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Celiac disease (CD) is an autoimmune disease of the small bowel characterized by a strong genetic association with HLA - DQ2 and DQ8. Gluten is the etiological factor and the tissue enzyme transglutaminase (TGase) is its autoantigen. CD is associated with several autoimmune diseases such as type 1 diabetes, systemic lupus erythematous, rheumatoid arthritis, Sjögrens syndrome and autoimmune thyroid diseases. The aim of this study was to investigate the occurrence of serum IgA anti-endomysial and anti-human TGase antibodies in individuals with positive anti-thyroid antibody (ATA). The concordance between these two tests was also evaluated. Anti-endomysial antibodies were positive in 10 out of 456 (2.2%) and anti-human TGase were positive in 14 of 454 (3.1%) individuals with positive ATA. In control subjects they were positive in 1 of 197 (0.5%) and 2 of 198 (1%) for anti-endomysial and anti-human tissue TGase antibodies, respectively. The odds ratio (OR) for the anti-endomysial antibodies was 4.42 and for the anti-human TGase 3.12 in individuals with ATA when compared with controls. An elevated concordance index (k= 0.84) was observed between anti-endomisyal antibodies and anti-human TGase. We conclude that the determination of anti-TGase antibodies is a good test for DC screening.

An easy and convenient microwave-assisted synthesis of N-alkylated glycine methyl esters is described. Parallel and nonparallel combinatorial methods are described and compared. The described reactions are reductive alkylations of several aldehydes and glycine methyl ester in the presence of NaBH3CN. Good yields and short reaction times are the main aspects of these procedures.

Peptide models have been widely used to investigate conformational aspects of domains of proteins since the early 1950s. A pioneer in this field was Dr. Murray Goodman, who applied a battery of methodologies to study the onset of structure in homooligopeptides. This article reviews some of Dr. Goodman's contributions, and reports recent studies using linear and constrained peptides corresponding to the first extracellular loop and linear peptides corresponding to the sixth transmembrane domain of a G-protein coupled receptor from the yeast Saccharomyces cerevisiae. Peptides containing 30-40 residues were synthesized using solid-phase methods and purified to near homogeneity by reversed phase high performance liquid chromatography. CD and NMR analyses indicated that the first extracellular loop peptides were mostly flexible in water, and assumed some helical structure near the N-terminus in trifluoroethanol and in the presence of micelles. Comparison of oligolysines with native loop residues revealed that three lysines at each terminus of a peptide corresponding to the sixth transmembrane domain of the alpha-factor receptor resulted in better aqueous solubility and greater helicity than the native loop residues., (Copyright 2005 Wiley Periodicals, Inc)

An efficient, facile, and practical parallel combinatorial synthesis of substituted-benzoxazines under microwave irradiation was described. The procedure involved the use of a microwave oven especially designed for organic synthesis suitable for parallel synthesis of solution libraries. A demonstration 19-membered library of substituted N,N-dimethyl- and N-methyl-benzoxazine amide derivatives, structurally related to the potassium channel opener cromakalim, was generated by both conventional and microwave procedures, achieving a reduction from 7 h to 30-36 min in library generation time for the microwave approach. All the synthesized compounds were tested using the in vitro models of rat aorta and guinea pig trachea rings pre-contracted with phenylephrine and carbachol, respectively. All N,N-dimethyl amide derivatives showed a relaxant activity higher on guinea pig trachea rings than on rat aorta rings.

In the present paper, the role played by Na+/Ca2+ exchanger (NCX) in focal cerebral ischemia was investigated. To this aim, permanent middle cerebral artery occlusion (pMCAO) was performed in male rats. The effects on the infarct volume of some inhibitors, such as tyrosine-6 glycosylated form of the exchanger inhibitory peptide (GLU-XIP), benzamil derivative (CB-DMB) and diarylaminopropylamine derivative (bepridil), and of the NCX activator, FeCl3, were examined. FeCl3, CB-DMB, bepridil and GLU-XIP, a modified peptide synthesized in our laboratory in order to facilitate its entrance into the cells through the glucose transporter, were intracerebroventricularly (i.c.v.) infused. FeCl3 (10 microg/kg) was able to reduce the extension of brain infarct volume. This effect was counteracted by the concomitant icv administration of CB-DMB (120 microg/kg). All NCX inhibitors, GLU-XIP, CB-DMB and bepridil, caused a worsening of the brain infarct lesion. These results suggest that a stimulation of NCX activity may help neurons and glial cells that are not irreversibly damaged in the penumbral zone to survive, whereas its pharmacological blockade can compromise their survival.

