Quinolones may induce hepatitis.
Explore the source record for details and available documents.
Adult↗
Biomedical subjects
R H Smith
Publications and source records attributed to R H Smith.
A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation.
This study examines the intranasal instillation of lipopolysaccharide (LPS) into BALB/c mice causing acute pulmonary damage, due to neutrophil infiltration and sepsis. A dose response with LPS showed that an intranasal instillation of 167 microg/ml (10 microg/mouse) caused acute lung injury within 2-4 h and reached maximal damage at 24-48 h. We found the method of LPS administration for induction of acute pulmonary damage to be crucial. After 24 h post-LPS injection, a comparison showed a substantial increase in pulmonary damage with intranasal instillation of LPS. As for intravenous injection, it showed a baseline effect. This study indicates that LPS administered intranasally causes acute pulmonary damage, whereas with intravenous and intraperitoneal endotoxin administration a tissue-specific or similar degree of pulmonary injury may not develop.
Acute Disease↗
Detection of an abnormal aortic valve by intraoperative transesophageal echocardiography.
Explore the source record for details and available documents.
Aged↗
Intraoperative evaluation of a bicuspid aortic valve.
Explore the source record for details and available documents.
Aged↗
An interunit performance improvement program: can you get me to the OR on time?
To maximize clinical outcomes of patients served and to efficiently use resources, delays contributing to patients' readiness for surgery and delays in surgery start times were identified as performance improvement measures that were monitored and evaluated. Because delays in patient readiness for surgery impact negatively on operating room use and length of stay, an interdepartmental multi-disciplinary approach was taken to measure performance. Data collection tools were developed to capture unit specific information on patients undergoing ophthalmic surgery. Design of the data collection tools, the data collection process, outcomes, and corrective actions will be discussed in this article.
Appointments and Schedules↗
Molecular modeling of HIV-1 reverse transcriptase drug-resistant mutant strains: implications for the mechanism of polymerase action.
A computer model of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) either alone, or complexed with a non-nucleoside inhibitor (NNI), was constructed using crystal coordinate data from a subset of the protein surrounding the binding pocket region. Molecular mechanics calculations were carried out on solvated wild-type RT and RT that contained modifications corresponding to resistance-engendering mutations. Results from the calculations revealed that the r.m.s. difference between 12 modified proteins and that of wild-type RT could be qualitatively correlated with the measured polymerase activity of the enzyme in the presence of these mutations. In addition, the level of activity was related to the measured distance between the primer grip and dNTP binding regions of the protein. These data suggest a direct correlation between RT structure and function. Complexes of RT-8-C1 TIBO and RT-alpha-APA were also minimized in models containing modifications corresponding to key drug-resistant mutants. The variant complexes all showed weaker binding than wild-type RT, while giving rise to similar, but critical changes in the protein. Therefore, the design of new inhibitors should center on obtaining stronger binding drugs to key drug-resistant RT variants.
Anti-HIV Agents↗
The Rep78 gene product of adeno-associated virus (AAV) self-associates to form a hexameric complex in the presence of AAV ori sequences.
The Rep78 and Rep68 proteins of adeno-associated virus (AAV) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. Genetic and biochemical studies have identified Rep mutants that demonstrate a trans-dominant negative phenotype in vitro and in vivo, suggesting the possibility that multimerization of Rep is essential for certain replicative functions. In this study, we have investigated the ability of the largest of the Rep proteins, Rep78, to self-associate in vitro and in vivo. Self-association of Rep78 in vivo was demonstrated through the use of a mammalian two-hybrid system. Rep-Rep protein interaction was confirmed in vitro through coimmunoprecipitation experiments with a bacterially expressed maltose-binding protein-Rep78 fusion protein in combination with [35S]methionine-labeled Rep78 synthesized in a coupled in vitro transcription-translation system. Mapping studies with N- and C-terminal truncation mutant forms of Rep indicate that amino acid sequences required for maximal self-association occur between residues 164 and 484. Site-directed mutagenesis identified two essential motifs within this 321-amino-acid region: (i) a putative alpha-helix bearing a 3,4-hydrophobic heptad repeat reminiscent of those found in coiled-coil domains and (ii) a previously recognized nucleoside triphosphate-binding motif. Deletion of either of these regions from the full-length polypeptide resulted in severe impairment of Rep-Rep interaction. In addition, gel filtration chromatography and protein cross-linking experiments indicated that Rep78 forms a hexameric complex in the presence of AAV ori sequences.
Amino Acid Sequence↗
Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant.
