Ultrastructural localization of the iodination centre in the endostyle of the adult amphioxus (Branchiostoma lanceolatum)

Summary The site of iodination in the endostyle of the adult amphioxus was examined by light-and electron-microscopic autoradiography. In accordance with previous studies, light-microscopic autoradiography showed a distinct accumulation of autoradiographic grains at the apical end of epithelial cells in the lateral part of the endostyle. In the electron microscope two distinct cellular zones were identified in an approximate position of the light-microscopic zone 5. Zone 5a, not previously recognized, was adjacent to zone 4 and consisted of six to nine rows of cells free of characteristic granules. Cells in zone 5b contained large mucous granules and had, in previous ultrastructural studies, been identified as belonging to the typical zone 5. Four or less incomplete rows of granule-containing cells, not observed in previous studies, marked the border between zones 5b and 6. After incubation in ¹²⁵I for 5 min, electron-microscopic autoradiography showed a selective concentration of label to zone 5a, which, thus, corresponds to the iodination centre seen in the light microscope. The grains were associated with cilia and microvilli in the lumen. After longer incubation times (30, 60, 90 min) grains were still concentrated at the surface of zone 5a but were also associated with the surface of zones 5b and 6. Grains were also located over the cytoplasm of all three zones. They were associated with vesicles and lysosome-like structures, suggesting secondary uptake of labelled products by endocytosis. Methimazole, an inhibitor of peroxidase, abolished the autoradiographic reaction. In conclusion, the site of iodination in the endostyle of amphioxus is located in zone 5a, which has not previously been ultrastructurally defined. Iodination in the endostyle is an extracellular process, but secondary uptake by endocytosis appears to occur.     

Fine-grained secretory cells in the intestine of the lancelet,Branchiostoma (Amphioxus) lanceolatum,studied by light microscopy

Summary In the posterior part of the mid-gut epithelium in the lancelet, Branchiostoma lanceolatum, fine-grained cells occur. The appearance of the these cells is conspicuous with a basal and an apical swelling with small secretory granules. At the end of the secretion cycle the granular content is released into the gut lumen. The secretion product seems to consist of proteins and probably has an enzyme function. The restricted localization, the fine granulation, and the characteristic shape are features, that make these cells distinguishable from other secretory cells in the lancelet intestinal epithelium. A possible endocrine capacity of the cells is discussed.This work was supported by grant 2124-23 from the Swedish Natural Science Research Council and by grants from the Faculty of Science, University of Stockholm, Sweden.     

The fine structure of lamellate cells in the brain of amphioxus (Branchiostoma lanceolatum,Cephalochordata)

Summary The lamellate cells of amphioxus have round nuclei, and cytoplasm with many mitochondria and a large amount of glycogen. Each of these cells projects a highly modified, branched cilium into the central canal, where it characteristically forms lamellar structures. Primary branches and secondary lamellae often contain accessory microtubules that are not derived from the axonema. The functional and evolutionary significance of this cell type is discussed in relation to the ciliary photoreceptors found in other chordates.This work is dedicated to Professor A. Carrato, Universidad Complutense, on the occasion of his 80th birthday     

The connection between spinal cord and notochord in Amphioxus (Branchiostoma lanceolatum)

Summary Silver impregnation of nerves, histochemical reactions for acetylcholinesterase, and electron microscopy reveal an efferent innervation of the notochord in amphioxus. Extensions of the notochordal lamellae end in groups (the notochordal horns) just below the ventro-lateral surface of the spinal cord where they are opposed to large nerve terminals originating as short collaterals of axons running longitudinally in the nerve cord. This neurochordal junction resembles an ordinary neuro-muscular junction in several respects and is interpreted as a part of the muscular system found in the notochord itself. Acknowledgement. The author is indebted to the staffs at the Marine laboratory in Plymouth and the Biological station at Helgoland for supply of material. The expert advices and criticism of Q. Bone, Ph. D., Plymouth, and Dr. U. Welsch, Kiel, are also greatly appreciated.     

The infundibular organ of the lancelet (Branchiostoma lanceolatum,Acrania): an immunocytochemical study

Reissner's fibers are secretions produced by different ependymal areas of the chordate brain, viz., in adult vertebrates, by the dorsal subcommissural organ, and in all stages of cephalochordates (Branchiostoma lancelets), by the ventral infundibular organ. Fibers produced by these different organs are seemingly identical and the two fiber sources also share some immunocytochemical and lectin-binding properties. The secretions in these two glands are, however, not identical; the infundibular organ cells are strongly reactive with antibodies against vertebrate Reissner's fibers, but they do not react with antibodies raised against the source of the vertebrate fibers, viz., the subcommissural organ. The results support the possibility that, in adult vertebrates, the Reissner's fibers are composed of material not only from the subcommissural organ, but also from another, not yet identified, source that is identical or equivalent to the infundibular organ of the lancelet. There are indications that the infundibular organ is immunocytochemically closely akin to some secretory cells in the vertebrate embryonic brain and also to those that produce the juvenile vertebrate Reissner's fibers, viz., secretory cells in the flexural organ.     

