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Twelve novel conformationally constrained homologues of glutamic acid have been synthesized and pharmacologically characterized at ionotropic glutamate receptors (iGluRs). Synthesis of the target compounds involved 1,3-dipolar cycloaddition of nitrile oxides to suitable dipolarophiles. The structure to the compounds has been assigned by (1)H NMR and, in the case of derivatives (+/-)-4a, (+/-)-4b, (+/-)-5a, and (+/-)-5b, by means of an X-ray crystallographic analysis carried out on intermediate (+/-)-12a. The synthesized amino acids were found to be without affinity (K(i)/IC(50)>100microM) for iGluRs with the exception of compounds (+/-)-4b and (+/-)-5b, which showed a modest affinity for NMDA receptors (K(i)=34 and 13microM, respectively). The results indicate that the increased conformational constraints introduced by the cyclopropane ring and the spiro-attached proline ring are both detrimental to the pharmacological activity.

Aim: To investigate the effect of a new oral preparation, highly concentrated in fish cartilage, in a group of inflammatory bowel diseases (IBD) patients with chronic iron deficient anemia., Methods: In an open label pilot study, we supple-mented a group of 25 patients (11 with Crohn's disease and 14 with ulcerative colitis) in stable clinical conditions and chronic anemia with a food supplement which does not contain iron but contains a standardized fraction of fish cartilage glycosaminoglycans and a mixture of antioxidants (Captafer Medestea, Turin, Italy). Patients received 500 mg, twice a day during meals, for at least 4 mo. Patients were suggested to maintain their alimentary habit. At time 0 and after 2 and 4 mo, emocrome, sideremia and ferritin were examined. Paired data were analyzed with Student's t test., Results: Three patients relapsed during the study (2 in the 3rd mo, 1 in the 4th mo), two patients were lost to follow up and two patients dropped out (1 for orticaria, 1 for gastric burning). Of the remaining 18 patients, levels of serum iron started to rapidly increase within the 2nd mo of treatment, P < 0.05), whereas serum ferritin and hemoglobin needed a longer period to significantly improve their serum levels (mo 4) P < 0.05. The product was safe, easy to administer and well tolerated by patients., Conclusion: These data suggest a potential new treatment for IBD patients with iron deficiency chronic anemia and warrant further larger controlled studies.

Aim: To develop a new formulation with hydroxy propyl methyl cellulose and Shellac coating for extended and selective delivery of butyrate in the ileo-caecal region and colon., Methods: One-gram sodium butyrate coated tablets containing 13C-butyrate were orally administered to 12 healthy subjects and 12 Crohn's disease patients and the rate of 13C-butyrate absorption was evaluated by 13CO2 breath test analysis for eight hours. Tauroursodeoxycholic acid (500 mg) was co-administered as a biomarker of oro-ileal transit time to determine also the site of release and absorption of butyrate by the time of its serum maximum concentration., Results: The coated formulation delayed the 13C-butyrate release by 2-3 h with respect to the uncoated tablets. Sodium butyrate was delivered in the intestine of all subjects and a more variable transit time was found in Crohn's disease patients than in healthy subjects. The variability of the peak 13CO2 in the kinetic release of butyrate was explained by the inter-subject variability in transit time. However, the coating chosen ensured an efficient release of the active compound even in patients with a short transit time., Conclusion: Simultaneous evaluation of breath 13CO2 and tauroursodeoxycholic acid concentration-time curves has shown that the new oral formulation consistently releases sodium butyrate in the ileo-cecal region and colon both in healthy subjects and Crohn's disease patients with variable intestinal transit time. This formulation may be of therapeutic value in inflammatory bowel disease patients due to the appropriate release of the active compound.

