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BioMed Research International
Volume 2013, Article ID 903292, 12 pages
Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme from Lachesis muta rhombeata Snake Venom
1Department of Biochemistry, Institute of Biology, University of Campinas (UNICAMP), P.O. Box 6109, 13083-970 Campinas, SP, Brazil
2Department of Structural and Functional Biology, Institute of Biology, State University of Campinas (UNICAMP), P.O. Box 6109, 13083-865 Campinas, SP, Brazil
3Department of Pharmacology, State University of Campinas (UNICAMP), P.O. Box 6109, 13083-887 Campinas, SP, Brazil
Received 4 June 2013; Accepted 29 June 2013
Academic Editor: Saulo Luís da Silva
Copyright © 2013 Frank Denis Torres-Huaco et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, "ex vivo", a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).
1. Introduction
Snakes of the genus Lachesis, commonly known as bushmasters, or surucucu in Brazil, can be found in the tropical forests of Central and South Americas. There are three different Lachesis species: L. stenophrys, L. melanocephala (both found in Central America), and L. muta. In Brazil, two subspecies can be found: L. muta muta in the Amazon Forest and L. muta rhombeata in the Atlantic Forest along the eastern coast of Brazil [1].
Poisoning by Lachesis is characterized by local damage, pain, edema, hemorrhage, and myonecrosis, as well as systemic complications, such as coagulation disorders, hemolysis, neurotoxicity, renal failure, diarrhea, hypotension, and bradycardia, among others [2–5]. Lachesis m. rhombeata crude venom shows lower lethal and hemorrhagic activities when compared with L. m. muta venom, while both venoms induce similar edema-forming and myotoxic activities. However, L. m. rhombeata venom exhibits higher coagulant and defibrinogenating effects [6].
Snake venom serine proteinases (SVSPs) belong to the trypsin S1 family of SA clan, the largest family of peptidases [7, 8]. SVSPs are among the best-characterized venom enzymes affecting the haemostatic system. They act on a variety of components of the coagulation cascade, on the fibrinolytic and kallikrein-kinin systems, and on platelets to cause an imbalance of the haemostatic system of the prey [8].
So far, two serine proteinases were isolated from Lachesis m. rhombeata: a thrombin-like/gyroxin-like enzyme denominated LMR-47 [9] and LMR kininogenin [10]. However, none of these proteins were fully characterized biochemically or had their effects studied over the haemostatic system. Since the L. m. rhombeata crude venom induces strong effects over the haemostatic system, it was decided to investigate the participation of the SVSP LMR-47 in those effects induced by the envenomation of this snake species.
In this study, we report a one-step method of purification, prediction of primary structure, and "in vivo" effects over the coagulation cascade as well as over the platelet aggregation of SVTLE LMR-47, which will suggest its possible role in relation to the effects on the haemostatic system of Lachesis m. rhombeata crude venom. Also, we propose to rename this protein as Rhombeobin according to its thrombin-like activity.
2. Materials and Methods
2.1. Venom and Reagents
Lachesis m. rhombeata venom was gently donated by Dr. Rodrigo C. G. de Souza from Serra Grande Center for Lachesis muta Breeding, with IBAMA Authorization no. 24945-1. All chemicals and reagents were of analytical or sequencing grade. Bovine fibrinogen was purchased from Amour Pharmaceutical, CA, USA.
2.2. Animals
Four-five-week male Swiss mice were supplied by the Animal Services Unit of the State University of Campinas (UNICAMP). Mice were housed at room temperature on a 12 h light/dark cycle and had free access to food and water. All procedures were performed according to the general guidelines proposed by the Brazilian Council for Animal Experimentation (COBEA) and were approved by the university's Committee for Ethics in Animal Experimentation (CEEA/UNICAMP) no. 1790-1.
2.3. Venom Fractionation
Five mg of crude venom of L. m. rhombeata was dissolved in 0.2 mL of solvent A (0.1% (v/v) trifluoroacetic acid). The resulting solution was clarified by centrifugation, and the supernatant was further submitted to a reversed-phase chromatography on a C8 Discovery BIO Wide Pore (25 cm × 4.6 mm × 10 μm). Fractions were eluted using a stepwise gradient of solvent B (66% acetonitrile in solvent A) (0%–55% B for 26 min, followed by 55%–65% B over 20 min, and 65%–100% B over 14 min). The flow rate of 1.0 mL/min was constant, and the resulting fractions were manually collected. The elution profile was monitored at 280 nm, and the collected fractions were lyophilized and conserved at −20°C. The homogeneity of the final material was assessed by a rechromatography on the same column with a linear gradient (0%–100%) of solvent B.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on purified Rhombeobin according to Laemmli [20]. The molecular mass markers used were (in kDa)the following: phosphorylase B, 94; albumin, 67; ovalbumin, 43; carbonic anhydrase, 30; soybean trypsin inhibitor, 20; and lysozyme, 14.
2.5. Determination of Protein Concentration
Protein concentration was determined by the method of Bradford [21] and standardized with bovine serum albumin.
2.6. Amidolytic Activity and Determination of Kinetic Parameters
Amidolytic activity was measured using the synthetic substrate N-benzoyl-L-arginine ρ-nitroanilide (BAρNA) modified for 96-well plates. The standard assay mixture contained 50 μL of buffer (10 mM Tris-HCl, pH 8.0, 10 mM CaCl2, and 100 mM NaCl), 200 μL of substrate solution (1 mM), 15 μL of water, and 5 μL of fractions or enzyme (Rhombeobin) in a final volume of 270 μL. The reaction was carried out in a VERSAMAX microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA) for 30 min at 37°C, with the absorbance being read at 410 nm. The results were expressed as the initial velocity of the reaction () calculated based on the amount of ρ-nitroaniline released [22]. The temperature and pH effect on amidolytic activity of Rhombeobin was examined by incubating the overall mixture reaction at different temperatures (10–80°C) and pH (5–11). At different BAρNA concentrations (0.0095–10 mM), the and of Rhombeobin were determined under optimal pH and temperature. The effects of inhibitors EDTA (5 mM), EGTA (5 mM), PMSF (2 mM), SBT-I (1 mg/mL), and DTT (5 mM) were tested by preincubating the sample with these compounds for 30 min at 37°C prior to the standard test. The inhibition results were expressed as percentage of residual activity.
2.7. Fibrinogenolytic Activity
Fibrinogenolytic activity was determined by mixing 10 μL of Rhombeobin with 200 μL of bovine fibrinogen solution (2 mg/mL in 10 mM Tris-HCl, pH 7.4, 10 mM CaCl2, and 100 mM NaCl) at 37°C, at different incubation times (5 min–24 h) and different concentrations of enzyme (0.632–10 μg). The reaction was stopped with 150 μL of denaturizing solution (Tris-HCl 0.05 M, pH 6.5, urea 10 M, b-mercaptoethanol 10% (v/v), glycerol 10% (v/v), and bromophenol blue 0.05% (v/v)), incubated for 15 minutes at room temperature. Fibrinogenolytic activity was evaluated by SDS-PAGE using 12.5% polyacrylamide gels as described by Laemmli [20].
