Colonization of corn, Zea mays, by the entomopathogenic fungus Beauveria bassiana.

Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin into corn, Zea mays L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located, indicating that B. bassiana does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how B. bassiana travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that B. bassiana does not lose virulence toward the European corn borer, Ostrinia nubilalis (Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities for biological control, including the use of indigenous fungal inocula as insecticides.

Characterization of a CREB gain-of-function mutant with constitutive transcriptional activity in vivo.

The cyclic AMP (cAMP)-responsive factor CREB promotes cellular gene expression, following its phosphorylation at Ser133, via recruitment of the coactivator paralogs CREB-binding protein (CBP) and p300. CBP and p300, in turn, appear to mediate target gene induction via their association with RNA polymerase II complexes and via intrinsic histone acetyltransferase activities that mobilize promoter-bound nucleosomes. In addition to cAMP, a wide variety of stimuli, including hypoxia, UV irradiation, and growth factor addition, induce Ser133 phosphorylation with stoichiometry and kinetics comparable to those induced by cAMP. Yet a number of these signals are incapable of promoting target gene activation via CREB phosphorylation per se, suggesting the presence of additional regulatory events either at the level of CREB-CBP complex formation or in the subsequent recruitment of the transcriptional apparatus. Here we characterize a Tyr134Phe CREB mutant that behaves as a constitutive activator in vivo. Like protein kinase A (PKA)-stimulated wild-type CREB, the Tyr134Phe polypeptide was found to stimulate target gene expression via the Ser133-dependent recruitment of CBP and p300. Biochemical studies reveal that mutation of Tyr134 to Phe lowers the K(m) for PKA phosphorylation and thereby induces high levels of constitutive Ser133 phosphorylation in vivo. Consistent with its constitutive activity, Tyr134Phe CREB strongly promoted differentiation of PC12 cells in concert with suboptimal doses of nerve growth factor. Taken together, these results demonstrate that Ser133 phosphorylation is sufficient for cellular gene activation and that additional signal-dependent modifications of CBP or p300 are not required for recruitment of the transcriptional apparatus to the promoter.

Stimulus-specific interaction between activator-coactivator cognates revealed with a novel complex-specific antiserum.

A number of second messenger pathways propagate inductive signals via protein-protein interactions that are phosphorylation-dependent. The second messenger, cAMP, for example, promotes cellular gene expression via the protein kinase A-mediated phosphorylation of cAMP-response element-binding protein (CREB) at Ser(133), and this modification in turn stimulates the association of CREB with the co-activator, CREB-binding protein (CBP). The solution structure of the CREB.CBP complex, using relevant interaction domains, kinase inducible domain and kinase-induced domain interacting domain, referred to as KID and KIX, respectively, shows that KID undergoes a coil to helix transition, upon binding to KIX, that stabilizes complex formation. Whether such changes occur in the context of the full-length CREB and CBP proteins, however, is unclear. Here we characterize a novel antiserum that specifically binds to the CREB. CBP complex but to neither protein individually. Epitope mapping experiments demonstrate that the CREB.CBP antiserum detects residues in KID that undergo a conformational change upon binding to KIX. The ability of this antiserum to recognize full-length CREB.CBP complexes in a phospho-(Ser(133))-dependent manner demonstrates that the structural transition observed with the isolated KID domain also occurs in the context of the full-length CREB protein. To our knowledge, this is the first report documenting formation of endogenous cellular protein-protein complexes in situ.

The novel progesterone receptor antagonists RTI 3021-012 and RTI 3021-022 exhibit complex glucocorticoid receptor antagonist activities: implications for the development of dissociated antiprogestins.

We have identified two novel compounds (RTI 3021-012 and RTI 3021-022) that demonstrate similar affinities for human progesterone receptor (PR) and display equivalent antiprogestenic activity. As with most antiprogestins, such as RU486, RTI 3021-012, and RTI 3021-022 also bind to the glucocorticoid receptor (GR) with high affinity. Unexpectedly, when compared with RU486, the RTI antagonists manifest significantly less GR antagonist activity. This finding indicates that, with respect to antiglucocorticoid function, receptor binding affinity is not a good predictor of biological activity. We have determined that the lack of a clear correlation between the GR binding affinity of the RTI compounds and their antagonist activity reflects the unique manner in which they modulate GR signaling. Previously, we proposed a two step "active inhibition" model to explain steroid receptor antagonism: 1) competitive inhibition of agonist binding; and 2) competition of the antagonist bound receptor with that activated by agonists for DNA response elements within target gene promoters. Accordingly, we observed that RU486, RTI 3021-012, and RTI 3021-022, when assayed for PR antagonist activity, accomplished both of these steps. Thus, all three compounds are "active antagonists" of PR function. When assayed on GR, however, RU486 alone functioned as an active antagonist. RTI 3021-012 and RTI 3021-022, on the other hand, functioned solely as "competitive antagonists" since they were capable of high affinity GR binding, but the resulting ligand receptor complex was unable to bind DNA. These results have important pharmaceutical implications supporting the use of mechanism based approaches to identify nuclear receptor modulators. Of equal importance, RTI 3021-012 and RTI 3021-022 are two new antiprogestins that may have clinical utility and are likely to be useful as research reagents with which to separate the effects of antiprogestins and antiglucocorticoids in physiological systems.

