Developing an experimental necrotic enteritis model in turkeys - the impact of Clostridium perfringens, Eimeria meleagrimitis and host age on frequency of severe intestinal lesions.

BACKGROUND: Necrotic enteritis is a significant problem to the poultry industry globally and, in Norway up to 30% of Norwegian turkey grow-outs can be affected. However, despite an awareness that differences exist between necrotic enteritis in chickens and turkeys, little information exists concerning the pathogenesis, immunity, microbiota or experimental reproduction of necrotic enteritis in turkeys. In particular, it is important to determine the appearance of the gross lesions, the age dependency of the disease and the role of netB toxin of Clostridium perfringens. To this end, we report our findings in developing an in vivo experimental model of necrotic enteritis in turkeys. RESULTS: A four tier (0-3) scoring system with clearly defined degrees of severity of macroscopic intestinal lesions was developed, based on 2312 photographic images of opened intestines from 810 B.U.T. 10 or B.U.T. Premium turkeys examined in nine experiments. Loss of macroscopically recognizable villi in the anterior small intestine was established as the defining lesion qualifying for a score 3 (severe intestinal lesions). The developed scoring system was used to identify important factors in promoting high frequencies of turkeys with severe lesions: a combined Eimeria meleagrimitis and Clostridium perfringens challenge, challenge at five rather than 3 weeks of age, the use of an Eimeria meleagrimitis dose level of at least 5000 oocysts per bird and finally, examination of the intestines of 5-week-old turkeys at 125 to 145 h after Eimeria meleagrimitis inoculation. Numbers of oocysts excreted were not influenced by Clostridium perfringens inoculation or turkey age. Among three different lesion score outcomes tested, frequency of severe lesions proved superior in discriminating between impact of four combinations of Clostridium perfringens inoculation and turkey age at challenge. CONCLUSIONS: This study provides details for the successful establishment of an in vivo model of necrotic enteritis in turkeys.

Analysis of Campylobacter jejuni infection in the gnotobiotic piglet and genome-wide identification of bacterial factors required for infection.

To investigate how Campylobacter jejuni causes the clinical symptoms of diarrhoeal disease in humans, use of a relevant animal model is essential. Such a model should mimic the human disease closely in terms of host physiology, incubation period before onset of disease, clinical signs and a comparable outcome of disease. In this study, we used a gnotobiotic piglet model to study determinants of pathogenicity of C. jejuni. In this model, C. jejuni successfully established infection and piglets developed an increased temperature with watery diarrhoea, which was caused by a leaky epithelium and reduced bile re-absorption in the intestines. Further, we assessed the C. jejuni genes required for infection of the porcine gastrointestinal tract utilising a transposon (Tn) mutant library screen. A total of 123 genes of which Tn mutants showed attenuated piglet infection were identified. Our screen highlighted a crucial role for motility and chemotaxis, as well as central metabolism. In addition, Tn mutants of 14 genes displayed enhanced piglet infection. This study gives a unique insight into the mechanisms of C. jejuni disease in terms of host physiology and contributing bacterial factors.

Multivariate evaluation of the effectiveness of treatment efficacy of cypermethrin against sea lice (Lepeophtheirus salmonis) in Atlantic salmon (Salmo salar).

BACKGROUND: The sea louse Lepeophtheirus salmonis is the most important ectoparasite of farmed Atlantic salmon (Salmo salar) in Norwegian aquaculture. Control of sea lice is primarily dependent on the use of delousing chemotherapeutants, which are both expensive, and toxic to other wildlife. The method most commonly used for monitoring treatment effectiveness relies on measuring the percentage reduction in the mobile stages of Lepeophtheirus salmonis only. However, this does not account for changes in the other sea lice stages and may result in misleading or incomplete interpretation regarding the effectiveness of treatment. With the aim of improving the evaluation of delousing treatments, we explored multivariate analyses of bath treatments using the topical pyrethroid, cypermethrin, in salmon pens at five Norwegian production sites. RESULTS: Conventional univariate analysis indicated reductions of over 90% in mobile stages at all sites. In contrast, multivariate analyses indicated differing treatment effectiveness between sites (p-value < 0.01) based on changes in the proportion and abundance of the chalimus and PAAM (pre-adult and adult males) stages. Low water temperatures and shortened intervals between sampling after treatment may account for the differences in the composition of chalimus and PAAM stage groups following treatment. Using multivariate analysis, such factors could be separated from those which were attributable to inadequate treatment or chemotherapeutant failure. CONCLUSIONS: Multivariate analyses for evaluation of treatment effectiveness against multiple life cycle stages of L. salmonis yield additional information beyond that derivable from univariate methods. This can aid in the identification of causes of apparent treatment failure in salmon aquaculture.

