Peptide antagonists of the human estrogen receptor.

Estrogen receptor alpha transcriptional activity is regulated by distinct conformational states that are the result of ligand binding. Phage display was used to identify peptides that interact specifically with either estradiol- or tamoxifen-activated estrogen receptor alpha. When these peptides were coexpressed with estrogen receptor alpha in cells, they functioned as ligand-specific antagonists, indicating that estradiol-agonist and tamoxifen-partial agonist activities do not occur by the same mechanism. The ability to regulate estrogen receptor alpha transcriptional activity by targeting sites outside of the ligand-binding pocket has implications for the development of estrogen receptor alpha antagonists for the treatment of tamoxifen-refractory breast cancers.

Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta.

Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the alpha-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 10(8) variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERalpha) as a target and found three different classes of peptides. All of these peptides interacted with ERalpha in an agonist-dependent manner and disrupted ERalpha-mediated transcriptional activity when introduced into target cells. Using a series of ERalpha-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERbeta- but not ERalpha-mediated reporter gene expression. This novel ERbeta-specific antagonist may be useful in identifying and characterizing the ERbeta-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.

Yeast alpha mating factor structure-activity relationship derived from genetically selected peptide agonists and antagonists of Ste2p.

alpha-Factor, a 13-amino-acid pheromone secreted by haploid alpha cells of Saccharomyces cerevisiae, binds to Ste2p, a seven-transmembrane, G-protein-coupled receptor present on haploid alpha cells, to activate a signal transduction pathway required for conjugation and mating. To determine the structural requirements for alpha-factor activity, we developed a genetic screen to identify from random and semirandom libraries novel peptides that function as agonists or antagonists of Ste2p. The selection scheme was based on autocrine strains constructed to secrete random peptides and respond by growth to those that were either agonists or antagonists of Ste2p. Analysis of a number of peptides obtained by this selection procedure indicates that Trp1, Trp3, Pro8, and Gly9 are important for agonist activity specifically. His2, Leu4, Leu6, Pro10, a hydrophobic residue 12, and an aromatic residue 13 are important for both agonist and antagonist activity. Our results also show that activation of Ste2p can be achieved with novel, unanticipated combinations of amino acids. Finally, the results suggest the utility of this selection scheme for identifying novel ligands for mammalian G-protein-coupled receptors heterologously expressed in S. cerevisiae.

Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins.

Based on the prevalence of modular protein domains, such as Src homology domain 3 and 2 (SH3 and SH2), among important signaling molecules, we have sought to identify new SH3 domain-containing proteins. However, modest sequence similarity among these domains restricts the use of DNA-based methods for this purpose. To circumvent this limitation, we have developed a functional screen that permits the rapid cloning of modular domains based on their ligand-binding activity. Using operationally defined SH3 ligands from combinatorial peptide libraries, we screened a series of mouse and human cDNA expression libraries. We found that 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, 10 of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome analysis and in bringing new targets into drug discovery programs.

Evolution and structure of the fibrinogen genes. Random insertion of introns or selective loss?

Chromosomal linkage as well as sequence homologies provide unequivocal evidence that the genes for the alpha, beta and gamma chains of fibrinogen arose by successive duplication of a single ancestral gene. Yet, when the three fibrinogen chains are aligned by amino acid homology, the positions of intervening sequences coincide at only two positions for all three chains. While one additional intron occurs at a homologous site in the beta and gamma chains, none of the positions of the remaining 11 introns in the three genes is shared. This arrangement of introns in the three fibrinogen genes suggests that either introns were selectively lost, implying that there is essential information in the retained introns, or the common introns were present in the ancestral fibrinogen gene and introns have been randomly inserted since the triplication of the original gene. The more likely possibility of selective loss of introns implies that the ancestral gene, as it existed about one billion years ago, must have been composed of numerous small exons.

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries.

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.

Analysis of the heterogeneity of the biological responses to native and mutant human interleukin-6.

