Innervation of cockroach cercal receptors as revealed by horseradish peroxidase (Insecta,Blattodea)

Summary The innervation of cerci of a desert burrowing cockroach, Arenivaga sp., was determined by horseradish peroxidase backfilling of the cercal nerve and histochemistry. The procedure yielded a high percentage of successful fills and in many cases every neuron filled completely, including dendrites and axons of less than one m. The innervation of the cerci was found to be highly ordered. Upon entering the cercus, the cercal nerve splits into bilateral branches, one on each side of the midline. The nerves branch again at each segment to form fascicles of sensory neurons which innervate the trichobothria, sensilla chaetica and tricholiths, each with a single bipolar neuron. While the cell bodies of neurons are of similar dimensions, the dendrites to the tricholiths are much longer and terminate on the midline side of the sensilla socket where the tricholith shaft attaches.     

Retinal projections in the catfish,Mystus vittatus (Bloch) as revealed by tracer studies with horseradish peroxidase

Summary The retinal efferents of the catfish, Mystus vittatus, were investigated with the use of the horseradish peroxidase (HRP) technique. Most retinal fibres extended contralateral to the eye that had received HRP label, while a few fascicles projected to the ipsilateral side without decussation in the optic chiasma. The contralateral fibres projected to the suprachiasmatic nucleus, the nucleus opticus dorsolateralis, the nucleus of the posterior commissure, the nucleus geniculatus lateralis, pretectal nuclear complex, and to two layers of the optic tectum, i.e., stratum fibrosum et griseum superficiale and stratum griseum centrale. The accessory optic tract arose from the inner area of the optic tract and extended ventromedially to the accessory optic nucleus. The ipsilateral fascicles projected to almost all the above mentioned nuclei, but these projections were comparatively sparse. The ipsilateral retinal projection was restricted to the rostral tectum.     

Hypophysiotropic neurons in the goldfish hypothalamus demonstrated by retrograde transport of horseradish peroxidase

Summary The horseradish-peroxidase (HRP) technique was used to visualize the cell bodies of axons projecting to the goldfish pituitary. Following intravenous injections of HRP, HRP reaction products were observed in axons of the rostral pars distalis, proximal pars distalis, neurointermediate lobe, pituitary stalk and in axons coursing from the pituitary into the hypothalamus. HRP-labelled cells in the brain were localized in two regions only — the nucleus preopticus (NPO) pars magnocellularis and pars parvocellularis, and the nucleus lateralis tuberis (NLT) of the hypothalamus. These observations suggest that the NPO and NLT are the source of the neurosecretory innervation of the goldfish pituitary.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicertranchus labrax (Pisces, Teleostei)

The morphological and biochemical characteristics of pigment accumulations found in the kidney, liver, spleen, and mesentery of three different species of teleost fishes have been studied. There are significant differences in number, distribution, and morphology of pigment accumulations in different organs of the three species. Biochemical studies have shown the existence of tyrosinase activity in the mesentery of Mugil cephalus and in the kidney and mesentery of Sparus auratus. No tyrosinase activity was found in any internal organs of Dicertranchus labrax. That activity was assayed using three methods: tyrosine hidroxylation, dopa oxidation, and melanin formation. The morphological and biochemical observations are in agreement. In those organs in which we have demonstrated melanin synthetic activity, the pigment cells are morphologically and like melanophores, while in the organs that show no melanin synthetic activity, the pigment cells resemble macrophages.     

The trochlear motoneurons of lampreys (Lampetra fluviatilis): location,morphology and numbers as revealed with horseradish peroxidase

Summary The cells of origin of the trochlear nerve of Lampetra fluviatilis have been labelled with horseradish peroxidase (HRP) in order to compare the location and morphology of trochlear motoneurons with those of other vertebrates and to gain insight into the phylogenetic changes of the trochlear system. About 126 bipolar and tripolar trochlear motoneuron perikarya are found in a dorsal tegmental position close to the trochlear root. Only 16% of the labelled cells are on the ipsilateral side of the brain, i.e. they lie predominantly contralateral as in gnathostome vertebrates. Dorsally directed dendrites reach the area of lateral-line and retinofugal fibres, and may establish functional contacts. In addition, each motoneuron has a ventral dendrite that extends towards the fasciculus longitudinalis medialis and to the ventral tegmentum. The dendrites branch close to the oculomotor root. Lampreys show a low muscle fibre to motoneuron ratio (4.51), i.e., they resemble amniotic vertebrates more than other anamniotic vertebrates. These data demonstrate both closer resemblance and larger differences of cyclostome and gnathostome trochlear motoneurons than previously suggested.     