Type C and C/D toxigenic Clostridium botulinum is not normally present in the intestine of healthy broilers.

Toxigenic Clostridium botulinum spores are widely distributed in wetland environments and are frequently recovered from healthy wild birds, where ingestion of toxic maggots from carcasses is considered a major initiating factor for botulism outbreaks. Toxic carcasses can perpetuate an existing outbreak also in poultry, but their importance as an initiating factor for poultry botulism is less clear. Although toxigenic C. botulinum can be recovered from healthy broilers during outbreaks, there are almost no data on the prevalence in healthy broiler flocks (and, by extension, carcasses) and their environment. To test the hypothesis that toxigenic C. botulinum is frequently present in healthy broilers, we examined 100 healthy broiler flocks and environmental samples from 30 broiler houses 3 to 4 years after an epidemic of botulism in the broiler population. None of the 100 flocks yielded type C or C/D botulinum toxin genes using real time PCR whereas a flock house that had remained empty since the most recent outbreak yielded type C (or C/D) toxin genes. The absence of toxigenic C. botulinum in all examined flocks indicates that botulism is a sporadic and exogenously acquired event in this broiler population and unlikely to mirror the epidemiology in wild birds.

Structural and functional analysis of the pore-forming toxin NetB from Clostridium perfringens.

Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a ß-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a ß-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.

Inhibition of cytotoxicity by the Nhe cytotoxin of Bacillus cereus through the interaction of dodecyl maltoside with the NheB component.

Nhe ('nonhaemolytic enterotoxin') is a three-component cytotoxin implicated in the pathogenesis of diarrhoea by Bacillus cereus. Nhe forms pores in pure lipid bilayers, but the function of the individual components (NheA, NheB and NheC) remains unclear. NheB and NheC are structural homologues of ClyA, a pore-forming cytotoxin of Escherichia coli. The non-ionic detergent dodecyl maltoside (DDM) has been shown to inhibit haemolysis of ClyA. We used DDM as a probe to examine the response of the Nhe proteins to DDM micelles. At its critical micellar concentration (0.2 mM), DDM inhibited propidium uptake by the native Nhe complex in Vero and HT29 cell suspensions. Pre-incubation of NheC with DDM did not inhibit cytotoxicity. NheB exhibited marked changes in 1-anilinonaphthalene-8-sulphonic acid (ANS) fluorescence after pre-exposure to DDM. Pre-incubation of NheB with DDM resulted in large molecular weight complexes as detected by size exclusion chromatography and diffusion through sized dialysis membranes and prevented binding of NheB to Vero cell monolayers. These data support a model in which conformational changes and oligomerization of NheB are prerequisite events in the process of pore formation.

Relative entropy differences in bacterial chromosomes, plasmids, phages and genomic islands.

BACKGROUND: We sought to assess whether the concept of relative entropy (information capacity), could aid our understanding of the process of horizontal gene transfer in microbes. We analyzed the differences in information capacity between prokaryotic chromosomes, genomic islands (GI), phages, and plasmids. Relative entropy was estimated using the Kullback-Leibler measure. RESULTS: Relative entropy was highest in bacterial chromosomes and had the sequence chromosomes > GI > phage > plasmid. There was an association between relative entropy and AT content in chromosomes, phages, plasmids and GIs with the strongest association being in phages. Relative entropy was also found to be lower in the obligate intracellular Mycobacterium leprae than in the related M. tuberculosis when measured on a shared set of highly conserved genes. CONCLUSIONS: We argue that relative entropy differences reflect how plasmids, phages and GIs interact with microbial host chromosomes and that all these biological entities are, or have been, subjected to different selective pressures. The rate at which amelioration of horizontally acquired DNA occurs within the chromosome is likely to account for the small differences between chromosomes and stably incorporated GIs compared to the transient or independent replicons such as phages and plasmids.

The use of a porcine intestinal cell model system for evaluating the food safety risk of Bacillus cereus probiotics and the implications for assessing enterotoxigenicity.

