The importance of mRNA structure in determining the pathogenicity of synonymous and non-synonymous mutations in haemophilia.

INTRODUCTION: Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled insight into genotype-phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non-synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half-life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated. AIM: To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease-associated mutations in haemophilia. METHODS: Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease-associated and synonymous vs. non-synonymous point mutations in F8, F9 and a third X-linked gene, DMD (dystrophin). RESULTS: In F8 and DMD, disease-associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101-151 nucleotide fragment length appears to be a feasible range for structuring future studies. CONCLUSION: mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease-associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications.

Vitreous-induced modulation of integrins in retinal pigment epithelial cells: effects of fibroblast growth factor-2.

Growth in the presence of vitreous results in transformation of human RPE cells from an epithelioid to a fibroblast-like appearance and leads to an elevation of the expression of alpha(5) and alpha(2) integrins, while the level of alpha(3) integrin is reduced. These changes are inhibited by the presence of FGF-2. Vitreous treatment increases mobility, as does antibody neutralization of FGF-2 or antibody blockade of FGF receptors. The vitreous-induced rise in mobility depends on an increase in alpha(5) integrin expression since it is inhibited by anti-alpha(5) integrin antibodies. Expression of alpha(5) integrin as a result of infection of RPE cells with an alpha(5) integrin-encoding adenovirus induced morphological transformation and an increase in mobility similar to that seen with vitreous. It is concluded that a decrease in FGF-2 plays an important role in vitreous-induced alterations of RPE cell morphology, integrin expression and mobility. High FGF-2 levels prevent at least some of the increased mobility of RPE cells induced by vitreous. This is mediated via extracellular FGF-2 binding to FGF receptor(s) since antibodies to FGF-2 or to its receptor(s) mimic the effects of vitreous. Changes in mobility and morphology involve altered alpha(5) integrin expression since mobility is blocked by antibodies against these proteins while elevated alpha(5) integrin expression increases mobility and leads to morphological changes.

The effect of road improvements on motor vehicle crash injury and mortality in a developing country.

The developing island-nation of Grenada undertook a major road improvement project between 1995 and 1998. In order to determine the effect of those improvements on crash injury and mortality, we reviewed the number of crash-related injuries and deaths for the years 1994 and 1999. After the road improvements, the proportion of crashes that resulted in injury decreased from 22% to 15%. The raw number of crashes (818 vs. 1233), deaths (4 vs. 12) and deaths per 100,000 population (4.1 vs. 11.9) increased, but the proportion of crashes that resulted in death (0.5% vs. 0.7%) remained effectively unchanged.

Use of local climatic data to determine if weather precludes the operation of an air medical system.

INTRODUCTION: Weather is one of many factors that affect safety in an air medical program. Syracuse, New York, has notoriously bad weather, and some have questioned whether an air medical service is practical given central New York's climate. This study was undertaken to determine the extent to which the area's climate could be expected to limit the availability of an air medical service. METHODS: CAMTS weather minimums for rotor-wing programs were compared with 1996-1997 hourly weather observations from the Northeastern Regional Climate Center (NRCC) and sunrise/sunset data from the United States Naval Observatory to determine how frequently weather conditions could be expected to preclude an air medical response in the greater Syracuse area. RESULTS: Exactly 17,544 hourly observations were made. CAMTS weather minimums would have precluded local flights for 606 (3.5%) of these hours and cross-country flights for 1111 (6.3%) hours. Cross-country flights were more likely to be precluded than local flights (P = .001), and both local and cross-country flights were more likely to be precluded at nighttime than in the daytime (P = .001). All flights were more likely to be precluded during winter months than during summer months (P = .000). CONCLUSION: The weather in central New York generally does not preclude the operation of an air medical services system.

Rural vs urban motor vehicle crash death rates: 20 years of FARS data.

