Antibacterial activity of chemically defined chitosans: influence of molecular weight, degree of acetylation and test organism.

Chitosans, polysaccharides obtained from the exoskeleton of crustaceans, have been shown to exert antibacterial activity in vitro and their use as a food preservative is of growing interest. However, beyond a consensus that chitosan appears to disrupt the bacterial cell membrane, published data are inconsistent on the chemical characteristics that confer the antibacterial activity of chitosan. While most authors agree that the net charge density of the polymer (reflected in the fraction of positively charged amino groups at the C-2 position of the glucosamine unit) is an important factor in antibacterial activity, conflicting data have been reported on the effect of molecular weight and on the susceptibility among different bacterial species to chitosan. Therefore, we prepared batches of water-soluble hydrochloride salts of chitosans with weight average molecular weights (M(w)) of 2-224kDa and degree of acetylation of 0.16 and 0.48. Their antibacterial activity was evaluated using tube inhibition assays and membrane integrity assays (N-Phenyl-1-naphthylamine fluorescence and potassium release) against Bacillus cereus, Escherichia coli, Salmonella Typhimurium and three lipopolysaccharide mutants of E. coli and S. Typhimurium. Chitosans with lower degree of acetylation (F(A)=0.16) were more active than the more acetylated chitosans (F(A)=0.48). No trends in antibacterial action related to increasing or decreasing M(w) were observed although one of the chitosans (M(w) 28.4kDa, F(A)=0.16) was more active than the other chitosans, inhibiting growth and permeabilizing the membrane of all the test strains included. The test strains varied in their susceptibility to the different chitosans with wild type S. Typhimurium more resistant than the wild type E. coli. Salmonellae lipopolysaccharide mutants were more susceptible than the matched wild type strain. Our results show that the chitosan preparation details are critically important in identifying the antibacterial features that target different test organisms.

Antibacterial activity of sphagnum acid and other phenolic compounds found in Sphagnum papillosum against food-borne bacteria.

AIMS: To identify the phenolic compounds in the leaves of Sphagnum papillosum and examine their antibacterial activity at pH appropriate for the undissociated forms. METHODS AND RESULTS: Bacterial counts of overnight cultures showed that whilst growth of Staphylococcus aureus 50084 was impaired in the presence of milled leaves, the phenol-free fraction of holocellulose of S. papillosum had no bacteriostatic effect. Liquid chromatography-mass spectrometry analysis of an acetone-methanol extract of the leaves detected eight phenolic compounds. Antibacterial activity of the four dominating phenols specific to Sphagnum leaves, when assessed in vitro as minimal inhibitory concentrations (MICs), were generally >2.5 mg ml(-1). MIC values of the Sphagnum-specific compound 'sphagnum acid' [p-hydroxy-beta-(carboxymethyl)-cinnamic acid] were >5 mg ml(-1). No synergistic or antagonistic effects of the four dominating phenols were detected in plate assays. CONCLUSIONS: Sphagnum-derived phenolics exhibit antibacterial activity in vitro only at concentrations far in excess of those found in the leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: We have both identified the phenolic compounds in S. papillosum and assessed their antibacterial activity. Our data indicate that phenolic compounds in isolation are not potent antibacterial agents and we question their potency against food-borne pathogens.

Early municipal bacteriology in Brighton, Aberdeen and Bristol: blessing or burden?

In contrast to the idea that bacteriology was introduced as a tool for the diagnosis and management of the individual patient, this review highlights the work of the municipal bacteriological laboratory in the United Kingdom to illustrate how bacteriological laboratories were introduced as means to control community epidemic disease. Using the examples of municipal laboratories in Brighton, Bristol and Aberdeen, it shows how public health considerations of community infectious diseases such as diphtheria and typhoid dominated the early development and workload of the municipal laboratory, rather than examination of patients with pathological states of uncertain aetiology. It argues that this public health focus of the Medical Officer of Health limited the range of diagnostic tests carried out in such laboratories for over two decades. The growing number of pathogenic microbes being discovered in the late 19th century appears to have had little impact on the tests being carried out in the municipal laboratory. Municipal bacteriological facilities in three towns, a central municipal laboratory (in Brighton), a central university pathological department (Aberdeen) or a combination of both (Bristol) all provided the same limited set of tests. This restricted set of bacteriological examinations is likely to have diminished the value and status of bacteriology in what should have been a period of increasing scope.

