Biomedical subjects

J L Patterson

Publications and source records attributed to J L Patterson.

At least 55 records · Page 3Linked to original sources

PubMed1989

Molecular analysis of the inhibitory effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro polymerase reaction.

The effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro transcription reaction was examined. Analysis of the kinetics observed when the concentrations of nucleoside triphosphates were varied was performed with vesicular stomatitis virus wild-type standard virions. Double-reciprocal and Eadie-Hofstee plots showed competitive inhibition with all natural nucleoside triphosphates when both ribavirin diphosphate (RDP) and ribavirin triphosphate (RTP) were used. The Km values for ATP obtained for the wild-type polymerase were similar to those reported previously. To further characterize the observed inhibition kinetics, in vitro transcription products synthesized in the presence or absence of RDP and RTP were purified by CsCl centrifugation and were primer extended with oligonucleotides specific for either positive-sense leader or nucleocapsid mRNA transcripts. The ratios of leader to nucleocapsid mRNA were measured from primer-extended in vitro transcription products. It was found that the addition of RDP or RTP did not significantly change the in vitro ratio, suggesting that the polymerase is blocked before it enters the 3' end of the template.

Antiviral Agents↗

PubMed1989

Mechanism of La Crosse virus inhibition by ribavirin.

The effect of ribavirin on the growth and replication of La Crosse virus was examined. The data suggest that low concentrations of ribavirin have a marked effect on the initial steps of La Crosse virus transcription. The therapeutic potential of ribavirin in the treatment of human California encephalitis serotype infections is discussed in light of these findings.

Bunyaviridae↗

PubMed1989

Effect of indomethacin pretreatment on acute mortality in experimental brain injury.

The effect of indomethacin administration on the mortality rate of brain-injured rats was studied in four groups of animals subjected to a level of injury with a fluid-percussion apparatus predetermined to cause 50% mortality (50% lethal dose, or LD50). There were 24 animals in each of the following groups: 1) a control group, on which the LD50 was evaluated; 2) an ethanol-treated group with a mean blood serum level of 0.32 +/- 0.03 gm% (+/- standard error of the mean); 3) an indomethacin-treated group at a dose level of 3 mg/kg body weight administered intraperitoneally 10 to 15 minutes before injury; and 4) an indomethacin/ethanol-treated group. Significant differences in mortality rates were found in these experimental groups; namely, 50%, 58%, 8.3% (p less than 0.005), and 25% (p less than 0.05), respectively. The predetermined LD50 level of a 2.5- to 2.6-atm peak pressure pulse produced immediate apnea in all animals, which was either sustained (Type III), followed by temporary respiratory recovery (Type II), or followed by permanent resumption of breathing (Type I). The most important effect of indomethacin on respiratory function was manifested by a much higher percentage of Type I respiratory responses and a much lower percentage of Type II and III responses (hence a lower mortality rate). There was also a more rapid return to normal breathing in the postapneic period of recovery. Suppression of prostaglandin synthesis and of superoxide anion production at the of trauma may explain, at least in part, these favorable effects of indomethacin.

Animals↗

PubMed1989

Combined effect of respirator-induced ventilation and superoxide dismutase in experimental brain injury.

The function-specific enzyme superoxide dismutase (SOD) was tested for its protective effect in severe experimental fluid-percussion brain injury (4.45 +/- 0.10 atm) in 30 of 60 randomly selected male Sprague-Dawley rats. A respirator was used only in the event of need. The number of animals with permanent resumption of spontaneous breathing (Type I respiratory response) remained essentially the same in each group. However, when Type II apnea (cannot maintain recovery) and Type III apnea (never recovers from the initial apnea) were terminated with a respirator, all rats with Type II responses from each group were successfully converted to a state of sustained spontaneous breathing. In contrast, only five (41.7%) of the 12 rats with Type III response were salvaged in the control group while five (83.3%) of six Type III rats in the SOD-treated group were saved. The results reveal the nature of the therapeutic effectiveness of superoxide radical scavengers in the overall outcome of head injury in this animal model. While SOD alone did not increase the number of spontaneous survivors, the drug shifted a number of animals from the critically injured rats with Type III respiratory response to the less critical Type II condition. Whereas induced respiration as the sole therapy in the control group lowered the mortality rate to 23.3%, respiratory assistance together with SOD treatment reduced the "mortality" to a single animal with Type III apnea (3.3%) which was alive but still required the respirator after 2 hours (p less than 0.001). The results show that respiratory assistance alone accounted for a 33% decrease in mortality rate and that SOD, given in addition to induced ventilation, further decreased mortality by 20%. Since SOD enzymes are reactively specific for superoxide, the increased survival rate of the brain-injured rat must have been due either to preventing or to minimizing pathophysiological changes, probably in the brain stem, caused by oxygen free radicals.

