Search

Objective: This study describes the pharmacokinetics of parent pimobendan (PIM) and its active metabolite, o-desmethyl-pimobendan (ODMP), after oral and rectal administration of pimobendan to healthy dogs. Animals: A total of eight healthy privately owned dogs were used in this study. Procedures: The dogs received a single dose (0.5 mg/kg) of a commercially available pimobendan tablet per os (PO). Twelve blood samples were collected over a 12-h period for pharmacokinetic analysis. After a 24-h washout period, the dogs received the same dose of pimobendan solution per rectum (PR), and samples were obtained at the same time for analysis. Results: For PIM, PO vs. PR, respectively, the mean maximum plasma concentration (Cmax, ng/ml) was 49.1 ± 28.7 vs. 10.1 ± 2, the time to reach a maximum concentration (Tmax, h) was 2.1 ± 0.9 vs. 1 ± 0.4, the disappearance half-life (t1/2, h) was 1.8 ± 0.8 vs. 2.2 ± 0.6, and the area under the concentration–time curve (AUC, ng*h/ml) was 148.4 ± 71.6 vs. 31.1 ± 11.9, with relative bioavailability (F, %) of 25 ± 8. For ODMP, PO vs. PR, respectively, Cmax was 30.9 ± 10.4 vs. 8.8 ± 4.8, Tmax was 3.2 ± 1.6 vs. 1.7 ± 1.1, and t1/2 was 5.0 ± 2.7 vs. 8.3 ± 4.8, with AUC of 167.8 ± 36.2 vs. 50.1 ± 19.2 and F of 28 ± 6. The differences between PO and PR were significant (P < 0.03) for AUC and Cmax for both PIM and ODMP. Conclusions and Clinical Relevance: The pharmacokinetics of PIM and ODMP were described following PO and PR administration. The findings suggest that pimobendan PR might achieve effective concentrations and, as such, warrant future studies of clinical effectiveness in treating dogs with congestive heart failure and which are unable to receive medication PO.

Background: Drug-coated balloons (DCBs), which deliver anti-proliferative drugs with the aid of excipients, have emerged as a new endovascular therapy for the treatment of peripheral arterial disease. In this study, we evaluated the use of keratose (KOS) as a novel DCB-coating excipient to deliver and retain paclitaxel. Methods: A custom coating method was developed to deposit KOS and paclitaxel on uncoated angioplasty balloons. The retention of the KOS-paclitaxel coating, in comparison to a commercially available DCB, was evaluated using a novel vascular-motion simulating ex vivo flow model at 1 h and 3 days. Additionally, the locoregional biological response of the KOS-paclitaxel coating was evaluated in a rabbit ilio-femoral injury model at 14 days. Results: The KOS coating exhibited greater retention of the paclitaxel at 3 days under pulsatile conditions with vascular motion as compared to the commercially available DCB (14.89 &plusmn, 4.12 ng/mg vs. 0.60 &plusmn, 0.26 ng/mg, p = 0.018). Histological analysis of the KOS&ndash, paclitaxel-treated arteries demonstrated a significant reduction in neointimal thickness as compared to the uncoated balloons, KOS-only balloon and paclitaxel-only balloon. Conclusions: The ability to enhance drug delivery and retention in targeted arterial segments can ultimately improve clinical peripheral endovascular outcomes.

An 8-year-old intact female Chihuahua was presented for evaluation and possible occlusion of a previously diagnosed patent ductus arteriosus (PDA). Transthoracic echocardiography revealed left ventricular and left atrial enlargement, enlargement of the main pulmonary artery, and a PDA with bidirectional shunting. Tricuspid regurgitant velocities suggested moderate pulmonary hypertension. The PDA was occluded with an Amplatz® Canine Duct Occluder using a transarterial approach on the following day. No immediate complications were observed other than an acute decrease in left ventricular systolic function. One day after the PDA occlusion transthoracic echocardiography revealed no residual ductal flow, but there was spontaneous echocardiographic contrast in the left ventricle. The patient was discharged with sildenafil, pimobendan, and clopidogrel. Five weeks later when the patient was presented for a recheck examination, the previously documented spontaneous echocardiographic contrast was no longer present. Finding spontaneous echocardiographic contrast in the dog has not previously been reported in association with PDA occlusion.

Background The effects of sacubitril/valsartan (S/V) on the renin-angiotensin-aldosterone system (RAAS) in dogs with cardiomegaly secondary to myxomatous mitral valve disease (MMVD) are currently unknown. Objectives To determine the pharmacodynamic effects of S/V on the RAAS, natriuretic peptide concentrations, systolic arterial pressure (SAP), tests of renal function, and serum electrolyte concentrations in dogs with cardiomegaly secondary to MMVD. Animals Thirteen client-owned dogs weighing 4-15 kg with American College of Veterinary Internal Medicine (ACVIM) Stage B2 MMVD. Methods Prospective, randomized, double-blind, placebo-controlled pilot study of S/V in dogs with ACVIM Stage B2 MMVD. Results Thirteen dogs were recruited: S/V (n = 7) and placebo (n = 6). The median percentage increase in urinary aldosterone to creatinine ratio (UAldo : C) between day 0 and day 30 was significantly lower in the S/V group (12%; P = .032) as compared with the placebo group (195%). The median percentage decrease of NT-proBNP concentration from day 0 to day 30 was not statistically different between groups (P = .68). No statistical differences were seen in echocardiographic, thoracic radiographic, SAP, or serum biochemical test results measured at any time point between groups. No adverse events were observed for dogs in either group. Conclusion and clinical importance Sacubitril/valsartan may provide a new pharmaceutical method to effectively inhibit the RAAS in dogs with ACVIM Stage B2 MMVD.

Background Dirofilaria immitis infection occurs in dogs and cats, both of which species are clinically affected by mature adult infections. Cats are uniquely affected by immature-adult infections with an inflammatory pulmonary disease called Heartworm-Associated Respiratory Disease (HARD). D. immitis infection causes pulmonary parenchymal and vascular pathology in the dog and cat. Dogs develop pulmonary hypertension and cor pulmonale, whereas the development of pulmonary hypertension is rare in the cat. D. immitis infection in the dog causes alteration of the right ventricular (RV) extracellular matrix, including a decrease in myocardial collagen. In this study, the RV myocardial changes of cats infected with adult and immature-adult D. immitis were assessed. Methods The cardiopulmonary systems of six groups of SPF cats (n = 9-10 per group) were examined 8 or 18 months after infection with L3 D. immitis. Two groups were untreated and allowed to develop adult HW; two groups were treated with ivermectin starting 3 months post infection, thus allowing HARD but no mature adult heartworms; and two groups were treated with selamectin beginning 1 month post infection, preventing development of L5 or adult heartworms. A group of specific pathogen free (SPF) normal cats was utilized as a negative control (n = 12). Lung pathologic lesions were objectively assessed, and both RV and left ventricular (LV) weights were obtained to calculate an RV/LV ratio. Intramural RV myocardial collagen content was quantitatively assessed. Results RV/LV weight ratios were not different between groups. Negative control cats had significantly greater RV collagen content than all other affected groups (P = 0.032). Analysis of the RV/LV ratios and collagen content revealed no significant relationship (r = 0.03, P = 0.723, respectively). Collagen content had a modest, but significant, negative correlation, however, with both pulmonary vascular pathology (r = −0.25, P = 0.032) as well as the total pulmonary parenchymal and vascular pathology (r = −0.26, P = 0.025). Conclusions Cats infected with mature and immature D. immitis did not develop RV hypertrophy but did demonstrate loss of RV myocardial collagen content. The collagen loss was present at 8 and 18 months after infection in all infected cats. This loss of RV myocardial collagen was correlated with the severity of pulmonary parenchymal and vascular pathology.