In fibroblasts, thrombin induces collagen deposition through activation of a G-protein-coupled receptor, proteinase-activated receptor 1 (PAR(1)). In the current study, we examined whether PAR(1) antagonism inhibits hepatic stellate cell (HSC) activation in vitro and whether it protects against fibrosis development in a rodent model of cirrhosis. A rat HSC line was used for in vitro studies whereas cirrhosis was induced by bile duct ligation (BDL). The current results demonstrated that HSCs express PAR(1), as well as proteinase-activated receptors 2 (PAR(2)) and 4 (PAR(4)), and that all three PARs were up-regulated in response to exposure to growth factor in vitro. Exposure to thrombin and to SFLLRN-(SF)-NH(2), a PAR(1) agonist, and GYPGKF (GY)-NH(2), a PAR(4) agonist, triggered HSC proliferation and contraction, as well as monocyte chemotactic protein-1 (MCP-1) production and collagen I synthesis and release. These effects were inhibited by the PAR(1) antagonist. Administration of this antagonist, 1.5 mg/kg/d, to BDL rats reduced liver type I collagen messenger RNA (mRNA) expression and surface collagen by 63%, as measured by quantitative morphometric analysis. Similarly, hepatic and urinary excretion of hydroxyproline was reduced significantly by the PAR(1) antagonist. In conclusion, PAR(s) regulates HSC activity; development of PAR antagonists might be a feasible therapeutic strategy for protecting against fibrosis in patients with chronic liver diseases.

Objectives: Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor proteolytically activated by trypsin, tryptase or factor Xa. Alternatively, PAR2 can be activated by synthetic peptides whose sequence mimics the tethered ligand exposed after receptor cleavage. It is known that PAR2 modulates vascular reactivity, both in vitro and in vivo. The present study was designed to investigate the role of basal nitric oxide and cyclic nucleotides, adenosine 3'5'cyclic monophosphate (cAMP) and guanosine 3'5' cyclic monophosphate (cGMP), in the vasorelaxation induced by a PAR2-activating peptide (PAR2-AP; SLIGRL-NH(2)) on rat aorta in vitro., Methods: A concentration-response curve to PAR2-AP was performed on rat thoracic aorta with or without a functional endothelium, and the effect of inhibitors was evaluated. The effect of PAR2-AP (10(-7)-3 x 10(-5) M) on endothelium-denuded aorta was also evaluated after tissue incubation with sodium nitroprusside., Results: PAR2-AP (10(-7)-3 x 10(-5) M) caused an endothelium-dependent relaxation abolished by N(omega)-nitro-L-arginine methyl ester, L-NAME, and by the guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), but unaffected by geldanamycin. Vasorelaxant effect of PAR2-AP was only partially inhibited by the adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), and it was increased by tissue incubation with the phosphodiesterase inhibitor, rolipram. On tissue without endothelium, PAR2-AP did not cause vasorelaxation, up to a concentration of 10(-4) M. However, after tissue incubation with sodium nitroprusside (SNP, 3 x 10(-9) M), the vasorelaxant effect of PAR2-AP was restored. Following tissue incubation with PAR2-AP, cAMP levels were significantly increased compared to control values., Conclusions: Our results suggest that vasorelaxation induced by PAR2-AP is modulated by basal nitric oxide with an involvement of both cyclic nucleotides, cGMP and cAMP.

As part of a search for new potassium channel openers, the synthesis and vasorelaxant activity of new 1,4-benzoxazine derivatives derived from transformation of the benzopyran skeleton of cromakalim were described. Several new 1,4-benzoxazine derivatives were provided with significant vasorelaxant activity with an overall pharmacological behavior similar to CRK (1f, 1i, 2d, 2e, 2f and 2i).

A series of novel 1,2,3-4-benzoyltriazole and saccharine derivatives were designed and synthesized by microwave heating. They were evaluated on a battery of receptors, including serotonin 5-HT(1A,) 5-HT(2A) and 5-HT(2C), and the most interesting compounds were further evaluated on dopaminergic D(1), D(2) and adrenergic alpha(1), alpha(2) receptors. Conventional and microwave heating of the reactions were compared. Synthesis by microwave heating gave the desired compounds in better yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. All compounds displayed moderate affinity for 5-HT(1A) receptor. The most interesting compound 33 showed a high affinity (K(i)=93 nM) which was combined with no affinity on the other receptors considered.

We explored the unique substrate specificity of the primary S, subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from Phenylacetyl-Phe-Ser-Arg-EDDnp, a previously described inhibitor with analgesic and anti-inflammatory activities [Emim et al., Br. J. Pharmacol. 130 (2000), 1099-1107]. Phe was substituted by amino acids containing larger aliphatic or aromatic side chains as well as by non-natural basic amino acids, which were designed to combine a large hydrophobic and/or aromatic group with a positively-charged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the non-natural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.

