Basal Endothelial Nitric Oxide Release Is Preserved in Overweight and Obese Adults

Objective: Impaired basal nitric oxide release is associated with a number of cardiovascular disorders including hypertension, arterial spasm, and myocardial infarction. We determined whether basal endothelial nitric oxide release is reduced in otherwise healthy overweight and obese adult humans. Research Methods and Procedures: Seventy sedentary adults were studied: 32 normal weight (BMI <25 kg/m²), 24 overweight (BMI ≥ 25 < 30 kg/m²), and 14 obese (BMI ≥ 30 kg/m²). Forearm blood flow (FBF) responses to intra‐arterial infusions of N^g‐monomethyl‐l ‐arginine (5 mg/min), a nitric oxide synthase inhibitor, were used as an index of basal nitric oxide release. Results: N^g‐monomethyl‐l ‐arginine elicited significant reductions in FBF in the normal weight (from 4.1 ± 0.2 to 2.7 ± 0.2 mL/100 mL tissue/min), overweight (4.1 ± 0.1 to 2.8 ± 0.2 mL/100 mL tissue/min), and obese (3.9 ± 0.3 to 2.7 ± 0.2 mL/100 mL tissue/min) subjects. Importantly, the magnitude of reduction in FBF (～30%) was similar among the groups. Discussion: These results indicate that the capacity of the endothelium to release nitric oxide under basal conditions is not compromised in overweight and obese adults.     

Application of MOSFET detectors for dosimetry in small animal radiography using short exposure times

Digital subtraction angiography (DSA) X-ray imaging for small animals can be used for functional phenotyping given its ability to capture rapid physiological changes at high spatial and temporal resolution. The higher temporal and spatial requirements for small-animal imaging drive the need for short, high-flux X-ray pulses. However, high doses of ionizing radiation can affect the physiology. The purpose of this study was to verify and apply metal oxide semiconductor field effect transistor (MOSFET) technology to dosimetry for small-animal diagnostic imaging. A tungsten anode X-ray source was used to expose a tissue-equivalent mouse phantom. Dose measurements were made on the phantom surface and interior. The MOSFETs were verified with thermoluminescence dosimeters (TLDs). Bland-Altman analysis showed that the MOSFET results agreed with the TLD results (bias, 0.0625). Using typical small animal DSA scan parameters, the dose ranged from 0.7 to 2.2 cGy. Application of the MOSFETs in the small animal environment provided two main benefits: (1) the availability of results in near real-time instead of the hours needed for TLD processes and (2) the ability to support multiple exposures with different X-ray techniques (various of kVp, mA and ms) using the same MOSFET. This MOSFET technology has proven to be a fast, reliable small animal dosimetry method for DSA imaging and is a good system for dose monitoring for serial and gene expression studies.     

Ebolavirus and Marburgvirus: insight the Filoviridae family

Ebolavirus and Marburgvirus (belonging to the Filoviridae family) emerged four decades ago and cause epidemics of haemorrhagic fever with high case-fatality rates. The genome of filoviruses encodes seven proteins. No significant homology is observed between filovirus proteins and any known macromolecule. Moreover, Marburgvirus and Ebolavirus show significant differences in protein homology. The natural maintenance cycle of filoviruses is unknown, the natural reservoir, the mode of transmission, the epidemic disease generation, and temporal dynamics are unclear. Lastly, Ebolavirus and Marburgvirus are considered as potential biological weapons. Vaccine appears the unique therapeutic frontier. Here, molecular and clinical aspects of filoviral haemorrhagic fevers are summarized.     

Phylogenetic relationships of Loxosceles and Sicarius spiders are consistent with Western Gondwanan vicariance

The modern geographic distribution of the spider family Sicariidae is consistent with an evolutionary origin on Western Gondwana. Both sicariid genera, Loxosceles and Sicarius are diverse in Africa and South/Central America. Loxosceles are also diverse in North America and the West Indies, and have species described from Mediterranean Europe and China. We tested vicariance hypotheses using molecular phylogenetics and molecular dating analyses of 28S, COI, 16S, and NADHI sequences. We recover reciprocal monophyly of African and South American Sicarius, paraphyletic Southern African Loxosceles and monophyletic New World Loxosceles within which an Old World species group that includes L. rufescens is derived. These patterns are consistent with a sicariid common ancestor on Western Gondwana. North American Loxosceles are monophyletic, sister to Caribbean taxa, and resolved in a larger clade with South American Loxosceles. With fossil data this pattern is consistent with colonization of North America via a land bridge predating the modern Isthmus of Panama.     

