Review article: oral ulceration--aetiopathogenesis, clinical diagnosis and management in the gastrointestinal clinic

Oral ulceration is a common complaint of patients attending out‐patient clinics. The aim of this review is to provide the gastroenterologist with a differential diagnosis of oral ulceration, and a practical guide for the management of recurrent aphthous stomatitis, including topical and systemic therapy. The association of recurrent aphthous stomatitis with Behçet's disease and other systemic disorders, including coeliac disease, is discussed. Recent evidence concerning the immunopathogenesis of Behçet's disease is reviewed, including renewed interest in the role of Streptococcus sanguis and possible infectious triggering of an inappropriate immunoinflammatory response, resulting in tissue damage. The efficacy and limitations of conventional treatment for this mutisystem disorder are outlined together with the potential role of novel biological agents, such as anti‐tumour necrosis factor‐α therapy. Oral ulceration, as a manifestation of inflammatory bowel disease and a complication of drug therapy, is described. Guidance is given concerning indications for referral of patients with oral ulceration to an oral physician/surgeon for further investigations, including biopsy if appropriate.     

Characterizing the Free‐Energy Landscape of MDM2 Protein–Ligand Interactions by Steered Molecular Dynamics Simulations

Inhibition of p53–MDM2 interaction by small molecules is considered to be a promising approach to re‐activate wild‐type p53 for tumor suppression. Several inhibitors of the MDM2–p53 interaction were designed and studied by the experimental methods and the molecular dynamics simulation. However, the unbinding mechanism was still unclear. The steered molecular dynamics simulations combined with Brownian dynamics fluctuation–dissipation theorem were employed to obtain the free‐energy landscape of unbinding between MDM2 and their four ligands. It was shown that compounds 4 and 8 dissociate faster than compounds 5 and 7 . The absolute binding free energies for these four ligands are in close agreement with experimental results. The open movement of helix II and helix IV in the MDM2 protein‐binding pocket upon unbinding is also consistent with experimental MDM2‐unbound conformation. We further found that different binding mechanisms among different ligands are associated with H‐bond with Lys51 and Glu25. These mechanistic results may be useful for improving ligand design.     

Induction of carbohydrate‐specific antibodies in HLA‐DR transgenic mice by a synthetic glycopeptide: a potential anti cancer vaccine for human use

Abstract: Over the last few years, anticancer immunotherapy has emerged as a new exciting area for controlling tumors. In particular, vaccination using synthetic tumor‐associated antigens (TAA), such as carbohydrate antigens hold promise for generating a specific antitumor response by targeting the immune system to cancer cells. However, development of synthetic vaccines for human use is hampered by the extreme polymorphism of human leukocyte‐associated antigens (HLA). In order to stimulate a T‐cell dependent anticarbohydrate response, and to bypass the HLA polymorphism of the human population, we designed and synthesized a glycopeptide vaccine containing a cluster of a carbohydrate TAA B‐cell epitope (Tn antigen: α‐GalNAc‐Ser) covalently linked to peptides corresponding to the Pan DR ‘universal’ T‐helper epitope (PADRE) and to a cytotoxic T lymphocyte (CTL) epitope from the carcinoembryonic antigen (CEA). The immunogenicity of the construct was evaluated in outbred mice as well as in HLA transgenic mice (HLA‐DR1, and HLA‐DR4). A strong T‐cell dependent antibody response specific for the Tn antigen was elicited in both outbred and HLA transgenic mice. The antibodies induced by the glycopeptide construct efficiently recognized a human tumor cell line underlying the biological relevance of the response. The rational design and synthesis of the glycopeptide construct presented herein, together with its efficacy to induce antibodies specific for native tumor carbohydrate antigens, demonstrate the potential of a such synthetic molecule as an anticancer vaccine candidate for human use.     

