Using near-infrared enhanced thermozyme and scFv dual-conjugated Au nanorods for detection and targeted photothermal treatment of Alzheimer's disease.

Investigation of targeting inhibitors of Aß aggregation, heme-Aß peroxidase-like activity and efficient detectors of Aß aggregation, are of therapeutic value and diagnostics significance for the treatment of Alzheimer's disease (AD). Due to the complex pathogenesis of AD, theranostics treatment with multiple functions are necessary. Herein we constructed the NIR absorption property of gold nanorods (GNRs) loaded with single chain variable fragment (scFv) 12B4 and thermophilic acylpeptide hydrolase (APH) ST0779 as a smart theranostic complex (GNRs-APH-scFv, GAS), which possesses both rapid detection of Aß aggregates and NIR photothermal treatment that effectively disassembles Aß aggregates and inhibits Aß-mediated toxicity. Methods: We screened targeting anti-Aß scFv 12B4 and thermophilic acylpeptide hydrolase as amyloid-degrading enzyme, synthesized GAS gold nanorods complex. The GAS was evalued by Aß inhibition and disaggregation assays, Aß detection assays, Aß mediated toxicity assays in vitro. In vivo, delaying Aß-induced paralysis in AD model of Caenorhabditis elegans was also tested by GAS. Results: In vitro, GAS has a synergistic effect to inhibit and disassociate Aß aggregates, in addition to decrease heme-Aß peroxidase-like activity. In cultured cells, treatment with GAS reduces Aß-induced cytotoxicity, while also delaying Aß-mediated paralysis in CL4176 C.elegans model of AD. Furthermore, the photothermal effect of the GAS upon NIR laser irradiation not only helps disassociate the Aß aggregates but also boosts APH activity to clear Aß. The GAS, as a targeting detector and inhibitor, allows real-time detection of Aß aggregates. Conclusion: These results firstly highlight the combination of scFv, APH and nanoparticles to be theranostic AD drugs. Taken together, our strategy provides a new thought into the design of smart compounds for use as efficiently therapeutic and preventive agents against AD. Moreover, our design provides broad prospects of biomedical strategy for further theranostics application in those diseases caused by abnormal protein.

[The effect of magnetic fields on the activity of proteinases and glycosidases in the intestine of the crucian carp Carassius carassius].

It has been demonstrated by the example of the crucian carp (Carassius carassius) that a 1-hour stay of fish in a combined magnetic field with resonance parameters for calcium ions decreases the proteolytic and amylolytic activities of their intestinal enzymes. It has been found that a 1-hour exposure to a combined magnetic field with resonance parameters for potassium ions has almost no effect on the activity of proteinases, but it decreases the amylolytic activity. It has been noted that the activity of proteases and glycosidases is lower under hypomagnetic conditions. Upon the inversion of the vertical component of the geomagnetic field, the proteolytic activity of the intestinal mucosa in C. carassius decreases, while the amylolytic activity becomes higher compared to the control. Possible effects of magnetic fields on the activity of digestive hydrolases in fish are discussed.

[The effects of geomagnetic storms on proteinase and glycosidase activities in fish intestinal mucosa].

It has been demonstrated that the glycosidase activity of cyprinoid fishes (carp and crucian carp) exposed to a geomagnetic storm for up to 20 h considerably decreases; however, the proteinase activity is weakly altered (a statistically significant decrease in the enzyme activity has been observed only in fasting fish). An in vitro study of the effects of individual half hour intervals of the geomagnetic storm that correspond to the main and recovery phases on the same enzyme activities demonstrates the opposite trend. Independently of the experimental conditions, geomagnetic storms have been shown to influence the enzyme system of fasting fish negatively.

[The effect of weak low-frequency magnetic fields on the intracellular calcium-dependent proteinases of fish].

The in vivo and in vitro effects of weak, low-frequency magnetic fields with resonance parameters for calcium ions upon intracellular calcium-dependent proteinases (calpains) in the crucian carp (Carassius carassius (L.) and roach (Rutilus rutilus (L.) were studied. It has been revealed that the impact of a weak low-frequency magnetic field leads to considerable decrease in the activity of calpains in the fish skeletal muscles and brain.

