Intestinal metaplasia in endoscopic biopsy specimens of gastric mucosa

A total of 1412 consecutive cases of endoscopic gastric biopsy, carried out over a four year period, were reviewed and specimens were examined histochemically to determine the prevalence of intestinal metaplasia and its variants. Three types were characterised: complete intestinal metaplasia and two classes of incomplete intestinal metaplasia (type IIa and type IIb) depending on the absence or presence, respectively, of sulphomucins within mucin secreting columnar cells. Type IIb intestinal metaplasia was significantly more common in patients with gastric carcinoma (p less than 0.001) and in those with dysplasia (p less than 0.001) than in patients with benign gastric pathology. No such association was found with either type I or type IIa intestinal metaplasia. In addition to those present in the columnar cells of type IIb intestinal metaplasia, sulphomucins were also commonly found in goblet cells of all three types of metaplasia. The presence of sulphomucins in goblet cells, however, was not significantly associated with gastric carcinoma or dysplasia. The significance of the different types of intestinal metaplasia in relation to the pathological findings is discussed.     

Remodelling of gap junctions and connexin expression in diseased myocardium

Gap junctions form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart: connexin43 (Cx43), Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally-specialized subsets of cardiac myocyte. Mutations in genes that encode connexins have only rarely been identified as being a cause of human cardiac disease, but remodelling of connexin expression and gap junction organization are well documented in acquired adult heart disease, notably ischaemic heart disease and heart failure. Remodelling may take the form of alterations in (i) the distribution of gap junctions and (ii) the amount and type of connexins expressed. Heterogeneous reduction in Cx43 expression and disordering in gap junction distribution feature in human ventricular disease and correlate with electrophysiologically identified arrhythmic changes and contractile dysfunction in animal models. Disease-related alterations in Cx45 and Cx40 expression have also been reported, and some of the functional implications of these are beginning to emerge. Apart from ventricular disease, various features of gap junction organization and connexin expression have been implicated in the initiation and persistence of the most common form of atrial arrhythmia, atrial fibrillation, though the disparate findings in this area remain to be clarified. Other major tasks ahead focus on the Purkinje/working ventricular myocyte interface and its role in normal and abnormal impulse propagation, connexin-interacting proteins and their regulatory functions, and on defining the precise functional properties conferred by the distinctive connexin co-expression patterns of different myocyte types in health and disease.     

Dietetic intervention for inpatients on fluid‐only diets helps to achieve nutritional requirements

Aim

The present study aimed to assess whether dietetic intervention helps patients on fluid‐only diets to meet their energy and protein requirements. This topic has not been previously investigated.

Methods

A quasi‐experimental study of 57 patients receiving fluid‐only diets was conducted at The Townsville Hospital. The fluid consumption of participants was observed over 24 hours and was used to calculate total energy and protein intakes. The percentage of protein and energy requirements met was compared between patients receiving dietetic intervention and patients who were not.

Results

Patients receiving dietetic interventions met a higher percentage of their energy requirements (75.88) than the control group (18.10) based on median intakes (P < 0.001). Patients receiving dietetic intervention also met a higher percentage of their protein requirements (75.99) than the control group (13.80) based on median intakes (P < 0.001). Stratification for age, body mass index (BMI) and fluid diet type showed no change in effect.

Conclusions

This study shows that dietetic intervention enabled patients on fluid‐only diets to meet up to 80% more of their energy requirements and up to 95% more of their protein requirements. These results were consistent across age, BMI and fluid diet type. The significance of these differences has resulted in a change of clinical practice at the study hospital. All patients on fluid‐only diets for three days or longer are now blanket referred for dietetic intervention.     

Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes

A key feature of immune evasion for African trypanosomes is the functional specialization of their surface membrane in an invagination known as the flagellar pocket (FP), the cell''s sole site of endocytosis and exocytosis. The FP membrane is biochemically distinct yet continuous with those of the cell body and the flagellum. The structural features maintaining this individuality are not known, and we lack a clear understanding of how extracellular components gain access to the FP. Here, we have defined domains and boundaries on these surface membranes and identified their association with internal cytoskeletal features. The FP membrane appears largely homogeneous and uniformly involved in endocytosis. However, when endocytosis is blocked, receptor-mediated and fluid-phase endocytic markers accumulate specifically on membrane associated with four specialized microtubules in the FP region. These microtubules traverse a distinct boundary and associate with a channel that connects the FP lumen to the extracellular space, suggesting that the channel is the major transport route into the FP.     

