Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we describe the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution. (C) 1997 Elsevier Science B.V.

Presentations of physical illness in people with developmental disability: the example of gastro-oesophageal reflux

People with developmental disabilities are becoming an important part of the general practice population. Although they have a similar range of medical conditions to the general population, there are some important differences in prevalence, risk factors, presentation and management of particular conditions. We use gastro-oesophageal reflux to illustrate how developmental disability may affect the presentation, assessment and management of a common condition.

Interactions among smoking, obesity, and symptoms of acid reflux in Barrett's esophagus

Background: Barrett's esophagus, a metaplastic precursor to esophageal adenocarcinoma, is becoming increasingly prevalent in many populations. Clinical studies suggest acid reflux causes Barrett's esophagus; however, no population-based estimates of risk have been reported, and the role of other health factors in modifying risk is unclear. Methods: We conducted a population-based case-control study in Brisbane, Australia. Cases were 167 patients with histologically confirmed Barrett's esophagus diagnosed between February and December 2003. Age-matched and sex-matched controls (n = 261) were randomly selected from a population register. Data on exposure to self-reported symptoms of acid reflux, smoking, obesity, and other factors were collected through self-completed questionnaires followed by telephone interview. Risks of Barrett's esophagus and Barrett's esophagus with dysplasia associated with these exposures were estimated by the odds ratio (OR) and 95% confidence interval (95% Cl), both crude and adjusted for other factors. Results: Self-reported weekly episodes of acid reflux were associated with greatly increased risks of Barrett's esophagus (adjusted OR, 29.7; 95% CI, 12.2-72.6) and Barrett's esophagus with dysplasia (OR, 59.7; 95% CI, 18.5-193). Smoking was also associated with risk of Barrett's esophagus. We found evidence of interactions between symptoms of acid reflux and smoking and obesity. Obese people with self-reported symptoms of acid reflux had markedly higher risks of Barrett's esophagus (OR, 34.4; 95% CI, 6.3-188) than people with reflux alone (OR, 9.3; 95% CI, 1.4-62.2) or obesity alone (OR, 0.7,95% CI, 0.2-2.4). Similarly, those reporting both acid reflux symptoms and smoking were at substantially higher risks of Barrett's esophagus (OR, 51.4; 95% CI, 14.1-188) than those reporting acid reflux or smoking alone. Conclusions: Although history of symptoms of acid reflux is the principle factor associated with Barrett's esophagus, risks are substantially increased by obesity and smoking.

'Free' surfactant in gastric aspirates and bronchoalveolar lavage in children with and without reflux oesophagitis

Aim: Dipalmitoylphosphatidycholine (DPPC) is the characteristic and main constituent of surfactant. Adsorption of surfactant to epithelial surfaces may be important in the masking of receptors. The aims of the study were to (i) compare the quantity of free DPPC in the airways and gastric aspirates of children with gastroesophageal reflux disease (GORD) to those without and (ii) describe the association between free DPPC levels with airway cellular profile and capsaicin cough sensitivity. Methods: Children aged < 14 years were defined as 'coughers' if a history of cough in association with their GORD symptoms was elicited before gastric aspirates and nonbronchoscopic bronchoalveolar lavage (BAL) were obtained during elective flexible upper gastrointestinal endoscopy. GORD was defined as histological presence of reflux oesophagitis. Spirometry and capsaicin cough-sensitivity test was carried out in children aged > 6 years before the endoscopy. Results: Median age of the 68 children was 9 years (interquartile range (IQR) 7.2). Median DPPC level in BAL of children with cough (72.7 mu g/mL) was similar to noncoughers (88.5). There was also no significant difference in DPPC levels in both BAL and gastric aspirates of children classified according to presence of GORD. There was no correlation between DPPC levels and cellular counts or capsaicin cough-sensitivity outcome measures. Conclusion: We conclude that free DPPC levels in the airways and gastric aspirate is not influenced by presence of cough or GORD defined by histological presence of reflux oesophagitis. Whether quantification of adsorbed surfactant differs in these groups remain unknown. Free DPPC is unlikely to have a role in masking of airway receptors.

Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins. (C) 2003 Elsevier Science (USA). All rights reserved.

The effect of hearing impairment on the vocal characteristics of older people

Measures of vocal intensity, frequency and harshness were compared for 19 hearing-impaired and 21 normal-hearing people over 60 years of age. Significantly greater comfortable intensity levels were found in the hearing-impaired group, but the other measures of frequency and harshness were not significantly different. A large proportion of the subjects in both groups reported a history of gastro-oesophageal reflux (GER), a condition associated with vocal fold pathology and hoarseness. Comparison of the GER and non-GER subjects on the measures of vocal function showed that the female GER speaker exhibited lower frequency on the vowel /u/ than the non-GER subjects. Clinicians need to be aware of the effect of highly prevalent disorders such as hearing impairment and GER on the voices of elderly speakers.