Significantly different contact patterns between Aβ40 and Aβ42 monomers involving the N‐terminal region

Aβ42 peptide, with two additional residues at C‐terminus, aggregates much faster than Aβ40. We performed equilibrium replica‐exchange molecular dynamics simulations of their monomers using our residue‐specific force field. Simulated ³J_HNHα‐coupling constants agree excellently with experimental data. Aβ40 and Aβ42 have very similar local conformational features, with considerable β‐strand structures in the segments: A2‐H6 ( A ), L17‐A21 ( B ), A30‐V36 ( C ) of both peptides and V39‐I41 ( D ) of Aβ42. Both peptides have abundant A‐B and B‐C contacts, but Aβ40 has much more contacts between A and C than Aβ42, which may retard its aggregation. Only Aβ42 has considerable A ‐ B ‐ C ‐ D topology. Decreased probability of A ‐ C contact in Aβ42 relates to the competition from C ‐ D contact. Increased A ‐ C contact probability may also explain the slower aggregation of A2T and A2V mutants of Aβ42.     

Design of potent B‐RafV600E inhibitors by multiple copy simulation search strategy

B‐Raf kinase is a vital intermedium in the mitogen‐activated protein kinase (MAPK) signaling pathway, which transforms extracellular signals into cellular mechanisms. Mutations in this kinase, for instance, the most common V600E mutation, can lead to the ERK signaling pathologically activated and hence cause severe diseases such as somatic tumors. So far, the development of B‐Raf inhibitors has made remarkable progress. However, the resistance and relapse of approved Raf drugs have been widely reported, and the optimization for old drugs and the discovery for new inhibitors still remain a significant task. In this study, we designed and evaluated a series of novel B‐Raf^V600E inhibitors. A fragment library has been established before the docking simulation carried out using the MCSS strategy (multicopy simulation search). The appropriate fragments were reassembled to provide new candidate compounds, which were further screened by iterative docking simulations and molecular dynamics. Bioassays were carried out to evaluate the pharmacological profile of the compounds identified and synthesized. The result showed that compound 5n had an impressive enzyme inhibitory and antiproliferation activity, suggesting a promising potential in the future study.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

Molecular dynamics insights on the role β‐augmentation of the peptide N‐terminus with binding site β‐hairpin of proprotein convertase subtilisin/kexin 9

PCSK9, a member of the proprotein convertase family, is a key negative regulator of hepatic low‐density lipoprotein receptor (LDLR) concentrations in the blood plasma and is associated with the risk of coronary artery disease (CAD). Peptide inhibitors designed to block PCSK9‐LDLR interactions could reduce the risk of CAD. We present a study of the interaction of a PCSK9 bound peptide and its design through modification by phosphorylation using molecular dynamics simulations. Extensive explicit solvent simulations of PCSK9 and its mutant (Asp374 → Tyr374) with designed peptides provide insights into the mechanism of peptide binding at the protein interface. We establish that β‐augmentation is the key mechanism of peptide association with PCSK9. Position‐specific phosphorylation of threonine residues is observed to have noticeable effect in modulating protein–peptide association. This study provides a handle to explore and improve the design of peptides bound to PCSK9 by incorporating knowledge‐derived functional motifs into designing potent binders.     

Folding study of an Aib‐rich peptide in DMSO by molecular dynamics simulations

Abstract: To evaluate the ability of molecular dynamics (MD) simulations using atomic force‐fields to correctly predict stable folded conformations of a peptide in solution, we show results from MD simulations of the reversible folding of an octapeptide rich in α‐aminoisobutyric acid (2‐amino‐2‐methyl‐propanoic acid, Aib) solvated in di‐methyl‐sulfoxide (DMSO). This solvent generally prevents the formation of secondary structure, whereas Aib‐rich peptides show a high propensity to form secondary structural elements, in particular 3₁₀‐ and α‐helical structures. Aib is, moreover, achiral, so that Aib‐rich peptides can form left‐ or right‐handed helices depending on the overall composition of the peptide, the temperature, and the solvation conditions. This makes the system an interesting case to study the ensembles of peptide conformations as a function of temperature by MD simulation. Simulations involving the folding and unfolding of the peptide were performed starting from two initial structures, a right‐handed α‐helical structure and an extended structure, at three temperatures, 298 K, 340 K, and 380 K, and the results are compared with experimental nuclear magnetic resonance (NMR) data measured at 298 K and 340 K. The simulations generally reproduce the available experimental nuclear Overhauser effect (NOE) data, even when a wide range of conformations is sampled at each temperature. The importance of adequate statistical sampling in order to reliably interpret the experimental data is discussed.     