ObjectiveConstrictive extracellular matrix (ECM) remodeling contributes significantly to restenosis after arterial reconstruction, but its molecular regulation is poorly defined. Hyaluronan (HA) accumulates within ECM at sites of injury where it is thought to facilitate smooth muscle cell (SMC) trafficking and collagen remodeling analogous to its role in cutaneous wound healing. SMC receptors for HA include receptor for hyaluronan-mediated motility (RHAMM), which mediates HA-induced migration. We hypothesized RHAMM would also mediate SMC-matrix interactions to alter the extent of constrictive remodeling.MethodsWe studied the role of RHAMM in SMC attachment to collagen, migration, and contraction of collagen gels using blocking antibodies and SMC from RHAMM −/− knockout mice. We then determined the role of RHAMM in constrictive artery wall remodeling by comparing changes in wall geometry in RHAMM −/− vs wild-type (WT) RHAMM +/+ controls 1 month after carotid ligation.ResultsHA increased SMC attachment to collagen-coated plates, but blocking RHAMM reduced adhesion (P = .025). RHAMM −/− SMC also demonstrated reduced adhesion (% adherent: 36.1 ± 2.2 vs 76.3 ± 1.9; P < .05). SMC contraction of collagen gels was enhanced by HA and further increased by RHAMM blockade (P < .01) or knockout (gel diameter, mm: RHAMM −/−, 6.7 ± 0.1 vs WT 9.8 ± 0.1; P < .01). RHAMM promoted constrictive remodeling in vivo as carotid artery size was significantly larger in knockout mice 1 month after ligation. Neointimal thickening, however, was not affected in RHAMM −/− (P = NS vs WT), but lumen size was significantly larger (lumen area, μm2: 52.4 ± 1.4 × 103 vs 10.4 ± 1.8 × 103; P = .01) because artery size constricted less (external elastic lamina area, μm2: RHAMM −/−, 92.4 ± 4.7 × 103 vs WT, 51.3 ± 5.9 × 103; P = .015). Adventitial thickening and collagen deposition were also more extensive in ligated RHAMM −/− carotids (adventitial thickness, μm: 218 ± 12.2 vs 109 ± 7.9; P = .01).ConclusionsHA activation of RHAMM significantly impacts SMC-ECM adhesive interactions and contributes to constrictive artery wall remodeling in mice. Strategies to block RHAMM at sites of vessel injury may prove useful in the prevention of clinical restenosis.Clinical RelevanceConstrictive (inward) remodeling is a critical factor in the pathogenesis of restenosis following artery injury. Presently, no effective treatment has been developed to prevent constrictive remodeling in patients except a metal stent. In this study, we demonstrated that receptor for hyaluronan-mediated motility (RHAMM) interacts with hyaluronan and regulates smooth muscle cell attachment, motility, and remodeling of extracellular matrix in vitro. In a mouse carotid ligation model, we found that RHAMM promotes constrictive artery wall remodeling and lumen narrowing, as knockout of RHAMM significantly improves wall caliber and prevents lumen stenosis. These results suggested that pharmacological inhibitors of RHAMM or approaches to block RHAMM signaling could be effective components of a multimodal strategy to prevent restenosis following arterial reconstruction.

OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate–labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor-2, and von Willebrand factor) and hematopoietic (CD14) cell markers by use of indirect immunofluorescence assay (IFA) and flow cytometry. RESULTS Colonies with cobblestone morphology were isolated from 15 of 17 horses. Blood collected from the cephalic vein yielded colonies significantly more often (14/17 horses) than did blood collected from the jugular vein (8/17 horses). Of 14 cephalic blood samples with colonies, 13 were obtained with DGC and 8 with WBA. Of 8 jugular blood samples with colonies, 8 were obtained with DGC and 4 with WBA. Colony frequency (colonies per milliliter of blood) was significantly higher for cephalic blood samples and samples isolated with DGC. Cells formed vascular tubules, had uptake of DiI-Ac-LDL, and expressed endothelial markers by use of IFA and flow cytometry, which confirmed their identity as ECFCs. CONCLUSIONS AND CLINICAL RELEVANCE Maximum yield of ECFCs was obtained for blood samples collected from both the jugular and cephalic veins and use of DGC to isolate cells. Consistent yield of ECFCs from peripheral blood of horses will enable studies to evaluate diagnostic and therapeutic uses.

To avoid complications of prosthetic vascular grafts, engineered vascular constructs have been investigated as an alternative for vascular reconstruction. The scaffolds for vascular tissue engineering remain a cornerstone of these efforts and yet many currently available options are limited by issues of inconsistency, poor adherence of vascular cells, or inadequate biomechanical properties. In this study, we investigated whether PCL/collagen scaffolds could support cell growth and withstand physiologic conditions while maintaining patency in a rabbit aortoiliac bypass model. Our results indicate that electrospun scaffolds support adherence and growth of vascular cells under physiologic conditions and that endothelialized grafts resisted adherence of platelets when exposed to blood. When implanted in vivo, these scaffolds were able to retain their structural integrity over 1 month of implantation as demonstrated by serial ultrasonography. Further, at retrieval, these scaffolds continued to maintain biomechanical strength that was comparable to native artery. This study suggests that electrospun scaffolds combined with vascular cells may become an alternative to prosthetic vascular grafts for vascular reconstruction.

Cassini radar images show a variety of fluvial channels on Titan's surface, often several hundreds of kilometers in length. Some (predominantly at low- and mid-latitude) are radar-bright and braided, resembling desert washes where fines have been removed by energetic surface liquid flow, presumably from methane rainstorms. Others (predominantly at high latitudes) are radar-dark and meandering and drain into or connect polar lakes, suggesting slower-moving flow depositing fine-grained sediments. A third type, seen predominantly at mid- and high latitudes, have radar brightness patterns indicating topographic incision, with valley widths of up to 3 km across and depth of several hundred meters. These observations show that fluvial activity occurs at least occasionally at all latitudes, not only at the Huygens landing site, and can produce channels much larger in scale than those observed there. The areas in which channels are prominent so far amount to about 1% of Titan's surface, of which only a fraction is actually occupied by channels. The corresponding global sediment volume inferred is not enough to account for the extensive sand seas. Channels observed so far have a consistent large-scale flow pattern, tending to flow polewards and eastwards.

Uridine triphosphate (UTP) constricts cerebral arteries by activating transduction pathways that increase cytosolic [Ca2+] and myofilament Ca2+ sensitivity. The signaling proteins that comprise these pathways remain uncertain with recent studies implicating a role for several G proteins. To start clarifying which G proteins enable UTP-induced vasoconstriction, a small interfering RNA (siRNA) approach was developed to knock down specified targets in rat cerebral arteries. siRNA directed against Gq and RhoA was introduced into isolated cerebral arteries using reverse permeabilization. Following a defined period of organ culture, arteries were assayed for contractile function, mRNA levels, and protein expression. Targeted siRNA reduced RhoA or Gq mRNA expression by 60–70%, which correlated with a reduction in RhoA but not Gq protein expression. UTP-induced constriction was abolished in RhoA-depleted arteries, but this was not due to a reduction in myosin light chain phosphorylation. UTP-induced actin polymerization was attenuated in RhoA-depleted arteries, which would explain the loss of agonist-induced constriction. In summary, this study illustrates that siRNA approaches can be effectively used on intact arteries to induce targeted knockdown given that the protein turnover rate is sufficiently high. It also demonstrates that the principal role of RhoA in agonist-induced constriction is to facilitate the formation of F-actin, the physical structure to which phosphorylated myosin binds to elicit arterial constriction.

