Specimen retrieval in laparoscopic cholecystectomy.
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Cholecystectomy, Laparoscopic↗
Biomedical subjects
J T Patton
Publications and source records attributed to J T Patton.
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↗
Endoscopic stenting for recurrent malignant gastric outlet obstruction.
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Aged↗
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↗
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↗
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↗
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↗
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↗
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↗
Structure and function of the rotavirus RNA-binding proteins.
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Amino Acid Sequence↗
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↗
The rotavirus RNA-binding protein NS35 (NSP2) forms 10S multimers and interacts with the viral RNA polymerase.
The gene 8 product of SA11 rotavirus, NS35 (NSP2), is a nonspecific RNA-binding protein that accumulates in cytoplasmic inclusions (viroplasms) and is required for genome replication. To gain additional information on the role of NS35 in virus replication, lysates of simian rotavirus SA11-infected cells were treated with the thio-cleavable crosslinking agent, dithiobis(succinimidyl propionate) (DSP). Gel electrophoresis of NS35-specific immunoprecipitates recovered from the crosslinked lysates indicated that infected cells contained NS35 multimers, the largest consisting of four or more molecules of the protein. Sedimentation analysis of NS35 expressed in rabbit reticulocyte lysates by cell-free translation and in vTF7-3-infected cells by transfection with a gene 8-containing transcription vector showed that NS35 assembles into multimers of approximately 10S and that the formation of the multimers does not require other viral proteins. The 10S multimers were also detected in rotavirus-infected cells, providing evidence that they function in virus replication. The lack of RNase sensitivity indicates that the 10S multimers probably lack an RNA component. However, by an NS35-specific RNA capture assay, the multimers were shown to possess the RNA-binding activity previously demonstrated for NS35. Despite its ability to multimerize and bind RNA, indirect immunofluorescence assays showed that when transiently expressed in cells, NS35 alone is not sufficient to induce the formation of viroplasms. DSP-crosslinking of infected cell lysates and immunoprecipitation also revealed that NS35 interacts with the putative viral RNA polymerase VP1. Analysis of cytoplasmic extracts resolved by sedimentation on glycerol gradients suggested that the VP1-NS35 complexes are soluble and RNA-free. Complexes formed from NS35 multimers, VP1, and viral messenger RNA may function to coordinate RNA packaging and the assembly of viral cores.
Cytoplasm↗
The carboxyl-half of the rotavirus nonstructural protein NS53 (NSP1) is not required for virus replication.
The rotavirus nonstructural protein NS53 (NSVP1), the product of genome segment 5, possesses RNA-binding activity and contains a highly conserved cysteine-rich motif located in the amino-terminal half of the protein. The genome of the bovine rotavirus variant, brvA, lacks a normal segment 5 but includes a novel dsRNA (gene A) of approximately 2600 basepairs (bp) that contains segment 5-specific sequences (F. Hundley, B. Biryahwaho, M. Gow, and U. Desselberger, Virology 143, 88-103, 1985). To gain information about the nature of the rearrangement in gene A and its capacity to encode a protein product, we prepared and sequenced complementary (c)DNA of the gene A RNA. The results showed that gene A is 2693 bp in size and contains a head-to-tail duplication of 1112 bp that originates from the open reading frame (ORF) of gene 5. The duplication begins at nucleotide (nt) 1454, which is 53 nt upstream from the end of the ORF for NS53. Gene A contains a point mutation at nt 808 which results in the presence of a nonsense codon near the middle of the ORF for NS53. Thus the predicted product of gene A is a truncated NS53 of 258 amino acids (aa) (31 kDa), approximately one-half the size of the authentic 491-aa NS53 (58 kDa). Examination of lysates from brvA-infected cells by Western blot assay using an NS53-specific antibody confirmed that the variant encodes only a truncated gene 5 product. Despite the truncation, analysis of the gene A product suggested that it, like full-length NS53, accumulated in association with the cytoskeleton of the infected cell, thus providing evidence that the subcellular localization signal in NS53 resides in the amino terminal half of the protein. Given that brvA is a viable, nondefective mutant, these results demonstrate that the carboxyl-terminal 233 aa of NS53 are not required for rotavirus replication in vitro.
Amino Acid Sequence↗
Temperature-sensitive lesions in the capsid proteins of the rotavirus mutants tsF and tsG that affect virion assembly.
The SA11 rotavirus mutants tsF and tsG contain temperature sensitive (ts) lesions in the capsid proteins VP2 and VP6, respectively, that interfere with their ability to assemble. To understand the nature of their lesions, full-length cDNAs of tsF gene 2 and tsG gene 6 were prepared from viral mRNA by reverse transcription and polymerase chain reaction. Comparative sequence analysis indicated that the ts phenotype of tsF VP2 is due to an Ala-->Asp substitution at position 387. The mutation falls outside of those regions of VP2 previously suggested to be of functional significance and therefore points to a previously unidentified site in VP2 that is important for the assembly of viral cores. Comparative sequence analysis showed that tsG VP6 contains two mutant amino acids, i.e., Thr-10 and His-13, and therefore one or both of these mutations are responsible for the ts phenotype of the mutant VP6. In the case of other group A and group C VP6 sequences, these residues are Ser and Asp, respectively. Characterization of tsG-infected cells by indirect immunofluorescence staining showed that while viroplasmic inclusions are formed at the nonpermissive temperature, the mutant VP6 accumulates in these structures only at the permissive temperature. While influencing intracellular accumulation, the Thr-10-->Ser and His-13-->Asp mutations in tsG VP6 are probably not directly involved in the interaction of VP6 with VP2, as VP6 deletion mutants lacking residues 10 and 13 retain the ability to bind VP2 in vitro. Analysis of VP6 failed to confirm previous reports that the protein was myristylated and thus excludes the possibility that this cotranslational modification is temperature-dependent for tsG VP6. Together, these data suggest that the amino terminus of VP6 plays an essential role in virus assembly in vivo, perhaps by being necessary for the movement of the protein to viroplasmic inclusions, the site of core and single-shelled particle formation.
