Deployment of response surface methodology to optimize microencapsulation of peroxidases from turnip roots (Brassica rapa L.) by double emulsion in PLA polymer.

In order to improve the preservation conditions and stability of peroxidase catalytic properties, a number of immobilization techniques have been widely developed. In this context, we set as objective, the optimization of synthesis and stability of microcapsules of peroxidases (POD) from turnip using polylactic acid (PLA) polymer with the double emulsion technique. The surfactant, polymer, and peroxidase concentrations were the optimized parameters. According to the results obtained using the Box-Behnken design, the optimal parameters found were 1.55% of PVA, 55 mg/mL of peroxidases, and 30 mg/mL of PLA polymer with an encapsulation efficiency of 57.29%. The scanning electron microscopy morphological characterization of the optimized microcapsules showed a regular spherical structure. Fourier transform infrared spectroscopy identified the specific functional groups and chemical bonds before and after microencapsulation. The elaborated microcapsules were characterized by an average size of 200 µm (mainly from 150 to 500 µm) with a low residual moisture content (2.26%) and the encapsulated peroxidases showed better thermal stability. The in vitro release of peroxidases confirmed that the microcapsules have an excellent sustained release in simulated gastric digestion. Encapsulated peroxidases' storage under 25 and 4 °C displays a good residual POD activity with about 60% of initial activities during 80 days of storage, whereas free POD losses its initial activity within 15 and 30 days, respectively. The obtained results are promising for the development of effective therapeutic treatment of some intestinal troubles due to oxidative stress. PRACTICAL APPLICATION: Brassica rapa L. root is well known for its richness on peroxidases and thus presents an interesting potential for developing high added value products. In order to preserve the activity of extracted peroxidases (POD) from turnip roots, microencapsulation was optimized using a polylactic acid polymer. The encapsulated POD showed the maintenance of its activity under the effect of different storage conditions (time and temperature) and demonstrated resistance to gastric acidity. According to the obtained results, the encapsulation of peroxidases opens up medicine and pharmaceutical applications such as intestinal and colic protection against inflammations.

Fetal rat intestinal absorption of horseradish peroxidase from swallowed amniotic fluid.

Horseradish peroxidase (HRP) injected into amniotic fluid is swallowed by rat fetuses and within 3-6 h reaches the gut lumen. This macromolecular protein is then absorbed by the columnar lining cells via a system of apical cytoplasmic tubules formed by invaginations of the plasma membrane. From cytoplasm subjacent to the brush border HRP is transported, within vacuoles, to the supranuclear region, where some is retained for at least 18 h, and to interepithelial spaces. Extracellular enzyme is then found throughout the epithelial basement membrane and between connective tissue cells of the mucosal and submucosal layers Finally, HRP can be detected within lumina of blood and lymphatic capillaries, strongly suggesting that it is transported from the intestine to the circulation.

Comparative analysis of the concentration of injected horseradish peroxidase in cytoplasmic granules of the kidney cortex, in the blood, urine, and liver.

The concentration of horseradish peroxidase in total particulate fractions from the kidney cortex did not change much during the first few hours after injection, as long as most of the injected protein was not yet cleared from the blood. It decreased at a rate of 6-8% per hr afterwards. The concentration of peroxidase in total particulate fractions increased in proportion to the load (dose) over a wide range, suggesting that a constant fraction of the protein was reabsorbed by micropinocytic vesicles into the tubule cells from the glomerular filtrate. The amount of peroxidase excreted in the urine also increased in proportion to the injected dose. The proportion of peroxidase taken up by the liver, however, decreased several times when the dose was increased. A marked decrease of protein uptake into the kidney cortex and an increase of urinary excretion were observed when rats received a second, equal dose of peroxidase 4 hr after the first injection, and the rate of clearance of peroxidase from the blood was decreased after the second injection. The liver, on the other hand, took up almost twice as much peroxidase after two injections as after one. The uptake of peroxidase by the kidney cortex increased with age. Cytochemical observations on the preferential absorption of peroxidase by certain cell types and segments of the renal tubules in relation to dose are reported.

