Biomedical subjects
Publications and source records attributed to P J McCann.
A series of N-(4-hydroxy-3-methylsulfonanilidoethanol)arylglycinamides were prepared and evaluated for their human beta3 adrenergic receptor agonist activity. SAR studies led to the identification of BMS-201620 (39), a potent beta3 full agonist (Ki = 93 nM, 93% activation). Based on its favorable safety profile, BMS-201620 was chosen for clinical evaluation.
An absorption spectrometer equipped with a IV-VI semiconductor tunable mid-IR diode laser was used to make sensitive measurements of benzene (C(6)H(6)) gas in the 5.1-microm spectral range. Wavelength modulation coupled with second-harmonic detection achieved accurate real-time quantification of benzene concentrations down to a minimum detection limit of 1 ppmv with an integration time of 4 s. A variety of calibrated benzene-sensing measurements were made, including the determination of the benzene concentrations in vehicle exhaust and headspace vapors from unleaded gasoline and other liquids. Kinetic phenomena, including the monitoring of benzene evaporation and absorption/desorption by granulated activated carbon were observed with the instrument. Measurements were performed that allowed experimental determination of the activation energy for desorption of benzene from activated carbon, which was found to be 198 meV/molecule (19.0 kJ/mol).
Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)
A series of 4-hydroxy-3-methylsulfonanilido-1,2-diarylethylamines were prepared and evaluated for their human beta(3) adrenergic receptor agonist activity. SAR studies led to the identification of BMS-196085 (25), a potent beta(3) full agonist (K(i)=21 nM, 95% activation) with partial agonist (45%) activity at the beta(1) receptor. Based on its desirable in vitro and in vivo properties, BMS-196085 was chosen for clinical evaluation.
Twenty-two patients were referred to the maxillofacial surgical unit for assessment and management of suspected fractures of the zygomatico-orbital complex. In each case, both routine plain radiographic and ultrasound examinations were made. The aim of the study was to investigate the sensitivity and reliability of ultrasound to detect such fractures. Imaging with ultrasound was carried out at five areas: the infraorbital margin; lateral wall of the maxillary sinus; zygomatic arch; frontozygomatic process; and orbital floor. Both radiographic and ultrasound findings were correlated with the findings at operation. An overall agreement of 85% between radiographs and ultrasound scans was found. Ultrasound imaging was most reliable at the lateral wall of the maxillary sinus, where the sensitivity was 94% and specificity 100%. The positive predictive value at this area was 100% compared with radiographic findings. We conclude that ultrasound is a useful tool in imaging facial trauma as an initial investigation, and can help to reduce the total number of radiographs required for the diagnosis of fractures of the zygomatico-orbital complex.
The herpes simplex virus type 1 UL12 gene product, alkaline nuclease (AN), appears to be involved in viral DNA processing and capsid egress from the nucleus (Shao, L., Rapp, L. M., and Weller, S. K., Virology 196, 146-162, 1993). Although the HSV-1 AN is not absolutely essential for viral replication in tissue culture, conservation of the AN gene in all herpesviruses suggests an important role in the life cycle of herpesviruses. The counterpart of HSV-1 AN for human cytomegalovirus (HCMV) is the UL98 gene product. To examine whether the HCMV AN could substitute for HSV-1 AN, we performed trans-complementation experiments using a HSV-1 amplicon plasmid carrying the HCMV UL98 gene. Our results indicate (i) HCMV AN can complement the growth of the HSV-1 AN deletion mutant UL12lacZ virus in trans; (ii) a new recombinant virus, UL12laZcUL98/99, appears to be generated by the integration of the HCMV UL98 gene into the HSV-1 UL12lacZ viral genome; (iii) in contrast to its parental HSV-1 UL12lacZ virus, capsids formed in UL12lacZUL98/99-infected Vero cells were able to transport from the nucleus to the cytoplasm and mature into infectious viruses. Our results demonstrate a functional conservation of AN between HSV-1 and HCMV.
