Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein.

Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.

Epidermal growth factor receptor binding is affected by structural determinants in the toxin domain of transforming growth factor-alpha-Pseudomonas exotoxin fusion proteins.

TGF-alpha-PE40 is a hybrid protein composed of transforming growth factor-alpha (TGF-alpha) fused to a 40,000-dalton segment of Pseudomonas exotoxin A (PE40). This hybrid protein possesses the receptor-binding activity of TGF-alpha and the cell-killing properties of PE40. These properties enable TGF-alpha-PE40 to bind to and kill tumor cells that possess epidermal growth factor (EGF) receptors. Unexpectedly, TGF-alpha-PE40 binds approximately 100-fold less effectively to EGF receptors than does native TGF-alpha (receptor-binding inhibition IC50 = 540 and 5.5 nM, respectively). To understand the factors governing receptor binding, deletions and site-specific substitutions were introduced into the PE40 domain of TGF-alpha-PE40. Removal of the N-terminal 59 or 130 amino acids from the PE40 domain of TGF-alpha-PE40 improved receptor binding (IC50 = 340 and 180 nM, respectively) but decreased cell-killing activity. Substitution of alanines for cysteines at positions 265 and 287 within the PE40 domain dramatically improved receptor binding (IC50 = 37 nM) but also decreased cell-killing activity. Similar substitutions of alanines for cysteines at positions 372 and 379 within the PE40 domain did not significantly affect receptor-binding or cell-killing activities. These studies indicate that the PE40 domain of TGF-alpha-PE40 interferes with EGF receptor binding. The cysteine residues at positions 265 and 287 of PE40 are responsible for a major part of this interference.

Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.

Long-term captopril therapy: evolving hemodynamic effects.

Nine patients with resistant hypertension received captopril for 12 months. Five received captopril alone, four required additional therapy. In the former, mean blood pressure fell from 109 +/- 4.2 mm Hg to 84 +/- 7.5 mm Hg (P less than 0.025) after seven days. A rise to 101 +/- 19 mm Hg was noted at six and 12 months. Total peripheral resistance fell at seven days but returned to levels above control at six and 12 months. Cardiac index was 3.21 +/- 0.55 liters/min/m2 before treatment, 3.27 +/- 0.56 liters/min/m2 at seven days, and 2.17 +/- 4.0 liters/min/m2 (P less than 0.025) at 12 months. However, forearm blood flow rose from subnormal levels during the 12 months of observation, suggesting a persistent effect on the arterioles of the extremities. Plasma converting enzyme activity was significantly reduced at seven days but was above control levels at six and 12 months. However, plasma renin activity remained elevated, and plasma aldosterone concentration was significantly reduced. The fall in mean blood pressure was not related to the change in plasma converting enzyme activity in patients receiving captopril alone (five patients) or with diuretic (two patients). In the presence of beta-adrenergic blockade and volume depletion (two patients), changes in mean blood pressure appeared to be related to changes in converting enzyme activity. The data suggest that patients with essential hypertension whose blood pressure was not adequately controlled by previous medications may initially respond to captopril with a fall in blood pressure and total peripheral resistance. However, in certain individuals, these effects diminish with time despite addition of diuretics and beta-adrenergic receptor blocking agents.

Resorcylic acid lactones: naturally occurring potent and selective inhibitors of MEK.

A resorcylic acid lactone, L-783,277, isolated from a Phoma sp. (ATCC 74403) which came from the fruitbody of Helvella acetabulum, is a potent and specific inhibitor of MEK (Map kinase kinase). L-783,277 inhibits MEK with an IC50 value of 4 nM. It weakly inhibits Lck and is inactive against Raf, PKA and PKC. L-783,277 is an irreversible inhibitor of MEK and is competitive with respect to ATP. L-783,290, the trans-isomer of L-783,277, was isolated from the same culture and evaluated together with several semi-synthetic resorcylic acid lactone analogs. A preliminary structure-activity relationship is presented. Several independent cell-based assays have been carried out to study the biological activities of these resorcylic acid lactone compounds and a brief result summary from these studies is presented.

Achieving anterior aesthetics with an anti-rotational abutment.

Single-tooth implants of various minerals and metals have been used, with some success, since ancient times. However, single-tooth implant survival was unpredictable until Branemark and his co-workers developed the theory and technique for obtaining a predictable direct bone to implant interface, which has been termed "osseointegration." Retrospective studies suggest excellent survival of single-tooth implants that predictably achieve this direct bone to implant interface. Case reports of single-tooth implants in the anterior and posterior areas of the jaws suggest the efficacy of this treatment, although scientific prospective studies evaluating the safety and efficacy of this treatment have only recently begun.

Management of microsystems at Forbes Health System.

Support for purchase and operation of microsystems hardware and software at Forbes Health System in Pittsburgh, Pennsylvania is provided by Management Engineering, part of the Management Systems Division. Five key areas of support are described in this paper: Purchase Control, Installation Management, Operational Support, Development Services and Training Management. Discussion of multiple training options, centralized hardware support and specialized levels of end user operational support are included. Some future directions in software and hardware utilization are also presented.

