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J L Patterson

Publications and source records attributed to J L Patterson.

At least 37 records · Page 2Linked to original sources
PubMed1994

Identification of a short viral transcript in Leishmania RNA virus-infected cells.

Certain strains of Leishmania guyanensis carry persistently infecting double-stranded RNA viruses. The viral polymerase has been shown to have both transcriptase and replicase activity. To date, only full-length RNA transcription of minus and plus strands have been reported. This report describes the synthesis of a 320 nt transcript which is complementary to the 3' end of the minus strand RNA. This plus-stranded short transcript was first detected in an in vitro polymerase assay. It was also detected in virus-infected Leishmania cells by a reverse transcription-polymerase chain reaction assay and by Northern analysis of infected Leishmania cell RNA.

Animals↗
PubMed1994

Complete sequence of Leishmania RNA virus 1-4 and identification of conserved sequences.

In order to understand the coding strategies and identify potential cis-acting sequences in Leishmania RNA virus 1 (LRV1), a complete cDNA sequence was obtained for LRV1-4 and compared to the sequence reported for LRV1-1. The results show that the 5' end of LRV1 is conserved at the nucleotide level while open reading frames (ORFs) 2 and 3 are conserved at the amino acid level. A simple translation initiation consensus sequence is conserved at the 5' end of ORF2 but absent from ORF3, consistent with a possibility that ORF3 is expressed as a gag-pol fusion protein. Comparison of secondary structure predictions obtained for both isolates identified nucleotide sequences capable of forming conserved stem-loops at the virus termini and in the putative frameshift region between ORF2 and ORF3. Although direct evidence is lacking, the appearance of compensatory nucleotide substitutions suggests that the structures may form in vivo. Possible functions for the conserved structures are discussed.

Amino Acid Sequence↗
PubMed1994

Synthesis of viruslike particles by expression of the putative capsid protein of Leishmania RNA virus in a recombinant baculovirus expression system.

The putative capsid open reading frame (ORF2) of the Leishmania RNA virus LRV1-4 was expressed in a baculovirus expression system. The expressed protein was identified by Western immunoblot analysis with polyclonal antiserum raised to purified LRV1-4 virus. Electron microscopy and sedimentation analysis indicated that the expressed protein self-assembles into empty viruslike particles of similar size and shape to authentic virus particles, thus confirming that ORF2 encodes the viral capsid. The expressed particles are present exclusively in the cytoplasm of infected SF9 cells and are able to assemble in the absence of LRV1-4 RNA, viral polymerase, or any Leishmania host factors.

Animals↗
PubMed1994

Mutational analysis of the capsid protein of Leishmania RNA virus LRV1-4.

The virion of Leishmania RNA virus is predicted to be composed of a 742-amino-acid major capsid protein and a small percentage of capsid-polymerase fusion molecules. Recently, the capsid protein alone was expressed and shown to spontaneously assemble into viruslike particles. Since the major structural protein of the virion shell self-assembles into viruslike particles when expressed in the baculovirus expression system, assembly of the virion can be studied by mutational analysis and expression of a single open reading frame. In this study, several deletions and one addition of the capsid protein of Leishmania RNA virus LRV1-4 were generated. These mutants show different degrees of assembly. Assembly domains are being identified such that the capsid protein may be used as a macromolecular packaging and delivery system for Leishmania species.

Animals↗
PubMed1994

Cultivation of Leishmania braziliensis in an economical serum-free medium containing human urine.

Leishmania braziliensis cells are difficult to culture in vitro and usually require media supplemented with serum for sustained cell division. Fresh, sterile urine is an inexpensive substitute for serum in the culture of 2 strains of L. braziliensis, 1 infected with Leishmania RNA virus 1, and 1 uninfected. In the presence of urine, both the infected and the uninfected strains grew to the same final cell density as the same strains grown in the presence of serum. One strain of Leishmania major was also successfully cultured in urine-supplemented media.

Animals↗
PubMed1993

Successful transient introduction of Leishmania RNA virus into a virally infected and an uninfected strain of Leishmania.

Viruses of Leishmania have recently been identified and characterized. These viruses are consistently double-stranded RNA viruses of approximately 5 kb. To date, they have not been reported to exist outside their protozoan host, nor have they been shown to be infectious. We report here the ability to transiently transfer these viruses to two strains of Leishmania, one previously infected and one that did not previously carry a virus. A PCR-based assay was used to detect viral negative-stranded RNA. Input RNA was ruled out as the source of template because a replication-incompetent (UV inactivated) virus was not detectable after transfer into Leishmania.

