Nitrate and bisulfide: monitoring and patterns in Onondaga Lake, New York, following implementation of nitrification treatment.

Spatial and temporal patterns of nitrate (NO3(-)) and bisulfide (HS(-)) are documented in mercury-polluted, culturally eutrophic, Onondaga Lake, New York, following implementation of year-round nitrification treatment at a domestic wastewater treatment plant (WWTP). Measurements of NO3(-) and HS in the lake were made with a rapid-profiling, high-resolution, in situ ultraviolet spectrophotometer (ISUS) and were validated by standard laboratory wet chemistry analyses. A nearly 2-fold increase in epilimnetic NO3(-) concentrations, prolonged presence of NO3(-), and delay of the onset of HS(-) accumulations in the hypolimnion by approximately 1 month are demonstrated. Detailed vertical patterns resolved within the anoxic hypolimnion first depict operation of the thermodynamically favored NO3(-) reduction process(es) and, subsequently, sulfate (SO4(2-)) reduction and the localization of these processes in the lake's sediments. Variations in the effective depth of entry of WWTP discharge into the lake's water column, ranging from surface waters to metalimnetic depths, are demonstrated. Two- and three-dimensional patterns of NO3(-) from ISUS profiles depict substantial spatial structure mediated primarily by hydrodynamic processes. In situ ultraviolet spectrophotometer measurements of NO3(-) and HS(-) will play an important role in ongoing rehabilitation programs for the lake.

In vitro resistance study of rupintrivir, a novel inhibitor of human rhinovirus 3C protease.

Rupintrivir (formerly AG7088) is an irreversible inhibitor of the human rhinovirus (HRV) 3C protease that has been demonstrated to have in vitro activity against all HRVs tested, consistent with its interaction with a strictly conserved subset of amino acids in the 3C protease. The potential for resistance was studied following in vitro serial passage of HRV serotypes 14, 2, 39, and Hanks in the presence of increasing rupintrivir concentrations. HRV variants with reduced susceptibilities to rupintrivir (sevenfold for HRV 14) or with no significant reductions in susceptibility but genotypic changes (HRV 2, 39, and Hanks) were initially isolated following 14 to 40 cumulative days in culture (three to six passages). Sequence analysis of the 3C protease identified one to three substitutions in diverse patterns but with common features (T129T/A, T131T/A, and T143P/S in HRV 14; N165T in HRV 2; N130N/K and L136L/F in HRV 39; T130A in HRV Hanks). Notably, three of the four HRV variants contained a substitution at residue 130 (residue 129 in HRV 14). Continued selection in the presence of escalating concentrations of rupintrivir (40 to 72 days) resulted in the accumulation of additional mutations (A121A/V and Y139Y/H in HRV 14, E3E/G and A103A/V in HRV 2, S105T in HRV 39), with only minimal further reductions in susceptibility (up to fivefold). The ability of specific substitutions to confer resistance was examined by susceptibility testing of HRV 14 variants constructed to contain 3C protease mutations. In summary, the slow accumulation of multiple amino acid substitutions with only minimal to moderate reductions in susceptibility highlight the advantages of 3C protease as an antiviral target.

Conservation of amino acids in human rhinovirus 3C protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus 3C protease inhibitor.

The picornavirus 3C protease is required for the majority of proteolytic cleavages that occur during the viral life cycle. Comparisons of published amino acid sequences from 6 human rhinoviruses (HRV) and 20 human enteroviruses (HEV) show considerable variability in the 3C protease-coding region but strict conservation of the catalytic triad residues. Rupintrivir (formerly AG7088) is an irreversible inhibitor of HRV 3C protease with potent in vitro activity against all HRV serotypes (48 of 48), HEV strains (4 of 4), and untyped HRV field isolates (46 of 46) tested. To better understand the relationship between in vitro antiviral activity and 3C protease-rupintrivir binding interactions, we performed nucleotide sequence analyses on an additional 21 HRV serotypes and 11 HRV clinical isolates. Antiviral activity was also determined for 23 HRV clinical isolates and four additional HEV strains. Sequence comparison of 3C proteases (n = 58) show that 13 and 11 of the 14 amino acids that are involved in side chain interactions with rupintrivir are strictly conserved among HRV and HEV, respectively. These sequence analyses are consistent with the comparable in vitro antiviral potencies of rupintrivir against all HRV serotypes, HRV isolates, and HEV strains tested (50% effective concentration range, 3 to 183 nM; n = 125). In summary, the conservation of critical amino acid residues in 3C protease and the observation of potent, broad-spectrum antipicornavirus activity of rupintrivir highlight the advantages of 3C protease as an antiviral target.

