Rat long chain acyl-CoA synthetase 5, but not 1, 2, 3, or 4, complements Escherichia coli fadD

Long chain fatty acids are converted to acyl-CoAs by acyl-CoA synthetase (fatty acid CoA ligase: AMP forming, E.C. 6.2.1.3; ACS). Escherichia coli has a single ACS, FadD, that is essential for growth when fatty acids are the sole carbon and energy source. Rodents have five ACS isoforms that differ in substrate specificity, tissue expression, and subcellular localization and are believed to channel fatty acids toward distinct metabolic pathways. We expressed rat ACS isoforms 1-5 in an E. coli strain that lacked FadD. All rat ACS isoforms were expressed in E. coli fadD or fadDfadR and had ACS specific activities that were 1.6-20-fold higher than the wild type control strain expressing FadD. In the fadD background, the rat ACS isoforms 1, 2, 3, 4 and 5 oxidized [(14)C]oleate at 5 to 25% of the wild type levels, but only ACS5 restored growth on oleate as the sole carbon source. To ensure that enzymes of beta-oxidation were not limiting, assays of ACS activity, beta-oxidation, fatty acid transport, and phospholipid synthesis were also examined in a fadD fadR strain, thereby eliminating FadR repression of the transporter FadL and the enzymes of beta-oxidation. In this strain, fatty acid transport levels were low but detectable for ACS1, 2, 3, and 4 and were nearly 50% of wild type levels for ACS5. Despite increases in beta-oxidation, only ACS5 transformants were able to grow on oleate. These studies show that although ACS isoforms 1-4 variably supported moderate transport activity, beta-oxidation, and phospholipid synthesis and although their in vitro specific activities were greater than that of chromosomally encoded FadD, they were unable to substitute functionally for FadD regarding growth. Thus, membrane composition and protein-protein interactions may be critical in reconstituting bacterial ACS function.     

Host cell specificity of minute virus of mice in the developing mouse embryo

Productive infection by the murine autonomous parvovirus minute virus of mice (MVM) depends on a dividing cell population and its differentiation state. We have extended the in vivo analysis of the MVM host cell type range into the developing embryo by in utero inoculation followed by further gestation. The fibrotropic p strain (MVMp) and the lymphotropic i strain (MVMi) did not productively infect the early mouse embryo but were able to infect overlapping sets of cell types in the mid- or late-gestation embryo. Both MVMp and MVMi infected developing bone primordia, notochord, central nervous system, and dorsal root ganglia. MVMp exhibited extensive infection in fibroblasts, in the epithelia of lung and developing nose, and, to a lesser extent, in the gut. MVMi also infected endothelium. The data indicated that the host ranges of the two MVM strains consist of overlapping sets of cell types that are broader than previously known from neonate and in vitro infection experiments. The correlation between MVM host cell types and the cell types that activate the transgenic P4 promoter is consistent with the hypothesis that activation of the incoming viral P4 promoter by the host cell is one of the host range determinants of MVM.     

The effect of short, high intensity magnetic field pulses on the healing of skin wounds in rats

The object of this study was to examine the effect of high intensity, short duration pulsed electromagnetic fields (PEMF) on the healing of full thickness skin wounds in rats. Full thickness skin wounds were surgically created in two groups of Sprague-Dawley male rats. The rats were randomly divided into two groups, each containing 20 rats. Animals in the treatment group received treatments with the PEMF device on day 0, 3, 7, 9, 12, 14, 17, and 22, while the rats in the control group were subjected to the same procedure, but with the PEMF device not activated. Photographs of the surgically created wounds were obtained on day 0, 3, 7, 9, 12, 14, 17, and 22. Wound contraction (WC), wound epithelialization (WE), non-healed wound, and contraction-epithelialization (CE) ratio were calculated for each wound. No significant difference was found between the two groups for the parameters of WC, WE, non-healed wound, and CE ratio. A significant group x time interaction was found for WE and CE ratio. This type of PEMF did not have a significantly beneficial effect on wound healing. Wounds in the PEMF treated group were relatively less contracted and showed a compensatory increase in epithelialization in the early stages of wound repair.     

Defective nucleotide excision repair in xpc mutant mice and its association with cancer predisposition

Mice that are genetically engineered are becoming increasingly more powerful tools for understanding the molecular pathology of many human hereditary diseases, especially those that confer an increased predisposition to cancer. We have generated mouse strains defective in the Xpc gene, which is required for nucleotide excision repair (NER) of DNA. Homozygous mutant mice are highly prone to skin cancer following exposure to UVB radiation, and to liver and lung cancer following exposure to the chemical carcinogen acetylaminofluorene (AAF). Skin cancer predisposition is significantly augmented when mice are additionally defective in Trp53 (p53) gene function. We also present the results of studies with mice that are heterozygous mutant in the Apex (Hap1, Ref-1) gene required for base excision repair and with mice that are defective in the mismatch repair gene Msh2. Double and triple mutant mice mutated in multiple DNA repair genes have revealed several interesting overlapping roles of DNA repair pathways in the prevention of mutation and cancer.     

Fluid structure interaction with contact surface methodology for evaluation of endovascular carotid implants for drug-resistant hypertension treatment

Drug-resistant hypertensive patients may be treated by mechanical stimulation of stretch-sensitive baroreceptors located in the sinus of carotid arteries. To evaluate the efficacy of endovascular devices to stretch the carotid sinus such that the induced strain might trigger baroreceptors to increase action potential firing rate and thereby reduce systemic blood pressure, numerical simulations were conducted of devices deployed in subject-specific carotid models. Two models were chosen--a typical physiologic carotid and a diminutive atypical physiologic model representing a clinically worst case scenario--to evaluate the effects of device deployment in normal and extreme cases, respectively. Based on the anatomical dimensions of the carotids, two different device sizes were chosen out of five total device sizes available. A fluid structure interaction (FSI) simulation methodology with contact surface between the device and the arterial wall was implemented for resolving the stresses and strains induced by device deployment. Results indicate that device deployment in the carotid sinus of the physiologic model induces an increase of 2.5% and 7.5% in circumferential and longitudinal wall stretch, respectively, and a maximum of 54% increase in von Mises arterial stress at the sinus wall baroreceptor region. The second device, deployed in the diminutive carotid model, induces an increase of 6% in both circumferential and longitudinal stretch and a 50% maximum increase in von Mises stress at the sinus wall baroreceptor region. Device deployment has a minimal effect on blood-flow patterns, indicating that it does not adversely affect carotid bifurcation hemodynamics in the physiologic model. In the smaller carotid model, deployment of the device lowers wall shear stress at sinus by 16% while accelerating flow entering the external carotid artery branch. Our FSI simulations of carotid arteries with deployed device show that the device induces localized increase in wall stretch at the sinus, suggesting that this will activate baroreceptors and subsequently may control hypertension in drug-resistant hypertensive patients, with no consequential deleterious effects on the carotid sinus hemodynamics.