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Biomedical subjects

R Mark Payne

Publications and source records attributed to R Mark Payne.

13 recordsLinked to original sources
PubMed2006

Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth.

Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophies. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of the cardiac working load. However, the functional significance of this biological event has not been determined, mainly because of the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (alphaMHC-BMP10) in which the cardiac-specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These alphaMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. alphaMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in alphaMHC-BMP10 mice was solely because of defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the responses of these hearts to both physiological (e.g. exercise-induced hypertrophy) and pathological hypertrophic stimuli remain normal. In addition, the alphaMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by four weeks of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophies.

Animals↗
PubMed2006

Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria.

Supporting a co-translational model of protein import into mitochondria, we have previously shown that ribosome-nascent chain complexes (RNCs) specifically bind to mitochondria. When producing RNCs using the rabbit reticulocyte lysate in vitro translation system, it was necessary to maximize ribosome loading with truncated nascent proteins because it had a direct impact on RNC binding. We describe here the optimal conditions for preparing RNCs. We show that translation temperature and reaction time are two critical factors, with 30 degrees Celsius and 15min being optimal, respectively. We also show that transcription reactions can be used directly in the translation reaction to create RNCs.

Animals↗
PubMed2006

The pathogenesis of pulmonary contusion: an open chest model in the rat.

BACKGROUND: Chemokines direct leukocytes to areas of inflammation or injury. In general, CC chemokines (MCP-1, MIP-1alpha, RANTES) are chemoattractants for mononuclear cells and CXC (CINC-1, MIP-2alpha) for polymorphonuclear cells (PMNs). Herein we describe an open chest model of pulmonary contusion (PC) in a rodent (rat) and have identified a possible role for CC and CXC chemokines in the pathogenesis of PC. METHODS: Sprague-Dawley rats (350 g) underwent thoracotomy. The exposed lung was struck with a piston at 5.2 m/s (150 J/M2). Blood, bronchoalveolar lavage (BAL), and lung tissue were collected at 3 hours and 24 hours after injury. PaO2/FiO2 (P/F) ratio was calculated at 15-minute intervals for 3 hours after contusion. Serum was evaluated for cytokine and chemokine expression using ELISA. Cell count/differential was performed on BAL, and lung tissue was obtained for histologic analysis, protein expression and wet to dry weights. Data are reported as pg/mL +/- SE. Data were analyzed using Student's t test to identify significant differences (p < or = 0.05 significant) between sham and injured animals. RESULTS: Piston impact caused PC based upon morphologic and histologic criteria. BAL cell count and lung wet to dry weights were increased and P/F ratio was decreased after PC. Systemic levels of IL-ra, MCP-1, and the CXC chemokines MIP-2alpha and CINC-1 were significantly elevated at 3 hours when sham and injured animals were compared. All chemokines were found to be significantly elevated at 24 hours, consistent with the early PMN and subsequent mononuclear infiltration observed in the contused lung. Pulmonary expression of TNF-alpha, IL-1beta, CINC-1, MIP-2alpha, ICAM-1, and elastase were increased and activated systemic neutrophils showed increased CD-11b. CONCLUSION: A model of PC is described in which innate inflammation is activated locally and systemically. Systemic levels of CC and CXC chemokines are increased after PC. This correlates with elevated PMN CD-11b expression, enhanced pulmonary ICAM-1 expression, and mononuclear and PMN infiltration into the lung and alveolar space. Elevated levels of CC and CXC chemokines are seen after PC and may be involved in the lung's inflammatory response to injury.

Animals↗
PubMed2005

Development of a finite element-based injury metric for pulmonary contusion part I: model development and validation.