Anaplastic carcinoma presenting with cervical lymphadenopathy

We describe 50 patients with anaplastic carcinoma presenting with a mass in the neck. The diagnosis of anaplastic carcinoma was confirmed by immunocytochemistry to exclude very poorly differentiated squamous carcinomas, amelanotic melanoma, and non-Hodgkin's lymphoma. The primary site was established immediately in 26 patients (25 in the head and neck; 1 in the lung); a further 4 had radiological evidence of a primary tumour in the lung. The primary site was established later in 1 patient, in the ethmoid sinuses. In 20 patients the primary site was never established. The commonest primary site was the nasopharynx. The basic treatment policy was radiotherapy, although 20% of patients with a known primary tumour, and 50% of those without, were untreated. The 2-year survival was about 30% in both groups, and did not differ significantly. Prognostic factors for survival were age, performance status, and T status of the primary tumour. Sex, node status, node level, and laterality of nodes, were not.     

Escherichia coli succinate dehydrogenase variant lacking the heme b

The Escherichia coli enzyme succinate:ubiquinone oxidoreductase [(succinate dehydrogenase (SdhCDAB)] couples succinate oxidation to ubiquinone reduction and is structurally and functionally equivalent to mitochondrial complex II, an essential component of the aerobic respiratory chain and tricarboxylic acid cycle. All such enzymes contain a heme within their membrane anchor domain with a highly contentious, but as-yet-undetermined, function. Here, we report the generation of a complex II that lacks heme, which is confirmed by both optical and EPR spectroscopy. Despite the absence of heme, this mutant still assembles properly and retains physiological activity. However, the mutants lacking heme are highly sensitive to the presence of detergent. In addition, the heme does not appear to be involved in reactive oxygen species suppression. Our results indicate that redox cycling of the heme in complex II is not essential for the enzyme's ubiquinol reductase activity.     

Relative expression of immunolocalized connexins 40 and 43 correlates with human atrial conduction properties.

OBJECTIVES: The aim of this study was to determine the relationship between immunolocalized gap-junctional proteins and human atrial conduction. BACKGROUND: As a determinant of intercellular conductance, gap-junctional coupling is considered to influence myocardial conduction velocity. This study tested the hypothesis that the quantity of immunodetectable atrial gap-junctional proteins, connexin40 (Cx40) and connexin43 (Cx43), are related to atrial conduction velocity in humans. METHODS: Epicardial mapping was performed on 16 patients undergoing cardiac surgery using an array of 56 unipolar electrodes. The conduction velocity was measured over the right atrial free wall during sinus rhythm and at a paced cycle length 500 ms. A biopsy from this region was excised for quantitative confocal immunodetection of Cx40 and Cx43. RESULTS: There was no correlation between conduction velocity and Cx43 signal or total connexin signal (Cx40 + Cx43). Connexin40 signal was inversely correlated with conduction velocity (p = 0.036). However, the relative quantity of connexin immunolabeling (expressed as Cx40/[Cx40+Cx43] or the inverse equivalent Cx43/[Cx40+Cx43]) was strongly associated with conduction velocity during sinus rhythm, such that, as the proportion of Cx40 signal increased (and that for Cx43 decreased), the conduction velocity decreased (p < 0.005, r = -0.66). Furthermore, with paced atrial activation at 500 ms cycle length, the relative quantity of connexin labeling (Cx40/[Cx40+Cx43]) correlated with the rate-related change in atrial conduction velocity (p < 0.02, r = 0.59). CONCLUSIONS: In human right atrium, conduction velocity is inversely related to immunodetectable Cx40 levels. The relative level of connexins 40 and 43 signal is strongly associated with atrial conduction properties, suggesting that interactions between the two connexins may result in novel coupling properties.     

