Oesophageal causes of dysphagia localised only to the pharynx: Implications for the suspected head and neck cancer pathway.

OBJECTIVES: Dysphagia is a presenting symptom of both pharyngeal and oesophageal cancers. The referral pathway choice is determined by whether it is thought to be oropharyngeal or oesophageal, and this is in turn influenced by whether dysphagia is perceived to be above or below the suprasternal notch. We studied the concordance between the presence of pharynx-localised dysphagia (PLD) and the location of the underlying disease processes. DESIGN: A subset analysis of the Dysphagia Hotline Cohort, collected between 2004 and 2015, of patients with PLD and a structural diagnosis. MAIN OUTCOME MEASURES: Information about patient demography and presenting symptoms were recorded. The incisor-to-pathology distance, and the nature of the pathology, were recorded. Logistic regression analysis was used to identify independent predictors of malignancy. RESULTS: The study included 177 patients. There were 92 males, and mean age at presentation was 74 years. The commonest benign pathologies were cricopharyngeal dysfunction with or without pharyngeal pouch (n = 67), peptic stricture (n = 44) and Schatzki's ring (n = 11). There were 49 cases of cancer, including one hypopharyngeal cancer, one cervical oesophageal cancer, 28 cancers of the upper/mid-thoracic oesophagus, 15 cancers of the lower thoracic oesophagus and 4 cardio-oesophageal cancers. In 105 (59%) patients, PLD was caused by oesophageal disease. Independent predictors of malignancy were weight-change (loss >2.7 kg), a short history (<12 weeks) and presence of odynophagia. Nineteen (39%) of oesophageal cancers that presented with dysphagia that was localised only to the pharynx would have been beyond the reach of rigid oesophagoscopy. CONCLUSIONS: Pharynx-localised dysphagia is more likely to be a referred symptom of structural oesophageal disease, including cancer, than a primary symptom of structural pharyngeal disease. Absence of additional alarm symptoms such as a short history, weight-loss, and odynophagia, do not adequately exclude the possibility of oesophageal cancer. When the differential diagnosis of PLD includes malignancy, cancer should be presumed to be arising from the oesophagus or the cardio-oesophageal region until proven otherwise. This requires direct visualisation of the mucosal surfaces of the oesophagus and the cardio-oesophageal region, using either transoral or transnasal flexible endoscopy, irrespective of whether the initial assessment occurs within head and neck or upper gastrointestinal suspected cancer pathways.

An unusual complication of oesophageal stent deployment.

Insertion of a self-expanding metal stent is commonly used to palliate dysphagia secondary to oesophageal carcinoma. Displacement of the stent itself is a recognized complication which can result in perforation of the upper gastrointestinal tract. We report the first case of bowel obstruction and perforation resulting from the olive of the deployment system. This was probably due to peritoneal deposits on the small intestine obstructing passage of the olive.

Effect of a gluten-free diet on plasma nitric oxide products in coeliac disease.

BACKGROUND: Inducible nitric oxide synthase is expressed in the small intestine of patients with coeliac disease. This produces increased plasma concentration of nitric oxide end products (NOx), most marked in those ingesting gluten. The time-course of change in NOx with a gluten-free diet (GFD) and its correlation with histology and coeliac serology were studied. METHODS: Fasting plasma NOx was determined by the Greiss reaction in 20 coeliac patients at diagnosis and 2, 4 and 6 months after commencing a GFD. Endomysial and gliadin antibodies were checked at the same time. Duodenal biopsies were taken at diagnosis and at 6 months, and then graded according to the Marsh classification. RESULTS: Plasma NOx fell rapidly following the introduction of a GFD (mean before GFD 95.8 microM to 61.5 microM at 2 months), and further still by 6 months (mean = 37.0 microM). Reductions at 2 and 6 months were statistically significant compared with baseline (P < 0.01 and P < 0.005, respectively: Wilcoxon signed ranks test). Plasma NOx was correlated with histological grade initially (P = 0.03: Kruskal-Wallis) but not after 6 months on a GFD (P = 0.24). Coeliac serology correlated poorly with histology. CONCLUSIONS: Plasma NOx falls rapidly following GFD in coeliac disease and is related to histological grade initially. However, values vary widely between individuals, which may limit its use as a clinical tool.

Retrospective audit of the value of the pancreolauryl test in a district general hospital.