2.8. Determination of Minimum Coagulant Dose (MCD)
The MCD over bovine fibrinogen (MCD-F) or plasma (MCD-P) of Rhombeobin was determined according to Theakston and Reid [23]. Briefly, 0.25 to 10 μg of Rhombeobin (20 μL PBS) were added to 200 μL of bovine fibrinogen solution (2 mg/mL) or mice citrated plasma at 37°C. The solutions were mixed thoroughly, and the clotting times were recorded. The MCDs for bovine fibrinogen and plasma were calculated by plotting Log of Rhombeobin concentration versus Log of the clotting times. The MCD was defined as the least amount of toxin (in mg of dry weight per liter of test solution) that clots citrated plasma or bovine fibrinogen solution in 60 seconds at 37°C.
2.9. Determination of the Molecular Mass of the Purified Protein by Mass Spectrometry
An aliquot (4.5 μL, 0,1% formic acid) of Rhombeobin (10 μg) was injected by C18 (100 mm × 100 mm) RP-UPLC (nanoACQUITY UPLC, Waters) coupled with nanoelectrospray tandem mass spectrometry on a Q-Tof Ultima API mass spectrometer (MicroMass/Waters) at a flow rate of 600 nL/min. The gradient was 0%–50% acetonitrile in 0.1% formic acid over 45 min. The instrument operation and the processing parameters were set according to Damico et al. [24].
2.10. Sequencing Procedures
The reduced (5 mM dTT for 25 min at 56°C) and alkylated (14 mM iodoacetamide, 30 min) protein was digested with trypsin (Promega-Sequencing Grade Modified) or SV-8 (Promega-Sequencing Grade Modified). The digestion procedures, the peptide separation, and the mass analysis of the resulting peptides were made following the methodology described by Damico et al. [24].
Raw data files from LC-MS/MS runs were processed using Masslynx 4.1 software package (Waters) and were analyzed using the MASCOT search engine version 2.3 (Matrix Science, Ltd.) against the snakes database, using the following parameters: peptide mass tolerance of ±0.1 Da, fragment mass tolerance of ±0.1 Da, oxidation as variable modification in methionine, and trypsin as enzyme.
2.11. Evaluation of Gyroxin Syndrome
Rhombeobin gyroxin-like activity was determined according to Seki et al. [25]. Briefly, 25 μL of enzyme (1–20 μg protein in PBS) was injected iv. into adult mice weighing 25 ± 2 g. After injection, it was determined whether the animals showed the characteristic equilibrium loss and, eventually, typical rolling movements as described by Barrabin et al. [26]. The animals were observed 24 h after injection in order to rule out the contamination of the purified fraction with lethal toxins.
2.12. Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT)
Male Swiss mice were anesthetized (xylazine, 2%–16% mg/kg; ketamine, 10%–100% mg/kg), and blood was collected from cava vein in citrate 3.2% and and centrifuged at 1500 ×g for 15 min at 25°C; after blood withdrawn was mice were euthanized by anesthesia deepening. For APTT, a 50 μL, of aliquot plasma was warmed to 37°C for 2 min, 50 μL, of APTT reagent was added, of, after a 2 min incubation at 37°C, 0.25 M of CaCl2 was added, and the clotting time was determined. For the PT test, 100 μL of CLOT PT reagent was incubated for 4 min at 37°C, and 50 μL of plasma was added, triggering the reaction. These analyses were performed in triplicate, using the APTT and PT Kit CLOT BIOS diagnosis (CLOT, Brazil) in a CLOTimer coagulometer (CLOT, Brazil). To assay APTT and PT in vitro, mice plasma (45 μL) was preincubated with 5 μL of a different Rhombeobin (2, 5, 10, and 20 μg/mL); then the standard analyses for APTT and PT were followed. To assay APTT and PT ex vivo, Swiss mice were injected i.v. with Rhombeobin (0.1 and 1 μg/animal), and after 30, 60, and 90 min, blood was withdrawn and processed as described above.
2.13. Effects of Rhombeobin on the Mice Plasma Fibrinogen
The ex vivo test was performed according to Maruñak et al. [27] and modified as follows. Groups of mice with different exposure times (30, 60, and 90 min) and one control group, each one composite of four mice weighing 25 ± 2 g, were injected i.v. with 25 μL of PBS (control group) or Rhombeobin (0.1 and 1 μg/animal). After the times of exposure, blood samples and poor platelet plasma were obtained as described in Section 2.12. The fibrinogen content was determined using the Wiener diagnostic kit, and the results were expressed as percentage of the remaining fibrinogen compared with the control group.
2.14. Platelet Aggregation Assay
Washed platelets suspensions from mice were obtained according to Theakston and Reid [23]. Platelet aggregation was performed in an optical aggregometer (Chrono-log, Kordia Life Sciences, Leiden, Belgium) at 37°C with 400 μL of washed platelets placed in glass cuvettes containing a disposable stir bar for constant stirring. Platelet aggregation was induced using ADP (20 μM) or thrombin (0.05 U/mL) as agonist. The ex vivo effects of Rhombeobin over platelet aggregation were studied by injecting i.v. 25 μL of PBS or enzyme (0.1 or 1 μg/animal) in adult mice (25 ± 2 g). After 1 h, the mice were anesthetized, and washed platelets were obtained.
2.15. Statistical Analyses
Results were reported as mean ± SEM. The significance of differences among means was assessed by analysis of variance followed by Tuckey's test. The statistical analyses were made using the Origin 8 SR2 v8.0891 (B891; OriginLab Corporation, Northampton, MA, USA). Program was considered statistically significant.
3. Results
The fractionation of Lachesis muta rhombeata crude venom in an RP-HPLC system (C8 column) results in twenty-three main fractions named F1 to F23 (Figure 1(a)). Each fraction was assayed for amidolytic and coagulant activities, and both were found only in F-16 fraction. Rechromatography of F-16 fraction in an RP-HPLC, with a linear gradient, results in a single symmetric peak eluted with 57% of buffer B and a retention time of 36.23 ± 2.2 min (Figure 1(b)); SDS-PAGE showed that this fraction in nonreduced and reduced conditions is a single-chain protein, with a relative molecular mass of 45 kDa (Figure 1(b): insert). The protein homogeneity of F-16 was confirmed by ESI-MS, and it was shown that fraction F-16 is protein with a real molecular mass of 47035.49 Da (Figure 1(c)).
The fraction F-16 was named Rhombeobin, and it showed an arginine amidase activity toward the chromogenic substrate BApNA with and of 0.8 × 10−3 M and 1.06 ± 0.106 nmol/min/mg, respectively (Figure 2(b)). The optimum temperature was between 40 and 50°C, and the remarkable stability in the pH was in the range of 7 to 10. The serineproteinase inhibitor PMSF and the reducing agent DTT completely abrogated Rhombeobin enzymatic activity, while SBT-I partially reduced () the enzymatic activity (Figure 2(c)). However, EDTA and EGTA did not show any effects (Figure 2(c)).
Rhombeobin is a strong thrombin-like enzyme since it can clot a bovine fibrinogen solution with an MCD-F of 18.3 mg/L of mice citrated plasma, with an MCD-P of 7.3 mg/L (Figure 2(d)).
Rhombeobin showed a concentration and time-dependent fibrinogenolytic activity. This enzyme completely degraded fibrinogen α-chain with a concentration of 1.25 μg with 15 min of incubation. Rhombeobin degraded fibrinogen β-chain at high concentrations and long incubation times; on the other hand, the enzyme did not show any activity over fibrinogen γ-chain (Figures 3(a) and 3(b)).
