Instability of Insulators near Quantum Phase Transitions.

Thin films of amorphous indium oxide undergo a magnetic field driven superconducting to insulator quantum phase transition. In the insulating phase, the current-voltage characteristics show large current discontinuities due to overheating of electrons. We show that the onset voltage for the discontinuities vanishes as we approach the quantum critical point. As a result, the insulating phase becomes unstable with respect to any applied voltage making it, at least experimentally, immeasurable. We emphasize that unlike previous reports of the absence of linear response near quantum phase transitions, in our system, the departure from equilibrium is discontinuous. Because the conditions for these discontinuities are satisfied in most insulators at low temperatures, and due to the decay of all characteristic energy scales near quantum phase transitions, we believe that this instability is general and should occur in various systems while approaching their quantum critical point. Accounting for this instability is crucial for determining the critical behavior of systems near the transition.

Nonequilibrium Second-Order Phase Transition in a Cooper-Pair Insulator.

In certain disordered superconductors, upon increasing the magnetic field, superconductivity terminates with a direct transition into an insulating phase. This phase is comprised of localized Cooper pairs and is termed a Cooper-pair insulator. The current-voltage characteristics measured in this insulating phase are highly nonlinear and, at low temperatures, exhibit abrupt current jumps. Increasing the temperature diminishes the jumps until the current-voltage characteristics become continuous. We show that a direct correspondence exists between our system and systems that undergo an equilibrium, second-order, phase transition. We illustrate this correspondence by comparing our results to the van der Waals equation of state for the liquid-gas mixture. We use the similarities to identify a critical point where an out of equilibrium second-order-like phase transition occurs in our system. Approaching the critical point, we find a power-law behavior with critical exponents that characterizes the transition.

Little-Parks oscillations in an insulator.

We present the results of a magnetoresistance study of the disorder-induced superconductor-insulator transition in an amorphous indium-oxide thin film patterned by a nanoscale periodic array of holes. We observed Little-Parks-like oscillations over our entire range of disorder spanning the transition. The period of oscillations was unchanged and corresponded to the superconducting flux quantum in the superconducting as well as in the insulating phases. Our results provide direct evidence for electron pairing in the insulator bordering with superconductivity.

The neutralization mechanism of Vipera palaestinae neurotoxin by a purified factor from homologous serum.

The serum of Vipera palaestinae contains two separate factors which neutralize the hemorrhagic and the neurotoxic activities of its venom. The purified antineurotoxic factor shows one major band in disc electrophoresis and an 800-fold increase in specific activity. Its molecular weight is approximately 56 000 and the isoelectric point is 4.0. These data, together with the failure to form precipitin lines in immuno-diffusion tests, suggest that the antineurotoxic factor of Vipera palaestinae serum is probably an albumin-like or alpha-globulin fraction rather than an immunoglobulin fraction. In vivo experiments show that the neutralizing protein does not protect or block the neurotoxin target sites. When the neurotoxic fraction is injected into mice intravenously at different intervals following injection of the snake serum, the neutralizing activity decreases with time. Experiments carried out in vitro show that the antineurotoxic factor is inactivated upon heating at 95 degrees C for 10 min but resists boiling when mixed with the neurotoxic component, suggesting the formation of a thermostable complex between the two components. This assumed complex dissociates at pH 2.5 and the neurotoxic activity reappears. Further investigation of the reaction with 131I-labeled neurotoxin and neurotoxin components shows that a stable complex is formed between the neutralizing protein and one of the synergistic components of the neurotoxic fraction.

Isolation, characterization and mode of neutralization of a potent antihemorrhagic factor from the serum of the snake Bothrops asper.

