Permeability of submandibular salivary glands in dogs to blood-borne horseradish peroxidase (HRP)

Summary Horseradish peroxidase (HRP) was administered to the submandibular glands of dogs by close-arterial bolus-type injections, and its localisation was examined histochemically by light and electron microscopy. The HRP became widespread in the interstices of the glands and reached many central acinar lumina via scattered localised parts of their tight junctional complexes. Reaction product was less often found in the lumina of demilunes, which suggested that the intercellular junctions there were less leaky. HRP was often found in sizeable spaces between myoepithelial cells and the underlying parenchymal cells; such large spaces have not been observed in this situation in other species. The possibility that permeability pathways may arise intermittently at different sites in the adhering mechanisms between the acinar cells is discussed.It is concluded that potential paracellular permeability pathways for macromolecules exist in these glands and, if the concentration gradient is sufficiently high, molecules even as large as those of HRP can to some extent permeate passively from the interstices to the saliva. In resting glands the principal permeability site is between the central acinar cells.Supported by Grants from the M.R.C. and the V.R.T. King's College HospitalWe wish to acknowledge the technical help of Mr. K.J. Davies and Mr. P.S.A. Rowley     

HRP在模拟人体胃肠环境中的稳定性研究

以中国药典中的人工胃液和人工肠液模拟人体胃肠环境,对过辣根氧化物酶的稳定性进行了一系列单独及综合研究。结果表明,辣根过氧化物酶在人工胃液中受到很强的破坏,20min后,其活性仅存1.1%,主要由胃酸破坏,而在人工肠液中则较为稳定,1.5h后,其活性仍存在89%,同时还发现,天然结构的辣根过氧化物酶对胰蛋白酶具有较强的耐受力。     

Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig

Glial cells of the myenteric plexus from guinea pig small intestine were intracellulary filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid, somata (85±0.7 m equivalent diameter, i.e., about 330 m³ volume), and send up to 20 thin and short processes (less than 26 to about 110 m in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly, branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of holes in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of 2000 m², almost 90% of which are contributed by the cell processes. This geometry may enable an intense exchange of metabolites and ions between neurons, glial cells, and the capillaries and/or environment of enteric ganglia.     

Mandibular movement patterns relative to food types in common tree shrews (Tupaia glis)

Although common tree shrews have long been considered a model system for early eutherian mastication, little information on mandibular movement patterns relative to specific food types has been reported. Detailed analysis of mandibular movement patterns when related to resulting attrition facets may permit more accurate extrapolations regarding the dietary habits of primitive mammals. Marker beads were sewn to chins of five animals that were placed in a restraint system and filmed while they fully masticated mealworm larvae and standardized pieces of banana, almond, and commercial cat chow. These sequences were divided into early, middle, and late thirds of food reduction. Mandibular positions from both frontal and lateral perspectives were digitized frame by frame to yield plots of orbits in three dimensions as well as graphic display of displacements, velocities, and accelerations. Plot coordinates were averaged to generate composite orbital shapes. Significant (p < 0.01) findings included: (1) shortest orbital durations and greatest peak closing velocities and accelerations in early third of reduction; (2) smallest maximum gape, smallest maximum lateral excursion from midline, and longest duration of powerstroke relative to orbital duration in late third of reduction; (3) shortest orbital durations and smallest maximum gape during mastication of chow; (4) greatest maximum lateral excursion during mastication of mealworm larvae; and (5) smallest peak closing accelerations during mastication of banana. Significant differences were also found among subjects for all parameters examined. Capacity for complex jaw movement may have been critical for allowing primitive molars to be used for trituration of a variety of food types, and may have preceded evolution of more specialized molar forms.     