Immunohistochemical investigation of neuropeptides in the central nervous system of the amphioxus,Branchiostoma belcheri

The immunohistochemical localization of nine different neuropeptides was studied in the central nervous system of the amphioxus, Branchiostoma belcheri. In the brain, perikarya immunoreactive for urotensin I and FMRFamide were localized in the vicinity of the central canal. One of the processes of each of these perikarya was found to cross the dorso ventral slit-like lumen of the central canal. Oxytocin-immunoreactive short fibers, but not perikarya, were detected in the ventral part of the brain. Perikarya immunoreactive for arginine vasopressin/vasotocin, oxytocin and FMRFamide were widely distributed in the spinal cord. Arginine vasopressin/vasotocin-immunoreactive fibers often made contacts with Rohde cell axons. Angiotensin II-immunoreactive perikarya were observed in the posterior half of the spinal cord, and urotensin I-immunoreactive perikarya were found in the caudal region of the spinal cord. Cholecystokinin/gastrin-immunoreactive fibers, but not perikarya, were detected in the spinal cord; some extended as far as the ependymal layer of the cerebral ventricle. No colocalization of the peptides examined was observed. No immunoreactivity for atrial and brain natriuretic peptides nor for urotensin II was detected. The present study indicates that there are at least six separate neuronal systems that contain different peptides, respectively, in the central nervous system of the amphioxus. Their functions remain to be determined.Part of this investigation has previously been presented in abstract form (Uemura et al. 1989)     

Occurrence of different secretin-like cells in the digestive tract of the ascidian Styela plicata (Urochordata,Ascidiacea)

Summary Secretin-like cells have been detected in the digestive tract of the ascidian Styela plicata by means of immunofluorescent and immunocytochemical methods.Especially, in the esophageal epithelium there are immunoreactive cells (S₂) in which a biogenic amine (5-HT) and a regulatory peptide (secretin) occur together. In the gastric epithelium only secretin-like cells (S₁) are present.Tests of cross-reactivity performed with glucagon, GIF and VIP, have confirmed the presence of a secretin-like molecule only in the S₁ and S₂ cells.     

Immunohistochemical localization of chromogranin A and B in endocrine cells of the alimentary tract of the adult lizard Podarcis sicula

The distribution, argyrophilia, and the possible amine/peptide co-localizations in endocrine cells immunoreactive (IR) to antisera against chromogranin A (CgA) and chromogranin B (CgB) in the alimentary tract of the lizard Podarcis sicula have been investigated using novel monoclonal antibodies. Many CgA-IR and CgB-IR cells were found in the tract, except in the distal small intestine. Almost all chromogranin-IR cells (Cgs-IR) were also argyrophilic with parallel intensity. Some CgA-IR and CgB-IR cells did not display co-localized amines or peptides. CgA or CgB or both were found co-localized, with some local differences, in almost all serotonin-IR, histamine-IR, substance P-IR and gastric peptide tyrosine tyrosine (PYY)-IR cells. Moreover, both Cgs were co-localized only in some somatostatin-IR cells, whereas neurotensin-IR, gastrin/cholecystokinin-IR, pancreatic polypeptide-IR and intestinal PYY-IR cells did not show any co-localization with Cgs. The presence of Cgs in the endocrine cells was heterogeneous with regard to the complex interrelationship with their amine/peptide content. Consequently, Cgs cannot be considered as universal markers of all endocrine cell types.     

Immunohistochemical detection of adenohypophyseal cells containing hormones in Rana ridibunda

Summary The pars distalis of the pituitary of Rana ridibunda captured throughout the spring and summer was examined with immunofluorescence techniques using antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, four different cell types are recognized: 1) cells immunoreactive to anti-bovine LTH, 2) cells immunoreactive to anti-bovine STH, 3) cells immunoreactive to anti-ovine LH, and 4) cells immunoreactive to anti-synthetic ACTH (1–24). Their distribution and morphology as well as their staining characteristics (classical histological techniques) are reported in this study.     

Immunohistochemical localization of chromogranin A and B in the endocrine cells of the alimentary tract of the green frog,Rana esculenta

Novel monoclonal antibodies to human chromogranin A (CgA) and chromogranin B (CgB) were used to investigate the presence of immunoreactive (-IR) elements in the alimentary tract of the green frog Rana esculenta. Numerous CgA-IR and a few CgB-IR endocrine cells were found within the gut mucosa, from the oesophagus to the cloaca, with some local differences in density. Co-localization studies demonstrated that they were costored in almost all the serotonin-IR, the amylin-IR or islet amyloid polypeptide-IR cells and in the peptide tyrosine tyrosine-IR cells located proximal to the pylorus, but not in those located in more caudal tracts. No other co-localization was demonstrated; substances investigated included somatostatin, substance P, gastrin/cholecystokin, glucagon, glycentin, bombesin, secretin and neurotensin. CgA-IR and CgB-IR cells nearly always displayed argyrophilia with the Grimelius silver method     

Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 degrees C to 35 degrees C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.     