The protein digestion (Rhombeobin) with trypsin and protease SV-8, followed by LC/MS/MS, identified nine (T1–T9) and seven peptides (S1–S7), respectively. The deduced sequence and the measured masses of alkylated peptides are summarized in Tables 1 and 2, covering approximately 94% of the protein sequence. Each peptide was submitted separately to the SNAKE database using the protein search program BLAST-p. Using the position matches of the "de novo" sequenced peptides with homologous proteins present in the database, it was possible to deduce their original position on the unknown protein. Thus, it was deduced that peptides T-1, S-3, and T-8 contain the catalytic triad histidine, aspartic acid, and serine, respectively. Also, the digested peptides contain the twelve cysteine residues characteristic of the snake venom serineproteinase family. Rhombeobin showed homology (Figure 4) with other thrombin-like SVSPs from Lachesis m. muta, Crotalus durissus terrificus, Agkistrodon bilineatus, Bothrops jararaca, Bothrops jararacussu, Bothrops atrox, Agkistrodon acutus, and Agkistrodon rhodostoma, sharing high-sequence identity with them, especially with LM-TL from Lachesis m. muta (75.6%).
Rhombeobin induced "gyroxin syndrome" when injected into the mice tail vein at dose of 0.2 μg/kg of bodyweight. The treated animals were progressively hypoactive over a period of 1–3 min, followed by a loss of the righting reflex, opisthotonos, and rotations around the long axis, lasting up to 30 minutes. All treated animals lost the ability to right themselves. Two hours following Rhombeobin injection, the animal's behavior returned to normal. Gyroxin syndrome was not observed with lower doses, in which the main effects were flaccid paralysis (mainly of inferior extremities) and hypoactivity.
In vitro, Rhombeobin showed a strong procoagulant activity with a dose-dependent effect on citrated mice plasma, shortening significantly both APPT and PT tests (Figures 5(a) and 5(b)).
Interestingly, ex vivo, Rhombeobin treatment produced both anticoagulant and procoagulant effects. The i.v. injection, with 1 μg of protein, renders unclottable blood after 30 minutes of exposure. This effect was sustained through the whole time of the experiment (Figures 5(c) and 5(d)). On the other hand, Rhombeobin shortened the APTT and PT tests in mice treated with a dose ten times lower; this was a rapid and sustained effect on APTT test (Figure 5(c)); although on PT test the shortening effect was rapid, one hour after injection this effect was not observed (Figure 5(d)).
Mice plasma fibrinogen levels were determined after Rhombeobin i.v. injection. At 1 μg dose, the protein showed a strong defibrinogenating effect, with no detectable plasma fibrinogen levels after thirty minutes after injection (Figure 6(a)). On the other hand, with a dose ten times lower, only partial reduction of plasma fibrinogen levels was observed (Figure 6(a)). These results corroborate with the fibrinogenolytic activity on bovine fibrinogen analyzed by SDS-PAGE. While 0.1 μg of the protein lightly degraded fibrinogen α-chain (Figure 6(b)), 1 μg, after 90 minutes of incubation, almost completely degraded fibrinogen α-chain, producing fibrinopeptide A (Figure 6(c)).
When mice were treated with Rhombeobin i.v. at 0.1/animal and after one hour platelet suspensions were obtained, a significant reduction () of the platelet aggregating response to ADP as agonist was observed. However, no effect over the platelet aggregating response was observed when Thrombin was used as agonist (Figures 7(a) and 7(b)).
4. Discussion
This work reports an efficient and simple procedure for the purification of LMR-47: a thrombin-like/gyroxin-like enzyme from Lachesis muta rhombeata venom, which we renamed as Rhombeobin. In our approach, using a simple chromatographic step based on RP-HPLC showed that the L. m. rhombeata venom can be separate in twenty-three main fractions. From all of them, only fraction sixteen presents amidolytic and thrombin-like activities. The purification of thrombin-like enzymes from the venom is usually made using methods based on molecular-size exclusion followed by either ion-exchange or affinity-binding (benzamidine- or arginine-Sepharose) chromatography [8], but this approach requires several steps, considerable time, and loss of material by the dialysis procedures prior to obtaining a lyophilized sample. Asingle chromatographic step using HPLC systems provided more purified protein samples than other conventional methods reducing time and material loss. de-Simone et al. [14] reported the purification of LMR-47 (Rhombeobin) with a single affinity chromatography step on HPLC system using two columns in tandem with the same ligand; although the authors were able to obtain a purified protein, the purification method was rather complicated by the time require and did not improve the protein recovery observed with conventional methods [9]. In comparison, with the purification procedure described here, we were able to recover near 5% of the purified protein (data not shown), twice more efficient in terms of protein recovery.
The molecular homogeneity of Rhombeobin was assessed by RP-HPLC (Figure 1(b)) showing a single and symmetric peak with elution and retention time typical for the snake venom serineproteinase family [28–30]. The SDS-PAGE analysis (Figure 1(b), insert) showed a single band with an identical electrophoretic mobility and relative molecular mass of ~45 kDa shown, by LMR-47 [9]. ESI-MS analysis showed that the molecular mass of Rhombeobin was 47035.49 Da.
Rhombeobin showed a high amidolytic activity upon BApNA with a Michaelian enzymatic behavior (Figure 2(a)). The calculated values of , and (Figure 2(b)) were similar to those exhibited for LMR-47 over the same substrate [9]. As with other snake venom serine proteinases [31–33], Rhombeobin loses all enzymatic activity at high temperatures. Interestingly, this enzyme showed a broad range of optimum pH; the maximal enzymatic activity remained unaltered along of neutral and mild basic conditions (pH 7–10). The high enzymatic activity observed for Rhombeobin at the range of pH has been described for other snake venom serine proteinases with thrombin-like activity like bothrops protease A (Bothrops jararaca) [34]. PMSF completely inhibited enzymatic activity of Rhombeobin. On the other hand, the partially inhibitory effect of Kunitz-type inhibitor SBT-I over the enzymatic activity of this enzyme has been described for other snake venom serine proteinases, although complete inhibitions were achieved, in some cases, at high concentrations [8]. As a thrombin-like enzyme, Rhombeobin showed a high clotting activity upon bovine fibrinogen, and its activity was higher over citrated plasma (Figure 2(d)). This enzyme showed an α-fibrigenonolytic behavior when incubated with bovine fibrinogen, and it degraded fibrinogen β-chain at higher doses and prolonged incubation times (Figures 3(a) and 3(b)). The thrombin-like activity and the pattern found on the fibrinogenolytic activity of Rhombeobin were similarly shown by SVSP LMR-47 [9].
The biochemical, enzymatic, and biological data obtained in this work strongly suggest that Rhombeobin is the same protein LMR-47 thrombin-like/gyroxin-analog characterized by Aguiar et al. [9]. According to the Scientific and Standardization Subcommittee of the International Society on Thrombosis and Haemostasis [11], we propose to rename LMR-47 as Rhombeobin, which is the result of the combination of the subspecies name rhombeata with the suffix "obin".