Retrograde axonal transport in the central nervous system.

When horseradish peroxidase is injected into the optic tectum of a chick, axons of ganglion cells transport it centripetally to their cell bodies in the retina at a rate of about 72 millimeters per day. After intraocular injections in the young chick, the peroxidase is transported centripetally along efferent axons, and is concentrated in cell bodies within the isthmo-optic nucleus. This retrograde movement of protein from axon terminal to cell body suggests a possible mechanism by which neurons respond to their target areas.

Protein absorption by the intestine of the fetal rat in utero.

Horseradish peroxidase (molecular weight, about 40,000) injected into the amniotic sacs in pregnant rats has been identified ultrastructurally, 6 to 18 hours later, within the fetal intestine in the absorptive cells and the underlying vascular endothelium. This indicates that macromolecular protein within amniotic fluid swallowed by the fetus can be absorbed and transported by fetal intestine, and may indicate that physiological compounds can be transported by this enteric route to contribute to fetal development.

Intestinal uptake of macromolecules: effect of oral immunization.

Animals were orally immunized with horseradish peroxidase and bovine serum albumin, and absorption of these antigens was studied. In comparison with controls, a consistent and significant decrease in peroxidase uptake was noted in both germ-free and conventional rats immunized with peroxidase; a similar decrease in serum albumin uptake was also noted in animals immunized with serum albumin. There was no difference in the uptake of an unrelated macromolecule. These observations suggest that local immunization interferes specifically with the intestinal uptake of macromolecular antigens.

Recombinant Manganese Peroxidase Reduces A2E Burden in Age-Related and Stargardt's Macular Degeneration Models.

Macular degeneration is hallmarked by retinal accumulation of toxic retinoid species (e.g., A2E) for which there is no endogenous mechanism to eliminate it. This ultimately results in progressive dysfunction and loss of vision either in advanced age for genetically normal patients (age-related macular degeneration) or in adolescence for those with inherited genetic mutations (Stargardt's disease). In this article, we present a proof-of-concept study for an enzyme-based therapy to remove these retinoids, modeled on traditional enzyme replacement therapy. Recombinant manganese peroxidase (rMnP) is produced in Pichia pastoris. In vitro, we demonstrate that rMnP breaks down A2E and other lipofuscin fluorophores with limited cellular toxicity, and as this enzyme is mannosylated, it can be taken up into cells through mannose receptor-dependent endocytosis. In vivo, we demonstrate that rMnP can significantly reduce the A2E burden when administered by intravitreal injections. Together, these data provide encouraging results toward the development of an enzyme-based therapy for macular degeneration and indicate the need for additional work to characterize the molecular mechanism of A2E breakdown and to improve the pharmacological parameters of the enzyme.

A novel antibacterial agent based on AgNPs and Fe3O4 loaded chitin microspheres with peroxidase-like activity for synergistic antibacterial activity and wound-healing.

In this study, a novel antibacterial agent was developed based on chitin nanofibrous microspheres loaded with AgNPs and Fe3O4 nanoparticles (Ag-Fe3O4-NMs) for synergistic antibacterial activity and wound healing. Ag-Fe3O4-NMs was prepared via an in situ synthetic method which showed an excellent porosity and wettability. Moreover, Ag-Fe3O4-NMs were capable of sustained release of Ag+ and catalysed the decomposition of low H2O2 concentrations to generate hydroxyl radical (OH). The OH and Ag+ showed higher antibacterial activity and inhibited the toxicity with high dose of AgNPs and H2O2. In vitro biocompatibility results suggested that Ag-Fe3O4-NMs have low toxicity and low hemolysis. Thus, a novel antibacterial agent with enhanced synergistic antibacterial activity was obtained by combination of Ag-Fe3O4-NMs and H2O2 at a low and biologlically safe dosage, which could facilitate fibroblast growth, accelerate epithelialization, and promote the healing rate of infected wounds.

Endocytosis of microperoxidase in the marginal cells of stria vascularis.