Cytotoxicity in Bacillus mojavensis is abolished following loss of surfactin synthesis: implications for assessment of toxicity and food poisoning potential.

Bacillus subtilis and the closely related species Bacillus pumilus and Bacillus licheniformis have periodically been suggested to play a role in the aetiology of food poisoning despite the fact that the organisms do not possess the genes associated with enteropathogenicity in Bacillus cereus. We show here that Bacillus mojavensis, an organism closely related to B. subtilis, is able to produce toxic components which identify as a complex of three different surfactin analogues. These cyclic lipopeptides were soluble in methanol, heat stable after treatment in a boiling water bath for 10 min, resistant to enzymatic degradation by pepsin, trypsin, endoprotease V8 and proteinase K and formed pores in planar lipid bilayers. They were cytotoxic when tested in a series of commonly used laboratory cytotoxicity assays, namely, lactate dehydrogenase release, haemolysis, inhibition of both protein synthesis in Vero cells and motility in boar sperm. We show that such in vitro markers of enterotoxicity are due entirely to production of cyclic lipopeptides since deletion of sfp, a gene essential for surfactin synthesis which abolished the cytotoxicity to Vero cells, boar sperm motility and haemolytic activity. Thus, the relevance of cyclic lipopeptides as food poisoning toxins needs to be evaluated in assays other than the cell cytotoxicity assays in common use.

Appearance of bacteriology in the British medical school curriculum.

Published histories of bacteriology concentrate on the scientific concepts, exemplified by Louis Pasteur and Robert Koch. Arguably, the early British bacteriological studies are headed by Lord Lister, whereas other notables such as Ronald Ross, Robert Bruce and Patrick Manson are honoured for their discoveries of 'tropical' microbes, accomplished abroad. What then was happening in Great Britain? The introduction of bacteriology into the medical school curriculum is examined according to the published lectures in The Lancet between 1889 and 1901 and the dates are reviewed in light of other published sources. The names of the people delivering bacteriology at the medical schools in Great Britain and Ireland provide a guide to the relevance of crediting Lister as the leading light for microbiology in the UK. The diversity of names and backgrounds suggests that a critical reassessment of the perceived late and limited start of UK medical bacteriology is needed.

Okadaic acid-sensitive activation of Maxi Cl(-) channels by triphenylethylene antioestrogens in C1300 mouse neuroblastoma cells.

1. The regulation of Maxi Cl(-) channels by 17beta-oestradiol and non-steroidal triphenylethylene antioestrogens represents a rapid, non-classical effect of these compounds. In the present study we have investigated the signalling pathways used for the regulation of Maxi Cl(-) channel activity by oestrogens and antioestrogens in C1300 neuroblastoma cells. 2. Whole-cell Maxi Cl(-) currents were readily and reversibly activated by tamoxifen, toremifene and the membrane-impermeant ethyl-bromide tamoxifen, only when applied to the extracellular medium. 3. Pre-treatment of C1300 cells with oestrogen or cAMP prevented the antioestrogen-induced activation of Maxi Cl(-) channels. The inhibitory effect of 17beta-oestradiol and cAMP was abolished by the kinase inhibitor staurosporine. 4. Current activation was unaffected by the removal of intracellular Ca(2+) and Mg(2+), but was completely abolished in the presence of okadaic acid. These results are consistent with the participation of an okadaic acid-sensitive serine/threonine protein phosphatase in the activation of Maxi Cl(-) channels. However, neither oestrogen or antioestrogen treatment modified the total activity of the two major serine/threonine phosphatases, PP1 and PP2A, in C1300 cells. 5. Although the role of these Maxi Cl(-) channels remains unknown, our findings suggest strongly that their modulation by oestrogens and antioestrogens is linked to intracellular signalling pathways.

Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins.

Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain. Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability. In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins. The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.

CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia.

CytK is a cytotoxin isolated from a strain of Bacillus cereus cultured from cases of necrotic enteritis and the amino acid sequence of the protein suggests that it may belong to the family of beta-barrel pore-forming toxins. We show here in planar lipid bilayers the toxin is able to form pores which are weakly anion selective and exhibit an open channel probability close to one. The predicted minimum pore diameter is approximately 7 A. We also show that cytK is a potent cytotoxin against human intestinal Caco-2 epithelia. CytK, like other beta-barrel pore-forming toxins, spontaneously forms oligomers which are resistant to sodium dodecyl sulphate (SDS), but not to boiling. CytK represents a pore-forming toxin linked with human cases of necrotic enteritis.

Volume--activated chloride currents in HeLa cells are blocked by tamoxifen but not by a membrane impermeant quaternary analogue.

BACKGROUND/AIMS: Tamoxifen has been shown to inhibit volume activated chloride currents in many cell types. Tamoxifen has also been reported to inhibit a number of cation channels as well as cytosolic proteins such as calmodulin. The mechanism of channel block by tamoxifen is not known but three hypotheses can be proposed: i) a direct effect following binding to the channel protein from the aqueous environment or ii) a direct effect on the channel protein after partitioning into the lipid membrane or iii) an indirect mechanism via binding to intracellular regulatory proteins after diffusion across the lipid membrane. The aim of these experiments was to distinguish between these hypotheses using membrane permeant and impermeant antioestrogens. METHODS: Volume activated chloride currents were recorded from single HeLa cells using whole cell patch clamp technique. The ability of tamoxifen and its membrane impermeant quaternary derivative ethyl bromide tamoxifen (EBT) to inhibit these currents was examined. RESULTS: Extracellular tamoxifen at 3 microM inhibited volume activated chloride currents in HeLa cells whereas EBT had no effect up to 10 microM when applied either to the extracellular bathing solution or the intracellular solution via the patch pipette. CONCLUSION: Eliminating the ability of tamoxifen to cross the plasma membrane abolishes its channel blocking activity against volume activated chloride channels in HeLa cells.

Membrane impermeant antioestrogens discriminate between ligand- and voltage-gated cation channels in NG108-15 cells.

Native 5-HT(3) and AChR ligand-gated cation channels can be inhibited (blocked) by the non-steroidal antioestrogen tamoxifen. However, the exact site and mechanism of inhibition by tamoxifen on these channels remain unclear. We have investigated the action of the membrane impermeant quaternary derivative, ethylbromide tamoxifen (EBT), on native ligand-gated 5-HT(3) receptor channels and voltage-gated K(+) channels in NG108-15 cells using whole cell patch clamp. Extracellular EBT inhibited whole cell cationic currents of 5-HT(3) receptors with IC(50) of 0.22+/-0.4 microM (n(H)=1.05+/-0.2). The channel block was characterised by voltage independent and use independent behaviour (similar to that of tamoxifen). EBT was unable to inhibit voltage-gated K(+) currents in NG108-15 cells. This was in contrast to the inhibition by tamoxifen which, at similar concentrations, accelerated the apparent inactivation of these outward K(+) currents. The inhibition of 5-HT(3) receptors by a membrane impermeant derivative of tamoxifen supports the view that the binding site for antioestrogens is extracellular and the inhibition is not mediated through genomic/transcriptional activity.

Expression of nucleotide-regulated Cl(-) currents in CF and normal mouse tracheal epithelial cell lines.

The dominant route for Cl(-) secretion in mouse tracheal epithelium is via Cl(-) channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(-/-)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/-)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10(-4) M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current (I(sc)) and resistance of both cell lines, with larger increases in I(sc) being elicited by ionomycin (10(-6) M). Both cell lines expressed P(2)Y(2) receptors and responded to the purinergic agonists ATP, UTP, and 5'-adenylylimidodiphosphate (10(-4) M) with an increase in I(sc). This response could be inhibited by DIDS and was abolished in the presence of Cl(-)-free Ringer solution. Reducing the mucosal Cl(-) concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(-/-) cells. Pretreatment of these cells with thapsigargin caused a direct increase in I(sc) and inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl(-) currents and may prove valuable tools in studying the properties of this pathway.