HDL lowers the risk for atherosclerotic cardiovascular disease by promoting cholesterol efflux from macrophage foam cells. However, other antiatherosclerotic properties of HDL are poorly understood. To test the hypothesis that the lipoprotein carries proteins that might have novel cardioprotective activities, we used shotgun proteomics to investigate the composition of HDL isolated from healthy subjects and subjects with coronary artery disease (CAD). Unexpectedly, our analytical strategy identified multiple complement-regulatory proteins and a diverse array of distinct serpins with serine-type endopeptidase inhibitor activity. Many acute-phase response proteins were also detected, supporting the proposal that HDL is of central importance in inflammation. Mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with CAD was selectively enriched in apoE, raising the possibility that HDL carries a unique cargo of proteins in humans with clinically significant cardiovascular disease. Collectively, our observations suggest that HDL plays previously unsuspected roles in regulating the complement system and protecting tissue from proteolysis and that the protein cargo of HDL contributes to its antiinflammatory and antiatherogenic properties.

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 +/- 178 micromol/mol Tyr) than that in circulating HDL (104 +/- 11 micromol/mol Tyr; p0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.

Purpose This retrospective review describes the surgical management and clinical outcome for renal artery aneurysms (RAAs) in 62 consecutive patients. Methods From January 1987 through July 2003, 804 patients had operative renal artery (RA) repair involving 1206 kidneys at our center. A subgroup of 62 patients (42 women, 20 men; mean age 46 ± 18 years) received repair of 72 RAAs. Demographic data, comorbidity, and surgical technique were examined. Blood pressure and renal function response were determined. Patency of repair was evaluated by renal duplex sonography. Primary patency and patient survival were estimated by life-table methods. Tests of association were performed using χ 2 and the Student t tests. Results Seventy-two RAs were repaired for RAA with a mean diameter of 2.6 cm (range, 1.3 to 5.5 cm). Bilateral RAAs were present in 21 patients. Associated conditions included fibromuscular dysplasia, atherosclerosis, and arteritis in 54%, 35%, and 7%, respectively. Hypertension was present in 89% (mean blood pressure, 171 ± 35/95 ± 19 mm Hg; mean medications, 2.2 ± 1.2 drugs) and renal insufficiency was present in 8% (mean serum creatinine, 1.9 ± 0.6 mg/dL). RAA repair included bypass (67%), aneurysmorrhaphy (15%), or a combination (17%). One planned nephrectomy (1%) was performed for un-reconstructable disease. Branch RA reconstruction in 78% used ex vivo cold perfusion in 50%, in situ cold perfusion in 29%, and warm in situ repair in 21%. Of 9 bilateral RAA repairs, 7 (78%) were staged and 2 (22%) were simultaneous. Combined aortic reconstruction was required in 6 (10%) patients. Perioperative death occurred in 1 patient (1.6%), and significant morbidity was observed in 8 patients (12%). Hypertension was considered improved in 54%, cured in 21%, and unchanged in 25% at mean follow-up of 48 months (range, 1-156 months). Among patients with renal insufficiency, renal function was improved in 3 (60%), unchanged in 1 (20%), and declined in 1 (20%). Follow-up patency (mean, 33 months; range, 1-118 months) was determined for 64 (91%) RA reconstructions. Product-limit estimate of primary patency at 48 months was 96%. Product-limit estimate of survival was 91% at 120 months. Conclusion RAAs were repaired with low morbidity and mortality. Complex branch RAA repair using cold perfusion preservation and ex vivo techniques resulted in no unplanned nephrectomy, with an estimated primary patency of 96% at 48 months. Beneficial blood pressure response was observed in the majority of hypertensive patients. These results support selective surgical management of RAA.

* BACKGROUND AND OBJECTIVE: The goal of this study was to quantify retinal hemodynamics using the freeze-frame technique for determining volumetric retinal blood flow. * MATERIALS AND METHODS: Leukocytes and erythrocytes were removed from the circulation of rats, stained with fluorescent dyes, returned to the animals' circulation, and excited to fluorescence using the scanning laser ophthalmoscope. The retinal circulation was videotaped and viewed frame-byframe to count the total number of visible stained cells passing through the field of view during a known period of time while excluding repeated appearances of cells in multiple frames. The total number of cells visualized, in conjunction with cell density data collected in a coincident in vitro study, was used to calculate in vivo volumetric blood flow for both ceil types. * RESULTS: Measurements of volumetric blood flow based on fluorescent leukocytes (34.2 ± 5.9 nL/sec [mean ± standard deviation]; n = 30 rats) and fluorescent erythrocytes (35.1 ±6.1 nL/sec; n = 30 rats) were not significantly different, indicating that the freeze-frame technique provides a blood flow measurement independent of cell type. Repeated blood flow measurements obtained from an individual rat over time showed little difference (P < .0001), demonstrating the reproducibility of the technique. * CONCLUSIONS: The freeze-frame technique offers a promising method for calculating volumetric blood flow independent of individual cell velocity, size, or type. [Ophthalmic Surg Lasers Imaging 2003;34:505514]

The polymerization of acrylamide (AAm) and N,N‘-methylenebis(acrylamide) (BIS) in the presence of high concentrations of tetradecyltrimethylammonium bromide (TTAB) results in formation of macroporous gels. Prior to polymerization, the presence of AAm monomer shifts the TTAB micelle to the columnar phase transition boundary. The combination of dynamic viscosity measurements and X-ray diffraction shows for high TTAB concentrations that during polymerization TTAB micelles are driven into nanodomains of the hexagonal−columnar phase. X-ray diffraction from lower concentration TTAB samples shows that micelles are limited to a closest separation of 10 nm, apparently due to the presence of the polyacrylamide network.

A modified actinomycin D was prepared with a hydroxyl group that replaced the amino group at the chromophore 2-position, a substitution known to strongly reduce affinity for double-stranded DNA. Interactions of the modified drug on single-stranded DNAs of the defined sequence were investigated. Competition assays showed that 2-hydroxyactinomycin D has low affinity for two oligonucleotides that have high affinities (K(a) = 5-10 x 10(6) M(-1) oligomer) for 7-aminoactinomycin D and actinomycin D. Primer extension inhibition assays performed on several single-stranded DNA templates totaling around 1000 nt in length detected a single high affinity site for 2-hydroxyactinomycin D, while many high affinity binding sites of unmodified actinomycin D were found on the same templates. The sequence selectivity of 2-hydroxyactinomycin D binding is unusually high and approximates the selectivity of restriction endonucleases. Binding appears to require a complex structure, including residues well removed from the polymerase pause site.

Electrophoretic mobilities of DNA molecules ranging in length from 200 to 48 502 base pairs (bp) were measured in agarose gels with concentrations T = 0.5% to 1.3% at electric fields from E = 0.71 to 5.0 V/cm. This broad data set determines a range of conditions over which the new interpolation equation nu(L) = (beta+alpha(1+exp(-L/gamma))(-1) can be used to relate mobility to length with high accuracy. Mobility data were fit with chi(2) > 0.999 for all gel concentrations and fields ranging from 2.5 to 5 V/cm, and for lower fields at low gel concentrations. Analyses using so-called reptation plots (Rousseau, J., Drouin, G., Slater, G. W., Phys. Rev. Lett. 1997, 79, 1945-1948) indicate that this simple exponential relation is obeyed well when there is a smooth transition from the Ogston sieving regime to the reptation regime with increasing DNA length. Deviations from this equation occur when DNA migration is hindered, apparently by entropic-trapping, which is favored at low fields and high gel concentrations in the ranges examined.