The change of selectivity and the induction of antagonism by the insertion of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the second position of several opioid peptides have led to the interpretation of Tyr-Tic as a specific message domain for delta-opioid antagonists and to the discovery of dipeptides with substantial opioid activity. Selectivity and activity increase enormously when Tyr is substituted by 2',6'-dimethyl tyrosine (Dmt), hinting that the side chain of Dmt fits a hydrophobic cavity of the receptor very tightly and precisely. We have investigated the specificity of this fit by systematic changes of the substituents on the aromatic ring of ryr. Mono- and disubstitutions different from 2',6'- invariably lead to catastrophic decreases of activity. The only substitution compatible with retention of substantial antagonism is 2-methyl. An analysis of the conformational properties of all analogues reveals that substitutions do not affect the global shape of the molecule significantly. Accordingly, it is possible to use the shape of the different side chains to map the hydrophobic cavity of the receptor. The resulting complementary image is funnel shaped.

SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, anti-inflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure-function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule.

Parsalmide (5-amino-N-butyl-2-(2-propynyloxy) benzamide) (5a), is a non-steroidal anti-inflammatory drug (NSAID), commercialised in Italy until 1985 with the brand name of Synovial(R), that has been widely used to treat arthritic patient. In addition, it was shown to spare gastric mucosa. Here we have synthesised a series of novel substituted benzamides, related to Parsalmide, and have evaluated their activity in vitro on COX-1 and COX-2 as well as in vivo in the carrageenin-induced rat paw edema, a classical in vivo anti-inflammatory assay. Compounds 5b, 11a and 11b, which showed a favourable profile in vitro and in vivo, were screened in comparison with Parsalmide for gastrointestinal (GI) tolerability in vivo in the rat. Results obtained showed that Parsalmide and compound 11b inhibited both COX-1 and COX-2 in vitro as well as they were active in vivo. Both compounds were devoid of gastric effect at the efficacious dose. In addition, both prevented indomethacin-induced gastric damage. Thus, these compounds may guide the definition of a new leading structure with anti-inflammatory activity that may allow designing new safer NSAIDs.

Nitric oxide synthase (NOS) catalyses the conversion of L-arginine to nitric oxide (NO) which plays an important role in the regulation of cellular functions and intracellular communications. Three distinct isoforms of NOS have so far been identified, two constitutive and one inducible. All three mammalian isoforms of NOS contain putative CaM-binding domains with the canonical composition. In this paper we report the synthesis and the inhibitory activity on rat neuronal and lung inducible NOS of antisense peptides corresponding to the antisense strand read in 3' to 5' (CALM 1) or 5' to 3' (CALM 2) direction of the region encoding for the CaM-binding domain of the inducible NOS isoform (residues 503-522). CALM 1 inhibited, at all the concentrations tested (0.01-1 mM), both the inducible and constitutive NOS (IC(50) 98 microM and 56 microM, respectively), while CALM 2 (0.01-1 mM) was ineffective on both isoforms. The acetylation of CALM 1 at its amino terminal (CALM 8) completely abolished its inhibitory activity. We also synthesized and analysed the activity of amino terminal truncated analogues (CALM 3-7) of CALM 1, which selectively inhibited the inducible isoform, although less potently than the parent compound. The pentapeptides (CALM A-D) deriving from the cleavage of CALM 1 were ineffective, except the pentapeptide CALM C corresponding to the residues 513-517, which was as potent as the parent compound (IC(50) 65 microM).

A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT1A receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and alpha1-, alpha2-adrenergic receptors. A o-OCH3 derivative (2e) bound at 5-HT1A sites with subnanomolar affinity (IC50 = 0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents.

A study was performed on the structure-activity relationships of a series of phenol derivatives, CVFM analogs, derived from the two most active compounds of a first series (1A and 1B) of inhibitors of Ras farnesyl transferase (FTase) that we have recently described. We report the synthesis and the activity of a second series of compounds in which the phenylalanine residue was replaced by unconventional aromatic and non-aromatic amino acids, with varying electronic, lipophilic, steric and conformational properties. The compounds showed to be significantly less active than reference compounds against FT, with the only exception of derivative 3A (IC50 = 3 microM), which is slightly more active than 1A but not 1B. Subsequently we tested the effects of compounds 1A, 1B and 3A, 3B on the anchorage-dependent growth of two epithelial cell lines of rats, FRTL-5 and the same line v-Ha-ras transformed. Compound 3A derived from lead compound 1A, showed an appreciable selectivity against transformed cells. In contrast, compounds derived from derivative 1B had only a modest cellular activity.