[Analysis of hydrolytic enzyme activities on sludge aerobic/anoxic digestion after ultrasonic pretreatment].

In order to evaluate the function of sludge aerobic/anoxic digestibility by ultrasonic pretreatment. The SS, VSS and hydrolytic enzyme activities (amylase, glucosidase, protease, phosphatase) were measured before and after ultrasonic pretreatment (28 kHz, 0.15 kW x L(-1), 10 min). The results showed that the performances of aerobic/anoxic were greatly improved after ultrasonic pretreatment, the removal efficiency of VSS went to 44.3%, 7.8% better than of traditional aerobic/anoxic digestion. The variational trend of sludge hydrolytic enzyme activities increased firstly and then fell off during 13d digestion, the maximum of amylase activity and glucosidase activity in ultrasonic sludge, appeared in the 5 d, amylase activity was 0.104 micromol x g(-1) and glucosidase activity was 0.637 (micromol x g(-1). The maximum of intracellular protease activity and extracellular proteases activity in ultrasonic sludge, appeared in the 7 d, intracellular protease activity was 23.68 micromol x g(-1), higher than extracellular proteases activity, and it was playing a leading role in sludge digestion. The acid phosphatase activity of ultrasonic sludge was higher than the control sludge, and the alkaline phosphatase was sensitive to environment. So the alkaline phosphatase activity reduced when the internal properties of sludge was changed.

Hydrolytic enzymes in activated sludge: extraction of protease and lipase by stirring and ultrasonication.

Hydrolytic enzymes released by the microorganisms in activated sludge are responsible for the organic matter degradation; however, the optimal extraction procedure of this valuable resource has not been well established until now. The present study evaluates the recovery of protease and lipase from the activated sludge by using stirring and ultrasonication, varying different parameters such as extraction time, concentration of additives (Triton X100, Cation Exchange Resin and Tris buffer), stirring velocity, ultrasonic power and sludge source. Sludge was collected from two urban wastewater treatment plants located in Prague (Czech Republic) and Reus (Spain). It was found that stirring using 2% v/v Triton X100 for 1h was enough to extract 57.4 protease units/g VSS, and that the same method using a combination of 10mM Tris pH 7.5+0.48 g/mL CER+0.5% TX100 as an additive allowed to extract 15.5 lipase units/g VSS from sludge collected from Reus Wastewater Treatment Plant. Ultrasonication allowed reducing the extraction time to 10 min for protease (using 2% v/v Triton X100 yielding 52.9 units/g VSS) and to 20 min for lipase (without any additive yielding nearly 21.4 units/g VSS), which makes this method appropriate for the extraction of enzymes from the activated sludge, and suitable to be scaled up for its application in the industry.

5-Aminolevulinic acid-based photodynamic therapy suppressed survival factors and activated proteases for apoptosis in human glioblastoma U87MG cells.

Glioblastoma is the most common astrocytic brain tumor in humans. Current therapies for this malignancy are mostly ineffective. Photodynamic therapy (PDT), an exciting treatment strategy based on activation of a photosensitizer, has not yet been extensively explored for treating glioblastoma. We used 5-aminolevulinic acid (5-ALA) as a photosensitizer for PDT to induce apoptosis in human malignant glioblastoma U87MG cells and to understand the underlying molecular mechanisms. Trypan blue dye exclusion test showed a decrease in cell viability after exposure to increasing doses of 5-ALA for 4h followed by PDT with a broad spectrum blue light (400-550 nm) at a dose of 18J/cm(2) for 1h and then incubation at 37 degrees C for 4h. Following 0.5 and 1mM 5-ALA-based PDT (5-ALA-PDT), Wright staining and ApopTag assay showed occurrence of apoptosis morphologically and biochemically, respectively. After 5-ALA-PDT, down regulation of nuclear factor kappa B (NFkappaB) and baculovirus inhibitor-of-apoptosis repeat containing-3 (BIRC-3) protein indicated inhibition of survival signals. Besides, 5-ALA-PDT caused increase in Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF). Activation of calpain, caspase-9, and caspase-3 occurred in course of apoptosis. Calpain and caspase-3 activities cleaved alpha-spectrin at specific sites generating 145kD spectrin breakdown product (SBDP) and 120kD SBDP, respectively. The results suggested that 5-ALA-PDT induced apoptosis in U87MG cells by suppression of survival signals and activation of proteolytic pathways. Thus, 5-ALA-PDT can be an effective strategy for inducing apoptosis in glioblastoma.

