Biomedical subjects
Publications and source records attributed to R Parker.
The 3' untranslated region (3' UTR) can control gene expression by affecting the localization, stability and translation of mRNAs. The recent finding that 3' UTRs can control the decapping rate of mRNAs, in combination with their ability to influence the initiation of translation, suggests that 3' UTRs act through a direct or indirect interaction between the 3' and 5' ends of mRNAs.
The effect of fluctuating temperatures on microbial growth is important in the passage of foods from production to consumption. Suspensions of Salmonella typhimurium have been subjected to sinusoidally time-varying temperatures of periods from 60 to 240 min between 4 degrees and 22 degrees C, that is within and below the growth temperature range. The suspensions were prepared with two concentrations of sodium chloride and adjusted to two different values of pH. The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data. Generally, the experimental average generation times exceeded the predictions by not more than 10%. In enumerating viable bacteria in the suspensions containing 3.5% (w/v) sodium chloride it was necessary to use sodium chloride in the diluent and recovery medium in order to recover the bacteria quantitatively.
The RNA14 and RNA15 gene products have been implicated in a variety of cellular processes. Mutations in these genes lead to faster decay of some mRNAs and yield extracts that are deficient in cleavage and polyadenylation in vitro. These results suggest that the RNA14 and RNA15 gene products may be involved in both adenylation and deadenylation in vivo. To explore the roles of these gene products in vivo, we examined the site of adenylation and the rate of deadenylation for individual mRNAs in rna14 and rna15 mutant strains. We observed that the rates of deadenylation are not affected by lesions in either the RNA14 or the RNA15 gene. This result suggests that the proteins encoded by these genes are not involved in regulation of the deadenylation rate. In contrast, we observed that the site of adenylation for the ACT1 transcript can be altered in these mutants. Interestingly, we also observed that mutation of the poly(A) polymerase gene altered the site of ACT1 polyadenylation. These observations suggest that the RNA14, RNA15, and PAP1 proteins are involved in poly(A) site choice. This alteration in poly(A) site choice in the rna14 mutant can be corrected by the ssm4 suppressor, indicating that this suppression acts at the level of polyadenylation and not by slowing mRNA degradation.
The first step in the decay of several yeast mRNAs is the shortening of the poly(A) tail, which for the MFA2 transcript triggers decapping and 5'-to-3' degradation. To understand the basis for differences in mRNA decay rates, it is important to determine if deadenylation-dependent decapping is specific to the unstable MFA2 transcript or is a general mechanism of mRNA degradation. To this end, we analyzed the turnover of the stable PGK1 mRNA by monitoring the decay of a pulse of newly synthesized transcripts while using two strategies to trap decay intermediates. First, we used strains deleted for the XRN1 gene, which encodes a major 5'-to-3' exonuclease in Saccharomyces cerevisiae. In xrn1 delta cells, PGK1 transcripts lacking the 5' cap structure and a few nucleotides at the 5' end were detected after deadenylation. Second, we inserted into the PGK1 5' untranslated region strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates. These secondary structures led to the accumulation of PGK1 mRNA fragments, following deadenylation, trimmed from the 5' end to the site of the secondary structure. The insertion of strong secondary structures into the 5' untranslated region also inhibited translation of the mRNA and greatly stimulated the decay of the PGK1 transcripts, suggesting that translation of the PGK1 mRNA is required for its normally slow rate of decay. These results suggest that one mechanism of degradation of the PGK1 transcript is deadenylation followed by decapping and subsequent 5'-to-3' exonucleolytic degradation. In addition, by blocking the 5'-to-3' degradation process, we observed PGK1 mRNA fragments that are consistent with a 3'-to-5' pathway of mRNA turnover that is slightly slower than the decapping/5'-to-3' decay pathway. These observations indicate that there are multiple mechanisms by which an individual transcript can be degraded following deadenylation.