The use of porcine intestinal cell lines in assessing toxicity of Bacillus cereus probiotics in conjunction with animal challenge trials with toxigenic B. cereus was investigated. Toxigenic and toxin deletion mutants of B. cereus and two probiotic strains (Paciflor and Toyocerin) were examined for bacterial attachment, cytotoxicity and ability to induce nitric oxide as markers of toxicity. Both cytotoxicity and production of nitric oxide were detected in wild-type toxigenic strains and the Paciflor probiotic strain but not Toyocerin. Attachment of B. cereus was low (less than 1%) in all strains. Discrimination between toxigenic B. cereus and the probiotic strains was possible semi-quantitatively via dilution. Despite cytotoxicity in vitro, challenge experiments using 10(8)-10(9) spores of the toxigenic B. cereus NVH75/95 in weaned piglets did not induce diarrhoea or intestinal lesions. Thus, the pig small intestinal epithelial intestinal cell line PSI is appropriate for identification of potential toxicity in B. cereus strains and sets a low threshold for risk of enterotoxicity to humans.

Formation of very large conductance channels by Bacillus cereus Nhe in Vero and GH(4) cells identifies NheA + B as the inherent pore-forming structure.

The nonhemolytic enterotoxin (Nhe) produced by Bacillus cereus is a pore-forming toxin consisting of three components, NheA, -B and -C. We have studied effects of Nhe on primate epithelial cells (Vero) and rodent pituitary cells (GH(4)) by measuring release of lactate dehydrogenase (LDH), K(+) efflux and the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Plasma membrane channel events were monitored by patch-clamp recordings. Using strains of B. cereus lacking either NheA or -C, we examined the functional role of the various components. In both cell types, NheA + B + C induced release of LDH and K(+) as well as Ca(2+) influx. A specific monoclonal antibody against NheB abolished LDH release and elevation of [Ca(2+)](i). Exposure to NheA + B caused a similar K(+) efflux and elevation of [Ca(2+)](i) as NheA + B + C in GH(4) cells, whereas in Vero cells the rate of K(+) efflux was reduced by 50% and [Ca(2+)](i) was unaffected. NheB + C had no effect on either cell type. Exposure to NheA + B + C induced large-conductance steps in both cell types, and similar channel insertions were observed in GH(4) cells exposed to NheA + B. In Vero cells, NheA + B induced channels of much smaller conductance. NheB + C failed to insert membrane channels. The conductance of the large channels in GH(4) cells was about 10 nS. This is the largest channel conductance reported in cell membranes under quasi-physiological conditions. In conclusion, NheA and NheB are necessary and sufficient for formation of large-conductance channels in GH(4) cells, whereas in Vero cells such large-conductance channels are in addition dependent on NheC.

Analysis of intra-genomic GC content homogeneity within prokaryotes.

BACKGROUND: Bacterial genomes possess varying GC content (total guanines (Gs) and cytosines (Cs) per total of the four bases within the genome) but within a given genome, GC content can vary locally along the chromosome, with some regions significantly more or less GC rich than on average. We have examined how the GC content varies within microbial genomes to assess whether this property can be associated with certain biological functions related to the organism's environment and phylogeny. We utilize a new quantity GCVAR, the intra-genomic GC content variability with respect to the average GC content of the total genome. A low GCVAR indicates intra-genomic GC homogeneity and high GCVAR heterogeneity. RESULTS: The regression analyses indicated that GCVAR was significantly associated with domain (i.e. archaea or bacteria), phylum, and oxygen requirement. GCVAR was significantly higher among anaerobes than both aerobic and facultative microbes. Although an association has previously been found between mean genomic GC content and oxygen requirement, our analysis suggests that no such association exits when phylogenetic bias is accounted for. A significant association between GCVAR and mean GC content was also found but appears to be non-linear and varies greatly among phyla. CONCLUSIONS: Our findings show that GCVAR is linked with oxygen requirement, while mean genomic GC content is not. We therefore suggest that GCVAR should be used as a complement to mean GC content.

Cytotoxicity of the Bacillus cereus Nhe enterotoxin requires specific binding order of its three exoprotein components.

This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addition of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in solution, excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addition of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted beta-tongue were replaced with glycine (NheCcys-) or where the entire hydrophobic stretch was deleted (NheChr-), the predicted hydrophobic beta-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in solution. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is associated with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.

Stretches of alternating pyrimidine/purines and purines are respectively linked with pathogenicity and growth temperature in prokaryotes.