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target for chemotherapeutic agents used in the treatment of AIDS; the TIBO compounds are potent non-nucleoside inhibitors of HIV-1 RT (NNRTIs). Crystal structures of HIV-1 RT complexed with 8-Cl TIBO (R86183, IC50 = 4.6 nM) and 9-Cl TIBO (R82913, IC50 = 33 nM) have been determined at 3.0 A resolution. Mutant HIV-1 RT, containing Cys in place of Tyr at position 181 (Tyrl81Cys), is highly resistant to many NNRTIs and HIV-1 variants containing this mutation have been selected in both cell culture and clinical trials. We also report the crystal structure of Tyrl81Cys HIV-1 RT in complex with 8-Cl TIBO (IC50 = 130 nM) determined at 3.2 A resolution. Averaging of the electron density maps computed for different HIV-1 RT/NNRTI complexes and from diffraction datasets obtained using a synchrotron source from frozen (-165 degrees C) and cooled (-10 degrees C) crystals of the same complex was employed to improve the quality of electron density maps and to reduce model bias. The overall locations and conformations of the bound inhibitors in the complexes containing wild-type HIV-1 RT and the two TIBO inhibitors are very similar, as are the overall shapes and volumes of the non-nucleoside inhibitor-binding pocket (NNIBP). The major differences between the two wild-type HIV-1 RT/TIBO complexes occur in the vicinity of the TIBO chlorine substituents and involve the polypeptide segments around the beta5-beta6 connecting loop (residues 95 to 105) and the beta13-beta14 hairpin (residues 235 and 236). In all known structures of HIV-1 RT/NNRTI complexes, including these two, the position of the beta12-beta13 hairpin or the "primer grip" is significantly displaced relative to the position in the structure of HIV-1 RT complexed with a double-stranded DNA and in unliganded HIV-1 RT structures. Since the primer grip helps to position the template-primer, this displacement suggests that binding of NNRTIs would affect the relative positions of the primer terminus and the polymerase active site. This could explain biochemical data showing that NNRTI binding to HIV-1 RT reduces efficiency of the chemical step of DNA polymerization, but does not prevent binding of either dNTPs or DNA. When the structure of the Tyr181Cys mutant HIV-1 RT in complex with 8-Cl TIBO is compared with the corresponding structure containing wild-type HIV-1 RT, the overall conformations of Tyr181Cys and wild-type HIV-1 RT and of the 8-Cl TIBO inhibitors are very similar. Some positional changes in the polypeptide backbone of the beta6-beta10-beta9 sheet containing residue 181 are observed when the Tyr181Cys and wild-type complexes are compared, particularlty near residue Val179 of beta9. In the p51 subunit, the Cys181 side-chain is oriented in a similar direction to the Tyr181 side-chain in the wild-type complex. However, the electron density corresponding to the sulfur of the Cys181 side-chain in the p66 subunit is very weak, indicating that the thiol group is disordered, presumably because there is no significant interaction with either 8-Cl TIBO or nearby amino acid residues. In the mutant complex, there are slight rearrangements of the side-chains of other amino acid residues in the NNIBP and of the flexible dimethylallyl group of 8-Cl TIBO; these conformational changes could potentially compensate for the interactions that were lost when the relatively large tyrosine at position 181 was replaced by a less bulky cysteine residue. In the corresponding wild-type complex, Tyr181 iin the p66 subunit has significant interactions with the bound inhibitor and the position of the Tyr181 side-chain is well defined in both subunits. Apparently the Tyr181 --> Cys mutation eliminates favorable contacts of the aromatic ring of the tyrosine and the bou
Antiviral Agents↗
The roles of AAV Rep proteins in gene expression and targeted integration.
Explore the source record for details and available documents.
Animals↗
T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices.
This paper establishes that the isolated shoot meristem of monocotyledons can be infected and transformed using Agrobacterium. Since this explant from nearly any cereal cultivar can rapidly regenerate into a plant, using this explant effectively eliminates the genotype regeneration restrictions to cereal crop transformation allowing direct transformation of elite germplasm. Shoot apices of Oryza sativa L. Tropical Japonica, cv. Maybelle were explants used for cocultivation, and gene transfer was accomplished using Agrobacterium containing plasmids for the bar gene expression driven by the CaMV 35S promoter or by the rice actin 1 promoter. Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinate-ammonium (PPT), established that no shoot apices survived on 0.5 or 1.0 mg/l PPT. After shoot apices were cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT. Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells appeared to be most effective in promoting transformation. Integration, expression and transmission of the transferred foreign genes in primary, R1 and R2 generation plants were confirmed by molecular analyses and herbicide application tests. A germination test of R2 progeny from one of the transgenic plants (R1) established a phenotype segregation ratio showing a non-Mendelian inheritance pattern. Inactivation of the transferred foreign gene in R2 progeny appeared to result from transgene methylation.
Aminobutyrates↗
Specificity of DNA alkylation by 1-(2-chloroethyl)-3-alkyl-3-acyltriazenes depends on the structure of the acyl group: kinetic and product studies.
