Horseradish peroxidase study of the associative connections of the motor region of the cerebral cortex in cats

Somatotopic organization of the body representation in the motor cortex of the cat's brain and a high degree of differentiation in the organization of associative projections between the motor and the somatosensory cortex was shown by means of retrograde transport of horse-radish peroxidase. Pyramid and stellate neurones of the second and the third cortical layers participate in the formation of these projections and interconnections.     

Cortico-cortical connections in the motor cortex of the brain; a study using the peroxidase method]

Using a method based on retrograde axonal transport of horse-radish peroxidase (HRP), the cortico-cortical afferents of the motor cortex were studied. After enzyme injection into the posterior sigmoideus gyrus, the HRP product was found in the first and the second somatic sensory areas and in parietal cortex (fields 5a, 5b). The HRP-positive neurons occurred in layers II, III and V of the cortex and belonged to the pyramidal cells.     

Horseradish peroxidase. XXXVI. On the difference between peroxidase and metmyoglobin.

A mechanism for the reaction of hydrogen peroxide with horseradish peroxidase is proposed which involves the catalytic activity of the carboxylate side chain of aspartate residue 43. The corresponding residue in the active site of metmyoglobin is glycine E8, which explains the inability of metmyoglobin to form compound I. Certain aspects of the proposed peroxidase mechanism may be relevant to the catalytic triad for the serine proteases.     

Horseradish peroxidase as a label of injured cells

Synopsis The present study is concerned with artifacts likely to occur in a horseradish peroxidase exclusion test. Incubation of murine peritoneal macrophages and lymphocytes with the peroxidase showed a close relationship between the number of living cells and the percentage of cells excluding the tracer. The penetration of the cytoplasm by horseradish peroxidase is attributed to an increase in the permeability of the cell membrane during the incubation (ranging from 10 to 120 min). It was not increased by the presence of tracer throughout the incubation period. However, concomitant fixation of the cell in the presence of horseradish peroxidase caused an increase in the influx of the tracer. The horseradish peroxidase exclusion test applied to the guinea-pig organ of Corti has proved to be valid provided that: (a) mechanical lesions prior to the tracer incubation are avoided; (b) incubation is terminated by removal of the extracellular tracer; (c) fixation is carried out as soon as possible; (d) a low concentration of horseradish peroxidase is used; and (e) specimens are incubated in diaminobenzidine-H₂O₂ medium for the shortest possible period.Although fixation-induced cytoplasmic infiltration by horseradish peroxidase was not detected in cochlear specimens, the findings call attention to possible sources of error and define the level of significance of the test. Horseradish peroxidase does not appear to be a cytotoxic agent under the conditions used.     

Electrophysiological study of connections between the entorhinal cortex and the neocortex

In acute experiments in rabbits immobilized by d-tubocurarine, stimulation of the entorhinal area with rectangular electric impulses led to the appearance of evoked potentials (EP) with a latent period of 6–12 msec in the occipital, temporal, parietal, and cingular areas of the neocortex. The amplitude of the positive response component was 500 µV, and its duration 25–50 msec. The negative component was not always discernible. When rhythmic stimulation was used, these EPs followed stimulation frequencies not exceeding 20 per sec. Stimulation of the medial parts of the entorhinal area with a frequency of one to three per sec was accompanied by recruitment of the EP in the occipital and temporal neocortex areas. Nembutal depressed the amplitude of the neocortex EP appearing in response to stimulation of the entorhinal cortex. With the aid of double stimulation it could be established that, after conditioning stimulation of the entorhinal area, the positive component of the primary response (PR) evoked by stimulation of the contralateral sciatic nerve in the projection zone of the somatosensory cortex is strengthened during the first 50 msec, and subsequently after 80–120 msec. In these cases, the negative component was depressed. These findings are discussed with a view to the influence of limbic structures on the neocortex.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 1, pp. 73–78, January–February, 1970.     

Horseradish peroxidase catalyzed degradation of industrially important dyes.

