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R A Collins

Publications and source records attributed to R A Collins.

At least 73 records · Page 4Linked to original sources
PubMed1996

Cloning and biologic activities of a bovine interferon-alpha isolated from the epithelium of a rotavirus-infected calf.

A cDNA encoding a distinct bovine (Bo) interferon (IFN) alpha, designated BoIFN-alpha E, was generated from gut epithelial cells isolated from a rotavirus-infected calf. The BoIFN-alpha E cDNA sequence shared a greater than 90% identity with the other BoIFN-alpha subtypes. The cDNA encoding BoIFN-alpha E has been expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Insect cells infected with recombinant virus secreted a protein with a relative molecular mass of 19,500 into the culture medium not observed in cells infected with wild-type AcMNPV. Supernatants harvested from cultures of insect cells infected with the recombinant AcMNPV encoding IFN-alpha E inhibited the replication of Semliki Forest virus in a bovine cell line and typically showed 10(6) dilution units/ml of antiviral activity. However, differences were observed between the activities of recombinant BoIFN-alpha E and BoIFN-alpha 1 1 on the proliferation of WC1+ gamma/delta T cells. Purified ( > 99%) WC1+ gamma/delta T cells failed to proliferate to IFN-alpha 1 1 or concanavalin A and IFN-alpha E acted as a weak proliferative signal to these cells, demonstrating a functional difference between two closely related BoIFN-alpha subtypes.

Amino Acid Sequence↗
PubMed1996

Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase.

A mutant form of pyruvate kinase in which serine 384 has been mutated to proline has been engineered in the yeast Saccharomyces cerevisiae. Residue 384 is located in a helix in a subunit interface of the tetrameric enzyme, and the mutation was anticipated to alter the conformation of the helix and hence destabilize the interface. Previous results indicate that the mutant favours the T quaternary conformation over the R conformation, and this is confirmed by the results presented here. Addition of phosphoenol-pyruvate (PEP), ADP and fructose-1, 6-bisphosphate (Fru-1.6-P2) singly to the wild-type and mutant enzymes results in a significant quenching of tryptophan fluorescence (12-44%), and for Fru-1,6-P2, a red shift of 15 nm in the emission maximum. Fluorescence titration experiments showed that PEP, ADP and Fru-1,6-P2 induce conformations which have similar ligand-binding properties in the wild-type and mutant enzymes. However, the Fru-1,6-P2 induced conformation is demonstrably different from those induced by either ADP or PEP. The enzymes differ in their susceptibility to trypsin digestion and N-ethylmaleimide inhibition. The thermal stability of the enzyme is unaltered by the mutation. Far-UV CD spectra show that both enzymes adopt a similar overall secondary structure in solution. Taken together, the results suggest that the Ser384-Pro mutation causes the enzyme to adopt a different tertiary and/or quaternary structure from the wild-type enzyme and affects the type and extent of the conformational changes induced in the enzyme upon ligand binding. A simplified minimal reaction mechanism is proposed in which the R and T states differ in both affinity and kcat. Thus, in terms of the models of cooperativity and allosteric interaction, pyruvate kinase is both a K and a V system.

Adenosine Diphosphate↗
PubMed1996

Bovine interleukins 2 and 4 expressed in recombinant bovine herpesvirus 1 are biologically active secreted glycoproteins.

The open reading frames encoding bovine interleukin 2 (boIL-2) and bovine interleukin 4 (boIL-4) were integrated into the unique short segment of the genome of bovine herpesvirus 1 (BHV-1) and expressed under control of the murine cytomegalovirus (MCMV) immediate-early 1 (ie1) enhancer-promoter element or the MCMV early 1 (e1) promoter. Madin-Darby bovine kidney cells infected with the recombinant viruses secreted boIL-2 or boIL-4 into the culture medium. Secretion was inhibited by the presence of brefeldin A during the infection, indicating that export from the cells was dependent on a functional Golgi apparatus. Treatment of the secreted interleukins with N-glycosidase F reduced the apparent molecular mass of recombinant BHV-1-expressed boIL-2 from 22 kDa to 16 kDa and that of boIL-4 from 20 kDa to 13 kDa, which demonstrated that both cytokines contain N-linked oligosaccharides. Digestion with neuraminidase and O-glycosidase had no detectable effect on the apparent molecular masses, suggesting that BHV-1-expressed boIL-2 and boIL-4 are not, or only slightly, O-glycosylated. In vitro experiments demonstrated the biological activity of recombinant BHV-1-expressed boIL-2 and boIL-4 by their ability to maintain the proliferation of bovine 4325 T cells and activated bovine B cells, respectively. In conclusion, we show that boIL-2 and boIL-4 are secreted from recombinant BHV-1-infected cells as biologically active glycoproteins.