BACKGROUND: The genomic fractions of purine (RR) and alternating pyrimidine/purine (YR) stretches of 10 base pairs or more, have been linked to genomic AT content, the formation of different DNA helices, strand-biased gene distribution, DNA structure, and more. Although some of these factors are a consequence of the chemical properties of purines and pyrimidines, a thorough statistical examination of the distributions of YR/RR stretches in sequenced prokaryotic chromosomes has to the best of our knowledge, not been undertaken. The aim of this study is to expand upon previous research by using regression analysis to investigate how AT content, habitat, growth temperature, pathogenicity, phyla, oxygen requirement and halotolerance correlated with the distribution of RR and YR stretches in prokaryotes. RESULTS: Our results indicate that RR and YR-stretches are differently distributed in prokaryotic phyla. RR stretches are overrepresented in all phyla except for the Actinobacteria and beta-Proteobacteria. In contrast, YR tracts are underrepresented in all phyla except for the beta-Proteobacterial group. YR-stretches are associated with phylum, pathogenicity and habitat, whilst RR-tracts are associated with phylum, AT content, oxygen requirement, growth temperature and halotolerance. All associations described were statistically significant with p < 0.001. CONCLUSION: Analysis of chromosomal distributions of RR/YR sequences in prokaryotes reveals a set of associations with environmental factors not observed with mono- and oligonucleotide frequencies. This implies that important information can be found in the distribution of RR/YR stretches that is more difficult to obtain from genomic mono- and oligonucleotide frequencies. The association between pathogenicity and fractions of YR stretches is assumed to be linked to recombination and horizontal transfer.

Demonstration of a cholesterol-dependent cytolysin in a noninsecticidal Bacillus sphaericus strain and evidence for widespread distribution of the toxin within the species.

During the course of screening Bacillus species from food and water in Norway, we isolated a strain of Bacillus sphaericus of DNA homology group V, not previously recognized to contain entomopathogenic strains, that was cytotoxic to Vero cell epithelia. Peptide mass fingerprinting of a protein purified from the culture supernatant of B. sphaericus B354 identified a cholesterol-dependent cytolysin (CDC) with high amino acid sequence identity with sphaericolysin, a CDC identified recently in B. sphaericus DNA homology group IIA. The toxin was haemolytic against erythrocytes from a range of species. Haemolysis was potentiated by dithiothreitol and inhibited by preincubation with cholesterol. The toxin induced lactate dehydrogenase release from Vero cells and formed pores in planar lipid bilayers. The distribution of CDC genes in B. sphaericus was examined, with CDC gene products obtained in 13 out of 17 strains representing four of the six DNA homology groups. Thus, we demonstrate the presence of a CDC in a nonentomopathogenic DNA homology group of B. sphaericus (group V) with typical CDC characteristics. CDCs appear to be present in a high proportion of B. sphaericus strains and are not restricted to group IIA insecticidal strains.

Bacillus cereus Nhe is a pore-forming toxin with structural and functional properties similar to the ClyA (HlyE, SheA) family of haemolysins, able to induce osmotic lysis in epithelia.

The mechanism by which Bacillus cereus causes diarrhoea is unknown. Three putative enterotoxins have been proposed, haemolysin BL (Hbl), cytotoxin K and non-haemolytic enterotoxin (Nhe). Both Hbl and Nhe are three-component cytotoxins and maximal cytotoxicity of Nhe against epithelia is dependent on all three components. However, little is known of the mechanism of cytotoxicity. Markers of plasma membrane disruption, namely propidium iodide uptake, loss of cellular ATP and release of lactate dehydrogenase (LDH), were observed in epithelia exposed to Nhe from culture supernatants of B. cereus, but not in those exposed to supernatants from a mutant strain lacking NheB and NheC. Consistent with an exogenous cause of membrane damage, purified Nhe components combined to form large conductance pores in planar lipid bilayers. The inhibition of LDH release by osmotic protectants and the increase in cell size caused by Nhe indicate that epithelia lyse following osmotic swelling. Nhe and Hbl show sequence homology, and Hbl component B has remarkable structural similarities to cytolysin A (ClyA), with both structures possessing an alpha-helix bundle and a unique subdomain containing a hydrophobic beta-hairpin. Correspondingly, we show that Nhe has haemolytic activity against erythrocytes from a variety of species. We propose that the common structural and functional properties indicate that the Hbl/Nhe and ClyA families of toxins constitute a superfamily of pore-forming cytotoxins.