Timely topic: Premalignant lesions associated with adenocarcinoma of the upper gastrointestinal tract

The changing incidence of adenocarcinomas, particularly in the oesophagus and gastric cardia, has led to the rapid expansion of screening programmes aimed at detecting the precursor lesion of dysplasia before adenocarcinoma develops. The pathologist now has an important role in first diagnosing patients at risk for developing dysplasia, and then correctly classifying dysplasia when it occurs. Barrett's oesophagus has had different diagnostic criteria in previous years but is currently diagnosed by the presence of intestinal metaplasia of any length in the true oesophagus. Intestinal metaplasia confined only to the gastro-oesophageal junction or cardia is of uncertain significance but is probably common, with less risk of progressing to dysplasia or malignancy. In the stomach, patients with autoimmune atrophic gastritis and Helicobacter-associated multifocal atrophic gastritis have an increased risk of adenocarcinoma, but screening protocols are not well-developed compared with those used for Barrett's oesophagus. Dysplasia of glandular epithelium can be classified using well-described criteria. Low grade dysplasia is the most common type and regresses or remains stable in the majority of patients. High grade dysplasia is more ominous clinically, with a propensity to coexist with or progress to adenocarcinoma.

The oral medicine of tooth wear

This review illustrates, through a series of case histories, how oral medicine insights aid the diagnosis and management of patients with excessive tooth wear. The cases reviewed are drawn from the records of 500 southeast Queensland patients referred to the author over a 12 year period. Patients most at risk of dental erosion have work and sports dehydration, caffeine addiction, gastro-oesophageal reflux, asthma, diabetes mellitus, hypertension or other systemic diseases or syndromes that predispose to xerostomia. Saliva protects the teeth from the extrinsic and intrinsic acids which cause dental erosion. Erosion, exacerbated by attrition and abrasion, is the main cause of tooth wear. These cases illustrate that teeth, oral mucosa, salivary glands, skin and eyes should be examined for evidence of salivary hypofunction and attendant medical conditions. Based on comprehensive oral medicine, dietary analyses and advice, it would seem patients need self-management plans to deal with incipient chronic tooth wear. The alternative is the expensive treatment of pain, occlusal damage and pulp death required to repair the effects of acute severe tooth wear.

Localization of small esophageal cancers for radiation planning using endoscopic contrast injection: report on a series of eight cases

Recently, Barrett's esophagus and early adenocarcinomas have been detected increasingly frequently in routine follow-up of patients with gastroesophageal reflux. Although surgery is the treatment of choice, some patients are medically unfit for esophagectomy and, in this case, the only alternative curative therapy is radical chemoradiation therapy. In addition, some patients who present with symptoms have small tumors that cannot be localized accurately using routine imaging techniques. This report describes a series of eight patients with small esophageal cancers in whom the tumors were successfully localized following endoscopic injection of contrast, and treated with chemoradiation therapy. The treatment was successful in seven patients. This method of tumor localization demonstrated that conventional techniques are mostly, unreliable when applied to very early cancers.

Healthy ageing - Adults with intellectual disabilities: Physical health issues

This report has been prepared by the Ageing Special Interest Research Group of the International Association for the Scientific Study of Intellectual Disabilities (IASSID) in collaboration with the Department of Mental Health and Substance Dependence and the Programme on Ageing and Health, World Health Organization (WHO), Geneva, Switzerland, and all rights are reserved by the above mentioned organization. The document may, however, be freely reviewed, abstracted, reproduced or translated in part, but not for sale or use in conjunction with commercial purposes. It may also be reproduced in full by non-commercial entities for information or for educational purposes with prior permission from WHO/IASSID. The document is likely to be available in other languages also.

Dental erosion patterns from intrinsic acid regurgitation and vomiting

Background: The distribution of lesions from dental erosion due to intrinsic acid regurgitation and vomiting may be different from patterns of dental erosion due to extrinsic acids. To date studies have failed to validate this assumption. This study described the sites and nature of lesions from dental erosion in cases of intrinsic acid regurgitation, and compared them with the distribution of lesions occurring in age and sex matched controls, whose lesions are due to extrinsic acids. Methods: The University of Queensland tooth wear clinic patients were screened to select 30 cases, 21 self-identified bulimics and nine medically diagnosed chronic gastric acid regurgitators, and 30 controls. Epoxy resin models of the subjects' dentition were examined under stereoscopic light microscope at magnification 16 to 40. The patterns and sites of tooth wear were recorded for teeth representative of 20 tooth sites in every subject. Results: While the incisal edges of maxillary and mandibular anterior teeth of acid regurgitators were more frequently affected by erosion, incisal attrition was more common on controls' teeth. Cervical lesions were more commonly found in association with incisal attrition in the controls, and in association with incisal erosion in the cases. In 10 per cent of sites in case subjects, cervical lesions associated with incisal erosion were found on the lingual aspects of their mandibular incisors, canines and premolars. These lesions were almost exclusive to the case subjects. Conclusions: These results validate that lingual cervical lesions associated with incisal erosion on the mandibular anterior teeth are strong discriminators between tooth wear in patients with bulimia nervosa or chronic gastro-oesophageal reflux and those whose dental erosion is due to extrinsic acids.