Animals↗
PubMed1995

A subunit interface mutant of yeast pyruvate kinase requires the allosteric activator fructose 1,6-bisphosphate for activity.

A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mutated to proline has been engineered in order to study the allosteric properties of this enzyme. Both the mutant and wild-type enzymes were overexpressed in a strain of yeast in which the genomic copy of the pyruvate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effect fructose 1,6-bisphosphate with concomitant reduction in co-operativity. In contrast, the mutant was found to be dependent on the presence of the effector for catalytic activity and was inactive in its absence. The fully activated mutant enzyme had a kcat. 1.6 times greater than that of the wild-type enzyme. The mutation introduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although kcat. is also affected. The S0.5 for PEP and S0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic parameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0.07 in the presence of the effector, compared with a value of 0.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectroscopy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiary and quaternary structures such as intersubunit and interdomain interactions may be modified. An improved purification procedure has been devised that allows large quantities of enzyme to be rapidly prepared.

Allosteric Regulation↗
PubMed1995

Enhancement of Neurospora VS ribozyme cleavage by tuberactinomycin antibiotics.

Several examples of inhibition of the function of a ribozyme or RNA-protein complex have shown that certain antibiotics can interact specifically with RNA. There are, however, few examples of antibiotics that have a positive, rather than a negative, effect on the function of an RNA. We have found that micromolar concentrations of viomycin, a basic, cyclic peptide antibiotic of the tuberactinomycin group, enhance the cleavage of a ribozyme derived from Neurospora VS RNA. Viomycin decreases by an order of magnitude the concentration of magnesium required for cleavage. It also stimulates an otherwise insignificant transcleavage reaction by enhancing interactions between RNA molecules. The ability of viomycin to enhance some RNA-mediated reactions but inhibit others, including translation and Group I intron splicing, demonstrates the potential for natural selection by small molecules during evolution in the 'RNA world' and may have broader implications with respect to ribozyme expression and activity in contemporary cells.

Enviomycin↗
PubMed1995

A secondary-structure model for the self-cleaving region of Neurospora VS RNA.

Neurospora VS RNA performs an RNA-mediated self-cleavage reaction whose products contain 2',3'-cyclic phosphate and 5'-hydroxyl termini. This reaction is similar to those of hammerhead, hairpin, and hepatitis delta virus ribozymes; however, VS RNA is not similar in sequence to these other self-cleaving motifs. Here we propose a model for the secondary structure of the self-cleaving region of VS RNA, supported by site-directed mutagenesis and chemical modification structure probing data. The secondary structure of VS RNA is distinct from those of the other naturally occurring RNA self-cleaving domains. In addition to a unique secondary structure, several Mg-dependent interactions occur during the folding of VS RNA into its active tertiary conformation.

Cloning, Molecular↗
PubMed1995

The VS catalytic RNA replicates by reverse transcription as a satellite of a retroplasmid.