AIMS: We investigated the therapeutic efficacy of low doses of X-rays on different in-vivo models of monoarticular arthritis which have been developed for the investigation of anti-inflammatory drugs. MATERIAL AND METHODS: Zymosan or heat-inactivated mycobacterium tuberculosis was injected into 1 knee joint of Wistar rats to produce, via different pathogenetic mechanisms, an acute monoarticular arthritis. Five days later, the amount of joint swelling, bone destruction and cartilage catabolism were measured. Immediately after arthritis induction, the knees were irradiated with a single dose of 5 Gy or with 4 daily fractions of 1 Gy. RESULTS: X-irradiation with daily doses of 1 Gy significantly reduced bone loss and cartilage degradation in Zymosan-induced arthritis and joint swelling in mycobacterium tuberculosis induced arthritis. However, a single high radiation dose significantly increased bone loss in mycobacterium tuberculosis induced arthritis. CONCLUSIONS: These data confirm the hypothesis of an anti-inflammatory effect of low radiation doses which so far has been based only on clinical experience. By using an established model of monoarticular arthritis we have now the opportunity to study the mechanism of the anti-inflammatory radiation effect in comparison to that of anti-inflammatory drugs. This way, we hope to provide a scientific basis for the use of radiotherapy in various painful degenerative joint disorders.
The cytokine interleukin-1 alpha (IL-1 alpha) showed a cytostatic effect on human ovarian carcinoma cells and significantly enhanced the antiproliferative activity of cis-diamminedichloroplatinum(II) (cisplatin) toward the NIH:OVCAR-3 tumor cell line in culture. The factor of sensitization was 15-20-fold. The maximum levels of sensitization were observed both with simultaneous exposure to cisplatin and IL-1 alpha and with 24-hr pretreatment with IL-1 alpha. Synergy between these agents was diminished when cells were pretreated with an IL-1 alpha-specific receptor antagonist, indicating that synergistic interaction was receptor mediated. Using atomic absorption spectroscopy, we evaluated the cellular accumulation of cisplatin and the DNA platination; the results showed that IL-1 alpha increased cellular accumulation of cisplatin and DNA platination. Cisplatin did not affect IL-1 alpha accumulation in NIH:OVCAR-3 cells. Further studies showed that IL-1 alpha reduced the removal of platinum from DNA. These results strongly suggest that IL-1 alpha inhibits DNA repair, and this decrease in DNA repair may explain, in part, the strong synergistic interaction between IL-1 alpha and cisplatin in NIH:OVCAR-3 cells.
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The degradation of messenger RNA in eukaryotic cells is initiated by endonucleolytic cleavage or by shortening of the poly(A) tail, which for some mRNAs activates a deadenylation-dependent decapping reaction. One type of rapid mRNA degradation in eukaryotes is caused by premature termination of translation. This turnover process prevents the translation of aberrant mRNAs, may affect the abundance and splicing pattern of nuclear transcripts, and may be involved in the aetiology of human genetic disease. Here we show that premature translational termination in yeast triggers decapping, independent of deadenylation, thereby exposing the transcript to 5'-to-3' degradation. Inactivation of the 5'-to-3' exonuclease reveals an additional 3'-to-5' pathway of mRNA turnover. These observations provide in vivo evidence for two new mechanisms of mRNA decay.
The first step in the decay of some eukaryotic mRNAs is the shortening of the poly(A) tail. To examine how the transcript body was degraded after deadenylation, we followed the decay of a pulse of newly synthesized MFA2 transcripts while utilizing two strategies to trap intermediates in the degradation pathway. First, we inserted strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates, into the MFA2 5' UTR. Following deadenylation, fragments of the MFA2 mRNA trimmed from the 5' end to the site of secondary structure accumulated as full-length mRNA levels decreased. In addition, in cells deleted for the XRN1 gene, which encodes a major 5' to 3' exonuclease in yeast, the MFA2 transcript is deadenylated normally but persists as a full-length mRNA lacking the 5' cap structure. These results define a mRNA decay pathway in which deadenylation leads to decapping of the mRNA followed by 5'-->3' exonucleolytic degradation of the transcript body. Because the poly(A) tail and the cap structure are found on essentially all mRNAs, this pathway could be a general mechanism for the decay of many eukaryotic transcripts.