The identification of new biomarkers or a disease-related protein fingerprint for inflammatory bowel diseases (IBDs) represents a major task in the diagnosis, prognosis and pharmacological therapy. To address these issues, a simple and rapid analytical proteomic method for serum protein profiling based on selective beads-based solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and chemometric data analysis was developed. Serum proteins from healthy subjects (22) and patients with Crohn's disease (15) and ulcerative colitis (26) were selectively extracted according to reversed-phase (C18), strong anion-exchange (SAX) and metal ion affinity (IDA-Cu(II)) principles. This approach allowed enrichment of serum proteins/peptides due to the high interaction surface between analytes and the solid phase and high recovery due to the elution step performed directly on the MALDI-target plate. The MALDI-TOF MS serum protein profiles were acquired and, after a data pre-processing step, analyzed by linear discriminant analysis (LDA), a chemometric classification technique, in order to classify serum samples among healthy subjects and patients with inflammatory bowel diseases (IBDs). Since the high number of variables in the MALDI spectra (more than 16000 m/z values) prevents the use of LDA, the variables were reduced to 10-20 by features selection, thus allowing the evaluation of a pattern of m/z values with high discriminant power. Serum protein profiles obtained by reversed-phase extraction and the selection of 20 m/z values gave the best overall prediction ability (96.9%). The recognition of these m/z values may allow the identification of protein biomarkers involved in IBDs., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Background & Aims: Secreted immunoglobulins play an integral role in host defense at mucosal surfaces, and recent evidence shows that IgG can participate in antigen sampling from the intestinal lumen. We examined whether IgE also could facilitate transepithelial antigen sampling., Methods: Stool samples from food-allergic patients undergoing oral food challenge were analyzed for CD23 and food-specific IgE. CD23 isoform expression on primary human intestinal epithelial cells (IEC) was analyzed by polymerase chain reaction. The role of CD23 isoforms in transcytosis of antigen and IgE-antigen complexes was assessed using polarized human T84 cells retrovirally transfected with CD23a or CD23b., Results: CD23 was expressed constitutively on IECs, and food-allergic patients had increased levels of soluble CD23 and food-specific IgE in the stool after challenge. CD23a, but not CD23b, was expressed by primary human IECs. We show in transcytosis assays that CD23a, but not CD23b, acts as a bidirectional transporter of IgE. In addition, specific IgE facilitated the uptake of antigen from the apical surface of an epithelial monolayer by diverting antigen from delivery to lysosomes. Finally, delivery of antigen-IgE complexes across the epithelial barrier could induce the degranulation of rat basophil leukemia cells transfected with the human high-affinity IgE receptor., Conclusions: These studies show that CD23a is expressed normally on human IECs, and in the presence of IgE can function as an antigen-sampling mechanism capable of activating subepithelial mast cells. IgE may serve as a secretory immunoglobulin that in concert with CD23 participates in food-induced pathophysiology of the gastrointestinal tract.

This paper describes the synthesis of racemic 3,5-dihydro-5-methyl-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one (+/-)-5, attempted stereoselective synthesis of its enantiomers, chiral HPLC resolution of the racemate, and assignment of the absolute configuration. Enantiomer (5S)-(-)-5 is provided with an in vivo anticonvulsant activity 8 times higher than its enantiomer (5R)-(+)-5. This result is confirmed in the in vitro test by the ability to inhibit the kainate-induced increase of the [Ca(2+)](i) in a primary culture of rat cerebellar granule cells which express alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. Binding affinity of compound (+/-)-5 at the AMPA and N-methyl-d-aspartic acid (NMDA) receptors was also evaluated.

The four stereoisomers of 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid(+)-4, (-)-4, (+)-5, and (-)-5 were prepared by stereoselective synthesis of two pairs of enantiomers, which were subsequently resolved by enzymatic procedures. These four stereoisomers and the four stereoisomers of the bicyclic analogue 5-amino-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-3,5-dicarboxylic acid (+)-2, (-)-2, (+)-3, and (-)-3 were tested at ionotropic and metabotropic glutamate receptor subtypes. The most potent NMDA receptor antagonists [(+)-2, (-)-4, and (+)-5] showed a significant neuroprotective effect when tested in an oxygen glucose deprivation (OGD) cell culture test. The same compounds were preliminarily assayed using Xenopus oocytes expressing cloned rat NMDA receptors containing the NR1 subunit in combination with either NR2A, NR2B, NR2C, or NR2D subunit. In this assay, all three derivatives showed high antagonist potency with preference for the NR2A and NR2B subtypes, with derivative (-)-4 behaving as the most potent antagonist. The biological data are discussed on the basis of homology models reported in the literature for NMDA receptors and mGluRs.

N1-substituted bicyclic pyrazole amino acids (S)-9a-9c and (R)-9a-9c, which are conformationally constrained analogues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested for activity at ionotropic and metabotropic glutamate receptors. Some of them turned out to be selective for the NMDA receptors, where they behaved as weak antagonists. The biological activity is mainly due to the interaction with the glutamate binding site, and not with the glycine co-agonist site.