Horseradish peroxidase (HRP) is known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols. Here, for the first time we have shown HRP to be effective in degrading and precipitating industrially important azo dyes. For Remazol blue, the enzyme activity was found to be far better at pH 2.5 than at neutral pH. In addition, Remazol blue acts as a strong competitive inhibitor of HRP at neutral pH. Horseradish peroxidase shows broad substrate specificity toward a variety of azo dyes. Kinetic constants (K(m)(app) and V(max)(app)) for two different dyes have been determined. In addition to providing a systematic analysis of the potential of HRP in degradation of dyes, this study opens up a new area on exploration of commercial dyes as inhibitors of enzymes. 2001 John Wiley & Sons, Inc.     

Reactivation of Horseradish peroxidase in organic solvent using extraction

A novel extraction method was developed aiming at increasing the stability of enzymes in organic solvent media. Horseradish peroxidase (HRP), inactivated in a tetrahydrofuran (THF)/water (1:1, v/v), regained and maintained its activity when HRP was extracted by adding a THF/benzene mixture to the original solution. However, the HRP activity was drastically lowered in the enzyme-free blank solution that had been formed by employing the same extraction procedure. As a result, the reactivation after the extraction is believed to depend on enzyme history, and might be arisen from an irreversible structural change of the enzyme.     

Horseradish peroxidase for the removal of carcinogenic aromatic amines from water

A new method has been developed for the removal of carcinogenic aromatic amines from industrial aqueous effluents. It includes the treatment of aqueous solutions containing the carcinogens with horseradish peroxidase and hydrogen peroxide. Such treatment results in a nearly complete precipitation of carcinogenic aromatic amines from water due to enzymatic crosslinking. This method was used to remove ten recognized human carcinogens from water: benzidine and its derivatives, naphthylamines, 4-aminobiphenyl, and p-phenylazoaniline. The dependence of the removal efficiency of the peroxidase treatment on the concentrations of the enzyme, H₂O₂ and a carcinogen and also on pH and the duration of the treatment was studied. The enzymatic removal of carcinogens from water was confirmed by both chemical and toxicological assays.     

Horseradish peroxidase. XXXII. pH dependence of the oxidation of L-(-)-tyrosine by compound I.

The rate of oxidation of L-(-)-tyrosine by horseradish peroxidase compound 1 has been studied as a function of pH at 25 degrees C and ionic strength 0.11. Over the pH range of 3.20--11.23 major effects of three ionizations were observed. The pKa values of the phenolic (pKa = 10.10) and amino (pKa = 9.21) dissociations of tyrosine and a single enzyme ionization (pKa = 5.42) were determined from nonlinear least squares analysis of the log rate versus pH profile. It was noted that the less acidic form of the enzyme was most reactive; hence, the reaction is described as base catalyzed. The rate of tyrosine oxidation falls rapidly with the deprotonation of the phenolic group.     

Horseradish peroxidase: a modern view of a classic enzyme

Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed evolution techniques are now used routinely to investigate the structure and function of horseradish peroxidase and offer the opportunity to develop engineered enzymes for practical applications in natural product and fine chemicals synthesis, medical diagnostics and bioremediation. A combination of horseradish peroxidase and indole-3-acetic acid or its derivatives is currently being evaluated as an agent for use in targeted cancer therapies. Physiological roles traditionally associated with the enzyme that include indole-3-acetic acid metabolism, cross-linking of biological polymers and lignification are becoming better understood at the molecular level, but the involvement of specific horseradish peroxidase isoenzymes in these processes is not yet clearly defined. Progress in this area should result from the identification of the entire peroxidase gene family of Arabidopsis thaliana, which has now been completed.     

Intracortical connections between neuron groups in the somatosensory cortex studied by the retrograde horseradish peroxidase transport method in rats

Short corticocortical connections between specialized groups of neurons (so-called barrels) were studied in the somatosensory cortex. After microinjections of horseradish peroxidase into a definite "barrel" labeled neurons were found in nearby groups within a radius of up to 400 µ. Labeled neurons were located chiefly in cortical layers V and III; 90% of them were pyramidal cells. Intracortical connection of labeled neurons were 1.6 times more numerous than thalamocortical connections. It is postulated that connections between neighboring cortical neuron groups are effected through their output cells, i.e., through pyramidal neurons of layers V and III.     

