Ankle fusion in hemophilic patients.

INTRODUCTION: Hemophilic arthropathy is painful and disabling. We report a retrospective study of ankle fusion with intra- and peri-operative clotting factor perfusion. The objective was to assess the efficacy of maintaining perioperative clotting factor rates close to 100%, and report long-term results. The study hypothesis was that results would be good, without early hemorrhagic complications. MATERIAL AND METHOD: Between 2000 and 2013, 12 ankle fusions were performed in 9 patients, with a mean age of 39years (range, 19-58years). Anti-hemophilic factor perfusion was controlled by the reference physician of the Regional Hemophilia Treatment Center. Clinical AOFAS and Olerud scores and the Pettersson radiologic score were used for assessment. Mean preoperative AOFAS score was 22 (range, 2-55) and mean Olerud score 7 (range, 5-12). Mean preoperative factor VIII concentration was <1% (range, <1-3%). RESULTS: Mean follow-up was 8years (range, 2-16years). Mean AOFAS score at follow-up was 69 (range, 35-92) and mean Olerud score 70 (range, 30-100). Improvement mainly concerned the Pain dimension. Statistical analysis found a significant difference between pre- and post-operative clinical scores (AOFAS, P=0.004; Olerud, P=0.004). Mean factor VIII concentration at surgery was 90% (range, 24-117%), and 109% (range, 75-152%) the day following surgery. There were no cases of hematoma or surgical site infection. Radiologic fusion was systematic at a mean 3.5 months (range, 3-4months). CONCLUSION: The study hypothesis was confirmed. Ankle fusion in advanced hemophilic arthropathy improved function and quality of life. Perioperative clotting factor perfusion contributed to these good results, providing supplementary prevention of hemorrhagic risk. LEVEL OF EVIDENCE: IV, retrospective study.

Efficient electron heating in relativistic shocks and gamma-ray-burst afterglow.

Electrons in shocks are efficiently energized due to the cross-shock potential, which develops because of differential deflection of electrons and ions by the magnetic field in the shock front. The electron energization is necessarily accompanied by scattering and thermalization. The mechanism is efficient in both magnetized and nonmagnetized relativistic electron-ion shocks. It is proposed that the synchrotron emission from the heated electrons in a layer of strongly enhanced magnetic field is responsible for gamma-ray-burst afterglows.

An expanding radio nebula produced by a giant flare from the magnetar SGR 1806-20.

Soft gamma-ray repeaters (SGRs) are 'magnetars', a small class of slowly spinning neutron stars with extreme surface magnetic fields, B approximately 10(15) gauss (refs 1 , 2 -3). On 27 December 2004, a giant flare was detected from the magnetar SGR 1806-20 (ref. 2), only the third such event recorded. This burst of energy was detected by a variety of instruments and even caused an ionospheric disturbance in the Earth's upper atmosphere that was recorded around the globe. Here we report the detection of a fading radio afterglow produced by this outburst, with a luminosity 500 times larger than the only other detection of a similar source. From day 6 to day 19 after the flare from SGR 1806-20, a resolved, linearly polarized, radio nebula was seen, expanding at approximately a quarter of the speed of light. To create this nebula, at least 4 x 10(43) ergs of energy must have been emitted by the giant flare in the form of magnetic fields and relativistic particles.

A giant gamma-ray flare from the magnetar SGR 1806-20.

Two classes of rotating neutron stars-soft gamma-ray repeaters (SGRs) and anomalous X-ray pulsars-are magnetars, whose X-ray emission is powered by a very strong magnetic field (B approximately 10(15) G). SGRs occasionally become 'active', producing many short X-ray bursts. Extremely rarely, an SGR emits a giant flare with a total energy about a thousand times higher than in a typical burst. Here we report that SGR 1806-20 emitted a giant flare on 27 December 2004. The total (isotropic) flare energy is 2 x 10(46) erg, which is about a hundred times higher than the other two previously observed giant flares. The energy release probably occurred during a catastrophic reconfiguration of the neutron star's magnetic field. If the event had occurred at a larger distance, but within 40 megaparsecs, it would have resembled a short, hard gamma-ray burst, suggesting that flares from extragalactic SGRs may form a subclass of such bursts.

Slow proliferation as a survival advantage: an attempt to resolve a paradox.

A model is proposed for immunological high zone- and self-tolerance that is based on the concept of overstimulation of replication: It is demonstrated that if proliferation is hindered by delayed response to nutritional deficiency, then under competitive conditions slower proliferation can become a survival advantage. This offers a resolution of the paradox that self-antigens promote both positive and negative selection in the development of lymphocytes. It also suggests that maintaining nutrition and caloric intake at a minimal level can prolong the survival of cancer patients if the cancer cells replicate more rapidly than healthy ones.

Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase.