OBJECTIVES: To audit the specificity and value of the pancreolauryl test (PLT) for the diagnosis of pancreatic insufficiency. DESIGN: A retrospective case note review of 47 patients who had a PLT during three consecutive years. SETTING: A 650 bedded district general hospital. MAIN OUTCOME MEASURES: Patient demographics, symptoms, baseline blood, endoscopic and radiological investigations, and alcohol consumption were related to the results of the PLT to determine if the test could be better targeted. Response to therapy and final diagnoses were noted to determine the specificity of the test. RESULTS: Forty seven patients had a PLT during the three years studied. All successfully performed the test as outpatients. Seven inconclusive tests were repeated. Thirty eight patients (81%) had diarrhoea, of whom 10 (21%) described steatorrhoea; 23 (49%) had an abnormal PLT. All with an abnormal test had diarrhoea, more frequently describing watery stools than steatorrhoea. Those treated with pancreatic enzyme supplements had an excellent response, although two of these had a final diagnosis of coeliac disease. Nineteen patients with an abnormal PLT were thought to be suffering from pancreatic insufficiency. All patients with a normal PLT were clinically thought not to have pancreatic insufficiency. Age, sex, and alcohol intake correlated poorly with final diagnosis. CONCLUSIONS: The PLT can be used in a district general hospital setting as an outpatient test for pancreatic insufficiency and has a specificity of 83%-91%. Its use should be restricted to those presenting with diarrhoea, not necessarily steatorrhoea, and not restricted to those with a history of high alcohol consumption.

Intestinal disaccharidase deficiency without villous atrophy may represent early celiac disease.

BACKGROUND: Intestinal disaccharidase activities are decreased in untreated celiac disease and also in other conditions without villous atrophy. Of 908 patients examined for suspected malabsorption, 37 (4.1%) had generalized disaccharidase deficiency without villous atrophy. The aim was to determine if generalized disaccharidase deficiency without villous atrophy represented latent celiac disease. METHODS: Case notes and histology of the 37 patients were reviewed. History and blood investigations including antigliadin and endomysial antibodies were checked. Where celiac disease was suspected, endoscopic duodenal biopsies for histology and disaccharidase estimation were repeated. RESULTS: Of the initial 37 patients, 6 patients had had repeat endoscopic biopsies; one having celiac disease. A further 18 patients were reviewed. The remainder declined further investigation. Eight had repeat endoscopic duodenal biopsies; one had celiac disease. Two with positive celiac serology also had enteroscopy with jejunal biopsies; both had celiac disease. CONCLUSIONS: At least 11% of patients with generalized disaccharidase deficiency without villous atrophy develop celiac disease. Enteroscopic biopsies from distal duodenum and proximal jejunum should be considered as the next investigation if endomysial or antigliadin antibodies are positive.

Wild-type and met-65-->Leu variants of human cystatin A are functionally and structurally identical.

The solution structure of an N-terminally truncated and mutant form (M65L(2-98)) of the human cysteine protease inhibitor cystatin A has been reported that reveals extensive structural differences when compared to the previously published structure of full-length wild-type (WT) cystatin A. On the basis of the M65L(2-98) structure, a model of the inhibitory mechanism of cystatin A was proposed wherein specific interactions between the N- and C-terminal regions of cystatin A are invoked as critical determinants of protease binding. To test this model and to account for the reported differences between the two structures, we undertook additional structural and mechanistic analyses of WT and mutant forms of human cystatin A. These show that modification at the C-terminus of cystatin A by the addition of nine amino acids has no effect upon the affinity of papain inhibition (K(D) = 0.18+/-0.02 pM) and the consequences of such modification are not propagated to other parts of the structure. These findings indicate that perturbation of the C-terminus can be achieved without any measurable effect on the N-terminus or the proteinase binding loops. In addition, introduction of the methionine-65 --> leucine substitution into cystatin A that retains the N-terminal methionine (M65L(1-98)) has no significant effect upon papain binding (K(D) = 0.34+/-0.02 pM). Analyses of the structures of WT and M65L(1-98) using (1)H NMR chemical shifts and residual dipolar couplings in a partially aligning medium do not reveal any evidence of significant differences between the two inhibitors. Many of the differences between the published structures correspond to major violations by M65L(2-98) of the WT constraints list, notably in relation to the position of the N-terminal region of the inhibitor, one of three structural motifs indicated by crystallographic studies to be involved in protease binding by cystatins. In the WT structure, and consistent with the crystallographic data, this region is positioned adjacent to another inhibitory motif (the first binding loop), whereas in M65L(2-98) there is no proximity of these two motifs. As the NMR data for both WT9C and M65L(1-98) are wholly consistent with the published structure of WT cystatin A and incompatible with that of M65L(2-98), we conclude that the former represents the most reliable structural model of this protease inhibitor.