Bisphenol A interacts with the estrogen receptor alpha in a distinct manner from estradiol.

We investigated the interaction of bisphenol A (BPA, an estrogenic environmental contaminant used in the manufacture of plastics) with the estrogen receptor alpha (ERalpha) transfected into the human HepG2 hepatoma cell line and expanded the study in vivo to examine the effect of BPA on the immature rat uterus. Bisphenol A was 26-fold less potent in activating ER-WT and was a partial agonist with the ERalpha compared to E2. The use of ERalpha mutants in which the AF1 or AF2 regions were inactivated has permitted the classification of ER ligands into mechanistically distinct groups. The pattern of activity of BPA with the ERalpha mutants differed from the activity observed with weak estrogens (estrone and estriol), partial ERalpha agonists (raloxifene or 4-OH-tamoxifen), or a pure antagonist (ICI 182, 780). Intact immature female Sprague-Dawley rats were exposed to BPA alone or with E2 for 3 days. Unlike E2, BPA had no effect on uterine weight; however, like E2, both peroxidase activity and PR levels were elevated, though not to the level induced by E2. Following simultaneous administration, BPA antagonized the E2 stimulatory effects on both peroxidase activity and PR levels but did not inhibit E2-induced increases of uterine weight. These results demonstrate that BPA is not merely a weak estrogen mimic but exhibits a distinct mechanism of action at the ERalpha.

The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor.

Previously, we defined a novel class of ligands for the human progesterone receptor (PR) which function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components which distinguish between different ligand-induced PR conformations, we have determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending upon the class of ligand bound to the receptor. Specifically, we observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner which is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, we show that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis revealed that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, we propose that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part, to a disruption of the interaction between PR and the corepressors NCoR and SMRT. Cumulatively, these results suggest that NCoR and SMRT expression may play a pivotal role in PR pharmacology.

Dissection of the molecular mechanism of action of GW5638, a novel estrogen receptor ligand, provides insights into the role of estrogen receptor in bone.

The estrogen receptor (ER) mixed agonists tamoxifen and raloxifene have been shown to protect against bone loss in ovariectomized rats. However, the mechanism by which these compounds manifest their activity in bone is unknown. We have used a series of in vitro screens to select for compounds that are mechanistically distinct from tamoxifen and raloxifene in an effort to define the properties of an ER modulator required for bone protection. Using this approach, we identified a novel high affinity ER antagonist, GW5638, which when assayed in vitro functions as an ER antagonist, inhibiting the agonist activity of estrogen, tamoxifen, and raloxifene and reversing the "inverse agonist" activity of the pure antiestrogen ICI182,780. Thus, GW5638 appears to function as an antagonist in these in vitro systems, although in a manner distinct from other known ER modulators. Predictably, therefore, GW5638 alone displays minimal uterotropic activity in ovariectomized rats, but will inhibit the agonist activity of estradiol in this environment. Unexpectedly, however, this compound functions as a full ER agonist in bone and the cardiovascular system. These data suggest that the mechanism by which ER operates in different cells is not identical, and that classical agonist activity is not required for the bone protective activity of ER modulators.

Simplified therapy for Asherman's syndrome.

OBJECTIVE: To evaluate a technique that converts a blind hysteroscopic procedure to a "septum" division. DESIGN: Open noncomparative clinical study. SETTING: Tertiary care center. PATIENT(S): Six women with Asherman's syndrome; five with complete and one with incomplete obliteration of the uterine cavity. INTERVENTION(S): The patients underwent recreation of the uterine cavity by the hysteroscopic-laparoscopic technique described to establish the correct dissection plane. MAIN OUTCOME MEASURE(S): The ability to reestablish the uterine cavity; postoperative resumption of menses and fertility. RESULT(S): In all patients, the cavity of the uterus was restored; menses resumed in all women who were previously amenorrheic; and 5 women conceived, of whom four had live births and one a missed abortion. At hysteroscopy, two women incurred perforations and in another hemorrhage occurred. CONCLUSION(S): This technique appears to be effective and safe for the reconstruction of a functional endometrial cavity in women with Asherman's syndrome.

Identification of the sequences within the human complement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity.

