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S K Murphy

Publications and source records attributed to S K Murphy.

9 recordsLinked to original sources
PubMed1999

RNA replication for the paramyxovirus simian virus 5 requires an internal repeated (CGNNNN) sequence motif.

A functional RNA replication promoter for the paramyxovirus simian virus 5 (SV5) requires two essential and discontinuous elements: 19 bases at the 3' terminus (conserved region I) and an 18-base internal region (conserved region II [CRII]) that is contained within the coding region of the L protein gene. A reverse-genetics system was used to determine the sequence requirements for the internal CRII element to function in RNA replication. A series of copyback defective interfering (DI) RNA analogs were constructed to contain point mutations in the 18 nucleotides composing CRII, and their relative replication levels were analyzed. The results indicated that SV5 DI RNA replication was reduced by substitutions for two CG dinucleotides, which in the nucleocapsid template are in the first two positions of the first two hexamers of CRII nucleotides. Substitutions for other bases within CRII did not reduce RNA synthesis. Thus, two consecutive 5'-CGNNNN-3' hexamers form an important sequence in the SV5 CRII promoter element. The position of the CG dinucleotide within the SV5 leader and antitrailer promoters was highly conserved among other members of the Rubulavirus genus, but this motif differed significantly in both sequence and position from that previously identified for Sendai virus. The possible roles of the CRII internal promoter element in paramyxovirus RNA replication are discussed.

Promoter Regions, Genetic↗
PubMed1998

A functional antigenomic promoter for the paramyxovirus simian virus 5 requires proper spacing between an essential internal segment and the 3' terminus.

A previous analysis of naturally occurring defective interfering (DI) RNA genomes of the prototypic paramyxovirus simian virus 5 (SV5) indicated that 113 bases at the 3' terminus of the antigenome were sufficient to direct RNA encapsidation and replication. A nucleotide sequence alignment of the antigenomic 3'-terminal 113 bases of members of the Rubulavirus genus of the Paramyxoviridae family identified two regions of sequence identity: bases 1 to 19 at the 3' terminus (conserved region I [CRI]) and a more distal region consisting of antigenome bases 73 to 90 (CRII) that was contained within the 3' coding region of the L protein gene. To determine whether these regions of the antigenome were essential for SV5 RNA replication, a reverse genetics system was used to analyze the replication of copyback DI RNA analogs that contained a foreign gene (GL, encoding green fluorescence protein) flanked by 113 5'-terminal bases and various amounts of SV5 3'-terminal antigenomic sequences. Results from a deletion analysis showed that efficient encapsidation and replication of SV5-GL DI RNA analogs occurred when the 90 3'-terminal bases of the SV5 antigenomic RNA were retained, but replication was reduced approximately 5- to 14-fold in the case of truncated antigenomes that lacked the 3'-end CRII sequences. A chimeric copyback DI RNA containing the 3'-terminal 98 bases including the CRI and CRII sequences from the human parainfluenza virus type 2 (HPIV2) antigenome in place of the corresponding SV5 sequences was efficiently replicated by SV5 cDNA-derived components. However, replication was reduced approximately 20-fold for a truncated SV5-HPIV2 chimeric RNA that lacked the HPIV2 CRII sequences between antigenome bases 72 and 90. Progressive deletions of 6 to 18 bases in the region located between the SV5 antigenomic CRI and CRII segments (3'-end nucleotides 21 to 38) resulted in a approximately 25-fold decrease in SV5-GL RNA synthesis. Surprisingly, replication was restored to wild-type levels when these length alterations between CRI and CRII were corrected by replacing the deleted bases with nonviral sequences. Together, these data suggest that a functional SV5 antigenomic promoter requires proper spacing between an essential internal region and the 3' terminus. A model is presented for the structure of the 3' end of the SV5 antigenome which proposes that positioning of CRI and CRII along the same face of the helical nucleocapsid is an essential feature of a functional antigenomic promoter.

Animals↗
PubMed1997

Genome nucleotide lengths that are divisible by six are not essential but enhance replication of defective interfering RNAs of the paramyxovirus simian virus 5.