Animals↗

PubMed1988

Effect of phosphorylated ribavirin on vesicular stomatitis virus transcription.

The effect of phosphorylated ribavirin on a vesicular stomatitis virus (VSV) in vitro transcription reaction was examined. Viral mRNA synthesized in the presence of the 5' mono-, di-, and triphosphorylated forms of the drug translated with equal efficiencies under the test conditions. However, all three phosphorylated species inhibited VSV transcription. The mono- and diphosphorylated forms of the drug possessed approximately two to three times the inhibitory activity as the triphosphorylated form. Transcripts synthesized in the presence of drug were full length and were absent of incorporated drug. Inhibition by ribavirin 5'-diphosphate could be reversed by the addition of UTP, CTP, and GTP, while the addition of GDP to the reaction did not reverse inhibition. Ribavirin diphosphate was added to a La Crosse virus in vitro transcription assay to determine whether an inhibitory effect could be established in a viral system that was more sensitive to ribavirin than was VSV; it led to profound inhibition of RNA synthesis at concentrations as low as 0.1 microgram/ml. These data suggest that ribavirin has an effect on the initial steps of transcription by some RNA-dependent RNA polymerases and that this effect may be mediated by several phosphorylated forms.

Nucleotides↗

PubMed1988

Further characterization of the soluble form of the G glycoprotein of respiratory syncytial virus.

A soluble form of the G glycoprotein, the attachment protein, of respiratory syncytial virus is shed from infected HEp-2 cells. The Gs proteins of the Long and 18537 strains have apparent molecular sizes of 82 and 71 kilodaltons, respectively, 6 to 9 kilodaltons smaller than the virion-associated forms (Gv). The Gs protein of the Long strain was further characterized. Approximately one in six of all of the radiolabeled G molecules in these cultures at 24 h postinfection was present as the Gs protein. The Gs protein was clearly evident in culture fluids at 6 h postinfection, but the Gv protein could not be discerned until 12 h after infection, an observation that is consistent with the 12-h eclipse period for respiratory syncytial virus. Therefore, the Gs protein is shed, in part at least, from intact, infected cells and before the appearance of progeny virus. The appearance of a smaller Gs protein (74 kilodaltons) in fluids of infected calls which were incubated with tunicamycin shows that addition of N-linked oligosaccharides is not required for the genesis and shedding of the Gs protein. Sequencing of the amino terminus of purified Gs protein revealed two different termini, whose generations are consistent with cleavages of the full-length G protein between amino acids 65 and 66 and between residues 74 and 75. This result suggests that the Gs protein is present in two different forms which lack the proposed intracytoplasmic and transmembrane domains of the full-length G protein.

Amino Acid Sequence↗

PubMed1987

Appearance of a soluble form of the G protein of respiratory syncytial virus in fluids of infected cells.

We examined the antigenic reactivities and virion associations of glycoproteins that were released into the culture fluids of cells infected with respiratory syncytial (RS) virus. Culture fluids and cell extracts were obtained from cells 24 to 30 h after they were infected with the Long strain of RS virus. Radioimmune precipitation of [3H]glucosamine-labelled glycoproteins by large glycoprotein (G)-specific or fusion protein (F)-specific monoclonal antibodies (MAbs) revealed that the G, F1 and F2 proteins were present in cell extracts but only the G protein was clearly evident in culture fluids. A glycoprotein (Mr 43K) which may be a precursor or a breakdown product of the G protein was also precipitated by the G-specific MAb from cell extracts and culture fluids. The G protein in culture fluids was slightly smaller (Mr 82K) than the G protein in cell extracts (Mr 88K). An abundant or heavily labelled, 18K glycoprotein in the fluids of virus-infected but not of mock-infected cells was weakly precipitated by the F-specific MAb; this suggested that the 18K protein shares epitopes with the fusion protein of RS virus. The absence of F1 and F2 polypeptides from culture fluids is evidence that the cells, which contained an abundance of these proteins, were intact. To determine whether any of the viral glycoproteins released by infected cells were soluble (non-virion-associated), culture fluid was subjected to rate zonal centrifugation in a 10 to 50% sucrose gradient. An assay of fractions using a MAb-capture ELISA for the nucleocapsid (N) and F proteins revealed a peak of activity, due to virions, in the centre of the gradient, and a strong signal for the N protein at the top of the gradient suggesting that N protein was released from intact cells. Radioimmune precipitation of glycoproteins from the fractions at the top of the gradient using a hyperimmune guinea-pig serum revealed the G protein and a heterogeneous band which had the electrophoretic mobility of the 43K protein. Neither the F1 nor the F2 protein was present in these fractions thus suggesting that virions had remained intact. These results showed that a soluble form of the G protein of RS virus is released into the culture fluids of intact, infected cells. Several theories concerning viral and non-viral origins for the 18K protein are discussed.