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY,a mononuclear Mo enzyme

A long-standing contradiction in the field of mononuclear Mo enzyme research is that small-molecule chemistry on active-site mimic compounds predicts ligand participation in the electron transfer reactions, but biochemical measurements only suggest metal-centered catalytic electron transfer. With the simultaneous measurement of substrate turnover and reversible electron transfer that is provided by Fourier-transformed alternating-current voltammetry, we show that Escherichia coli YedY is a mononuclear Mo enzyme that reconciles this conflict. In YedY, addition of three protons and three electrons to the well-characterized “as-isolated” Mo(V) oxidation state is needed to initiate the catalytic reduction of either dimethyl sulfoxide or trimethylamine N-oxide. Based on comparison with earlier studies and our UV-vis redox titration data, we assign the reversible one-proton and one-electron reduction process centered around +174 mV vs. standard hydrogen electrode at pH 7 to a Mo(V)-to-Mo(IV) conversion but ascribe the two-proton and two-electron transition occurring at negative potential to the organic pyranopterin ligand system. We predict that a dihydro-to-tetrahydro transition is needed to generate the catalytically active state of the enzyme. This is a previously unidentified mechanism, suggested by the structural simplicity of YedY, a protein in which Mo is the only metal site.Most living species require a Mo enzyme (1), and apart from nitrogenase, all of these Mo-containing proteins are part of the large family of “mononuclear Mo” enzymes. The general ability of mononuclear Mo enzymes to catalyze two-electron oxygen atom transfer reactions has been attributed to the Mo(IV)/Mo(V)/Mo(VI) oxidation state cycling of the active site, and this mechanism is a common part of undergraduate syllabuses (1, 2). Escherichia coli YedY is a mononuclear Mo enzyme (3), and based on sequence homology, the majority of sequenced Gram-negative bacterial genomes encode a YedY-like protein (3–5). Uniquely for a mononuclear Mo enzyme, it has not been possible to form the YedY Mo(VI) state in experiments using ferricyanide as an oxidizing agent, and an unusually positive reduction potential for the Mo(V/IV) transition [+132 mV vs. standard hydrogen electrode (SHE) at pH 7] was determined from EPR experiments (6). Although the physiological substrate of YedY is unknown, a possible role in the reduction of reactive nitrogen species is suggested by experiments on the pathogen Campylobacter jejuni, where deletion of the Cj0379 YedY homolog generated a mutant that is deficient in chicken colonization and has a nitrosative stress phenotype (4). YedY catalysis can be assayed by measuring the two-electron reduction of either dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) (3), but the inaccessibility of the YedY Mo(VI) means the enzyme mechanism does not proceed via the common two-electron Mo-redox cycle. In small-molecule analogs of mononuclear Mo enzymes, the pterin ligands are described as “noninnocent,” meaning that the redox processes could be ligand or metal based (7). This study explores the possibility that ligand-based redox chemistry plays a role in YedY catalysis.YedY has been structurally characterized via both X-ray crystallography and X-ray absorption spectroscopy (XAS) (3, 8, 9). In most mononuclear Mo enzymes, heme groups and iron sulfur clusters are found within the same protein as the Mo center, but the only metal site in YedY is Mo, making this enzyme a helpfully simple system for studying redox chemistry (Fig. 1) (1, 3). Within the active site, the X-ray structure was interpreted to show Mo(V) in a square pyramidal environment (3), identical to other members of the “sulfite oxidase” family of mononuclear Mo enzymes. In contrast, XAS has suggested a pseudooctahedral Mo center (8, 9) with an additional O (from Glu104) or N (from Asn45) axial ligand coordinating trans to the apical oxo group (Fig. 1) (8). The equatorial ligation is provided by one oxygen-containing ligand and three sulfur donor atoms, one provided by cysteine (Cys102) and two from the pyranopterin cofactor, which binds to the Mo in a bidentate fashion via the enedithiolate side chain (3).Open in a separate windowFig. 1.Structure of Escherichia coli YedY. (A and B) The protein structure, PDB ID code 1XDQ (3). (C) The active site in the as-isolated Mo(V) state containing the dihydro form of pyranopterin.A 2012 computational study provided evidence that two different oxidation states can be accessed by protein-bound pyranopterin ligands (10). Conformational analysis and electronic structure calculations were used to assign redox states to the pyranopterin ligands in all known mononuclear Mo enzyme structures (10). It was concluded that, although enzymes from the sulfite oxidase family (such as YedY) contain pyranopterin ligands in the “dihydro” form, the xanthine dehydrogenase family of enzymes contain the two-proton, two-electron more reduced “tetrahydro” form of the pyranopterin (10).Traditionally, redox potential measurements of enzymes have required substrate-free conditions to either permit a solution equilibrium to be established (spectroscopic redox titrations) or to prevent catalytic signals from masking the noncatalytic response (film electrochemistry). Fourier-transformed alternating-current voltammetry (FTacV) is a technique that offers the ability to measure catalytic chemical redox reactions and reversible electron transfer processes in a single experiment (11, 12). In the FTacV measurement, a large-amplitude sine wave of frequency f is superimposed on a linear voltage–time sweep (11, 13–15) and the resulting current–time response is measured and then Fourier transformed (FT) into the frequency domain to give a power spectrum of harmonic contributions at frequencies f, 2f, 3f, etc. Band selection of the individual harmonics followed by inverse FT resolves the data back into the time domain. The higher harmonic components only arise from fast, reversible redox reactions, devoid of catalysis and baseline contributions, but the aperiodic (dc) component (f = 0) gives the same catalytic information as a traditional dc cyclic voltammetry (dcV) experiment, and can therefore show catalytic turnover (13, 14, 16).In this study, we both discover previously unidentified mononuclear Mo enzyme redox chemistry as well as demonstrate the significant advantages of using FTacV to probe the mechanism of a redox active enzyme.