Activities Of Non-specific Enzymes Research Articles

Targeted Thrombolysis with Magnetic Nanotherapeutics: A Translational Assessment.

Plasminogen activators, such as recombinant tissue-type plasminogen activators (rtPAs), while effective in treating thromboembolic diseases, often induce hemorrhagic complications due to non-specific enzyme activities in the systemic circulation. This study evaluated the targeting efficiency, efficacy, biodistribution, and potential toxicity of a rtPA covalently attached to chitosan-coated magnetic nanoparticles (chitosan-MNP-rtPA). The thrombolytic activity of a chitosan-MNP-rtPA was preserved by protection from an endogenous plasminogen activator inhibitor-1 (PAI-1) in whole blood and after circulation in vivo, as examined by thromboelastometry. Single-photon emission computed tomography (SPECT) demonstrated real-time retention of a 99mTc-MNP-rtPA induced by magnet application in a rat embolic model; an 80% reduction in rtPA dosage for a chitosan-MNP-rtPA with magnetic guidance was shown to restore blood flow. After treatment, iron deposition was observed in the reticuloendothelial systems, with portal edema and neutrophil infiltration in the liver at a ten-fold higher dose but not the regular dose. Nevertheless, no liver or renal toxicity was observed at this higher dose. In conclusion, the liver may still be the major deposit site of rtPA nanocomposites after targeted delivery; chitosan-coated MNPs are potentially amenable to target therapeutics with parenteral administration.

A nanocarrier immersion vaccine encoding surface immunogenic protein confers cross-immunoprotection against Streptococcus agalactiae and Streptococcus iniae infection in tilapia

Streptococcosis is a highly contagious aquatic bacterial disease that poses a significant threat to tilapia. Vaccination is a well-known effective measure to prevent and control fish bacterial diseases. Among the various immunization methods, immersion vaccination is simple and can be widely used in aquaculture. Besides, nanocarrier delivery technology has been reported as an effective solution to improve the immune effect of immersion vaccine. In this study, the surface immunogenic protein (Sip) was proved to be conserved and potential to provide cross-immunoprotection for both Streptococcus agalactiae (S. agalactiae) and Streptococcus iniae (S. iniae) by multiple sequences alignment and Western blotting analysis. On this basis, we expressed and obtained the recombinant protein rSip and connected it with functionalized carbon nanotubes (CNT) to construct the nanocarrier vaccine system CNT-rSip. After immersion immunization, the immune effect of CNT-rSip against above two streptococcus infections was evaluated in tilapia based on some aspects including the serum specific antibody level, non-specific enzyme activities, immune-related genes expression and relative percent survival (RPS) after bacteria challenge. The results showed that compared with control group, CNT-rSip significantly (P < 0.05) increased the serum antibody levels, related enzyme activities including acid phosphatase, alkaline phosphatase, lysozyme and total antioxidant capacity activities, as well as the expression levels of immune-related genes from 2 to 4 weeks post immunization (wpi), and all these indexes peaked at 3 wpi. Besides, the above indexes of CNT-rSip were higher than those of rSip group with different extend during the experiment. Furthermore, the challenge test indicated that CNT-rSip provided cross-immunoprotection against S. agalactiae and S. iniae infection with RPS of 75 % and 72.41 %, respectively, which were much higher than those of other groups. Our study indicated that the nanocarrier immersion vaccine CNT-rSip could significantly improve the antibody titer and confer cross-immuneprotection against S. agalactiae and S. iniae infection in tilapia.

