The carboxyl-terminal region of the human papillomavirus type 16 E1 protein determines E2 protein specificity during DNA replication

The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.     

Activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.     

The 2.5 A resolution structure of the jel42 Fab fragment/HPr complex

The tertiary structure of Jel42 Fab fragment complexed with HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli, has been determined at 2.5 A resolution. X-ray diffraction from a larger crystal provided 22,067 unique reflections as compared to 14,763 unique reflections (2.8 A resolution), which were obtained previously from a smaller crystal. The higher resolution allowed for more precise location of amino acid side-chains and for the location of well-ordered water molecules. Five more residues in the Fab fragment are found to be involved in binding HPr and two additional residues are identified as part of the epitope, bringing the totals to 24 and 16, respectively. At least nine water molecules are found at the interface between the two proteins, and these mediate hydrogen bonding interactions between the Fab fragment and HPr. Three additional hydrogen bonds have been identified (bringing the total to ten) and one salt-bridge occurs between LysL50 of the L2 complementarity-determining region (CDR) and GluP66 of HPr. This salt-bridge is the only interaction between HPr and CDRL2; thus all six CDRs are involved in binding. Inspection and empirical energy minimization of mutant HPrs in the complex indicate that, in some cases in the binding interaction, water molecules may compensate for residue alterations. Binding to the mutant SerP64Tyr HPr may require a movement of the HPr main chain. The active centre region of HPr, which is not involved in binding the antibody, and which was not resolved in the 2.8 A resolution structure of the complex, was determined. This active centre determined at pH 5.8, which is completely free of intermolecular contacts due to crystal packing, shows a potential hydrogen bond between the AsnP12 OD1 atom and the HisP15 NE2 atom, and no involvement of the C terminus with HisP15. The HisP15 ND1 atom is the site of phosphorylation in HPr. Although a specific amino acid at residue 12 is not conserved in HPr molecules from all species, a hydrogen bond between the side-chains of residue 12 and HisP15 may be a conserved feature of the active centres.     

Controlled evaluation of a bandage contact lens and a topical nonsteroidal anti-inflammatory drug in treating traumatic corneal abrasions

BACKGROUND: Treating traumatic corneal abrasions is a common problem for the ophthalmologist. Traditional management has been the use of a pressure patch. Three different therapeutic modalities were evaluated for their efficacy in treating traumatic corneal abrasions. METHODS: Forty-seven consecutive patients with traumatic corneal abrasions were randomized prospectively in a single-masked, controlled clinical trial which compared the efficacy of (1) pressure patching, (2) a bandage contact lens, and (3) a bandage contact lens with a topical nonsteroidal anti-inflammatory drug (0.5% ketorolac tromethamine). RESULTS: There was no significant difference in the healing time of the three groups. However, psychometric analysis showed a significant decrease in pain in the group that received a bandage contact lens with a topical nonsteroidal anti-inflammatory drug. There was a significant difference in the ability to return to normal activities in both contact lens groups compared with the pressure-patch group. There was no significant difference among the three groups with respect to photophobia, redness, ocular irritation, headache, or tearing. CONCLUSION: Use of a bandage contact lens significantly shortens the time required for a patient to return to normal activities. Moreover, addition of a nonsteroidal anti-inflammatory drug to a treatment regimen significantly decreases the pain associated with traumatic corneal abrasions. Use of a bandage contact lens with a topical nonsteroidal anti-inflammatory may prove to be an effective adjunct in treating traumatic corneal abrasions.     

In vitro integration of retrotransposon Ty1: a direct physical assay

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.     

A simple immunohistochemical method for the detection of prostatic acid phosphatase

A simplified immunohistochemical method was developed to identify prostatic cells in paraffin sections for the diagnosis of primary or metastatic prostatic carcinoma. By incubating each section with a specific antiserum, followed by incubation with a specific acid phosphatase isoenzyme of the prostate, the antibody binding site is visualized by staining for acid phosphatase activity in the glandular epithelial cells of the prostate and in the metastatic prostate carcinoma cells that involve the lymph node. The present method is simpler and more specific than the previously described indirect immunoperoxidase method.     

Development of a highly infective Babesia bigemina vaccine of reduced virulence

The virulence of a strain of Babesia bigemina was reduced by syringe passaging at 3 to 16-week intervals in a series of 7 calves. The calves were splenectomised 1 to 14 weeks after inoculation to induce the relapse parasitaemias used for passaging. Parasites taken at relapse from the last 3 calves in the series were inoculated into splenectomised calves from which highly parasitised blood for vaccine was obtained. The vaccine produced mild infections in 32 recipient cattle. When challenged either 5 weeks or 7 months after vaccination, the cattle had substantial immunity to a heterologous strain of B. bigemina.