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T D Brown

Publications and source records attributed to T D Brown.

At least 91 records · Page 5Linked to original sources
PubMed1996

Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame.

The construction of a full-length clone of the avian coronavirus infectious bronchitis virus (IBV) defective RNA (D-RNA), CD-91 (9,080 nucleotides [Z. Penzes et al., Virology 203:286-293]), downstream of the bacteriophage T7 promoter is described. Electroporation of in vitro T7-transcribed CD-91 RNA into IBV helper virus-infected primary chick kidney cells resulted in the production of CD-91 RNA as a replicating D-RNA in subsequent passages. Three CD-91 deletion mutants were constructed--CD-44, CD-58, and CD-61--in which 4,639, 3,236, and 2,953 nucleotides, respectively, were removed from CD-91, resulting in the truncation of the CD-91 long open reading frame (ORF) from 6,465 to 1,311, 1,263, or 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively. Electroporation of in vitro T7-transcribed RNA from the three constructs into IBV helper virus-infected cells resulted in the replication and packaging of CD-58 and CD-61 but not CD-44 RNA. The ORF of CD-61 was further truncated by the insertion of stop codons into the CD-61 sequence by PCR mutagenesis, resulting in constructs CD-61T11 (ORF: nucleotides 996 to 1,058, encoding 20 amino acids), CD-61T22 (ORF: nucleotides 996 to 2,294, encoding 432 amino acids), and CD-61T24 (ORF: nucleotides 996 to 2,450, encoding 484 amino acids), all of which were replicated and packaged to the same levels as observed for either CD-61 or CD-91. Analysis of the D-RNAs showed that the CD-91- or CD-61-specific long ORFs had not been restored. Our data indicate that IBV D-RNAs based on the natural D-RNA, CD-91, do not require a long ORF for efficient replication. In addition, a 1.4-kb sequence, corresponding to IBV sequence at the 5' end of the 1b gene, may be involved in the packaging of IBV D-RNAs or form part of a cis-acting replication element.

Animals↗
PubMed1996

Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus.

A region of the infectious bronchitis virus (IBV) genome between nucleotide positions 8693 and 10927 which encodes the predicted 3C-like proteinase (3CLP) domain and several potential cleavage sites has been clones into a T7 transcription vector. In vitro translation of synthetic transcripts generated from this plasmid was not accompanied by detectable processing activity of the nascent polypeptide unless the translation was carried out in the presence of microsomal membrane preparations. The processed products so obtained closely resembled in size those expected from cleavage at predicted glutamine-serine (Q/S) dipeptides and included a protein with a size of 35 kDa (p35) that corresponds to the predicted size of 3CLP. Efficient processing was dependent on the presence of membranes during translation; processing was found to occur when microsomes were added posttranslationally, but only after extended periods of incubation. C-terminal deletion analysis of the encoded polyprotein fragment revealed that cleavage activity was dependent on the presence of most but not all of the downstream and adjacent hydrophobic region MP2. Dysfunctional mutagenesis of the putative active-site cysteine residue of 3CLP to either serine or alanine resulted in polypeptides that were impaired for processing, while mutagenesis at the predicted Q/S release sites implicated them in the release of the p35 protein. Processed products of the wild-type protein were active in trans cleavage assays, which were used to demonstrate that the IBV 3CLP is sensitive to inhibition by both serine and cysteine protease class-specific inhibitors. These data reveal the identity of the IBV 3C-like proteinase, which exhibits characteristics in common with the 3C proteinases of picornaviruses.

Animals↗
PubMed1996

Coronal plane rotation of the first metatarsal.

A method for measuring first metatarsal rotation on weightbearing tangential radiographs is described. Under controlled conditions using cadaver specimens, 5 degree changes in first metatarsal rotation were associated with a mean change in radiographically measured rotation of 5.4 degrees (SD = 1.7 degrees). A clinical study of 30 hallux valgus and 30 control patients was undertaken to assess the reliability of the method of measurement. The overall reliability was high for both hallux valgus and control patients (r = 0.93 and 0.95, respectively). In these two groups, no significant difference was found between the mean values for first metatarsal rotation. Likewise, we found no correlation between first metatarsal rotation measured on tangential standing ("sesamoid" view) radiographs and the first metatarsophalangeal angle or the intermetatarsal 1-2 angle measured on anteroposterior standing radiographs.

Analysis of Variance↗
PubMed1996

The Frank Stinchfield Award. 3-Dimensional sliding/contact computational simulation of total hip wear.