Animals↗
PubMed1993

The current status of Leishmania RNA virus I.

Viruses of Leishmania have recently been identified and characterized. These viruses are consistently double-stranded RNA viruses of approximately 5 kb. They have not been shown to exist outside their protozoan host, persistently infecting these parasites. The laboratory of jean Patterson has been interested in characterizing and identifying viruses of protozoans in order to use them as molecular probes of the unique gene expression mechanisms of protozoan parasites.

Journal Article↗
PubMed1993

Detection of Leishmania RNA virus 1 proteins.

Polyclonal antiserum was raised against the peak viral fraction of a sucrose gradient from LRV1-4-infected cells and used in Western immunoblot analysis to identify viral proteins from various isolates. Consistent with this result, in vitro-translated protein from cloned RNA was immunoprecipitated with the same antiserum. The putative capsid at times appeared as a doublet; relative amounts of the two species varied, depending on the method of purification.

Animals↗
PubMed1993

Endothelium-dependent relaxation competes with alpha 1- and alpha 2-adrenergic constriction in the canine epicardial coronary microcirculation.

BACKGROUND: The purpose of this study was to determine whether endothelium-dependent relaxation competes with alpha 1- and alpha 2-adrenergic coronary microvascular constriction in the beating heart in vivo. METHODS AND RESULTS: Coronary microvascular diameters were measured using stroboscopic epi-illumination and intravital microscopy during fluorescein microangiography in open-chested dogs (n = 20). Both alpha 1- and alpha 2-adrenergic receptors were selectively activated by intracoronary infusions of norepinephrine (0.05 and 0.2 microgram.kg-1 x min-1) in the presence of the alpha 2-adrenergic antagonist rauwolscine (0.2 mg/kg) or the alpha 1-adrenergic antagonist prazosin (0.75 mg/kg) during beta-adrenergic blockade (1 mg/kg propranolol). Microvascular diameters during selective alpha-adrenergic receptor activation were measured under baseline conditions and after inhibition of endogenous nitric oxide synthesis by an analogue of L-arginine, either NG-nitro-L-arginine (L-NA, 30 mg/kg) or NG-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg). Under baseline conditions, alpha 1-adrenergic activation constricted small arteries (vessels with diameters between 100 and 300 microns) (4 +/- 1% and 5 +/- 1% decrease in diameter for the low and high doses of norepinephrine, respectively, both p < 0.05) but did not change the diameter of arterioles (vessels with diameters < 100 microns). In contrast, alpha 2-adrenergic activation by the lower but not the higher dose of norepinephrine induced constriction of arterioles (6 +/- 2% and 3 +/- 4% decrease in diameter, p < 0.05 and NS, respectively) but not small arteries. Inhibition of nitric oxide synthase activity by either L-NA or L-NAME produced constriction of small coronary arteries (9 +/- 2% decrease in diameter, p < 0.01) and arterioles (6 +/- 1% decrease in diameter, p < 0.05). The dilatation of small arteries and arterioles by acetylcholine (0.05 microgram-1 x kg-1 x min-1 intracoronary infusion; 10 +/- 1% increase in diameter under baseline conditions, p < 0.05) was abolished by either analogue. Both alpha 1- and alpha 2-adrenergic coronary microvascular constriction were markedly potentiated after L-NA or L-NAME. alpha 1-Adrenergic constriction was unmasked in arterioles (7 +/- 3% and 10 +/- 4% decrease in diameter, p < 0.05), although it was not significantly increased in small arteries. Conversely, alpha 2-adrenergic constriction was unmasked in small arteries (8 +/- 1% and 6 +/- 2% decrease in diameter, both p < 0.05) and potentiated in arterioles (12 +/- 1% and 8 +/- 4% decrease in diameter, both p < 0.05). After L-NA or L-NAME, microvessels retained the ability to dilate to sodium nitroprusside (0.1 microgram.kg-1 x min-1 intracoronary infusion; 10 +/- 2% increase in diameter, p < 0.05). alpha-Adrenergic constriction was not accentuated by increased tone alone, since it was either attenuated or converted to dilatation during a similar degree of preconstriction by the endothelium-independent vasoconstrictor angiotensin II (p < 0.05 for both alpha 1- and alpha 2-adrenergic activation). CONCLUSIONS: These data confirm that alpha-adrenergic receptors are widespread in the coronary microcirculation, with the baseline functional responses to alpha 1-adrenergic activation predominating in small arteries and those to alpha 2-adrenergic activation predominating in arterioles. Furthermore, coronary microvascular constriction caused by both alpha 1- and alpha 2-adrenergic receptor activation is significantly modulated by endothelium-dependent relaxation, being markedly potentiated by inhibition of nitric oxide synthase activity. The data imply that alpha-adrenergic activation will assume considerable importance as a determinant of coronary microvascular resistance in pathophysiological situations associated with coronary endothelial impairment.