Access of horseradish peroxidase (HRP) to the extracellular spaces of the maturation zone of the rat incisor enamel organ

Adult rats received a single dose of HRP intravenously and were killed from 10 min to 6 hr after injection. Following fixation with glutaraldehyde, the enamel organs were treated with a Graham-Karnovsky-type procedure for peroxidase activity, post-osmicated, and embedded in plastic. Sections were studied with light and electron microscopes. Ten minutes after injection, reaction product was found in all extra-cellular spaces of the enamel organ, at the enamel-ameloblast interface over smooth-ended and intermediate ameloblasts, and in apical surface invaginations and vesicles of the latter cell types. The enamel-ameloblast interface over the ruffle-ended aemlo-blasts and the extracellular spaces within the ruffled border were free of reaction product and remained so for up to 6 hr. The apical terminal bars of the ruffle-ended ameloblasts functioned as a barrier to HRP. The basal terminal bars of the smooth-ended ameloblasts likewise seemed to prevent the passage of the HRP. Possibly, HRP flows in a lateral direction from groups of ruffle-ended into groups of smooth-ended ameloblasts. Between 10 min and 6 hr, HRP was cleared more rapidly from the extra-cellular spaces of the papillary layer than from those of the ameloblast layer, and there was little backflow of tracer from the ameloblast into the papillary layer. Eventually, tracer was cleared also from the extracellular spaces of the ameloblast layer, probably mainly through micropinocytosis by the ameloblasts. A working model is proposed regarding the handling of large molecules by the enamel organ in the maturation zone.     

Distribution of horseradish peroxidase (HRP)-anti-HRP immune complexes in mouse spleen with special reference to follicular dendritic cells

The distribution of immune complexes has been studied in mouse spleen stimulated to contain many germinal centers (GC's). Horseradish peroxidase (HRP)-anti-HRP complexes were used as an appropriately precise and sensitive model. We were primarily interested in the relative abilities of three cell types to interact with complexes: lymphocytes, macrophages, and follicular dendritic cells (FDC's). The latter are distinctive, nonendocytic, stellate cells located primarily at the transition of mantle and GC zones of 2 degrees lymphoid follicles (Chen, L. L., J. C. Adams, and R. M. Steinman, 1978, J. Cell Biol. 77:148). Binding of immune complexes to lymphocytes could not be visualized in situ. Macrophages avidly interiorized complexes into lysosomes, but did not retain them extracellularly. In contrast, FDC's could retain HRP-anti-HRP extracellularly under appropriate conditions, but did not endocytose them. Cytochemical reactivity accumulated progressively on FDC's 1--6 h after administration of complexes i.v., remained stable in amount and location for 1 day, and then was progressively lost over a 1- to 5-day period. Several variables in the association of complexes with macrophages and FDC's were pursued. Only 1 microgram of complexed HRP had to be administered to visualize binding to both cell types. Macrophages interiorized complexes formed in a wide range of HRP/anti-HRP ratios, while FDC's associated with complexes formed in HRP excess only. Quantitative studies with [125I]HRP-anti-HRP demonstrated that 20% of the splenic load of HRP associated with FDC's. Complexes formed with an F(ab')2 anti-HRP were distributed primarily in macrophages. When the levels of the third component of serum complement were depleted by prior treatment with cobra venom factor, uptake of complexes by macrophages was reduced some 50% whereas association with FDC's was abolished. The fact that antigen excess complexes are retained extracellularly strengthens the idea that they are immunogenic. Finally, the association of complexes with FDC's seems to retard the entry of antigen into the GC proper.     

Comparison of accessory olfactory bulb volumes in the common tree shrew (Tupaia glis)

The volume size of the accessory olfactory bulb (AOB) of 40 common tree shrews (Tupaia glis) was compared with regard to differences between left and right sides, males and females, and animals born in the wild and those born and raised in captivity. There were no statistically significant differences between the two sides and the two sexes, but a significant reduction of the AOB from wild to captive animals was apparent. This reduction was more pronounced in females than in males and somewhat more pronounced in the inner granular layer (layer 6) than in the other measured components (layers 1 + 2, layers 3-5). No well-founded explanation for this reduction could be given.     

Afferent projections from the brainstem to the area hypothalamica dorsalis: a horseradish peroxidase study in the cat

Experiments using the retrograde transport of horseradish peroxidase were performed in order to identify the cells of origin the ascending projections from different brainstem regions to the area hypothalamica dorsalis (aHd) in the cat. The afferent inputs to this area originate mainly from the midbrain and medulla oblongata regions. The main afferent source of the area hypothalamica dorsalis arises from the substantia grisea centralis, where a large number of labeled cells were observed bilaterally, although more abundant on the ipsilateral side. Substantial afferents reach the aHd from the nuclei vestibularis medialis and inferior and the formatio reticularis mesencephali. A modest number of peroxidase-labeled neurons were observed in the nuclei ruber, interpeduncularis, substantia nigra, reticularis gigantocellularis, vestibularis lateralis, cuneatus and gracilis. From the pons, the nucleus raphe magnus sends a weak projection to the aHd. These anatomical data suggest that such area could be involved in visceral, sexual, nociceptive somatosensorial, sleep-waking and motor mechanisms.     