The structure-function relationships of the biological activities of mutant varieties of the pleiotropic cytokine interleukin-6 (human) were measured by three assays: induction of immunoglobulin M (IgM) secretion from an Epstein-Barr virus-transformed human B cell line and induction of fibrinogen secretion from either a human hepatoma cell line or a rat hepatoma cell line. The biological effects of the cytokine were characterized by three parameters as determined by a novel analysis: effectiveness (the maximal response attainable), efficiency (the concentration yielding a half-maximal response), and complexity (a measure of heterogeneity and feedback control). Substitution of serine for cysteine was associated with a reduction in the effectiveness of interleukin-6 in both fibrinogen secretion assays. In the assay with human hepatoma cells, there was also a profound reduction in efficiency. Serine substitution in the human IgM synthesis assay appears mainly to reduce the efficiency. Deletion of amino acids 4 to 23 increased the efficiency in the rat hepatoma assay. The complexity parameter suggests the presence of multiple receptor classes or negative feedback in all three assays. Use of the proposed sequential approach to the analysis of dose-response relations in bioassays provides a more useful quantitative assessment of activities as well as more insight into the complexity of the reactions.

Comparative analyses of mechanistic differences among antiestrogens.

Antiestrogens such as tamoxifen are one of the most effective methods of treating estrogen receptor (ERalpha) positive breast cancers; however, the effectiveness of this therapy is limited by the almost universal development of resistance to the drug. If antiestrogens are recognized differently by the cell as it has been suggested, then in disease conditions where tamoxifen fails to function effectively, a mechanistically different antiestrogen might yield successful results. Although many antiestrogens have been developed, a direct comparison of their mechanisms of action is lacking, thus limiting their utility. Therefore, to determine if there are mechanistic differences among available antiestrogens, we have carried out a comprehensive analysis of the molecular mechanisms of action of 4-hydroxy-tamoxifen (40HT), idoxifene, raloxifene, GW7604, and ICI 182,780. Using a novel set of peptides that recognize different surfaces on ERalpha, we have found that following binding to ERalpha, each ligand induces a distinct ERalpha-ligand conformation. Furthermore, transcriptional assays indicate that each ERalpha-ligand complex is recognized distinctly by the transcription machinery, and consequently, antiestrogens vary in their ability to inhibit estradiol- and 40HT-mediated activities. Relative binding assays have shown that the affinity of these ligands for ERalpha is not always representative of their inhibitory activity. Using this assay, we have also shown that the pharmacology of each antiestrogen is influenced differently by hormone binding proteins. Furthermore, GW7604, like ICI 182,780, but unlike the other antiestrogens evaluated, decreases the stability of the receptor. Overall, our results indicate that there are clear mechanistic distinctions among each of the antiestrogens studied. However, GW7604 and ICI 182,780 differ more significantly from tamoxifen than idoxifene and raloxifene. These data, which reveal differences among antiestrogens, should assist in the selection of compounds for the clinical regulation of ERalpha function.

An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets.

We have examined the potential of isolating novel ligands from a library of M13 pIII-fusion phage displaying peptides composed of 38 random amino acids (aa). The library was panned with streptavidin (SA) and a polyclonal goat antimouse IgG Fc antibody (Ab) preparation coupled to paramagnetic beads. SA selected two classes of phage from the library. One class exhibited the aa motif, HP(Q/M) theta (where theta signifies a non-polar aa), similar to the motif identified by Devlin et al. [Science 249 (1990) 404-406] using a 15-aa random peptide library displayed on phage. The other class of phage had no discernible motif. In binding experiments, the non-HP(Q/M) theta phage had a slightly higher affinity for SA than did the motif phage. Both classes of SA-binding phage failed to bind native and non-glycosylated forms of avidin, even though SA and avidin are structurally similar and both proteins possess extraordinary affinities for biotin. The polyclonal goat anti-mouse IgG Fc Ab preparation selected phage displaying sequences similar to a region of the mouse IgG Fc. Thus, a single immunodominant epitope on the mouse IgG Fc was identified. Furthermore, a second phage displaying peptides with no discernible sequence similarities to mouse IgG Fc was isolated. Thus, an M13 library displaying 38-aa peptides can yield phage with affinity for various targets. Finally, we have observed a biological bias against odd numbers of Cys residues in the displayed peptides.

Construction and functional analysis of hybrid interleukin-6 variants. Characterization of the role of the C-terminus for species specificity.

We have constructed several hybrid human interleukin-6 (IL-6) variants in which the carboxyl-terminus, which includes a receptor binding site of IL-6 has been replaced with the C-terminus of various proteins homologous to human IL-6. IL-6 hybrids with the C-terminus of human growth hormone and human granulocyte-colony stimulating factor maintain part of the biological activity of human IL-6. Replacing the C-terminus of human IL-6 with the C-terminus of mouse and rat IL-6 resulted in a normal or increased activity on a mouse cell line; however, this gave a low (to 200-fold less) activity on a human cell line compared to wild-type human IL-6. We therefore conclude that the C-terminus of IL-6 plays an important role in the species specificity of IL-6.