For some members of the Paramyxoviridae family of negative strand RNA viruses, efficient genome replication only occurs when the total genome length is a multiple of six (6N length, where N is any integer). To determine if this "rule of six" requirement applied to the replication of the prototype paramyxovirus simian virus 5 (SV5), defective interfering (DI) RNA genomes were generated by sequential undiluted passage of virus in tissue culture. Molecular cloning and nucleotide sequence analysis of 10 RNA genomes revealed a series of copyback DI RNAs with chain lengths between 449 and 1365 bases, but only 4 of the 10 naturally occurring RNA genomes were of 6N length. Many of the cloned DI genomes could be grouped into two distinct nested sets, with the members of each set having the same polymerase crossover junctions and extent of terminal complementarity but differing from each other by internal deletions. One of these nested sets of genomes consisted of novel DI RNAs that contained a pentameric stretch of nontemplated adenosine residues inserted precisely at the polymerase crossover junction. A reverse genetics system was established in which SV5 DI genomes were replicated in vivo entirely by cDNA-derived components. Using this system, two naturally occurring SV5 DI RNAs were examined in a mutational analysis to determine the role of genome length on SV5 RNA replication. The progressive insertion of one to six nucleotides into a 6N length DI genome (852 bases) resulted in a reduction in replication for RNAs that contained one to four additional bases (approximately 35-50% of WT levels), followed by an increase back to WT replication levels for genomes that were altered by five and six base insertions (approximately 70 and 100% of WT levels, respectively). An insertion of five nucleotides into a second non-6N length DI RNA (499 total bases) created a genome length that was a multiple of six (504 bases) and led to a approximately 10-fold stimulation of replication over that of the unaltered genome. Together, these results indicate that there was a clear influence of 6N genome length on SV5 DI RNA replication, but the stringency of this replication requirement appeared to be less than that found previously for other paramyxoviruses. This work completes the testing of the rule of six replication requirement for representatives of each of the four genera of the Paramyxoviridae family and indicates that the preference for replication of 6N length RNA genomes varies between the individual paramyxoviruses.

Animals↗
PubMed1994

Alteration of phosphoinositide metabolism by attenuation of cAMP resulting from expression of the H-ras oncogene.

Most studies characterizing H-ras have been conducted in constitutively expressing cell lines. To explore the early interaction between H-ras p21 and signal transduction systems we have utilized an NIH3T3 fibroblast line transfected with a steroid inducible MMTV H-ras vector. Exposure to dexamethasone resulted in transcription of H-ras accompanied by an increase in PI turnover. Addition of cAMP analogs restored PI metabolism to control level. We postulate that these effects are due to the regulatory action of PKA on PLC and that H-ras may interfere with cAMP metabolism, negating its regulatory effect on PLC. Therefore, we investigated the role of p21 in cAMP metabolism and PLC activity. We demonstrated that after p21 reached maximal level of expression, cAMP synthesis was reduced to 45% of the control. Radioimmunoassay of cAMP also indicated H-ras acts to inhibit adenylate cyclase activity. Further, we found a 4-fold increase in PLC activity in H-ras expressing cells that could be reversed by elevation of cAMP. Incubation with the PKA inhibitor, KT5720, resulted in activity similar to that observed in H-ras expressing cells. Additionally, we have shown no correlation between H-ras expression and GTP gamma s stimulated PI metabolism, indicating that H-ras is not functioning as a G protein in PLC activation. These results imply that H-ras functions, in this system, to decrease levels of cAMP, thus negating the regulatory effect of PKA on PLC.

3T3 Cells↗
PubMed1992

Effects of temperature abuse on survival of Vibrio vulnificus in oysters.