Although normal intracellular levels of arginine are well above the K(m), and should be sufficient to saturate nitric oxide synthase in vascular endothelial cells, nitric oxide production can, nonetheless, be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox," has suggested the existence of a separate pool of arginine directed to nitric oxide synthesis. In this study, we demonstrate that exogenous citrulline was as effective as exogenous arginine in stimulating nitric oxide production and that citrulline in the presence of excess intracellular and extracellular arginine further enhanced bradykinin stimulated endothelial nitric oxide production. The enhancement of nitric oxide production by exogenous citrulline could therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regeneration of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but also suggested channeling of substrates that would require a functional association between nitric oxide production and arginine regeneration. To support this proposal, we showed that nitric oxide synthase, and the enzymes involved in arginine regeneration, argininosuccinate synthase and argininosuccinate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production that is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine.

Insulin regulates alternative splicing of protein kinase C beta II through a phosphatidylinositol 3-kinase-dependent pathway involving the nuclear serine/arginine-rich splicing factor, SRp40, in skeletal muscle cells.

Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) betaII mRNA. In this report, we show that insulin regulates this exon inclusion (alternative splicing) via the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway through the phosphorylation state of SRp40, a factor required for insulin-regulated splice site selection for PKCbetaII mRNA. By taking advantage of a well known inhibitor of PI 3-kinase, LY294002, we demonstrated that pretreatment of L6 myotubes with LY294002 blocked insulin-induced PKCbetaII exon inclusion as well as phosphorylation of SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells mimicked insulin-induced exon inclusion. When antisense oligonucleotides targeted to a putative SRp40-binding sequence in the betaII-betaI intron were transfected into L6 cells, insulin effects on splicing and glucose uptake were blocked. Taken together, these results demonstrate a role for SRp40 in insulin-mediated alternative splicing independent of changes in SRp40 concentration but dependent on serine phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This switch in PKC isozyme expression is important for increases in the glucose transport effect of insulin. Significantly, insulin regulation of PKCbetaII exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCbetaII mRNA in L6 cells occurs in response to a metabolic change.

Optical search for extraterrestrial intelligence with Air Cerenkov telescopes.

We propose using large Air Cerenkov telescopes (ACTs) to search for optical, pulsed signals from extraterrestrial intelligence. Such dishes collect tens of photons from a nanosecond-scale pulse of isotropic equivalent power of tens of solar luminosities at a distance of 100 pc. The field of view for giant ACTs can be on the order of 10 square degrees, and they will be able to monitor 10-100 stars simultaneously for nanosecond pulses of about 6th magnitude or brighter. Using the Earth's diameter as a baseline, orbital motion of the planet could be detected by timing the pulse arrivals.

Landau excitation of spiral density waves in an inhomogeneous disk of stars

Drift-resonant Landau excitation of spiral density waves in a stellar disk of flat galaxies is proposed. This excitation of waves is suggested as a mechanism for the formation of structural features such as spiral arms and the slow dynamical relaxation of galaxies in a regime of hydrodynamical Jeans-type stability.

Nonthermal optical transients from relativistic fireballs

A general upper bound is derived on the total energy in incoherent nonthermal transients at frequency nu from relativistic fireballs with bulk Lorentz factors gamma and observed duration Deltat, and shown to be about 10(-2)[gammanuDeltat](3) ergs. It is suggested that detection in the optical can be achieved with the next generation of ground based gamma ray telescopes and/or small optical telescopes. Phenomena within the Galaxy such as accretion disk flares and neutron star magnetospheric discharges might be discovered in this way.

Hydrodynamic collimation of gamma-ray-burst fireballs

Analytic solutions are presented for the hydrodynamic collimation of a relativistic fireball by a surrounding baryonic wind emanating from a torus. The opening angle is shown to be the ratio of the power output of the inner fireball to that of the exterior baryonic wind. The gamma ray burst 990123 might thus be interpreted as a baryon-poor jet (BPJ) with an energy output of order 10(50) erg or less, collimated by a baryonic wind from a torus with an energy output of order 10(52.5) erg, roughly the geometric mean of the BPJ and its isotropic equivalent.

Stimulation of receptor-mediated nitric oxide production by vanadate.

