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The Early Growth Response protein (Egr-1) is a C(2)H(2)-zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation-reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H(2)O(2) stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H(2)O(2) stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.

Vaginal innate immunity in response to microbial perturbation is still poorly understood and could be crucial for protection from adverse outcomes. We investigated the relationship between interleukin (IL)-8, IL-1beta and neutrophils in vaginal fluid obtained from 60 healthy women and 51 women who were bacterial vaginosis (BV) positive. Concentrations of IL-8 and IL-1beta were highly correlated with counts of neutrophils in vaginal fluid of the entire population examined (111 subjects). Vaginal IL-1beta concentrations were significantly higher (P0.001) in BV positive women. There was no significant difference in IL-8 levels or number of neutrophils between healthy controls and BV positive women. None of the healthy controls with high neutrophil counts (or =75th percentile, 14 average count per field) had high concentrations of IL-1beta (or =75th percentile, 220 pg/ml), whereas 84% of BV positive women with high neutrophil counts had high IL-1beta concentrations (P0.001). On the contrary, no difference in the percentage of subjects with elevated concentrations of IL-8 (or =75th percentile, 2842 pg/ml) was found between healthy and BV positive women with high numbers of neutrophils (55.5% of healthy versus 53% of BV positive women). Our findings show that BV causes a large increase in IL-1beta concentrations which is not paralleled by an increase in IL-8 concentrations in vaginal fluid, suggesting that BV-associated factors more specifically dampen IL-8 rather than IL-1beta. The lack of an increase in IL-8 may explain the absence of an increase in neutrophil numbers in most women exposed to abnormal vaginal colonization (BV).

A nested case-control study of low birth weight and preterm delivery was performed with singleton women. Immunoglobulin A (IgA) against the Gardnerella vaginalis hemolysin (anti-Gvh IgA) and sialidase and prolidase activities were determined in vaginal fluid at 17 weeks of gestation. Sialidase positivity and bacterial vaginosis with high prolidase activity were associated with 2- and 11-fold increased risks for low birth weight, respectively. No woman with bacterial vaginosis plus a strong anti-Gvh IgA response had an adverse outcome.

Mucosal immune system activation may represent a critical determinant of adverse consequences associated with bacterial vaginosis (BV), such as sexual human immunodeficiency virus transmission, upper genital tract infections, postsurgical infections, and adverse pregnancy outcomes. Concentrations of sialidase, prolidase, and anti-Gardnerella vaginalis hemolysin (Gvh) immunoglobulin A (IgA) were higher in vaginal fluids of 75 fertile women with BV, compared with concentrations in vaginal fluids of 85 healthy control subjects. Interleukin (IL)-8 levels were positively associated with anti-Gvh IgA response and inversely correlated with high levels of prolidase and sialidase in women with BV. IL-8 concentration was strongly associated with leukocyte count in both healthy and BV-positive women. The absence of leukocytes in most women with BV likely is due to lack of IL-8 induction. Parallel impairment of innate and adaptive mucosal immune factors, likely through microbial hydrolytic effects, may allow for the ascent of microorganisms to the upper genital tract and may facilitate viral infections.

Inhibin production has been demonstrated in malignant epithelial ovarian tumours, but secretion of inhibins by benign cystadenoma has not yet been reported. The present study evaluated the concentrations of inhibin A and inhibin B and the relationship with oestradiol and nitric oxide metabolites in fluid collected from benign ovarian serous cystadenomas (n = 15). In addition, follicular fluid samples (n = 14) from women with regular ovulatory cycles undergoing ovarian stimulation for IVF were studied as a reference group. High concentrations of inhibin A (median = 89.3 ng/ml) and inhibin B (median = 116.1 ng/ml) were found in the cystic fluid of ovarian serous cystadenomas. These inhibin concentrations were even higher than in follicular fluid of stimulated follicles (inhibins A and B = 41.2 and 46.8 ng/ml respectively; P: < 0.001), whereas oestradiol was approximately 18-fold lower in cystic fluid than in follicular fluid (median = 34 versus 622 pg/ml, P: < 0.001). In ovarian cysts, the concentrations of inhibin A and oestradiol were inversely correlated (r = -0.678, P: = 0.008). Cystic fluid samples containing the highest concentrations of NO(2)(-)/NO(3)(-) (45-60 micromol/l) had lower inhibin A and higher oestradiol concentrations than those samples containing lower concentrations (10-25 micromol/l) of NO(2)(-)/NO(3)(-). It is concluded that high amounts of dimeric inhibins are present in ovarian serous cystadenoma. The source of inhibins and the determinants of the inverse association of inhibin A with oestradiol and nitric oxide remain to be determined.