A subunit vaccine encoding glycoprotein holds potential to protect snakehead (Channa argus) from snakehead vesiculovirus infection

Snakehead vesiculovirus (SHVV) is one of the most harmful viral diseases to the snakehead farming industry. Subunit vaccines with highly protective effects strongly depend on the type of vaccine antigen. In order to screen the potential antigen proteins with efficient immunogenicity and reactogenicity, the glycoprotein (G), matrix protein (M), nucleoprotein (N) and phosphoprotein (P) of SHVV were molecular cloned, sequences analyzed and prokaryotic expressed in this study, then four recombinant proteins were served as subunit vaccines to immunize snakehead by intraperitoneal injection to evaluate the immune effects based on the indexes including the serum antibody level, nonspecific enzyme activity, immune-related gene expression and relative percent survivals (RPS). The results showed that compared with control group, the serum antibody levels and T-AOC activities were significantly higher in all four subunit vaccine groups, the serum ACP and AKP activities in four subunit vaccine groups were higher with different degree, and the detected immune-related genes expression levels were remarkably higher in four subunit vaccine groups except IL-8 gene in spleen of rP group. Among four subunit vaccines, rG protein displayed better immune effect than other three proteins. Besides, the RPS of rG group was highest with 83.3%, followed by rM (66.7%), rN (50.0%) and rP (8.3%), respectively. The above results indicated that glycoprotein was a better candidate protective antigen of SHVV and held potential to protect Snakehead from SHVV infection in the type of subunit vaccine.

Aedes aegypti vector resistance status on malation and activity of non specific esteration enzymes in Tembalang district, Semarang city

Background: Controlling of dengue vectors around dengue haemorrhagic fever cases is often conducted by using insecticides, including a malathion insecticide in the city of Semarang. A research on susceptibility status of Ae. aegypti to insecticides, used for fogging and based on a high number of hemorrhagic fever cases in Tembalang district, Semarang city, needs to be observed. Objective: This study aimed to determine resistance status of Ae. aegypti mosquitoes based on bioassay and biochemical tests of esterase non-specific enzyme activity in the Tembalang District. Methods: This study used a cross sectional design, it was conducted in June-September 2018 in a population of Aedes sp. from Tembalang District, Semarang City. Samples of F2 generations of female Ae. aegypti were obtained by ovitraps in the Tembalang District consisting of 12 urban villages, and their resistance status were tested by impregnated paper bioassay with malathion at a diagnostic dose of 0.8%. Esterase enzyme activity in the Ae. aegypti mosquito body of this population was tested biochemically to prove an increase in a non-specific esterase enzyme on the a-naphthyl acetate substrate. Results: The bioassay results showed that mortality rates of Ae. aegypti at the 12 villages in Tembalang district after 24 hourtest by impregnated paper indicated a range of 0-62%. Mechanism of resistance indicating an increase of non-specific enzyme esterase activity on a-naphthyl acetate at Rowosari, Kedungmundu, Sambiroto, and Meteseh could not be seen, It began to be seen in a low percentage (15%) at Sendang Mulyo, Tandang, Sendangguwo and Bubusan, and in a moderate percentage (45-75%) in Tembalang, Jangli and Mangunharjo. The mechanism of high esterase enzyme activity could be seen in the Ae. aegypti population at Kramas with AV=0,700-0,900 by a percentage of 20%, and AV=0,900 by a percentage of 80%. Conclusion: This study provided information about some Ae. aegypti mosquitoes from Tembalang District that showed resistance to the malathion insecticide with an elevation of non-specific esterase enzyme activity ona-naphthyl acetate substrate in several villages except Kelurahan Rowosari, Kedungmundu, Sambiroto, and Meteseh.

Effects and transcriptional responses in the hepatopancreas of red claw crayfish Cherax quadricarinatus under cold stress.