Release of prostaglandin I2-like activity from the rat aorta: effect of captopril, furosemide, and sodium.

The effect of captopril, furosemide, indomethacin and intake of sodium on the production of PGI2-like material was studied in the rat aorta. Release of PGI2-like material from these vessels was estimated by its ability to inhibit ADP-induced platelet aggregation. Pretreatment with indomethacin (15 mg/kg/day) reduced the capacity of the aorta to release PGI2-like material. Pretreatment with captopril (10 mg/kg/day) had no effect. Intravenous furosemide (60 microgram/ml plasma volume) increased the capacity of the aorta to inhibit by 28% (p less than 0.25). The inhibitory capacity of aorta removed from rats on a low sodium diet did not differ from those on a high sodium diet. We conclude that the action of furosemide in reducing vascular tone may be related to stimulation of PGI2 synthesis in blood vessels whereas the effect of captopril and sodium in reducing vascular tone may involve a mechanism unrelated to PGI2 synthesis or may involve the synthesis of a prostaglandin other than PGI2.

Identification of a novel retinoblastoma gene product binding site on human papillomavirus type 16 E7 protein.

Transformation of mammalian cells by human papillomavirus type 16 appears to require binding of the viral E7 protein to the cellular retinoblastoma growth suppressor gene product (pRB). Binding of E7 protein to pRB inhibits several of pRB's biochemical properties, including association with the transcription factor E2F. Fragments of E7 protein derived from its conserved region 2 (CR2) domain bind to pRB and are sufficient to inhibit binding of full-length E7 protein to pRB. However, these CR2 fragments exhibit reduced affinity for pRB compared to the full-length protein and do not inhibit formation of the pRB-E2F complex. These observations suggest the existence of additional contact sites between the E7 protein and pRB. In the current study we have identified a region of E7, distinct from the CR2 domain, which is sufficient to bind pRB. This new pRB binding motif encompasses the zinc-binding conserved region 3 (CR3) domain of E7. Studies with a series of pRB deletion mutants suggest that pRB residues between amino acids 803 and 841 are necessary for binding to the E7 CR3 domain. An E7 CR3 peptide inhibits binding of E2F to pRB, indicating that E2F and E7(31-98) bind to pRB at the same or overlapping sites. These results are consistent with a model in which optimal binding of E7 to pRB requires at least two distinct contact sites: the previously identified high affinity interaction between the E7 CR2 domain and the pRB "pocket" region, and a second interaction between the E7 CR3 domain and the COOH-terminal region of pRB. The latter interaction is sufficient for E7's inhibition of E2F binding to pRB.

Residual moisture determines the level of touch-contact-associated bacterial transfer following hand washing.

We report here a new and critical determinant of the effectiveness of hand hygiene procedures, namely the amount of residual moisture left on the hands after washing and drying. When samples of skin, food and utilities were touched with wet, undried hands, microbial numbers in the order of 68000, 31000 and 1900 respectively translocated to these representative surfaces. Bacterial numbers translocating on touch contact decreased progressively as drying with an air or cloth towel system removed residual moisture from the hands. A 10 s cloth towel-20 s air towel protocol reduced the bacterial numbers translocating to skin, food and utilities on touch contact to 140, 655 and 28 respectively and achieved a 99.8, 94 and 99% reduction in the level of bacterial translocation associated with wet hands. Careful hand drying is a critical factor determining the level of touch-contact-associated bacterial transfer after hand washing and its recognition could make a significant contribution towards improving handcare practices in clinical and public health sectors.

Characterization of functional HPV-16 E7 protein produced in Escherichia coli.

Human papillomaviruses (HPVs) are the etiologic agents responsible for genital warts and are contributing factors in the pathogenesis of human cervical cancer. The HPV E7 gene is transcriptionally active in these diseases and has been shown to transform mammalian cells in vitro. We have expressed and purified the HPV-16 E7 gene product in Escherichia coli. The isolated E7 protein contains zinc in a 1:1 molar ratio. X-ray absorption fine structure studies demonstrated that the zinc is coordinated by 4 sulfur ligands. We sequentially derivatized the E7 cysteines to differentiate between solvent-exposed, metal-bound, and disulfide-associated cysteines. Our results demonstrate that Cys24 and Cys68 are accessible to solvent, while cysteines in the two conserved Cys-X-X-Cys motifs are likely involved in binding zinc. We observed no evidence for the existence of disulfide bonds in recombinant E7 protein under the conditions tested.

Transcription factor E2F binds DNA as a heterodimer.

