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R C Bates

Publications and source records attributed to R C Bates.

At least 19 recordsLinked to original sources
PubMed1992

Adhesion to thrombospondin by human embryonic fibroblasts is mediated by multiple receptors and includes a role for glycoprotein 88 (CD36).

Fetal embryonic fibroblasts attach and spread on thrombospondin (TSP). Adhesion is tight and focal adhesion plaques and "spots" are formed. We have investigated the receptors responsible for this adhesion. Unstimulated cells express the vitronectin receptor on their surface and this beta 3 integrin molecule contributes to adhesion. Another putative receptor for TSP, termed glycoprotein (GP) 88, which exists as a cytoplasmic pool in unstimulated cells becomes surface expressed when these cells are plated on TSP and localizes to areas of cell adhesion. Western blot analysis of cell lysate confirms GP88 as a TSP binding protein. Studies with fucoidan indicate that the heparan sulfate proteoglycan, known to function as a receptor for TSP, appears to contribute substantially to the TSP attachment of these cells and may be the receptor most important in the initial phases of TSP interaction.

Antibodies, Monoclonal↗
PubMed1991

Individual embryonic fibroblasts express multiple beta chains in association with the alpha v integrin subunit. Loss of beta 3 expression with cell confluence.

The alpha chain of the vitronectin receptor, alpha v, has been found in association with the integrin subunits beta 1, beta 3, or beta 5 on different cell types. We show here that cultured embryonic fibroblasts simultaneously display alpha v beta 3, alpha v beta 1, and alpha v in association with two other beta subunits, one of which is probably beta 5. Polymerase chain reaction analysis of single cells isolated by micromanipulation identified mRNA for alpha v, beta 1, beta 3, and beta 5 in six of eight clones. Immunoprecipitation of iodinated cell surface proteins with a monoclonal antibody to alpha v indicated that the relative proportions of the different beta chains in association with alpha v varied, particularly between two different cell lines. The cytokines platelet-derived growth factor, transforming growth factor beta 1, and tumor necrosis factor alpha did not appear to alter this ratio although tumor necrosis factor alpha increased the surface expression of the alpha v-associated integrins; but overnight culture in basic fibroblast growth factor caused a lower expression of alpha v beta 1 and alpha v beta 5 with no reduction in alpha v beta 3 expression. When the cell cultures were grown to complete confluence, surface expression of beta 3 was abolished, and the expression of an unknown beta chain (beta u) became more prominent. This effect was not overcome by culturing confluent cells with basic fibroblast growth factor. Affinity column chromatography showed that alpha v beta 5 bound to vitronectin but alpha v beta 1 did not, whereas alpha v beta 1 but not alpha v beta 5 bound to fibronectin. These results suggest that, on individual cells, the beta subunits found in association with alpha v may vary according to the proliferative capacity of the cell and that the promiscuous beta 3 subunit is progressively replaced by beta subunits of individual ligand specificity.

Base Sequence↗
PubMed1991

Arg-Gly-Asp-containing peptides expose novel collagen receptors on fibroblasts: implications for wound healing.

Integrins are a family of cell-surface receptors intimately involved in the interactions of cells with their extracellular matrix. These receptors comprise an alpha and beta subunit in noncovalent association and many have been shown to recognize and bind an arginine-glycine-aspartate (RGD) sequence contained within their specific extracellular matrix ligand. Fibroblasts express integrin receptors belonging to two major subfamilies. Some of the members within the subfamily defined by beta 1 (VLA) are receptors for collagen but, perhaps surprisingly, the other major subfamily of integrins on fibroblasts--that defined by the alpha chain of the vitronectin receptor, alpha v--all appear to bind primarily vitronectin and/or fibronectin. In the present study we show that RGD-containing peptides expose cryptic binding sites on the alpha v-associated integrins enabling them to function as collagen receptors. The addition of RGD-containing peptides to fibroblasts cultured on type I collagen induced dramatic cell elongation and, when the cells were contained within collagen matrices, the peptides induced marked contraction of the gels. These processes were inhibited by Fab fragments of a monoclonal antibody against an alpha v integrin. Also, alpha v-associated integrins from cell lysates bound to collagen I affinity columns in the presence, but not in the absence, of RGD-containing peptides. These data suggest a novel regulatory control for integrin function. In addition, because the cryptic collagen receptors were shown to be implicated in the contraction of collagen gels, the generation of such binding forces suggests that this may be the major biological role for these integrins in processes such as wound healing.