Association connections of the cat's parietal cortex

Intercortical connections of primary sensory (visual, auditory, somatosensory) areas with the parietal association cortex were studied in cats by the retrograde axonal transport of horseradish peroxidase and the Fink-Heimer silver impregnation of degenerated fibers techniques. This combined study revealed the shape, size, and intracortical location of cells connecting the primary sensory areas monosynaptically with the parietal cortex and also the distribution of preterminals and terminals of the fibers of these cells in the parietal association cortex. The greatest number of cells forming connections with area 7 of the parietal association cortex was shown to occur in visual area V1, and with area 5 in somatosensory area S1. Besides pyramidal neurons tagged with horseradish peroxidase, which were located mainly in layers II–IV, a few tagged stellate and fusiform cells also were found. The results supplement and confirm data on afferent connections of the parietal association cortex in cats.M. Gor'kii Donetsk Medical Institute. Translated from Neirofiziologiya, Vol. 13, No. 1, pp. 3–6, January, 1981.     

Associative connections of the cat parietal cortex

Interneuronal connections of area 7 of the cat parietal cortex with projection areas of the visual, auditory, and somatosensory cortex were analyzed by orthograde degeneration and retrograde transport of horseradish peroxidase methods. By combined investigation the cortico-cortical sources of afferentation of parietal area 7 could be precisely identified and concentration sites of neurons sending their axons into this area identified, and the morphological characteristics of these neurons could also be determined.A. A. Ukhtomskii Physiological Institute, A. A. Zhdanov Leningrad State University. Donetsk Medical Institute. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 13–17, January–February, 1980.     

Tests on the locomotion of the elongate and limbless reptile Ophisaurus apodus (Sauna: Anguidae)

A series of Ophisaurus apodus was filmed while traversing plane surfaces, fields of nails and pins at different spacings, and channels of different diameter. Small individuals can practise slow lateral undulation on very rough surfaces, but with increased speed, all shift to slide-pushing, using either constrained bends of the body or very wide swings of the tail. In fields of closely-spaced pins, they travel by undulation, often pushing at a limited number of sites and pulling and pushing the trunk among these. Amid pins of wider spacing, the undulation involves some repositioning of curves. In channels, they utilize continuous bend concertina movement with the initial bend formed anteriorly and subsequent ones added, either to the level of the cloaca or on to the tail as well. The cloacal region cannot be established on the basis of locomotor pattern, as the propulsive waves pass smoothly from head to tail. Maximum voluntary velocities (of the centre of gravity) were 13 cm/s for slide-pushing, 55 cm/s for lateral undulation, and 3 cm/s for concertina movement. Propulsion is entirely effected by bending of the trunk; tests gave no evidence that the dorsal and ventral portions of the integumentary armour show significant anteroposterior displacement relative to each other.     

Horseradish peroxidase catalyzed hydroxylation of phenol: II. Kinetic model

A numeric kinetic model of the horseradish peroxidase catalyzed hydroxylation of phenol is proposed to complete the previous thermodynamic analysis. As previously stated, the basic role of HRP is to catalyze the production of DHF* radicals. These further form hydroxyl radicals that hydroxylate phenol via noncatalyzed reactions. The transient differential equations of the model are solved numerically. Several kinetic constants are adjusted to fit basic experimental data. This set of values is then kept constant to simulate additive experiments carried out under different conditions. Predictions of the model concerning the effects of HRP concentration, temperature variation, and presence of catalase and superoxide dismutase are consistent with the experimental results. The quantitative kinetic approach consequently fully confirmed the previous thermodynamic conclusions.     

Non-isothermal kinetic modelling of potassium indigo-trisulfonate dye discolouration by Horseradish peroxidase

Abstract

Textile industries account for two-thirds of the total dyestuff market been responsible for the production of a large volume of effluent with unfixed dye. Aromatic dyes as potassium indigo-trisulfonate dye (PIT) are characterized as a chemically stable and complex molecule with high heat and light stability and with high toxicity even at low concentration. In order to evaluate an environmentally friendly method to remove potassium indigo-trisulfonate dye from aqueous solution, a commercial peroxidase (horseradish peroxidase, HPR) was used in the experimental and the data were modelled by the Arrhenius equation. According to the results, the best reaction conditions were obtained using 80?mg.L^?1 of dye concentration, 45?°C, pH 5.0, 349.35?U.mL^?1, and 4.5?mM hydrogen peroxide concentration leading to a 96% of dye discolouration.