Intestinal trehalase activity in a UK population: establishing a normal range and the effect of disease.

Trehalose is a disaccharide, the main dietary source being mushrooms. It has been approved as an additive in the preparation of dried food. Isolated intestinal trehalase deficiency is found in 8% of Greenlanders, but is rare elsewhere. The normal range of trehalase activity and the incidence of isolated trehalase deficiency in the UK have not been reported. Patients (n 400) were investigated for suspected malabsorption. Endoscopic distal duodenal biopsies were taken for histological assessment and maltase, sucrase, lactase and trehalase estimation. Disaccharidase activities were determined by Dahlqvist's technique (Dahlqvist, 1968). Most patients (n 369) had normal duodenal histology. In these, square root transformation of trehalase activity produced a normal distribution. The normal range (mean +/- 2 SD) was 4.79-37.12 U/g protein. One patient had an isolated borderline trehalase deficiency. The thirty-one patients with villous atrophy had significantly reduced disaccharidase activities. With ingestion of a gluten-free diet, maltase, sucrase and trehalase activities recovered to normal in most patients, whereas lactase activity did not. The normal range and very low incidence of isolated enzyme deficiency is comparable with that described in populations from the USA and mainland Europe. Activity is significantly reduced in untreated coeliac disease and recovers with treatment with a gluten-free diet. There is no place for routine determination of trehalase activity in the UK population and there should be no concern over the introduction of trehalose-containing dried foods.

Interaction of glucocorticoid receptor isoforms with transcription factors AP-1 and NF-kappaB: lack of effect of glucocorticoid receptor beta.

Glucocorticoids act through the glucocorticoid receptor (GR) to enhance or repress transcription of glucocorticoid responsive genes depending on the promoter context and cellular background. The human GR primary transcript is alternatively spliced resulting in hGR alpha and hGR beta isoforms. Transactivation and transrepression are mediated by hGR alpha and while it has been demonstrated that hGR beta, can act as a dominant negative inhibitor of hGR alpha mediated transactivation, its effects on transrepression are not known. To investigate hGR beta actions, we used GR-deficient COS-7 and HEK-293 cells. When hGR alpha (0.5 microg 10(6) cells(-1)) was transfected into COS-7 cells dexamethasone (150 nM) inhibited TNF alpha (80 U ml(-1)) effects on a NF-kappaB responsive reporter gene by 40%. There was no evidence of a dominant negative effect when hGR beta (1-10 microg) was co-transfected with hGR alpha up to ratios of 10:1. Similarly hGR beta had no effect on hGR alpha inhibition of a phorbol ester stimulated Ap-1-responsive reporter gene in COS-7 or HEK-293 cells. In comparison, an apparent dominant negative effect of hGR beta on hGR alpha-mediated transactivation was found to be attributable to non-specific transcriptional squelching in COS-7 cells. In summary, the potential for hGR beta, to act as a dominant negative inhibitor of hGR alpha-mediated transactivation remains controversial, but our data suggest that hGR beta, was unable to act as a dominant negative inhibitor of either hGR alpha-mediated transrepression or transactivation in these promoter and cell contexts.

Steroid recognition by chloramphenicol acetyltransferase: engineering and structural analysis of a high affinity fusidic acid binding site.