The mitochondria of certain natural isolates of Neurospora contain both the Varkud plasmid, which encodes a reverse transcriptase, and a small unrelated RNA (VS RNA) that performs RNA-mediated self-cleavage and ligation reactions. Here, we show that VS RNA is transcribed from a VS plasmid DNA template by the Neurospora mitochondrial RNA polymerase using a promoter located immediately upstream of the RNA self-cleavage site that generates monomeric transcripts. VS RNA is then reverse transcribed by the Varkud plasmid reverse transcriptase to yield a full-length (-) strand cDNA, a predicted replication intermediate. Combined with previous genetic evidence, our results indicate that the VS plasmid replicates by reverse transcription as a satellite of the Varkud plasmid. This mode of replication, unprecedented for a satellite RNA, likely reflects the promiscuity of the Varkud plasmid reverse transcriptase, which does not require a specific primer to initiate cDNA synthesis. Our findings indicate how primitive reverse transcriptases with similar relaxed specificity could have facilitated the evolution of new retroelements.

Base Sequence↗
PubMed1995

Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from neurospora VS RNA.

We have constructed a ribozyme containing 144 nucleotides of Neurospora VS RNA that can catalyze the cleavage of a separate RNA in a true enzymatic manner (Km approximately 0.13 microM, kcat approximately 0.7/min). Comparison of the rates of cis- and trans-cleavage, as well as the lack of effect of pH on the rate of cleavage, suggest that a rate-limiting step, possibly a conformational change, occurs prior to cleavage. The minimum contiguous substrate sequence required for cleavage consists of one nucleotide upstream and 19 nucleotides downstream of the cleavage site. Unlike most other ribozymes which interact with long single-stranded regions of their substrates, the minimal substrate for the VS ribozyme consists mostly of a stable stem-loop, which would appear to preclude its recognition simply via extensive Watson-Crick base pairing.

Base Sequence↗
PubMed1995

Effect of recombinant bovine IL-1 and IL-2 on B cell proliferation and differentiation.

The roles of interleukin (IL)-1 and IL-2 in bovine B cell function were investigated by determining the ability of each cytokine to support proliferation and differentiation. Recombinant bovine (rb)IL-2, but not rbIL-1 beta, induced the proliferation of B cells. The effect of IL-2 was dependent on the state of B cell activation. Only low and medium density B cells, but not high density, proliferated in response to IL-2. IL-1 enhanced the IL-2-induced proliferation; this effect was maintained if addition of IL-2 was delayed 48 h. IL-1 did not induce the differentiation of B cells into immunoglobulin-secreting cells. IL-2 was an inducer of secretion in all classes of immunoglobulin. As with proliferative responses, only activated B cells were responsive and IL-2 could not induce immunoglobulin secretion by high density B cells. IL-1 acted as a modulator of IL-2-induced immunoglobulin secretion, an effect which was influenced by the timing of the addition of IL-1 and the target B cell population. Simultaneous addition of IL-1 and IL-2 resulted in the inhibition of IL-2-induced IgM secretion, whilst delayed addition of IL-2 inhibited the amount of IgG1 secreted by purified B cells but had no effect on IgM, IgG2 and IgA secretion. IL-1 had no effect on the ability of low- or high-density B cells to secrete immunoglobulins. However, simultaneous or delayed addition of IL-1 maintained or enhanced the production of immunoglobulins by medium-density B cells in response to IL-2.

Animals↗
PubMed1995

CD45RO expression on bovine T cells: relation to biological function.

The 180,000 MW isoform of CD45 (CD45RO) has been identified in cattle with a novel monoclonal antibody (mAb) (IL-A116). This has allowed a more precise analysis of T-cell function in relation to CD45 isoform expression. Within the CD4+ and CD8+ T-cell populations, CD45RO+ and CD45RO- subsets were evident. Most CD4+ and CD8+ T cells that expressed the CD45RO isoform did not express the 220,000 and 205,000 MW isoforms recognized by mAb CC76. In contrast, the WC1+, CD2-, CD4-, CD8-, gamma delta T-cell receptor (TCR)+ T cells in bovine peripheral blood mononuclear cells (PBMC) were all CD45RO+. Monocytes and granulocytes were CD45RO+ but B cells were CD45RO-. Sorting experiments with CD4+ T cells from an immunized calf demonstrated that proliferative responses to ovalbumin (OVA) were entirely within the CD45RO+ subset. Following stimulation with concanavalin A (Con A) the CD45RO- subset of CD4+ T cells produced transcripts for interleukin-2 (IL-2) but not IL-4 or interferon-gamma (IFN-gamma), while the CD45RO+ subset produced mRNA for IL-2, IL-4 and IFN-gamma. Biologically active IL-2 was present in supernatants from both CD45RO+ and CD45RO-, CD4+ T cells, and IFN-gamma protein was identified by ELISA in supernatants from the CD45RO+ subset, confirming the production of cytokines implied by polymerase chain reaction (PCR). In contrast, sorting experiments with CD8+ T cells from animals immune to the protozoan parasite Theileria parva revealed substantial numbers of cytotoxic T-lymphocyte precursors in both the CD45RO+ and CD45RO- subsets. Thus it appears that although all antigenically primed CD4+ T cells remain CD45RO+, and expression of this molecule consequently identifies memory cells within PBMC, antigenically primed CD8+ T cells down-regulate CD45RO expression after activation.