Precursor of human adenovirus core polypeptide Mu targets the nucleolus and modulates the expression of E2 proteins.

We have examined the subcellular localization properties of human adenovirus 2 (HAdV-2) preMu and mature Mu (pX) proteins as fusions with enhanced green fluorescence protein (EGFP). We determined that preMu is exclusively a nucleolar protein with a single nucleolar accumulation signal within the Mu sequence. In addition, we noted that both preMu-EGFP and Mu-EGFP are excluded from adenovirus DNA-binding protein (DBP)-rich replication centres in adenovirus-infected cells. Surprisingly, we observed that cells in which preMu-EGFP (but not Mu-EGFP) is transiently expressed prior to or shortly after infection with Ad2 did not express late adenovirus genes. Further investigation suggested this might be due to a failure to express pre-terminal protein (preTP) from the E2 region, despite expression of another E2 protein, DBP. Deletion mutagenesis identified a highly conserved region in the C terminus of preMu responsible for these observations. Thus our data suggest that preMu may play a role in modulating accumulation of proteins from the E2 region.

Nuclear perturbations following adenovirus infection.

Adenoviruses are processed and assembled in the nuclei of infected cells and thereby produce significant perturbations to their structure and function. As the complex interactions that occur in the nuclei of uninfected cells are not yet fully understood many of the changes seen on infection have been described mainly in morphological terms. This chapter attempts to place more recent findings into this context and demonstrates that adenoviruses are able to hijack many cellular processes and enzymes to their advantage. In particular, modifications to nuclear PODs and nucleoli have more recently been explored in greater detail.

Design and synthesis of irreversible depsipeptidyl human rhinovirus 3C protease inhibitors.

Novel tripeptidyl C-terminal Michael acceptors with an ester replacement of the P(2)-P(3) amide bond were investigated as irreversible inhibitors of the human rhinovirus (HRV) 3C protease (3CP). When screened against HRV serotype-14 the best compound was shown to have very good 3CP inhibition (k(obs)/[I]=270,000M(-1)s(-1)) and potent in vitro antiviral activity (EC(50)=7.0nM).

Enhanced cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the adenovirus core.

Promising advances in nonviral gene transfer have been made as a result of the production of cationic liposomes formulated with synthetic cationic lipids (cytofectins) that are able to transfect cells. However few cationic liposome systems have been examined for their ability to transfect CNS cells. Building upon our earlier use of cationic liposomes formulated from 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidyl-ethanolamine (DOPE), we describe studies using two cationic viral peptides, mu (mu) and Vp1, as potential enhancers for cationic liposome-mediated transfection. Mu is derived from the condensed core of the adenovirus and was selected to be a powerful nucleic acid charge neutralising and condensing agent. Vp1 derives from the polyomavirus and harbours a classical nuclear localisation signal (NLS). Vp1 proved disappointing but lipopolyplex mixtures formulated from pCMVbeta plasmid, mu peptide and DC-Chol/DOPE cationic liposomes were able to transfect an undifferentiated neuronal ND7 cell line with beta-galactosidase reporter gene five-fold more effectively than lipoplex mixtures prepared from pCMVbeta plasmid and DC-Chol/DOPE cationic liposomes. Mu was found to give an identical enhancement to cationic liposome-mediated transfection of ND7 cells as poly-L-lysine (pLL) or protamine sulfate (PA). The enhancing effects of mu were found to be even greater (six- to 10-fold) when differentiated ND7 cells were transfected with mu-containing lipopolyplex mixtures. Differentiated ND7 cells represent a simple ex vivo-like post-mitotic CNS cell system. Successful transfection of these cells bodes well for transfection of primary neurons and CNS cells in vivo. These findings have implications for experimental and therapeutic uses of cationic liposome-mediated delivery of nucleic acids to CNS cells.