The antibiotic fusidic acid and certain closely related steroidal compounds are potent competitive inhibitors of the type I variant of chloramphenicol acetyltransferase (CATI). In the absence of crystallographic data for CATI, the structural determinants of steroid binding were identified by (1) construction in vitro of genes encoding chimaeric enzymes containing segments of CATI and the related type III variant (CATIII) and (2) site-directed mutagenesis of the gene encoding CATIII, followed by kinetic characterisation of the substituted variants. Replacement of four residues of CATIII (Gln92, Asn146, Tyr168 and Ile172) by their equivalents from CATI yields an enzyme variant that is susceptible to competitive inhibition by fusidate with respect to chloramphenicol (Ki = 5.4 microM). The structure of the complex of fusidate and the Q92C/N146F/Y168F/I172V variant, determined at 2.2 A resolution by X-ray crystallography, reveals the inhibitor bound deep within the chloramphenicol binding site and in close proximity to the side-chain of His195, an essential catalytic residue. The aromatic side-chain of Phe146 provides a critical hydrophobic surface which interacts with non-polar substituents of the steroid. The remaining three substitutions act in concert both to maintain the appropriate orientation of Phe 146 and via additional interactions with the bound inhibitor. The substitution of Gln92 by Cys eliminates a critical hydrogen bond interaction which constrains a surface loop (residues 137 to 142) of wild-type CATIII which must move in order for fusidate to bind to the enzyme. Only two hydrogen bonds are observed in the CAT-fusidate complex, involving the 3-alpha-hydroxyl of the A-ring and both hydroxyl of Tyr25 and NE2 of His195, both of which are also involved in hydrogen bonds with substrate in the CATIII-chloramphenicol complex. In the acetyl transfer reaction catalysed by CAT, NE2, of His195 serves as a general base in the abstraction of a proton from the 3-hydroxyl of chloramphenicol as the first chemical step in catalysis. The structure of the CAT-inhibitor complex suggests that deprotonation of the 3-alpha-hydroxyl of bound fusidate by this mechanism could produce an oxyanion nucleophile analogous to that seen with chloramphenicol, but one which is incorrectly positioned to attack the thioester carbonyl of acetyl-CoA, accounting for the observed failure of CAT to acetylate fusidate.

Properties of hybrid active sites in oligomeric proteins: kinetic and ligand binding studies with chloramphenicol acetyltransferase trimers.

Alteration of the charge of surface lysyl residues of chloramphenicol acetyltransferase (CAT) by site-directed mutagenesis was used to increase the charge difference between the subunits of two naturally occurring enzyme variants (CATI and CATIII). The introduced charge change greatly facilitates the purification of CATI/CATIII and CATIII/CATIII hybrid trimers by ion-exchange chromatography. Hybrids containing only one functional active site per trimer were generated in vitro by reversible denaturation of mixtures of "active" subunits (retention of a catalytic histidine at position 195) and "inactive" subunits (with alanine replacing histidine 195). Such hybrids were used (1) to demonstrate that the previously observed novel binding of a steroidal antibiotic (fusidic acid) by CATI involves amino acid residues at each subunit interface and (2) to identify specific residues contributing to such interactions. A pre-steady-state kinetic characterization of homotrimers containing the H195A substitution also revealed that fusidate binding to CATI may, like chloramphenicol binding, involve a hydrogen bond with the catalytic histidine residue. In addition, confirmation of the fact that His-195 interacts with chloramphenicol in CATI as well as in CATIII makes it likely that it is essential for the catalytic mechanism of all naturally occurring variants of CAT, as first suggested by structural evidence for the type III enzyme (Leslie, 1990).

Analysis of hydrogen bonding in enzyme-substrate complexes of chloramphenicol acetyltransferase by infrared spectroscopy and site-directed mutagenesis.

Chloramphenicol acetyltransferase (CAT) reversibly transfers an acetyl group between CoA and the 3-hydroxyl of either chloramphenicol (Cm) or 1-acetylchloramphenicol (1AcCm). The products of the forward reactions, 3-acetylchloramphenicol (3-AcCm) and 1,3-diacetylchloramphenicol (1,3Ac2-Cm), are the substrates for the reverse reaction. The role of the 3-acetyl carbonyl group in the binding of the substrates 3AcCm and 1,3Ac2Cm to CAT has been investigated using infrared spectroscopy. Comparison of difference spectra (3-[12C = O]acetyl- minus 3-[13C = O]acetyl-) obtained for the binary complexes of 3AcCm with wild-type CAT, and with a variant wherein serine-148 is replaced by alanine (S148A), reveals a large (9 cm-1) down frequency shift for the 3-acetyl carbonyl stretch in the wild-type complex, indicative of a hydrogen bond between this carbonyl and the hydroxyl group of Ser-148. The carbonyl bandwidth in the wild-type complex is reduced by 33% compared to that for the complex with S148A, indicating restriction of carbonyl mobility and dispersion in the former, an observation consistent with the proposed hydrogen bond between the ester carbonyl and the hydroxyl of Ser-148. Repetition of the experiment using 1,3Ac2Cm as the ligand reveals a frequency shift of only 3 cm-1 between wild-type and S148A complexes, indicating only a small change in the strength of carbonyl interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Replacement of catalytic histidine-195 of chloramphenicol acetyltransferase: evidence for a general base role for glutamate.