Campylobacter jejuni inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular tight junctions.

Caco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in 'domes' forming in differentiated monolayers. Here, the effect of Campylobacter jejuni on this process has been examined. C. jejuni caused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized that C. jejuni could inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease. C. jejuni infection of fluid-transporting ('doming') Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants of C. jejuni also reduced dome numbers in infected monolayers. C. jejuni also altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism of C. jejuni.

Single channel evidence for innate pore-formation by Vibrio parahaemolyticus thermostable direct haemolysin (TDH) in phospholipid bilayers.

Vibrio parahaemolyticus thermostable direct haemolysin (TDH) is widely considered to be a pore-forming toxin. The protein has no significant homology to other known pore-forming toxins and its mechanism of action in vivo remains undefined. We demonstrate single channel pore-forming activity of V. parahaemolyticus TDH in planar lipid bilayers. Channel conductance ranged between 30-450 pS in 0.5 M KCl with a calculated cation selectivity (P(K)/P(Cl)) of 2.7. Channels were formed in NaCl and choline-Cl with and without cholesterol present and in the presence of neutral or negatively charged phospholipids. Zinc ions did not block pore formation. Whilst various techniques have previously suggested that TDH is a pore-forming toxin, the data in this study provide direct single channel evidence and indicate several features of pore formation in synthetic phospholipid membranes.

Genetic and functional analysis of the cytK family of genes in Bacillus cereus.

CytK is a pore-forming toxin of Bacillus cereus that has been linked to a case of necrotic enteritis. PCR products of the expected size were generated with cytK primers in 13 of 29 strains. Six strains were PCR-positive for the related gene hly-II, which encodes haemolysin II, a protein that is 37 % identical to the original CytK. Five of the strains were positive for both genes. The DNA sequences of putative cytK genes from three positive strains were determined, and the deduced amino acid sequences were 89 % identical to that of the original CytK. The authors have designated this new cytK variant cytK-2, and refer to the original cytK as cytK-1. The CytK-2 proteins from these three strains were isolated, and their identity was verified by N-terminal sequencing. blast analysis using the cytK-2 gene sequences revealed very high homology with two cytK-2 sequences in the genomes of B. cereus strains ATCC 14579 and ATCC 10987. The differences between CytK-1 and the CytK-2 proteins were clustered to certain regions of the proteins. The isolated CytK-2 proteins were haemolytic and toxic towards human intestinal Caco-2 cells and Vero cells, although their toxicity was about 20 % of that of CytK-1. Both native and recombinant CytK-2 proteins from B. cereus 1230-88 were able to form pores in planar lipid bilayers, but the majority of the channels observed were of lower conductance than those created by CytK-1. It is likely that CytK-2 toxins contribute to the enterotoxicity of several strains of B. cereus, although not all of the CytK-2 toxins may be as harmful as the CytK-1 originally isolated.

Activation of Maxi Cl(-) channels by antiestrogens and phenothiazines in NIH3T3 fibroblasts.

The identification of alternative estrogen actions has been accumulating steadily over the past two decades. Typically, these novel actions are not directly related to nuclear transcriptional events but related to the interaction of estrogens with sites present at plasma membrane or cytosolic locations. These alternative effects, widely known as non-genomic effects, range from the modulation of plasma membrane ion channel activity to the regulation of different intracellular signalling cascades. In the present study we have investigated the modulation of a large conductance chloride channel (Maxi Cl(-)) by estrogens, non-steroidal triphenylethylene antiestrogens and phenothiazines in NIH3T3 fibroblasts and the dependence on guanosine triphosphate (GTP) of the Maxi Cl(-) activation. Our data identifies the non-steroidal antiestrogens toremifene and tamoxifen, and the phenothiazines chlorpromazine and triflupromazine as activators of Maxi Cl(-) channels. In contrast, 17 beta-estradiol and cAMP, added prior to the exposure to antiestrogens, prevent channel activation. The pure antiestrogen ICI 182780 did not activate the channel nor prevent its activation by non-steroidal antiestrogens. The activation of Maxi Cl(-) channels by toremifene and tamoxifen required the presence of intracellular nucleotides and was inhibited by the stable analog, GDP beta -S, suggesting the participation of a G-protein in the activation process. Little is known about the physiological relevance of Maxi Cl(-) channels. However, that fact that its regulation by estrogens and antiestrogens is shared by different cell types might imply a common role which needs to be identified.