Advantages of percutaneous hollow needle technique for iliac bone harvest in alveolar cleft grafting.

OBJECTIVE: To evaluate the percutaneous hollow needle technique for bone harvest to determine if morbidity from the bone donor site can be reduced significantly. METHODS: A retrospective chart review was performed evaluating all patients undergoing alveolar bone grafting at our institution from January 1992 through December 1996. Patients who underwent additional major procedures were excluded. Group I consisted of 12 patients in whom the percutaneous technique was utilized. The patients had an average age of 11.1 years (range: 8 to 15 years). Six were male and six were female. One had a bilateral cleft. Group II consisted of 15 patients in whom the conventional open technique for iliac crest bone harvest was used. They had an average age of 13.1 years (range: 7 to 31 years). Six were male and nine were female. Two had bilateral clefts. Minimum follow-up was 6 months. We evaluated intraoperative blood loss, total postoperative analgesia requirement, and length of hospital stay based on a retrospective hospital chart review. RESULTS: A significant difference was found between the two groups regarding intraoperative blood loss (group I: 83.3 cm3, group II: 208 cm3; p = .0015), postoperative total analgesia requirement (group I: 0.04 mg/kg, range: 0 to 0.17 mg/kg; group II: 0.34 mg/kg, range: 0.03 to 0.74 mg/kg; p = .0002), and length of hospital stay (group I: 1.0 days, group II: 2.13 days; p = .0001). There was no significant change in these results when bilateral clefts were excluded. CONCLUSION: Iliac bone graft harvest using the percutaneous hollow needle technique results in less blood loss, decreased postoperative pain, and shorter hospital stays compared with the open technique.

Cationic currents induced by Clostridium perfringens type A enterotoxin in human intestinal CaCO-2 cells.

Clostridium perfringens type A produces an enterotoxin that induces diarrhoea experimentally in man and animals. The enterotoxin causes increased membrane permeability in susceptible cells which is thought to be due to pore formation in the host cell membrane. The effect of purified C. perfringens enterotoxin on intact intestinal CaCO-2 monolayers was examined in Ussing chambers and on single cells by whole-cell patch clamp. Mucosal application of C. perfringens enterotoxin resulted in prompt increases in short-circuit current coupled with a reduction in transepithelial resistance consistent with movement of sodium and other cations smaller than diethanolamine from mucosa to serosa. These changes were independent of extracellular calcium. Increases in short-circuit current were also observed in the apical membranes of CaCO-2 monolayers permeabilised across the basolateral membrane with nystatin. Currents were blocked by subsequent exposure to mucosal barium and zinc. Zinc also prevented the development of the current increases in apical membranes. Cationic currents were also observed following exposure of single CaCO-2 cells in whole-cell patch clamp recordings. These data indicate that C. perfringens enterotoxin is able to form cation permeant pores in the apical membrane of human intestinal CaCO-2 epithelia and the increases in short-circuit current can be prevented by pre-exposure to zinc ions.

Inhibition of ligand-gated cation-selective channels by tamoxifen.

The nonsteroidal antioestrogen tamoxifen has been shown to block a number of voltage-gated cation-selective channels but its effect on ligand-gated cation-selective channels has not been studied. We have investigated the action of tamoxifen and the related derivative toremifene on ligand-gated cationic nicotinic acetylcholine and 5-HT3 receptor channels. Tamoxifen and toremifene both inhibited cationic currents of adult-type human muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes with similar IC50 values of 1.2 +/- 0.03 microM (nH = 0.84 +/- 0.02) and 1.2 +/- 0.1 microM (nH = 1.1 +/- 0.1), respectively. Tamoxifen could also block native 5-HT3 receptors in NG108-15 neuroblastoma/glioma hybrid cells with IC50 = 0.81 +/- 0.15 microM and nH of 1.3 +/- 0.3. The characteristics of block by tamoxifen at the 5-HT3 receptor were voltage- and use-independent. The inhibition of the 5-HT-evoked currents were not overcome by increasing concentrations of 5-HT consistent with a noncompetitive mechanism of block.

