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J T Patton

Publications and source records attributed to J T Patton.

At least 37 records · Page 2Linked to original sources
PubMed2000

Late fracture of the hip after reamed intramedullary nailing of the femur.

In a consecutive series of 498 patients with 528 fractures of the femur treated by conventional interlocking intramedullary nailing, 14 fractures of the femoral neck (2.7%) occurred in 13 patients. The fracture of the hip was not apparent either before operation or on the immediate postoperative radiographs. It was diagnosed in the first two weeks after operation in three patients and after three months in the remainder. Age over 60 years at the time of the femoral fracture and female gender were significantly predictive of hip fracture on bivariate logistic regression analysis, but on multivariate analysis only the location of the original fracture in the proximal third of the femur (p = 0.0022, odds ratio = 6.96, 95% CI 2.01 to 24.14), low-energy transfer (p = 0.0264, odds ratio = 15.56, 95% CI 1.38 to 75.48) and the severity of osteopenia on radiographs (p = 0.0128, odds ratio = 7.55, 95% CI 1.54 to 37.07) were significant independent predictors of later fracture. Five of the 19 women aged over 60 years, who sustained an osteoporotic proximal diaphyseal fracture of the femur during a simple fall, subsequently developed a fracture of the neck. Eleven of the hip fractures were displaced and intracapsular and, in view of the advanced age of most of these patients, were usually treated by replacement arthroplasty. Reduction and internal fixation was used to treat the remaining three intertrochanteric fractures. Three patients developed complications requiring further surgery; five died within two years of their fracture.

Adolescent↗
PubMed1999

Rotavirus open cores catalyze 5'-capping and methylation of exogenous RNA: evidence that VP3 is a methyltransferase.

Rotavirus open cores prepared from purified virions consist of three proteins: the RNA-dependent RNA polymerase, VP1; the core shell protein, VP2; and the guanylyltransferase, VP3. In addition to RNA polymerase activity, open cores have been shown to contain a nonspecific guanylyltransferase activity that caps viral and nonviral RNAs in vitro. In this study, we examined the structure of RNA caps made by open cores and have analyzed open cores for other capping-related enzymatic activities. Utilizing RNase digestion and thin-layer chromatography, we found that the majority ( approximately 70%) of caps made by open cores contain the tetraphosphate linkage, GppppG, rather than the triphosphate linkage, GpppG, found on mRNAs made by rotavirus double-layered particles. Enzymatic analysis indicated that the GppppG caps resulted from the lack of a functional RNA 5'-triphosphatase in open cores, to remove the gamma-phosphate from the RNA prior to capping. RNA 5'-triphosphatases commonly exhibit an associated nucleoside triphosphatase activity, and this too was not detected in open cores. Caps of some RNAs contained an extra GMP moiety (underlined) and had the structure 3'-GpGp(p)ppGpGpC-RNA-3'. The origin of the extra GMP is not known but may reflect the cap serving as a primer for RNA synthesis. Methylated caps were produced in the presence of the substrate, S-adenosyl-l-methionine (SAM), indicating that open cores contain methyltransferase activity. UV cross-linking showed that VP3 specifically binds SAM. Combined with the results of earlier studies, our results suggest that the viral guanylyltransferase and methyltransferase are both components of VP3 and, therefore, that VP3 is a multifunctional capping enzyme.

Acid Anhydride Hydrolases↗
PubMed1999

Synthesis and biological evaluation of a potent E-selectin antagonist.

An early step of the inflammatory response-the rolling of leukocytes on activated endothelial cells-is mediated by selectin/carbohydrate interactions. The tetrasaccharide sialyl Lewis(x) (sLe(x)) 1 is a ligand for E-, P-, and L-selectin and, therefore, serves as a lead structure to develop analogues which allow the control of acute and chronic inflammation. Here we describe the efficient synthesis (10 linear steps) of the potent sLe(x) mimetic 2. Compared to sLe(x), compound 2 showed a 30-fold improved affinity in a static, cell-free E-selectin-ligand binding assay (IC(50) = 36 microM). These data were confirmed by a marked inhibition in an in vitro cell-cell rolling assay which simulates in vivo conditions (IC(50) approximately 40 microM). The assays are predictive for the in vivo efficacy of test compounds as indicated by a marked inhibitory effect of 2 in a thioglycollate induced peritonitis model of acute inflammation in mice (ED(50) approximately 15 mg/kg).