The reactions of calf thymus DNA with ten 1-(2-chloroethyl)-3-alkyl-3-acyltriazenes of varying acyl side chain structure were studied alone, or in the presence of porcine liver esterase in pH 7.0 phosphate buffer. In several of the key triazenes, the acyl substituent contained a free carboxylic acid group. With esterase present in the reaction mixture, the resultant levels of DNA alkylation could be correlated with the kinetic rates of decomposition of the triazenes. Under these conditions, the predominant pathway of decomposition involved deacylation of the parent triazene and eventual production of an alkanediazonium ion. This intermediate subsequently alkylated DNA--guanine to give 7-alkylguanine as the principal reaction product. In the absence of esterase, the order of DNA alkylation for all of the acyltriazenes did not correlate with their respective rates of decomposition, leading to the conclusion that the triazenes did not decompose by the expected mode of uncatalyzed N(2)-N(3) heterolyic cleavage. The major DNA alkylation product from the N(3)-methyltriazenes was 7-methylguanine, instead of the expected 7-(chloroethyl)- and 7-(hydroxyethyl)guanine products, which suggested that the acyl group was being hydrolyzed. However, acyltriazenes with an N(3)-benzyl group rather than a methyl in this position produced very little 7-benzylguanine product, contrary to prediction. An alternative mechanism involving internally assisted hydrolysis of the side chain ester is proposed to explain these results. NMR product analysis and computational studies were carried out to lend support to the postulated mechanism.
Alkylating Agents↗
Association of increased left ventricular mass in the absence of electrocardiographic left ventricular hypertrophy with ST depression during exercise.
Explore the source record for details and available documents.
Aged↗
Characterization of DNA binding properties of the immediate-early gene product of equine herpesvirus type 1.
The equine herpesvirus type 1 (EHV-1) immediate-early (IE) gene encodes a phosphoprotein that is essential for the activation of transcription from viral early and late promoters and that regulates the transcription from its own promoter. Employment of EHV-1 IE promoter DNA probes and glutathione S-transferase fusion proteins harboring truncated portions of the IE gene product in gel shift assays, super shift assays with anti-IE monoclonal antibodies, and DNase I footprinting analyses revealed: (1) amino acid residues 422 to 597 within the 1487-amino-acid IE protein are sufficient for sequence-specific DNA binding; (2) the IE protein binds to EHV-1 DNA at sequences from -11 to +14 that overlap the transcription initiation site (+1); (3) the conserved pentanucleotide 5'-ATCGT-3' in the IE promoter located at nucleotides (nt) -6 to -2, relative to the transcription initiation site (+1), is critical for IE protein binding; (4) a weak binding site for the IE protein is also present at nt -92 to -82 of the IE gene within the sequence (-86)ATCGA(-82) in which four of the five nt in the consensus binding sequence are conserved; (5) the IE protein binds to sequences in EHV-1 early and late promoters that contain a degenerate version of the consensus sequence 5'-ATCGT-3'; and (6) mutation of the C or G nt in the pentanucleotide 5'-ATCGT-3' prevents sequence-specific binding of the IE protein, whereas mutation of each of the other three nt only reduces binding. These results suggest that the IE protein can recognize the sites which differ slightly from the proposed consensus sequence. Overall, these findings suggest that formation of a specific complex between an IE protein and its own gene promoter may be a common mechanism used by Alphaherpesvirinae to autoregulate transcription of an essential IE gene. In addition, the finding that the DNA binding domain of the IE protein maps within amino acids 422 to 597, a domain conserved in the IR2 early protein that is a truncated form of the IE protein, suggests that the IR2 protein plays a role in the regulation of the IE gene expression.
Animals↗
Characterization of the regulatory function of the ICP22 protein of equine herpesvirus type 1.
The IR4 gene (inverted repeat gene 4) of equine herpesvirus type 1 (EHV-), the homolog of the herpes simplex virus type 1 ICP22 gene, is differentially expressed as a 1.4-kb early transcript and a 1.7-kb late transcript that encode a series of proteins that migrate between 42 to 47 kDa, localize to the nucleus of EHV-1-infected cells, and become packaged within EHV-1 virions (V. R. Holden, G. B. Caughman, Y. Zhao, R. N. Harty, and D. J. O'Callaghan, J. Virol. 68, 4329-4340, 1994). To assess the role of the IR4 protein in EHV-1 gene regulation, an IR4 expression vector was cotransfected with EHV-1 chimeric promoter-CAT reporter constructs and EHV-1 effector plasmids to determine the effects of the IR4 protein on the expression of immediate-early (IE), early, and late promoters. These studies revealed that the IR4 protein: (i) minimally trans-activates EHV-1 promoters, (ii) acts synergistically with the UL3 (ICP27) gene product to trans-activate the IE promoter, (iii) does not interfere with the trans-repression of the IE promoter by the IE protein, (iv) enhances transactivation of early promoters by the IE protein, (v) enhances the transactivation of both early and late promoters by the IE and UL3 proteins, and (vi) interacts synergistically with the IE protein to trans-activate the heterologous HSV-1 ICP4 promoter. These data suggest that the IR4 gene product plays a significant role in EHV-1 gene regulation.