Donor hepatitis C virus status does not adversely affect short-term outcomes in HCV+ recipients in renal transplantation.

BACKGROUND: Recipient hepatitis C virus (HCV) seropositivity has been associated with inferior outcomes in renal transplantation (RTx). We sought to determine whether donor HCV+ status influenced the incidence of rejection, liver dysfunction, and graft survival in HCV+ recipients. METHODS: We reviewed 44 HCV+ recipients (R+) receiving RTx from HCV+ (D+) and HCV- (D-) donors between February 1991 and September 1996. All patients were followed to the end of the study period (mean=36 months, range=12-60 months). We compared the R+ group with a demographically matched cohort of 44 HCV- recipients (R-). RESULTS: Of the 44 R+, 25 (57%) had a total of 48 rejection episodes. Among the 44 R-, 32 (73%) had 58 rejection episodes (P>0.1). Within the R+ group, 28 were D+/R+; of these 14 (50%) had 27 rejection episodes, whereas among the 16 D-/R+, 11 (68%) had 21 rejection episodes (P>0.3). Graft and patient survival was similar in both the groups (86.4% and 91%, respectively). Liver dysfunction was slightly increased in the R+ group (4/44 vs. 0/44, P>0.1), with one death due to liver failure in this group. CONCLUSION: Donor HCV+ status had no influence on outcomes in HCV+ recipients after kidney transplantation in the short term. The incidence of rejection, graft loss, and mortality was comparable between the D+/R+ and D-/R+ groups. Furthermore, rejection, graft loss, and death were identical in R+ and R-groups throughout the 5-year study period. We therefore conclude that HCV+ recipients can safely receive kidney transplants without concern about donor HCV status or fear of adverse events from their own HCV+ status.

Convenient uses of polymerase chain reaction in analyzing recombinant cDNA clones.

We have used polymerase chain reaction to accelerate our analysis of recombinant lambda-cDNA clones. We have amplified the inserts of lambda gt10 or lambda gt11 recombinants starting with bacteriophage in cored plaques or isolated DNA. The amplifications made with simple or complex oligonucleotide primers have allowed convenient sizing, subcloning and translation of the phage inserts into protein.

Multipurpose vectors for peptide expression on the M13 viral surface.

We have developed a set of three cloning vectors for the expression of polypeptides on the surface of the M13 viral coat. The M13mp8 genome has been engineered for expression of foreign protein sequences near the NH2-terminus of the mature pIII protein, which is present in five copies on the outside of each M13 viral particle. All three of the vectors carry the same two useful restriction sites for directed cloning of inserts in the pIII coding region; in addition, one vector carries the bacterial gene conferring resistance to the antibiotic tetracycline, and another expresses the lacZ' polypeptide that allows functional complementation of beta-galactosidase activity within the host bacterial cell. All of these vectors propagate well in E. coli DH5 alpha F' cells and do not require helper phage. We demonstrate that a bacteriophage, expressing an eleven amino acid epitope (from human c-myc) at the NH2-terminus of pIII in one of our vectors, can be purified from a vast mixture of other M13 phage through panning techniques. In particular, we find that the c-myc-expressing viral particles can be easily recovered from phage mixtures with the biotinylated form of the monoclonal antibody, 9E10, and streptavidin-coated MagneSphere beads.

Endogenous circadian rhythm in electroretinogram of free-moving lizards.

An endogenous circadian rhythm in the electroretinogram (ERG) of free-moving lizards, Anolis carolinensis, can be demonstrated in experiments lasting up to eight days. The rhythm does not appear to arise from processes that could modulate the amount of light reaching the photoreceptors. Moreover, since the rhythm is well-developed in the b-wave, but not a-wave, the direct modulation of neural processes proximal to the photoreceptors may be involved. Lastly, this rhythm may have ecological significance for this diurnally active species, since the largest responses occur at projected midday.

Is laparoscopic donor nephrectomy the new criterion standard?