Several observations in eukaryotic cells suggest that the processes of translation and mRNA turnover are interrelated. To understand this relationship, we examined the effects of translational inhibition on the decay of the unstable yeast MFA2 mRNA, which is degraded in a 5' to 3' direction following deadenylation (1). Although inhibition of translation in cis stabilizes several unstable mammalian transcripts, inhibiting translation of the MFA2 mRNA in cis, by the insertion of a large stem-loop structure in the 5'-untranslated region (UTR), did not affect the half-life, deadenylation rate, or appearance of specific decay intermediates. Therefore, efficient translational elongation on the MFA2 mRNA is not a requirement for the normal rate, or mechanism, of degradation of this transcript. In contrast, inhibition of translation in trans, by the addition of cycloheximide, stabilized the deadenylated form of MFA2 mRNA. Furthermore, the MFA2 transcripts that were not translated due to a stem-loop in the 5'-UTR were also stabilized in the presence of cycloheximide, suggesting that cycloheximide is likely to affect mRNA stability indirectly. These results suggest possible relationships between the mechanisms of mRNA decay and the translational process.
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Nasopharyngeal carcinoma recurrent following primary radiation therapy has been treated with surgery and reirradiation. Reirradiation is often limited by the tolerance of structures previously treated. Radiosurgery was used to boost the recurrent site while avoiding critical structures. Seven patients were evaluated for treatment. Three patients met requirements for treatment. The lesions invaded the parapharyngeal region, the base of skull, cavernous sinus, cranial nerves, or carotid artery. Treatment included a radiosurgery boost utilizing multiple isocenters, noncoplanar arcs, and arc weighting, to yield a plan conforming to the tumors while avoiding critical anatomical structures. The patients tolerated the procedure well with minor acute side effects. Follow-up included magnetic resonance imaging (MRI) and positron emission tomography (PET). Two lesions responded, and one had no significant change. One patient had a regional recurrence. Two patients had distance recurrence. Long term side effects include trismus, parotiditis, ear fullness, hemorrhage, and pain. Radiosurgery may improve the local control rate of such lesions, however, with the severe long term complications of single fraction radiosurgery in the head and neck region this procedure may be more beneficial if the treatment is fractionated.
Analysis of 1657 lost-time logging accidents in the New Zealand logging industry (1985-1991) indicates that 17.5% were as a result of slips, trips and falls and a total of 2870 days were lost. Most (56%) of these slipping, tripping and falling accidents occurred in the felling and delimbing phase of the logging operation, where 37% of the workforce are employed. In an attempt to reduce the number of slipping injuries to loggers employed in felling and delimbing, a study of the effectiveness of spike-soled (caulk) boots was undertaken. Four loggers were intensively observed at work, by continuous time-study methods, while wearing their conventional rubber-soled boots and then spike-soled boots. The number of slips, work methods used, physiological workload and productivity were compared for loggers wearing the two footwear types. Results indicated that spike-soled boots were associated with a significant reduction in the frequency of slips and had no adverse effect on work methods, physiological workload or productivity. Spike-soled boots are now being promoted for use by loggers in New Zealand as a simple method to reduce slipping, tripping and falling accidents.
Recent experiments have identified distinct mechanisms of eukaryotic RNA turnover. In one mechanism, deadenylation triggers decapping, exposing the messenger RNA to 5' to 3' degradation. This pathway may act at different rates on the majority of messenger RNAs. There are also degradation mechanisms, such as endonucleolytic cleavage, limited to messenger RNAs containing specific sequence elements.