Photic regulation of melatonin in rat retina and the role of proteasomal proteolysis.

Several investigations have shown that illumination at night reduces melatonin level in the mammalian pineal, but the effect of night illumination on the retina is not known. In this study retinas were cultured in a flow-through apparatus and then were exposed to light at ZT 18. Light exposure reduced melatonin levels to the daytime level within 30 min. The reduction of melatonin levels was due to a rapid decrease in the activity of the enzyme AA-NAT; AA-NAT mRNA levels were not affected by illumination. Pre-incubation with lactacystin (25 microM) prevented light-induced reduction of AA-NAT activity and melatonin levels. These results demonstrate that melatonin levels in the mammalian retina are affected by light exposure at night, via proteosomal proteolysis of AA-NAT.

Protective effect of oral phosphatidylcholine on radiation-induced release of intestinal peptidases in rats.

PURPOSE: To investigate whether phosphatidylcholine (PPC) has a protective effect on mucosa-irradiated rats. METHODS: The rats were orally fed with 25, 50, and 100 mg PPC/kg body weight (b.w.), respectively, for 3 weeks before irradiation. After administering the medication and 1 day after irradiation, a 20 cm segment of the proximal jejunum was perfused in situ and peptidase activities, as well as the concentrations of the membrane components, were assayed. RESULTS: We have shown that the application of a low dose of 25 mg PPC/kg b.w. daily for 21 days can prevent damage to membranes induced by 2.0 Gy as represented in the peptidase release profiles during the perfusion of the proximal jejunum of rats. Higher dose levels did not increase the protective effect. CONCLUSIONS: These findings suggest that a low dosage of exogenous PPC is capable of hindering the impairment of membranes induced by a small dose of radiation.

Ionizing radiation affects 26s proteasome function and associated molecular responses, even at low doses.

BACKGROUND AND PURPOSE: Ionizing radiation is known to activate certain signal transduction pathways, the regulation of which could involve post-transcriptional as well as transcriptional mechanisms. One of the most important post-transcriptional pathways in eukaryotic cells is the ATP- and ubiquitin-dependent degradation of proteins by the 26s proteasome. This process controls initiation of many cellular stress responses, as well as inflammatory responses under control of the transcription factor NF-kappaB. The literature on the relationship between radiation and inflammation seems somewhat paradoxical. At high doses, radiation is generally pro-inflammatory. On the other hand, low dose radiation has a long history of use in the treatment of inflammatory disease. This suggests the involvement of multiple mechanisms that may operate differentially at different dose levels. MATERIALS AND METHODS: In this paper, the ability of different doses of ionizing radiation to directly affect 26s proteasome activity was tested in ECV 304 cells. Proteasome activity, IkappaBalpha protein levels, and NF-kappaB activation were monitored. RESULTS: Inhibition of chymotrypsin-like 20s and 26s proteasome activity was observed immediately after low- and high-dose irradiation either of cells or purified proteasomes. The inhibitory effect was independent of the availability of the known endogenous proteasome inhibitor heat shock protein 90 (hsp90). Levels of IkappaBalpha, a physiological 26s proteasome substrate, were increased only at low doses (0.25 Gy) and unaltered at higher doses whereas only the highest doses (8 and 20 Gy) activated NF-kappaB. CONCLUSIONS: We conclude that the proteasome is a direct target of ionizing radiation and suggest that inhibition of proteasome function provides a molecular framework within which low dose anti-inflammatory effects of radiation, and radiation-induced molecular responses in general, should be considered.