Amino Acid Sequence↗
PubMed1990

Analysis of the kinetic hairpin transfer model for parvoviral DNA replication.

All linear DNA molecules face special problems in replicating their 5' ends, as DNA polymerases add nucleotides only to pre-existing strands with free 3'-OH groups. Parvoviruses, a group of small animal viruses with a linear single-stranded DNA genome, cope with this problem by having palindromic terminal sequences that can fold back on themselves to form hairpin structures essential in priming DNA replication. The 3' terminal sequence that initiates replication becomes reversed in orientation during the process, and if the palindrome is imperfect, two different, reverse-complementary terminal sequences are generated. The relative abundances of the terminal sequence orientations at each end of the DNA molecules can be measured and give information about the replication process. From such clues, we developed a "kinetic hairpin transfer model" based on differential rates of hairpin formation and inversion processes depending on the conformations of the 3' termini. Numerical studies showed that this simple idea can account for the diverse pattern of DNA distributions observed in the family Parvoviridae. In this paper, we simplify the model to a set of coupled linear first-order ordinary differential equations in order to delineate its essential properties by Perron-Frobenius theory. Secondly, we examine our assumption of linear kinetics by modeling enzyme catalysis of the component steps of the hairpin transfer process. We show that the rate-determining step of the process is the binding of initiation complex to the self-priming hairpin structures. Furthermore, we find that if the replication machinery is saturated by DNA substrate late in an infection, the differential equations become non-linear but the steady-state DNA distribution is still given by the solution of our original linear equations.

Computer Simulation↗
PubMed1990

Interaction of virally coded protein and a cell cycle-regulated cellular protein with the bovine parvovirus left terminus ori.

Replication of parvoviruses requires cis signals located in terminal palindromes that function as origins of replication in conjunction with trans-acting viral and cellular proteins. A gel retardation assay was used to identify proteins in crude nuclear extracts of bovine parvovirus (BPV)-infected bovine fetal lung cells that interact with the hairpinned left end (3' OH terminus of the viral minus strand in the flop conformation) of BPV. Three specific DNA-protein complexes formed. One complex was shown to involve a BPV structural protein(s) by inhibiting its formation when antiserum specific for these BPV proteins was used. By specific competition with serum containing antibodies against the BPV nonstructural proteins, a second complex was shown to involve a BPV nonstructural protein. A third complex contained protein of cellular origin and was also formed with extracts of uninfected bovine fetal lung cells. DNA competition assays suggest that the viral proteins do not bind to the right hairpin, which differs in sequence and secondary structure from the left terminus, or to a BPV terminus that lacks the first 52 nucleotides, preventing formation of the stem of the hairpin. The cellular protein is regulated in a cell cycle-dependent fashion, with its binding activity increased in uninfected, actively dividing cells compared with contact-inhibited cells. Since autonomous parvovirus replication requires an S-phase factor for progeny formation, the terminal binding protein demonstrated here is a candidate for this factor.

Animals↗
PubMed1989

A kinetic hairpin transfer model for parvoviral DNA replication.

The DNAs encapsidated by parvoviruses show distinctly different patterns with respect to the ratio of plus-to-minus strands and sequence heterogeneity at the ends. A kinetic model, based on differential rates of hairpin transfer at 3' termini, is described and shown to account for all known parvoviral DNA distributions.

Base Sequence↗
PubMed1989

Identical ends are not required for the equal encapsidation of plus- and minus-strand parvovirus LuIII DNA.

Sequence analyses of the left and right termini of LuIII virus show they are nonidentical imperfect palindromes of 122 and 211 nucleotides, respectively. The left terminus of the minus strand of LuIII DNA, uniquely in the flip conformation, can assume a T-shaped structure. The right terminus of the minus strand of LuIII DNA can assume a U-shaped structure, and it exists in either the flip or flop conformation. The termini of LuIII shared a high degree of sequence homology and showed conserved secondary structure with those of the rodent parvoviruses MVMp and H-1. LuIII, like adeno-associated virus, encapsidates equal amounts of plus- and minus-strand DNA. However, the sequence data for LuIII virus demonstrate that identical termini are not required for this encapsidation pattern.

Base Sequence↗
PubMed1989

Natural parvovirus infection in laboratory rabbits.