The results obtained from the sequences provided several peptides derived of trypsin (Table 1) and SV-8 (Table 2) enzyme digestion; from these peptides, we were able to deduce almost the entire Rhombeobin protein sequence. Furthermore, the peptides named T-1, S-1, and S-2, combined, contain a 46 N-terminal amino acid sequence which showed 100% identity with the 30 N-terminal of LMR-47 [9], confirming that Rhombeobin and LMR-47 are the same protein.
The homology study (Figure 4) showed that Rhombeobin shared a high degree of sequence identity with SVSP with thrombin-like activity [12, 13, 15–18, 28, 35] especially with LM-TL (Lachesis muta muta). Detailed analysis showed small but important differences between Rhombeobin and LM-TL. In both enzymes, the primary (D176) and secondary (G199) specificity sites are conserved, while the tertiary specificity site (position 210) in LM-TL is a Gly residue, while in Rhombeobin it is an Ala residue. Another difference is observed in part of the hydrophobic site [19], LM-TL has Phe, Val, and Phe residues in positions 82, 196, and 197, respectively, while Rhombeobin shows Lys, Ala, and Ser in the same positions. These substitutions suggest that these proteins could have differences in their affinity for natural and synthetic substrates [36]. Also, there are some modifications in the region called "90 Loop" (position 79 to 85 in our numbering), in which Phe82 and Trp85 are responsible for the resistance of LM-TL to the inhibitory effect of Kunitz-type inhibitors by obstruction of the inhibitor binding region and sterically hindering other residues (Lys80, Arg180, and Ser233) [19]. The substitution of Phe82 by Lys82 in Rhombeobin could have allowed better interaction with the binding site of SBT-I inhibitor as suggested by the results of the inhibitory studies (Figure 2(c)).
In general, snake venom serine proteinases (SVSPs) affect the coagulation cascade by activation or proteolytic degradation of specifically coagulation factors [8, 36]. A number of snake venom serine proteinases with thrombin-like activity showed procoagulant activity "in vitro" by direct activation of factor V [37–39], factor VIII [28, 40], and factor X [41]. In vitro, Rhombeobin acts as a procoagulant SVSP shortening both APPT and PT tests (Figures 5(a) and 5(b)). These results suggest that this enzyme could act over the common coagulation pathway, likely by activating coagulation factor V and/or factor X. However, the fact that Rhombeobin induces a more marked shortening of APPT than PT test could be an indicator of activation of other specific intrinsic pathway factors.
Venom-induced consumptive coagulopathy or VICC [42] is characterized by activation of the coagulation pathway and consumption of coagulation factors, mainly fibrinogen, resulting in multiple factor deficiencies in snakebitten patients [42–44]. In laboratory, it is characterized by the alteration of basic clotting tests like WBTC, APPT, and PT tests [42, 43]. In this regard, Rhombeobin (0.1 μg/animal) activates both the intrinsic and extrinsic coagulation pathways (Figures 5(c) and 5(d)), but it does not induce defibrinogenation (Figure 6(a)) because, at this concentration, Rhombeobin did not effectively degrade fibrinogen (Figure 6(b)). However, with 1 μg/animal, Rhombeobin rendered mice blood unclottable (Figures 5(c) and 5(d)), by activating the blood coagulation factors and mainly by a rapid and sustained defibrinogenation (Figure 6(a)) due to a proteolytic degradation (Figure 6(c)). Thus, Rhombeobin appears to be a key player in the induction of the severe coagulopathy induced by L. m. rhombeata venom [6].
Snake venom serine proteinases also act on platelet-rich plasma or washed platelet suspensions promoting platelet-aggregation and platelet release reactions [8, 36]. This activation is mediated by the proteolytic activation of the membrane receptors PAR1 and PAR4 [45], or by interacting with glycoprotein GPIb or GPIIbIIIa [41].
However, when Rhombeobin was injected i.v. in adult mice, we observed a reduction on platelet aggregation in response to ADP (20 μM, Figures 7(a) and 7(b)) as agonist. This impairment of platelet function was found to be well correlated with a decrease in plasma fibrinogen concentration, an effect previously described for other SVSPs with thrombin-like activities [46]. Interestingly, Rhombeobin did not show any effect over the activity of thrombin as platelet aggregation agonist.
5. Conclusion
Our results showed that venom components, like Rhombeobin, can evoke a venom-induced consumptive coagulopathy by itself, mainly through procoagulant effect mainly through the intrinsic and common pathway that involves catalytic activation of coagulation factors and the enzymatically hydrolyzing plasma fibrinogen. Thus, Rhombeobin has a main role in the coagulopathies induced by Lachesis muta rhombeata snake venom. The mechanisms by which Rhombeobin interacts with platelet membrane receptors in vivo are different from those described in the literature, and, therefore, necessary additional studies for better understanding of the "in vivo" mechanism of this enzyme that delays the thrombus formations are needed.
Conflict of Interests
The authors do not have any conflict of interests.
The authors acknowledge the Mass Spectrometry Laboratory at Brazilian Biosciences National Laboratory, CNPEM-ABTLUS, Campinas, Brazil, for its support with the mass spectrometric analyses. The authors thank Mr. Paulo A. Baldasso for general technical assistance. This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Process no. 09/02299-8.
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Sept 22 (Reuters) - Amvig Holdings Ltd
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text:
We specialize in Photography & Video that captures the essence of your wedding day in Savannah, whether it's a beach wedding or a family portrait in. Take a look around our site and if you like what you see, please feel free to get in touch with us.
Jack Bolyston, a.k.a Tybee Jack, is a retired teacher, coach and sports marketing manager who has lived most of his life on Tybee Island. Tybee Jack has performed more than 100 wedding ceremonies on the beach and at sites around historic Savannah.
From an engagement session that reflects your passions and pursuits to wedding day coverage that reflects your style & meets your every photography need, Shannon provides a one-of-a-kind experience in Savannah GA.
Tybee Wedding Chapel is the premier wedding facility on Tybee Island and newest wedding and reception facility in the Savannah/Coastal area. Reminiscent of a 1908 Seaside Chapel designed with all the amenities of a true special events facility for today's weddings.
Thrive offers a refreshing menu for all your special events. We are happy to assist in any rehearsal dinner, weddings, welcome parties or farewell brunches. Accommodating numbers from 20 to 500+, from casual lunches to formal plated dinners, Thrive will satisfy everyone's needs for an event no one will forget.
Designing & planning your Savannah wedding from start to finish the way you envision it is our main priority while at the same time giving special attention to every detail. Every event we plan is carefully designed to fit your needs. OC can turn your dreams & visions into a reality!
CQ's has the feeling of an old, cozy, and comfortable inn.
Serving the "Best Savannah BBQ" (voted by Savannah Magazine and Connect Savannah (2010 and 2011), plus Best BBQ Ribs (2011) in the Savannah area. Janet and Wiley will help you make your special occasion one to remember!
Don't just remember your wedding day - RELIVE IT by allowing us to capture and produce a wedding video that you'll cherish forever.
Marriott's Grande Ocean welcomes you with an impeccable location on Hilton Head Island, South Carolina and luxury accommodations.
If you have been to Europe and enjoy small cramped dining areas and a patio that is in a parking lot, then you will enjoy dining at Vine. If not, then there are many other fine establishments on Hilton Head that you will likely enjoy better.Our food is ordinary, our service sub standard, lights too low to see, and music ...