A low dose of ionizing radiation increases luminal release of intestinal peptidases in rats.

PURPOSE: Mucosal inflammation in the small intestine is a potentially hazardous side effect of abdominal irradiation. In an effort to develop a quantitative method of evaluating mucosal damage, the luminal release of brush border enzymes in response to ionizing radiation was examined using two investigational strategies. METHODS: First, a 20 cm segment of the proximal jejunum was perfused in situ and enzymatic activities within the perfusates were evaluated. In a second approach, enzymatic activities were directly evaluated in isolated brush border membranes from the jejunal mucosa. RESULTS: Most of the peptidase activities measured were increased in the perfusates 1 day after irradiation and had returned to control levels at 4 days. In the brush border membranes, some enzyme activities decreased at 1 day and were, with the exception of leucineaminopeptidase (LAP), similar to control levels at 4 days. CONCLUSIONS: LAP is more strongly affected by radiation than the transmembranously bounded enzymes.

[Immobilization of the lytic enzyme complex lysoricephine]. / Immobilizatsiia liticheskogo fermentnogo kompleksa lizoritsefina.

Entrapping of the lysoenzyme complex of lysoricephine in solutions of hydrophilic polymers and its immobilization on dressing materials were performed. Immobilized preparations that retained 80-100% of the lytic activity and stable during storage were obtained. Properties of the immobilized preparations: dependence on pH and temperature, stability in acidic medium, and effect of gamma-radiation were studied. It was shown that coimmobilization with protease C induced a 1.5-1.7-fold increase in the lytic activity of the immobilized preparation compared to the native enzyme complex of lysoricephine.

[Coimmobilization of proteolytic and bacteriolytic enzymes and properties of isolated materials]. / Issledovanie sovmestnoi immobilizatsii proteoliticheskogo i bakterioliticheskogo fermentov i svoistv poluchennykh materialov.

Coimmobilization of proteolytic and bacteriolytic enzymes on a carboxyl-containing grafted cellulose copolymer was studied. The resistance of lysozyme to gamma-sterilization significantly increased after coimmobilization with a proteolytic enzyme. The resulting material accelerated the cleansing of wounds.

An in vitro evaluation of the effect of laser irradiation on the thrombogenicity of thrombus.

The effect of laser irradiation on the thrombogenicity of thrombus was evaluated by treating thrombi, formed in-vitro from canine blood, with two different doses of cw Nd:YAG laser energy at 1064 nm. The thrombi were then incubated with whole blood, and the plasma levels of fibrinogen and thrombin-antithrombin III-complexes were measured. A statistically significant decrease (p < 0.05) in the thrombogenicity was indicated by a reduction in both fibrinogen consumption and levels of thrombin-antithrombin III-complexes in the high dose group (600 joules, 100 degrees C peak temperature) in comparison to the low dose group (300 joules, 70 degrees C peak temperature) and the untreated thrombi. These findings suggest that laser irradiation of thrombus at an appropriate dose may substantially reduce its thrombogenicity and ability to modulate hemostasis.

[A method for selecting Streptomyces spheroides mutants--producers of an exoprotease with fibrinolytic activity]. / Metod otbora mutantov Streptomyces spheroides--produtsentov ékzoproteazy s fibrinoliticheskoi aktivnost'iu.

The paper present a description of a fast method to obtain mutants of Streptomyces spheroides--supersynthetics of exoprotease. The primary culture spores being irradiated by UV-rays, the mutants are formed with presence in the cultural medium of own proteolytic enzyme added from without. The described method is based on the phenomenon of growth suppression in the primary culture spore by high concentrations of the own proteolytic enzyme. It permits selecting producers with high proteolytic activity of fibrinolytic proteases from the small number of mutants grown in these colonies.

[Chromatin degradation in the death of thymic lymphocytes induced by radiation or dexamethasone: the necessity for a stage of preliminary proteolysis]. / Degradatsiia khromatina pri gibeli limfotsitov timusa, indutsirovannoi radiatsiei ili deksametazonom: neobkhodimost' étapa predvaritel'nogo proteoliza.

