EPCRA: a retrospective on the environmental Right-to-Know Act.

October 2011 marked the 25th Anniversary of the Emergency Planning and Community Right-to-Know Act (EPCRA), which was celebrated for its "significant role in protecting human health and the environment over the last quarter century by providing communities and emergency planners with valuable information on toxic chemical releases in their area." This Note aims to evaluate the effectiveness of three important provisions of the statute-the Toxics Release Inventory, the emergency planning mandate, and the citizen suit provision-through a case study of their implementation in Institute, West Virginia, the site of an industrial accident that prompted the enactment of EPCRA in 1986. This Note argues that although EPCRA made significant improvements to industry transparency in terms of its production and release of hazardous substances, there remain significant barriers concerning adequate resources, informational tools, and enforcement measures.

5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83), a selective anti-human immunodeficiency virus agent with an improved metabolic and toxicological profile.

5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83) is a selective anti-human immunodeficiency virus (HIV) agent. When tested in phytohemagglutinin-stimulated normal human peripheral blood lymphocytes against fresh clinical isolates of HIV type 1 (HIV-1) obtained from patients naive to AZT (3'-azido-3'-deoxythymidine [zidovudine]), 935U83 inhibited virus growth with an average 50% inhibitory concentration (IC50) of 1.8 microM; corresponding IC50s were 0.

Review of research leading to new anti-herpesvirus agents in clinical development: valaciclovir hydrochloride (256U, the L-valyl ester of acyclovir) and 882C, a specific agent for varicella zoster virus.

Research leading to the new anti-herpesvirus compounds discussed here has come from three approaches. The first approach was directed towards improving the bioavailability of acyclovir by examining the potential of a variety of prodrugs, leading to the new compound valaciclovir hydrochloride. The second approach was to examine a large number of 5-substituted pyrimidines for activity against those viruses which were not as potently inhibited by acyclovir as are herpes simplex viruses, i.

Random mutagenesis of the thymidine kinase gene of varicella-zoster virus.

To understand the relationship between the primary structure and function of varicella-zoster virus thymidine kinase (VZV TK; EC 2.7.1.

6-Methoxypurine arabinoside as a selective and potent inhibitor of varicella-zoster virus.

Seven 6-alkoxypurine arabinosides were synthesized and evaluated for in vitro activity against varicella-zoster virus (VZV). The simplest of the series, 6-methoxypurine arabinoside (ara-M), was the most potent, with 50% inhibitory concentrations ranging from 0.5 to 3 microM against eight strains of VZV.

Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction.

We have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants.

High-level expression of the Epstein-Barr virus alkaline deoxyribonuclease using a recombinant baculovirus: application to the diagnosis of nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). Infection of the insect cell line Spodoptera frugiperda (SF9) with the recombinant virus led to the expression of an enzymatically active alkaline DNase. The recombinant EBV alkaline DNase was highly soluble, and the recombinant baculovirus produced approximately 10-20 mg of EBV DNase per 1 X 10(9) cells.

Immunological studies on the Epstein-Barr virus encoded alkaline deoxyribonuclease found in virus-producing lymphoblastoid cells.

Antisera were raised against a purified recombinant form of the Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) expressed in Escherichia coli. These sera were shown to be reactive with lymphoblastoid cells expressing EBV antigens (B95-8, P3HR-1 and Raji). Immunostaining studies of cells expressing EBV antigens revealed that the DNase was a component of the restricted early antigen complex of EBV.

The characterization of the EBV alkaline deoxyribonuclease cloned and expressed in E. coli.

Studies of nucleic acid homology suggest the BGLF5 open reading frame of Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase) sharing some homology with that of herpes simplex virus. We report here the expression of the BGLF5 open reading frame in E. coli and the expression of high levels of a novel alkaline DNase activity in induced cells.

Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity.

There is considerable interest in the potential of human immunodeficiency virus type 1 (HIV-1) to develop drug resistance, especially as 3'-azido-3'-deoxythymidine (Retrovir) is now in widespread clinical use to treat people with AIDS and AIDS-related complex (ARC). To address this possibility, mutations in the HIV reverse transcriptase [deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins.

HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis.

Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions.

Characterization of human immunodeficiency virus type 1 reverse transcriptase by using monoclonal antibodies: role of the C terminus in antibody reactivity and enzyme function.

We describe the production of eight monoclonal antibodies reactive with human immunodeficiency virus type 1 reverse transcriptase (RT) by immunization of mice with purified recombinant RT. These antibodies were found to react with one or the other of two regions of the enzyme and were found to be useful in immunodeficiency purification of large amounts of the enzyme. One epitope located at the C terminus of the enzyme was of particular interest, since it was present in only the larger, 66-kilodalton (kDa) RT species and not its smaller, 51-kDa counterpart.

Structural studies of the acquired immunodeficiency syndrome virus reverse transcriptase.

The clinical success of zidovudine has established the human immunodeficiency virus (HIV) reverse transcriptase (RT) as a valid target for the design of drugs to treat acquired immunodeficiency syndrome. In order to facilitate structural studies of this enzyme, expression systems in Escherichia coli, which allow the production of large amounts of RT, have been established. Using this recombinant material the RT has been purified and crystallized.

