An efficient algorithm for generating the internal branches of a Kingman coalescent.

Coalescent simulations are a widely used approach for simulating sample genealogies, but can become computationally burdensome in large samples. Methods exist to analytically calculate a sample's expected frequency spectrum without simulating full genealogies. However, statistics that rely on the distribution of the length of internal coalescent branches, such as the probability that two mutations of equal size arose on the same genealogical branch, have previously required full coalescent simulations to estimate. Here, we present a sampling method capable of efficiently generating limited portions of sample genealogies using a series of analytic equations that give probabilities for the number, start, and end of internal branches conditional on the number of final samples they subtend. These equations are independent of the coalescent waiting times and need only be calculated a single time, lending themselves to efficient computation. We compare our method with full coalescent simulations to show the resulting distribution of branch lengths and summary statistics are equivalent, but that for many conditions our method is at least 10 times faster.

Safety, pharmacokinetics and pharmacodynamics of the anti-hepcidin Spiegelmer lexaptepid pegol in healthy subjects.

BACKGROUND AND PURPOSE: Anaemia of chronic disease is characterized by impaired erythropoiesis due to functional iron deficiency, often caused by excessive hepcidin. Lexaptepid pegol, a pegylated structured l-oligoribonucleotide, binds and inactivates hepcidin. EXPERIMENTAL APPROACH: We conducted a placebo-controlled study on the safety, pharmacokinetics and pharmacodynamics of lexaptepid after single and repeated i.v. and s.c. administration to 64 healthy subjects at doses from 0.3 to 4.8 mg·kg(-1) . KEY RESULTS: After treatment with lexaptepid, serum iron concentration and transferrin increased dose-dependently. Iron increased from approximately 20 µmol·L(-1) at baseline by 67% at 8 h after i.v. infusion of 1.2 mg·kg(-1) lexaptepid. The pharmacokinetics showed dose-proportional increases in peak plasma concentrations and moderately over-proportional increases in systemic exposure. Lexaptepid had no effect on hepcidin production or anti-drug antibodies. Treatment with lexaptepid was generally safe and well tolerated, with mild and transient transaminase increases at doses ≥2.4 mg·kg(-1) and with local injection site reactions after s.c. but not after i.v. administration. CONCLUSIONS AND IMPLICATIONS: Lexaptepid pegol inhibited hepcidin and dose-dependently raised serum iron and transferrin saturation. The compound is being further developed to treat anaemia of chronic disease.

Pharmacological characteristics of a novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP).

BACKGROUND: Recombinant factor VIIa (rFVIIa) is approved for use in controlling bleeding episodes in people with hemophilia who have developed inhibitors to replacement therapy. Due to its short half-life (t½), frequent injections are required, limiting its use as a prophylactic treatment. A novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) has been developed to extend the t(½) of rFVIIa. OBJECTIVES: The aim of our studies was to investigate the pharmacokinetic/pharmacodynamic characteristics of rVIIa-FP in preclinical animal species. METHODS: Pharmacokinetic (PK) parameters were derived after single intravenous dosing in hemophilia A mice, rats, rabbits and monkeys. PK analysis was based on human FVII plasma levels determined by measuring FVII antigen levels by ELISA in mice and rats, and FVIIa activity using STACLOT® VIIa-rTF in rabbits and monkeys. Induction of thrombin generation was investigated in mice, while hemostatic activity was assessed by thrombus formation in rabbits. RESULTS: Compared with rFVIIa, rVIIa-FP displayed a prolonged t(½), enhanced in vivo recovery and reduced clearance in all species investigated. In mice, 16 h after treatment with rVIIa-FP, thrombin levels were quantifiable, indicating prolonged efficacy, whereas values had approached baseline at this time after treatment with rFVIIa. After 12 h, hemostatic efficacy was negligible in rFVIIa-treated rabbits, but sustained in animals receiving rVIIa-FP. CONCLUSIONS: These studies indicate that the longer t(½) of rVIIa-FP compared with rFVIIa translates into extended activity. These findings suggest that rVIIa-FP has the potential to be administered less frequently than rFVIIa-containing concentrates in clinical use.