Animals↗
PubMed1994

Revision of the nucleotide sequence and RNA splicing pathway of the Neurospora mitochondrial gene encoding ATPase subunit 6.

Previous sequence analysis of the Neurospora oli2 (ATP6) mitochondrial gene suggested that, in addition to a typical Group-I intron, it contained an unusual, mostly-palindromic, 93-nucleotide intron. We report here revisions of the nucleotide sequence and analysis of the size and sequence of reverse-transcriptase PCR products that show: (1) the Group-I intron splice sites are located as predicted by previous DNA sequence analysis; (2) the putative 93-nt intron is not excised from the mature mRNA, and most of this sequence is actually in the 5' untranslated region. We conclude that the Neurospora ATP6 gene contains only one intron. Analysis of the cDNA sequence also confirms the non-universal nature of the Neurospora mitochondrial genetic code: a TGA codon inferred from the DNA sequence is present as UGA in the mRNA. This provides direct evidence that this codon is not altered, for example by RNA editing, to conform to the universal code.

Adenosine Triphosphatases↗
PubMed1994

Cloning and expression of bovine and porcine interleukin-2 in baculovirus and analysis of species cross-reactivity.

The cDNAs encoding bovine and porcine interleukin-2 (IL-2) have been expressed using the baculovirus Autographa californica nuclear polyhedrosis virus as a vector in insect cells. Insect cells infected with recombinant viruses secreted bovine and porcine IL-2 into the culture medium, with biological activities for maintaining the proliferation of homologous cells. When the activities of these two IL-2 proteins and commercially available human IL-2 were tested on heterologous cells differences were found. Recombinant bovine (rb)IL-2 only supported the growth of bovine lymphocytes and was not active on human, mouse or porcine lymphocytes. Recombinant porcine (rp)IL-2 and recombinant human (rh)IL-2 supported the proliferation of human, bovine, porcine and murine cells. However, the proliferative response of human lymphocytes to rpIL-2 was only 50% of that seen with rhIL-2. Sequence differences at the predicted p55 and p75 contact binding sites may explain this.

Amino Acid Sequence↗
PubMed1994

Value of serial myoglobin levels in the early diagnosis of patients admitted for acute myocardial infarction.

STUDY OBJECTIVE: The potential for missing the diagnosis of acute myocardial infarction (AMI) and the need for appropriate use of ICU beds make early and accurate diagnostic tests to assist in this diagnosis valuable. We studied the use of serial myoglobin determinations for patients evaluated in the emergency department and admitted for possible AMI. DESIGN: Over a 3.5-month period, all patients presenting to the ED and admitted for suspected cardiac symptoms had serial cardiac enzymes obtained prospectively at admission and 2, 3, 4, and 6 hours after the onset of symptoms. SETTING: Large urban community hospital. PARTICIPANTS: One hundred thirty-three consecutive patients admitted to treat or rule out AMI. RESULTS: Twenty-one of 22 patients with an initially normal myoglobin that doubled within 1 to 2 hours after presentation were positive for AMI (specificity, 95%). Sensitivity of myoglobin at 2 hours after the onset of symptoms was 37% and rose to 86% at 6 hours, with 95% specificity. The negative predictive value if myoglobin was normal at 6 hours and had not doubled within 2 hours was 97% (positive predictive value, 88%). CONCLUSION: A repeat myoglobin level that doubled within 1 to 2 hours after the initial value, even if still within the normal range, was highly specific for AMI. Serial myoglobin levels may be useful in earlier identification of AMI to help prevent inappropriate discharge from the ED and for appropriate placement in ICU beds.

