Vibrio illness in Florida, 1998-2007.

This study characterized the current epidemiology of vibrio infections in Florida and examined cases reported from 1998 to 2007. Logistic regression was used to determine risk of death. There were 834 vibrio infections in 825 individuals (average annual incidence rate 4·8/1,000,000). Common Vibrio species reported were Vibrio vulnificus (33%), V. parahaemolyticus (29%), and V. alginolyticus (16%). Most exposures were attributed to wounds (42%), and the most common clinical syndromes were wound infections (45%) and gastroenteritis (42%). Almost half of individuals reported an underlying health condition. Risk of death was associated with any underlying condition and increased with the number of conditions (P<0·0001). In Florida, incidence of vibriosis associated with raw oyster consumption has decreased while incidence associated with wound infections has increased. Most prevention efforts to date have focused on oyster consumption. New educational messages focusing on the risk of vibriosis from wound infections should target high-risk populations.

Transcription activation by the human estrogen receptor subtype beta (ER beta) studied with ER beta and ER alpha receptor chimeras.

We have studied the two estrogen receptor (ER) subtypes, ER alpha and ER beta, and chimeric constructs with ER alpha and ER beta to examine the bioactivities of these receptors and their responses to estrogen and antiestrogen ligands. Transcriptional activity of ER beta is highly dependent on cell/promoter context and on the nature of the ligand. ER beta activated significant levels of transcription in response to estrogens in certain cell types, but showed only moderate activity compared with ER alpha in others. Antiestrogens such as tamoxifen and 2-phenylbenzofuran, which show some agonistic activity with ER alpha, exhibit no agonistic activity with ER beta. Alteration of the amino-terminal A/B receptor domain can result in a dramatic change in cell type- and ligand-specific transcriptional activity of ER beta. Upon replacing the A/B domain of ER beta with the A/B domain of ER alpha, this receptor chimera not only exhibits an improved transcriptional response to estrogens, but also is now able to activate transcription upon treatment with these antiestrogens. As antiestrogen agonism was lacking in ER beta and the ER beta/alpha chimera containing the amino-terminal A/B domain of ER beta fused to domains C through F of ER alpha, but was restored in an ER alpha/beta chimera containing the A/B domain of ER alpha, antiestrogen agonism was shown to depend on the A/B domain (activation function-1-containing region) of ER alpha. Together, these results indicate that the differences in the amino-terminal regions of ER alpha and ER beta contribute to the cell- and promoter-specific differences in transcriptional activity of these receptors, and their ability to respond to different ligands, thus providing a mechanism for differentially regulated transcription by these two ERs.

Different residues of the human estrogen receptor are involved in the recognition of structurally diverse estrogens and antiestrogens.

We have previously examined, by alanine scanning mutagenesis, amino acids 515-535 of the estrogen receptor (ER) ligand binding domain to determine which of these residues are important in estradiol binding. Mutation at four sites that potentially lie along one face of an alpha-helix, Gly521, His524, Leu525, and Met528, all significantly impaired estradiol binding by the ER (Ekena, K., Weis, K. E., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1996) J. Biol. Chem. 271, 20053-20059). In this report, we compare the pattern of residues that are important in the recognition of several structurally diverse estrogen agonists and antagonists (the synthetic nonsteroidal agonist hexestrol, an agonist derived from the mold metabolite zearalenone, P1496, and the partial agonist-antagonist trans-hydroxytamoxifen) with those that are predicted to contact estradiol in the receptor-ligand complex. Although there are some similarities in the pattern of residue recognition among all four ligands, each ligand showed distinct differences as well. Interestingly, alanine substitution at only one residue, the leucine at position 525, was found to inhibit binding of all the ligands tested. Another residue, His524, was found to be important in the recognition of three different agonists but not trans-hydroxytamoxifen (the only ligand lacking a second hydroxyl group). The recognition of estradiol and another agonist, P1496, was impaired by the G521A mutation, whereas ligand-induced activity by the two compounds that lack B- and C-rings, hexestrol and trans-hydroxytamoxifen, was unaffected. Our findings demonstrate that these ligands fit into the ER ligand binding pocket differently and that each contacts a distinct set of amino acids. The smaller ligands (estradiol and hexestrol) have a narrower footprint of interacting residues than the larger ligands (P1496 and trans-hydroxytamoxifen). This pattern of interaction is most consistent with the amino acids within this region being in contact with the portion of these ligands that corresponds to the D-ring end of estradiol. The interplay between the shape of an ER ligand and the residues that support its binding to ER may potentially underlie the selective actions of different ER ligands in various cell and promoter contexts.