OBJECTIVE: Endocytosis has been thought to control entry into the cell and play a crucial role in the development, immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. We investigated the basic properties of endocytosis in the marginal cells of stria vascularis (SV) to discuss whether marginal cells have a potential to maintain the endolymph homeostasis. METHODS: We perfused microperoxidase (MPO), an endocytosis tracer, into the cochlear duct. After 5-60 min of endolymphatic perfusion, the tissues were fixed and the distribution of MPO within the marginal cell was observed by transmission electron microscopy. RESULTS: Endocytosis started already at 5 min after MPO perfusion. Small MPO-loaded endosomes were observed up to 30 min after MPO perfusion. The small tubulovesicular endosomes and the plasma membrane invagination were not decorated by an electron dense bristle structure. After endocytosis, MPO labeled preendosomes were quickly transported to the large vacuolar endosomes that connected with tubular endosomes. At 60 min after MPO perfusion, MPO-loaded large vesicles that have small vesicles in the lumen were observed. CONCLUSION: The time-course of MPO-loaded endosomes was similar to that of CF-loaded endosomes in the marginal cells of SV. The strial marginal cells have vigorous endocytotic activity both in clathrin-independent and clathrin-dependent endocytosis. This high activity of endocytosis in SV seems to be needed to maintain the homeostasis of endolymph via membranous channels and/or receptors regulations.

Horseradish and radish peroxidases eaten with fish could help explain observed associations between fish consumption and protection from age-related dementia.

A juxtaposition of regional cuisines and recent prospective studies of fish consumption in China and Japan points to fresh horseradish and/or radish (HRR) as possible contributors to delaying age-related dementia. The hypothesis is that the inverse association found sometimes between fish intake and cognitive decline is partially due to exposure of the oral cavity to active peroxidases from HRR served in conjunction with fish. This hypothesis can be tested by specifically looking at whether HRR is consumed with fish and whether such HRR is prepared in a way that preserves activity of HRR peroxidases. It is possible that by putting active HRR peroxidases in their mouths, elderly people supplement their age-diminished salivary antioxidant capacity and break down additional hydrogen peroxide (H2O2) in the oral cavity before it can migrate into the brain, thus decreasing the incidence of brain cell death induction by chronically-elevated H2O2. Intentional exposure of the oral cavity to active HRR peroxidases could be a prophylactic for delaying dementia. Because vegetable peroxidases are inactivated by gastric juices, it will be difficult to obtain benefit from HRR peroxidases' antioxidant effect via ingestion in encapsulated dietary supplements.

Immunotoxins containing glucose oxidase and lactoperoxidase with tumoricidal properties: in vitro killing effectiveness in a mouse plasmacytoma cell model.

We have tested the tumoricidal potency of enzyme immunotoxins constructed of antibodies conjugated to glucose oxidase and to lactoperoxidase. Murine plasmacytoma cells were targeted in vitro with the use of affinity-purified rabbit anti-plasmacytoma membrane antibodies (conjugated to glucose oxidase or lactoperoxidase) or rabbit serum raised against plasmacytoma microsome membranes followed by goat anti-rabbit immunoglobulin conjugates (to glucose oxidase or lactoperoxidase). Cytotoxicity was generated subsequently by incubation of the washed cells in a medium supplemented with glucose and sodium iodide, which were the substrates of these enzymes. This resulted in the presumed metabolic release of highly toxic reduced oxygen species and iodinated derivatives. Targeting of tumor cells with both conjugates, as opposed to one of them alone, produced a synergistic killing effect. The gain of specific versus unspecific cytotoxicity was upwards of 10,000-fold. The killing rates were elevated (t10 values less than 30 min) and linear over time. The resultant reduction in tumor cell viability was in the order of 5 to 6 logs after only 20 to 90 min of incubation in the glucose/NaI medium. Cytotoxicity was enhanced by the gamma-glutamyl cysteine synthetase inhibitor buthionine-S,R-sulfoximine and by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, while catalase was inhibitory. The results suggest that these enzyme immunotoxins may be suitable for the ex vivo purging of autologous bone marrow grafts.