Combined 5‐hydroxymethylcytosine content of human leucocyte antigen‐B and human leucocyte antigen‐DQB1 as novel biomarker for anti‐tuberculosis drug‐induced liver injury

This study investigated the diagnostic value of 5‐methylcytosine (5‐mC) and 5‐hydroxymethylcytosine (5‐hmC) contents of human leucocyte antigen (HLA)‐B and HLA‐DQB1 in anti‐tuberculosis drug‐induced liver injury (ADLI). In total, 110 ADLI patients and 120 patients without ADLI controls were enrolled. Enzyme‐linked immunosorbent assay (ELISA) was used to detect the 5‐mC and 5‐hmC content in DNA from peripheral blood leucocytes. The univariate analysis showed that smoking, drinking, and 5‐mC and 5‐hmC content of HLA‐B and HLA‐DQB1 were significantly associated with ADLI. After adjusting for drinking and smoking, we found that 5‐mC content of HLA‐B and HLA‐DQB1 were associated with ADLI (odds ratio [OR] = 0.251 and 0.347, respectively) and 5‐hmC contents of HLA‐B and HLA‐DQB1 were also associated with ADLI (OR = 1.848 and 4.705, respectively). Receiver operating characteristic (ROC) analysis indicated that the 5‐hmC contents of both HLA‐B and HLA‐DQB1 were more clinically significant than the 5‐mC contents were. The combined 5‐hmC level of HLA‐B and HLA‐DQB1 was the best diagnostic biomarker for ADLI, with the highest areas under the curve (AUC) for 0.953, sensitivity for 0.900 and specificity for 0.875. Therefore, combined 5‐hmC levels of HLA‐B and HLA‐DQB1 could be significant evidence for diagnosis of ADLI.     

Human leucocyte antigens (HLA) and rheumatic diseases: HLA class ii antigen-associated diseases

The association between genes of the major histocompatibility complex, HLA in man, and disease susceptibility is one of the most intriguing discoveries of modern genetics. These studies are important for understanding the genetic basis, pathogenesis and aetiology of human diseases. With progress in these areas, new and better forms of treatment as well as disease prevention will become possible. In the previous paper, we reviewed the literature on associations between HLA Class I (A,B,C) genes and rheumatic disease. In this article, we will now concentrate on rheumatic disorders associated with genes in the HLA Class II region, including DR, DQ, DP and the transporter associated with antigen processing (TAP) genes. In addition, the role of the pathogenic mechanism(s), which results in an aberrant expression(s) of HLA molecules on the cell surface, in causing disease susceptibility will be examined.     

Antifungal screening and in silico mechanistic studies of an in‐house azole library

Systemic Candida infections pose a serious public health problem with high morbidity and mortality. C. albicans is the major pathogen identified in candidiasis; however, non‐albicans Candida spp. with antifungal resistance are now more prevalent. Azoles are first‐choice antifungal drugs for candidiasis; however, they are ineffective for certain infections caused by the resistant strains. Azoles block ergosterol synthesis by inhibiting fungal CYP51, which leads to disruption of fungal membrane permeability. In this study, we screened for antifungal activity of an in‐house azole library of 65 compounds to identify hit matter followed by a molecular modeling study for their CYP51 inhibition mechanism. Antifungal susceptibility tests against standard Candida spp. including C. albicans revealed derivatives 12 and 13 as highly active. Furthermore, they showed potent antibiofilm activity as well as neglectable cytotoxicity in a mouse fibroblast assay. According to molecular docking studies, 12 and 13 have the necessary binding characteristics for effective inhibition of CYP51. Finally, molecular dynamics simulations of the C. albicans CYP51 (CACYP51) homology model's catalytic site complexed with 13 were stable demonstrating excellent binding.     