The two diastereomeric pairs of acidic amino acids 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid (8A/8B) and 4-(2-amino-2-carboxyethyl)-5,5-dimethyl-4,5-dihydroisoxazole-3-carboxylic acid (10A/10B) were prepared via a strategy based on a 1,3-dipolar cycloaddition. The four amino acids were tested at ionotropic and metabotropic glutamate receptors. None of the compounds was active, neither as agonists nor as antagonists, at 1 mM on metabotropic receptors (mGluR1, -2, -4, and -5 expressed in CHO cell lines). Conversely, the pair of stereoisomers 8A/8B showed a remarkable affinity, antagonist potency, and selectivity for NMDA receptors, when tested on ionotropic glutamate receptors. The affinity of 8A proved to be 5 times higher than that of diastereomer 8B (K(i) values 0.21 and 0.96 microM, respectively). Furthermore, compounds 8A and 8B exhibited a noteworthy anticonvulsant activity in in vivo tests on DBA/2 mice. Derivative 10A was inactive at all ionotropic glutamate receptors, whereas its stereoisomer 10B displayed a seizable binding to both NMDA and AMPA receptors.

A set of racemic conformationally constrained analogues of the antitumor antibiotic acivicin (+)-1 has been prepared through a strategy based on 1,3-dipolar cycloaddition of bromonitrile oxide to suitable dipolarophiles. The bromo analogue (2) of acivicin was also synthesized and tested as a reference compound, together with its stereoisomer 3. The antitumor properties of novel amino acids 4-7 were evaluated in vitro against human tumor cell lines. Their efficacy to inhibit glutamate synthase (GltS) from Azospirillum brasilense was also assayed. None of the studied compounds, but 2, showed significant activity.

We compared the intra-operative diagnostic value of CDS vs IA-DSA in identifying major and minor technical defects after CEA. Between August 1997 and December 1998, 138 consecutive patients undergoing 141 carotid endarterectomies were intra-operatively investigated with colour duplex scan and intra-arterial digital subtraction angiography. Thirty-six (25.5%) technical defects were identified. Four (11.1%) major defects were detected by both methods and they were immediately corrected. Fifteen (41.6%) minor defects were detected by both methods, thirteen (36.1%) minor defects were detected by colour duplex but ignored by angiography. Angiography detected four (11.1%) kinkings missed with the colour duplex. The overall sensitivity of both methods for major defect was 100%. The sensitivity of colour duplex for minor defects was 87% vs 59% for angiography. On the basis of our study, colour duplex could be considered the choice method for quality control after carotid endarterectomy.

Endomyocardial biopsy is an essential procedure for the diagnosis and grading of rejection in heart transplant patients. Direct control of the bioptome positioning has classically been obtained by fluoroscopy. Starting in June 1988, at our institution an alternative approach involving the use of two-dimensional echocardiography was introduced in clinical practice. In 125 patients 1591 biopsies have been performed: 445 under echographic control and 1146 under fluoroscopic control with 3.6 and 4.5 samples/biopsy, respectively. The percentages of inadequate samples caused by biopsy site sampling were 0.4% and 1.3%, respectively, in the two groups. Cardiac perforation has occurred twice in the fluoroscopic group; it has not been observed in the echographic group. One case of iatrogenic tricuspid regurgitation was detected in each group. We now consider echocardiography the method of choice to guide the bioptome. We prefer it to fluoroscopy because it eliminates the risks of x-ray exposure, increases the number of sampling sites in cases of echocardiographic evidence of rejection, can be easily performed as a bedside procedure, allows choice and variation of sampling sites, and permits monitoring of cardiac complications during and after the procedure. A randomized clinical trial is probably needed to assess with statistical significance the superiority of the echographic-controlled biopsy.

A brain specific protein, 14-3-2, has been isolated from bovine brain by the use of ammonium sulfate precipitation, gel filtration on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose and DEAE-Sephadex. It is an acidic protein, in agreement with the high content of glutamic and aspartic acid, that is composed of a single polypeptide chain of mol. wt. 50,000. Immunochemical tests using antiserum to purified 14-3-2 showed that the protein is present in at least 100-fold greater amounts in brain than in any other rat tissue. Furthermore, 14-3-2 was found in brains of a number of vertebrate species, although the antigen is apparently not entirely identical in all species tested. The protein 14-3-2 can be considered to be a species non-specific protein which is neuronal in origin.