Intracellular labelling of rat subthalamic neurones with horseradish peroxidase: computer analysis of dendrites and characterization of axon arborization.

Neurones of the rat subthalamic nucleus were identified by their response to cortical stimulation and then intracellularly labelled with horseradish peroxidase. After fixation, the brains were cut serially in sagittal plane and processed by the cobalt chloride-diaminobenzidine procedure. The morphology of nine of the twenty stained neurones strictly located inside the subthalamic nucleus is described by means of quantitative parameters following light-microscopic examination and three-dimensional computer reconstruction. They were all identified as Golgi type I neurones. The somata were ovoidal in shape. A mean of four dendritic stems arose from the soma and gave rise to a mean of 27 tips. The dendrites were thin with long and pedunculated spines. The dendritic fields were ellipsoidal in shape (100 x 600 x 300 micrometer) and were parallel to the principal plane of the nucleus. The dimensions of the dendritic fields are very close to those of the nucleus, and some dendrites cross its limits. The axons gave off two branches, one going caudally and the other rostrally. The caudal-going branch of the axon of one neurone, followed into the substantia nigra, divided into several collaterals coursing dorsoventrally. The rostral-going branch was never followed up to its termination. An intranuclear axonal collateral was observed in only one case. The present data are compared with those obtained from the primate subthalamic neurons. In spite of slight differences in the pattern of dendritic branching, the neurones are similar in both species. However, major differences in the internal organization of the dendritic fields are observed. Dendrites mixing with other neuronal populations were never observed in the primate. Moreover, the relative sizes of the dendritic fields and of the nucleus are strikingly different. This gives to the primate subthalamic nucleus specific and more precisely organized afferent connections.

The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics.

Avidin has an extraordinary affinity for the small-molecule vitamin biotin. Covalently coupling biotin or avidin to peroxidase molecules does not interfere with their normal biochemical functions. The avidin or biotin molecules, either peroxidase conjugated or unconjugated, can be brought to the antigen sites by means of an antiavidin antibody. Several immunohistochemical staining technics based on this principal have been described. The method utilizing an avidin-biotin-peroxidase complex was found to be more sensitive than the unlabeled antibody (PAP) method. This method involved four sequential staining procedures: (1) primary antibody (goat anti-human antigen); (2) secondary antibody (rabbit antigoat IgG) added in relative excess; (3) goat antiavidin antibody; (4) avidin-biotin-peroxidase complex. The applications of this technic are discussed.

A method for intracellular injection of horseradish peroxidase by pressure.

A technique for intracellular injection of horseradish peroxidase and other tracers into neurones is described. The method utilises gas pressure to force the tracer solution out of glass micropipettes and allows electrophysiological recordings to be made simultaneously with the injection process. Constructional details of the simple and inexpensive equipment are given. The method has the advantages of being equally suitable for charged and uncharged tracer molecules, and of providing reliable indication of the success of the injection at the time of the injection attempt.

Lysophosphatidyl choline facilitates labeling of CNS projections with horseradish peroxidase.

Previously, it has been difficult to label sensory terminations in the CNS by back-filling peripheral sensory nerves with horseradish peroxidase (HRP), apparently because the amount of HRP transported along the axons is insufficient. Labeling of central sensory projections in axolotls and frogs can be improved by agents known to disrupt membranes. Of those tested, L-alpha-lysophosphatidyl choline is the most effective. The addition of this detergent ot the HRP solution dramatically increases both the number of axons labeled and the density of label within single axons.

Uptake sites of horseradish peroxidase after injection into peritoneal structures: defining some pitfalls.