Electrophoretic mobilities of DNA molecules ranging in length from 100 to 10 000 base pairs (bp) were measured in gels of eleven concentrations of agarose from 0.5 to 1.5%. Excellent fits of the dependence of mobility on DNA length were obtained with the relationship 1/μ(L) = 1/μl - (1/μl - 1/μs)e-L/γ showing an e-L/γ crossover, where L is the length of a DNA fragment and γ is a crossover length ranging from 8000 to 12 000 bp. The other parameters in the fit are μs the mobility of short DNA with unit charge in the limit as length is extrapolated to zero, and μl, the mobility of long DNA as length is extrapolated to infinity. This exponential relationship should be a useful interpolation function for determining DNA lengths over a wide range. The simplicity of this relationship may be of more fundamental significance and suggests that some common feature dominates the electrophoresis of double stranded DNA fragments in agarose gels, regardless of length.

Triton is the major moon of Neptune. It was discovered shortly after Neptune's discovery and orbits in a retrograde, or backward, sense compared with Neptune's rotation. Composed of a rock and metal core and a massive icy mantle, and probably an internal ocean, Triton displays a variety of active geological processes including cryo-, or cold, volcanism; tectonic deformation; and active plume eruptions. The latter are thought to be driven by solar heating of a predominantly nitrogen ice surface. Along with methane and other exotic ices, this extremely cold nitrogen ice maintains a thin atmosphere in vapor pressure equilibrium. This tenuous atmosphere supports hazes and clouds and high winds and is subject to strong seasons. Triton is thought to be a captured body from the Kuiper belt beyond Neptune, and as such offers a glimpse of what major bodies in the Kuiper belt, such as Pluto and Eris, may be like.

Small functional RNAs required for structure studies are often prepared by in vitro transcription. Capillary electrophoresis in liquid crystalline gels of Pluronic F127 was used to analyze unfractionated in vitro transcription reactions and anion-exchange high-performance liquid chromatography (HPLC) fractions from transcription reactions. Guanosine monophosphate (GMP), the four nucleoside triphosphates (NTPs), abortive transcripts, and transcripts with lengths near the desired product length were simultaneously resolved and quantified in a single run. Oligonucleotides up to at least 35 nucleotides were resolved to baseline within 10 min using a moderate field (185 V/cm) and short effective capillary length (7.6 cm) for electrophoresis in 20% Pluronic F127 at pH 8.3 in Tris-borate-EDTA (TBE) buffer (30°C). Nucleotide migration times were 4—5 min, in the order UTP+CTP (unresolved)

Abstract —Remodeling contributes to restenosis when cells shrink the artery wall at sites of injury. This may be analogous to wound healing, where tissue remodeling achieves wound contraction. Hyaluronan (HA) is prominent in wound matrix and inhibits fetal scarring. HA is also produced in the artery wall after angioplasty, where it may inhibit constrictive remodeling. This hypothesis was tested in vitro using a model of matrix contraction. Primate aortic smooth muscle cells and adventitial fibroblasts were seeded into collagen I gels containing increasing amounts of HA (0% to 50%, wt/wt). Both cell types reduced the diameter of collagen alone ≈65% at 18 hours. HA significantly increased gel contraction (diameter in mm: 0% HA, 7.7±0.9; 2%, 7.1±0.7; 10%, 6.7±0.5; 50%, 5.6±0.9; P 1 -integrins completely inhibited contraction of collagen, but gels containing HA required CD44 and β 1 -integrin blockade for complete inhibition. Enhanced collagen reorganization and contraction were not attributable to increased collagenase activity, because the metalloproteinase inhibitor batimastat had no effect. In summary, HA enhanced collagen reorganization by the cell types most likely to mediate constrictive remodeling after angioplasty. These effects were CD44-dependent, thus providing a potential target for therapies to prevent constrictive remodeling and restenosis.

Pulsed field gradient nuclear magnetic resonance (PFGNMR) measurements of oligonucleotide (poly-thymidine, T1 to T30) self-diffusion coefficients in gels of Pluronic [poly(ethylene oxide) (PEO)−poly(propylene oxide) (PPO)−poly(ethylene oxide) (PEO)] micelles follow the Zimm scaling theory that predicts the diffusion coefficient to vary with solute molecular weight to the −0.5 power. The ratio of the diffusion coefficients in a 20% Pluronic gel-like liquid crystal phase to those in buffer solutions (without Pluronics) was approximately equal to 0.5 for all molecule sizes tested. PFGNMR water self-diffusion coefficient measurements in the gel phase and the solution phase of Pluronic micelles were interpreted within the context of volume averaging theory and known structural features of Pluronic micelles for various Pluronic concentrations. The result of combining the volume averaging theory and measurements of water and oligonucleotide self-diffusion indicates that the geometrical constraints of the Pluroni...

The liquid crystalline gel phases of solutions of Pluronic F127, a triblock copolymer, were recently introduced as an alternative to disordered solutions of random coil polymers as replaceable media for capillary gel electrophoresis (CGE). Pluronic F127, from BASF, is a copolymer of poly(ethylene oxide) and poly(propylene oxide) with the approximate formula (EO)106 (PO)70 (EO)106. Polymer chains aggregate into spherical micelles in aqueous solutions, with poly(propylene oxide) chains creating a hydrophobic core surrounded by brushes of hydrated poly(ethylene oxide) tails. Crowding at high concentrations promotes ordering of micelles. Solutions in the range of about 14–24 % polymer are self-supporting, gel-like cubic liquid crystals at 25–30°C, but when cooled they become low viscosity liquids that are easily loaded into capillaries. This article reviews applications of Pluronic F127 media for capillary gel electrophoresis separations of nucleic acids of several types including oligonucleotides, double stranded DNA fragments, and supercoiled plasmid DNAs.

Purpose: We investigated whether control of constitutive endothelial cell nitric oxide synthase (cNOS) and nitric oxide (NO) by changes in shear stress might be important for the regulation of smooth muscle cell (SMC) growth and vascular diameter. Methods: Bilateral femoral arteriovenous fistulas were placed in baboons to increase the blood flow in the external iliac arteries. At 2 months, the fistula was ligated on one side to restore normal flow (flow switch). Results: In response to flow switch and a decrease in shear stress, iliac artery lumenal area decreased and SMC proliferation was induced. A decline in NO production, cNOS messenger RNA (mRNA), and protein were associated with these biological effects. In a subset of animals with iliac arteries under high flow, infusion of Nω-nitro-l-arginine, an inhibitor of cNOS, did not induce proliferation. Conclusion: Shear stress can regulate cNOS, vasoconstriction, and SMC proliferation. A decrease in nitric oxide may be necessary, but is not sufficient to induce SMC proliferation in response to a decrease in blood flow. (J Vasc Surg 1998:28:514-21.)