Acute Disease↗
PubMed1999

Open reading frame in rotavirus mRNA specifically promotes synthesis of double-stranded RNA: template size also affects replication efficiency.

The 11 rotavirus mRNAs are capped, but not polyadenylated, have a high AU content, and serve as templates for the synthesis of double-stranded (ds)RNA. Earlier studies using a cell-free replication system showed that the 5'- and 3'-untranslated regions (UTRs) of the mRNAs have cis-acting signals that promote minus-strand synthesis. To identify additional factors that affect RNA replication, chimeric RNAs were made that consisted of portions of the gene 8 mRNA of SA11 rotavirus and of the gene for green fluorescent protein (gfp) or for the N protein of respiratory syncytial virus. Analysis of the chimeras in the cell-free replication system under noncompetitive conditions showed that the open reading frame (ORF) of viral mRNAs contains information that specifically promotes minus-strand synthesis. Results were also obtained indicating that a high AU content may increase the replication efficiency of RNAs and that, in general, an inverse correlation exists between replication efficiency and the length of the RNA template. Replication assays performed under competitive conditions showed that nonviral RNAs can interfere significantly with the replication of viral mRNAs, mostly likely by sequestering nonspecific RNA-binding proteins that are of limited concentration in the replication system and that are essential for dsRNA synthesis. In summary, rotavirus dsRNA synthesis is affected by many factors including cis-acting replication signals located in the 5'-UTR, 3'-UTR, and ORF of the mRNA as well as the size and possibly the AU content of the mRNA.

5' Untranslated Regions↗
PubMed1999

Multimers formed by the rotavirus nonstructural protein NSP2 bind to RNA and have nucleoside triphosphatase activity.

The nonstructural protein NSP2 is a component of rotavirus replication intermediates and accumulates in cytoplasmic inclusions (viroplasms), sites of genome RNA replication and the assembly of subviral particles. To better understand the structure and function of the protein, C-terminally His-tagged NSP2 was expressed in bacteria and purified to homogeneity. In its purified form, the protein did not exist as a monomer but rather was present as an 8S-10S homomultimer consisting of 6 +/- 2 subunits of recombinant NSP2 (rNSP2). As shown by gel mobility shift assays, the rNSP2 multimers bound to RNA in discrete cooperative steps to form higher-order RNA-protein complexes. The RNA-binding activity of the rNSP2 multimers was determined to be nonspecific and to have a strong preference for single-stranded RNA over double-stranded RNA, for which it displayed little affinity. Enzymatic analysis revealed that rNSP2 possessed an associated nucleoside triphosphatase (NTPase) activity in vitro, which in the presence of Mg(2+) catalyzed the hydrolysis of each of the four NTPs to NDPs with equal efficiency. Evidence indicating that the hydrolysis of NTP resulted in the covalent linkage of the gamma-phosphate to rNSP2 was obtained. Additional experiments showed that NSP2 expressed transiently in MA014 cells is phosphorylated. We propose that NSP2 functions as a molecular motor, catalyzing the packaging of viral mRNA into core-like replication intermediates through the energy derived from its NTPase activity.

Acid Anhydride Hydrolases↗
PubMed1999

RNA-binding and capping activities of proteins in rotavirus open cores.

Guanylyltransferases are members of the nucleotidyltransferase family and function in mRNA capping by transferring GMP to the phosphate end of nascent RNAs. Although numerous guanylyltransferases have been identified, studies which define the nature of the interaction between the capping enzymes of any origin and their RNA substrates have been limited. Here, we have characterized the RNA-binding activity of VP3, a minor protein component of the core of rotavirions that has been proposed to function as the viral guanylyltransferase and to direct the capping of the 11 transcripts synthesized from the segmented double-stranded RNA (dsRNA) genome of these viruses. Gel shift analysis performed with disrupted (open) virion-derived cores and virus-specific RNA probes showed that VP3 has affinity for single-stranded RNA (ssRNA) but not for dsRNA. While the ssRNA-binding activity of VP3 was found to be sequence independent, the protein does exhibit preferential affinity for uncapped over capped RNA. Like the RNA-binding activity, RNA capping assays performed with open cores indicates that the guanylyltransferase activity of VP3 is nonspecific and is able to cap RNAs initiating with a G or an A residue. These data establish that all three rotavirus core proteins, VP1, the RNA polymerase; VP2, the core capsid protein; and VP3, the guanylyltransferase, have affinity for RNA but that only in the case of the RNA polymerase is the affinity sequence specific.