Following injection of horseradish peroxidase (HRP) either into the jejunal wall or the peritoneal cavity, neurons in the dorsal motor nucleus of the vagus nerve, celiac, nodose and spinal ganglia, and ventral and lateral horns of the spinal cord from the mid-thoracic to lumbar segments were labeled. When HRP was injected into the wall of the exteriorized gut, neurons of the spinal cord were not labeled. Furthermore, there was a significant decrease in the number of labeled neurons in the dorsal motor nucleus and ganglia examined. These results indicate that HRP injected into the intestinal wall could leak into the peritoneal cavity and be taken up and transported to neuronal cell bodies by nerve fibers not terminating in the injection area. The leakage of HRP to nearby abdominal structures and its subsequent uptake by nerve fibers is attributed to the lack of a diffusion barrier across the surfaces of the intestinal wall and the abdominal structures. It is suggested that in applying the HRP techniques for the study of neuronal connections in the peripheral nervous system, it is essential that carefully pla-ned control experiments be undertaken which can overcome the problem of mislabeling due to diffusion from the injection site.

A new microelectrophoretic procedure for delivery of horseradish peroxidase.

A new procedure for microelectrophoretic delivery of horseradish peroxidase (HRP) is described. A dilute solution (1.5--3% w/v) of fluorescein isothiocyanate-conjugated HRP is deposited according to a standard microelectrophoretic method and the tissue reacted according to a sensitive histochemical procedure. A major advantage of this method is the reduced secondary diffusion of the enzyme present at the injection site.

Evidence for sublaminar organization of the cells of origin of the geniculo-striate pathway in the cat: a novel method for cortical deposition of HRP.

A novel technique for retrograde labelling was used to trace the axonal projections from the lateral geniculate nucleus (LGN) to the striate cortex in the cat. Cortical deposits of horseradish peroxidase were made in the form of long straight lines oblique to layer IV. The projections along the lengths of these linear deposits were inferred, by retinotopic correspondence, from the distributions of the retrogradely labelled cells in the LGN. The results are consistent with an earlier report, by Humphrey et al., that the depths of axon terminations in layer IV reflect the depths of the parent cell bodies in layers A and A1 of the LGN. The pattern observed in the present experiments--border cells in the A layers project mainly to cortical layers IVa and centre cells to IVb--suggests that: (1) the sublaminar segregation of the ON and OFF pathways that occurs in the cat LGN is reversed by convergent projections to IVa from the dorsal (ON-rich) and ventral (OFF-rich) borders of the A layers, and (2) the paucity of Y input to IVb is due to the near absence of Y cells in the narrow central sublaminae of the A layers that supply the main input to IVb.

Observations of HRP labeling following injection through a chronically implanted cannula--a method to avoid diffusion of HRP into injured fibers.

One of the limitations of the horseradish peroxidase (HRP) tracer method is the diffusion of HRP into injured axons resulting in unintended labeling of neurons not terminating in the injection area. To overcome this limitation, an experiment was designed to inject the HRP through an implanted cannula after degeneration and healing had taken place. It was shown that implantation of a cannula into the internal capsule significantly decreased the number of labeled axons in the injection site, thus limiting the unintended labeling of neurons from that injection. When injections followed implantation of the cannula by 24 h or more, fibers damaged by the cannula had healed or degenerated sufficiently that intraaxonal diffusion of HRP into those injured fibers did not occur. A significant difference between control (without the cannula) and experimental (with the cannula) injections was observed. Extensive axonal and neuronal labeling following the control injections was seen at the injection site and caudate nucleus, and in the thalamus and parietal cortex, respectively. Experimental injections resulted in sparse axonal and neuronal labeling evident mostly with the larger injections of HRP.

Use of retrograde peroxidase flooding as an effective tool for morphogenic analysis.

Retrograde horseradish peroxidase in conjunction with dimethyl sulfoxide was employed to flood the neonatal rat hypoglossal neuronal somata as well as their proximal and distal dendritic segments. Through such an approach total inundation of the hypoglossal neurons was achieved and as such these flooded cells compared favorably with Golgi preparations made in a parallel study. Such cellular flooding provided for the easy recognition at both the light and electron microscopic level of all hypoglossal neuronal appendages. It is suggested that the use of peroxidase with dimethyl sulfoxide is an effective tool for the analysis of various aspects of morphogenesis, particularly in relation to the distal dendritic segments.