We have previously identified phosphodiester oligonucleotides exclusively made of G and T bases, named GTn, that significantly inhibit human cancer cell growth and recognize specific nuclear single-stranded DNA binding proteins. We wished to examine the ability of the modified GTn oligonucleotides with different degrees of phosphorothioate modifications to bind specifically to the same nuclear proteins recognized by the GTn phosphodiester analogues and their cytotoxic effect on the human T-lymphoblastic CCRF-CEM cell line. We showed that the full phosphorothioate GTn oligonucleotide was neither able to specifically recognize those nuclear proteins, nor cytotoxic. In contrast, the 3'-phosphorothioate-protected GTn oligonucleotides can maintain the specific protein-binding activity. The end-modified phosphorothioate oligonucleotides were also able to elicit the dose-dependent cell growth inhibition effect, but a loss in the cytotoxic ability was observed increasing the extent of sulphur modification of the sequences. Our results indicate that phosphorothioate oligonucleotides directed at specific single-stranded DNA-binding proteins should contain a number of phosphorothioate end-linkages which should be related to the length of the sequence, in order to maintain the same biological activities exerted by their phosphodiester analogues.

The functional importance of APE1 nucleolar accumulation is described. It is shown that acetylation of Lys27–35, affecting local conformation, regulates APE1 function by 1) controlling its interaction with NPM1 and rRNA and its nucleolar accumulation, 2) modulating K6/K7 acetylation status, and 3) promoting APE1 BER activity in cells., Apurinic/apyrimidinic endonuclease 1 (APE1) is the main abasic endonuclease in the base excision repair (BER) pathway of DNA lesions caused by oxidation/alkylation in mammalian cells; within nucleoli it interacts with nucleophosmin and rRNA through N-terminal Lys residues, some of which (K27/K31/K32/K35) may undergo acetylation in vivo. Here we study the functional role of these modifications during genotoxic damage and their in vivo relevance. We demonstrate that cells expressing a specific K-to-A multiple mutant are APE1 nucleolar deficient and are more resistant to genotoxic treatment than those expressing the wild type, although they show impaired proliferation. Of interest, we find that genotoxic treatment induces acetylation at these K residues. We also find that the charged status of K27/K31/K32/K35 modulates acetylation at K6/K7 residues that are known to be involved in the coordination of BER activity through a mechanism regulated by the sirtuin 1 deacetylase. Of note, structural studies show that acetylation at K27/K31/K32/K35 may account for local conformational changes on APE1 protein structure. These results highlight the emerging role of acetylation of critical Lys residues in regulating APE1 functions. They also suggest the existence of cross-talk between different Lys residues of APE1 occurring upon genotoxic damage, which may modulate APE1 subnuclear distribution and enzymatic activity in vivo.

The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF-1HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1HD alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF-1HD within the protein-DNA complex. TTF-1HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5'-TAAT-3', recognized by Antennapedia, and 5'-CAAG-3', preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5'-TAAT-3' or 5'-CAAG-3' indicate that only in the presence of the latter motif the Gln50 in TTF-1 HD is utilized for DNA recognition. Since the Gln at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5'-TAAT-3' containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts.

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein-protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

To discover new potential biomarkers of HCC, we used 2-DE gel separation and MALDI-TOF-MS analysis of partially enriched nuclear fractions from liver biopsies of 20 different patients. We obtained a proteomic map of subfractioned liver samples including about 200 common protein spots, among which identified components corresponded to expression products of 52 different genes. A differential analysis of proteins from tumoral and control tissues revealed a significant change in the expression level of 16 proteins associated to cytoskeletal, stress response and metabolic functions. These data may provide novel candidate biomarkers for HCC and useful insights for understanding the mechanisms of HCC pathogenesis and progression.