Unlike many jewelry stores, we do everything right on the premises - design, carve waxes, and complete the finished creation.
Let us create an event your family and friends will always remember.
Rob Kaufman is a sought after Destination Wedding Photographer based out of Hilton Head Island, SC. His wife, Samantha, creates "MTV-style" Fusion Videos to capture the motion and moments of your wedding day. Call us...we would love to sit down and get to know you. Congratulations!
The Sea Pines Resort is located on the south end of Hilton Head Island and is the perfect venue to host your Wedding Ceremony, Reception and Wedding weekend! With numerous ceremony and reception sites, we can host intimate to large weddings. Special Events, Special Places, Special Memories...The Sea Pines Resort.
Michael Anthony's Cucina Italiana offers traditional regional Italian cuisine combining innovative presentations and style. The award winning culinary team presents a menu using the freshest ingredients for an unforgettable culinary experience featuring homemade gnocchi, succulent veal,daily selections of fresh fish and ...
Authentic Lowcountry Style Cuisine prepared the way it used to be. All homemade, delicious Crab Cakes, Fried Green Tomato BLT's, Banana pudding, Seafood, Soups, Salads and sandwiches, just to name a few. Fresh desserts and cakes prepared daily. Dine inside, or outside in a classic, Lowcountry Backyard garden complete with ...
Java Burrito Co. Is a family- owned and operated Mexican Grill & Coffee Bar in Hilton Head Island, South Carolina. We've only been around since 2013 yet the family history in the restaurant industry goes back over 100 years!! We use the finest ingredients and traditional methods to produce the most exceptional ...
Celebrated Hilton Head Chef Michael Cirafesi & Legendary Maestro of Italian Cuisine Nunzio Patruno bring you OMBRA Cucina Rustica, located in the beautiful Village at Wexford on the south end of Hilton Head Island. Come and enjoy Classic Regional Italian cuisine along with an extensive wine list including Italian ...
The jazz corner is a perfect venue for any of your private events and including birthdays, anniversaries. We also book the music of your choice.


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The Lightweight Directory Access Protocol (LDAP), which allows you to access and search a directory (another name for a specialized database or data repository that stores typed information about objects), is becoming a common component in Linux environments. If you're looking for a Linux option to search a directory with LDAP, check out Luma.
I got Luma directly from the Packman openSUSE repository, but you can probably find it in all distributions. The latest stable version is 2.3-0, dated March 2006, but the Luma site offers release candidate version 2.4, which is almost a year old. If you want to install it manually, you'll need Python, Qt, PyQt, and python-ldap, all of which are easily available. Luma is available under the GPLv2.
When you start it, you'll see a brief splash screen featuring a Shrek-like bluish figure, followed by the main screen, which offers several plugins that provide the program's functionality. Luma is organized around plugins, including browsers (both for data and schemas), an address book, user management (including massive user creation), and a general search function. More functions may become available in the future.
Before you can do anything with Luma, you first need to define which directory server you want to access. There's no limit as to how many servers you might want to connect to. Navigate to Settings -> Edit Server List -> Add, to add a new server to the list. Click on the newly added server, then sequentially click on each available option. Network Options is where you define the hostname, port, and encryption options; Authentification (yes, that's how they spell it) is where you define the login method; and LDAP Options is where you select whether you want to "Follow Aliases" (a concept similar to symbolic links) or "Use Base DNs as defined by the server" (usually a good option). After you define your server characteristics, click on Summary to check whether everything is OK, and then on Save. You should then be ready to start browsing or searching the whole directory.
The Browser plugin is a good starting point for getting to know the server. Click on your server name (you may notice a delay here, since Luma has to establish a connection to the server and download some information) to traverse the whole directory in a tree-like fashion. Click on the + symbols to open the nodes up and get more detail, and click on the - symbol to close them down again. When you reach a leaf node (that is, a node with no dependent nodes), Luma displays all its attributes and values. If you have enough rights, you can edit an attribute value (by clicking on the pencil icon), remove an attribute or class (by clicking on the red X), or add a new attribute (the star icon at the top) by selecting it from a list of allowed attributes and then entering its value by editing. Your changes aren't saved automatically; you need to click on Save on the top menu or confirm the changes when you move to a different node.
If you want to add new objects with the Browser plugin, you will need a Template, and for this you have to use the Template plugin. Click on Add, and you will be able to add a new template: give it a name and a description. Then, add object classes you want the template to apply to, and additional attributes for them. You cannot change the attribute characteristics (if it is mandatory or binary, for example) but you can give it a default value.
If you're curious about the directory's schema, try out the Schema Browser plugin. It provides full information about the complete data scheme, including classes, attributes, and dependencies. Select a server and wait until Luma connects to it and gets its structural information. Then click on ObjectClasses to learn what kind of objects are stored in the directory, or click on Attributes to get more details about the meaning of each.
The Address Book is a simpler plugin that allows you to maintain a list of names and addresses. Click on Add to add a new entry, Delete to remove one, or search by using the filter. Navigate to Settings -> Configure Plugins, and click on Address Book to see the attributes that will be searched; you can easily add a new one or delete an old one.
The Search plugin provides a more powerful way to search. Pick the server and the base distinguished name (DN, a globally unique identifier for entries), then click on the Filter Wizard to create a query string. If you're knowledgeable about searching, you can skip the wizard and just type in something like (&(objectclass=person)(ou=accounting)) in the Filter field. In either case, you must have some general ideas about LDAP searching; you might want to bone up on search specifics.
The Admin Utilities plugin provides a bunch of functions that aren't LDAP-specific, including password generation and encryption and date/time calculations; they're nice but seemingly irrelevant.
Finally, the User Management and Massive User Creation plugins allow you to create users, either one at the time or many simultaneously. You should be familiar with creating Linux or Unix users before using these plugins (the documentation frankly allows this plugin is not general, but rather quite Unix oriented) because you need to specify several administrative options, such as the user shell, home prefix, and user ID range. Note, however, that the tool won't create the home directories for the user: you will have to do that on your own, or set up your system to use pam_mkhomedir so the required directories will be automatically created the first time the user logs in.
Even if you have little experience in LDAP matters, using Luma can quickly help you put your theoretical knowledge into practice. Luma could use some user documentation (the Help button provides only About information) or, at the very least, pointers to Web-based information. But if you have to deal with LDAP servers, Luma can provide much-needed aid.


text:
1051 Gaffney Rd # 5, Fort Wainwright, AK 99703
(907) 356-7813
Compliment this business
Street address:
1051 Gaffney Rd # 5
City / suburb:
Fort Wainwright
Fairbanks North Star
ZIP code:
(907) 356-7813
Contact name:
Kim Herman
Contact title:
(907) 356-7815
GPS Longitude:
GPS Latitude:
Places nearby
0.04 miles away
Law Firms and Law Offices
0.04 miles away
Law Firms and Law Offices
General information
- Consumer Products & Services Consumer Services Direct Selling Establishments Direct Selling Establishments, Newspaper Delivery, Door to Door Cosmetic Sales
- Retail Trade Miscellaneous Retail Direct Selling Establishments
Business data
|Standard industrial classification code:
|Estimated number of employees:
|Annual sales estimate:
|City / suburb:
|Fort Wainwright, AK
|Fairbanks North Star
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More details about ACORN
According to our records, this business is located at 1051 Gaffney Rd # 5 in Fort Wainwright (in Fairbanks North Star County), Alaska 99703, the location GPS coordinates are: 37.568239 (latitude), -84.4957449 (longitude). ACORN is categorized under Direct Selling Establishments (SIC code 5963). Known organization manager is Kim Herman. Current estimates show that the unit has a sales volume of $7,800,000 and staff of approximately 50 people. You can contact the company by phone at (907) 356-7813; the following fax number is also provided: (907) 356-7815.