Micellar, liquid crystalline solutions of poly(ethylene oxide) (EO)–poly(propylene oxide) (PO)–poly(ethylene oxide) copolymers are alternatives to solutions of random coil polymers as replaceable media for capillary `gel' electrophoresis. Excellent capillary gel electrophoresis separations of oligonucleotides were obtained in the liquid crystalline phase of 18–30% solutions of Pluronic F127 (approximate molecular formula [EO]106[PO]70[EO]106). These solutions are isotropic and flow freely into capillaries at reduced temperatures (0–5°C), but rapidly transform into a gel-like, cubic liquid crystalline phase of spherical micelles at room temperature and above. Separations on Pluronic gels can be tuned because polymer chain packing in the liquid crystalline phase is sensitive to polymer concentration and temperature. Optimum resolution of short oligonucleotides of sizes typically used for DNA sequencing primers or oligonucleotide therapies (≤40 nucleotides) was obtained by capillary gel electrophoresis in 25% Pluronic F127 at 30°C with an applied voltage of 500 V/cm. Decreasing the Pluronic concentration to 20% and increasing the temperature to 50°C substantially reduced analysis time with a modest decrease in resolution. Individual nucleotides in dT12–18 and dT19–24 standards were separated in 8 min with baseline resolution. The complex dependencies of resolution and peak shape on oligonucleotide size, Pluronic concentration, temperature, and applied voltage are described.

The pore structures of cross-linked polyacrylamide gels can be altered by polymerizing in the presence of high concentrations of unreactive, micellar surfactant cosolutes which act as "templates". Removal of surfactant after polymerization is expected to leave pores with the approximate shape and dimensions of the surfactant micelles. A simple model was developed to simulate gel permeation chromatography (GPC) separations of globular proteins on templated gels. The model assumes that the partition coefficient for sieving of a protein is equal to the fraction of gel volume accessible to a sphere with a radius equal to the protein Stokes radius. The total gel volume is considered to include a fraction that is a conventional, random gel matrix and a remaining fraction contributed by templated pores. The pore size distribution of the conventional gel was estimated using the Ogston equation, which approximates the matrix as a random collection of long, thin, rigid fibers. Templated pores were assumed to have a Gaussian distribution of radii centered about some mean determined by the micelle radius. In comparison to conventional media, gels with templated pores are predicted to exhibit more sharply defined exclusion limits and improved resolution over a narrow size range centered on the mean templated pore size. Selectivity and resolution are expected to increase as the volume fraction of templated pores is increased and as the dispersion of templated pore radius is decreased. Small changes in template radius lead to large changes in the molecular weight range of optimal separation of globular proteins. It should be possible to create a series of GPC media that collectively offer high resolution over the molecular weight range of most globular proteins of interest.

Abstract Blood flow and the tractive force shear stress are important determinants of artery caliber, and reduced shear predisposes arteries to intimal thickening and atherosclerosis. The molecular basis for shear-induced changes in artery wall structure is poorly defined. A number of factors associated with normal and pathological artery wall remodeling are induced by shear stress in endothelial cell cultures. These include platelet-derived growth factor (PDGF), a potent mitogen, chemoattractant, and vasoconstrictor. To determine whether similar changes occur in vivo, we examined the effects of reduced blood flow on endothelial cell PDGF expression and proliferation in the rat carotid artery. Branches of the right internal and external carotid arteries were ligated, reducing common carotid artery blood flow from 8.0±0.6 to 0.5±0.1 mL/min while increasing flow in the left carotid from 7.1±0.6 to 10.8±0.7 mL/min. Shear stress following the procedure was 1.4±0.2 and 33.4±1.1 dyne/cm 2 in carotids with reduced blood flow (RF) and increased blood flow (IF), respectively. Arteries were harvested 6, 24, 48, or 72 hours after ligation, perfusion-fixed, and opened longitudinally. Endothelial cell proliferation (bromodeoxyuridine [BrdU] labeling) was assessed en face at 24, 48, and 72 hours; expression of mRNA for PDGF-A and -B chains and PDGF α- and β-receptors (in situ hybridization) was determined at 6, 48, and 72 hours after unilateral flow reduction. RF induced endothelial cell proliferation, which peaked at 48 hours (RF BrdU labeling: 24 hours, 0.4±0.2%; 48 hours, 7.2±2.0%; and 72 hours, 4.1±0.6%; n=5). PDGF-B expression increased in RF compared with IF endothelium within 48 hours and persisted at 72 hours (percent labeling [RF/IF×100]: 6 hours, 76±20%; 48 hours, 395±179%; and 72 hours, 208±44%; n=3). PDGF-A expression was similarly increased in RF endothelium. In contrast, expression of PDGF α- and β-receptors was undetectable in RF and IF endothelium at all times. We conclude that endothelial cell PDGF ligand expression is induced by reduced shear stress in vivo and may play an important role in flow-mediated remodeling and atherogenesis.

Alkylamine-substituted naphthalene imides and diimides bind DNA by intercalation and have applications as anticancer agents. The unique structures of these imides in which two adjacent carbonyl groups lie coplanar to an extended aromatic ring system allow the possibility of sequence-selective interactions between the intercalated chromophore and guanine amino groups situated in the DNA minor groove. The binding affinities of N-[3-(dimethylamino)propyl amine]-1,8-naphthalenedicarboxylic imide (N-DMPrNI) and N,N'-bis [3,3'-(dimethylamino)propylamine]-naphthalene-1,4,5,8-tetracarboxylic diimide (N-BDMPrNDI) for natural DNAs of differing base composition were determined spectroscopically and by equilibrium dialysis. In agreement with the above proposition, binding studies indicated that both the naphthalene imide and diimide strongly prefer to intercalate into steps containing at least one G:C base pair. The dependencies of association constants on DNA base composition are consistent with a requirement for one G:C pair in the binding site of the monomide, and two G:C pairs in binding sites of the diimide. These selectivities are comparable to or exceed that of actinomycin D, a classic G:C-selective drug. Protection footprinting with DNase I confirmed that the naphthalene monoiimide (N-DMPrNI) prefers to bind adjacent to G:C base pairs, with a most consistent preference for "mixed" steps containing both a G:C and an A:T pair, excepting GA:TC. Several 5'-CG-3' steps were also good binding sites as indicated by nuclease protection, but few GC:GC or GG:CC steps were protected. The naphthalene diimide inhibited DNase I digestion, but did not yield a footprint. The base recognition ability and versatile chemistry make naphthalene imides and diimides attractive building blocks for design of highly sequence-specific, DNA-directed drug candidates including conjugated oligonucleotides or oligopeptides.

An approach is described for the synthesis of nanostructured hydrogels with defined size channels or pores for separations of biological macromolecules. Polyacrylamide gels (15-20% acrylamide) were cast in the presence of high concentrations (5%-28%) of hydrophilic, macromolecular cosolutes either semirigid, rod-like polyelectrolytes (short fragments of DNA or xanthan) or spherical micelles of sodium dodecyl sulfate (SDS). Polyanionic cosolutes were then removed by a combination of diffusion and electrophoresis. These processes are expected to yield gels with 'templated' channels having dimensions near those of the double helical polymers (diameters approximately 2-3 nm, lengths of 50-200 nm) or pores near the size of the spherical SDS micelles (approximately 4-5 nm). This hypothesis was tested by comparing the relative electrophoretic mobilities of proteins (3400 to 43,000 Da) complexed with SDS on templated and conventional gels. Differences in electrophoretic mobilities were observed between all templated gels and control normal gels, demonstrating that the templating process altered the polyacrylamide network. No evidence was found for phase separation of cosolutes from the polyacrylamide network during polymerization. Templated pores are expected to enhance the mobilities of molecules in a particular size range relative to smaller and larger molecules. Gels templated with DNA or xanthan (8-10% final concentration) exhibited little size selectivity, but selectivity was observed for gels templated with SDS varied with polyacrylamide concentration in a manner consistent with the creation of templated pores approximately the size of SDS micelles and larger than the average pore size in a surrounding polyacrylamide network.