The promoter of the human C3 gene has been shown to be responsive to stimulation by both estrogen and tamoxifen-activated estrogen receptor (ER) in transcriptional assays reconstituted in mammalian cells. Using a series of deletions and point mutations, we have determined that the agonist activity of these two compounds was dependent upon the direct interaction of ER with each of three estrogen response elements (EREs) contained within this promoter. One of these sequences, ERE1 resembles the canonical vitellogenin A2-ERE whereas the other two, ERE2 and ERE3, do not display significant homology to known EREs. Using gene transfer studies it was shown that these sequences are necessary and sufficient for ER-mediated transcription. Interestingly, using in vitro receptor/DNA-binding assays we demonstrated that neither ERE1, ERE2, or ERE3 alone formed high-affinity complexes with purified ER; however when a promoter fragment containing all three sequences was used, specific, high-affinity ER-DNA interactions were observed. It was not surprising, therefore, that, when assayed individually on a heterologous promoter, these sequences function as weak EREs but together they act in a synergistic manner to create a strong ER-dependent enhancer. It has been suggested that tamoxifen mediates its partial agonist activity through AP-1 at target promoters. However, the fact that purified ER can bind directly to the estrogen-responsive sequences within the C3 promoter, and that tamoxifen activity on this promoter is unaffected by AP-1 coexpression, indicates that at least on some promoters tamoxifen can manifest partial agonist activity through a classical ER/ ERE- mediated mechanism.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Continuous 12-lead ST-segment recovery analysis in the TAMI 7 study. Performance of a noninvasive method for real-time detection of failed myocardial reperfusion.

BACKGROUND: If a practical, reliable, noninvasive marker of failed reperfusion was available in real time, the benefits of further therapy in this patient subgroup could be tested. We developed a method of 12-lead ST-segment recovery analysis using continuously updated reference points to provide such a marker. METHODS AND RESULTS: In this study, our method was prospectively tested in 144 patients given thrombolytic therapy early in myocardial infarction. All patients had 12-lead continuous ST-segment monitoring and acute angiography, each analyzed in an independent, blinded core laboratory. ST-segment recovery and re-elevation were analyzed up to the moment of angiography, at which time patency was predicted. Predictions were correlated to angiographic infarct artery flow, with TIMI flow 0 to 1 as occluded and TIMI flow 2 to 3 as patent. Infarct artery occlusion was seen on first injection in 27% of patients. The positive predictive value of incomplete ST recovery or ST re-elevation by our method was 71%, negative predictive value 87%, with 90% specificity and 64% sensitivity for coronary occlusion. ST recovery analysis predicted patency in 94% of patients with TIMI 3 flow versus 81% of patients with TIMI 2 flow and predicted occlusion in 57% of patients with collateralized occlusion versus 72% of patients with non-collateralized occlusion. In a regression model including other noninvasive clinical descriptors, ST recovery alone contained the vast majority of predictive information about patency. CONCLUSIONS: In a blinded, prospective, angiographically correlated study design, 12-lead continuous ST-segment recovery analysis shows promise as a practical noninvasive marker of failed reperfusion that may contribute substantially to currently available bedside assessment. Our data also suggest that patients with TIMI 2 flow or with collateralized occlusions may represent a physiological spectrum definable with ST-segment recovery analysis.

Continuously updated 12-lead ST-segment recovery analysis for myocardial infarct artery patency assessment and its correlation with multiple simultaneous early angiographic observations.

Early angiography may not adequately subgroup patients with myocardial infarction if cyclic changes in coronary flow occur frequently. From a pilot experience using a new 12-lead ST-segment monitor, a continuously updated, self-referenced ST-recovery analysis method was developed to quantify both instantaneous recovery, as a noninvasive marker of patency, and cumulative ST recovery over time, as a marker of the speed, stability and duration of reperfusion. In 22 patients with acute infarction in whom 44 observations of unique angiographic patency were noted within 6 hours of presentation, serial patency assessments simultaneous with all angiographic observations predicted coronary occlusion with 90% sensitivity and 92% specificity. Of the 22 patients, 11 (50%) had multiple ST trend transitions suggesting cyclic changes in coronary flow before catheterization. Speed, stability and duration of ST-segment recovery were defined by the time to first 50% ST recovery, total number of ST-trend transitions and patent physiology index (percentage of monitoring period showing ST recovery), respectively. Subgrouped angiographically, the median (interquartile range) for cumulative ST parameters with patent (n = 8) versus occluded (n = 14) arteries were, respectively--time to 50% recovery, 1.57 (1.16, 1.70) versus 0.17 (-0.47, 0.32) hours; number of reelevation/recovery events, 1.5 (1, 3) versus 3 (1, 3); and patent physiology index, 52 (47, 59) versus 50 (5, 73). Thus, continuous ST-segment recovery analysis appears to predict simultaneous angiographic patency over serial assessments, whereas cumulative parameters appear to contain independent information, probably because of patency changes before or after angiography.