A potent antihemorrhagic factor (BaSAH1) was isolated from the serum of the snake Bothrops asper by ammonium sulfate precipitation at 40-60%, Sephacryl S-200 and Sephadex G-50 gel filtration, DEAE-Sepharose, and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 5.2 and a molecular weight of 66 kDa. 4 micrograms of the purified factor BaSAH were needed to neutralize the hemorrhagic dose of B. asper whole venom compared to 60 micrograms of the clinically used horse polyvalent immunoglobulins. Moreover, 0.35 microgram of BaSAH were sufficient to achieve complete neutralization of the main hemorrhagic toxin (BaH1), with a molar ratio of 2:1. The antihemorrhagic activity was stable between pH 1.5-9 and up to 60 degrees C but lost activity completely after 30 min of heating at 70 degrees C. BaSAH did not digest the hemorrhagic toxin BaH1 or formed a precipitin line with it, nor with the whole venom. Both ELISA experiments and chromatography of BaSAH after incubation with the 125I-labeled hemorrhagic toxin BaH1 demonstrated that the mechanism of the neutralization involves a formation of an inactive soluble complex between the natural antihemorrhagin and the main hemorrhagin of B. asper venom.

A potent antihemorrhagin in the serum of the non-poisonous water snake Natrix tessellata: isolation, characterization and mechanism of neutralization.

The main natural antihemorrhagic factor (NtAH), which inhibits the hemorrhagic activity of Bothrops asper snake venom, was isolated from the serum of the non-poisonous water snake Natrix tessellata by ammonium sulfate precipitation at 35-55%, Sephadex G-75 gel filtration, ion exchange chromatography on DEAE-Sepharose and CM-Sepharose and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 4.5 and a molecular mass of about 880 kDa. The antihemorrhagic activity was stable between pH 5.5-11.7 and up to 50 degrees C, but lost activity after 20 min at 60 degrees C. It did not form a precipitin line with the main hemorrhagin of Bothrops asper snake venom (BaH1), nor with the whole venom, which suggests that the antihemorrhagic factor is not an immunoglobulin. The mechanism of neutralization by the isolated antihemorrhagic factor NtAH did not include digestion of the hemorrhagic toxin BaH1. Chromatography of NtAH with active 125I-labeled BaH1 toxin as well as ELISA experiments demonstrated that the mechanism of neutralization involves formation of an inactive soluble complex between the natural NtAH of the non-poisonous water snake and the main hemorrhagin of Bothrops asper venom.

Evidence for a Finite-Temperature Insulator.

In superconductors the zero-resistance current-flow is protected from dissipation at finite temperatures (T) by virtue of the short-circuit condition maintained by the electrons that remain in the condensed state. The recently suggested finite-T insulator and the "superinsulating" phase are different because any residual mechanism of conduction will eventually become dominant as the finite-T insulator sets-in. If the residual conduction is small it may be possible to observe the transition to these intriguing states. We show that the conductivity of the high magnetic-field insulator terminating superconductivity in amorphous indium-oxide exhibits an abrupt drop, and seem to approach a zero conductance at T < 0.04 K. We discuss our results in the light of theories that lead to a finite-T insulator.

Echinhibin-1--an inhibitor of Sendai virus isolated from the venom of the snake Echis coloratus.

The snake venom of Echis coloratus was found to abolish the hemagglutinating activity, hemolytic activity and in vivo infectivity of Sendai virus. The active factor (Echinhibin-1) was purified by gel filtration on Sephadex G-50, followed by chromatography on DEAE-Sepharose and CM-Sepharose. Echinhibin-1 is a protease with a molecular weight of about 25 kDa, an isoelectric point of 7 and is stained by PAS, indicating that it is a glycoprotein. It showed a strong azocollase activity that was stable up to 68 degrees C and at pH values of 4.5-10.5. Ten micrograms/ml were sufficient to abolish the hemolytic effect of the virus on human erythrocytes when incubation was at 37 degrees C for 2 h, while 20 micrograms/ml abolished the hemagglutinating activity. Addition of Echinhibin-1 after the adsorption of Sendai virions onto washed erythrocytes at 4 degrees C did not inhibit the subsequently hemolytic activity at 37 degrees C, indicating that Echinhibin-1 interferes with virus adsorption to the cells. Of various protease inhibitors, only Na2 EDTA and o-phenanthroline inhibited the antiviral activity of the purified factor, indicating that it is a metalloproteinase. In vivo, mice inoculated intranasally with the virus pretreated with Echinhibin-1 developed well and gained weight, whereas untreated virus-infected mice lost weight and died within 1 week. Intravenous administrations of the purified factor up to 80 micrograms/mouse produced no signs of toxicity and subcutaneous injections caused no hemorrhagic activity, while the whole venom is very hemorrhagic with an LD50 of 250 micrograms/kg for mice.