OBJECTIVES: Historically, motor vehicle crash (MVC)-related mortality is higher in rural areas than in urban areas. The authors evaluated whether the difference in rural and urban MVC deaths is persisting, and whether the frequency of patients being found dead at the scene, particularly in rural areas, is increasing. METHODS: Fatal Accident Reporting System (FARS) data for 1977 through 1996 were reviewed. The authors determined the frequency with which crash deaths occurred, and calculated population-based and vehicle-miles-traveled-based crash death rates. They compared rates for urban and rural areas. RESULTS: A total of 875,405 crash deaths were included in the analysis. Both population-based and vehicle-miles-traveled-based MVC deaths have decreased over the last 20 years, but rural rates remain significantly higher than urban rates. Dead-at-scene rates may be increasing, and the rural dead-at-scene rate is higher than the urban rate. CONCLUSION: While MVC death rates are declining, the rural MVC death rate is still higher than the urban rate. Although these data may indicate some positive movement in the area of MVC-related deaths, differences in the rural and urban rates and the number of patients found dead on-scene remain as issues that require attention.

Do warning lights and sirens reduce ambulance response times?

OBJECTIVE: To determine the time saving associated with lights and siren (L&S) use during emergency response in an urban EMS system. METHODS: This prospective study evaluated ambulance response times from the location at time of dispatch to the scene of an emergency in an urban area. A control group of responses using L&S was compared with an experimental group that did not use L&S. An observer was assigned to ride along with ambulance crews and record actual times for all L&S responses. At a later date, an observer and an off-duty paramedic in an identical ambulance retraced the route--at the same time of day on the same day of the week--without using L&S and recorded the travel time. Response times for the two groups were compared using paired t-test. RESULTS: The 32 responses with L&S averaged 105.8 seconds (1 minute, 46 seconds) faster than those without (95% confidence interval: 60.2 to 151.5 seconds, p = 0.0001). The time difference ranged from 425 seconds (7 minutes, 5 seconds) faster with L&S to 210 seconds (3 minutes, 30 seconds) slower with L&S. CONCLUSION: In this urban EMS system, L&S reduce ambulance response times by an average of 1 minute, 46 seconds. Although statistically significant, this time saving is likely to be clinically relevant in only a very few cases. A large-scale multicenter L&S trial may help address this issue on a national level.

Motor vehicle safety: current concepts and challenges for emergency physicians.

Motor vehicle travel is the primary means of transportation in the United States, providing freedom in travel and enterprise for many people. Motor vehicle crashes are the leading cause of death for the young and contribute to a high degree of morbidity and mortality for all ages. Motor vehicle crashes produce an enormous burden for society in terms of suffering, disability, death, and costs. Motor vehicle crash injury prevention is developing as a focused discipline to implement proven interventions involving technology and behavior known to prevent or reduce the severity of motor vehicle crash injury. Emergency physicians have an important role in advocating motor vehicle safety and injury prevention, both in the emergency department and within the community.

Expression of antioxidant protective proteins in the rat retina during prenatal and postnatal development.

PURPOSE: In retinopathy of prematurity, capillary growth in the retina is attenuated. Subsequent cyclic elevation of oxygen levels leads to renewed capillary growth that may eventually result in retinal detachment. It is hypothesized that the sensitivity of the premature retina to oxidative shock results from the absence of antioxidant protective proteins. METHODS: The expression of heme oxygenase-1, metallothionein, superoxide dismutase, and catalase mRNAs was measured in retinas of rats from 6 days before birth to 4 days after birth using in situ hybridization and semiquantitative reverse transcription-polymerase chain reaction with Southern blot analysis. RESULTS: Superoxide dismutase mRNA was expressed to a similar extent at all time points. Metallothionein mRNA expression, which was high at embryonic days (E) 16 and 18, decreased to low levels by the time of birth and remained low at least until 4 days after birth. Catalase mRNA expression was low until birth and increased until at least postnatal day 4. Heme oxygenase-1 mRNA showed low expression at E16 and E18, increased before birth, and then diminished. CONCLUSIONS: Four antioxidant protein mRNAs showed very different patterns of expression in the rat retina. Two of these proteins, heme oxygenase-1 and catalase, were expressed at relatively low levels until approximately the time of birth. The former is important in protection against heme-mediated generation of reactive oxygen species, whereas the latter protects against hydrogen peroxide-generated damage. As a result of the low expression of these mRNAs, and presumably the proteins encoded by them, the premature rat (and probably the premature human) is likely to be born without a full complement of antioxidant defenses.