The time-course of appearance and net accumulation of horseradish peroxidase (HRP) presented orally to rainbow trout Salmo gairdneri (Richardson)

1. A sensitive enzyme-linked immunosorbent assay (ELISA) technique was used in order to determine horseradish peroxidase (HRP) uptake from the rainbow trout gut. 2. HRP was detected in blood plasma and various tissues within 15 min of oral intubation. 3. The time-course of net accumulation (uptake-degradation) over a 75 min period was recorded. 4. The presence of HRP reached a maximum in the body tissues approximately 45 min after intubation and on a ng/g weight basis the order of accumulation within the tissues was liver greater than spleen = kidney greater than plasma greater than heart. 5. The total organ accumulation (net) was in the order liver greater than plasma greater than kidney greater than spleen greater than heart.     

A perfusion-fixation procedure for the concurrent demonstration of Timm's, horseradish peroxidase (HRP), and acethycholinesterase (AChE) histochemistry

The sulfide-silver method of Timm has been a widely used histochemical technique to demonstrate the presence of heavy metals in biological tissue, particularly in the central nervous system. However, the use of this method or its several modifications results in less than optimal morphological preservation and requires embedding the tissue in paraffin or freezing it and cutting it directly onto slides with a cryostat. These procedures can decrease the sensitivity and limit the application of other histochemical procedures, particularly when experiments necessitate processing large specimens or reaction procedures require techniques using free-floating sections. A perfusion-fixation protocol is described that yields sufficient fixation to cut whole frozen blocks of tissue with a sliding microtome, permits the use of free-floating sections, and allows the concurrent demonstration of horseradish peroxidase and acetylcholinesterase histochemistry without loss of sensitivity. The method consists of a short initial exposure to a sodium sulfide solution followed by a prolonged exposure to a combined sulfide-aldehyde fixative solution.     

The time-course of appearance and net accumulation of horseradish peroxidase (HRP) presented orally to juvenile carp Cyprinus carpio (L.)

A sensitive enzyme-linked immunosorbent assay (ELISA) technique was used in order to determine horseradish peroxidase (HRP) uptake from the carp gut. HRP was detected in blood plasma and various tissues within 15 min of oral intubation. The time-course of net accumulation (uptake-degradation) over a 2 hr period was recorded. The presence of HRP reached a maximum in the body tissues approximately 1 hr after intubation and on a microgram/g wet weight basis the order of accumulation within the tissues was spleen greater than kidney greater than liver. The total organ accumulation (net) was in the order liver greater than kidney greater than spleen.     

Microvasculature of the thyroid gland in the common tree shrew (Tupaia glis): microvascular corrosion cast/scanning electron microscopy study.

A thyroid vascular cast of the common tree shrew (Tupaia glis) was obtained by injection of Batson's No. 17 plastic mixture into the ascending aorta. The cast was studied under the scanning electron microscope. It was found that each half of the gland is supplied by a large superior and a rather small inferior thyroid artery. After plunging into the gland, the arteries divide into smaller branches that are the interlobular, intralobular and follicular arteries (afferent vessels). The basket-like capillaries arising from the follicular arteries and encapsulating thyroid follicles are of large diameter and are arranged in a single layer. The follicular side of the capillary casts was observed to contain numerous small and some large projecting knobs compatible with the presence of fenestrations in the endothelial cells. On the other hand, endothelial nuclear imprints were found mainly on the stromal surface of the follicular capillary casts. Transfollicular capillaries connecting the adjacent follicular capillary networks were also observed. Blood from the follicular capillaries either drains into the follicular veins (efferent vessels) or abruptly drains into the intralobular veins before proceeding to intralobular and interlobular veins, respectively. The interlobular veins are collected into a few small superior, a few larger middle and a few even larger inferior thyroid veins. These veins drain directly into the laryngeal vein lying adjacent to the deep surface of the thyroid gland before joining the jugular vein. Venous valves were identified outside the thyroid gland. In addition, the glomerular capillary island of the parathyroid gland was often seen at the cranioanterolateral and sometimes at the cranioposterolateral aspect of the thyroid gland.     