Determinants for the repression of estrogen receptor transcriptional activity by ligand-occupied progestin receptors.

There is considerable evidence for cross-talk between the estrogen and progestin signaling pathways, including examples of repression or attenuation of estrogen-stimulated endpoints by progestin receptor (PR) agonists and antagonists. We have previously described an experimental system for examining aspects of this cross-talk, namely the repression of estrogen receptor (ER) transcriptional activity by liganded PR (Kraus, W. L., Weis, K. E., Katzenellenbogen, B. S., Mol. Cell. Biol. 15 (1995) 1847-1857). Under promoter and cell type conditions where liganded PR was not a good activator of transcription, PR isoforms were shown to act as potent ligand-dependent repressors of ER transcriptional activity. In the current study, we have identified multiple determinants of this repression by systematically manipulating potentially important variables in this system (e.g. PR A:PR B ratio, sequence of the response elements, receptor structure, and ligand type). Alterations in several of these parameters had profound effects on the ability of PR to repress the activity of ER. Decreases in the PR A:PR B ratio and changes in the sequence of the progestin response element in the reporter gene construct abolished the repressive action of agonist-occupied PR A on ER transcriptional activity. In addition, point or deletion mutations in the amino-terminal A/B region of ER, including a triple point mutation which eliminates phosphorylation sites previously shown to be important in the activity of the receptor, made the ER more sensitive to the repressive actions of liganded PR. The PR ligands that promoted the most potent repression of ER activity were those with 11beta phenyl substitutions, suggesting that the phenyl moiety in the 11beta position is the important structural feature leading to strong repression. Interestingly, changes in the structure of the ER ligand and the sequence of the estrogen response element did not influence the magnitude of repression by PR. The fact that alterations in these check points along the estrogen signaling pathway had little or no effect on the magnitude of repression suggests that liganded PR interferes with the ability of ER to interact productively with the transcriptional machinery; in other words, PR-mediated repression occurs downstream of the events leading to the ligand-dependent conversion of ER to a transcriptionally active form. Our results indicate that a number of parameters which are naturally varied in vivo, such as the sequence of PR DNA binding sites and the PR A:PR B ratio, can dramatically alter the repression of ER activity by liganded PR, and may explain the differential affects of progestin-occupied PR on the expression of different estrogen regulated genes.

Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein.

To better understand structure-activity relationships in the human estrogen receptor (ER), we examined the role of tyrosine 537 in the transcriptional response of the receptor, since this residue is close to a region of the hormone-binding domain shown previously to be important in hormone-dependent transcriptional activity and because this amino acid has been proposed to be a tyrosine kinase phosphorylation site important in the activity of the ER. We substituted five amino acids at this position (alanine, phenylalanine, glutamic acid, lysine, or serine) and screened these mutants for their biological activities in the presence and absence of estradiol. Two of the ER mutants, Y537A and Y537S, displayed estrogen-independent constitutive activity that was approximately 20% or 100%, respectively, of the activity of the wild type receptor with estradiol, when assessed in two different cell backgrounds using three different estrogen-responsive promoters. In some circumstances, the Y537E and Y537K proteins also exhibited some low level of constitutive activity. The constitutive activity of the mutants, as well as their activity in the presence of E2, was fully suppressed by antiestrogen. The extent of interaction of the constitutively active ERs with the steroid receptor coactivator-1 (SRC-1) closely parallel the magnitude of transcriptional activity of the receptor. Whereas wild type ER showed interaction with SRC-1 only in the presence of estrogen, Y537A and Y537S ER showed moderate or full interaction in the absence of ligand, an interaction that was blocked by antiestrogen, and the magnitude of interaction was increased to or remained at 100% upon estradiol treatment, implying that the ability of an ER to associate with SRC-1 is a good indicator of a transcriptionally active conformational state of the receptor. Our findings indicate that tyrosine 537 is in a region important in the ligand regulation of ER transcriptional activity and that the presence of certain amino acids at this position can shift ER into a conformation that is active even without ligand. However, tyrosine is not required at this site for estrogen binding or transcriptional response to estrogen in the systems investigated. Our findings, interpreted in light of the recently published x-ray crystal structure of the ligand-binding domains of three related receptors of the nuclear receptor superfamily, suggest that some of the amino acid substitutions introduced at position 537 may facilitate the shift of helix 12 of the ER into an active conformation and/or allow for differential stabilization of the receptor in its active form.