Capsid↗

PubMed1984

Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes.

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.

Animals↗

PubMed1984

Characterization of La Crosse virus small-genome transcripts.

With restriction fragments from DNA clones of the La Crosse virus S genome segment, the 3' end of the S mRNA was located by S1 nuclease mapping near a polyuridine tract, approximately 100 nucleotides, from the end of the S genome. Genome replication in La Crosse virus-infected cells was abolished by the drug cycloheximide, similar to other negative-strand RNA viruses. However, the synthesis of S mRNA could not be detected in cells pretreated with cycloheximide, suggesting that ongoing protein synthesis is required for La Crosse virus genome transcription and replication. Primer extension experiments in the presence of chain-terminating nucleoside triphosphates demonstrated that the 5' end of the La Crosse virus S mRNA begins 10 to 14 nucleotides before the 3' end of the S genome segment, suggesting that the La Crosse virus S mRNA is initiated on a host primer. A hypothesis consistent with these unexpected findings is presented.

Animals↗

PubMed1984

La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease.

Purified La Crosse virions in vitro were found to transcribe their negative polarity (-)RNA genomes. This polymerase activity was stimulated by oligonucleotides such as (A)nG, cap analogs such as m7GpppAm, and natural mRNAs such as alfalfa mosaic virus RNA 4. For (A)nG- and alfalfa mosaic virus RNA 4-stimulated reactions, evidence is presented that these RNAs stimulate activity by acting as primers for viral transcription. The cap analogs appear to stimulate activity via an alternative mechanism. Purified La Crosse virions were also found to contain an endonuclease which specifically cleaves alfalfa mosaic virus RNA 4 when this RNA contains a methylated cap group.

Animals↗

PubMed1983

Multiple leader RNAs and messenger RNAs are transcribed from the La Crosse virus small genome segment.

Nucleotide sequencing has demonstrated that the Small genome segment of Bunyaviruses contains the genetic information for two viral proteins (N and NSs) in overlapping reading frames (Akashi and Bishop, 1983; Cabradilla et al., in press). Using 3' end-labeled genome probes, La Crosse virus (LAC) infected cells were shown to contain three leader RNAs, which start at position 1 and terminate at approximate positions 74, 95, and 115 from the 3' end of the genome. Primer extension and S1 mapping studies have shown that LAC-infected cells contain seven mRNAs, five of which initiate at positions 1, 74, 101, 117, and 123 and two more which initiate at approximate positions 147 and 159 from the 3' end of the Small genome segment. The existence of multiple leader RNAs and mRNAs from the Small genome segment may allow access to both overlapping reading frames by ribosomes that still initiate only at 5' proximal AUGs, without the need to splice the mRNA.

Bunyaviridae↗

PubMed1983

Isolation of the ends of La Crosse virus small RNA as a double-stranded structure.

The genome of La Crosse virus, a member of the Bunyaviridae, is made up of three molecules. Circular nucleocapsid structures, in three size classes, have been isolated from La Crosse virus (Obijeski et al. J. Virol. 20:664-675, 1976). Recently, Obijeski et al. (Nucleic Acids Res. 8:2431-2438) have found that the 5' and 3' ends of each segment are complementary in sequence. We determined that a 5' and 3' end complementary structure, predicted by the rules of Tinoco et al. (Nature [London] 230:362-367), can and will anneal under certain conditions. This structure is resistant to RNase in high-salt medium and can be isolated in a reasonably high yield.

Base Sequence↗

PubMed1981

Mechanism of cerebral arteriolar abnormalities after acute hypertension.

Acute severe hypertension induced by intravenous norepinephrine or angiotensin in anesthetized cats equipped with a cranial window caused prolonged arteriolar vasodilation associated with reduced responsiveness to arterial hypercapnia or hypocapnia and passive response to changes in arterial blood pressure. Scanning and transmission electron microscopy of such pial arterioles showed discrete destructive endothelial lesions the density of which correlated with the degree of vasodilation. Abnormalities of the vascular smooth muscle were seen in all dilated arterioles but affected only a small number of smooth muscle cells. The oxygen consumption of pial arterioles from cats subjected to hypertension was significantly reduced in comparison to that of vessels from normal animals. The arteriolar abnormalities induced by hypertension were inhibited by pretreatment with inhibitors of cyclooxygenase (indomethacin or AHR-5850) or by topical application on the brain surface of scavengers of free oxygen radicals (mannitol or superoxide dismutase). The results suggest that the mechanism of the arteriolar abnormalities from acute hypertension involves a sudden increase in prostaglandin synthesis that leads to generation of free oxygen radicals.

Animals↗

PubMed1981

Oxidative metabolic activity of cerebral cortex after fluid-percussion head injury in the cat.