Splenic primary sensory afferents in the guinea pig demonstrated with anterogradely transported wheat-germ agglutinin conjugated to horseradish peroxidase

Summary The distribution of primary visceral afferents to the spleen of the guinea pig was studied after injections of wheat-germ agglutinin conjugated to horseradish peroxidase (HRP) into the left dorsal root ganglia at levels T7–T12. After anterograde transport of the tracer, labeled fibers were found in the nerves around the splenic artery in the hilus region and in the splenic parenchyma. The majority of labeled fibers in the spleen were detected in the white pulp. HRP-positive fibers were also observed in the red pulp and in the trabeculae. The distribution of the HRP-labeled fibers was in part similar to those of substance P-immunoreactive and calcitonin gene-related peptide-immunoreactive nerve structures. The results show that the anterograde tracing technique can be used successfully to investigate splenic primary afferent innervation.     

Binding of thiocyanate and cyanide to manganese(III)-reconstituted horseradish peroxidase: a 15N nuclear magnetic resonance study

Binding of thiocyanate and cyanide ions to Mn(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by relaxation rate measurements (at 50.68 MHz) of 15N resonance of SC15N- and C15N-. At pH = 4.0 the apparent dissociation constant (KD) for thiocyanate and cyanide binding to Mn(III)HRP was deduced to be 156 and 42 mM, respectively. The pH dependence of the 15N line width as well as apparent dissociation constant for thiocyanate and cyanide binding were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate and cyanide in deprotonated form bind to the enzyme in a protonated form. The binding of thiocyanate and cyanide to Mn(III)HRP was found to be facilitated by protonation of an ionizable group on the enzyme [Mn(III)HRP] with a pKa = 4.0. From competitive binding studies it was shown that iodide, thiocyanate and cyanide bind to Mn(III)HRP at the same site; however, the binding site for resorcinol is different. The apparent dissociation constant for iodide binding deduced from competitive binding studies was found to be 117 mM, which agrees very well with the iodide binding to ferric HRP. The binding of thiocyanate and cyanide was shown to be away from the metal center and the distance of the 15N of thiocyanate and cyanide from the paramagnetic manganese ion in Mn(III)HRP was found to be 6.9 and 6.6 A, respectively. Except for cyanide binding, these observations parallel with the iodide and thiocyanate ion binding to native Fe(III)HRP. Water proton relaxivity measurements showed the presence of a coordinated water molecule to Mn(III)HRP with the distance of Mn-H2O being calculated to be 2.6 A. The slow reactivity of H2O2 towards Mn(III)HRP could be attributed to the presence of water at the sixth coordination position of the manganese ion.     

Localization of horseradish peroxidase (HRP)-anti-HRP complexes in cryostat sections: Influence of endotoxin on trapping of immune complexes in the spleen of the rat

Substance P-like immunoreactivity (SPLI) was studied by immunocytochemistry and radioimmunoassay in the cerebral arteries, choroid plexus and dura mater of the guinea-pig, rabbit, cat and man. The highest concentrations were found in cerebral blood vessels: 6.1 +/- 2.3 pmol/g (guinea-pig), 9.0 +/- 1.1 pmol/g (rabbit), 7.1 +/- 0.4 pmol/g (cat), and 2.4 +/- 0.9 pmol/g (man). Lower levels were obtained in the choroid plexus and dura mater. The distribution of substance P (SP)-immunoreactive nerve fibres found in various regions of the guinea-pig correlated well with the amount of SPLI measured. Sympathectomy did not alter the concentration of SPLI in the dura mater or in cerebral blood vessels. Electrical field stimulation or 124 mM potassium enhanced the spontaneous efflux of SPLI by 10 and 20%, respectively, from superfused pial arteries in vitro. These data are in support of a functional role of perivascular SP within the cranial circulation.     

Selective illumination of single photoreceptors in the house fly retina: local membrane turnover and uptake of extracellular horseradish peroxidase (HRP) and Lucifer Yellow

Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.