The red claw crayfish, Cherax quadricarinatus, is an economically important freshwater crustacean that cannot tolerate low temperature, which diminishes survival via unknown mechanisms. Herein, physiological regulation of C. quadricarinatus was investigated following exposure to low temperature stress at 9 ± 2 °C for 4 weeks. Hepatopancreas tissue was tested for nonspecific enzyme activity, histological structure, and transcriptome sequencing analyses. The results showed that the activities of nonspecific enzymes were inhibited following low temperature stress. Ultrastructural observation revealed that the hepatopancreas structure was oxidatively damaged at low temperature, with numerous autophagic vesicles visible. Apoptosis in the hepatopancreas was significantly increased in the cold stress group, indicating diminished function. Transcriptome sequencing identified 2615 differentially expressed genes (DEGs) following low temperature stress, of which 1147 and 1468 were up- and down-regulated, respectively. Functional analysis of DEGs indicated involvement in substance metabolism, antioxidant defences, signal transduction, and immune responses. Therefore, chronic cold stress can suppress metabolism and cause oxidative damage and immune deficiency in crayfish. The findings provide fundamental molecular information for further study of the regulatory mechanisms of cold tolerance in red claw crayfish.

Allosteric interaction between 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4isomerase and cytochrome b5influences cofactor binding

The biosynthesis of steroid hormones, essential to the survival of all mammals, is dependent on the activity of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD). 3βHSD activity is, in turn, influenced by cytochrome-b(5) (Cyt-b(5)). However, the mechanism through which this occurs is unknown. In this study, we investigated this mechanism by evaluating the influence of Cyt-b(5) on the dehydrogenase and isomerase activities of 3βHSD. Capra hircus 3βHSD was overexpressed in SF-9 cells, using a baculovirus expression system, and purified. Substrate and cofactor kinetics were determined spectrophotometrically in the presence and absence of purified Ovis aries liver Cyt-b(5). Nonspecific enzyme activity was evaluated by zero-enzyme, -substrate, and -cofactor blanks. Fusion proteins, 3βHSD-eCFP, and Cyt-b(5)-eYFP were subsequently coexpressed in COS-1 cells and analyzed for FRET. A CFP-YFP fusion protein served as positive control, while coexpression of 3βHSD-eCFP and cytochrome P450 17α-hydroxylase/17,20 lyase-eYFP (CYP17A1-eYFP) served as negative control. Results showed Cyt-b(5) to decrease the K(m,)(NAD(+)) value of 3βHSD ≈3.5-fold while increasing the V(max,app) of the dehydrogenase reaction ≈17%. FRET analysis showed COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b(5)-eYFP to exhibit a FRET signal ≈9-fold greater than that of the negative control. These results indicate that Cyt-b(5) augments 3βHSD activity via an allosteric mechanism by increasing the affinity of the enzyme toward NAD(+).

Measuring the Quantity and Activity of Mitochondrial Electron Transport Chain Complexes in Tissues of Central Nervous System Using Blue Native Polyacrylamide Gel Electrophoresis

Mitochondrial dysfunction and degeneration are associated with many neurodegenerative disorders. A dysfunctional mitochondrial electron transport chain (ETC) impairs ATP production and accelerates the generation of free radicals. To evaluate mitochondrial function, reliable methods are needed. Conventional spectrophotometric assays may not eliminate interference from nonspecific enzyme activities and do not measure quantities of specific ETC complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to resolve mitochondrial ETC complexes. Combined with histochemical staining, it has also been applied to measure ETC enzyme activities in muscles. The current study is to determine (1) whether BN-PAGE can be used to detect ETC complexes from different regions of the central nervous system (CNS) and (2) the quantitative range of BN-PAGE in measuring the amounts and activities of different ETC complexes. By systematically varying the protein amount and the time of histochemical reactions, we have found linear ranges comparable to spectrophotometric assays for measuring enzyme activities of several ETC complexes. In addition, we found linear ranges for measuring protein quantities in several ETC complexes. These results demonstrate that BN-PAGE can be used to measure the amount and activity of the ETC enzymes from the nerve tissues and, thus, can be applied to evaluate the functional changes of mitochondria in neurodegenerative disorders.