Production of antibodies of predetermined specificity against herpes simplex virus DNA polymerase and their use in characterization of the enzyme.

Peptides from preselected regions of the herpes simplex virus DNA polymerase were used to generate monospecific antisera to defined regions of the enzyme. The antisera were used to localize the polymerase within the infected cell and to determine the time of synthesis during productive infection. Comparison with a neutralizing polyclonal antiserum was used to show the specificity of the peptide antisera.

AIDS virus reverse transcriptase defined by high level expression in Escherichia coli.

The causative agent of AIDS the human immunodeficiency virus (HIV) encodes as part of its pol gene a reverse transcriptase (RT) which has a key role in the replication of the virus and thus constitutes an ideal target for antiviral chemotherapy. The purified HIV RT from virus particles consists of two related polypeptides of 66 and 51 kd mol. wt and similar polypeptides are found on expression of the complete HIV pol gene using prokaryotic systems.

Site-specific mutagenesis of AIDS virus reverse transcriptase.

Human immunodeficiency virus (HIV) is the causative agent of AIDS (acquired immune deficiency syndrome) a disease which poses a serious challenge to modern medicine. If we are to conquer this disease we will need a protective vaccine or effective drugs able to block the life cycle of the virus. An early stage in the invasion of the host cell is the conversion of the RNA genome of the virus to a double-stranded DNA intermediate which subsequently becomes integrated into the host cell chromosome.

Molecular basis of the glycoprotein C-negative phenotypes of herpes simplex virus type 1 mutants selected with a virus-neutralizing monoclonal antibody.

Previously (Holland et al., J. Virol.

DNA-binding protein associated with herpes simplex virus DNA polymerase.

Purified preparations of herpes simplex virus type 2 DNA polymerase made by many different laboratories always contain at least two polypeptides. The major one, of about 150,000 molecular weight, has been associated with the polymerase activity. The second protein, of about 54,000 molecular weight, which we previously designated ICSP 34, 35, has now been purified.

Studies on the herpes simplex virus alkaline nuclease: detection of type-common and type-specific epitopes on the enzyme.

Five monoclonal antibodies to the alkaline nuclease of herpes simplex virus (HSV) types 1 and 2 have been used in immunoperoxidase tests to demonstrate the nuclear localization of the enzyme within HSV-1- and HSV-2-infected cells and to purify the enzyme from cells infected with either virus by immunoadsorbant chromatography. Affinity chromatography with a 32P-labelled extract of HSV-2-infected cells has enabled us to demonstrate that the nuclease eluting from the immunoadsorbant is a phosphoprotein, hence confirming the nuclease to be identical to the phosphorylated polypeptide previously referred to as ICSP 22 (HSV-2) or ICP 19 (HSV-1). In addition, the results clearly demonstrate that monoclonal antibodies Q1, CC1 and CH2 are directed against HSV type-common epitopes while V1 and T2T1 antibodies are against HSV-2-specific epitopes on the enzyme.

Interactions between herpes simplex virus DNA-binding proteins.

Monoclonal antibodies directed against several herpes simplex virus (HSV)-induced DNA-binding proteins were used to investigate protein interactions in HSV-infected cells. Q1 monoclonal antibody, which is specific for the HSV-induced alkaline nuclease, when used in an immunoadsorbant column resulted in the purification of the alkaline nuclease, to which large quantities of the major DNA-binding protein were bound. Conversely, when a monoclonal antibody to the major DNA-binding protein was used in affinity chromatography other polypeptides (including the DNA polymerase and alkaline nuclease) were eluted in addition to the major DNA-binding protein.

Single mutations at many sites within the DNA polymerase locus of herpes simplex viruses can confer hypersensitivity to aphidicolin and resistance to phosphonoacetic acid.

Aphidicolin, a tetracyclic diterpenoid which inhibits the DNA polymerase-alpha activities of many eukaryotic cells, inhibited herpes simplex virus growth and DNA synthesis in infected cultures and the activity of the virus DNA polymerase in vitro. A wide range of stable aphidicolin sensitivities was represented amongst a collection of virus strains with no prior exposure to this drug, but viruses with polymerase mutations selected for resistance to phosphonoacetic acid (PAA) or to acycloguanosine typically showed increased sensitivity to aphidicolin. Of 16 unrelated PAA-resistant variants, 7 were hypersensitive to aphidicolin.

Herpes simplex virus non-structural proteins. IV. Purification of the virus-induced deoxyribonuclease and characterization of the enzyme using monoclonal antibodies.

The alkaline nucleases induced by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) have been purified from high salt extracts of virus-infected cells. The purification used three types of column chromatography and resulted in apparently homogeneous DNase preparations with good recovery. The enzyme from HSV-2-infected cells has been characterized.

Herpes simplex virus non-structural proteins. III. Function of the major DNA-binding protein.

The herpes simplex virus type 2 major DNA-binding protein has been functionally characterized using temperature-sensitive mutants in the complementation group 2-2. The mutants were shown to be defective in the DNA-binding protein gene by mapping the mutants to the area of the genome known to code for the protein, and by demonstrating alterations in the major DNA-binding protein induced in mutant-infected cells. The mutants were shown to be defective in the replication of virus DNA.