Nitric oxide (NO) production by endothelial cells in response to bradykinin (Bk) treatment was markedly and synergistically enhanced by cotreatment with sodium orthovanadate (vanadate), a phosphotyrosine phosphatase inhibitor. This enhancement was blocked by tyrosine kinase inhibitors. Calcium ionophore- (A23187) activated production of NO was also enhanced by cotreatment with vanadate. No significant changes were found in total endothelial NO synthase (eNOS) protein or in eNOS distribution between membrane (caveolae) and cytosolic fractions in response to the various treatments. Vanadate had no direct effect on eNOS activity, and lysates prepared from cells treated with vanadate showed little change in specific activity of eNOS. Western blots of immunoprecipitated eNOS showed the presence of a major tyrosine-phosphorylated protein band at a mass corresponding to approximately 125 kDa and 2 minor bands corresponding to approximately 105 and 75 kDa after treatment with vanadate/Bk. No tyrosine phosphorylation of eNOS after treatment with vanadate/Bk was observed. Geldanamycin, an inhibitor of heat shock protein 90, also inhibited the enhancement of NO production by vanadate/Bk or vanadate/A23187, and there was an increase in the amount of heat shock protein 90 that coimmunoprecipitated with eNOS after treatment with vanadate/Bk. These results show that there is a clear link between tyrosine phosphorylation and stimulation of eNO production, which does not appear to involve direct modification of eNOS, changes in eNOS levels, or compartmentation, but rather appears to be due to changes in proteins associating with eNOS, thereby enhancing the state of activation of eNOS.

Leptin binding activity (LBA) in plasma of nondiabetic and diabetic adolescents and obese children: relation to auxologic and hormonal data.

Leptin circulates in serum bound to high molecular weight proteins. Hypothesizing that leptin binding proteins may regulate the functional efficiency of leptin, we characterized auxologic and hormonal factors that influence leptin binding in three disparate groups: normal adolescents, obese children, and teenagers with type I diabetes mellitus (IDDM). Specific leptin binding activity (sLBA) was assessed by column chromatography after incubation of serum with 125I-leptin in the presence and absence of excess unlabeled leptin. Mean sLBA was 17.0 +/- 7% (SD) in the healthy adolescents (n=41), 6.6 +/-4.3% in the obese children (n=26), and 14.9 +/-7.3% in the diabetic teenagers (n=17). At any value of sLBA, obese children had higher serum leptin levels than non-obese adolescents or diabetic teenagers, consistent with "leptin resistance" in the obese group. sLBA was higher in males than in females only in those with diabetes (18.6 +/- 7.3 vs 10.9 +/- 5.1%, p<0.05). sLBA correlated inversely with serum insulin-like growth factor-I values in the normal group (r= -0.45, p<0.01) and with insulin in the obese children (r= -0.53, p<0.01). There was no correlation between sLBA or serum leptin values and HbA1c, in the diabetic group. The serum leptin concentration was the principal determinant explaining the total variability of sLBA in all three cohorts. However, body mass index (BMI = weight/ height2) accounted for more of the total variability of percent specific binding in the healthy adolescents than in the other groups. We conclude that sLBA reflects circulating leptin levels, body composition, and hormonal milieu. Thus, in addition to leptin, qualitative and quantitative characteristics of leptin binding may play a physiological role in the regulation of appetite and in the "leptin resistance" of obesity.

Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCbetaII mRNA in vascular smooth muscle cells.

Acute hyperglycemia may contribute to the progression of atherosclerosis by regulating protein kinase C (PKC) isozymes and by accelerating vascular smooth muscle cell (VSMC) proliferation. We investigated acute glucose regulation of PKCbeta gene expression in A10 cells, a rat aortic smooth muscle cell line. Western blot analysis showed that PKCbetaII protein levels decreased with high glucose (25 mM) compared to normal glucose (5.5 mM), whereas PKCbetaI levels were unaltered. PKCbeta mRNA levels were depleted by 60-75% in hyperglycemic conditions. To elucidate whether high glucose regulated PKCbeta expression via the common promoter for PKCbetaI and PKCbetaII, deletion constructs of the PKCbeta promoter ligated to CAT as reporter gene were transfected into A10 cells. Construct D (-411 to +179CAT) showed quenching in high glucose (25 mM) and suggested the involvement of a carbohydrate response element in the 5' promoter region of the PKCbeta gene. In actinomycin D-treated A10 cells, a 60% decrease in PKCbeta mRNA with high glucose treatment indicated that posttranscriptional destabilization by glucose was also occurring. We have demonstrated that glucose-induced posttranscriptional destabilization of PKCbetaII message is mediated via a nuclease activity present in the cytosol. The specificity of glucose to posttranscriptionally destabilize PKCbetaII mRNA, but not the PKCbetaI mRNA, was confirmed in both A10 cells and primary cultures from human aorta.

A spun-column assay for determination of leptin binding in serum.

Serum collected from 27 patients was assayed simultaneously using a spun-column assay (SPC) and a traditional exclusion gel-filtration assay (GFC) to determine specific leptin binding. The levels of serum leptin binding determined by either assay correlated inversely with serum leptin levels (SPC, r = 0.63, P < 0.001; GFC, r = 0.79, P < 0.0001). Although specific leptin binding as determined by the traditional exclusion gel-filtration assay was generally higher than that obtained by the spun-column assay (mean = 18.3% vs 14.0%, P < 0. 02, respectively); the values obtained between the two assay methods were highly correlative (r = 0.89, P < 0.0001). By varying either the amount of 125I-leptin or the amount of competitor, analysis was carried out using the spun-column assay to determine the intrinsic properties of serum leptin binding. Results yielded a Kd = 0.3 nM, where each variable amount of leptin or competitor was carried out in duplicate. The complete analysis was carried out in the time that it typically takes for a single sample determination by the traditional exclusion gel-filtration assay. We conclude that the "spun-column" assay is a useful method for rapid and accurate quantification of leptin binding in serum.