Identification of amino acids in the hormone binding domain of the human estrogen receptor important in estrogen binding.

The initial step in the regulation of the transcriptional activity of the estrogen receptor (ER) is the binding of hormone. Previous studies have suggested that the region between amino acids 515 and 535 near the C terminus of the human ER is likely to be important in ligand binding. In order to explicitly define which amino acids in this region are critical for ligand recognition and binding, we have utilized alanine-scanning mutagenesis over the complete 515-535 region. The ability of these 21 mutants to activate transcription in response to the natural estrogen, 17beta-estradiol (E2), was evaluated in cell co-transfection assays with estrogen-responsive reporter genes. In addition, their ability to bind E2 was also tested. Mutations at four sites in the 521-528 region had the greatest effects on E2-induced transcription, with L525A reducing responsiveness 250-fold, G521A and H524A 35-fold, and M528A 11-fold. Mutations at other sites had either no effect or a 4-fold or lesser reduction in sensitivity to E2 (M517A, Y526A, N532A, and P535A). Three of the mutants most affected in their transcriptional response, G521A, H524A, and M528A, showed a coordinate reduction in E2 binding affinity. E2 binding by the most affected mutant, L525A, could not be detected. Thus, the altered transcriptional response of these ER mutants appears to derive solely from an alteration in their affinity for the ligand E2. The four sites most affected by alanine substitution, 521, 524, 525, and 528, follow an alpha-helical periodicity, such that they would be positioned on one face of an alpha-helix. Furthermore, they correspond precisely to residues in an alpha-helix shown to be in contact with ligand in the recently described x-ray crystal structures of two other members of the nuclear hormone receptor superfamily, namely the retinoic acid receptor- and thyroid hormone receptor-ligand complexes. Our findings, which broaden observations to the steroid receptor family within the superfamily of nuclear receptors, suggest that this region of the estrogen receptor is in contact with its cognate ligand in a similar fashion.

Inhibitory cross-talk between steroid hormone receptors: differential targeting of estrogen receptor in the repression of its transcriptional activity by agonist- and antagonist-occupied progestin receptors.

Although estrogen receptor (ER) and progestin receptor (PR) are members of different steroid hormone receptor subfamilies, there is considerable biological evidence for cross-talk between the estrogen and progestin hormone-receptor signaling pathways. We have developed a model system to analyze the mechanisms underlying this cross-talk, specifically the repression of ER-mediated transcriptional activity by PR complexed with agonistic or antagonistic ligands. Estrogen- and progestin-responsive reporter vectors containing a variety of promoters were transfected into primary cultures of rat uterine cells and 3T3 mouse fibroblasts with expression vectors for PR (the A and/or B isoforms) as well as ER. Our results demonstrate that both PR isoforms can act as potent ligand-dependent repressors of ER activity. The magnitude of the repression was dependent on the PR isoform (i.e., PR A or PR B), ligand type (i.e., agonist or antagonist), PR levels, and ligand concentration but was unaffected by the ER levels. The promoter context was important in determining both the magnitude and PR isoform specificity of the repression for agonist-occupied PR but not for antagonist-occupied PR. Ligand-occupied PR A was a stronger repressor of ER-mediated transcriptional activity than was ligand-occupied PR B, and antagonist-occupied PR was a more effective repressor than agonist-occupied PR. Mechanistic studies suggest that liganded PR represses ER activity by interfering with its ability to interact productively with the transcriptional machinery, a process known as quenching. The data do not support competitive repression, direct repression, or squelching as the mechanism of PR's inhibitory effect. Experiments with ER mutants demonstrated that the N-terminal portion of ER was required for repression by agonist-occupied PR but not by antagonist-occupied PR. These results, as well as other differences between the two PR-ligand complexes, suggest that they differentially target ER when repressing ER transcriptional activity. These findings underscore the mounting evidence for the importance of interactions between members of the steroid hormone receptor family.