Monoclonal antibody to rat uterine peroxidase and its use in identification of the peroxidase as being of eosinophil origin

Peroxidase was purified from uteri of estrogen-treated rats by calcium chloride extraction, affinity chromatography on concanavalin A-Sepharose and hydrophobic interaction chromatography on phenyl-Sepharose. An overall purification of greater than 1700-fold was achieved with a final recovery of 27%. Monoclonal antibodies to peroxidase were subsequently prepared by immunization of male C57BL/10J mice with the highly purified peroxidase from rat uterus. Spleen and lymph node cells from the mice were fused with Sp2/0-Ag 14 mouse myeloma cells. The resultant hybrid cells were screened for production of antibody using a solid-phase, double antibody radioimmunoassay. The mature rat spleen, shown previously to be abundant in eosinophils, contains high peroxidase activity. Spleen peroxidase purified by the same procedure as the uterine enzyme cross-reacted with a monoclonal antibody, designated IgG-107B, used in all subsequent studies. Peroxidase extracted from isolated rat eosinophils also cross-reacted with the antibody and yielded identical titers as the spleen and uterine peroxidases. Spleen, uterine and horse eosinophil peroxidase had the same apparent molecular weight, 57 000, as determined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. Following electrophoretic transfer to nitrocellulose, spleen, uterine and eosinophil peroxidase reacted with monoclonal antibody, using an immunoblotting technique. These results provide biochemical and immunological evidence that the majority of the calcium chloride-extractable peroxidase activity from the uteri of estrogen-treated rats is derived from infiltrating eosinophils.     

The role of Arginine 38 in horseradish peroxidase enzyme revisited: A computational investigation

Molecular dynamics simulations on hydrogen peroxide complex with wild-type (WT) and Arg38Leu mutated (R38L) Horseradish Peroxidase (HRP) were carried out over nanoseconds timescale in water solution at 300 K. Comparison of the results provides interesting insights about the role of highly conserved Arg38 and His42 residues in the chemical features of HRP, underlying its biological activity which initiates with Compound0 (Cpd0). In the WT-HRP enzyme current molecular dynamics simulations show, for the first time, that Arg38 residue: i) prevents the entrance of water inside the reaction cavity, hence providing a hydrophobic reactive scenario, ii) it maintains the distance between His42 and heme–H₂O₂ complex suitable for the occurrence of proton transfer reaction leading, thereafter, to heme–H₂O₂ disruption according to Poulos-Kraut mechanism. On the other hand, R38L mutant can be considered as a “wet enzyme” where the presence of water solvent molecules in the heme reaction pocket, unfavoring the initial heme–H₂O₂ complex formation, decreases the catalytic efficiency in agreement with experimental kinetics measurements. Furthermore, we note that Arg38Leu mutation pushes the His42 residue far from the heme–H₂O₂ complex, making unlikely a direct proton transfer and suggesting that, in the mutant, a solvent water molecule could be involved in the first step of the Poulos-Kraut mechanism.     

The paraventriculo-infundibular corticotropin releasing factor (CRF) pathway as revealed by immunocytochemistry in long-term hypophysectomized or adrenalectomized rats

The immunocytochemical localization of corticotropin releasing factor (CRF)-containing pathways projecting from the paraventricular nucleus (PVN) to the external layer of the median eminence (ME) in long-term hypophysectomized or adrenalectomized rats is described. Immunocytochemistry was followed by silver intensification of the diaminobenzidine end-product. In comparison with untreated control rats, both hypophysectomy and adrenalectomy resulted in a dramatic increase in immunostaining of the CRF-containing perikarya and fibers, particularly those originating from the PVN and terminating in the ME. The staining was more intense in adrenalectomized than in hypophysectomized rats. The CRF-positive fibers emerging from the PVN form a medial, an intermediate and a lateral fiber pathway. The lateral and intermediate CRF tracts leave the dorsolateral part of the PVN and course laterally and medially of the fornix, respectively, then ventrally toward the optic tract. Just dorsal to the optic tract they turn in caudal direction and run parallel with and very close to the basal surface of the hypothalamus; individual fibers then turn medially to terminate in the external layer of the ME. Only a few fibers originate from the medial-ventral part of the PVN (medial pathway). These fibers run in ventral direction along the walls of the 3rd ventricle and terminate in the ME. Thus the majority of CRF fibers, similarly to other peptidergic systems, reach the medial basal hypothalamus from the anterolateral direction.     