Laboratory rabbits from various commercial and private sources were found to have high serum antibody titers specific for lapine parvovirus (LPV). By both immunofluorescence and hemagglutination inhibition assays, 75% of these sera were positive for LPV. This finding, together with the recovery of LPV from kidneys of neonatal rabbits, suggested that LPV infection is common in commercially available rabbits in the United States. It was concluded that use of infected rabbits could interfere with research in which rabbit cell cultures or in vitro immunologic assays are used.

Animals↗
PubMed1988

Analysis of the termini of the DNA of bovine parvovirus: demonstration of sequence inversion at the left terminus and its implication for the replication model.

The distribution of terminal-sequence orientations in the viral DNA of bovine parvovirus (BPV), an autonomous parvovirus, was studied by end labeling and restriction enzyme digestion and also by cloning. The left (3') end of the minus strand of BPV was found in two alternative sequence orientations (designated as flip and flop, which are reverse complements of each other), with a 10-fold excess of flip. This is in contrast to the autonomous rodent parvoviruses which encapsidate minus-strand DNA with only the flip orientation at this end. The right (5') end of the minus strand of BPV contained both sequence orientations with equal frequencies, as in the rodent parvoviruses. Sequence inversions were also detected at both ends of the plus strand, which makes up about 10% of the encapsidated BPV DNA. Each terminus of BPV DNA had a characteristic ratio of flip to flop forms, and this ratio was restored in the progeny DNA resulting from transfection with genomic clones of different defined terminal conformations. Replicative-form DNA showed the same distribution of terminal-sequence orientations as the reannealed plus and minus virion DNAs, suggesting that the distribution of flip and flop forms observed in virion DNA is not due to selective encapsidation, but rather to the specific distribution of replicative forms. The current replication model for autonomous parvoviruses, which was based on the available data for the rodent parvoviruses, cannot account for the observed distribution of BPV DNA. An alternative model is suggested.

Animals↗
PubMed1988

Genomic clones of bovine parvovirus: construction and effect of deletions and terminal sequence inversions on infectivity.

Genomic clones of the autonomous parvovirus bovine parvovirus (BPV) were constructed by blunt-end ligation of reannealed virion plus and minus DNA strands into the plasmid pUC8. These clones were stable during propagation in Escherichia coli JM107. All clones tested were found to be infectious by the criteria of plaque titer and progressive cytopathic effect after transfection into bovine fetal lung cells. Sequencing of the recombinant plasmids demonstrated that all of the BPV inserts had left-end (3')-terminal deletions of up to 34 bases. DNA isolated from progeny virions arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogenous, cloned DNA. Full-length genomic clones with 3' flip and 3' flop conformations were constructed and were found to have equal infectivity. Analysis of low-molecular-weight DNA isolated from lysates of cells transfected with these clones demonstrated that rescue and replication of BPV DNA could be detected 3 to 8 days after transfection. Expression of capsid proteins from transfected genomes was demonstrated by hemagglutination, indirect immunofluorescence, and immunoprecipitation of [35S]methionine-labeled cell lysates. Use of appropriate antiserum for immunoprecipitation showed the synthesis of BPV capsid and noncapsid proteins after transfection. Independently, a series of genomic clones with increasingly larger 3'-terminal deletions was prepared from separately subcloned 3'-terminal fragments. Transfection of these clones into bovine fetal lung cells revealed that deletions of up to 34 bases at the 3' end lowered but did not abolish infectivity, while deletions of greater than 52 bases were lethal. End-label analysis showed that the 34-base deletion was repaired to wild-type length in the progeny virus.

Amino Acid Sequence↗
PubMed1987

Bovine parvovirus DNA-binding proteins: identification by a combined DNA hybridization and immunodetection assay.

We have investigated the interaction between bovine parvovirus (BPV) capsid and non-capsid proteins and restriction fragments of the BPV genome by a combined DNA hybridization and immunodetection assay. 32P-labelled DNA was bound to nitrocellulose membranes bearing lysates of mock-infected and virus-infected cells whose proteins had been separated by SDS-polyacrylamide gel electrophoresis. The position of bound DNA was determined by autoradiography. The proteins on the membrane were still accessible to specific antibodies, allowing confirmation of the DNA-binding species by an immunodetection reaction. In 0.2 M-NaCl, BPV capsid proteins VP2 (72,000 daltons) and VP3 (62,000 daltons) bound the 0 to 16 map unit EcoRI fragment of BPV DNA which contained label in either the minus or plus strand. At higher salt concentration (0.5 M), only VP2 still bound DNA. Within this fragment, the capsid protein binding was restricted to those nucleotides between map units 0 and 4. No binding to capsid proteins was seen with the fragment spanning the middle of the genome and minor binding to VP3 was seen with the 5' end. Binding to the BPV non-capsid protein NP-1 was observed with the 0 to 16 map unit fragment when label was in the virion strand and to other possibly BPV-coded proteins when label was in the plus strand. The NP-1 binding was localized to map units 4 to 16. We did not detect binding to the BPV homologue(s) of the autonomous parvovirus non-capsid protein NS1, due in part to its low concentration in the cell lysates used. Points of the parvovirus replication cycle at which DNA-binding proteins may serve controlling functions are discussed.