Polyethylene wear in total hip replacements is a complex, multifactorial process. A tribologically grounded finite element formulation was developed to make quantitative estimates of polyethylene wear in total hip arthroplasty, incorporating the combined influences of contact stress, sliding distance, and a surface specific wear coefficient. For loading and sliding distance inputs taken directly from human gait data, the computational model showed a strong direct proportionality between femoral head size and volumetric wear rate. Other factors being equal, reducing the thickness of the polyethylene liner led to increases in the computed wear rates, but the effect was far less pronounced than the strong increases in wear rate that accompanied head size increases. Compared with human gait inputs, the load and sliding distance inputs for a 23 degrees biaxial rocking hip simulator led to computed wear rates that were 1.7 times as large, and in which the direction of wear was near the cup apex rather than within the posterosuperolateral quadrant. In general, the finite element model's results emphasize the importance of articulation kinematics, especially sliding distance, in the complex process of polyethylene wear in total hip arthroplasty.

Computer Simulation↗
PubMed1995

Characterisation and mutational analysis of an ORF 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein.

Coronavirus gene expression involves proteolytic processing of the mRNA 1-encoded polyproteins by viral and cellular proteinases. Recently, we have demonstrated that an ORF 1b-encoded 100-kDa protein is proteolytically cleaved from the 1a/1b fusion polyprotein by a viral-specific proteinase of the picornavirus 3C proteinase group (3C-like proteinase). In this report, the 3C-like proteinase has been further analysed by internal deletion of a 2.3-kb fragment between the 3C-like proteinase-encoding region and ORF 1b and by substitution mutations of its catalytic centre as well as the two predicted cleavage sites flanking the 100-kDa protein. The results show that internal deletion of ORF 1a sequences from nucleotide 9911 to 12227 does not influence the catalytic activity of the proteinase in processing of the 1a/1b polyprotein to the 100-kDa protein species. Site-directed mutagenesis studies have confirmed that the predicted nucleophilic cysteine residue (Cys2922) and a histidine residue encoded by ORF 1a from nucleotide 8985 to 8987 (His2820) are essential for the catalytic activity of the proteinase, and that the QS(G) dipeptide bonds are its target cleavage sites. Substitution mutations of the third component of the putative catalytic triad, the glutamic acid 2843 (Glu2843) residue, however, do not affect the processing to the 100-kDa protein. In addition, cotransfection experiment shows that the 3C-like proteinase is capable of trans-cleavage of the 1a/1b polyprotein. These studies have confirmed the involvement of the 3C-like proteinase domain in processing of the 1a/1b polyprotein, the predicted catalytic centre of the proteinase, and its cleavage sites.

Amino Acid Sequence↗
PubMed1995

Identification, expression, and processing of an 87-kDa polypeptide encoded by ORF 1a of the coronavirus infectious bronchitis virus.

Nucleotide sequence analysis has shown previously that the genomic-length mRNA (mRNA1) of the coronavirus infectious bronchitis virus (IBV) contains two large open reading frames (ORFs), 1a and 1b, with the potential to encode polyproteins of approximately 441 and 300 kDa, respectively. We have characterized the specificity of a set of region-specific antisera raised against the 5'-portion of ORF 1a by immunoprecipitation of in vitro-synthesized, C-terminally truncated 1a polypeptides and used these antisera to detect virus-specific proteins in IBV-infected Vero cells. Two antisera, which had specificity for IBV sequences from nucleotides 710 to 2079 and 1355 to 2433, respectively, immunoprecipitated a polypeptide of approximately 87 kDa from IBV-infected Vero cells. In vitro translation of ORF 1a sequence terminating at nucleotide 5763 did not produce this protein unless the in vitro translation products were incubated with Vero cell S10 extracts prepared from either IBV-infected or mock-infected Vero cells. However, processing of the 87-kDa protein was also observed when the same region was expressed in Vero cells using the vaccinia virus/T7 expression system. This observation indicates that the 87-kDa polypeptide is encoded within the 5'-most 3000 nucleotides of mRNA 1 and that it might be cleaved from the 1a polyprotein by viral and cellular proteinases.

Animals↗
PubMed1995

Three-dimensional geometric and structural symmetry of the turkey ulna.