Animals↗
PubMed1993

Effect of neonatal capsaicin treatment on neurogenic pulmonary edema from fluid-percussion brain injury in the adult rat.

The frequent occurrence of acute death from pulmonary failure in experimental head injury studies on Sprague-Dawley rats prompted an investigation into the manner in which acute neurogenic pulmonary edema develops in these animals as a result of an applied fluid pressure pulse to the cerebral hemispheres. Studies were performed in adult animals using histamine H1 and H2 blocking agents, or in adult animals treated as neonates with capsaicin to destroy unmyelinated C-fibers. Recordings were made of either the pulmonary arterial or the right ventricular pressure, and the left atrial and femoral arterial pressures before, during, and after injury to provide a record of the hemodynamic response throughout the development of neurogenic pulmonary edema. Head injury triggered the almost immediate development of pressure transients with and without neurogenic pulmonary edema. All rats, regardless of treatment, reacted with nearly identical systemic arterial pressure responses; however, the pulmonary responses followed a time course that was independent of systemic arterial pressure changes. Acute neurogenic pulmonary edema was always associated with a substantial increase in pulmonary arterial and left atrial pressures; conversely, pressure increases of similar magnitude were not always associated with edema. Histamine H1 and H2 blockers significantly reduced the pulmonary pressure surges only in rats free of neurogenic pulmonary edema. All capsaicin-treated rats showed suppressed pulmonary pressure responses, normal lung water content, elevated lung surface tension, and significantly reduced levels of immunoreactive substance P in the spinal cord and vagus nerve. While the pressures cannot clarify how edema influences the observed hemodynamics, they do not support the view that edema is the direct consequence of pulmonary hypertension. It is proposed that neurogenic pulmonary edema is a functional disturbance provoked by adverse stimuli from outside the lungs and that in the rat the primary afferent fiber is essential to the production of this entity.

Animals↗
PubMed1992

Transcribing and replicating particles in a double-stranded RNA virus from Leishmania.

During the replicative cycle of many double-stranded RNA viruses, transcription of particles with a double-stranded RNA genome alternates with replication of particles containing a single-stranded genome. In virions infecting some strains of Leishmania guyanensis the putative transcriptase and replicase activities of the RNA-dependent RNA polymerase were previously detected in vitro. Northern hybridization to RNA of known polarity demonstrates that the single-stranded RNA products are of positive polarity and, by definition, are the products of the viral transcriptase. Re-evaluation of previously published data in the light of these findings suggests that transcription in Leishmania viruses is conservative. Sedimentation in sucrose gradients revealed two types of viral particles; single-stranded RNA particles comprised a small fraction of the virus population and sedimented more slowly than the peak of double-stranded RNA particles. In agreement with the replicative model of other dsRNA viruses, these single-stranded particles co-purified with the viral replicase activity that resulted in double-stranded RNA synthesis. In virus-infected promastigote extracts replicase activity decreased with increasing parasite density in culture, suggesting a correlation between cell division and viral replication.

Animals↗
PubMed1991

Genomic structure and RNA polymerase activity in Leishmania virus.

Viral particles infecting some stocks of the protozoan parasite Leishmania braziliensis subsp. guyanensis contain a double-stranded RNA genome of ca. 5 kbp and are associated with an RNA-dependent RNA polymerase which synthesizes in vitro double-stranded and single-stranded, genome-length transcripts. The majority of viral transcripts are single-stranded and templated from one genomic strand. The putative replicase generates double-stranded RNA by synthesizing the opposite strand on a preexisting RNA template. These data are compatible with a replicative cycle proposed for the yeast viruses. Purification of the Leishmania virus on CsCl yields virus without double-strand synthesis activity, while this activity is consistently present in unpurified virus and in particles from sucrose gradients. The deficiency in double-strand synthesis in CsCl-derived virions correlates with the accessibility of the viral polymerase and genomic RNA to exogenously added enzymes, indicative of a structural modification of the viral capsid.

Animals↗
PubMed1991

External loading comparisons between able-bodied and below-knee-amputee children during walking.