Food items of the grey mullet Mugil cephalus in the Banc d'Arguin area (Mauritania)

The stomach and gut content of 22 Mugil cephalus from the Banc d'Arguin shallows (Mauritanian coast, West Africa) is examined. Quartz grains are the predominant material found in the stomach together with small portions of benthic diatoms and flakes, aggregates of fine-grained inorganic and organic particles. As the composition of this material is modified by a sorting procedure during feeding it is difficult to conclude, whether seagrass stands (muddy and mixed sediments with sand fraction) or sandy flats are preferred as feeding habitats. The animals studied did not or not relevantly utilize the non-diatom microphytes, though the environment of the Banc d'Arguin is rich in microbial mats (cyanobacteria), Vaucheria beds and epiphytic vegetation of seagrass leaves.     

Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing

The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo-and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (alpha-, beta-, gamma-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0----S1 (0----0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by approximately 20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only beta-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0----0 transition energy for the tautomeric forms of MP and an increase in the 0----0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the delta v = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for beta-RE. In the same energy range, however, the transition into the delta nu = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA less than NHA less than HQ less than RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0----0 energies (from 60-70 to approximately 30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.     

Perifusion system: its use in the study of the neuroendocrine control of human pituitary tumoral cells

Conclusion Taken together, these results show the usefulness of the perifusion technique both in the studyof hormone regulation and in the physiopathology of the human pituitary. It allows the studyof dynamic changes in hormone release, relationships between in vivo and in vitro responses, relationships between hormone response and receptor status. Furthermore, we could use this approach to demonstrate release of pituitary neuropeptides and the relation between secretoryprofiles of neuropeptides and those of pituitary hormones. It is another approach to all thesedifferent points than long term culture that needs enzymatic dispersion, and several days ofrecovery before any experiment can be performed on the cells.     

One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (<Emphasis Type="Italic">Armoracia rusticana</Emphasis>) treated with Tb(III)

One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (Armoracia rusticana) treated with Tb(III) was investigated using some biophysical and biochemical methods. Firstly, it was found that a large amount of Tb(III) can be distributed on the cell wall, that some Tb(III) can enter into the horseradish cell, indicating that peroxidase was mainly distributed on cell wall, and thus that Tb(III) would interact with horseradish peroxidase (HRP) in the plant. In addition, peroxidase bioactivity was decreased in the presence of Tb(III). Secondly, a new peroxidase-containing Tb(III) complex (Tb–HRP) was obtained from horseradish after treatment with Tb(III); the molecular mass of Tb–HRP is near 44 kDa and the pI is about 8.80. Thirdly, the electrocatalytic activity of Tb–HRP is much lower than that of HRP obtained from horseradish without treatment with Tb(III). The decrease in the activity of Tb–HRP is due to the destruction (unfolding) of the conformation in Tb–HRP. The planarity of the heme active center in the Tb–HRP molecule was increased and the extent of exposure of Fe(III) in heme was decreased, leading to inhibition of the electron transfer. The microstructure change in Tb–HRP might be the result of the inhibition effect of Tb(III) on peroxidase activity in horseradish.     

The oxidation of oxytocin and vasopressin by peroxidase/H2O2 system

Summary Oxytocin and vasopressin are oxidized by horseradish peroxidase and by lactoperoxidase, in the presence of hydrogen peroxide. Spectrophotometric measurements are indicative of the formation of dityrosine. Kinetic parameters indicate that the affinity of horseradish peroxidase is slightly higher for oxytocin with respect to vasopressin and that the two hormones are better substrates for both peroxidases than free tyrosine.