Renal Ewing tumors.

BACKGROUND: Renal Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET) is extremely rare. Clinical symptoms are nonspecific presenting abdominal pain, palpable mass, and hematuria. Owing to advanced technology demonstrating the ES-specific EWS/ETS translocation, this differential diagnosis has become feasible. PATIENTS AND METHODS: The German database of GPOH Ewing's sarcoma trials from 1980 to 2009 was searched for kidney as primary site. Twenty-four patients were identified and analyzed. The median time of observation was 3.71 years (range 0.27-8.75 years). Additionally, we carried out a Medline search for renal ES/PNET. RESULTS: The median age was 24.9 years (range 11-60 years). In 37.5%, patients presented with primary metastases. Tumor thrombi in the adjacent renal vessels occurred in 56.2%. In 90.9%, rearrangements of t(11;22) were found. All patients received a combined chemotherapy according to the EURO-E.W.I.N.G.99 protocol. In accordance, local control consisted predominantly of combined modality surgery and radiation (47%). At 3 years, overall survival (OS) was 0.80 (SE = 0.09), and event-free survival (EFS) 0.66 (SE = 0.11). CONCLUSIONS: ES/PNET should be considered in the differential diagnosis of renal tumors. Patients with renal ES/PNET respond to and benefit from conventional ES treatment according to ES study protocols. Therefore, an accurate diagnostic approach and a guideline-adapted therapy should be facilitated.

Hematopoietic stem and progenitor cell mobilization in mice and humans by a first-in-class mirror-image oligonucleotide inhibitor of CXCL12.

NOX-A12 is a PEGylated mirror-image oligonucleotide (a so-called Spiegelmer) that binds to CXCL12 (stromal cell-derived factor-1, SDF-1) with high affinity thereby inhibiting CXCL12 signaling on both its receptors, CXCR4 and CXCR7. In animals, NOX-A12 mobilized white blood cells (WBCs) and hematopoietic stem and progenitor cells (HSCs) into peripheral blood (PB). In healthy volunteers, single doses of NOX-A12 had a benign safety profile and also dose-dependently mobilized WBCs and HSCs into PB. HSC peak mobilization reached a plateau at five times the baseline level at an i.v. dose of 5.4 mg/kg. In accordance with the plasma half-life of 38 h, the duration of the WBC and HSC mobilization was long lasting and increased dose-dependently to more than 4 days at the highest dose (10.8 mg/kg). In conclusion, NOX-A12 may be appropriate for therapeutic use in and beyond mobilization of HSCs, e.g., in long-lasting mobilization and chemosensitization of hematological cancer cells.

Enrichment of cis-regulatory gene expression SNPs and methylation quantitative trait loci among bipolar disorder susceptibility variants.

We conducted a systematic study of top susceptibility variants from a genome-wide association (GWA) study of bipolar disorder to gain insight into the functional consequences of genetic variation influencing disease risk. We report here the results of experiments to explore the effects of these susceptibility variants on DNA methylation and mRNA expression in human cerebellum samples. Among the top susceptibility variants, we identified an enrichment of cis regulatory loci on mRNA expression (eQTLs), and a significant excess of quantitative trait loci for DNA CpG methylation, hereafter referred to as methylation quantitative trait loci (mQTLs). Bipolar disorder susceptibility variants that cis regulate both cerebellar expression and methylation of the same gene are a very small proportion of bipolar disorder susceptibility variants. This finding suggests that mQTLs and eQTLs provide orthogonal ways of functionally annotating genetic variation within the context of studies of pathophysiology in brain. No lymphocyte mQTL enrichment was found, suggesting that mQTL enrichment was specific to the cerebellum, in contrast to eQTLs. Separately, we found that using mQTL information to restrict the number of single-nucleotide polymorphisms studied enhances our ability to detect a significant association. With this restriction a priori informed by the observed functional enrichment, we identified a significant association (rs12618769, P(bonferroni)<0.05) from two other GWA studies (TGen+GAIN; 2191 cases and 1434 controls) of bipolar disorder, which we replicated in an independent GWA study (WTCCC). Collectively, our findings highlight the importance of integrating functional annotation of genetic variants for gene expression and DNA methylation to advance the biological understanding of bipolar disorder.