HYPOTHESIS: The posttransplantation renal function outcomes between consecutive open donor and laparoscopic donor nephrectomies (LDNs) are similar and affect living donation. DESIGN: Using the medical records of renal living donor-recipient pairs, 36 consecutive open donor nephrectomies were compared with the subsequent 100 LDNs. Data collected on donor characteristics included demographics (age, race, sex, weight, and height), renal vascular and ureteral anatomical features, surgical information (blood loss, number of blood transfusions, operating time, warm ischemia time, and renal injury), complications, and length of hospital stay. Recipients' data also included renal function information (serum creatinine level on postoperative days 7 and 30) and ureteral complications during the initial hospital stay. SETTING: A not-for-profit tertiary care teaching hospital in a metropolitan area. PATIENTS: Adults who had end-stage renal disease and received a living donation kidney. MAIN OUTCOME MEASURES: Operative time, warm ischemia time, blood loss, and posttransplantation serum creatinine level. RESULTS: Patient characteristics were not significantly different between the open donor nephrectomy and LDN groups. No right kidney LDNs were done because of the shortness of the right renal vein; and, after the initial experience, left kidneys with more than 2 arteries were excluded. Warm ischemia time was recorded only for LDN, and it was found that a warm ischemia time of 10 minutes or longer was associated with difficulty in extraction and was uniformly associated with elevated mean serum creatinine levels on postoperative day 7. CONCLUSIONS: The length of hospital stay was decreased and cosmetic result enhanced. The number of living donors has increased from 28 in 1997 to 53 in 1998 and to 63 in 1999 at our institution. The length of hospital stay, incidence of complications, and comparable kidney quality indicate that LDN should be the initiating procedure for most patients.

Evidence by scanning electron microscopy for an association between spermatozoa and T-mycoplasmas in men of infertile marriage.

The morphology of T-mycoplasmas in culture and in semen from men of infertile marriage was examined by scanning electron microscopy. In cultures obtained from semen of infected individuals, T-mycoplasmas were recognized as spherical particles, 160 to 200 nm in diameter, frequently interconnected by short, straight fibrils. Numerous T-mycoplasmas and associated fibrils with similar morphologic characteristics were also observed adhering to spermatozoa obtained from ejaculates of the infected donors. Similar granulofibrillar aggregates were never observed on control specimens. These observations indicate a physical association between the microorganisms and spermatozoa in semen of infected men. This association may contribute to the decreased motility of spermatozoa in semen from which T-mycoplasmas can be cultured. The binding of T-mycoplasmas to human spermatozoa may provide an effective mechanism for transfer of the microorganisms past the normal microbial barrier of the cervix.

T-mycoplasmas and human infertility: correlation of infection with alterations in seminal parameters.

The presence of T-mycoplasmas in semen specimens from 625 men with infertility of unknown etiology was correlated with seminal cytologic findings. A change in the distribution toward higher ejaculate volumes and lower counts was demonstrated in the 246 patients with positive T-mycoplasma cultures, compared with the 379 patients with negative cultures. There was a significant increase in the number of tapering forms and spermatids in the T-mycoplasma-positive group. The increases in aberrant forms occurred at the expense of the oval and microcephalic forms. This change in the cytologic picture is consistent with the "stress" pattern seen in viral infections and allergic reactions, with the exception of the specific decrease in microcephalic forms. Relatively fewer patients with T-mycoplasmas in their semen had a high-level spermatozoal motility, compared with those lacking T-mycoplasmas. More than half again as many patients with T-mycoplasma infections had the poorest levels of spermatozoal motility. Infertile patients with T-mycoplasma infections had an over-all decrease in semen quality compared with those lacking demonstrable T-mycoplasmas.

Conceptual numerousness judgments by squirrel monkeys.

Two monkeys were reinforced for responding to the card which displayed fewer number of entities (three randomly selected sizes of filled circles) than the other card in any given pair. Area and brightness cues were controlled (at least for the successive numerousness discriminations), as were specific-pattern learning cues. Training proceeded in the order 2 versus 7 (2:7), 2:6, 2:5...2:3, 3:7, 3:6...3:4, etc., until it was judged that the monkeys were unlikely to attain the stringent criteria for discrimination. Both monkeys met criteria on the 7:8 discrimination, and one monkey met criteria on the 8:9 discrimination. It was concluded that the monkeys' numerousness judgments were made on a conceptual basis and that, among nonhuman animals, the evidence for such judgments appears to be limited to apes and monkeys.