Cell Line↗
Effect of proteolytic and glycolytic enzymes on a factor in Sorghum bicolor that induces mycelial growth in the smut fungus, Sporisorium reilianum.
Proteins obtained from seedling shoots and floral meristems of Sorghum bicolor (L.) Moench cv. NK 1210 induced mycelial growth in the smut fungus, Sporisorium reilianum in vitro. Proteins precipitated with trichloroacetic acid and ammonium sulfate were equally effective as inducers, although there were minor variations in the pattern of mycelial growth. Hydrolysis of the protein fraction with the proteolytic enzyme pronase E resulted in considerable reduction in the proteins' ability to induce mycelial growth. Digestion of the protein fraction with driselase, resulted in a slight enhancement of biological activity. The results suggest that amino sugar moieties in glycoproteins may act as inducers of mycelial growth in Sporisorium reilianum.
Edible Grain↗
Utilization of sialic acid-binding synthetic peptide sequences derived from pertussis toxin as novel anti-inflammatory agents.
Pertussis toxin, a virulence factor produced by the organism Bordetella pertussis, has been shown to have functional similarities with selectins and to bind to similar sialic acid-containing oligosaccharides structures. Previously, we demonstrated that the amino-terminal region of the S2 subunit of pertussis toxin contained a short six amino acid sequence (SPYGRC) which displayed reasonable homology to a sequence that constitutes a portion of the sialic acid binding site in wheat germ agglutinin. Synthetic peptides containing this hexapeptide motif had the ability to bind to sialic acid-containing glycoconjugates including the putative oligosaccharide receptors (sialyl Lewis X and sialyl Lewis A) for selectins. Control peptides containing randomized sequences were inactive at inhibiting binding, indicating that the hexapeptide motif is important for interacting with sialic acid. Since pertussis toxin-derived peptides demonstrated the ability to interact with selectin receptors, we speculated that they should antagonize selectin-mediated inflammatory activity. To test this hypothesis, we evaluated the peptides for the ability to reduce neutrophil binding to activated endothelial cells as well as the anti-inflammatory activity in the mouse footpad swelling assay. Both S2 peptides were active at reducing neutrophil binding and footpad swelling, while the randomized control peptides were inactive.
Amino Acid Sequence↗
Regulatory function of the equine herpesvirus 1 ICP27 gene product.
The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides -123 to +20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.
Animals↗
Detection and intracellular localization of equine herpesvirus 1 IR1 and IR2 gene products by using monoclonal antibodies.
During lytic infection, two transcripts arise from the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene (IR1): a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early mRNA (IR2). Previous studies demonstrated that transiently expressed IR1 and IR2 gene products are potent transcriptional regulators: IR1 proteins are capable of trans activating representative EHV-1 early and late promoters, while both IR1 proteins and the IR2 product, which lacks IR1 amino acid residues 1 to 322, trans repress the IR1 promoter. In the present study, monoclonal antibodies (MAbs) against the major IE protein, IE1, were developed, characterized as to their ability to detect IR1 and IR2 products, and used to examine extracellular virions for the presence of IE1-related proteins and to define the IR1 and IR2 protein synthesis and intracellular distribution in EHV-1-infected cells. The results demonstrated that (i) anti-IE1 MAbs representing three noncompetitive epitope-binding groups reacted with multiple IE protein species, as well as with a 146-kDa early protein identified as the putative IR2 gene product; (ii) the three reactive epitopes mapped to a region spanning amino acids 323 to 552 of IR1; (iii) anti-IE1 MAbs reacted with the 144-kDa in vitro-translated IR2 product and with a transiently expressed IR2 product similar in size; (iv) small amounts of IE1 and the 146-kDa protein were associated with the nucleocapsid-tegument fraction of mature virions; (v) in immunofluorescence assays of lytically infected cells, IR1-IR2 gene products were first detectable between 1 and 2 h postinfection as discrete, punctate, intranuclear foci; (vi) as the infection progressed, the intranuclear reactivity increased and redistributed into large, intensely stained nuclear compartments which corresponded to the sites of active viral DNA synthesis; (vii) fibrillar, as well as more generalized cytoplasmic staining, first observed at about 5 h postinfection, increased throughout infection; and (viii) while viral DNA synthesis was required for the progressive intranuclear redistribution, the cytoplasmic accumulation of IR1-IR2 proteins occurred subsequent to early infection events.
Animals↗