloned and sequenced. The gene is 903 nucleotides in length and contains a long open reading frame (ORF) capable of encoding a polypeptide of 295 amino acid residues. A smaller, second, overlapping ORF encoding a polypeptide 137 amino acids in length was also present. The large ORF directed the synthesis of a 39-kDa polypeptide and four additional polypeptides with masses of 37 kDa, 26 kDa, 23 kDa, and 16 kDa in vitro. The smaller polypeptides were generated by internal initiation on in-frame AUG initiation codons to generate carboxy co-terminal products. Western immunoblot analysis indicated that at least two of these proteins and several other related polypeptides are present in infected cells, and the possible origins of these are discussed. Western blot analysis using antiserum raised against a synthetic peptide and specific for the predicted second ORF product identified a polypeptide of 23 kDa in PVM-infected cells. The pattern of PVM P gene expression is unlike that of the closely related respiratory syncytial virus and is reminiscent of that of paramyxoviruses such as Sendai virus. This is the first example of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo.

Toxicity of non-abeta component of Alzheimer's disease amyloid, and N-terminal fragments thereof, correlates to formation of beta-sheet structure and fibrils.

The non-Abeta component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NAC forms beta-sheet structures and fibrils [El-Agnaf, O.M.A., Bodles, A.M., Guthrie, D.J.S., Harriott, P. & Irvine, G.B. (1998) Eur. J. Biochem. 258, 157-163]. As a measure of their neurotoxic potential we have examined the ability of fresh and aged NAC and fragments thereof to inhibit the reduction of the redox dye 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide by rat pheochromocytoma PC12 cells. Micromolar concentrations of NAC and fragments thereof display varying degrees of toxicity. On immediate dissolution and after an incubation period for 3 days at 37 degrees C the full-length peptide and fragments NAC(3-18) and NAC(1-18) scrambled sequence [NAC(1-18 s)] were toxic, whereas fragments NAC(1-13) and NAC(6-14) were not. CD indicates that NAC(3-18) and NAC(1-18 s) exhibit beta-sheet secondary structure in aqueous solution, whereas NAC(1-13) and NAC(6-14) do not. NAC(3-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days measured by an HPLC assay, and forms fibrils, as determined by electron microscopy. However, although some fibrils were detected for NAC(1-18 s) it does not come out of solution to a significant degree. Fragments NAC(1-13) and NAC(6-14) form few fibrils and remain in solution. These findings indicate that the ability of the central region of NAC to form beta-sheet secondary structures is important for determining the toxicity of the peptide. This contrasts with what has been reported previously for most Abeta peptides as their toxicity appears to require the peptide to have formed fibrillary aggregates as well as displaying beta-sheet. These results suggest that an intermediate, which exhibits beta-sheet structure, may be responsible for the toxic properties of NAC and provides further evidence for the role of NAC in the pathogenesis of AD, PD and DLB.

Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1.

Streptokinase activates platelets, limiting its effectiveness as a thrombolytic agent. The role of antistreptokinase antibodies and proteases in streptokinase-induced platelet activation was investigated. Streptokinase induced localization of human IgG to the platelet surface, platelet aggregation, and thromboxane A(2) production. These effects were inhibited by a monoclonal antibody to the platelet Fc receptor, IV.3. The platelet response to streptokinase was also blocked by an antibody directed against the cleavage site of the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), but not by hirudin or an active site thrombin inhibitor, Ro46-6240. In plasma depleted of plasminogen, exogenous wild-type plasminogen, but not an inactive mutant protein, S(741)A plasminogen, supported platelet aggregation, suggesting that the protease cleaving PAR-1 was streptokinase-plasminogen. Streptokinase-plasminogen cleaved a synthetic peptide corresponding to PAR-1, resulting in generation of PAR-1 tethered ligand sequence and selectively reduced binding of a cleavage-sensitive PAR-1 antibody in intact cells. A combination of streptokinase, plasminogen, and antistreptokinase antibodies activated human erythroleukemic cells and was inhibited by pretreatment with IV.3 or pretreating the cells with the PAR-1 agonist SFLLRN, suggesting Fc receptor and PAR-1 interactions are necessary for cell activation in this system also. Streptokinase-induced platelet activation is dependent on both antistreptokinase-Fc receptor interactions and cleavage of PAR-1. (Blood. 2000;95:1301-1308)

Protease-activated receptors and their role in IL-6 and NF-IL-6 expression in human gingival fibroblasts.

The serine protease thrombin is formed at sites of coagulation and inflammation and has been shown to have important proinflammatory cellular effects relevant to the pathogenesis of periodontal disease. Thrombin acts via specific cell surface receptors termed protease-activated receptor-1 (PAR-1) and PAR-3, which have a distinctive method of activation. Proteolytic cleavage of the extracellular domain by thrombin reveals a hidden amino terminus which then acts as a "tethered ligand". A short synthetic peptide (SFLLRN) can also mimic the tethered ligand and activate PAR-1 but not PAR-3. Also, a trypsin-sensitive receptor termed PAR-2 has been described which is activated by the PAR-1 activating peptide SFLLRN. Here we show conclusively by flow cytometric and Northern blot analysis that human gingival fibroblasts (HGF) express PAR-1 but not PAR-2. In functional studies we also show that thrombin and SFLLRN stimulated increased expression of mRNA encoding nuclear transcription factor NF-IL-6 and IL-6 in vitro. At optimal concentrations, thrombin (10(-7) M) induced 7.6 +/- 0.01 ng/ml immunoactive IL-6 and PAR-1 activating peptide (5 x 10(-5) M) induced 2.2 +/- 0.2 ng/ml (mean +/- standard error of mean). A proteolytically inactive recombinant thrombin (serine 195 to alanine) was without activity. These data show that HGF express PAR-1 and suggest that PAR-1 activation stimulates increased NF-IL-6 and IL-6 gene expression and IL-6 secretion by HGF in vitro. Whether HGF express PAR-3 is unknown, but the fact that SFLLRN was not a complete replacement for thrombin raises the possibility that HGF may express additional thrombin receptors. These findings add weight to the importance of the cytokine-like role played by thrombin and raise the possibility that protease-activated receptors may play a role in the pathogenesis of inflammatory periodontal disease.