Pulmonary contusion is the most commonly identified thoracic soft tissue injury in an automobile crash and after blunt chest trauma and affects 10-17% of all trauma admissions. The mortality associated with pulmonary contusions is significant and is estimated to be 10-25%. Thus, there is a need to develop a finite element model based injury metric for pulmonary contusion for the purpose of predicting outcome. This will enable current and future finite element models of the lung to incorporate an understanding of how stress and strain may be related to contusion injuries. This study utilizes 14 impacts onto male Sprague-Dawley rats. In 5 of these tests, a calibrated weight (46 g) is dropped from a height of 44 cm directly onto the lungs of intubated, anesthetized rats in situ. Contused volume is estimated from MicroPET scans of the lung and normalized on the basis of liver uptake of 18F-FDG. The lungs are scanned at 24 hours, 7 days, and 28 days (15 scans), and the contused volume is measured. In addition, 9 controlled mechanical tests on in situ rat lung are used for model development and validation. Identical impacts are performed on a finite element model of the rat lung. The finite element model is developed from CT scans of normal rat and scaled to represent average rat lung volume. First principal strain is chosen as a candidate injury metric for pulmonary contusion. The volume of contused tissue at the three time points measured using PET is compared to the strain level achieved by a corresponding volume in the finite element model. For PET scans (n=5 scans per time point), the average contusion volume was 4.2 cm3 at 24 hours, 2.8 cm3 at 7 days, and 0.39 cm3 at 28 days. These volumes were used to identify threshold peak first principal strain levels measured by the finite element model. Maximum first principal strain from the finite element model for the three volume levels (4.2, 2.8, and 0.39 cm3) was 3.5%, 8.8%, and 35% strain, respectively. Furthermore, the lung model exhibited exponential decay in principal strain threshold as more of the lung volume was considered, correlating to the precise and well defined volume of the contusion as it healed. The results of this study may be used to establish an injury metric to predict pulmonary contusion due to an impact to the lungs. The results may be used to improve finite element models of the human body, which may then be used to tune stiffnesses of interior components of automobiles and tune safety systems for maximum mitigation of this serious injury.

Journal Article↗
PubMed2004

Transactivator of transcription fusion protein transduction causes membrane inversion.

The transactivator of transcription (TAT) protein transduction domain is an 11-amino acid positively charged peptide that has been shown to pull diverse molecules across cell membranes in vitro and in vivo. Fusion proteins constructed with TAT rapidly enter and exit cells and have been shown to cross intracellular membranes as well. Electrostatic interactions between TAT and the cell membrane have been implicated as a part of the mechanism of transduction. Here, we report that TAT transduction causes membrane phospholipid rearrangement as evidenced by detection of phosphatidylserine on the outer surface of the cell membrane. Furthermore, these rearrangements can be blocked by positively charged polylysine, further implicating electrostatic interactions as a part of the mechanism. Neither apoptosis nor necrosis is induced in these cells after exposure to TAT. We conclude that the process of TAT.GFP transduction causes phosphatidylserine to translocate from the inner to the outer leaflet of the plasma membrane. These results provide insight into the mechanism of TAT protein transduction domain transduction.

Animals↗
PubMed2003

Ribosomes specifically bind to mammalian mitochondria via protease-sensitive proteins on the outer membrane.

The interaction of ribosomes with specific components of membranes is one of the central themes to the co-translational targeting and import of proteins. To examine ribosome binding to mammalian mitochondria, we used ribosome-nascent chain complexes (RNCs) to follow the in vitro binding of ribosomes that correspond to the initial targeting stage of proteins. Mitochondria were found to contain a limited number of RNC binding sites on the outer membrane. It required more than twice the amount of non-translating ribosomes to inhibit RNC binding by one-half, indicating that RNCs have a competitive binding advantage. In addition, we found that RNCs bind mainly through the ribosomal component and not the nascent chain. RNCs bind via protease-sensitive proteins on the outer membrane, as well as by protease-insensitive components suggesting that two classes of receptors exist. We also show that binding is sensitive to cation conditions. Nearly all of the binding was inhibited in 0.5 m KCl, indicating that they interact with the membrane primarily through electrostatic interactions. In addition, disruption of RNC structure by removing magnesium causes the complete inhibition of binding under normal binding conditions indicating that it is the intact ribosome that is crucial for binding and not the nascent chain. These findings support the hypothesis that the outer mitochondrial membrane contains receptors specific for ribosomes, which would support the conditions necessary for co-translational import.