Improved kinetics of rIX-FP, a recombinant fusion protein linking factor IX with albumin, in cynomolgus monkeys and hemophilia B dogs.

BACKGROUND: Prophylaxis of hemophilia B, at present, requires multiple infusions of human factor (F)IX concentrates per week. A FIX molecule with a prolonged half-life has the potential to greatly improve the convenience of, and adherence to, prophylaxis. OBJECTIVES: The aim of our studies was to investigate the pharmacokinetic (PK) and pharmacodynamic (PD) profile of a recombinant fusion protein linking coagulation FIX with albumin (rIX-FP). METHODS: Cynomolgus monkeys and hemophilia B dogs received single intravenous doses of rIX-FP (50-500 IU kg(-1)). rIX-FP plasma levels were determined by an activity-based assay (dogs only) and anti-FIX ELISA methods. Additionally, activated partial thromboplastin time (APTT) was determined in hemophilia B dogs. Data were compared with a direct study comparator (recombinant FIX [rFIX]) or previously published data. RESULTS: The terminal half-life of rIX-FP was prolonged in both species compared with FIX reference data. In hemophilia B dogs, human FIX antigen levels remained above 0.05 IU mL(-1) more than three times longer after rIX-FP (7.3 days) compared with rFIX (2.3 days), whereas respective calculations based on activity levels confirmed the observed superior profile. Prolonged PDs of rIX-FP were demonstrated with APTT<60 s sustained around four times longer with rIX-FP (5.9 days) than rFIX (1.5 days). CONCLUSIONS: These studies indicate that the recombinant albumin fusion technology successfully improves the PK profile of FIX. Clinical studies will test whether the improved kinetics result in a significant half-life extension in patients with hemophilia B.

Combined superoxide dismutase mimetic and peroxynitrite scavenger protects against neointima formation after endarterectomy in association with decreased proliferation and nitro-oxidative stress.

OBJECTIVE: Reactive oxygen and nitrogen species (e.g., peroxynitrite) may trigger neointima formation leading to restenosis. In a rat carotid endarterectomy (CEA) model, we investigated the effects of the manganese(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP), a superoxide dismutase (SOD) mimetic and peroxynitrite scavenger on neointima formation. METHODS: CEA was performed in male Sprague-Dawley rats. Animals received either vehicle (control group; n=15) or 15 mg kg(-1) day(-1) MnTBAP intraperitoneally for 3 weeks (treatment group; n=13). Four groups of carotids were analysed: the left, uninjured carotids (sham) and the right, injured carotids (control CEA) from the control group, the right, injured carotids from the treatment group (CEA+MnTBAP) and an additional group of carotids that were harvested 1h following endarterectomy. The analysis of carotid arteries was performed by histology, immunohistochemistry and real-time polymerase chain reaction (PCR). Plasma malondialdehyde (MDA) levels were measured by lipid hydroperoxidase assay. RESULTS: Stenosis rate (10.5+/-8.1% vs. 45.4+/-28.3%), the percentage of proliferating cell nuclear antigen-positive cells (13.4+/-7.1% vs. 23.3+/-11.0%) and nitrotyrosine immunoreactivity (5.8+/-1.9 vs. 8.0+/-2.0) were significantly reduced in the vascular wall of the CEA+MnTBAP group compared with control CEA group. Ratio of Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL)-positive nuclei was significantly lower after antioxidant therapy (41.7+/-26.7% vs. 64.9+/-18.5%). Plasma MDA levels increased after endarterectomy (11.7+/-4.8 vs. 4.1+/-2.0 micromol l(-1)) and reduced in the treatment group (3.2+/-2.1 micromol l(-1)). No significant gene regulation after MnTBAP treatment could be noted. CONCLUSIONS: MnTBAP decreased neointima formation, which was associated with reduced vascular smooth muscle cell proliferation and attenuated local and systemic nitro-oxidative stress.

Genome-wide association study of bipolar disorder in European American and African American individuals.