The purpose of this investigation was to determine external loading variables that could describe any statistically significant differences between the limbs of below-knee-amputee (BKA) and able-bodied children. Eleven able-bodied children and four BKA children volunteered to participate in this investigation. Force platform data were collected for two consecutive foot falls during two experimental sessions. Significant external load differences existed between the prosthetic limbs and nonprosthetic limbs of BKA children and between limbs of the BKA and able-bodied children. The prosthetic limb generally displayed a subordinate role when compared to nonprosthetic and normal limbs. The nonprosthetic limbs displayed a dominant role when compared to the other limbs. These external loading characteristics of the prosthetic and nonprosthetic limbs may be a logical consequence of the morphologic and functional differences that exist between the groups. It was concluded that as long as the prosthetic limb functions differently from a normal limb, BKA children may have a difficult time walking the same as able-bodied children. It would appear to be advantageous to determine the most appropriate gait pattern for BKA children, given the influence of such factors as prosthetic design, construction, and alignment, rehabilitation, and joint loading.

Adolescent↗
PubMed1990

RNA polymerase activity is associated with viral particles isolated from Leishmania braziliensis subsp. guyanensis.

Viral particles purified from species of the protozoan parasite Leishmania braziliensis subsp. guyanensis by centrifugation in CsCl gradients were examined for the presence of viral polymerase. We demonstrated that RNA-dependent RNA polymerase is associated with viral particles. Viral transcription was studied in vitro with pulse-chase experiments and by assaying the RNase sensitivity of the viral transcripts. Viral polymerase synthesized full-length transcripts within 1 h. Double-strained, genome-length, and single-stranded RNAs were produced in this system. The nature of the RNA extracted from virions was also tested by RNase protection assays; both single-stranded and double-stranded RNAs were found.

Animals↗
PubMed1990

Ribavirin is an inhibitor of human immunodeficiency virus reverse transcriptase.

Ribavirin inhibits the human immunodeficiency virus reverse transcriptase in an in vitro reaction. Ribavirin-5'-diphosphate was close to 40% more inhibitory than ribavirin-5'-triphosphate. Unphosphorylated ribavirin had a reduced, but detectable, effect as an inhibitor, compared with the phosphorylated forms. The compounds seem to have a direct effect on the viral polymerase, and no chain termination was observed in the presence of ribavirin-5'-triphosphate. Combination of any of the ribavirin derivatives tested with 3'-azido-3'-deoxythymidine (zidovudine)-5'-triphosphate resulted in an increase of its anti-human immunodeficiency virus reverse transcriptase activity in the in vitro assay.

Avian Myeloblastosis Virus↗
PubMed1989

Characterization of a RNA virus from the parasite Leishmania.

We were interested in screening a series of isolates of the protozoan Leishmania for the presence of viruses. The experimental procedure we used was based on an enzymatic assay originally developed for viral RNA-dependent RNA polymerases. Simultaneously, total promastigote nucleic acid preparations were analyzed for the presence of viral genome and/or transcripts. Two isolates, both classified as L. braziliensis guyanensis, were found to be positive for RNA polymerase activity and to carry a large (6 kilobases) RNA species. The polymerase reaction products hybridized to the 6-kilobase RNA, believed to be the viral genome. In conjunction with electron microscopical observations these results indicate the presence of an RNA virus in these Leishmania isolates.

Animals↗
PubMed1989

Altered ATP function of a vesicular stomatitis virus mutant detected by kinetic analysis of the transcriptase using phosphorylated ribavirin.

We have studied the effect of phosphorylated ribavirin on the vesicular stomatitis virus (VSV) in vitro polymerase reaction by analysis of kinetic data obtained by varying the concentration of nucleoside triphosphates. The wild-type VSV had previously shown a competitive inhibition with the four natural nucleoside triphosphates with the use of ribavirin diphosphate (RDP) or ribavirin triphosphate (RTP). In contrast, when RDP (or RTP) was added to a transcription assay system using the polR1 mutant of VSV, a non-competitive or mixed type of inhibition was observed when the concentration of ATP was varied. Our results indicate that polR1 has an altered ATP function in addition to the previously described phenotypic characteristics of this mutant, which include synthesis of readthrough products of the leader/nucleocapsid (N) gene junction and a decreased ATP requirement for transcription. We have also studied CsCl-purified in vitro transcription products by primer-extending leader or N mRNA transcripts and found that the ratio of leader/N mRNA for VSV polR1 (1.3:1) was lower than values obtained previously for wild-type (3.7:1).

Adenosine Triphosphate↗