Structural and immunogenicity analysis of chimeric B‐cell epitope constructs derived from the gp46 and gp21 subunits of the envelope glycoproteins of HTLV‐1

Abstract: B‐cell epitopes were selected from the gp21 and gp46 subunits of the envelope glycoprotein of human T‐cell lymphotropic virus type 1 (HTLV‐1) by computer‐aided analyses of protein antigenicity. Molecular modeling was used to design and synthesize the epitopes as chimeric constructs with promiscuous T‐helper epitopes derived either from the tetanus toxoid (amino acids 947–967) or measles virus fusion protein (amino acids 288–302). Circular dichroism measurements revealed that the peptides had a secondary structure that correlated well with the crystal structure data or predicted structure. The chimeric peptides were then evaluated for their immunogenicity in rabbits or mice. Antibodies against one of the epitopes derived from the gp21 subunit were found to be neutralizing in its ability to inhibit the formation of virus‐induced syncytia. These studies underscore the importance of the gp21 transmembrane region for the development of vaccine candidates. The applicability of a chimeric approach is discussed in the context of recent findings regarding the role of gp21 transmembrane region in the viral fusion process.     

The Chemical Shift Index method applied to resin‐bound peptides

Abstract: The Chemical Shift Index (CSI) method proposed by Wishart et al. [Biochemistry (1992) 31 , 1647–1651] to evaluate the secondary structure of peptides in aqueous solution uses as its reference the chemical shift values of each of the 20 natural amino acids (X) in a typical nonstructured sequence GGXAGG (17–20). In order to apply the CSI method to protected resin‐bound peptides, we established a new database of chemical shift values for the same GGXAGG sequences in their protected form and anchored to a polystyrene resin swollen in DMF‐d₇. The predictive value of this new reference set in the CSI protocol was tested on different resin‐bound peptides that were previously characterized by a full NOE analysis.     

Preventive Effects of Unsei-in and Oren-gedoku-to,Chinese Traditional Medicines,Against Rat Paw Oedema and Abdominal Constriction in Mice

Oren-gedoku-to and Unsei-in are complex mixtures of ingredients derived from plants. These two drugs are clinically most frequently used in the treatment of Behçet's disease, we have investigated the anti-inflammatory and analgesic actions of Oren-gedoku-to and Unsei-in using a battery of tests; rat paw oedema induced by five different agents; abdominal constriction; mouse ear swelling; and dye leakage tests, designed to clarify the therapeutic potential of these medicines. The findings in this study that these medicines are able to inhibit the rat paw oedema, suppress the abdominal constriction and inhibit the increased dye permeability confirm the analgesic and anti-inflammatory effects of these compounds. From these results there seems a clear rationale for exploring the effectiveness of these Chinese medicines in the treatment øf chronic and acute inflammatory diseases.     

A θ‐defensin composed exclusively of d‐amino acids is active against HIV‐1

Abstract: The ability of certain θ‐defensins, including retrocyclin‐1, to protect human cells from infection by HIV‐1 marks them as potentially useful molecules. θ‐Defensins composed of l ‐amino acids are likely to be unstable in environments that contain host and microbial proteases. This study compared the properties of two enantiomeric θ‐defensins, retrocyclin‐1, and RC‐112. Although these peptides have identical sequences, RC‐112 is composed exclusively of d ‐amino acids, whereas retrocyclin‐1 contains only l ‐amino acids. We compared the ability of these peptides to protect JC53‐BL human cells from infection by 30 primary HIV‐1 isolates. JC53‐BL cells are modified HeLa cells that express surface CD4, CXCR4, and CCR5. They also contain reporter cassettes that are driven by the HIV‐1 LTR, and express β‐galactosidase and luciferase. The HIV‐1 isolates varied in co‐receptor specificity and included subtypes A, B, C, D, CRF01‐AE, and G. RC‐112 was several fold more potent than retrocyclin‐1 across the entire HIV‐1 panel. Although RC‐112 bound immobilized gp120 and CD4 with lower affinity than did retrocyclin‐1, surface plasmon resonance experiments performed with 1 μg/mL of RC‐112 and retrocyclin‐1 revealed that both glycoproteins were bound to a similar extent. The superior antiviral performance of RC‐112 most likely reflected its resistance to degradation by surface‐associated or secreted proteases of the JC53‐BL target cells. θ‐Defensins composed exclusively of d ‐amino acids merit consideration as starting points for designing microbicides for topical application to the vagina or rectum.     