Animals↗
PubMed1998

Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis.

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5' untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3' tail and the 5' stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5' stem-loop, mutations which caused complete or near-complete complementarity between the 5' end and the 3' tail significantly inhibited (>/=10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3' tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5' and 3' termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3' end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.

Base Sequence↗
PubMed1997

Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.

Animals↗
PubMed1996

The carbohydrate-recognition domain of E-selectin is sufficient for ligand binding under both static and flow conditions.

Selectins are a family of adhesion molecules with a well-defined domain structure comprised of a lectin or carbohydrate-recognition domain (CRD), an epidermal growth factor (EGF)-like motif, and a variable number of consensus repeats (CRs). While it is clear from various lines of evidence that the CRD plays a pivotal role in selectin-ligand interactions, little is known about the role of the non-lectin selectin domains. We expressed a series of soluble chimeric proteins with various domains switched between E- and L-selectin and measured binding of the resulting chimeras to sialyl Lewis(a) and sulfatide, two carbohydrate structures which are specific for the E- and L-CRDs, respectively. Both CRDs bind to their respective ligands with the same affinity regardless of the origin of the other domains they are attached to. The domain-switched chimeras were assayed for their ability to support static binding and rolling of various cell lines which bind specifically to E-selectin. In these assays, the E-CRD was indispensable for both static binding and rolling under physiological flow conditions. The E-CRD alone, when substituted into L-selectin, supported rolling without the requirement for additional ligand-recognition elements. We conclude that the EGF domain or the CRs of E- and L-selectin have no influence on the CRD's specificity to carbohydrates. Furthermore, at least in the case of E-selectin, they do not contribute to the specificity of binding to cell surface ligands.

Animals↗
PubMed1996

Identification of the minimal replicase and the minimal promoter of (-)-strand synthesis, functional in rotavirus RNA replication in vitro.

An in vitro replication system supporting the initiation and synthesis of complete rotavirus (-)-strands on (+)-strand template RNA (Chen et al., J Virol 68: 7030, 1994) was used to examine several parameters related to rotavirus RNA replication. Coexpression of VP1/2/3 in all possible combinations from baculovirus vectors revealed: [i] Virus-like particles (VLPs) were formed only if VP2 was present, and [ii] VP1/2 and VP1/2/3 VLPs had replicase activity in the in vitro system whereas VP2/3 and VP2 VLPs did not. Thus, the minimal replicase is composed of VP1 and VP2 and replicase activity is associated with VP1. In vitro replication reactions, using T7 transcripts of porcine rotavirus OSU genome segment 9 as reporter template, were performed to map cis-acting elements that regulate replication. Internal deletions and terminal truncations of the reporter RNA localized a replication signal, conferring full template activity, to the 5'-terminal 27 nucleotides (nt 1-27) and the 3'-terminal 26 nucleotides (nt 1037-1062). Further analysis showed that a minimal promoter of (-)-strand synthesis was contained in the 3'-terminal 7 nucleotides (nt 1056-1062); the sequence conserved at the 3'-terminus of all rotavirus genes. Hybrid constructs with this promoter had minimal, but detectable, template activity. This result indicated that upstream sequences between nucleotides 1037-1055 positively regulate the activity of the minimal promoter.

Animals↗
PubMed1996

The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis.