Insulin regulates protein kinase CbetaII expression through enhanced exon inclusion in L6 skeletal muscle cells. A novel mechanism of insulin- and insulin-like growth factor-i-induced 5' splice site selection.

The protein kinase Cbeta (PKCbeta) gene encodes two isoforms, PKCbetaI and PKCbetaII, as a result of alternative splicing. The unique mechanism that underlies insulin-induced alternative splicing of PKCbeta pre-mRNA was examined in L6 myotubes. Mature PKCbetaII mRNA and protein rapidly increased >3-fold following acute insulin treatment, while PKCbetaI mRNA and protein levels remained unchanged. Mature PKCbetaII mRNA resulted from inclusion of the PKCbetaII-specific exon rather than from selection of an alternative polyadenylation site. Increased PKCbetaII expression was also not likely accounted for by transcriptional activation of the gene or increased stabilization of the PKCbetaII mRNA, and suggest that PKCbetaII expression is regulated primarily at the level of alternative splicing. Insulin effects on exon inclusion were observed as early as 15 min after insulin treatment; by 20 min, a new 5'-splice site variant of PKCbetaII was also observed. After 30 min, the longer 5'-splice site variant became the predominate species through activation of a downstream 5' splice site. Similar results were obtained using IGF-I. Although the role of this new PKCbetaII mRNA species is presently unknown, inclusion of either PKCbetaII-specific exon results in the same PKCbetaII protein.

Demonstration of a leptin binding factor in human serum.

Serum leptin levels are elevated in subjects with exogenous obesity, indicating that obesity is associated with leptin resistance. Since in man no abnormalities have yet been found in either the genes for leptin or its receptor, the mechanism of leptin resistance in obesity remains unknown. To determine if resistance might be related to leptin binding by a serum component, we assessed the carrier status of leptin in serum. The presence of a specific leptin binding factor in human serum has been established by (1) demonstrating [125I]-leptin binding to a serum component that is saturable and specifically displaceable only by unlabeled leptin and not by human growth hormone, pork insulin, insulin-like growth factors I and II, luteinizing or follicle stimulating hormones, transforming growth factor-beta 1, interleukin-6, or leukemia inhibiting factor; (2) fractionating the leptin bound serum complex and the serum leptin binding component on a molecular sieving column revealing a mass of approximately 450 kDa; and (3) identifying an inverse correlation between the concentration of serum leptin and the quantity of the leptin binding component. It is suggested that binding of leptin by this serum component may influence the physiologic response to leptin.

Characterization of a nucleolar 2'-O-methyltransferase and its involvement in the methylation of mouse precursor ribosomal RNA.

A nucleolar 2'-O-methyltransferase, partially purified from isolated mouse nucleoli, catalyzes the methylation of each of the four nucleosides, although to different levels depending on the RNA substrate. Similar to most methyltransferases which use S-adenosyl-L-methionine (SAM) as the methyl donor, the nucleolar 2'-O-methyltransferase was shown to bind S-adenosyl-L-homocysteine (SAH) (Kd = 0.17 microM), a product of the transfer reaction, as tightly as SAM (Kd = 0.24 microM). Binding assays also demonstrated stereospecificity about the sulfonium center of SAM. The naturally occurring S-chiral form of SAM had a 10-fold higher binding affinity than the R-chiral form. In addition, the alpha-amino group of the methionine moiety and the 6-amino group of the adenine moiety were shown to be required for maximal binding. The relative high affinity for both SAM and SAH may reflect a mechanism by which ribosome biogenesis is, in part, coordinated with cell growth, since a decrease in SAM:SAH ratio correlates with decreasing levels of 2'-O-methylation. The availability of unmethylated, in vitro-derived rRNA transcripts has made it possible to explore questions relating to the specificity for the RNA substrate. Using an in vitro-derived 28S rRNA transcript, the enzyme selectively methylated the sequence AmGmCm that occurs in a single-stranded bridge spanning two highly conserved structural domains of 28S rRNA. These results demonstrated that the purified nucleolar 2'-O-methyltransferase was sufficient to accurately methylate this region of 28S rRNA, and were taken to support the involvement of this nucleolar enzyme in the posttranscriptional methylation of the 47S precursor ribosomal RNA transcript.(ABSTRACT TRUNCATED AT 250 WORDS)