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Health is more than just exercising and eating right total health is a foundation that supports every aspect of who you are and what you wish to accomplish in life. All I Am® provides a full spectrum of high-quality natural products to enhance your total health. We invite you to explore your health today and enjoy finding products in our store that will enrich your life.
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"It is joyful, jolly
like the flying.
Swimming in the rivers, tearing the clothes and burning the shoes
Angel of the nature; counting the grasses, touching the flower, teasing the birds"
― M.F. Moonzajer, LOVE, HATRED AND MADNESS
White Orchids are known as a symbol of innocence, elegance and beauty. What better ways to quietly give them, to the one you regard to have that angelic beauty in them? This hand bunch of 12 White orchids combined with blue spirals, delicately wrapped in an exotic blue paper is the sweetest way of complimenting someone for their purity in their personality.
- Once you or your loved ones receive the flowers, make sure to trim the stems and place them in water.
- No leaves should remain below the water and replenish daily.
- Do not place under direct sunlight.
The image displayed is in indicative nature. The actual product may vary in design or shape according to availability.
Angel's Beauty
- ₹1,749.00
- Ex Tax: ₹1,749.00


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Scores based on 194 reviews
Invoice history based on 151 reviews.
Trade price carpets
"The mobile shop with the wholesale prices"
We are a mobile independent carpet shop. We offer a complete service with over 40 years experience, so we can cover all of your flooring needs.
We also have thousands of samples which enables us to bring them to the comfort of your own home are opening times are Monday - Friday 8am-6pm Saturday 8am-12pm.
Portsmouth, Southsea, Havant, Fareham, Lee-on-the-Solent,


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The music of Edgard Varese has been cropping up with unusual frequency this season. Perhaps a reason is that as musicians have looked back over the 20th century, Varese, who died in 1965, appears as a beacon of inventiveness whose influence is greater now than it was in his lifetime.
In a way he was to contemporary music what Gesualdo was to the early Baroque. Both were composers who stood outside the stylistic battles of their time and followed their own peculiarly dissonant, deeply personal paths. Varese, alas, did not have Gesualdo's financial independence. But he left a body of works that, for all their noisy assertiveness, touch something in listeners' psyches.
Speculum Musicae devoted its entire Sunday afternoon concert at the Miller Theater to Varese's music, offering five of his works with discussions of them between Allen Blustine, the ensemble's clarinetist and president, and Cho Wen-Chung, Varese's student and assistant. Mr. Chou's anecdotes added a measure of personal color to what we know about Varese, the most salient being a sense of the frustration he felt at his inability to find someone to underwrite an electronic music laboratory where he could work out his theories of organized sound and fulfill his need to create new kinds of sounds.
His music, of course, shows that outlandish sounds were not beyond his grasp, no matter what the financial or technical impediments may have been. Included on the program was the ''Poeme Electronique,'' Varese's eerily pictorial 1958 essay in invented timbres and sonic atmospheres. Here and in the works for conventional instruments on the program, a listener had the impression that percussion battery was the meeting ground between acoustic and electronic sound: anything from a graceful bell tone to a thwack on a bass drum or the scream of a siren could be produced either way, and in works like ''Deserts'' (1950-54) for chamber orchestra and tape, one hears both.
''Deserts,'' which Mr. Chou described as the composer's magnum opus (some would argue for the orchestral ''Ameriques''), closed the program. Included as well were a lyrical account by Susan Nidel of the solo flute work ''Density 21.5'' (1936) and vigorous, tactile readings of ''Hyperprism'' (1922-23) and ''Octandre'' (1923).Continue reading the main story


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The Characters:
Colwyn - Heroic prince who wishes to free his world from the evil Beast. He is also made out of something fire retardant.
Lyssa - Princess who dearly loves Colwyn, even though she has known him for about one hour and it is an arranged marriage.
Ergo - Inept sorcerer who has some ego problems.
Ynyr - Ancient wise man with darn little common sense. Dies after he runs out of sand. (Go watch the movie and stop scratching your head over what that means.)
Torquil - Bandit and robber, he does not really exhibit any good qualities, but Colwyn likes him for some reason.
The Cyclops - Powerful warrior who can see into the future, unfortunately the only thing he can see is his own death. Ends up as a one eyed pancake.
Kegan - Liam Neeson! One of Torquil's fellow bandits who has a wife in almost every village. Shot by a Slayer.
The Widow of the Web - Once beloved of Ynyr, she has been sentenced to life imprisonment for killing his son. Eaten by a giant spider.
Turold - Colwyn's father, the late king.
Eirig - Lyssa's father, another late king. (In case you missed it, I'm not talking about punctuality.)
Rhun and the Merry Men - The rest of the bandits, just here so someone can die every other scene or so.
The Emerald Seer and Titch - Old man and his little apprentice, the seer is killed and replaced by a servant to the Beast.
The Slayers - Army of armored creatures which serve the Beast. When you kill one red special effects go off and it's skull splits open, then something nasty burrows into the ground.
The Beast - Conqueror of worlds, ruler of the known galaxy, but I've seen piles of sticks with better skill at romance. Roasted alive by Colwyn.
The Plot:
If you were any sort of fantasy film lover alive during the 80's it is impossible to imagine that "Krull" did not grab your attention at the video store. Not only does it have an impressive cast (so many of these people appeared in other films, though I only mentioned Liam with the "!"), there are swords, lasers, monsters, and even a remote control 10,000rpm throwing star! Coolness!
Along the lines of plot, well the film is a textbook example of some evil power stealing the hero's true love and him fighting to gain her back, with some sidetracks to show just how fantastic the setting is. Even though the Beast is a towering reptile monster it desires Lyssa to be his bride, she is a little unnerved by this. (Not the least of which would be consummating the event, yikes!) Slayers take her prisoner and leave Colwyn for dead, but the prince is saved by a wise old man and easily gathers a party of loyal friends to assist him in attacking an impregnable fortress.
The special effects range from downright ludicrous, such as the amazing flying Clydesdales scene, to exceptional. The stop motion spider that guards the Widow is a gorgeous piece of work. There is nothing like watching a convincing arachnid scaled to the size of a small house. Most of the acting I can do without, but Ergo was my favorite character. When not bumbling spells and turning himself into a goose, the little guy is extolling his own virtues, which leads to some amusing descriptive phrases.
No matter what "real" critics say, you can not get me to hate this film.
Things I Learned From This Movie:
Women can make water catch on fire.
Advanced alien armies, which have conquered the known galaxy, use horses for land transportation.
Heroes are made from asbestos.
When traveling as a flaming meteorite one should avoid ponds or other bodies of water.
Kings carry master keys to every lock in their kingdom. (Which probably work on chastity belts too.)