To assess the metabolic and vascular effects of head trauma, fluid-percussion pressure waves were transmitted to the brains of anesthetized, paralyzed, and artificially ventilated cats. Changes in the redox state of cytochrome a,a3, and relative local blood volume were measured in situ by dual-wavelength reflection spectrophotometry of the cortical surface viewed through an acrylic cranial window implanted within the closed skull. Initial fluid-percussion impacts of 0.5 to 2.8 atm peak pressure produced consistent transient oxidation of cytochrome a,a3 and increases of cortical blood volume. These changes occurred despite the presence of transient posttraumatic hypotension i some cases. Also, impact-induced alterations of vascular tone occurred, independent of the presence or absence of transient hypertension in the posttraumatic period. These data demonstrate that hypoxia does not play a role in the immediate posttraumatic period in cerebral cortex, and are consistent with the idea that after injury there is increased cortical energy conservation. These data also support the concept that head trauma alters the relationship of metabolism and cerebral circulation in the period immediately after injury.

Animals↗

PubMed1980

Isopiestic determination of water binding by fish antifreeze glycoproteins.

The effectiveness of water binding of antifreeze glycoproteins relative to hemoglobin, cytochrome c and polyvinylpyrrolidone was determined by analyzing results obtained in an isopiestic study at 25 degrees C. The net weight of water which moved from a protein/NaCl aqueous sample to a saturated NaCl reference solution increased in the order: antifreeze glycoprotein, hemoglobin, polyvinylpyrrolidone and cytochrome c. Since the glycoproteins were least effective in transporting water we conclude that, of the proteins studied, the glycoprotein was most effective in binding water under equilibrium conditions at 25 degrees C.

Animals↗

PubMed1980

Dependence of pial arteriolar response to hypercapnia on vessel size.

The responses of pial arterioles to changes in PaCO2 were studied in anesthetized cats equipped with cranial windows for the observation of the pial microcirculation. The percentile increase in vessel caliber induced by hypercapnia was size dependent, with the smaller vessels responding more vigorously than the larger ones. In contrast, the vasoconstrictor responses to arterial hypocapnia were size independent. Following topical application of phentolamine (10(-5) M), or after ipsilateral extirpation of the superior cervical ganglion, the increase in arteriolar caliber caused by moderate hypercapnia (5% CO2 inhalation) in the larger arterioles (greater than 100 micron in diameter) increased and became equal to that of the smaller arterioles (less than 100 micron in diameter). The response to hypocapnia was not affected by phentolamine. The results indicate that a major reason for the greater responsiveness of smaller vessels to moderate hypercapnia is the fact that the response of the larger vessels is limited by neurogenic vasoconstriction resulting from increased activity of adrenergic nerves originating from the cervical sympathetic chain. The absence of an influence of the sympathetic nervous system on the responses to hypocapnia is explained by the lack of significant resting neurogenic vasoconstrictor tone in pial arterioles of anesthetized cats.

Animals↗

PubMed1979

Monitoring the state of the human airways by analysis of respiratory sound.

A mechanism whereby sound is generated by the motion of vortices in the human lung is described. This mechanism is believed to be responsible for most of the sound which is generated both on inspiration and expiration in normal lungs. Mathematical expressions for the frequencies of sound generated, which depend only upon the axial flow velocity and diameters of the bronchi, are derived. This theory allows the location within the bronchial tree from which particular sounds emanate to be determined. Redistribution of pulmonary blood volume following transition from Earth gravity to the weightless state probably alters the caliber of certain airways and doubtless alters sound transmission properties of the lung. We believe that these changes can be monitored effectively and non-invasively by spectral analysis of pulmonary sound.

Adaptation, Physiological↗

PubMed1979

Oxygen consumption of pial arteries.

Pial arterioles and arteries, normally responsive to alterations in PaCO2, were obtained by microdissection from the parietal cortex of 53 anesthetized cats. The oxygen consumption of these vessels, determined with the Cartesian diver microrespirometer, was found to be size dependent. It increased progressively from vessels 60 mum in diameter to vessels 250 mum in diameter. In vessels 250--375 mum in diameter there was a progressive decrease in oxygen consumption with increasing vessel size. The oxygen consumption was not altered by increasing the oxygen concentration in the diver from 20% to 95%, or that of glucose from 1.1 to 2.0 mM. Planimetry of histological sections showed that the percentage of endothelium and smooth muscle increased as vessel diameter increased from 60 to 200 mum, and thereafter decreased with increasing vessel size. When these differences in the composition of the vessel wall were taken into account, there was still residual dependence of the oxygen consumption on vessel diameter. This may represent intrinsic differences in the metabolic rate of vascular smooth muscle in vessels of different size.

Animals↗