To identify bipolar disorder (BD) genetic susceptibility factors, we conducted two genome-wide association (GWA) studies: one involving a sample of individuals of European ancestry (EA; n=1001 cases; n=1033 controls), and one involving a sample of individuals of African ancestry (AA; n=345 cases; n=670 controls). For the EA sample, single-nucleotide polymorphisms (SNPs) with the strongest statistical evidence for association included rs5907577 in an intergenic region at Xq27.1 (P=1.6 x 10(-6)) and rs10193871 in NAP5 at 2q21.2 (P=9.8 x 10(-6)). For the AA sample, SNPs with the strongest statistical evidence for association included rs2111504 in DPY19L3 at 19q13.11 (P=1.5 x 10(-6)) and rs2769605 in NTRK2 at 9q21.33 (P=4.5 x 10(-5)). We also investigated whether we could provide support for three regions previously associated with BD, and we showed that the ANK3 region replicates in our sample, along with some support for C15Orf53; other evidence implicates BD candidate genes such as SLITRK2. We also tested the hypothesis that BD susceptibility variants exhibit genetic background-dependent effects. SNPs with the strongest statistical evidence for genetic background effects included rs11208285 in ROR1 at 1p31.3 (P=1.4 x 10(-6)), rs4657247 in RGS5 at 1q23.3 (P=4.1 x 10(-6)), and rs7078071 in BTBD16 at 10q26.13 (P=4.5 x 10(-6)). This study is the first to conduct GWA of BD in individuals of AA and suggests that genetic variations that contribute to BD may vary as a function of ancestry.

Singleton deletions throughout the genome increase risk of bipolar disorder.

An overall burden of rare structural genomic variants has not been reported in bipolar disorder (BD), although there have been reports of cases with microduplication and microdeletion. Here, we present a genome-wide copy number variant (CNV) survey of 1001 cases and 1034 controls using the Affymetrix single nucleotide polymorphism (SNP) 6.0 SNP and CNV platform. Singleton deletions (deletions that appear only once in the dataset) more than 100 kb in length are present in 16.2% of BD cases in contrast to 12.3% of controls (permutation P=0.007). This effect was more pronounced for age at onset of mania

Synthesis of silicon-based infrared semiconductors in the Ge-Sn system using molecular chemistry methods.

Growth reactions based on a newly developed deuterium-stabilized Sn hydride [(Ph)SnD(3)] with Ge(2)H(6) produce a new family of Ge-Sn semiconductors with tunable band gaps and potential applications in high-speed, high-efficiency infrared optoelectronics. Metastable diamond-cubic films of Ge(1-x)Sn(x) alloys are created by chemical vapor deposition at 350 degrees C on Si(100). These exhibit unprecedented thermal stability and superior crystallinity despite the 17% lattice mismatch between the constituent materials. The composition, crystal structure, electronic structure, and optical properties of these materials are characterized by Rutherford backscattering, high-resolution electron microscopy, and X-ray diffraction, as well as Raman, IR, and spectroscopic ellipsometry. Electron diffraction reveals monocrystalline and perfectly epitaxial layers with lattice constants intermediate between those of Ge and alpha-Sn. X-ray diffraction in the theta-2theta mode shows well-defined peaks corresponding to random alloys, and in-plane rocking scans of the (004) reflection confirm a tightly aligned spread of the crystal mosaics. RBS ion-channeling including angular scans confirm that Sn occupies substitutional lattice sites and also provide evidence of local ordering of the elements with increasing Sn concentration. The Raman spectra show bands corresponding to Ge-Ge and Sn-Ge vibrations with frequencies consistent with random tetrahedral alloys. Resonance Raman and ellipsometry spectra indicate a band-gap reduction relative to Ge. The IR transmission spectra suggest that the band gap decreases monotonically with increasing Sn fraction. The synthesis, characterization, and gas-phase electron diffraction structure of (Ph)SnD(3) are also reported.

Spirocyclopropanated bicyclopropylidenes: straightforward preparation, physical properites, and chemical transformations.