Structural models of long-bone preparations usually assume left-right symmetry of contralateral bones under normal (baseline) conditions. To obtain insight on how this assumption affects the detection of subtle changes (as from functional adaptation), we formally examined the three-dimensional geometric and structural symmetry of paired long bones, using contemporary image reconstruction and stress analysis techniques. Nine pairs of ulnae from normal male turkeys were reconstructed computationally from serial transverse images obtained by either (a) mechanical sectioning and digital photographic imaging or (b) computed tomography. Computed tomography scans allowed greater precision in reconstruction than did digitally imaged photographs. Left-right comparisons of parameters of geometric symmetry (from computed tomography reconstructions) revealed average differences in whole bone volume and whole bone principal moments of inertia of 3.6 and 3.0%, respectively. Differences in bone curvature were indexed as noncolinearity of left compared with (mirrored) right centroidal axes, giving a disparity of 0.7 +/- 0.3 mm. Within the longitudinal central 20% of the diaphysis (the customary region for histomorphometry), average left-right differences in cross-sectional area and area principal moments of inertia for computed tomography images were 4.7 and 5.0%, respectively. The overlap of longitudinally paired cross sections of the mid-diaphysis, aligned at common centroids and oriented in the respective principal inertial directions, was greatest (as much as 95%) in the central 20% of the diaphysis. Paired three-dimensional finite element models demonstrated nearly identical left and right stress/strain fields throughout the ulnar diaphyses for both compressive and torsional loading. Our data suggest that the assumption of contralateral geometric symmetry in long bones should be judged in the context of the specific attribute of symmetry under consideration; however, we conclude that for purposes of finite element modeling the assumption of symmetry is reasonable.

Adaptation, Physiological↗
PubMed1995

Generation of a defective RNA of avian coronavirus infectious bronchitis virus (IBV). Defective RNA of coronavirus IBV.

The Beaudette strain of IBV was passaged 16 times in chick kidney (CK) cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32P-labeled cDNA probes corresponding to the first 2 kb of the 5' end of the genome, but excluding the leader, and to the last 1.8 kb of the 3' end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage six. The defective RNA, present in total cell extract RNA and in oligo-(dT)30-selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5' end of the genome, 6322 from gene 1b corresponding to position 12423 to 18744 in the IBV genome and 1626 from the 3' end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.

Animals↗
PubMed1995

Chronic stress tolerance levels for human articular cartilage: two nonuniform contact models applied to long-term follow-up of CDH.

Two computer models of nonuniform contact stress on the articular surface of the human hip were used to study the relationship between chronically excessive articular cartilage contact stress and long-term clinical outcome in a series of patients with congenital dislocation of the hip (CDH). The analyzed database consisted of 409 stylus digitized radiographs from 83 patients with unilateral CDH, who had been treated by closed reduction, and whose average follow-up time was 29.2 y. The first model (nonuniform Legal) involved a three-dimensional contact stress distribution function whose pole was coincident with the resultant force acting through the hip, and which acted over a contact area whose borders were determined soley by bony landmarks. In the second model (Brinckmann), the direction of the pole of the contact stress distribution function was initially unknown; one border of the contact region was determined by radiographic landmarks, while the other border depended upon the pole of the iteratively determined contact stress distribution function. In both models, the contact stress distributions were converted to area engagement histograms, corresponding to the fractional areas of cartilage experiencing specific ranges of stress (0.5 MPa increments). These histograms were integrated over time to calculate a cumulative contact stress overdose, which was then compared to clinical outcome. Reasonable correlations (Spearman rho = 0.63-0.66) with patient outcomes were obtained for optimally chosen damage thresholds, although these thresholds were appreciably different (2.0 versus 4.5 MPa) due to the respective modelling assumptions.

Biomechanical Phenomena↗
PubMed1995

In vivo quantification of the cat patellofemoral joint contact stresses and areas.

A new method for in vivo measurement of patellofemoral joint contact areas, stresses, and patellar displacements, with joint loading approximating physiologic conditions was developed. Joint contact measurements were obtained using pressure sensitive film inserted directly between articular joint surfaces. Two-dimensional joint kinematics were measured using a high-speed video based motion analysis system. Joint loading, provided by quadriceps muscle stimulation, was measured with an implantable force transducer (IFT). Variations in joint mechanics as a function of joint flexion angle, joint loading and joint stability (anterior cruciate ligament intact or transected) were determined for four adult male cats. The contact measurements obtained with the pressure sensitive film displayed high repeatability with a standard error of +/- 6.8% of the mean value of the median pressure and +/- 4.4% of the mean contact area value. Substantial differences in joint mechanics were reliably detected with the new technique. The influence of experimental procedures, such as incisions in the joint capsule and insertion of pressure sensitive film between the articular surfaces, produced minimal changes in the joint kinematics during muscular contraction.

Animals↗
PubMed1995

Cloning, sequencing and expression of the S protein gene from two geographically distinct strains of canine coronavirus.