We used an in vitro template-dependent replicase assay (D. Chen, C. Zeng, M. Wentz, M. Gorziglia, M. Estes, and R. Ramig. J. Virol. 68:7030-7039, 1994) to identify the cis-acting signals required for replication of a genome segment 9 template from the group A rotavirus strain OSU. The replicase phenotypes for a panel of templates with internal deletions or 3'-terminal truncations indicated that no essential replication signals were present within the open reading frame and that key elements were present in the 5' and 3' noncoding regions. Chimeric constructs containing portions of viral sequence ligated to a nonviral backbone were generated to further map the regions required for in vitro replication of segment 9. The data from these constructs showed that the 3'-terminal seven nucleotides of the segment 9 mRNA provided the minimum requirement for replication (minimal promoter). Analysis of additional chimeric templates demonstrated that sequences capable of enhancing replication from the minimal promoter were located immediately upstream of the minimal promoter and at the extreme 5' terminus of the template. Mutational analysis of the minimal promoter revealed that the 3'-terminal -CC residues are required for efficient replication. Comparison of the replication levels for templates with guanosines and uridines at nucleotides -4 to -6 from the 3' terminus compared with levels for templates containing neither of these residues at these positions indicated that either or both residues must be present in this region for efficient replication in vitro.

Animals↗
PubMed1996

Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis.

Recent studies have shown that disrupted (open) rotavirus cores have an associated replicase activity which supports the synthesis of dsRNA from viral mRNA in a cell-free system (D. Chen, C. Q.-Y. Zeng, M. J. Wentz, M. Gorziglia, M. K. Estes, and R. F. Ramig, J. Virol. 68:7030-7039, 1994). To determine which of the core proteins, VP1, VP2, or VP3, recognizes the template mRNA during RNA replication, SA11 open cores were incubated with 32P-labeled RNA probes of viral and nonviral origin and the reaction mixtures were analyzed for the formation of RNA-protein complexes by gel mobility shift assay. In mixtures containing a probe representing the 3' end of SA11 gene 8 mRNA, two closely migrating RNA-protein complexes, designated s and f, were detected. The interaction between the RNA and protein of the s and f complexes was shown to be specific by competitive binding assay with tRNA and brome mosaic virus RNA. By electrophoretic analysis of RNA-protein complexes recovered from gels, VP1 was shown to be the only viral protein component of the complexes, thereby indicating that VP1 specifically recognizes the 3' end of gene 8 mRNA. Analysis of VP1 purified from open cores by glycerol gradient centrifugation verified that VP1 recognizes the 3' end of viral mRNA but also showed that in the absence of other viral proteins, VP1 lacks replicase activity. When reconstituted with VP2-rich portions of the gradient, VP1 stimulated levels of replicase activity severalfold. These data indicate that VP1 can bind to viral mRNA in the absence of any other viral proteins and suggest that VP2 must interact with the RNA-protein complex before VP1 gains replicase activity.

Cell Line↗
PubMed1996

cis-Acting signals that promote genome replication in rotavirus mRNA.

A previous study has shown that rotavirus cores have an associated replicase activity which can direct the synthesis of double-stranded RNA from viral mRNA in a cell-free system (D. Y. Chen, C. Q.-Y. Zeng, M. J. Wentz, M. Gorziglia, M. K. Estes, and R. F. Ramig, J. Virol. 68:7030-7039, 1994). To define the cis-acting signals in rotavirus mRNA that are important for RNA replication, gene 8 transcripts which contained internal and terminal deletions and chimeric transcripts which linked gene 8-specific 3'-terminal sequences to the ends of nonviral sequences were generated. Analysis of these RNAs in the cell-free system led to the identification of a cis-acting signal in the gene 8 mRNA which is essential for RNA replication and two cis-acting signals which, while not essential for replication, serve to enhance the process. The sequence of the essential replication signal is located at the extreme 3' end of the gene 8 mRNA and, because of its highly conserved nature, is probably a common feature of all 11 viral mRNAs. By site-specific mutagenesis of the gene 8 mRNA, residues at positions -1, -2, -5, -6, and -7 of the 3' essential signal were found to be particularly important for promoting RNA replication. One of the cis-acting signals shown to enhance the replication in the cell-free system was located near the 5' end of the 3' untranslated region (UTR) of the gene 8 mRNA, while remarkably the other was located in the 5' UTR of the message. The existence of an enhancement signal in the 5' UTR raises the possibility that the 5' and 3' ends of the rotavirus mRNA may interact with each other and/or with the viral replicase during genome replication.