Spider web sounds just like wind chimes when you shake it.
Beauty might not be skin deep, but love sure the heck is.
Cyclops are made out of Kevlar.
Boomerang throwing stars, that you intend on chucking at some slimy alien overlord, should have a nonstick coating.
Marriage vows are based on a grisly way to flame broil the bride and groom's enemies.
Stuff To Watch For:
10 mins - Advanced enough to have propane torches, but still using bows and swords...
16 mins - RANDOM ACT OF VIOLENCE AGAINST A HAY CART!
22 mins - Colwyn is really spry for having a laser lance pierce his shoulder. Oh, rock climbing now!
54 mins - Great idea Beast, keep traumatizing her with weird visions, I can see this being a successful marriage.
56 mins - Hehehe! He actually stood up in the quicksand for a moment.
58 mins - Everyone is darn clean after wading through all that quicksand.
74 mins - The phrase you are looking for is "Oh shit!" What did you expect was living in that huge web anyway?
84 mins - Why didn't you get a weekly schedule or something? That way they would know when the fortress was visiting some
91 mins - Holy blue screen Batman!
Audio clips in wav format
Starving actors speak out
Ynyr: "This it was given me to know, that many worlds have been enslaved by the Beast and his army, the Slayers."
Ergo: "I am Ergo the Magnificent! Short in stature, tall in power, narrow of purpose, and wide of vision, and I do not travel with peasants and beggars. Good bye!"
Torquil: "I don't kill men without good reason."
Ergo: "My name is no jest, beanpole. It's all very well to have a short name when you're twenty feet tall, but small people need large names to give them weight."
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Scenes from the movie
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Colwyn vs. the Beast.
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Re: Krull
Reply #49.
Posted on July 17, 2008, 11:10:43 PM by The Slayer
Come my children, seek the Slayer's wisdom at
Re: Krull
Reply #50.
Posted on November 01, 2008, 04:45:57 PM by Feisty Cadaver
Even after all these years, the princess is still hot!
Re: Krull
Reply #51.
Posted on November 09, 2008, 12:57:58 AM by Alec
Just watched this on TV what 25 YEARS after my mum took me to see it at the ABC cinema in Harrow!
I loved this film so much when I was a kid, I made up a game we used to play at breaktime called "Slayers" - basically it involved running around the playground at high speed and then fencing against your foe with one arm, aiming to score a hit and thereby win. Don't know what I was smoking but watching the movie brought back happy memories.
Watching as an adult, it's notable how BLEAK and NIHILISTIC the film is, as the hero's loyal companions get mown down in tragic and meaningless ways throughout - e.g. the guy who gets staked in that trap-room when he's just reaching for his dagger, or the guy who drowns in the quicksand, and obviously the cyclops who dies in the most horrendous and bloody awful way without remark!
The other thing that struck me is when the fire manes fly across the ravine near the end of the movie, it reminds me very much of Sigur Ros' Glosoli music video - I wonder if Krull was the influence for this beautiful scene?!
Re: Krull
Reply #52.
Posted on January 16, 2009, 06:38:11 PM by Wanderer
Love that review. This is oddly one of my favorite films. I knew all the lines in this movie at one time. Had my first born been a son his name was going to be Kegen Alexander.
Re: Krull
Reply #53.
Posted on April 22, 2009, 11:33:24 AM by Jennifer
I enjoyed this movie when I was a kid. I am sorta a sci-fi geek anyway. I love watching the old style animation. It's a bit cheesy but in 1983 I thought it was awesome! I watched it with my kids the other night and they really enjoyed it. My favorite part is when the slayers rise out of the water. It is so freaky. This movie has so many great actors in it and I think that is worth a look. Liam Neeson is so amazing. Anyway, this movie makes me feel young again.
Re: Krull
Reply #54.
Posted on September 17, 2009, 08:16:23 PM by Mister Gone
Sure it has Liam Neeson in it but no one recognized a young Robbie "Hagrid" Coletrain? He was the fat bandit! It's so great!
Re: Krull
Reply #55.
Posted on May 06, 2010, 07:41:25 PM by Jill
I love this movie, my mom and I watched it when I was about 10 and now I want my 10 year old to see it. I can't wait till it comes on tv I may just have to go rent it or buy it if I can find it. My favorite part is the flying clysdales with the flamming hooves. LOVE IT!
Re: Krull
Reply #56.
Posted on August 30, 2010, 12:42:58 PM by Chad
I didn't much care for Krull. It has the makings of a great camp classic, but I at least need an explanation for the happenings of the film to enjoy it. I ended up ranting about it to my parents for hours to the effect of...
"Where did the aliens come from?!"
"Why are there aliens in a fantasy film in the first place?!"
"Why are the families fighting each other when they should be fighting the aliens?!"
"Why is The Glave such a important symbol?!"
"Why does The Beast absolutely need to bang Colwyn's fiance?!"
"If the Firemares are supposed to be so damn fast, how are they so easily captured?!"
"Why does The Glave let Colwyn use the force to guide it?!"
"Since when was Colwyn a Firebender?!"
"How did everyone get back home if The Black Fortress is constantly teleporting?!"
"Why does this movie make less sense than the backstory of 'Highlander 2: The Quickening'?!"
I felt like Jim Carrey at the end of Batman Forever ("Too many questions... Too many questions...").
Bare in mind that I love bad movies (Yor, Forbidden Zone, and Rocky Horror top the list). I just want a little explanation. And if you don't explain it, you had better keep me placated enough so I don't start questioning it.
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The Coyle Free Library has an extensive genealogical/historical collection. We also just purchased Ancestry.com Library Edition for use within Coyle Free Library. Either stop in or contact Ruth Gembe at email@example.com or call 717-263-1054.
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You Can't Detect What You Can't See: Illuminating the Entire Kill Chain
On-Demand Webinar
Technical support for Linux, by and large, consisted of posting a query in one of the online Linux newsgroups. As more people begin using Linux for critical applications in the business world, however, that is beginning to change. Companies such as Red Hat and LinuxCare have jumped into the Linux support business, offering support packages aimed at corporations who want to use Linux in large or mission-critical applications. Support, such as this, is not cheap. Red Hat's Platinum support package, which offers unlimited, around the-clock phone support for your entire company, plus training, will set you back $55,000 a year.
Why pay $55,000 for information that you can get for free on the Web? "Customers can still post a question on the Web," says Carolyn Sparano, Director of Services for Red Hat. "But when you're running Linux in a business environment you don't always have the luxury of time to wait for an answer to come back."
Red Hat, which is based in Durham, North Carolina, has built up a call center with some 70 technicians. In October, it opened another, smaller center in the United Kingdom. Besides its Platinum support package, Red Hat offers a range of other support options, including "per incident" support, for $225 a call, remote dial-in administration, onsite support, as well as support targeted specifically for Linux developers.
Other Linux vendors provide similar offerings. Caldera, SuSe, Corel and TurboLinux all provide free installation support -- typically 60 or 90 days of email and phone support -- as well as optional longer term support contracts. Both Red Hat and LinuxCare offer support for Open Source applications which are frequently used with Linux, such as sendmail and the Apache Web server.
For users who don't want to be tied to a particular Linux vendor, LinuxCare, in San Francisco, offers a wide range of support options. Linuxcare supports all the major distributions of Linux with phone and email support, as well as on-site consulting. The company also offers courses on topics such as Linux system administration, Linux security, and a program to certify specific hardware and software products that will run using Linux.