OBJECTIVE: The pathogenesis of preterm birth and other adverse pregnancy outcomes linked with reproductive tract infection remains poorly understood. Mucolytic enzymes, including mucinases and sialidases (neuraminidase), are recognized virulence factors among enteropathogens and bacteria that cause periodontal infection. Perturbation of maternal cervicovaginal mucosa membrane host defenses by such enzyme-producing microorganisms may increase the risk of subclinical intrauterine infection during pregnancy and thus increase risks of preterm birth. STUDY DESIGN: We prospectively evaluated vaginal fluid mucinase and sialidase and selected cervicovaginal bacteria along with pregnancy outcomes in 271 women. Within this study, women with bacterial vaginosis (16 to 27 week' gestation) were treated with 2% clinadmycin vaginal cream or placebo. Enzyme, microbial findings, treatment effects, and pregnancy outcomes were compared among drug- and placebo-treated women and control women without bacterial vaginosis. RESULTS: Presence of bacterial vaginosis at intake was associated with increased risk of preterm birth (relative risk 3.3, 95% confidence interval 1.2 to 9.1, p = 0.02), premature rupture of membranes (relative risk 3.8, 95% confidence interval 1.6 to 9.0, p = 0.002), and preterm premature rupture of membranes. Mucinase and sialidase activities were more commonly identified, and they occurred in higher concentrations, if present, in women with bacterial vaginosis (mucinase: 44.3% with bacterial vaginosis vs 27.4% without, p = 0.007; sialidase: 45% with bacterial vaginosis vs 12% without p < 0.001). Sialidase activity was associated with bacterial vaginosis-linked organisms (Gardnerella vaginalis, Mobiluncus spp, and Mycoplasma hominis) and Chlamydia trachomatis and yeast species; mucinase activity was associated only with bacterial vaginosis-linked microorganisms. Clindamycin, 2% cream, was effective treatment for bacterial vaginosis and temporarily reduced mucinase and sialidase activities. Topical treatment of bacterial vaginosis did not reduce risks of perinatal morbidity. Women with persistent or recurrent sialidase 8 weeks after treatment were at increased risk of preterm birth (15.6% vs 7.4%) premature rupture of membranes (30% vs 15%), and low birth weight (20% vs 3%, relative risk 6.8, 95% confidence interval 1.6 to 28.1). CONCLUSIONS: Persistence of sialidase-producing vaginal microorganisms in numbers sufficient to increase vaginal fluid sialidase activity may be a risk factor for possibly preventable subclinical intrauterine infection and preterm birth. This study confirms and further informs our understanding of the association of bacterial vaginosis and preterm birth; studies to evaluate whether systemic treatment for bacterial vaginosis can effectively reduce vaginal mucolytic enzymes and risks of prematurity and other morbid outcomes are continuing.
The effect of fluctuating temperatures on microbial growth is important in the passage of foods through the food chain. Suspensions of Salmonella typhimurium were subjected to sinusoidally time-varying temperatures of periods from 40 to 480 min within their growth temperature range. The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data. The experimental average generation times exceeded the predictions by less than 30%, although the discrepancy increased with cycle frequency. Instantaneous growth rates obtained for the 480 min cycles were in agreement with those predicted from isothermal behaviour.
Treatment with zidovudine has been standard therapy for patients with advanced HIV infection, but intolerance is common. Previously, management of intolerance has consisted of symptomatic therapy, dose interruption/discontinuation, and, when appropriate, transfusion. The availability of new antiretroviral agents such as didanosine as well as adjunctive recombinant hematopoietic growth factors makes additional strategies possible for the zidovudine-intolerant patient. Because all of these agents are costly, we evaluated the cost implications of these various strategies for the management of zidovudine-intolerant individuals within a population of persons with advanced HIV disease. We performed a decision analysis using iterative algorithmic models of 1 year of antiretroviral care under various strategies. The real costs providing antiretroviral therapy were estimated by deflating medical center charges by specific Medi-Cal (Medicaid) charge-to-payment ratios. Clinical data were extracted from the medical literature, product package inserts, investigator updates, and personal communications. Sensitivity analysis was used to test the effect of error in the estimation of parameters. The models predict that a strategy of dose interruption and transfusion for zidovudine intolerance will provide an average of 46 weeks of therapy per year to the average patient at a cost of $5,555/year of therapy provided (1991 U.S. dollars). The models predict that a strategy of adding hematopoietic growth factors to the regimen of appropriate patients would increase the average amount of therapy provided to the average patient by 3 weeks (6%) and the costs attributable to therapy by 77% to $9,805/year of therapy provided.(ABSTRACT TRUNCATED AT 250 WORDS)
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