Base Sequence↗
PubMed1995

Human melanoma integrins contribute to arrest and stabilization potential while flowing over extracellular matrix.

To form distant metastases, tumour cells must stabilize adhesive interactions that prevent detachment at secondary sites. Primary receptor-ligand interactions alone may not maintain prolonged adhesive contacts without secondary events that lead to adhesion stabilization. Computerized imaging methods enable us to examine various substrates for: (i) the wall shear adhesion threshold (WSAT), a measure of the dynamic adhesive potential of tumour cells; (ii) the number of tumour cells that adhered; and (iii) the adhesion stabilization lag time (ASLT) or length of time required for tumour cells to stabilize adhesive contacts capable of withstanding high wall shear force (up to 100 dynes/cm2). The relative WSAT ratios found were: wheat germ agglutinin (WGA) > laminin > fibronectin > vitronectin > collagen I > collagen IV > von Willebrand factor (vWF) (the greater the shear rate the higher the adhesive potential). The relative stabilization ratios found were as follows: laminin < fibronectin < vitronectin < collagen IV < collagen I < vWF < WGA (shorter times correlate with greater stabilization potential). Stabilization data using fibronectin as a substrate correlated the best with metastatic potential. Using three melanoma lines of different metastatic potential semiquantitative reverse transcriptase-polymerase chain reaction (PCR) showed a two- to four-fold increase in alpha1, alpha3, alpha4, alpha5, alpha6, and ICAM-1 in the highly metastatic 70W cells compared to the MeWo and non-metastatic 3S5 melanoma cells. There were no differences in alphav, beta1 and beta3 levels among the three melanoma lines, and PCR products for alphaIIb, alpha2, CD36, or ICAM-2 were not detected. The 70W cells also had higher levels of alphax and beta2 (CD11/CD18 and p150 leukocyte antigen) than either the MeWo or 3S5 cells. The data indicate that melanoma cells exhibit differences in the adhesion properties under fluid shear and differences in the expression of adhesion components that correlate with their metastatic potential.

Base Sequence↗
PubMed1995

The timing of ossification of the limb bones, and growth rates of various long bones of the fore and hind limbs of the prenatal and early postnatal laboratory mouse.

In order to study the pattern of ossification of the skeletal components of the fore and hind limb of the mouse, intact embryos were isolated between days (d) 15 and 19 of pregnancy (the morning of finding a vaginal plug is termed d 1 of pregnancy), and postnatal animals isolated on d 1 (newborns), 7 and 14 after birth. The total number of fore and hind limbs studied for each day of pregnancy or postnatal day for the bone growth study is given in parentheses: d 15 (2), d 16, 17, 18 and 19 of pregnancy (5 specimens for each of these days), d 1 (newborn), wk 1 and 2, postnatal (4 specimens analysed at each of these times), since only the right limbs were studied. For the study involving the time of first appearance of ossification centres, either the right or the left limb of each of these prenatal and postnatal specimens was analysed. All specimens were fixed in 80% ethanol, bulk-stained using alizarin and Alcian blue, in order to stain ossification centres and cartilage, respectively, and cleared. The limbs were then disarticulated from the axial skeleton at the sternoclavicular and sacroiliac joints to facilitate (1) the determination of the sequential pattern of ossification in the various cartilage primordia analysed, and (2) the analysis of the pattern of growth of the humerus, ulna, femur and tibia. The latter values were plotted graphically, and the individual growth rate of each of the long bones studied was then deduced and also plotted graphically. The findings demonstrated that, with the exception of the femur and ulna, all of the long bones studied had significantly different growth patterns. The time of appearance of the various centres of ossification in the skeletal elements studied proceeded in a similar order to that described by previous authors, though there was some discrepancy in the exact time of first appearance of certain ossification centres. Of particular interest was the somewhat unusual pattern of ossification of the first digits of both the fore and hind limb compared with that of the other digits. The data presented here provide useful baseline information on the normal sequential pattern of ossification in the fore and hind limb, and the characteristic growth pattern of the individual long bones of the limbs in this species.

Animals↗