APE1/Ref-1 (also called HAP1 or APEX, and here referred to as APE1), the mammalian ortholog of Escherichia coli Xth (exonuclease III), is a vital protein (20) that acts as a master regulator of cellular response to oxidative stress conditions and contributes to the maintenance of genome stability (55, 56). APE1 is involved in both the base excision repair (BER) pathways of DNA lesions, acting as the major apurinic/apyrimidinic (AP) endonuclease, and in transcriptional regulation of gene expression as a redox coactivator of different transcription factors, such as early growth response protein 1 (Egr-1), NF-κB, and p53 (55, 56). These two biological activities are located in two functionally distinct protein domains. In fact, the N-terminal region, containing the nuclear localization signal (NLS) sequence, is principally devoted to redox activity through Cys65, while the C-terminal one exerts enzymatic activity on the abasic sites of DNA (56, 63). The protein C terminus is highly conserved during phylogenesis, while the N terminus is not. Except in mammals, which always show a high sequence conservation (more than 90%), and Danio, Drosophila, Xenopus, and Dictyostelium (presenting a sequence identity of less than 40%), the N-terminal region is mostly absent in other organisms. A third APE1 function, which is regulated by Lys6/Lys7 acetylation (7), is indirect binding to the negative calcium response elements (nCaRE) of some promoters (i.e., PTH and APE1 promoters), thus acting as a transcriptional repressor (12, 30). In different mammalian cell types, the APE1 subcellular distribution is mainly nuclear and is critical for controlling cellular proliferative rate (20, 25, 28). However, cytoplasmic, mitochondrial, and endoplasmic reticulum localization has also been reported (11, 22, 33, 50, 54). Interestingly, cytoplasmic expression of APE1 has been correlated with aggressiveness of different tumors (55, 56), although its role in tumorigenic processes is completely unknown. To date, no subnuclear distribution of APE1 has been reported. APE1 is an abundant protein (about 104 to 105 copies/cell) within eukaryotic cells and has a relatively long half-life (about 8 h). Thus, fine-tuning of the multiple APE1 functions may reside on its posttranslational modifications and on the modulation of its interactome under different conditions. While some posttranslational modifications have a functional role (i.e., Lys6/Lys7 acetylation) (7, 17), little information is available on APE1 protein interacting partners, except for those that are involved in BER function (38). Interestingly, proteolysis at residue Lys31 has recently been related to an enhanced immune cell death mediated by granzymes A and K (16, 23). This proteolytic event reduces APE1 accumulation within nuclei (16, 29) and its interaction with XRCC1 (60) and alters APE1 functions (16, 23). Recently, proteolysis occurring at Asn33 (giving rise to a protein form called NΔ33APE1) has also been described (11), suggesting that removal of the NLS may constitute a general mechanism for redirecting APE1 toward noncanonical subcellular compartments, such as mitochondria (11, 33, 54). Unfortunately, neither has the specific protease responsible for this cleavage been identified in nonimmune cells, nor has the mitochondrial localization signal been mapped yet. Mitochondrial localization of APE1 could be associated with a potential role in mtDNA repair of oxidized bases (11, 33, 54). However, since it is not clear whether NΔ33APE1 maintains its DNA repair activity in vivo (16) or acquires an aspecific endonuclease activity for double-stranded DNA (dsDNA) (66), at present it is not possible to drive any definitive conclusion. Moreover, as the truncated NΔ33APE1 form is associated with an apoptotic phenotype (23), it cannot be excluded that its generation may causatively be involved in the cytotoxic effect, driving proapoptotic triggering directly within mitochondria. The first 42 amino acids of APE1 are highly unordered in the protein crystallographic structure (3, 35), while the remainder of the protein has a globular fold (21). It is therefore presumable that the protein's N terminus is used for interacting with other partners, thus modulating the different APE1 functions. Interestingly, a similar bipartite arrangement for Rrp1, the Drosophila homologue of mammalian APE1, has been described, pointing out a functional role of the unstructured N-terminal domain in modulating protein-protein interactions (42, 52). By using an unbiased proteomic approach, in this work we have identified and characterized a novel APE1 complex. We found that APE1 N terminus is essential for binding to a number of proteins involved in ribosome biogenesis and RNA processing. Among the interacting partners, we focused on the nucleophosmin (NPM1)-APE1 interaction. NPM1 is an abundant protein which specifically resides within the granular region of the nucleolus and has been implicated in a variety of cellular processes, including centrosome duplication, maintenance of the genome's integrity, and ribosome biogenesis (19). NPM1 has a chaperone activity regulated by phosphorylation (51) and an endoribonuclease activity at a specific site of the spacer region between the 5.8S and the 28S rRNAs (43). Here, we demonstrate that the NPM1-APE1 interaction is required for APE1 subnuclear localization and for modulating the cleansing process of rRNA. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus possibly affecting gene expression through posttranscriptional mechanisms.