Is mechanism of injury dead?

Mechanism-of-injury data are frequently unavailable to the emergency physician. Furthermore, their value as a triage tool has recently been challenged. Motor vehicle crash patients have fewer external signs of injury, and commonly "look fine and feel fine" because of improvements in vehicle design, increased use of restraints, and airbag deployments. Yet forces involved in the crash may overcome all efforts at protecting the occupant, resulting in occult life-threatening injuries. Without data about the crash, physicians are blinded to violent crash forces that are ultimately transferred to the patient. Information technologies, such as on-scene photography by EMTs, automated collision notification systems, and telemedicine applications, may be used to deliver real-time crash information to aid the emergency physician in caring for these patients. Beyond improving patient care for any given encounter in emergency practice, collecting and evaluating these data will expand our knowledge about how to care for future motor vehicle crash patients.

Vitreous treatment of retinal pigment epithelial cells results in decreased expression of FGF-2.

PURPOSE: Changes in gene expression were investigated after treatment of cultured human retinal pigment epithelial (RPE) cells with vitreous. This may have implications for proliferative diseases such as proliferative vitreoretinopathy. METHODS: Cells were cultured in the presence or absence of human vitreous, and gene expression was examined using the differential display polymerase chain reaction technique. Differentially expressed RNAs were cloned, screened for differential expression, and sequenced. The expression of one of these RNAs (that for fibroblast growth factor [FGF]-2/basic FGF) was examined by in situ hybridization and ribonuclease protection assays. The level of FGF-2 protein was examined by immunoblot analysis. The effects of adding FGF-2 to cells cultured in the presence of vitreous were examined. RESULTS: Treatment of low passage human RPE cells with 25% vitreous resulted in the epithelial-to-fibroblast-like morphologic changes reported by others and in the decreased expression of FGF-2 mRNA and FGF-2 protein. Addition of FGF-2 to cultures at the same time as addition of vitreous prevented some of the effects of vitreous on these cells. CONCLUSIONS: Vitreous treatment of RPE cells in culture results in decreased expression of FGF-2 mRNA and protein. Because supplementation of FGF-2 prevents some of the vitreous-mediated effects, this may indicate that modulation of FGF-2 levels by the vitreous may play an important role in the phenotypic changes seen in RPE cells exposed to vitreous.

Expression of the protective proteins hemopexin and haptoglobin by cells of the neural retina.

The blood-retinal barrier, consisting of retinal pigment epithelial cells and retinal endothelial cells, prevents hemopexin and haptoglobin, anti-oxidant protective plasma proteins normally synthesized by the liver, from entering the neural retina. If present, these proteins must, therefore, be made locally. The cell types within the retina in which hemopexin and haptoglobin mRNAs are made have been investigated. RNA was extracted from both the neural retina and pigment epithelium obtained by dissection of human donor eyes as well as from cultured pigment epithelial and photoreceptor cells. The mRNAs for both haptoglobin and hemopexin were detected, using reverse-transcriptase polymerase chain reaction, in the neural retina and cultured photoreceptors but not in pigment epithelial cells. The cellular location of these mRNAs was determined using in situ hybridization of sections of human retina which revealed that haptoglobin mRNA was located principally in the photoreceptor cells, cells of the inner nuclear layer and some cells of the ganglion cell layer. Hemopexin mRNA, previously shown to be made in the human neural retina (Hunt et al., 1996. Journal of Cellular Physiology 168: 71-80), is expressed by most of the cells of neural retina including the photoreceptors and, notably, the ganglion cells.