Perspirocyclopropanated bicyclopropylidene (6) was prepared in three steps from 7-cyclopropylidenedispiro[2.0.2.1]heptane (4) (24% overall) or, more efficiently, through dehalogenative coupling of 7,7-dibromo[3]triangulane (15) (82%). This type of reductive dimerization turned out to be successful for the synthesis of (E)- and (Z)-bis(spiropentylidene) 14 (67%) and even of the "third-generation" spirocyclopropanated bicyclopropylidene 17 (17% overall from 15). Whereas the parent bicyclopropylidene 1 dimerized at 180 degrees C to yield [4]rotane, dimerization of 6 at 130 degrees C under 10 kbar pressure occured only with opening of one three-membered ring to yield the polyspirocyclopropanated (cyclopropylidene)cyclopentane derivative 19 (34% yield), and at the elevated temperature the polyspirocyclopropanated 2-cyclopropylidene[3.2.2]propellane derivative 20 (25 % yield). Perspirocyclopropanated bicyclopropylidene 6 and the "third-generation" bicyclopropylidene 17 gave addition of bromine, hydrogen bromide, and various dihalocarbenes without rearrangement. The functionally substituted branched [7]triangulane 28 and branched dichloro-C2v-[15]triangulane 32 were used to prepare the perspirocyclopropanated [3]rotane (D3h-[10]triangulane) 49 (six steps from 6, 1.4% overall yield) and the C2v-[15]triangulane 51 (two steps from 17, 41% overall). Upon catalytic hydrogenation, the perspirocyclopropanated bicyclopropylidene 6 yielded 7,7'-bis(dispiro[2.0.2.]-heptyl) (52) and, under more forcing conditions, 1,1'-bis(2,2,3,3-tetramethylcyclopropyl) (53). The bromofluorocarbene adduct 33 of 17 reacted with butyllithium to give the unexpected polyspirocyclopropanated 1,4-di-n-butyl-2-cyclopropylidenebicyclo[2.2.0]hexane derivative 37 as the main product (55% yield) along with the expected "third-generation" perspirocyclopropanated dicyclopropylidenemethane 38 (21% yield). Mechanistic aspects of this and the other unusual reactions are discussed. The structures of all new unusual hydrocarbons were proven by X-ray crystal structure analyses, and the most interesting structural and crystal packing features are presented.

Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells.

Sphingosine 1-phosphate (SPP) binds to members of the endothelial differentiation gene family (EDG) of receptors and leads to diverse signaling events including cell survival, growth, migration and differentiation. However, the mechanisms of how SPP activates these proangiogenic pathways are poorly understood. Here we show that SPP signals through the EDG-1 receptor to the heterotrimeric G protein G(i), leading to activation of the serine/threonine kinase Akt and phosphorylation of the Akt substrate, endothelial nitric-oxide synthase (eNOS). Inhibition of G(i) signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP also stimulates eNOS phosphorylation and NO release and these effects are also attenuated by inhibition of G(i) signaling, PI 3-kinase, and Akt. However, inhibition of NO production did not influence SPP-induced chemotaxis but effectively blocked the chemotactic actions of vascular endothelial growth factor. Thus, SPP signals through G(i) and PI 3-kinase leading to Akt activation and eNOS phosphorylation.

Synthesis, spectroscopic, and structural properties of spirocyclopropanated bicyclobutylidenes and their radical cations.