Proteolytic activity in a protein fraction of a rat thymocyte nuclear matrix was found to increase 1-2 h after gamma-irradiation or administration of dexamethazone. Cycloheximide did not prevent the observed protease activation. Neither histons nor thymocyte nuclear matrix proteins were subjected to proteolysis after exposure to radiation or the hormone. Such proteolysis inhibitors as phenylmethylsulfonyl fluorine, trasilol, and partly leupeptine inhibited nuclear DNA degradation in irradiated and dexamethazone treated thymus lymphocytes. In all appearance, this effect was not due to Ca/Mg-dependent endonuclease inactivation. The same was observed in the system of autolytic chromatin degradation in isolated thymocyte nuclei.

[The effect of x-ray radiation and hypoxia on the proteolytic activity of the normal rat spleen and during tumor growth]. / Vliianie rentgenovskogo izlucheniia i gipoksii na proteoliticheskuiu aktivnost' selezenki krys v norme i pri opukholevom roste.

The influence of X-radiation on activity of lysosomal enzymes (D, L, H cathepsins) in rat spleen tissue and in inoculated rat sarcoma 45 has been investigated. Intact rats and rats with tumors were subjected to whole-body and sarcoma 45 to local irradiation with doses of 0.155 C/kg and 0.31 C/kg in conditions of breathing gas hypoxic mixture containing 90% of nitrogen and 10% of oxygen (GHM-10). The combined exposure to radiation and GHM-10 was shown to produce a certain protective action (e.g. normalized cathepsin activity) in the spleen. In the tumor tissue the protective effect of GHM-10 was absent.

[Pro-oxidant-induced proteolysis in the postradiation interphase death of thymocytes]. / Prooksidantno-indutsirovannyi proteoliz v postluchevoi interfaznoi gibeli timotsitov.

Activity of intracellular alkaline proteinases increased and that of the trypsin-like proteinase inhibitors decreased in thymocytes at early times following whole-body X-irradiation of rats with a dose of 4 Gy. Pro-oxidant mechanisms of intracellular proteolysis activation are discussed.

[Metabolic indices of proteolysis of the blood serum in dogs in the early period following uniform whole-body gamma irradiation]. / Metabolicheskie pokazateli proteoliza v syvorotke krovi sobak v rannii period posle obshchego ravnomernogo gamma-oblucheniia.

Free amino acid concentration and proteinase inhibitor content were studied during the first 48 h following whole-body uniform gamma irradiation of dogs (LD30/50 and LD90/45). The contribution of metabolic profile features to individual radiosensitivity is discussed on the basis of the retrospective analysis of the initial level of metabolic indices in animals survived and died after irradiation. The comparison of the dynamics of changes in the indices under study in the animals died after exposure to different radiation doses permitted to suggest the important role that early hyperactivation of proteolysis played in the development of metabolism decompensation which promoted the fatal outcome of the affection.

[The action of gamma irradiation on terrilytin immobilized on cellulose-fiber materials]. / Deistvie gamma-oblucheniia na terrilitin, immobilizovannyi na tselliuloznykh voloknistnykh materialakh.

The effect of gamma-radiation on terrilytin, a proteolytic enzyme immobilized on modified and nonmodified cellulose materials was studied by EPR. Dialdehyde cellulose and graft copolymer of cellulose and polyacrylic acid were used as the modified cellulose materials. Dependence of the native and immobilized terrilytin activity and the content of free radicals in the irradiated samples on the irradiation dose was observed. It was shown that immobilization of the enzyme led to increasing of its stability to the effect of the ionizing radiation. This was due to transfer of the free valency from terrilytin to the carrying polymer which prevented radiation and chemical destruction of the enzyme. The proteolytic activity of native terrilytin subjected to gamma-irradiation markedly decreased because of intramolecular and intermolecular interactions during reactions of the terrilytin free radicals, since in this case there was no polymer as an acceptor of the enzyme free valency.