Doppler characteristics of normally functioning tilting disk prostheses in aortic position were studied in 55 patients (30 Medtronic-Hall and 25 Sorin) whose valvular function was considered normal using clinical and echocardiographic evaluation. Peak gradients, mean gradients and effective orifice area were estimated for different sizes of prostheses. The peak gradient calculated from maximal aortic velocity was 27.3 +/- 11.1 mmHg in Sorin and 21.1 +/- 9.7 mmHg in Medtronic-Hall valves; the mean gradients were 12.9 +/- 6.2 mmHg and 10.8 +/- 5.7 mmHg in Sorin and Medtronic-Hall valves respectively. The effective orifice area calculated by the continuity equation was 1.4 +/- 0.5 cm2 in Sorin and 1.5 +/- 0.57 cm2 in Medtronic-Hall prostheses; the performance index calculated as the ratio between functional area and manufactured area was 0.4-0.6 for Medtronic-Hall and 0.45-0.52 for Sorin prostheses. Prosthetic regurgitation was found in 64% of Sorin valves and 80% of Medtronic-Hall valves; prosthetic regurgitation was mild in 81% and moderate in 19% of cases. Doppler echocardiography is a reliable method for the characterization of the normal function of prosthetic aortic valves and provides information similar to cardiac catheterization.

Ten normal rhesus monkeys, five juvenile and five adult animals, were analyzed for symmetry in bone density across the cranioskeleton with computed tomography (CT). Fifty contiguous axial CT scans were done using 1.5-mm thick scans between the lower mandibular border and inferior orbital ridge, and 3-mm thick scans from the inferior orbital ridge to the rostral level of insertion of the temporalis muscle. Eleven regions comprising areas of the lower mandibular border, lower ramus, upper ramus with condyle, the coronoid process, the zygomatic arch, and cranial bone were analyzed for symmetry using small regions of interest. Only one region, the condyle, demonstrated a statistically significant difference between the two sides with all other measured sites demonstrating the same bone mineral density bilaterally. Large area analysis of five regions--the lower mandibular border, upper ramus with condyle, the coronoid process, the zygomatic arch, and upper cranium--demonstrated no significant difference in the distribution of CT numbers. This analysis of bilateral distribution of bone density in the primate craniofacial skeleton demonstrates a basic symmetry that can be used to study altered patterns of neuromuscular function and their effects on bone density.

Two aspects of the subunit structure of the bovine brain specific protein 14-3-2 have been examined. On the one hand, native 14-3-2 has been separated into two fractions by hydroxylapatite chromatography. One eluted at the same position when chromatographed on the same column, while the other redistributed into the same two fractions again. Amino acid analysis of these two forms of 14-3-2 gave results that were not significantly different under the condition of analysis. Furthermore, when each peak was subjected to cyanogen bromide cleavage, very similar elution profiles of the resultant fragments were obtained. The two pools also cross-reacted with antiserum to 14-3-2. Reaction of purified 14-3-2 with dimethylsuberimidate caused the formation of covalently bound protein units of 100,000 molecular weight when measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, as opposed to the 50,000 minimal molecular weight normally detected. On the other hand, analysis of the soluble tryptic peptides of S-[14C]carboxymethyl 14-3-2 yielded only three distinct radioactive peptides, each with one residue of S-carboxymethylcysteine whereas 8 are expected on the basis of the amino acid composition of the 50,000 molecular weight polypeptide chains. Thermolysin digestion of a similarly modified 14-3-2 preparation yielded all of the radioactivity in 5 S-carboxymethylcysteine-containing peptides. The partial amino acid sequence of these peptides indicates that they represent 4 unique areas of the polypeptide chain. Since 8 such peptides were expected, that is, double the number found, the minimum structural unit of the protein must be of 25,000 molecular weight. The results of these experiments do not permit distinction between a duplication of the structure within a single polypeptide chain or the alternate possibility of two polypeptide chains bound by unusually strong non-covalent bonds. These results suggest that 14-3-2 is a covalently linked dimer of 25,000 mol.wt. units that can aggregate to form larger species of 100,000 mol.wt. and higher.