Background Heparin is a potent inhibitor of smooth muscle cell (SMC) growth and intimal hyperplasia in animal models but has been ineffective in inhibiting restenosis in humans. This difference may relate to flaws in clinical study design or, alternatively, to interspecies differences in SMC response to heparin. To determine whether heparin could inhibit intimal hyperplasia in a species more closely related to humans, we studied the effect of a low-molecular-weight heparin (LMWH) on baboon SMC proliferation and migration in culture and in arteries subjected to experimental angioplasty. Methods and Results LMWH or saline was infused continuously after experimental angioplasty of baboon peripheral arteries (six animals per group). After 28 days, bromodeoxyuridine (BrdU) was given to label proliferating cells, and balloon-injured arteries were perfusion-fixed in situ and removed for analysis. All arteries had reendothelialized (Evans blue dye exclusion). LMWH increased partial thromboplastin time (LMWH, 81.7±8.4 seconds versus saline, 34.7±0.8 seconds [mean±SEM]; P =.004) but failed to inhibit intimal thickening or SMC proliferation (intimal area: LMWH, 0.19±0.03 mm 2 versus saline, 0.21±0.03 mm 2 ; BrdU labeling: LMWH, 2.9±0.6% versus saline, 2.4±0.4%; P =NS). In culture, LMWH and standard heparin (100 μg/mL) significantly inhibited serum-induced but not platelet-derived growth factor (PDGF-BB)–induced SMC proliferation (% control, serum: LMWH, 60.5±4.0%, P =.0002; standard heparin, 29.4±8.2%, P =.0001; % control, PDGF-BB: LMWH, 117.7±11.3%, P =NS; standard heparin, 90.9±14.4%, P =NS) and SMC migration (% control, serum: LMWH, 15.3±1.9%, P =.0198; standard heparin, 26.4±13.8%, P =.0032; % control, PDGF-BB: LMWH, 98.5±14.3%, P =NS; standard heparin, 100.0±13.5%, P =NS). Conclusions LMWH failed to inhibit intimal hyperplasia in a baboon angioplasty model. Furthermore, LMWH blocked serum-induced but not PDGF-BB–induced SMC proliferation and migration in culture. Thus, heparin-sensitive and -insensitive pathways exist for SMC activation. The relative importance of each pathway induced by injury may vary between species and thus account for different responses to heparin.

Venusian canali, outflow channels, and associated volcanic deposits resemble fluvial landforms more than they resmeble volcanic features on Earth and Mars. Some canali have meandering habits and features indicative of channel migration that are very similar to meandering river channels and flood plains on Earth, venusian outflow channels closely resemble water-carved outflow channels on Mars and the Channeled Scabland in Washington, collapsed terrains at the sources of some venusian channels resemble chaotic terrains at the sources of martian outflow channels, venusian lava deltas are similar to bird's-foot deltas such as the Mississippi delta, and venusian valley networks indicate sapping. We have developed an alternative possibility that the lava had a water-like rheology and a melting point slightly greater than Venus' surface temperature, thus accounting for the unusual behavior of the lava. Unlike silicate lavas, some carbonatites (including carbonate-sulfate-rich liquids) have these properties; thus they can flow great distances while retaining a high fluidity, significant mechanical erosiveness, and substantial capacity to transport and deposit sediment. Venusian geochemistry and petrology are consistent with extensive eruptions of carbonatite lavas, which could have crustal and/or mantle origins. Venus' atmosphere (especially CO2, HCl, and HF abundances) and rocks may be in local chemical equilibrium, which suggests that the upper crust contains large amounts of calcite, anhydrite, and other salts. Chemical analyses indicate, according to some models, that Venusian rocks may contain 4-19% calcite and anhydrite. Mixtures of crustal salts could melt at temperatures a few tens to a few hundred Kelvins higher than Venus' surface temperature; hence, melting may be induced by modest endogenetic or impact heating. Salts may have many of the same geologic roles on Venus as water and ice have on Mars. A molten salt (carbonatite) 'aquifer' may exist beneath a few hundred meters to several kilometers of solidified salt-rich 'permafrost.' Many geologic features can be explained by carbonatite magmatism: (1) impact melting of crustal salts can explain crater outflows, (2) small, sustained eruptions from molten salt aquifers can explain sapping valleys, (3) large, sustained eruptions may explain canali and their flood plans, and (4) catastrophic outbursts amy have formed outflow channels and chaotic terrain. Landforms created by carbonate-rich lavas would be thermally stable on Venus' surface, though some minerals may weather to other solid substances.

Giardia lamblia is dependent on the salvage of preformed purines and pyrimidines. This study investigated purine nucleoside and nucleobase transport utilizing rapid uptake determinations. Nucleoside substrate/velocity curves exhibited the hyperbolic kinetics of a saturable carrier-mediated system. Deoxynucleosides exhibited a much lower affinity for the transporter. Inhibition studies confirmed the relative carrier affinities of these ribonucleosides and deoxyribonucleosides. The nucleobase adenine did not exhibit saturation kinetics at a comparable substrate range, and did not inhibit nucleoside transport. Dipyridamole markedly inhibited nucleoside but not nucleobase transport, confirming the separate entry pathways. When cells were depleted of ATP, the velocity of nucleoside and nucleobase transport was unchanged, indicating that it is a non-energy-dependent process. Three nucleoside analogs, formycin A, adenine arabinoside and 7-deazaadenosine, were studied. Transport kinetics ranged widely among this group and could not completely account for their cytotoxic effect. When the apparent Km and Vmax of the nucleosides were compared, an approximately linear relationship (r2 = 0.95) was noted. This suggests that a high affinity of the nucleoside permease for the substrate retards disassociation of the substrate-carrier complex, slowing net influx.

The influences of base sequence on the thermodynamic properties associated with the interaction of actinomycin D with DNA are examined. It has been previously established that GpC steps of double-helical DNAs are highly preferred binding sites for actinomycin D. In this study, a series of oligonucleotides was designed and synthesized to probe the effects of flanking base sequence (adjacent to the GpC step) and novel non-GpC binding sites on the binding of actinomycin D. The use of these oligonucleotides provides a direct method for quantitating sequence specificities and actinomycin D binding energetics. Effects of different 5' and 3' flanking nucleotides on the interactions of actinomycin with the core GpC binding sites were examined using UV-visible spectrophotometric methods, and changes in binding energetics were quantitated. These studies demonstrate strong actinomycin D binding affinities to both classical GpC and an atypical non-GpC site. Enthalpy and entropy components of the DNA binding energetics for the GpC binding sites are compared and correlated with those determined for actinomycin D binding to the high-affinity non-GpC site of an 11-mer containing TGGGT as the central sequence. This TGGGT site, first suggested to be a high-affinity sequence in our earlier photoaffinity labeling studies, exhibits binding of actinomycin D comparable in strength to that of traditional actinomycin D binding sites (i.e., GpC steps). From these studies, the overall affinity and specific thermodynamic contributions (delta H degree, delta S degree) to binding of actinomycin D are demonstrated to be highly influenced both by the sequence at the intercalation site and by neighboring bases which flank the intercalation site.