Mitochondrial protein p32 can accumulate in the nucleus.

Human p32 was first isolated associated with the splicing factor ASF/SF-2. The p32 protein is translated as pre-protein from which a mitochondrial import signal is cleaved off to create the mature p32. The majority of p32 is consequently found in the mitochondria. In this study we investigated extramitochondrial p32. An increased nuclear localisation of endogenous p32 was demonstrated as a response to leptomycin B or actinomycin D treatment of cells. Mature p32 gene and deletion mutants were cloned into enhanced green fluorescence protein reporter plasmids. On transfection, EGFP-p32 protein was mainly localised to the cytoplasm and to a lesser extent to the nucleus of transfected COS cells. Upon treatment with actinomycin D or leptomycin B, the EGFP-p32 protein accumulated in the nucleus. Deletion analysis indicated which regions of EGFP-p32 are involved in nuclear export and nuclear import.

Adenovirus protein V induces redistribution of nucleolin and B23 from nucleolus to cytoplasm.

Adenovirus infection inhibits synthesis and processing of rRNA and redistributes nucleolar antigens. Adenovirus protein V associates with nucleoli in infected cells. This study delineates regions of protein V independently capable of nucleolar targeting. Also, evidence is presented that protein V has the unique property of relocating nucleolin and B23 to the cytoplasm when transiently expressed on its own in uninfected cells. Point mutation analysis indicates a role for the C terminus of protein V in the redirection of nucleolin and B23 to the cytoplasm. This is the first time an adenovirus protein has been shown to have a direct effect on nucleolar antigens in isolation from viral infection. Moreover, adenovirus protein V is the first protein demonstrated to be capable of redirecting nucleolin and B23 to the cytoplasm.

Responses of Onondaga Lake, New York, to early stages of rehabilitation: unanticipated ecosystem feedbacks.

Responses of polluted Onondaga Lake, New York, to early stages of a phased program to rehabilitate the lake from the effects of domestic waste inputs are documented. The analysis is based on more than 10 years of paired monitoring of the effluent (total ammonia and total phosphorus) of a wastewater treatment plant (WWTP) that discharges to the lake as well as the lake itself (including total ammonia, nitrite, total and dissolved forms of phosphorus, plankton biomass and composition, Secchi disc transparency, and zebra mussel density). Major reductions in total ammonia and total phosphorus loading relative to the preceding decade are reported for the WWTP for the November 1998 through October 1999 interval. Dramatic and, in some cases, unanticipated changes in the lake's water quality and biota in response to the reductions in loading are documented for the April to October interval of 1999 including: (1) major decreases in total ammonia concentrations and improved status with respect to ammonia toxicity standards, (2) development of dense populations of zebra mussels, (3) decreases in fall concentrations of nitrite and improved status with respect to the related toxicity standard, (4) decreases in total phosphorus and total dissolved phosphorus concentrations, and (5) a severe Microcystis (phytoplankton) bloom that caused nuisance conditions and poor clarity. The zebra mussel invasion is attributed to the reductions in total ammonia concentrations to below toxic levels. The Microcystis bloom was probably related to the abrupt increase in the zebra mussel population. Additional reductions in phosphorus loading from the WWTP will be required to limit phytoplankton production and avoid the potential for continued nuisance conditions. Potential complications in resolving lake responses to future reductions in loading associated with the zebra mussel invasion are considered.

Interaction between herpes simplex virus type 1 IE63 protein and cellular protein p32.

The herpes simplex virus type 1 (HSV-1) immediate-early gene IE63 (ICP27), the only HSV-1 regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far, is a multifunctional protein which regulates transcriptional and posttranscriptional processes. One of its posttranscriptional effects is the inhibition of splicing of viral and cellular transcripts. We previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and casein kinase 2 (CK2) as two protein partners of IE63 (H. Bryant et al., J. Biol. Chem. 274:28991-28998, 1999). Here, using a yeast two-hybrid assay, we identify another partner of IE63, the cellular protein p32. Confirmation of this interaction was provided by coimmunoprecipitation from virus-infected cells and recombinant p32 binding assays. A p32-hnRNP K-CK2 complex, which required IE63 to form, was isolated from HSV-1-infected cells, and coimmunoprecipitating p32 was phosphorylated by CK2. Expression of IE63 altered the cytoplasmic distribution of p32, with some now colocalizing with IE63 in the nuclei of infected and transfected cells. As p32 copurifies with splicing factors and can inhibit splicing, we propose that IE63 together with p32, possibly with other IE63 partner proteins, acts to disrupt or regulate pre-mRNA splicing. As well as contributing to host cell shutoff, this effect could facilitate splicing-independent nuclear export of viral transcripts.