The spirocyclopropanated bicyclobutylidenes 3-7 have been prepared by McMurry coupling of the corresponding spirocyclopropanated cyclobutanone (3 and 5), Staudinger-Pfenniger reaction (4), oxidative coupling of a Wittig ylide (4) or Wittig olefination of perspirocyclopropanated cyclobutanone (6 and 7). The structure of the parent 2a and the perspirocyclopropanated bicyclobutylidene 5 was determined by X-ray crystallography which disclosed considerable steric congestion around the double bond. As a result 5 did undergo addition of dichlorocarbene, epoxidation with meta-chloroperbenzoic acid, and cyclopropanation with CH2I2/ZnEt2, but did not add the more bulky dibromocarbene. The reaction of 5 with tetracyanoethene proceeded smoothly, but led to a formal [3+2] cycloadduct across the proximal single bond of one of the inner cyclopropane rings. The consecutive spirocyclopropanation of bicyclobutylidene led to a bathochromic shift in the UV spectra of 12 and 17nm, respectively, for each pair of beta- and alpha-spirocyclopropane groups. In the He(I)-photoelectron spectra of these bicyclobutylidenes, the effect of spirocyclopropanation upon their pi-ionization energies (pi-IE,) was found to be almost additive, leading to a lowering of 0.05 eV per any additional beta-spirocyclopropane, and 0.28-0.22 eV per additional alpha-spirocyclopropane group; this indicates an increasing nucleophilicity of the double bonds in the order 1 < 4 < 3 < 5. Following the radical cations of the three symmetrical bicyclobutylidenes without (2a, b) and with six (5) spiroannelated cyclopropane rings, the radical cations of two symmetrical bicyclobutylidenes with two (4) and four (3) such rings were studied by ESR spectroscopy. Whereas 2b.+, 3.+, and 5.+ could be generated by electrolytic oxidation of the corresponding hydrocarbons in solution, the spectra of 2a.+ and 4.+, with unsubstituted 2,2',4,4'-positions, were observed upon radiolysis of their neutral precursors in a Freon matrix. On going from 2a.+ to 4.+, the coupling constant [aH] of the eight beta protons in the 2,2',4,4'-positions of bicyclobutylidene increases from 2.62 to 3.08 mT, and that of the four gamma protons in the 3,3'-positions changes from 0.27 to 0.049 to 0.401 mT on passing from 2a.+ via 2b.+ to 3.+. Computations by means of the density functional theory (DFT) at the B3LYP/6-311+G*//B3LYP/6-31G* level reproduce well the experimental hyperfine data.

Changes of endothelial nitric oxide synthase level and activity during endothelial cell proliferation.

The goal of this study was to investigate the effect of endothelial cell proliferation on the expression and activity of endothelial nitric oxide synthase (eNOS). Bovine atrial endothelial cells (BAtEC) were studied between day 1 and 6 after seeding. During this period the number of cells in S-phase decreased progressively, while cell number and protein content increased, reaching a maximum at confluence (day 4). Expression of eNOS (determined by ELISA) and eNOS activity (determined by L-arginine to L-citrulline conversion) increased with culture duration with a maximum at confluence. Nitric oxide (*NO) release from BAtEC was determined after stimulation with Ca2+ ionophore A23187 (10 microM, 30 min) by .NO chemiluminescence in the absence of a chemical reduction system. Total *NO release (measured in the presence of 100 U/ml superoxide dismutase) did not change with state of cell proliferation/growth, whereas "bioavailable" *NO (measured in the absence of superoxide dismutase) was low in highly proliferating BAtEC. Relative eNOS activity (.NO and L-citrulline production per eNOS protein) was highest in proliferating BAtEC. The novel finding of this study is that the specific eNOS activity is upregulated in proliferating BAtEC and downregulated in quiescent BAtEC. The amount of "bioavailable" *NO is determined by eNOS activity and *NO inactivation (probably by superoxide), both high in proliferating BAtEC.

A coalescent approach to study linkage disequilibrium between single-nucleotide polymorphisms.

We present the results of extensive simulations that emulate the development and distribution of linkage disequilibrium (LD) between single-nucleotide polymorphisms (SNPs) and a gene locus that is phenotypically stratified into two classes (disease phenotype and wild-type phenotype). Our approach, based on coalescence theory, allows an explicit modeling of the demographic history of the population without conditioning on the age of the mutation, and serves as an efficient tool to carry out simulations. More specifically, we compare the influence that a constant population size or an exponentially growing population has on the amount of LD. These results indicate that attempts to locate single disease genes are most likely successful in small and constant populations. On the other hand, if we consider an exponentially growing population that started to expand from an initially constant population of reasonable size, then our simulations indicate a lower success rate. The power to detect association is enhanced if haplotypes constructed from several SNPs are used as markers. The versatility of the coalescence approach also allows the analysis of other relevant factors that influence the chances that a disease gene will be located. We show that several alleles leading to the same disease have no substantial influence on the amount of LD, as long as the differences between the disease-causing alleles are confined to the same region of the gene locus and as long as each allele occurs in an appreciable frequency. Our simulations indicate that mapping of less-frequent diseases is more likely to be successful. Moreover, we show that successful attempts to map complex diseases depend crucially on the phenotype-genotype correlations of all alleles at the disease locus. An analysis of lipoprotein lipase data indicates that our simulations capture the major features of LD occurring in biological data.