Amino Acid Sequence↗
Deletion mapping of the rotavirus metalloprotein NS53 (NSP1): the conserved cysteine-rich region is essential for virus-specific RNA binding.
NS53 (NSP1), the gene 5 product of the group A rotaviruses, is a minor nonstructural protein of 486 to 495 amino acids which binds zinc and contains an amino-terminal highly conserved cysteine-rich region that may form one or two zinc fingers. To study the structure-function of the gene 5 product, wild-type and mutant forms of NS53 were produced by using a recombinant baculovirus expression system and a recombinant vaccinia virus/T7 (vTF7-3) expression system. Analysis of the RNA-binding activity of the wild-type NS53 immobilized onto protein A-Sepharose beads with NS53-specific antiserum showed that the protein exhibited specific affinity for all 11 rotavirus mRNAs. The use of short virus-specific RNA probes indicated that NS53 specifically recognizes an element located near the 5' ends of viral mRNAs. Analysis of the RNA-binding activity of deletion mutants of NS53 showed that the RNA-binding domain resides within the first 81 amino acids of the protein and that the highly conserved cysteine-rich region within this region of the protein is essential for the activity. Gel electrophoresis and Western immunoblot analyses of intracellular fractions derived from infected cells revealed that large amounts of NS53 were present in the cytosol and in association with the cytoskeletal matrix. Indirect immunofluorescence analysis of cells programmed to transiently express mutant forms of NS53 using vTF7-3 indicated that the intracellular localization domain resides between amino acids 84 and 176 of NS53. Together, these data show that the RNA-binding domain and the intracellular localization domain lie upstream from the region of NS53 previously determined not to be essential for replication of rotaviruses in cell culture (J. Hua and J. T. Patton, Virology 198:567-576, 1994).
Amino Acid Sequence↗
Computerized analysis of tumor cells flowing in a parallel plate chamber to determine their adhesion stabilization lag time.
The importance of cell adhesion in a variety of physiological phenomena requires development of an understanding of the factors and molecular mechanisms underlying these behaviors. Cell adhesion is a multistep process involving primary receptor-ligand interactions followed by secondary events that may lead to the formation of focal contacts. Due to the lack of well-defined assays to study adhesion stabilization, little is known about this process, except that it may involve signaling events, receptor recruitment, and, as we have demonstrated, covalent peptide cross-linking by cell membrane-associated transglutaminase [Menter et al.: Cell Biophys. 18:123-143, 1992). To study the stabilization process we have developed a dynamic assay employing a parallel plate flow chamber coupled with video microscopy and digital image processing. Our studies utilize wheat germ agglutinin-selected human metastatic melanoma cell variants that exhibit differences in their experimental metastatic potential and expression of transglutaminase. Using this assay, quantifying cell-substrate stabilization was found to be quick, reliable, reproducible, and useful in evaluating agents that block this process.
Aged↗
Nucleotide and amino acid sequence analysis of the rotavirus nonstructural RNA-binding protein NS35.
NS35, a basic protein encoded by gene 8 of SA11 rotavirus, possesses RNA-binding activity and is essential for genome replication. To identify conserved regions in the NS35 gene and its protein product, we determined the nucleotide sequences of the NS35 gene for the mammalian and avian rotaviruses Wa, DS1, SA11 (Patton and Ramig strains), NCDV, and Ty-1 and compared them and their deduced amino acid sequences to those reported for SA11 (Both strain), OSU, and UK. The results indicated that the NS35 genes of the mammalian rotaviruses are 1058-1059 bases in length and encode proteins of 317 amino acids that exhibit high levels of sequence conservation (> or = 83%). The NS35 gene of the turkey rotavirus Ty-1 differed from those of the mammalian rotaviruses with respect to size of the predicted protein (315 amino acids) and of the gene (1042 bases). NS35 of Ty-1 exhibited a relatively low degree of amino acid homology (52-57%) with NS35 of the mammalian viruses. Phylogenetic analysis of the NS35 gene indicated that avian (TY-1) and mammalian rotaviruses are distantly related. Comparison of the predicted sequences of NS35 showed that all possessed a conserved basic domain of 37 amino acids at residues 205-241 that may serve as the RNA-binding domain. Electrophoretic examination showed that NS35 contains a disulfide bond probably located in the amino-terminal half of the protein. Comparison of NS35 genes at the nucleotide level revealed two regions of extensive conservation, (i) a 75-base (b) sequence that includes the 35-base 5'-noncoding region and the first 30 bases of the open reading frame for NS35, and (ii) a 28-b sequence in the 3'-noncoding region of the gene. Secondary structure predictions for the NS35 mRNA suggest that the 75-base sequence can fold to produce a stem double-loop structure. Such a structure may serve as a packaging signal for the assortment of NS35 mRNA into replicase particles.
Amino Acid Sequence↗