APE1/Ref-1, the mammalian ortholog of E. coli Xth, and a multifunctional protein possessing both DNA repair and transcriptional regulatory activities, has dual role in controlling cellular response to oxidative stress. It is rate-limiting in repair of oxidative DNA damage including strand breaks and also has co-transcriptional activity by modulating genes expression directly regulated by Egr-1 and p53 transcription factors. PTEN, a phosphoinositide phosphatase, acts as an 'off' switch in the PI-3 kinase/Akt signalling pathway and regulates cell growth and survival. It is shown here that transient alteration in the APE1 level in HeLa cells modulates PTEN expression and that acetylatable APE1 is required for the activation of the PTEN gene. Acetylation of APE1 enhances its binding to distinct trans-acting complexes involved in activation or repression. The acetylated protein is deacetylated in vivo by histone deacetylases. It was found that exposure of HeLa cells to H(2)O(2) and to histone deacetylase inhibitors increases acetylation of APE1 and induction of PTEN. The absence of such induction in APE1-downregulated HeLa cells confirmed APE1's role in regulating inducible PTEN expression. That APE1-dependent PTEN expression is mediated by Egr-1 was supported by experiments with cells ectopically expressing Egr-1. Thus, the data open new perspectives in the comprehension of the many functions exerted by APE1 in controlling cell response to oxidative stress.

Unconjugated bilirubin (UCB) is the major degradation product of the heme catabolism. A growing body of evidences suggests that UCB plays major biological effects by inhibiting cell proliferation in cancer cell lines and eliciting cell toxicity particularly in neurons and glial cells. Early molecular events responsible for bilirubin-induced cytotoxicity remain poorly understood. Using HeLa cells and mouse embryonic fibroblasts, we found that UCB at a concentration of free pigment (Bf) of 80 nM induced oxidative stress, promoting a significant increase in intracellular reactive oxygen species (ROS) and a decreased cell survival (by the MTT test). The ROS increase activated the antioxidant cell response through APE1/Ref-1, a master redox regulator in eukaryotic cells. Activation of APE1/Ref-1 was followed by a concomitant activation of Egr-1 transcription factor and by an upregulation of PTEN tumor suppressor, an Egr-1 target gene, leading to inhibition of cell growth. Blocking ROS generation with N-acetylcysteine pretreatment, restored cell survival, limited the upregulation of PTEN in response to UCB, and prevented the inhibition of cell proliferation. HeLa cells transfected with mutants of the PTEN promoter or silenced with APE1/Ref-1 small interference RNA confirmed that UCB modulates a signaling pathway involving APE1/Ref-1, Egr-1, and PTEN. These findings describe a new molecular pathway involved in the cytotoxic effects of UCB.

bstract: APE1/Ref-1, normally localized in the nucleus, is a regulator of the cellular response to oxidative stress. Cytoplasmic localization has been observed in several tumors and correlates with a poor prognosis. Because no data are available on liver tumors, we investigated APE1/Ref-1 subcellular localization and its correlation with survival in 47 consecutive patients undergoing hepatocellular carcinoma (HCC) resection, APE 1 /Ref-1 expression was determined by immunohistochemistry in HCC and surrounding liver cirrhosis (SLC) and compared with normal liver tissue. Survival probability was evaluated using Kaplan-Meier curves (log-rank test) and Cox regression. Cytoplasmic expression of APE 1 /Ref-1 was significantly higher in HCC than in SLC (P = 0.00001); normal liver showed only nuclear reactivity. Patients with poorly differentiated HCC showed a cytoplasmic expression three times higher than those with well-differentiated HCC (P = 0.03). Cytoplasmic localization was associated with a median survival time shorter than those with negative cytoplasmic reactivity (0.44 compared with 1.64 years, P = 0.003), and multivariable analysis confirmed that cytoplasmic APE I /Ref-1 localization is a predictor of survival. Cytoplasmic expression of APE 1 /Ref-1 is increased in HCC and is associated with a lower degree of differentiation and a shorter survival time, pointing to the use of the cytoplasmic localization of APE1/Ref-1 as a prognostic marker for HCC.

Dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production, by acting at the gene expression level. Molecular mechanisms through which mechanical forces are conversed into biochemical signalling in bone are still poorly understood. A growing body of evidence point to extracellular nucleotides (i.e., ATP and UTP) as soluble factors released in response to mechanical stimulation in different cell systems. Runx2, a fundamental transcription factor involved in controlling osteoblasts differentiation, has been recently identified as a target of mechanical signals in osteoblastic cells. We tested the hypothesis that these extracellular nucleotides could be able to activate Runx2 in the human osteoblastic HOBIT cell line. We found that ATP and UTP treatments, as well as hypotonic stress, promote a significant stimulation of Runx2 DNA-binding activity via a mechanism involving PKC and distinct mitogen-activated protein kinase cascades. In fact, by using the specific inhibitors SB203580 (specific for p38 MAPK) and PD98059 (specific for ERK-1/2 MAPK), we found that ERK-1/2, but not p38, play a major role in Runx2 activation. On the contrary, another important transcription factor, i.e., Egr-1, that we previously demonstrated being activated by extracellular released nucleotides in this osteoblastic cell line, demonstrated to be susceptible to both ERK-1/2 and p38 kinases. These data suggest a possible differential involvement of these two transcription factors in response to extracellularly released nucleotides. The biological relevance of our data is strengthened by the finding that a target gene of Runx2, i.e., Galectin-3, is up-regulated by ATP stimulation of HOBIT cells with a comparable kinetic of that found for Runx2. Since it is known that osteocytes are the primary mechanosensory cells of the bone, we hypothesize that they may signal mechanical loading to osteoblasts through release of extracellular nucleotides. Altogether, these data suggest a molecular mechanism explaining the purinoreceptors-mediated activation of specific gene expression in osteoblasts and could be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Our aim was to evaluate the prevalence of bacterial vaginosis and decrease in lactobacillus colonization in women 40 years old or older in relation to menopausal status by evaluation of Gram-stained smears. A total of 1,486 smears from Italian Caucasian women aged 40 to 79 years were examined. Women were classified as follows: fertile (regular cycles) ( n = 328), perimenopausal (irregular cycles) ( n = 237), and postmenopausal ( n = 921), including 331 women on estroprogestinic hormone replacement therapy (HRT). The prevalences of bacterial vaginosis (assessed as a Nugent score of ≥7) in fertile (9.8%) and perimenopausal (11.0%) women were not statistically different, whereas the prevalence was significantly lower overall in postmenopausal women (6.0%) ( P = 0.02). Specifically, 6.3% of postmenopausal women without HRT and 5.4% of postmenopausal women with HRT were positive for bacterial vaginosis. The Nugent score system was not adequate for evaluating the normal and intermediate vaginal flora in women over the age of 40 years. High numbers of peri- and postmenopausal women had no lactobacilli and no bacterial-vaginosis-associated microorganisms. This nonpathological absence of lactobacilli in women with a Nugent score of 4 was scored as 4∗, and this group was considered separately from the intermediate flora group. A score of 4∗ was obtained for 2.1% of fertile women, 11.4% of perimenopausal women, 44.1% of postmenopausal women without HRT, and 6.9% of postmenopausal women with HRT. The physiological reduction in lactobacillus colonization of the vagina in postmenopausal women does not cause an increase in bacterial-vaginosis prevalence. Reversion of lactobacillus flora to premenopausal levels due to HRT does not increase the prevalence of bacterial vaginosis in postmenopausal women.

The human mdr1 gene encodes a transmembrane glycoprotein the over-expression of which is associated with development of multidrug resistance in human tumor cells. A negative modulation of human mdr1 has been attempted via a 27-mer unmodified triple helix-forming oligonucleotide, named 1D, targeted to a homopurine sequence in the coding region of the gene. By administering 10 μM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug-resistant cell line CEM-VLB 100. This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.

The pleomorphic bacterium Gardnerella vaginalis releases in the culture broth a haemolytic exotoxin (Gvh) which is probably a virulence determinant of this unique bacterium, implicated in gynaecological and urological disorders. This 59 kDa cytolysin was purified to homogeneity in just one chromatographic step directly from the culture supernatant, a final specific activity up to 1.9 x 10(6) HU mg-1 being obtained. The toxin-induced lesion on human erythrocytes results from the formation of a pore whose radius is approximately 2.4 nm. The damage is inhibited by osmotic protectants and shows a sigmoidal dose-response profile suggesting an aggregation process of haemolysin molecules on the target membrane to create the functional lesion. The extent and the kinetics of haemolysis are strongly dependent on temperature and an activation energy of 64.0 kJ mol-1 has been derived. Lipid membranes can be very efficient inhibitors of Gvh-haemolysis, being able to bind the toxin quite avidly. The inhibitory effect requires the presence of cholesterol and it is stronger when cholesterol is mixed with negatively charged phospholipids rather than with zwitterionic phospholipids, suggesting that a negative surface potential increases the affinity of the toxin for the lipid bilayer. The functional properties of Gvh have been compared with those of Clostridium perfringens thetatoxin (PFO) and Escherichia coli haemolysin (HlyA), which are representative of widespread haemolysins produced by Gram-positive and Gram-negative bacteria, respectively. The toxin shares several features with the family of the so-called 'sulphydryl-activated' cytolysins produced by Gram-positive bacteria, although Gvh does not truly belong to this family, being deactivated by beta-mercaptoethanol and being antigenically distinct from them. We report here for the first time the detection in the vaginal fluid of infected women of a specific IgA response against the toxin.