Inhibition of local hemorrhage and dermonecrosis induced by Bothrops asper snake venom: effectiveness of early in situ administration of the peptidomimetic metalloproteinase inhibitor batimastat and the chelating agent CaNa2EDTA.

The effectiveness of the chelating agent CaNa2EDTA and the peptidomimetic matrix metalloproteinase inhibitor batimastat (BB-94) to inhibit local tissue damage induced by Bothrops asper snake venom was studied in mice. Both compounds totally inhibited proteolytic, hemorrhagic, and dermonecrotic effects, and partially reduced edema-forming activity, when incubated with venom prior to injection. Much lower concentrations of batimastat than of CaNa2EDTA were required to inhibit these effects. In addition, batimastat, but not CaNa2EDTA, partially reduced myotoxic activity of the venom. When the inhibitors were administered at various time intervals after envenomation at the same site of venom injection, both compounds were effective in neutralizing local hemorrhage and dermonecrosis if administered rapidly after venom. Inhibition was not as effective as the time lapse between venom and inhibitor injections increased. Owing to the relevance of metalloproteinases in the pathogenesis of local tissue damage induced by B. asper and other pit viper venoms, it is suggested that administration of peptidomimetic metalloproteinase inhibitors or CaNa2EDTA at the site of venom injection may represent a useful alternative to complement antivenoms in the neutralization of venom-induced local tissue damage.

Isolation and characterization of a hemorrhagic factor from the venom of the snake Atractaspis engaddensis (Atractaspididae).

The venom of Atractaspis is unique in its composition and contains both high and low molecular weight fractions. The first peak obtained by gel filtration on Sephadex G-50 (S1) induces hemorrhage in the skin of mice. The hemorrhagic activity is stable over a pH range of 6-9; at pH 5 or 9.5 the activity decreases to half of the original and it is destroyed when incubated at 56 degrees C for 15 min. The hemorrhagic fraction was further purified by ion exchange chromatography on DEAE-Sepharose followed by ammonium sulphate precipitation. The purified factor had a molecular weight of about 50,000 and, in acrylamide disc electrophoresis, showed an acidic band which strongly stained with Coomassie Brilliant Blue and Periodic Acid Schiff. The specific activity of the isolated hemorrhagin was about 12 times higher than that of the crude venom. It has no measurable protease activity on azocoll, casein or gelatin, but the hemorrhagic activity was inactivated by EDTA and was not restored by prolonged incubation with Ca2+ or Zn2+. This activity was also neutralized by sera of venomous and non-venomous snakes. Moreover, antibodies prepared against the venom of Vipera palaestinae neutralized the activity of Atractaspis hemorrhagin and formed one precipitation line in the immunodiffusion test. It is thus evident that Atractaspis, now considered to belong to a separate family, has a hemorrhagic factor which is similar to that of the venoms of the Viperidae.

Isolation and characterization of a proteolytic factor from the venom of Vipera palaestinae.

A proteolytic enzyme which is active on collagen and gelatin was isolated from the venom of Vipera palaestinae. The enzyme showed an optimal temperature of 45 degrees C and an optimal pH of 8.0. It was inhibited by snake blood serum, but not by EDTA or trypsin inhibitors. The enzyme was completely separated from one of the venom hemorrhagins, which accompanied it through the purification procedure. The possible evolution of hemorrhagins from proteolytic enzymes is discussed.

Inhibition of the proteolytic activity of hemorrhagin-e from Crotalus atrox venom by antihemorrhagins from homologous serum.