Cholinesterases in peripheral nervous system II. The motor, sensory and sympathetic nerves in the rabbit ear perichondrium and rat cremaster muscle

1. Cholinesterase activity has been demonstrated histochemically by Karnowsky and Roots' direct colouring thiocholine method in whole mounts of rabbit ear perichondrium and rat cremaster muscle. 2. The nerve elements stained exhibit mainly non-specific cholinesterase activity. This confirms previous results obtained with Gomori's modification of Koelle and Friedenwald's method. 3. Not all the nerve elements are stained, because the ferricyanide ion does not everywhere penetrate the perineurium and neither the ferricyanide nor the substrate penetrates the myelin sheath. 4. Observations on isolated nerves show specific cholinesterase activity in some myelinated axons in the rat and both specific and non-specific activity in the rabbit. Some nodes of Ranvier exhibit specific cholinesterase activity in the rat and both specific and non-specific activity in the rabbit. In both rat and rabbit, the unmyelinated fibres exhibit non-specific enzyme activity. 5. A network of fine lines (? outlining perineurial epithelial cells) is noted on the surface of the nerve bundles and also a plexus of fine unmyelinated fibres is described lining the inside of the perineurium. 6. Evidence is provided for both a motor and an autonomic nerve supply to the motor end-plates of the cremaster muscle.

Cholinesterases in peripheral nervous system: I. Mixed, motor and sensory trunks

By means of the direct-colouring thiocholine-ferricyanide method—combined with polarization microscopy—cholinesterase activity has been identified and localized in mixed, motor and sensory nerves. Motor axons (rat genitofemoral nerve) contain exclusively acetylcholinesterase, while sensory axons (rabbit great auricular nerve) exhibit only occasional acetyl- and no non-specific cholinesterase activity. Unmyelinated fibres in the sensory nerve contain only non-specific cholinesterase. Unmyelinated fibres in motor and mixed (sciatic) nerves appear to exhibit both acetyl- and non-specific enzyme activity.

INTRACELLULAR MITOCHONDRIAL VARIATION IN ENZYME ACTIVITY AS SHOWN BY HISTOCHEMICAL STUDIES USING LIGHT AND ELECTRON MICROSCOPY

Activities of reduced diphosphopyridine nucleotide (DPNH)-tetrazolium reductase, succinic dehydrogenase, and reduced triphosphopyridine nucleotide-tetrazolium reductase were studied using the substrates glutamate, iso-citrate, and DPNH for the first enzyme; succinate; and 6-phosphogluconate, glucose-1-phosphate, and TPNH for the last enzyme system. These activities were quantitated in light microscopical studies by counting the formazan particles in non-vacuolated epidermal meristematic cells of timothy grass seedling roots. Using Nitro-blue tetrazolium as the H+-acceptor, the average counts per cell were: 57, DPNH-tetrazolium reductase; 65.5, succinic dehydrogenase; 78.2, TPNH-tetrazolium reductase. These significantly different counts were higher than those found in an earlier study using neotetrazolium, but the three relative levels of activity were unchanged. One discrepancy was that 6-phosphogluconate and succinate gave the same activity levels although 6-phosphogluconate is believed to be oxidized via TPN-linked systems. INT also was tested, but the results suggested non-specific enzyme activity and those data were discarded. Electron microscopical studies were reported in which DPNH-tetrazolium reductase activity was shown to be restricted to the mitochondrial membranes in about half the mitochondrial population of individual cells. Since preliminary counts of mitochondrial profiles from electron micrographs indicated about 1000 mitochondria per cell, the absolute numbers of formazan particles using light microscopy probably is an unreliable measure. Also, clumping of mitochondria after histochemical incubation was demonstrated in some of the electron microgaphs. This suggested that lower brightfield counts might result since one or more mitochondria may have been associated as a single formazan deposit. Generally, the data verified the earlier conclusions that (a) mitochondria were the specific site of this oxidative enzyme activity, and (b) that only a fraction of the mitochondrial population of a cell was enzymatically active for this enzyme.