The method of substrate phage display was used to select a preferred substrate from three monovalent display libraries using the HSV-1 protease. The display libraries consisted of four random amino acids, six random amino acids, and a biased library containing four amino acids from the P side of the HSV-1 maturation site followed by four random amino acids. A series of consensus peptides was synthesized based upon the results from these screens and tested in peptide cleavage assays. An eight amino acids consensus peptide (LVLASSSF) derived from the phage results was cleaved as efficiently as a 20-mer maturation site peptide. The selected amino acid sequences also allowed the design of a four amino acid paranitroanilide substrate for continuous assay of HSV-1 protease. Similar to HCMV protease, these results define P4 to P1 as a minimal substrate recognition domain for the HSV-1 protease and suggest that P4 to P1 is the minimal substrate domain which all herpesvirus proteases recognize.
The herpes simplex virus type 1 (HSV-1) protease and its substrate, the assembly protein ICP35, are involved in virion maturation. Both proteins are encoded by a single open reading frame but are translated independently from 3'-coterminal mRNAs of different sizes and are in frame. The herpesvirus shell assembles around an internal scaffold which is subsequently lost during packaging of the viral genome. The scaffold is composed of ICP35, which is the major component, and autoproteolytically processed forms of the viral protease containing sequences common to ICP35 (Nb). In the baculovirus system, HSV-1 intact capsids can be formed in the presence of the protease or ICP35, indicating that the protease may substitute for ICP35 (Thomsen et al., J. Virol. 68:2442-2457, 1994). This is further supported by the fact that ICP35, in contrast to the protease, is not absolutely essential for viral growth. The processed intermediate of the protease analogous to ICP35 is the 388-amino-acid (aa) protein, Na, which is an N-terminal 59-aa extension of the 329-aa ICP35. To directly examine whether Na can functionally substitute for ICP35 during viral replication, we first constructed a mutant virus, Na delta35, in which 35 aa from the N terminus of Na were deleted. Phenotypic analysis of the mutant showed that this deletion had no effect on protease function. The function of Na was further examined by construction of a plasmid expressing Na alone and testing its ability to complement the growth of the mutant Prb virus in the absence of ICP35. Our results demonstrate that Na can functionally substitute for ICP35 during viral replication.
The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully processed Pra (N0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.
The herpes simplex virus type 1 (HSV-1) protease and its substrate, ICP35, are involved in the assembly of viral capsids and required for efficient viral growth. The full-length protease (Pra) consists of 635 amino acid (aa) residues and is autoproteolytically processed at the release (R) site and the maturation (M) site, releasing the catalytic domain No (VP24), Nb (VP21), and a 25-aa peptide. To understand the biological importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were performed to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mutant m100. Our results indicate that (i) expression of full-length protease is not required for viral replication, since a 514-aa protease molecule lacking the M site could support viral growth; and that (ii) elimination of the R site by changing the residue Ala-247 to Ser abolished viral replication. To better understand the functions that are mediated by proteolytic processing at the R site of the protease, we engineered an HSV-1 recombinant virus containing a mutation at this site. Analysis of the mutant A247S virus demonstrated that (i) the mutant protease retained the ability to cleave at the M site and to trans process ICP35 but failed to support viral growth on Vero cells, demonstrating that release of the catalytic domain No from Pra is required for viral replication; and that (ii) only empty capsid structures were observed by electron microscopy in thin sections of A247S-infected Vero cells, indicating that viral DNA was not encapsidated. Our results demonstrate that processing of ICP35 is not sufficient to support viral replication and provide genetic evidence that the HSV-1 protease has nuclear functions other than enzymatic activity.
The herpes simplex virus type 1 protease and its substrate, ICP35, are involved in the assembly of viral capsids. Both proteins are encoded by a single open reading frame from overlapping mRNAs. The protease is autoproteolytically processed at two sites. The protease cleaves itself at the C-terminal site (maturation site) and also cleaves ICP35 at an identical site, releasing a 25-amino-acid (aa) peptide from each protein. To determine whether these 25 aa play a role in capsid assembly, we constructed a mutant virus expressing only Prb, the protease without the C-terminal 25 aa. Phenotypic analysis of the Prb virus in the presence and absence of ICP35 shows the following: (i) Prb retains the functional activity of the wild-type protease which supports virus growth in the presence of ICP35; (ii) in contrast to the ICP35 null mutant delta ICP35 virus, the Prb virus fails to grow in the absence of ICP35; and (iii) trans-complementation experiments indicated that full-length ICP35 (ICP35 c,d), but not the cleaved form (ICP35 e,f), complements the growth of the Prb virus. The most striking phenotype of the Prb virus is that only unsealed aberrant capsid structures are observed by electron microscopy in mutant-infected Vero cells. Our results demonstrate that the growth of herpes simplex virus type 1 requires the C-terminal 25 aa of either the protease or its substrate, ICP35, and that the C-terminal 25 aa are involved in the formation of sealed capsids.