Substituted benzamide inhibitors of human rhinovirus 3C protease: structure-based design, synthesis, and biological evaluation.

A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease was identified using structure-based design. The design, synthesis, and biological evaluation of these inhibitors are reported. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3CP substrate. alpha,beta-Unsaturated cinnamate esters irreversibly inhibited the 3CP and displayed antiviral activity (EC(50) 0.60 microM, HRV-16 infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 A cocrystal structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.

Prayer and spirituality.

Many patients with arthritis are strongly influenced by religious beliefs and often participate in religious healing activities such as prayer and worship attendance. Scientific studies demonstrate, and most patients confirm, that faith and involvement in religious healing activities can be helpful in preventing and treating illness, recovering from surgery, reducing pain, and improving quality of life. To improve the care of patients, clinicians should develop a patient-centered, spiritually sensitive form of medical practice in which religious issues are addressed gently and appropriately with dignity, respect, and integrity.

Structure-based design of ketone-containing, tripeptidyl human rhinovirus 3C protease inhibitors.

Tripeptide-derived molecules incorporating C-terminal ketone electrophiles were evaluated as reversible inhibitors of the cysteine-containing human rhinovirus 3C protease (3CP). An optimized example of such compounds displayed potent 3CP inhibition activity (K = 0.0045 microM) and in vitro antiviral properties (EC50=0.34 microM) when tested against HRV serotype-14.

Effects of intercessory prayer on patients with rheumatoid arthritis.

BACKGROUND: Many individuals pray during times of illness, but the clinical effects of prayer are not well-understood. METHODS: We prospectively studied a cohort of 40 patients (mean age, 62 years; 100% white; 82% women) at a private rheumatology practice. All had class II or III rheumatoid arthritis and took stable doses of antirheumatic medications. All received a 3-day intervention, including 6 hours of education and 6 hours of direct-contact intercessory prayer. Nineteen randomly selected sample patients had 6 months of daily, supplemental intercessory prayer by individuals located elsewhere. Ten arthritis-specific outcome variables were measured at baseline and at 3-month intervals for 1 year. RESULTS: Patients receiving in-person intercessory prayer showed significant overall improvement during 1-year follow-up. No additional effects from supplemental, distant intercessory prayer were found. CONCLUSIONS: In-person intercessory prayer may be a useful adjunct to standard medical care for certain patients with rheumatoid arthritis. Supplemental, distant intercessory prayer offers no additional benefits.

In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3C protease.

AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C protease (inactivation rate constant (k(obs)/[I]) = 1,470,000 +/- 440,000 M(-1) s(-1) for HRV 14) that was discovered by protein structure-based drug design methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibited the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC(50)) of 0.023 microM (range, 0.003 to 0.081 microM) and a mean EC(90) of 0.082 microM (range, 0.018 to 0.261 microM) as well as that of related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. No significant reductions in the antiviral activity of AG7088 were observed when assays were performed in the presence of alpha(1)-acid glycoprotein or mucin, proteins present in nasal secretions. The 50% cytotoxic concentration of AG7088 was >1,000 microM, yielding a therapeutic index of >12,346 to >333,333. In a single-cycle, time-of-addition assay, AG7088 demonstrated antiviral activity when added up to 6 h after infection. In contrast, a compound targeting viral attachment and/or uncoating was effective only when added at the initiation of virus infection. Direct inhibition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled proteins, which showed a dose-dependent accumulation of viral precursor polyproteins and reduction of processed protein products. The broad spectrum of antiviral activity of AG7088, combined with its efficacy even when added late in the virus life cycle, highlights the advantages of 3C protease as a target and suggests that AG7088 will be a promising clinical candidate.

Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes.

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.