Coordination of nitric oxide by heme-hemopexin.

Hemopexin, which acts as an antioxidant by binding heme (Kd < 1 pM), is synthesized by hepatic parenchymal cells, by neurons of the central and peripheral nervous systems, and by human retinal ganglia. Two key regulatory molecules, nitric oxide (.NO) and carbon monoxide (CO), both bind to heme proteins and since ferroheme-hemopexin binds CO, the possible role of heme-hemopexin in binding .NO was investigated. .NO binds rapidly to hemopexin-bound ferroheme as shown by characteristic changes in the Soret and visible-region absorbance spectra. Circular dichroism spectra of .NO-ferroheme-hemopexin in the Soret region exhibit an unusual bisignate feature with a zero crossover at the absorbance wavelength maximum, showing that exciton coupling is occurring. Notably, the .NO complex of ferroheme-hemopexin is sufficiently avid and stable to allow hemopexin to bind this molecule in vivo and, thus, hemopexin may protect against NO-mediated toxicity especially in conditions of trauma and hemolysis.

Role of heme-hemopexin in human T-lymphocyte proliferation.

Heme-hemopexin supports and stimulates proliferation of human acute T-lymphoblastic (MOLT-3) cells, suggesting the participation of heme in cell growth and division. MOLT-3 cells express approximately 58,000 hemopexin receptors per cell (apparent Kd 20 nM), of which about 20% are on the cell surface. Binding is dose- and temperature-dependent, and growth in serum-free IMDM medium is stimulated by 100-1000 nM heme-hemopexin, consistent with the high affinity of the receptor for hemopexin, and maximal growth is seen in response to 500 nM complex. Growth was similar in defined minimal medium supplemented with either low concentrations of heme-hemopexin or iron-transferrin, and either of these complexes were about 80% as effective as a serum supplement. Heme-hemopexin, but not apo-hemopexin, reversed the growth inhibition caused by desferrioxamine showing that heme-iron derived from heme catabolism is used for cell growth. Cobalt-protoporphyrin (CoPP)-hemopexin, which binds to the receptor but is not transported intracellularly [Smith et al., (1993) J. Biol. Chem. 268, 7365], also stimulated cell proliferation in serum-free IMDM but did not "rescue" the cells from desferrioxamine. Furthermore, CoPP-hemopexin effectively competed for the hemopexin receptor with heme-hemopexin and diminished its growth stimulatory effects. In addition, protein kinase C (PKC) is translocated to the plasma membrane within 5 min after heme-hemopexin is added to the medium, reaches maximum activity within 5-10 min, and declines to unstimulated levels by 30 min. Heme-hemopexin and CoPP-hemopexin both augmented MOLT-3 cell growth stimulated by serum. Thus, heme-hemopexin not only functions as an iron source for T-cells but occupancy of the hemopexin receptor itself triggers signaling pathway(s) involved in the regulation of cell growth. The stimulation of growth of human T-lymphocytes by heme-hemopexin is likely to be a physiologically relevant mechanism at sites of injury, infection, and inflammation.

Photograph documentation of motor vehicle damage by EMTs at the scene: a prospective multicenter study in the United States.