The herpes simplex virus type 1 (HSV-1) protease is cleaved at two autoprocessing sites during viral maturation, one of which shares amino acid identity with its substrate, ICP35. Similar autoprocessing sites have been observed within other members of the Herpesviridae. Introduction of point mutations within the autoprocessing sites of the HSV-1 protease indicated that specificity resides within the P4-P1' region of the cleavage sites.
The herpes simplex virus type 1 protease and related proteins are involved in the assembly of viral capsids. The protease encoded by the UL26 gene can process itself and its substrate ICP35, encoded by the UL26.5 gene. To better understand the functions of the protease in infected cells, we have isolated a complementing cell line (BMS-MG22) and constructed and characterized a null UL26 mutant virus, m100. The mutant virus failed to grow on Vero cells and required a complementing cell line for its propagation, confirming that the UL26 gene product is essential for viral growth. Phenotypic analysis of m100 shows that (i) normal amounts of the c and d forms of ICP35 were produced, but they failed to be processed to the cleaved forms, e and f; (ii) viral DNA replication of the mutant proceeded at near wild-type levels, but DNA was not processed to unit length or encapsidated; (iii) capsid structures were observed in thin sections of m100-infected Vero cells by electron microscopy, but assembly of VP5 into hexons of the capsid structure was conformationally altered; and (iv) nuclear localizations of the protease and ICP35 are independent of each other, and the function(s) of Na, at least in part, is to direct the catalytic domain N(o) to the nucleus.
Herpes simplex virus type-1 (HSV-1) encodes a protease responsible for proteolytic processing of the virus assembly protein, ICP35 (infected cell protein 35). The coding region of ICP35 is contained within the gene that encodes the protease, and ICP35 shares amino acid identity with the carboxyl-terminal 329 amino acids of the protease. The HSV-1 protease was expressed in Escherichia coli as a fusion protein containing a unique epitope and the protein A Fc binding domain at its carboxyl terminus. The fusion protease underwent autoproteolytic cleavage at two distinct sites. The size of the cleavage products containing the carboxyl-terminal epitope mapped one cleavage site near the carboxyl terminus of the protease corresponding to the proteolytic processing site of ICP35, and the second site proximal to the amino terminus consistent with previous data. The carboxyl-terminal autoproteolytic cleavage products were partially purified on an IgG affinity column by virtue of the protein A Fc binding domain and subjected to direct amino-terminal sequence analysis. Protein sequencing revealed that cleavage occurs between the Ala and Ser residues at amino acids 610/611 and 247/248 of the HSV-1 protease. The flanking sequences share homology with each other and are highly conserved in homologous proteases of other herpes viruses.
The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.
The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a protease which is responsible for the C-terminal cleavage of the nucleocapsid-associated proteins, ICP35 c and d, to their posttranslationally modified counterparts, ICP35 e and f. To further characterize the HSV-1 protease, the UL26 gene product was expressed in Escherichia coli. The expressed protease underwent autoproteolytic processing at two independent sites. The first site is shared with ICP35 and results in removal of 25 amino acids from the C terminus of the protease. The second unique site gives rise to protein species consistent with deletion of a 28-kDa fragment at the N terminus. A mutant protease, which showed no activity in a mammalian cell cotransfection assay (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992), failed to exhibit autoproteolytic processing at either site when expressed in bacteria. The inactive mutant was able to serve as a substrate in a trans assay in which the substrate and protease were coexpressed in bacteria. This experiment demonstrated that the unique N-terminal processing was mediated exclusively by the HSV-1 protease. ICP35 c,d also served as a substrate in this assay and was correctly processed by HSV-1 protease in E. coli. This trans-cleavage assay will aid in the characterization of HSV-1 protease and assist in investigation of the role of proteolytic processing in the virus.