Immunomodulation of polyamine biosynthesis in tobacco plants has a significant impact on polyamine levels and generates a dwarf phenotype

Ornithine decarboxylase (ODC) is a cytosolic enzyme that catalyses the direct decarboxylation of l-ornithine to putrescine, one of the rate-limiting steps of polyamine biosynthesis in plants. In the present study, an ODC-specific murine single-chain antibody fragment (scFvODC1) was generated by phage display technology. To evaluate the effect of the recombinant antibody fragment on ODC activity and polyamine levels, we produced transgenic tobacco plants that accumulated scFvODC1 in the cytosol. Expression levels of up to 4% total soluble protein (TSP) were achieved, resulting in the inhibition of up to 90% of endogenous ODC activity. A significant reduction in putrescine, spermidine and spermine levels was observed in transgenic lines producing high levels of scFvODC1. Furthermore, these lines showed developmental abnormalities and a dwarf phenotype. We show that the immunomodulation of enzyme activity is a powerful approach that can be used to alter complex and tightly controlled metabolic pathways, allowing specific steps in the pathway to be blocked and the resulting physiological effects to be investigated.     

Microplate live cell assay system for early events in mechanotransduction

For studying mechanotransduction in cultured cells, we developed a microplate assay using a fluorescence/luminescence plate reader equipped with software-controlled injectors to deliver a reproducible mechanical stimulus (adjustable for both timing and force) and immediately measure adenosine 5(')-triphosphate (ATP) release and calcium mobilization. Suspension or adherent chondrocyte cultures in 96-well plates were incubated with firefly luciferase and luciferin for the ATP assay or loaded with Fluo-3-acetoxy methylester for intracellular calcium measurement. Steady state ATP release was measured in resting cells; then mechanical stimulation was delivered by injection of an equal volume of buffer into the wells. Serial integrations of 20 to 500ms allowed real-time analysis of the time course of ATP release. Luminescence increased within 500ms indicating the rapidity of ATP release in chondrocyte mechanotransduction. Subsequent injection of a cell lysis solution allowed quantitation of total cellular ATP as an internal control of cell viability and number. Intracellular calcium was also elevated within 500ms of fluid injection. This assay is easily adapted for changes in intracellular pH or other ions by use of different commercially available fluorescent indicators. The live-cell assay using fluid injection as a mechanical stimulus is a valuable tool for dissecting the role of signaling pathways in mechanotransduction.     

Influence of oral contraceptive use on endothelial t-PA release in healthy premenopausal women

We determined the influence of oral contraceptives (OC) on the capacity of the endothelium to release tissue-type plasminogen activator (t-PA). Twenty-three healthy premenopausal women were studied: 12 nonusers and 11 users of OC. Net endothelial release rates of t-PA were calculated as the product of the arteriovenous concentration gradient and forearm plasma flow in response to intra-arterial bradykinin (BK: 12.5-50 ng. 100 ml tissue(-1) x min(-1)) and sodium nitroprusside (SNP: 1.0-4.0 microg x 100 ml tissue(-1) x min(-1)). Net release of t-PA antigen and increment in t-PA activity across the forearm to BK increased (P < 0.01) in a dose-dependent fashion and to similar extents in the nonusers and users of OC. At the highest BK dose, net release of t-PA antigen was 64.5 +/- 8.2 and 66.2 +/- 15.4 ng x 100 ml tissue(-1) x min(-1) in the nonusers and users of OC, whereas the net increment in t-PA activity was 18.6 +/- 3.0 and 16.0 +/- 2.0 IU. 100 ml tissue(-1) x min(-1), respectively. There was no effect of SNP on t-PA release in either group. These results indicate that endothelial t-PA release is not altered in premenopausal women who use oral contraception.     

Effect of gangliosides on the copper-induced oxidation of human low-density lipoproteins

The role of gangliosides in the copper-induced oxidative modification of human low-density lipoprotein (LDL) was studied focusing on the early stage of LDL oxidation in which the concentration of conjugated dienes increases only weakly. The changes in the protein and lipid component were followed using fluorescence spectroscopy. The results indicate that binding of gangliosides to LDL causes slower destruction of tryptophan fluorescence and suppresses cross-linking between the reactive groups of the protein and the products of lipid peroxidation. The protective role of gangliosides could be assigned to their interference with the lipid-protein interaction in the LDL particle, which might be important for the maintenance of the native plasma antioxidant status in vivo.     

Complexes of Ag with mixed donor phenanthroline-containing macrocycles: spectrofluorimetric, spectrophotometric, conductometric and potentiometric studies

The reactions of Ag⁺ with five mixed donor phenanthroline-containing macrocycles (L¹-L⁵) having N₂S₃-, N₂S₂O-, N₂S₂-, N₃S₂-, and N₄S₂-donor sets, respectively, have been studied in MeCN by spectrofluorimetric, spectrophotometric, conductometric and potentiometric methods. All ligands form 1:1 [Ag(L)]⁺ complexes, and in the case of L⁴ and L⁵, formation of 1:2 [Ag(L)₂]⁺ species is also observed. The corresponding formation constants have been evaluated and their values allow a deeper insight into the role played by complexation process in the potentiometric selectivity for Ag⁺ of membrane electrodes (ISE), and in the selective transport of Ag⁺ through supported liquid membrane (SLM) systems based on these ligands. The X-ray crystal structure of the complex [Ag(L⁴)]BF₄ is also reported.