Animals↗
PubMed2003

Neuroprotection against hypoxia-ischemia in neonatal rat brain by novel superoxide dismutase mimetics.

We investigated the effects of two new low molecular weight nonpeptidyl superoxide dismutase (SOD) mimetics (M40403/M40401; MetaPhore Pharmaceuticals) on infarct volume after hypoxia-ischemia injury (H/I) in immature rats. Animals received vehicle or different doses of M40403 or M40401 i.p. 2 h before exposure to 3 h of 8% hypoxia. The infarct volume of the hemisphere ipsilateral to carotid ligation 24 h later was 73.9+/-8.9% in vehicle animals (n=9), and decreased to 39.7+/-7.2% (P<0.05, n=10) in animals treated with 3 mg/kg M40403 and to 37.2+/-6.4% for animals receiving 3 mg/kg M40401 (P<0.05, n=8). These data indicate that the SOD mimetics M40403 and M40401 have protective effects against hypoxic-ischemic brain injury, and suggest the involvement of superoxide anion in neuronal cell injury during H/I.

Animals↗
PubMed2003

Protective effect of a new nonpeptidyl mimetic of SOD, M40401, against focal cerebral ischemia in the rat.

We tested the neuroprotective effects of M40401, a new, low molecular weight (511.4 Da) maganese superoxide dismutase mimetic, against 90 min of middle cerebral artery occlusion (MCAO) in male Wistar rats. Animals received a single injection of vehicle (n=8), 1 mg/kg (n=6), or 3 mg/kg (n=7) 30 min before MCAO. Total lesion volume was reduced only in the group receiving 3 mg/kg M40401 (163.5+/-18.7 versus 43.4+/-7.0 mm(3), for vehicle and M40401, respectively; P<0.05), with almost complete reduction of lesion volume in the cortex but little protection in the basal ganglia. Neurological score was also improved in this group. The dose of 1 mg/kg M40401 had smaller and inconsistent effects on lesion parameters. Administration of a single dose of 3 mg/kg M40401 at 60 min of MCAO or at the end of MCAO (90 min) failed to significantly reduce lesion volume. A single dose of M40401 plus prolonged infusion into the post-MCAO period also failed to decrease lesion volume significantly. These data indicate that M40401 protects cerebral tissue from ischemic insult when administered before MCAO, probably by limiting damage mediated by detrimental actions of superoxide anion.

Animals↗
PubMed2003

Video-assisted ductal ligation in premature infants.

BACKGROUND: Video-assisted thoracic surgery has been shown to be a safe and effective method of closing the patent ductus arteriosus in infants and children. We have applied this technique in low birth weight premature infants and now report our experience. METHODS: Since 1996, we have used video-assisted thoracic surgery ligation as the treatment of choice for all patent ductus arteriosus, including 100 performed on premature infants (23 to 31 weeks' gestation, mean 25.6 weeks; 0.420 to 1.5 kg, mean 0.859 kg). A modification of our previously described technique was used with a three-port approach. All patients had some degree of symptoms of congestive failure with failure to wean from ventilatory support or oxygen dependency. Five infants had associated patent foramen, and 1 had a small ventricular septal defect. RESULTS: All 100 procedures were performed in the operating room. One infant was found to have a coarctation, and the procedure was aborted. The remaining 99 were successfully ligated, although three were converted to an open procedure (3%) because of coagulopathy, poor pulmonary compliance, or hemodynamic instability. There were no procedure-related deaths; however, 15 infants subsequently died of complications of prematurity, including enterocolitis, sepsis, and late respiratory failure. Six infants had chest tubes left in place for coagulopathy, effusions, suspected air leak, and existing empyema. There were six residual pneumothoraces, four requiring treatment. CONCLUSIONS: Video-assisted thoracic surgery is a safe and effective technique for patent ductus arteriosus ligation in premature infants, including those with very low and extremely low birth weight.

Cohort Studies↗
PubMed2003

A novel TAT-mitochondrial signal sequence fusion protein is processed, stays in mitochondria, and crosses the placenta.