Inhibition of voltage-gated cationic channels in rat embryonic hypothalamic neurones and C1300 neuroblastoma cells by triphenylethylene antioestrogens.

The effect of the non-steroidal antioestrogens tamoxifen and toremifene on voltage-gated cationic currents was examined in primary cultures of rat hypothalamic neurones and the C1300 mouse neuroblastoma cell line using the whole-cell patch clamp technique. When applied to the external bathing solution both tamoxifen and toremifene were able to inhibit TTX-sensitive sodium currents with IC50 values of 1-2 microM and delayed rectifier type potassium currents (IC50, 2-3 microM). However, only toremifene showed a significant inhibition of the I(A) current (IC50 3 microM). Inhibition of voltage-gated cationic currents was significantly impaired when tamoxifen was applied in a serum-containing solution. The steroidal antioestrogen ICI 182,780 did not inhibit any of the currents at 10 microM.

Ocular staphyloma associated with facial clefting.

A staphyloma is an uncommon ocular lesion consisting of an attenuation in the sclera, which, along with the underlying uveal tissue, bulges to form a raised pigmented area on the eye. The scleral defect predisposes the globe to rupture under conditions of increased intraocular pressure, which might occur while retracting the eye during cranio-orbital surgery. We report a case of a staphyloma in a child with bilateral facial clefts. Before hypertelorism correction, she underwent scleral repair with a cadaveric graft. Her orbital repositioning was performed without incident 10 months later. The significance of a possible association between facial clefting and staphyloma is discussed.

The frontonasal flap for increased exposure in posttraumatic nasal deformity: a technical overview.

Nasal reconstruction continues to be a surgical challenge. The prominent location of the nose, the unique quality and texture of its skin, and the intricacies of its cartilaginous and bony infrastructure demand careful attention to fine detail. Attempts to refine reconstructive techniques have resulted in a myriad of local flaps. The frontonasal flap is well-described and reliable, but it is infrequently used. A brief review of the literature is presented. The authors describe a unique case of a 64-year-old woman with posttraumatic nasal tip and dorsal deformity. The frontonasal flap provided soft tissue coverage for the nasal tip and allowed excellent exposure for reconstruction of the hard nasal framework with cartilage and bone grafts. It provides local tissue with excellent contour, color, and texture match, and can be performed in one stage.

Cycling Werner's syndrome fibroblasts display calcium-dependent potassium currents.

Werner's Syndrome (WS) fibroblasts undergo premature senescence. Two hypotheses have been proposed to explain this phenomenon: (i) the phenotype is due to the overexpression of senescence-specific proteins in every cell in the population. Such proteins are known to suppress calcium-dependent potassium currents. (ii) The WS mutation greatly increases the proportion of cells that stop cycling at each generation and become senescent. If hypothesis (i) is correct, such currents should be suppressed in all WS fibroblasts; whereas hypothesis (ii) predicts that they will be retained in the cycling fraction of the population. To distinguish between these hypotheses whole-cell patch-clamp currents were recorded from cycling cells. Slowly activating outward calcium-dependent potassium currents were detected in both cycling WS and control fibroblasts. These findings support hypothesis (ii): the premature senescence of WS fibroblasts is due to an increased rate of transition from cycling to senescence in the total cell population.

The multidrug resistance P-glycoprotein modulates cell regulatory volume decrease.

Cell volume is frequently down-regulated by the activation of anion channels. The role of cell swelling-activated chloride channels in cell volume regulation has been studied using the patch-clamp technique and a non-invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P-glycoprotein (P-gp), increased the rate of channel activation and the rate of RVD. In addition, P-gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P-gp. The data show that, in these cell types, swelling-activated chloride channels have a central role in RVD. Moreover, they clarify the role of P-gp in channel activation and provide direct evidence that P-gp, through its effect on chloride channel activation, enhances the ability of cells to down-regulate their volume.