E2F is a mammalian transcription factor that appears to play an important role in cell cycle control. DNA affinity column-purified E2F from HeLa cells reproducibly exhibits multiple protein bands when analyzed by SDS/PAGE. After electrophoretic purification, electroelution, and refolding of the individual protein components, the E2F DNA binding activity of the individual proteins was poor. However, upon mixing the individual components together, a dramatic (100- to 1000-fold) increase in specific DNA binding activity was observed. The five protein bands isolated can be separated into two groups based on apparent molecular mass. Optimal reconstitution of activity requires one of the two proteins found in the group of larger molecular mass (approximately 60 kDa) and one of the three proteins in the smaller-sized group (approximately 50 kDa). The reconstituted heterodimer is identical to authentic affinity-purified E2F by three criteria: DNA-binding specificity, DNA pattern, and binding to the retinoblastoma gene product. A recently cloned protein with E2F-like activity, RBP3/E2F-1, is related to the protein components of the group of larger molecular mass, as determined by Western blot analysis and reconstitution experiments. These data suggest that E2F, like many other transcription factors, binds DNA as an oligomeric complex composed of at least two distinct proteins.

Identification of HPV-16 E7 peptides that are potent antagonists of E7 binding to the retinoblastoma suppressor protein.

Complex formation between the human papilloma virus type-16 E7 protein (HPV-16 E7) and the retinoblastoma suppressor protein (pRB) is believed to be important in the process of cellular transformation that leads to cervical carcinoma. Utilizing an in vitro solution assay as well as a plate binding assay that measures the association between HPV-16 E7 and pRB proteins, we have examined a series of synthetic HPV-16 E7 peptides. HPV-16 E7 peptides which lie between amino acid residues 14 and 32 were found to be potent inhibitors of E7/pRB binding. The minimal peptide structure that possessed full antagonist activity was N-acetyl-E7-(21-29)-peptide amide. This peptide inhibited 100% of E7/pRB binding and exhibited an IC50 of 40 nM in the plate binding assay. A purified beta-galactosidase-E7 fusion protein exhibited an IC50 of 2 nM in the same assay. These results suggest that other regions of the E7 molecule in addition to amino acids 21-29 may contributed to E7/pRB interaction. Analysis of E7-(20-29)-peptides containing single amino acid substitutions suggests that Cys24, Tyr23, Tyr25, Asp21, and Glu26 are important residues for maintaining maximal antagonist activity. This series of peptides should prove useful in analyzing the biological consequences of E7/pRB binding in HPV-infected cells.

Transforming growth factor alpha-Pseudomonas exotoxin fusion protein prolongs survival of nude mice bearing tumor xenografts.

Transforming growth factor alpha (TGF alpha)-Pseudomonas exotoxin 40 (PE40) is a chimeric protein consisting of an N-terminal TGF alpha domain fused to a C-terminal 40-kDa segment of the Pseudomonas exotoxin A protein. TGF alpha-PE40 exhibits the receptor-binding activity of TGF alpha and the cell-killing activity of PE40. These properties make TGF alpha-PE40 an effective cytotoxic agent for cells that possess epidermal growth factor receptors (EGFR). However, the utility of this protein as an anticancer agent has been unclear because many normal tissues express EGFR and may be damaged by exposure to TGF alpha-PE40. To address this issue, we injected nude mice with a lethal inoculum of either A431 or HT29 human tumor cells that possess EGFR or with Chinese hamster ovary (CHO) tumor cells that lack EGFR. Animals were treated with a derivative of TGF alpha-PE40 in which the cysteine residues are replaced by alanine, termed "TGF alpha-PE40 delta cys," or with saline once a day for 5 days. Mice bearing EGFR+ tumor cells lived significantly (P less than 0.001) longer when treated with TGF alpha-PE40 delta cys compared with saline-treated controls (median survival: A431 cells, 51.5 vs. 25.5 days; HT29 cells, 101 vs. 47.5 days). TGF alpha-PE40 delta cys did not prolong the survival of mice bearing tumor cells that lack EGFR (median survival: CHO cells, 15.5 vs. 19.5 days). The only toxicity to normal tissues was mild periportal hepatic necrosis. These studies indicate that a therapeutic window exists in vivo for the use of some growth factor-toxin fusion proteins as anticancer agents.

Biological activity of a transforming growth factor-alpha--Pseudomonas exotoxin fusion protein in vitro and in vivo.

Transforming growth factor-alpha (TGF alpha)-pseudomonas exotoxin-40 (PE40) is a chimeric protein consisting of an N-terminal TGF alpha domain fused to a C-terminal 40-kDa segment of the pseudomonas exotoxin A protein. TGF alpha-PE40 exhibits the receptor binding activity of TGF alpha and the cell killing activity of PE40. In the current study, we report that a modified TGF alpha-PE40 derivative significantly prolongs the survival of nude mice bearing tumors derived from cell lines which express the epidermal growth factor receptor (EGFR). In addition, the therapeutic benefit of this protein is mediated by specific binding to the EGF receptor. These results indicate that a therapeutic window exists in vivo for the use of some growth factor--toxin fusion proteins as anticancer agents.