The imidazole N epsilon 2 of His-195 plays an essential part in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT), hydrogen bonding to and a abstracting a proton from the primary hydroxyl group of chloramphenicol. Replacement of His-195 by alanine or glutamine results in apparent decreases in kcat of (9 x 10(5)- and (3 x 10(5))-fold, respectively, whereas Km values for both substrates (chloramphenicol and acetyl-CoA) are similar to those of wild-type CAT. The structure of Gln-195 CAT has been solved at 2.5-A resolution and is largely isosteric with that of wild-type CAT. Substitution of His-195 by glutamate resulted in a (5 x 10(4))-fold decrease in kcat together with a 3-fold increase in the Km for chloramphenicol. Direct determination of binding constants for both substrates demonstrated that these substitutions result in only small decreases in the affinity of CAT for acetyl-CoA (Kd values increased 2- to 3-fold), whereas chloramphenicol Kd values are elevated 26-, 20-, and 53-fold for Ala-195 CAT, Gln-195 CAT and Glu-195 CAT, respectively. The pH dependence of kcat/Km yields apparent pKa values of 6.5 and 6.7 for Ala-195 CAT and Gln-195 CAT, respectively, which are very similar to that (6.6) determined for the ionization of His-195 in wild-type CAT. In contrast, the pH dependence of kcat/Km for Glu-195 CAT (pKa = 8.3) is very different from that of wild-type CAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrinsic fluorescence of chloramphenicol acetyltransferase: responses to ligand binding and assignment of the contributions of tryptophan residues by site-directed mutagenesis.

Replacement by tyrosine or phenylalanine was used to assign the additive contributions of each of the three tryptophan residues of chloramphenicol acetyltransferase (CAT) to its intrinsic fluorescence on excitation at 295 nm. During the assessment of the fluorescence responses of the wild-type enzyme to the binding of ligands, it was found that the overlapping absorption spectra of chloramphenicol and tryptophan, with an attendant inner filter effect, required the use of a displacement technique involving an alternative substrate (the p-cyano analogue of chloramphenicol) without significant absorption at 295 nm. By the use of two-Trp, one-Trp, and Trp-less variants, in combination with this displacement technique, it was possible to demonstrate that Trp-86 and Trp-152 are involved in the fluorescence quenching associated with the binding of chloramphenicol, most likely via nonradiative energy transfer from these residues to the bound substrate. Trp-152 is mainly responsible for the fluorescence enhancement accompanying the binding of acetyl-CoA (and CoA) through proximity effects and solvent exclusion on substrate association.

Stabilization of the imidazole ring of His-195 at the active site of chloramphenicol acetyltransferase.

The imidazole of His-195 plays an essential role in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT). The structure of the binary complex of CATIII and chloramphenicol suggests that two unusual interactions might determine the conformation of the side chain of His-195: (i) an intraresidue hydrogen bond between its main chain carbonyl and the protonated N delta 1 of the imidazole ring and (ii) face-to-face van der Waals contact between the His-195 imidazole group and the aromatic side chain of Tyr-25. Tyr-25 also makes a hydrogen bond, via its phenolic hydroxyl, to the carbonyl oxygen of the substrate chloramphenicol. Replacement of Tyr-25 of CATIII by phenylalanine results in a modest increase in the Km for chloramphenicol (from 11.6 to 14.6 microM) and a 2-fold fall in kcat (599 to 258 s-1), indicative of a free energy contribution to transition state binding of 0.6 kcal mol-1 for the hydrogen bond between Tyr-25 and chloramphenicol. In contrast, substitution of Tyr-25 by alanine yields an enzyme that is dramatically impaired in its ability to bind chloramphenicol (Km = 173 microM). As kcat for Ala-25 CAT is also reduced (130 s-1), the loss of the aryl group results in a 69-fold decrease in kcat/Km, corresponding to a free energy contribution to binding and catalysis of 2.5 kcal mol-1. In addition to the loss of the hydrogen bond between Tyr-25 and chloramphenicol, the loss of substrate affinity in Ala-25 CAT may be a direct consequence of reduced hydrophobicity of the chloramphenicol-binding site and/or the loss of critical constraints on the precise conformation of the catalytic imidazole. However, as with wild type CAT, inactivation of Ala-25 CAT by the affinity reagent 3-(bromoacetyl) chloramphenicol is accompanied by modification solely at N epsilon 2 of His-195. Hence, the results demonstrate that tautomeric stabilization of the imidazole ring persists in the absence of van der Waals interactions with the side chain of Tyr-25, probably as a consequence of hydrogen bonding between the protonated N delta 1 and the carbonyl oxygen of His-195.