Animals↗
PubMed1987

Detection of bovine parvovirus proteins homologous to the nonstructural NS-1 proteins of other autonomous parvoviruses.

Two nonstructural proteins of bovine parvovirus (BPV) with apparent molecular sizes of 75,000 and 83,000 daltons have been detected. The proteins were immunoprecipitated from lung cells infected with various isolates of BPV and from in vitro translations of infected cell mRNA. These proteins were expressed as nuclear phosphoproteins and were synthesized early in infection, before the peak of capsid protein synthesis. Early in infection, the 75-kilodalton-size species could be resolved into two bands of equal intensity, but later in infection, the lower-molecular-size form predominated. Antibodies directed against bacterial fusion proteins encoding amino acid sequences from a highly conserved region of the NS-1 polypeptides of two other parvoviruses, minute virus of mice and the human virus B19, gave specific nuclear fluorescence with BPV-infected cells, although the antibodies failed to immunoprecipitate any viral proteins. The noncapsid proteins appear to be homologous to the previously characterized NS-1 proteins of other autonomous parvoviruses.

Animals↗
PubMed1986

Possible sequences for nuclear accumulation of parvoviral proteins.

Parvoviral genomes have been searched for sequences which may code for the nuclear transport of viral proteins. Sequences similar to those which regulate the nuclear transport of T antigen and yeast mating type protein were detected within the sequences coding for capsid and non-capsid proteins.

Amino Acid Sequence↗
PubMed1986

Complete nucleotide sequence and genome organization of bovine parvovirus.

We determined the complete nucleotide sequence of bovine parvovirus (BPV), an autonomous parvovirus. The sequence is 5,491 nucleotides long. The terminal regions contain nonidentical imperfect palindromic sequences of 150 and 121 nucleotides. In the plus strand, there are three large open reading frames (left ORF, mid ORF, and right ORF) with coding capacities of 729, 255, and 685 amino acids, respectively. As with all parvoviruses studied to date, the left ORF of BPV codes for the nonstructural protein NS-1 and the right ORF codes for the major parts of the three capsid proteins. The mid ORF probably encodes the major part of the nonstructural protein NP-1. There are promoterlike sequences at map units 4.5, 12.8, and 38.7 and polyadenylation signals at map units 61.6, 64.6, and 98.5. BPV has little DNA homology with the defective parvovirus AAV, with the human autonomous parvovirus B19, or with the other autonomous parvoviruses sequenced (canine parvovirus, feline panleukopenia virus, H-1, and minute virus of mice). Even though the overall DNA homology of BPV with other parvoviruses is low, several small regions of high homology are observed when the amino acid sequences encoded by the left and right ORFs are compared. From these comparisons, it can be shown that the evolutionary relationship among the parvoviruses is B19 in equilibrium with AAV in equilibrium with BPV in equilibrium with MVM. The highly conserved amino acid sequences observed among all parvoviruses may be useful in the identification and detection of parvoviruses and in the design of a general parvovirus vaccine.

Amino Acid Sequence↗
PubMed1979

Transcription of the bovine parvovirus genome in isolated nuclei.

Transcription of the genome of the nondefective parvovirus BPV was examined in nuclei isolated from synchronized bovine fetal spleen cells. The relative levels of total RNA polymerase and RNA polymerase I, II, and III activities in nuclei isolated from BPV-infected and mock-infected cells were found to be similar throughout the course of infection. Hybridization of RNA synthesized in isolated nuceli indicated that BPV-specific RNA synthesis began during the period of 8 to 12 h postinfection and proceeded linearly until at least 20 h postinfection. By 20 h postinfection, 5% of the total RNA synthesized in nuclei from infected cells was virus specific. BPV-specific RNA synthesis was inhibited by 95% in the presence of 0.1 microgram of alpha-amanitin per ml, suggesting that the viral genome is transcribed by cellular RNA polymerase II.

Amanitins↗