Acute Disease↗
PubMed1993

Nucleotide sequence requirements for self-cleavage of Neurospora VS RNA.

We have used several complementary approaches to investigate the minimal contiguous sequence required for the in vitro self cleavage reaction performed by Neurospora VS RNA. Deletion analysis and site-directed mutagenesis revealed that only a single nucleotide is required upstream of the self-cleavage site, and that the identity of this nucleotide is not critical. This distinguishes VS RNA from all currently known ribozymes except hepatitis delta virus RNA. The shortest contiguous sequence capable of cleavage contains 153 nt downstream of the cleavage site. Linker insertion mutagenesis suggests that much of this downstream sequence is important for self-cleavage. Comparative sequence analysis of the VS plasmid from six natural isolates supports the importance in vivo of the minimal region determined by in vitro methods. Also, phylogenetic analysis raises the possibility of a recent horizontal transfer of the VS plasmid from Neurospora intermedia to Neurospora sitophila.

Base Sequence↗
PubMed1993

Reaction conditions and kinetics of self-cleavage of a ribozyme derived from Neurospora VS RNA.

We have investigated the self-cleavage reaction performed by a ribozyme that contains 164 nucleotides of Neurospora VS RNA. Self-cleavage requires a divalent cation, magnesium being more effective than manganese or calcium. Spermidine or monovalent cations stimulate the reaction but cannot replace magnesium. The temperature optimum is rather broad, around 40-50 degrees C. Unlike some other ribozymes, VS self-cleavage is inhibited by even low concentrations of urea or formamide. The rate of cleavage is the same from pH 5.5 to 8.9, suggesting either that hydroxide is not directly involved in the cleavage chemistry or that a step that precedes the actual cleavage event is rate-limiting.

Formamides↗
PubMed1993

Recombinant human interleukin 2 induces proliferation and immunoglobulin secretion by bovine B-cells: tissue differences and preferential enhancement of immunoglobulin A.

Recombinant human interleukin 2 (rhIL2) was found to have activity on bovine B-cells in vitro, inducing both their proliferation and differentiation into immunoglobulin secreting cells. Only low and medium density (activated) B-cells were responsive; high density (resting) B-cells did not respond to rhIL2. Proliferative responses by unfractionated and purified B-cell populations were similar for all tissues tested. In contrast, immunoglobulin secretion was stimulated to a greater degree in prescapular lymph node (PSLN) and bronchial lymph node (BLN) cells than in mesenteric lymph node (MLN) cells and splenic lymphocytes on culture with IL2. All isotypes were stimulated equally in the case of PSLN, BLN, and splenic lymphocytes. However, culture of unfractionated MLN lymphocytes with IL2 resulted in enhanced secretion of IgA relative to the other isotypes. Removal of T-cells and accessory cells from the MLN lymphocyte preparation resulted in this effect being lost.

Animals↗
PubMed1991

RNA-mediated ligation of self-cleavage products of a Neurospora mitochondrial plasmid transcript.

Neurospora VS RNA is a mitochondrial single-stranded RNA that combines certain features of catalytic RNAs and group I introns. We report here that monomeric VS RNA synthesized in vitro by self-cleavage of a multimeric transcript can perform an RNA-mediated self-ligation reaction producing circular RNAs indistinguishable from those isolated from mitochondria. We conclude that the active site for the ligation reaction is present in the RNA itself. Also, the mechanism for aligning the termini to be ligated may be different from mechanisms previously described. The lack of sequence similarity between VS RNA and previously characterized catalytic RNAs suggests that VS RNA is an independently evolved ribozyme capable of both cleavage and ligation.

Base Sequence↗