PBN spin trapping of free radicals in the reperfusion-injured heart. Limitations for pharmacological investigations.

Post-ischemic reperfusion causes cardiac dysfunction and radical-induced lipid peroxidation (LPO) detectable by ESR spin trapping. This study deals with the applicability of the spin trapping technique to pharmacological investigations during myocardial reperfusion injury. The use of the spin trap phenylbutylnitrone (PBN, 3 mM) in isolated rat hearts demonstrated the release of alkoxyl radicals (aN = 1.39 mT, aHbeta = 0.19 mT) formed particularly within the first 15 min of reperfusion following 30 min of ischemia. The decline of radicals, after 10 min of reperfusion, was accompanied by recovery of function in 80% of the hearts. The radical concentration in the coronary effluent (maximum after 7.5 min) was reduced by the infusion of 1 mM mercaptopropionylglycine (MPG, 2.7+/-0.5 U/ml, p < 0.001) or 5 microM vitamin E (11.7+/-0.8 U/ml, p < 0.001), compared to the (PBN-containing) control (29.7+/-4.3 U/ml). Moreover, functional recovery (left ventricular developed pressure, LVDP 91.6 +/-20% of pre-ischemic level, p < 0.05) was improved by the hydrophilic radical scavenger MPG, compared to the (PBN-containing) control (LVDP 50.5+/-15.7% of baseline). PBN alone led to higher functional recovery (p < 0.05) and reduced VF (duration of ventricular fibrillation; 7.10+/-0.36 min/30 min, p < 0.05), compared to the untreated (PBN-free) control (LVDP 26.6+/-11.8%; VF 19.42+/-3.64 min/30 min). The Ca antagonist verapamil (0.1 microM), MPG, and the lipophilic vitamin E showed cardioprotection in the absence of PBN: post-ischemic recovery of LVDP was 25.4+/-6.8% (p < 0.05), 39.6+/-12.7% (p < 0.05) and 52.4+/-2.6% (p < 0.01), respectively, compared to the corresponding untreated control (13.3+/-6.6%). Whereas verapamil and vitamin E were able to protect the heart when present alone, they offered no additive effect in the presence of PBN. Therefore, PBN can be used to estimate the radical scavenger properties of an agent in the heart. However, because of the protective properties of PBN itself, the results of simultaneous investigations of the effects of other compounds, such as Ca antagonists or lipophilic radical scavengers, on heart function may be limited.

Mapping genes through the use of linkage disequilibrium generated by genetic drift: 'drift mapping' in small populations with no demographic expansion.

Linkage disequilibrium has been a powerful tool in identifying rare disease alleles in human populations. To date, most research has been directed to isolated populations which have undergone a bottleneck followed by rapid exponential expansion. While this strategy works well for rare diseases in which all disease alleles in the population today are clonal copies of some common ancestral allele, for common disease genes with substantial allelic heterogeneity, this approach is not predicted to work. In this paper, we describe the dynamics of linkage disequilibrium in populations which have not undergone a demographic expansion. In these populations, it is shown that genetic drift creates disequilibrium over time, while in expanded populations, the disequilibrium decays with time. We propose that common disease alleles might be more efficiently identified by drift mapping - linkage disequilibrium mapping in small, old populations of constant size where the disequilibrium is the result of genetic drift, not founder effect. Theoretical models, empirical data, and simulated population models are presented as evidence for the utility of this approach.

Nitroxides increase the detectable amount of nitric oxide released from endothelial cells.

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on .NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of .NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 microM A23187 released a stable flux of .NO, as detected by .NO chemiluminescence. Addition of 100 units/ml SOD or 10 microM of the nitroxides 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of .NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 microM. EPR spin trapping in SIN-1 solutions revealed the formation of .OH adducts from spontaneous dismutation of O-2 and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the .OH adduct by accelerating the dismutation of O-2. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable .NO in vitro.