The gene encoding the spike (S) protein from two geographically distinct strains (American and British) of canine coronavirus (CCV) was cloned and sequenced. The nucleotide sequence revealed open reading frames of 1443 or 1453 amino acids, respectively. Structural features include an N-terminal hydrophobic signal sequence, a hydrophilic cysteine-rich cluster near the C-terminus, two heptad repeats and 29 or 33 potential N-glycosylation sites. Pairwise comparisons of S amino acid sequences from these isolates with other CCV strains (Insavc1 and K378) revealed that heterogeneity, found mostly in the form of conservative substitutions, is distributed throughout the canine sequences. However, 5 variable regions could be identified. Similar analysis with feline, porcine, murine, chicken and human coronavirus sequences revealed that the canine sequences are much more closely related to the feline S protein sequence than to the porcine S protein sequences even though they are all from the same antigenic group. Moreover, the sequence similarity between CCV isolates and the feline coronavirus, feline infectious peritonitis virus (FIPV) was comparable. Expression of the CCV or the transmissible gastroenteritis virus (TGEV) S gene using the vaccinia virus system produced a protein of the expected size which could induce extensive syncytia formation in infected canine A72 cells.

Amino Acid Sequence↗
PubMed1995

Effects of indomethacin on biologic fixation of porous-coated titanium implants.

The effect of perioperative administration of indomethacin on attachment strength and bone growth into porous-coated titanium implants was evaluated in the canine transcortical plug model. Various drug administration protocols simulating clinical use of indomethacin were studied. These included chronic treatment (starting 2 weeks prior to surgery), treatment immediately after surgery, and treatment 3, 6, 9, and 18 weeks following surgery. Indomethacin therapy was continued until sacrifice at 3, 6, 12, 18, or 24 postoperative weeks. Push-out testing was performed to determine the maximum bone-implant interface shear strength, and quantitative histologic analysis was used to determine percentage of bone ingrowth. When indomethacin was administered chronically or immediately after surgery, a statistically significant decrease in bone-implant interface attachment strength was seen at 3 postoperative weeks but not at later periods. No adverse effect was observed in any group after the 3-week period. Quantitative histologic analysis demonstrated no significant differences in percentage bone ingrowth among any of the treatment protocols at 3 or 6 weeks after surgery. No significant difference was observed between any of the groups at 18 or 24 weeks. The results of this study suggest that perioperative administration of indomethacin does not significantly affect attachment strength or bone ingrowth into porous-coated implants except at early periods, in which cases a transient decrease in attachment strength occurs.

Animals↗
PubMed1995

Reamed surface topography and component seating in press-fit cementless acetabular fixation.

Thirty-eight paired, size-matched, fresh-frozen cadaver acetabulae were used to study reamed surface topography and the seating of on-line versus oversized noncemented components. Four commonly used constructs were considered: on-line reamed and 2-mm oversized hemispherical cups, and on-line reamed and 1-mm oversized dual-radius cups. Reamed surface topography was indexed by three-dimensional surfaces mathematically fitted to digitized castings. Component seating was indexed by the distribution of bone-cup contact and the thickness of the gap between the component backing and the bone. The reamed surfaces were better approximated by slightly blunt ellipsoids than by spheres, especially for the smaller acetabulae. Seating of the on-line hemispherical cups was best near the pole and poorest near the equator, whereas the converse was true for the 1-mm oversized dual-radius cups. The on-line dual-radius cups offered better overall compromise between polar and equatorial contact than did the 2-mm oversized hemispherical components.

Acetabulum↗
PubMed1995

A randomized trial of two schedules of trimetrexate versus 5-fluorouracil in advanced colorectal cancer: a Southwest Oncology Group study.

Trimetrexate (TMQ), a non-classical folate antagonist, was studied in a randomized controlled trial in patients with advanced colorectal cancer and without prior chemotherapy. Patients were randomly assigned to one of three treatments: TMQ at 200 mg/m2 i.v. q 2 weeks, TMQ at 12 mg/m2 i.v. daily x 5 or 5-fluorouracil (5-FU) at 15 mg/kg i.v. weekly. Overall response rates were: 6% (four partial responses in 71 patients, 95% CI of 2-14%) for q 2 week TMQ, 0% (zero of 29, 95% CI of 0-29%) for daily x 5 TMQ and 18% (two complete and nine partial responses in 62 patients, 95% CI of 9-30%) for 5-FU. Median survival estimates were 10.3 months for the q 2 week TMQ schedule, 8.7 months for the daily x 5 TMQ schedule and 13.6 months for the 5-FU schedule. Grade < or = 3 toxicities were significantly more common with TMQ. TMQ does not appear to have significant antitumor activity against colorectal cancer.

Aged↗