HLA‐A*24:02 is associated with metronidazole‐induced cutaneous adverse drug reactions in Han Chinese individuals: A pilot case‐control study with both HLA gene and T cell receptor repertoire analysis

Metronidazole, a widely used drug for the treatment of infections with anaerobic and facultative anaerobic bacteria and protozoa, can frequently cause metronidazole‐induced cutaneous adverse reactions (McADRs). The aim of the present study was to investigate the association between human leucocyte antigen (HLA) alleles and McADRs in a Chinese Han population. The frequency of HLA‐B*24:02 carriers among the McADR patients was 73.3%, which was significantly higher than that of the population controls (32.16%, OR = 5.80, 95% CI = [1.80‐18.72], Pc = 0.004) and of the metronidazole‐tolerant patients (26.67%, OR = 7.56, 95% CI = [2.02‐28.35], Pc = 0.004). Molecular docking showed that metronidazole and one of its major metabolites had the potential to bind in the HLA groove and that there was a relatively stable binding state of the HLA‐B*24:02‐metronidazole/the metabolite complex. The CDR3 repertoires of both T cell receptor (TCR)Vα and Vβ of the patients showed a significantly skewed or an oligoclonal distribution. The TCRVβ CDR3 of the patients shared a similar motif, “CASSxxxxxxQxF.” The current study demonstrated that both the HLA‐A*24:02 allele and TCR are involved in the pathogenesis of McADRs.     

Treatment strategies for Behçet's disease

Background: Behçet's disease (BD) is a multisytemic vasculitis characterized by oral and genital ulceration, other skin lesions, uveitis and manifestations affecting the blood vessels, CNS and gastrointestinal system. It is rare in the Western world but is frequent in the Middle and Far East. Objective: The aim of this review is to discuss treatment strategies in BD. These might vary between simple reassurance and a combination of immunosuppressives. Methods: A systematic literature search was done using the Cochrane and Medline databases in June 2008. The EULAR recommendations for the management of BD were also taken into account. Conclusion: The last two decades have witnessed considerable improvement in eye disease and musculoskeletal involvement. However, the treatment of thrombophlebitis, CNS, and gastrointestinal manifestations remains problematic.     

Improved solid‐phase synthesis of α,α‐dialkylated amino acid‐rich peptides with antimicrobial activity*

Abstract: A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid‐phase synthetic techniques. Each nonapeptide was rich in α,α‐dialkylated amino acids [one 4‐aminopiperidine‐4‐carboxylic acid (Api) and six α‐aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9‐fluorenylmethyloxycarbonyl (Fmoc)‐Aib‐Aib‐OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3₁₀‐helical, amphipathic design of these peptides was born out most prominently in the N‐terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations ≤100 μm ) and the acetylated peptides (concentrations ≤200 μm ) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

Characteristic Structural Features of Indolicidin: Effects of the cis‐trans Isomerism on its Conformation

Indolicidin is an antimicrobial peptide showing a broad spectrum of antibacterial and antifungal activities, and according to the cis‐trans isomerism of three Xaa‐Pro peptide bonds, eight different stereoisomers could be distinguished for this peptide. As the cis‐trans isomerism about the Xaa‐Pro peptide bonds was not considered in previous studies, the structural features of distinct stereoisomeric forms were not characterized in detail, so far. In this theoretical study, the influences of cis‐trans isomerism on the conformation of indolicidin were investigated, as well as the typical structural properties of each stereoisomer were determined, focusing on the secondary structures and intramolecular interactions. Based on the results derived from the molecular dynamics simulations, it could be concluded that the eight different stereoisomeric forms of indolicidin adopted characteristic conformational features. Nevertheless, the appearance of various turn structures and intramolecular interactions proved to be dependent on the cis or trans nature of Xaa‐Pro peptide bonds, indicating the relevant role of Pro amino acids in determining the three‐dimensional structure of this peptide.     

A new B‐chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B‐chain structure

Abstract: The solution structure of a new B‐chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D‐NMR and molecular modeling. This structure is compared with that of the oxidized B‐chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res. 46 , 424–433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B‐chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B‐chain. 2D‐NMR experiments confirmed, among multiple conformations, that the B‐chain mutant presents defined secondary structures such as a α‐helix between residues B9 and B19, and a β‐turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B‐chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B‐chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B‐chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B‐chains.