Antihemorrhagic proteins from Crotalus atrox serum were tested for their ability to inhibit the proteolytic activity of the hemorrhagic toxin-e from Crotalus atrox venom and of several other proteolytic enzymes: trypsin, collagenase and thermolysin. The antihemorrhagic proteins inhibited the proteolytic activity of hemorrhagin-e when tested on gelatin type I and collagen type IV, the proteolytic activity of trypsin on photofilm gelatin and the proteolytic activity of whole venom when tested on azocollagen and photofilm gelatin. The antihemorrhagins failed to inhibit the proteolytic activity of trypsin when tested on the specific synthetic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester (APNE), the activity of microbial collagenase on N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) or on azocollagen and the activity of thermolysin on N-(3-[2-furyl]acryloyl)-Gly-Leu amide (FAGLA). It is tentatively suggested that the antihemorrhagins from snake blood serum are proteinase inhibitors that underwent specialization towards the neutralization of the proteolytic activity of hemorrhagic toxins.

Species specific sensitivity towards the hemorrhagin of Ophiophagus hannah (Elapidae).

The filtration pattern of Ophiophagus hannah venom on Sephadex G-75 shows several peaks. The first peak, S1, includes high molecular weight proteins and contains the hemorrhagic and proteolytic activities. The proteolytic fractions overlap the hemorrhagic fractions, but are not identical with them. The crude venom and the high molecular weight peak have caseinase, benzoyl-L-arginine ethyl ester hydrolase and kallikrein-like activities, but not collagenase, gelatinase, thrombin, plasmin or urokinase-like activities. The hemorrhagin of Ophiophagus hannah shows species specific differences in its hemorrhagic effects: it causes hemorrhages in rabbits and hares (Lagomorpha), but not in rats, mice or guinea-pigs (Rodentia).

Histopathological changes in WEHI-3B leukemia cells following intoxication by cytotoxin P4 from Naja nigricollis nigricollis venom.

The histopathological changes in WEHI-3B leukemia cells were followed by light and electronmicroscopy at different time intervals following exposure to cytotoxin P4 from Naja nigricollis nigricollis venom. At 1 hr after exposure to the toxin (2.5 x 10(-7) M) deformation was detected, primarily in the mitochondria, followed by vacuolization in the cytoplasm and an increase in lysosome number at 2 hr post-intoxication. Thereafter, the endoplasmic reticulum assumed a microsomal-like appearance, the plasma membrane was disrupted and, finally, the cells released their content to the culture medium. It is postulated that cytotoxin P4 may affect mitochondria either indirectly, by stimulating intracellular processes after binding to the cell membrane, or directly by interaction with the mitochondria after penetrating into the cell.

Antihemorrhagic factors from the blood serum of the western diamondback rattlesnake Crotalus atrox.

Several antihemorrhagic factors isolated from C. atrox serum are glycoproteins with mol. wt ranging from 65,000 to 80,000. The antihemorrhagic activity of these factors was stable at a pH range of 1.3-11.5 and at temperatures up to 85 degrees C, for 30 min. The isolated antihemorrhagins also neutralized the proteolytic activity of C. atrox venom, as tested with azocollagen and gelatin, and formed a complex with hemorrhagic toxin e isolated from the same venom. The neutralization capacity of the isolated antihemorrhagins was six times as high as the commercial polyvalent antivenom produced for clinical use.

Inhibition of the hemorrhagic activity of Bothrops asper venom by a novel neutralizing mixture.

This study screened 25 sera, 19 synthetic products and five antivenoms obtained after immunization for their ability to neutralize the hemorrhagic activity of venom from the snake Bothrops asper. Among the sera screened, the homologous serum of B. asper itself was found to possess the highest neutralizing capacity, abolishing the hemorrhagic effect of the venom at weight ratio of 3:1. It was more efficient than the antisera obtained by immunization. Among the synthetic compounds tested, only O-phenanthroline and EDTA salts inhibited the hemorrhagic activity at concentrations of 0.5-10 mM; however, only CaNa2EDTA was non-toxic at the concentrations studied. Intravenous injections and in situ administration of the non-toxic inhibitors revealed that a fraction of B. asper serum, the horse polyvalent antivenom and CaNa2EDTA were the most potent antihemorrhagic materials against B. asper venom, especially when administered in situ as a mixture. This work suggests that this neutralizing mixture could be highly useful in the neutralization of local and systemic hemorrhage developing after B. asper envenomation.