Opaque and translucent morphotypes of a TnphoA-containing strain of Vibrio vulnificus were fed to oysters, which were subsequently stored at temperatures ranging from 0.5 to 22 degrees C for 10 days. Samples of oysters were homogenized and plated at intervals to determine the cell density of V. vulnificus and total aerobic population of bacteria present. At all temperatures, the numbers of V. vulnificus (both morphotypes) declined over the 10-day study period. The same observation was made with a lower inoculum of V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus to levels below limits of detection. Little change in the total bacterial counts was observed in shellstock oysters at any of the test temperatures, whereas incubation at the higher temperatures (17 and 22 degrees C) resulted in large increases in total counts in shucked oysters. These data suggest that temperature abuse of oysters may not be a factor in increasing the public health risk of V. vulnificus through raw oyster consumption. However, the data also suggest that even with proper storage, indigenous levels of V. vulnificus may remain sufficiently higher in shellstock oysters to produce infection in compromised hosts.

Aerobiosis↗
PubMed1990

Modulation of phosphoinositide metabolism in aortic smooth muscle cells by allylamine.

Aortic smooth muscle cells (SMC) modulate from a contractile to a proliferative phenotype upon subchronic exposure to allylamine. The present studies were designed to determine if this phenotypic modulation is associated with alterations in the metabolism of membrane phosphoinositides. 32P incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) was lower by 31, 35, and 22%, respectively, in SMC from allylamine-treated animals relative to controls. In contrast, incorporation of [3H]myoinositol into inositol phosphates did not differ in allylamine cells relative to control cells. Exposure to dibutyryl (db) cAMP (0.2 mM) and theophylline (0.1 mM) reduced 32P incorporation into PIP and PIP2 in SMC from both experimental groups. Under these conditions, a decrease in [3H]myoinositol incorporation into inositol 1-phosphate was only observed in allylamine cells. The effects of db cAMP and theophylline in allylamine and control SMC correlated with a marked decrease in cellular proliferation. These results suggest that alterations in phosphoinositide synthesis and/or degradation contribute to the enhanced proliferation of SMC induced by allylamine. To further examine this concept, the effects of agents which modulate protein kinase C (PKC) activity were evaluated. Sphingosine (125-500 ng/ml), a PKC inhibitor, decreased SMC proliferation in allylamine, but not control cells. 12-O-Tetradecanoylphorbol-13-acetate (1-100 ng/ml), a PKC agonist, stimulated proliferation in control cells, but inhibited proliferation in cells from allylamine-treated animals. We conclude that allylamine-induced phenotypic modulation of SMC is associated with alterations in phosphoinositide metabolism.

Allylamine↗
PubMed1988

Retraction of bioprosthetic heart valve cusps: a cause of wide-open regurgitation in right-sided heart valves.

Most failures of bioprosthetic heart valves in children are due to stenosis secondary to thrombus, calcific deposits, or tissue ingrowth. Valve failures due to regurgitation typically involve cuspal detachment, tears, or perforations. We present four cases of prosthetic valve regurgitation in children caused by cuspal retraction without stenosis and describe the morphologic findings related to the valves at autopsy or explantation. A mononuclear cell and giant cell response to the cusps of the valve was a striking finding in one patient.

Adolescent↗
PubMed1987

Cellular signal transduction and the reversal of malignancy.

Animal cells contain only a few defined molecular systems that transduce hormonal and growth signals from the external environment to the intracellular milieu to regulate cellular growth and differentiation. Among the most ubiquitous of these "second messenger" pathways are those utilizing cyclic AMP and phosphatidylinositide turnover. The former activates protein kinase A, while the latter leads to the activation of protein kinase C and mobilization of intracellular calcium. Lesions induced by oncogenes in signal transduction systems may be responsible for the cancerous transformation of cells. In many tumor cell lines, including some transformed by the ras and sis oncogenes, activation of protein kinase A by elevation of cyclic AMP or activation of protein kinase C by addition of phorbol esters can restore many normal aspects of growth and morphology. Such "reverse transformation" is accompanied by the phosphorylation of unique cellular proteins and alterations in the phosphoinositide cycle. Molecular mechanisms by which activation of signal transduction systems can attenuate the malignant phenotype are considered in the context of cellular growth and differentiation.

Animals↗