Mutations in nuclear and mitochondrial genomes can lead to defects in mitochondrial function. To date, repair of these defects with exogenous proteins or gene transfer has been difficult with either viral or nonviral vectors. We hypothesized that TAT fusion proteins would cross both mitochondrial membranes and that incorporation of a mitochondrial signal sequence into a TAT fusion protein would allow processing and localization of exogenous proteins in mitochondria. A TAT-mitochondrial malate dehydrogenase signal sequence (mMDH)-enhanced green fluorescent protein (eGFP) fusion protein was constructed. TAT-mMDH-eGFP allowed rapid transduction and localization of fusion protein into mitochondria of multiple cell types. In contrast, TAT-GFP, without a mitochondrial signal sequence, rapidly transduced into cells and mitochondria, displayed pseudo-first-order kinetics, but did not remain there. Mice injected 5 days prior with TAT-mMDH-eGFP had detectable eGFP activity in multiple tissue types. Western blotting of cytosolic and mitochondrial fractions isolated from their livers confirmed eGFP localization to mitochondria and that the mMDH transit peptide was recognized and processed. Furthermore, TAT-mMDH-eGFP fusion protein injected into pregnant mice crossed the placenta and was detectable in both the fetus and the newborn pups. TAT fusion proteins containing a mitochondrial signal sequence are a viable method to localize proteins to mitochondria.

Animals↗
PubMedDate not supplied

Targeting proteins to mitochondria using TAT.

Disorders of mitochondrial function cause significant human disease and suffering. To date, correction of these mitochondria defects has depended on biochemical approaches and has not been achieved via gene therapies. Using previously described fusion proteins containing the transactivator of transcription (TAT) region from the HIV virus and green fluorescent protein (GFP), with and without a mitochondrial targeting sequence (MTS) from mitochondrial malate dehydrogenase (mMDH), we have investigated transduction across mitochondrial membranes. Both TAT-GFP and TAT-mMDH-GFP fusion proteins are protected from externally added protease when incubated with isolated mitochondria. Furthermore, both TAT fusion proteins rapidly enter cultured cells and transduce into mitochondria. However, the MTS allows processing of the fusion protein and is necessary for persistence in mitochondria over time. Neither degradation of import receptors nor disruption of the mitochondrial membrane potential or pH gradient inhibits protein transduction of either fusion protein. Furthermore, when injected into pregnant mice, TAT-mMDH-GFP is detectable throughout fetal and neonatal pups. These results indicate that TAT fusion proteins are able to traverse mitochondrial membranes through mechanisms that do not involve the regular import pathway, and that the addition of a MTS allows persistence of the fusion protein within mitochondria. TAT-MTS fusion proteins may represent a viable option as potential mitochondrial protein therapies.

Animals↗
PubMedDate not supplied

Evaluation of the quantitative capability of a high-resolution positron emission tomography scanner for small animal imaging.

OBJECTIVE: The quantitative capability of a positron emission tomography scanner for small animal imaging was evaluated in this study. METHODS: The microPET P4 (Concorde Microsystems, Knoxville, TN) scanner's capability for dynamic imaging and corrections for radioactive decay, dead time, and attenuation were evaluated. Rat brain and heart studies with and without attenuation correction were compared. A calibration approach to convert the data to nanocuries per milliliter was implemented. Calibration factors were determined using calibration phantoms of 2 sizes with and without attenuation correction. Quantitation was validated using the MiniPhantom (Data Spectrum, Chapel Hill, NC) with hot features (5:1 ratio) of different sizes (4, 6.4, 8, 13, and 16 mm). RESULTS: The microPET P4 scanner's ability to acquire dynamic studies and to correct for decay, dead time, and attenuation was demonstrated. The microPET P4 scanner provided accurate quantitation to within 6% for features larger than 10 mm. Sixty percent of object contrast was retained for features as small as 4 mm. CONCLUSIONS: The microPET P4 scanner can provide accurate quantitation.

Animals↗