The purpose of this study was to determine if emergency medical service (EMS) personnel could take instant photographs of motor vehicle damage at crash scenes depicting the area and severity of damage of the crash under adverse weather conditions, in different lighting, and quickly enough so as not to interfere with patient care. This prospective multicenter trial involved 35 ambulances responding to motor vehicle crash scenes in rural, suburban, and urban areas in five centers in four states. Emergency medical technicians (EMTs) reported their experience implementing a protocol for use of an instant camera to photograph vehicle damage at crash scenes. Time reported by EMTs to take the photographs was 1 minute or less in 204 of 288 (70.9%) of motor vehicle crashes and 2 minutes or longer in 12 of 288 (4.2%) of motor vehicle crashes. From one EMS agency in the study, 48 scene times during which photographs were taken were, on average, 1.5 minutes shorter than 48 scene times immediately before implementation of on-scene crash photography. Photographs were taken in different weather and lighting conditions. EMTs reported they were able to determine both area and severity of damage in 260 of 290 (92.5%) crash photographs, but they were unable to determine area and severity of damage in only 2 of 290 (0.7%) crash photographs.

Production of platelet-derived growth factor by interleukin-1 beta and transforming growth factor-beta-stimulated retinal pigment epithelial cells leads to contraction of collagen gels.

PURPOSE: In an in vitro model of the later contractile stages of proliferative vitreoretinopathy, interleukin-1 beta (IL-1 beta) and transforming growth factor-beta (TGF-beta) stimulate the contraction of collagen gels by retinal pigment epithelial (RPE) cells. This contraction occurs after a lag period and appears not to be a direct effect of the cytokines but is mediated by another factor produced in the presence of the two cytokines. The nature of this factor has been investigated. METHODS: Human RPE cells were seeded onto collagen gels in the presence of IL-1 beta and TGF-beta. After 24 hours, the conditioned medium was removed and added to new collagen gels seeded with RPE cells, and the diameter of the collagen gels was measured after various intervals. The ability of the conditioned medium to effect contraction was determined after various treatments, including size fractionation, heating, trypsin digestion, and binding to heparin-Sepharose. The involvement of platelet-derived growth factor (PDGF) as a stimulator of contraction was tested with neutralizing antibodies and by polymerase chain reaction analyses of specific mRNAs. RESULTS: IL-1 beta and TGF-beta cause RPE cells to contract after a delay of up to 24 hours, whereas conditioned medium from cytokine-treated cells results in immediate contraction in a manner similar to that of serum. The factor in the conditioned medium causing immediate contraction was found to be heat-stable, trypsin-sensitive, and resistant to extremes of pH. It has a size of between 30 and 50 kDa and binds heparin. The factor in conditioned medium from cytokine-treated cells does not act in the presence of C-kinase inhibitors or cycloheximide, suggesting that signaling is mediated by way of protein kinase C and new protein synthesis. Stimulation of contraction by conditioned medium is inhibited by anti-PDGF antibodies, and contraction is stimulated by human PDGF. CONCLUSIONS: Contraction in the presence of cytokines is mediated by the production of PDGF or a PDGF-like molecule. This factor could have implications in the pathogenesis of proliferative vitreoretinopathy.

Assessment of breath sounds during ambulance transport.

STUDY OBJECTIVE: To determine whether the environment of a moving ambulance affects the ability of our-of-hospital care providers to auscultate breath sounds. METHODS: Out-of-hospital care providers assessed breath sounds with a previously described breath-sounds model in a quiet environment (control) and in a moving ambulance. The setting was a nonurban emergency medical services system and an interhospital transport agency based at a 600-plus-bed tertiary care center. The participants were physicians, transport nurses, and advanced life support EMS providers routinely involved in the emergency out-of-hospital treatment and transportation of the ill and injured. The accuracy with which participants identified the presence or absence of breath sounds in the two environments was compared with the use of the chi 2 test, with the alpha-value set at .05. RESULTS: The accuracy of breath-sounds assessment in the control environment was 96% (251 of 260); the sensitivity was 96% and the specificity 97%. The accuracy of breath-sounds assessment in the experimental environment was 54% (140 of 260); the sensitivity was .09% and the specificity 98%. Participants were significantly less likely to hear breath sounds in the moving ambulance than in the quiet room (P < .001). CONCLUSION: Assessment of breath sounds is hampered by the environment of a moving ambulance.