Providing commercial support for Linux is a relatively new business, and many vendors are contracting out support services. Dell Computer Corp., for example, recently announced that it will make Linux available on its entire range of servers and will have contracts with LinuxCare for phone support for its Linux customers. LinuxCare also provides IBM's Linux customers with technical support for "level 3" problems, which are complicated issues that may involve changes to the Linux source code. VA Linux Systems, based in Sunnyvale, Calif., has hired nation-wide support provider DecisionOne, in Frazer, Penn, to provide phone support to its customers. Even Red Hat has contracted with client-server consulting group Collective Technologies, of Austin, Texas, for onsite support and consulting services.
As the use of Linux spreads, more companies are adding Linux expertise to their technical support offerings. Clearwater, Fla-based computer distributor Tech Data Corp. offers Red Hat, Caldera and Corel versions of Linux to its network of 100,000 computer resellers. These resellers provide consulting services and technical support to their customers, says Larry Armold, Tech Data's vice-president of sales support and operations. When the resellers need support, they call Tech Data -- which in turn, has an agreement with Red Hat for support on complicated technical support issues. So far, however, most of the questions fielded by Tech Data's ten Linux support technicians have been installation and configuration questions, says Armold.
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BRISTOL — A 23-year-old Bristol man accused of hiring a friend to kill a Waterbury man was arrested Monday and charged in the New Year's Eve shooting.
Anthony Harris, of 1175 Farmington Ave., is accused of offering to pay a friend $50,000 to kill a Waterbury man who he suspected had given police information leading to the arrest of his brother and uncle on drug charges, an affidavit said. Harris also suspected the Waterbury man had fired shots into his Bristol home, according to the affidavit.


text:
It's a classic from two French blokes who helped catapult house into the mainstream. Its 144 repetitions of the song's title made sure that no-one would ever need to ask them what that really cool song was called. It was named number 13 in NME's countdown of the top 25 dance anthem of all time. It's "Around The World" by Daft Punk, and it's THE definitive track from the late 90's when Paris was EDM's Mecca.
There were countless remixes of this track, some old, some new. Today I'll be posting three of the rarer mixes of "Around The World". Speaking of which, if ANYONE has that 14-minute Motorbass Miami Mix, PLEASE contact ilictronix and email it, we would much appreciate it.
First is one of four tracks from the "Around The World: Ricanstructed by Masters At Work" EP. Masters At Work are a house duo consisting of Kenny Dope and Louie Vega (both of whom are namechecked in DP's "Teachers"). They made 4 remixes of "Around The World" for this EP. All of them are chilled-out deep house epics, but the instrumental "Mellow" mix is my favourite. Enjoy :)
Daft Punk - Around The World (Mellow Mix) [Right Click To Download]
Next up is the I:Cube mix. This was on the flip of that Motorbass Miami Mix I was begging for earlier. Daft Punk also did a remix of I:Cube's "Disco Cubizm" which is some FUNKY SHIT, so check that out if you haven't already.
Daft Punk - Around The World (I:Cube Remix) [Right Click To Download]
And finally, a rare mix from Todd Terry. With added deep throated chanting and a bassline that jumps around more than a hostage in the trunk of a Citroen C4, this mix will get any party rocking.
Daft Punk - Around The World (Tee's Frozen Sun Mix) [Right Click To Download]
Keep it funky
- Joe
Thursday, 26 March 2009
Around The World.... Again
Article written by Attack Yourself! on 17:19
5 comments:
- Boba Fettuccini said...
Now ya need to find Around The World Again by Nicky Van She and Dangerous Dan. :D
- 26 March 2009 at 19:08
- prez jordan said...
Awesome, I only owned the mellow mix.
- 26 March 2009 at 19:09
- Joe said...
I actually used to have that NVS/DD track.
- 27 March 2009 at 14:38
- Gavin said...
to team ilicitronix,i have the coveted remix you are looking for.its on my first blog. if you want i will email it to u
- 27 March 2009 at 23:45
- Erik M said...
only DLed the todd terry mix because it is todd terry. too progressive to get a crowd going.
but applause for these harder to find remixes.
- 29 March 2009 at 03:30
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Photoshop turns 100-year-old woman into 30-year-old self
Photoshop can do some amazing things. It can make you look 100 years old, or if you're 100, it can make you look like you did when you were 30 again.
Life will go by before you know it. Every elderly person you see walking down the street or sitting on a bench was at once your age, with hopes and dreams of a future yet lived. But time flies, and 60 years will go by as though the years were weeks. When you see a frail 100 year old, it's hard to imagine what they looked like and how they were when they were vibrant and 30. And thanks to Photoshop, you don't have to imagine anything. All you have to do is just look at the photos.
When a prosecutor discovers he has months to live, can he turn his life around and tear down corruption? Find out in the gripping melodrama Punch:
If anything, these photos may make you appreciate your youth and the time you have to enjoy it. Because as wonderful as getting older and wiser is, we're never going to be the age we are today.
Check out the video below. By the end, you'll forget what she looked like in the beginning.


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The Miami Heat's annual free preseason open scrimmage will be 7 p.m. Oct.1 at AmericanAirlines Arena.
While there is no charge for the event, fans must register for tickets at Ticketmaster.com, with those tickets now available. Tickets will not be available at the AmericanAirlines Arena ticket office.
There is a maximum of four tickets per transaction. Doors for the event open at 6 p.m., with no on-site parking available.
As part of Breast Cancer Awareness Month, the Heat will incorporate the color pink into the scrimmage, with players wearing special-edition red-white-and-pink shooting shirts. The shirts then will be auctioned off...


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Louisville Shooting Ranges
Louisville Shooting Ranges - There's more there then world famous bats. Conveniently, you're also next to New Albany, Indiana - basically, two good areas for shooting joined together. We don't like to give preferential treatment to businesses, but as you're in Kentucky, the world famous Knob Creek Shooting Range, which has everything from Cowboy Action Shooting, to IDPA, to firearm certifications, and, of course, the famous Machine Gun shoot.
While you're having fun though, keep in mind the words of the late Hunter S. Thompson: never drink whiskey or bourbon while firing a machine gun, you'll spill your drink.
More seriously, always call in to the range ahead of time to check availability and rules; it can save you a lot of time. While most of the ranges are indoors, if you're going to an outdoor range like Knob Creek, check the weather report - shooting in a full downpour is never fun, and certain activities like shooting clays for trap or skeet are not going to happen in the rain or snow.
Give these businesses a try, and then leave a review - you'll be keeping them honest by telling your experiences.
Featured Louisville Shooting Ranges
Bluegrass Indoor RangeMarker A4402 Kiln Ct.
Louisville KY 40218-3042(502) 451-9088Shooting RangesFirearm Dealer/Gunsmith
Knob Creek Gun RangeMarker B690 Ritchey Ln
West Point KY 40177-6917(502) 922-4457Gun Shops, Shooting RangesFirearm Dealer/Gunsmith
Openrange Indoor PaintballMarker C6401 Cross Keys Blvd
Crestwood KY 40014-8723(502) 243-8282Shooting RangesFirearm Dealer/Gunsmith


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