Sepsis induces primary myocardial dysfunction. Yet, both hyper- and hypodynamic cardiac states characterize the sepsis syndrome, suggesting a modulatory role of septic mediators. Platelet activating factor (PAF), implicated in the pathogenesis of sepsis, is an endogenous phospholipid with diverse intracellular and extracellular effects. The purpose of this study was to investigate the influence of PAF (1) upon basal mechanical function of the heart, (2) upon receptor-coupled function of the heart, and (3) on basal and stimulated myocardial function at differing concentrations. In order to focus on the relationship between PAF and cardiac mechanical function, rat hearts were isolated and crystalloid perfused using a modified Langendorf preparation. Separate hearts received intracoronary vehicle (5% ethanol, 2.5% BSA) or PAF (20 or 40 μM) as a bolus, followed 10 min later by 0.25 μM isoproterenol (β-receptor agonist) infusion over 3 min. Both 20 and 40 μM PAF produced a rapid decrease in rate pressure product (RPP = HR × LVDPmax) relative to control (P 0.05) among the groups. Isoproterenol infusion increased (P 0.05). We conclude that PAF (1) induces rapid but reversible basal myocardial mechanical depression, (2) enhances receptor-coupled myocardial function when given in a low concentration (20 μM), and (3) appears to alter myocardial responsiveness to a β-adrenergic stimulus in a dose-specific manner. These findings demonstrate that PAF can both depress basal myocardial function as well as enhance the functional response to β-adrenergic stimulation in an isolated heart preparation.

Several types of cells made of Pyrex® ( nD20 = 1.474) capillaries were constructed for fluorimetry from the standpoint of waveguiding of the excitation source. Fluorescence intensity detected from a vortex cell is virtually linearly dependent on the refractive index of the solvent containing the fluorophore. This type of cell is applied as the fluorimctric detector for the flow injection analysis of the lead(II) ion. The lead ion was incorporated into a macrocyclic ligand, cryptand 2.2.2, and the resulting compound was ion-paired with eosine (fluorescent dye). This complex ([Pb(cryptand 2.2.2)]eosine) was extracted into chlorobenzene ( nD20 = 1.5248) and was introduced into the above vortex cell. An Ar+ laser operated at 514.5 nm was used as the light source, and the fluorescence of eosine at 600 nm was detected. Two ng/mL of Pb(II) ion was obtained as the detection limit.

Two nucleosides related to the known antiprotozoal agent 1-(beta-D-ribofuranosyl)-1,5-dihydro-4H-pyrazolo-[3,4-d]pyrimidine-4-one (allopurinol riboside, 1) were prepared and evaluated against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma gambiense. 3'-Deoxyinosine (2) exhibited potent antiprotozoal activity against the three protozoal pathogens with minimal toxicity for host cells. It was found to be especially effective against the Columbia strain of T. cruzi reported to be resistant to 1. The antiprotozoal activity of 2 appeared to be inversely related to the rate of cleavage of the glycosidic bond, as shown by metabolic profiles of 2 in the various pathogenic hemoflagellates and host cells. Combining the key structural elements of 1 and 2 led to the synthesis of 1-(3-deoxy-beta-D-erythro-pentofuranosyl)-1,5-dihydro-4H-pyrazolo[3,4-d] pyrimidin-4-one (3'-deoxy-allopurinol riboside, 3). which was found to be inactive as an antiprotozoal agent.

The noncovalent DNA binding of the bis(1,10-phenanthroline)copper(I) complex [(Phen)2CuI] was examined under anaerobic conditions by absorption and circular dichroism spectroscopy, and viscometry, as a function of phenanthroline concentration. Analyses according to the McGhee-von Hippel method indicated that binding exhibited both neighbor-exclusion and positive cooperativity effects, with a neighbor-exclusion parameter n approximately 2 and a cooperativity parameter omega approximately 4. The association constant for (Phen)2CuI binding decreased with increasing concentration of phenanthroline in excess over that required to stoichiometrically generate (Phen)2CuI, indicating that free phenanthroline was a weak competitive inhibitor of (Phen)2CuI binding. The maximal association constant for DNA binding of (Phen)2CuI in 0.2 M NaCl and 9.8% ethanol, extrapolated to zero concentration of excess phenanthroline, was 4.7 x 10(4) M-1 (DNA base pairs). The magnitude of the neighbor-exclusion parameter, the changes in spectral properties of (Phen)2CuI induced by DNA binding, and the increase in DNA solution viscosity upon (Phen)2CuI addition are consistent with a model for DNA binding by (Phen)2CuI involving partial intercalation of one phenanthroline ring of the complex between DNA base pairs in the minor groove as suggested previously [Veal & Rill (1989) Biochemistry 28, 3243-3250]. Viscosity measurements indicated that the mono(phenanthroline)copper(I) complex also binds to DNA by intercalation; however, no spectroscopic or viscometric evidence was found for DNA binding of free phenanthroline or the bis(2,9-dimethyl-1,10-phenanthroline)copper(I) complex. DNA binding of free phenanthroline may be cooperative and induced by prior binding of (Phen)2CuI.

Phyllosilicates have previously been detected in layered outcrops in and around the Martian outflow channel Mawrth Vallis. CRISM spectra of these outcrops exhibit features diagnostic of kaolinite, montmorillonite, and Fe/Mg-rich smectites, along with crystalline ferric oxide minerals such as hematite. These minerals occur in distinct stratigraphic horizons, implying changing environmental conditions and/or a variable sediment source for these layered deposits. Similar stratigraphic sequences occur on both sides of the outflow channel and on its floor, with Al-clay-bearing layers typically overlying Fe/Mg-clay-bearing layers. This pattern, combined with layer geometries measured using topographic data from HiRISE and HRSC, suggests that the Al-clay-bearing horizons at Mawrth Vallis postdate the outflow channel and may represent a later sedimentary or altered pyroclastic deposit that drapes the topography.

from

[1] Cassini RADAR observations now permit an initial assessment of the inventory of two classes, presumed to be organic, of Titan surface materials: polar lake liquids and equatorial dune sands. Several hundred lakes or seas have been observed, of which dozens are each estimated to contain more hydrocarbon liquid than the entire known oil and gas reserves on Earth. Dark dunes cover some 20% of Titan's surface, and comprise a volume of material several hundred times larger than Earth's coal reserves. Overall, however, the identified surface inventories (>3 × 104 km3 of liquid, and >2 × 105 km3 of dune sands) are small compared with estimated photochemical production on Titan over the age of the solar system. The sand volume is too large to be accounted for simply by erosion in observed river channels or ejecta from observed impact craters. The lakes are adequate in extent to buffer atmospheric methane against photolysis in the short term, but do not contain enough methane to sustain the atmosphere over geologic time. Unless frequent resupply from the interior buffers this greenhouse gas at exactly the right rate, dramatic climate change on Titan is likely in its past, present and future.

Methods are described for the synthesis of the α and β anomers of 7-D−ribofuranosylfuro[3,2-d]pyrimidines substituted at C-4 with either amino, oxo, thiono, or methylthio groups. The analogous 2,4-dithiono compounds are also described. The key steps involve the LDA-promoted cyclizations of the cyano ethers 3 and 14 to give, respectively, the substituted furan C-nucleosides A and 16. After separation of anomers, these compounds were subjected to a number of pyrimidine-ring-forming reactions to give the desired bicyclic C-nucleosides. The adenosine analogue 13 is markedly cytotoxic to mouse L1210 cells in vitro, and the inosine analogue 20 shows activity against the pathogenic protozoans Leishmania donovani and Trypanosoma gambiense.