Isolation and characterization of synergistic hemorrhagins from the venom of the snake Bothrops asper.

Three hemorrhagic factors (BaH1, BH2 and BH3) were isolated from the venom of Bothrops asper by gel filtration on Sephacryl S-200, DEAE-Sepharose chromatography, metal chelate affinity chromatography and hydrophobic interaction chromatography. They contain 55% of the total hemorrhagic activity of the whole venom when they are mixed, but lose almost half of the activity if they are separated, indicating a synergism between the three. The main hemorrhagin is BaH1 (Bothrops asper hemorrhagin 1); the other two are weak hemorrhagins but contribute to the synergism. They are acidic proteins with a pI of 4.5, 5.2 and 5; their mol. wt is 64,000, 26,000 and 55,000 respectively. The minimal hemorrhagic dose (MHD) of BaH1, BH2 and BH3 is 0.18, 2 and 1.6 micrograms, with a specific activity 55, 5 and 6.25 higher than that of the whole venom. The hemorrhagic activity of all three factors was inhibited by EDTA and ortho-phenathroline, indicating that the hemorrhagic activity is metal dependent. Phosphoramidon, soybean trypsin inhibitor, PMSF, pepstatin and aprotinin did not affect the hemorrhagic activity of the isolated factors.

In vitro activity of BaH1, the main hemorrhagic toxin of Bothrops asper snake venom on bovine endothelial cells.

Incubation of BaH1, the main hemorrhagic toxin purified from the venom of Bothrops asper, with endothelial cells caused the appearance of spaces among the cells. This effect became more noticeable with increasing hemorrhagin concentration and longer incubation time. Later, the cells became rounded and detached from the substrate into the medium. Augmentation of Trypan blue did not stain the detached cells, indicating their viability. Moreover, after washing the floating cells from the toxin they could be recultivated: they again spread on the substrate and proliferated, demonstrating that BaHl is not directly cytotoxic to the endothelial cells.

Immunological studies on BaH1 and BaP1, two hemorrhagic metalloproteinases from the venom of the snake Bothrops asper.

No immunological cross-reactivity was observed between BaH1 and BaP1, two hemorrhagic metalloproteinases isolated from B. asper venom, by gel immunodiffusion, Western blotting and neutralization studies. Cross-reactivity was detected with antisera against these toxins in several crotaline and viperine snake venoms by ELISA, whereas no reactivity was observed with either antiserum against the venoms of Bothrops nummifer, Crotalus durissus terrificus, Vipera russelli and several elapid venoms. Antiserum against native BaH1 neutralized hemorrhagic activity of the venoms of B. asper, B. atrox, B. jararaca, Crotalus atrox, C. durissus durissus, Echis carinatus and Trimeresurus flavoviridis, being ineffective against the venoms of Agkistrodon bilineatus and Lachesis muta.

Isolation and characterization of a metalloproteinase with weak hemorrhagic activity from the venom of the snake Bothrops asper (terciopelo).

A metalloproteinase, named BaP1, was purified to homogeneity from the venom of Bothrops asper (Pacific region) of Costa Rica by ion-exchange chromatography on CM-Sephadex and gel filtration on Sephacryl S-200. The enzyme has a mol. wt of 24,000 and contains few Cys and high numbers of Asp, Leu, Ser and Glu. BaP1 hydrolyzes casein, hide powder azure and fibrinogen, having an optimal pH of 8.0. It rapidly digests the A alpha-chain of fibrinogen and, later on, the B beta-chain, leaving the gamma-chain unaffected. Chelating agents (EDTA and 1,10-phenanthroline) inhibited proteolytic activity, whereas 2-mercaptoethanol and soybean trypsin inhibitor did not affect this activity. BaP1 has a weak hemorrhagic activity, with a minimum hemorrhagic dose of 20 micrograms; this activity was inhibited by EDTA and was abolished after incubation at 60 degrees C. In addition, BaP1 induces edema and a mild myotoxic effect, lacking coagulant, defibrinating and lethal effects.