Etude par microscopie optique polarisee et diffraction laser de l'influence des concentrations de l'ADN C D , de l'electrolyte et de la nature du contre-cation (Na + ou NH + 4 ) sur la valeur du pas de l'helice de l'ADN en solution electrolytique concentree. Contrairement a ce qui etait attendu, le pas de l'helice augmente avec C D au voisinage de la transition phase cholesterique→phase a haute densite

The surface of Saturn's haze-shrouded moon Titan has long been proposed to have oceans or lakes, on the basis of the stability of liquid methane at the surface1, 2. Initial visible3 and radar4, 5 imaging failed to find any evidence of an ocean, although abundant evidence was found that flowing liquids have existed on the surface5, 6. Here we provide definitive evidence for the presence of lakes on the surface of Titan, obtained during the Cassini Radar flyby of Titan on 22 July 2006 (T16). The radar imaging polewards of 70° north shows more than 75 circular to irregular radar-dark patches, in a region where liquid methane and ethane are expected to be abundant and stable on the surface2, 7. The radar-dark patches are interpreted as lakes on the basis of their very low radar reflectivity and morphological similarities to lakes, including associated channels and location in topographic depressions. Some of the lakes do not completely fill the depressions in which they lie, and apparently dry depressions are present. We interpret this to indicate that lakes are present in a number of states, including partly dry and liquid-filled. These northern-hemisphere lakes constitute the strongest evidence yet that a condensable-liquid hydrological cycle is active in Titan's surface and atmosphere, in which the lakes are filled through rainfall and/or intersection with the subsurface 'liquid methane' table.

International audience; The study of Titan, Saturn's largest satellite, is a major goal of the Cassini-Huygens mission. This joint project between NASA, the European Space Agency, and the Italian Space Agency consists of a Saturn orbiter (Cassini) and a Titan probe (Huygens). Since the mission's arrival at Saturn in July 2004, one of its most spectacular discoveries has been the finding of the first extraterrestrial nonmagmatic standing bodies of liquid: Titan's hydrocarbon lakes and seas. In July 2006, the first synthetic aperture radar (SAR) images of Titan's north polar region btained by the Cassini spacecraft showed dozens of lakes above latitudes of 70° [Stofan et al., 2007]. Subsequent SAR images obtained by Cassini have covered approximately 68% of Titan's north polar region at latitudes above 60 degrees. These images show more than 400 radar-dark areas that we interpret as being liquid lakes (shown in dark blue in Figure 1), including a few that are so large that they rightfully may be called seas. We discuss here the evidence for liquids on Titan, the distribution and morphology of lakes, and recent data that indicate the presence of lakes in the south polar regions.

A compact, dedicated cadmium zinc telluride (CZT) gamma camera coupled with a fully three-dimensional (3-D) acquisition system may serve as a secondary diagnostic tool for volumetric molecular imaging of breast cancers, particularly in cases when mammographic findings are inconclusive. The developed emission mammotomography system comprises a medium field-of-view, quantized CZT detector and 3-D positioning gantry. The intrinsic energy resolution, sensitivity and spatial resolution of the detector are evaluated with Tc-99m (140 keV) filled flood sources, capillary line sources, and a 3-D frequency-resolution phantom. To mimic realistic human pendant, uncompressed breast imaging, two different phantom shapes of an average sized breast, and three different lesion diameters are imaged to evaluate the system for 3-D mammotomography. Acquisition orbits not possible with conventional emission, or transmission, systems are designed to optimize the viewable breast volume while improving sampling of the breast and anterior chest wall. Complications in camera positioning about the patient necessitate a compromise in these two orbit design criteria. Image quality is evaluated with signal-to-noise ratios and contrasts of the lesions, both with and without additional torso phantom background. Reconstructed results indicate that 3-D mammotomography, incorporating a compact CZT detector, is a promising, dedicated breast imaging technique for visualization of tumors

International audience; The irreversible conversion of methane into higher hydrocarbons in Titan's stratosphere implies a surface or subsurface methane reservoir. Recent measurements from the cameras aboard the Cassini orbiter fail to see a global reservoir, but the methane and smog in Titan's atmosphere impedes the search for hydrocarbons on the surface. Here we report spectra and high-resolution images obtained by the Huygens Probe Descent Imager/Spectral Radiometer instrument in Titan's atmosphere. Although these images do not show liquid hydrocarbon pools on the surface, they do reveal the traces of once flowing liquid. Surprisingly like Earth, the brighter highland regions show complex systems draining into flat, dark lowlands. Images taken after landing are of a dry riverbed. The infrared reflectance spectrum measured for the surface is unlike any other in the Solar System; there is a red slope in the optical range that is consistent with an organic material such as tholins, and absorption from water ice is seen. However, a blue slope in the near-infrared suggests another, unknown constituent. The number density of haze particles increases by a factor of just a few from an altitude of 150 km to the surface, with no clear space below the tropopause. The methane relative humidity near the surface is 50 per cent.

Although oxidatively damaged lipoproteins are implicated in vascular injury, there is little information regarding the role of high-density lipoprotein (HDL) oxidation in atherogenesis. One potential pathway involves hypochlorous acid (HOCl) produced by myeloperoxidase (MPO), a heme protein secreted by phagocytes. We previously showed that 3-chlorotyrosine is a specific product of HOCl. Therefore, to explore the role of oxidized HDL in the pathogenesis of vascular disease, we used MS to quantify 3-chlorotyrosine in HDL isolated from plasma and atherosclerotic tissue. HDL from human aortic atherosclerotic intima had an 8-fold higher level of 3-chlorotyrosine than plasma HDL. Tandem MS analysis identified MPO as a component of lesion HDL, suggesting that the two interact in the artery wall. Moreover, immunohistochemical studies found that specific epitopes derived from HOCl colocalized with apolipoprotein A-I, the major protein of HDL. These observations strongly support the hypothesis that MPO promotes HDL oxidation in the human artery wall. Levels of 3-chlorotyrosine were elevated in HDL isolated from the blood of humans with established coronary artery disease, suggesting that circulating levels of oxidized HDL represent a unique marker for clinically significant atherosclerosis. HDL or lipid-free apolipoprotein A-I exposed to HOCl was less able to remove cholesterol from cultured cells by a pathway requiring the cell membrane transporter ATP-binding cassette transporter A1. The detection of 3-chlorotyrosine in HDL isolated from vascular lesions raises the possibility that MPO, by virtue of its ability to form HOCl, may promote atherogenesis by counteracting the established antiatherogenic effects of HDL and the ATP-binding cassette transporter A1 pathway.

Proteomics and peptidomics could benefit from simple methods for high-resolution separation of oligopeptides analogous to slab gel electrophoresis of proteins. Gels of Pluronic F127 copolymer surfactant were investigated as media for slab gel electrophoresis of oligopeptides using a trypsin digest of myoglobin. Concentrated solutions of Pluronic F127 are fluid at low temperatures (/= 5 degrees C), but become a gel-like micellar liquid crystal upon warming. Nucleic acids are well separated by electrophoresis in these gels as previously shown by Rill and Liu. Good separations of myoglobin tryptic peptides were accomplished by electrophoresis on slab gels of 24% Pluronic F127 or 15% polyacrylamide using the alkaline Laemmli buffer system (without sodium dodecyl sulfate). Labeling of peptides with the succinimidyl ester of Cascade Yellow (CY) prior to electrophoresis allowed sensitive detection with blacklight illumination at 365 nm. Labeled tryptic peptides were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. An inverse dependence of electrophoretic mobility on mass of peptides with charge Z = -1 was observed in both media. Two-dimensional (2-D) electrophoresis of myoglobin peptides on polyacrylamide, then on Pluronic media, at pH 8.3 indicated that the primary separation mechanism of most peptides was the same in both media. A few off-diagonal spots indicated that some peptides were preferentially retarded in Pluronic gels, perhaps due to hydrophobic effects. The ease of gel preparation and peptide recovery are advantages of Pluronic F127 gels for oligopeptide electrophoresis. The two media can be combined conveniently for 2-D electrophoresis, providing means to facilitate protein identification and peptidomics.