Immunocytochemical localization of hypocalcin in the endocrine cells of the corpuscles of Stannius in three teleost species (trout,flounder and goldfish)

Summary In order to identify the cell-type responsible for the production of hypocalcin (the recently isolated hypocalcemic hormone of teleost fish), the corpuscles of Stannius (CS) of trout, flounder and goldfish, were immunocytochemically stained with antisera raised against trout hypocalcin. The secretory granules of the type-1 cells of the CS, considered to be the hypocalcin-producing cells, showed intense immunoreactivity in all species examined. However, in trout and flounder, the secretory granules produced by the type-2 cells, which have been suggested to represent a functionally different cell-type, also showed an intense immunoreactivity. In goldfish, no type-2 cells were observed. We tentatively conclude that type-1 and type-2 cells represent structurally different forms of the same functional cell-type.     

Microflora and internal parasites of the digestive tract of Eurasian griffon vultures (Gyps fulvus) in Croatia

Faecal and vomit samples were collected from 12 griffon vultures (Gyps fulvus) in May 2002 and examined for bacteria, fungi and parasites. Bacteria present included Escherichia coli, Aeromonas salmonicidia, Proteus sp., Escherichia fergusonii, Providencia alcalifaciens, Micrococcus sp. and Streptococcus sp. Mycological examination revealed the presence of Candida sp., yeasts and in one case, Rhodotorula rubra. Strongyle eggs were found in several samples. In most faecal samples, ascarid eggs of Toxocara type were found. Tapeworm eggs of Moniezia type were present in one faecal sample. Both microbiologic and parasitic findings are most likely the result of the feeding habits of griffon vultures.     

Distribution patterns of neurotensin-like immunoreactive cells in the gastro-intestinal tract of higher vertebrates

Summary The endocrine system of the gastro-intestinal tract of selected species representing the five higher vertebrate classes was investigated with reference to occurrence and distribution of neurotensin-like immunoreactive cells. Using antibodies against C-terminal and N-terminal fragments of neurotensin and against the C-terminal sequence of xenopsin it was demonstrated that the intestine of all species studied contains endocrine, neurotensin-like immunoreactive cells. However, large differences in localization and frequency of these neurotensin-like immunoreactive cells were found. Except for a teleostean fish, neurotensin-like immunoreactive cells in the gastro-intestinal tract were more frequent in non-mammalian vertebrates than in mammals. In contrast to mammals, where the highest density of neurotensin-like immunoreactive cells was present in the ileal mucosa, in the non-mammalian vertebrates studied the corresponding cells were most abundant in the pyloric-duodenal junction. The exact mapping of neurotensin-like immunoreactive cells is presented throughout the entire gastro-intestinal tract of six species (Rattus, Coturnix, Lacerta, Rana, Xenopus, Carassius) including a quantitative evaluation of sequential serial sections.     

黑眉锦蛇消化道6种酶的组织化学定位

目的研究黑眉锦蛇消化道酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、过氧化物酶(POX)、非特异性酯酶(NSE)、腺苷三磷酸酶(ATPase)、琥珀酸脱氢酶(SDH)等酶的分布。方法消化道分8个部位取材,应用冰冻切片、石蜡切片、酶组织化学技术及光密度定量分析。结果 ACP主要分布于十二指肠至回肠的黏膜上皮,十二指肠和空肠酶活性显著较高(P<0.05);ALP分布于食管、十二指肠至回肠的黏膜上皮,十二指肠酶活性最高(P<0.05);POX和NSE在整个消化道黏膜上皮和黏膜固有层中均有分布,胃幽门和直肠酶活性较低(P<0.05);ATPase在消化道除直肠未检测到酶活性外,其它部位均有分布,以十二指肠酶活性最高(P<0.05);SDH除食管未检测到酶活性外,其它部位均有分布,胃中胃腺部酶活性较高(P<0.05)。结论黑眉锦蛇消化道黏膜酶的分布同其它动物有相似之处,也有其自身特点。不同酶的分布和消化道各部位的生理机能密切相关。     

The lysozyme locus in Drosophila melanogaster: an expanded gene family adapted for expression in the digestive tract

Lysozyme has been studied in insects as part of the system of inducible antibacterial defence in the haemolymph. We recently found two Drosophila lysozyme genes that are constitutively expressed in the digestive tract, and are probably involved in the digestion of bacteria in the food. To obtain an overview of the lysozyme genes in this species and their possible roles in immunity and digestion, we have now characterized all six lysozyme genes in the cloned part of the lysozyme locus at 61F, and a seventh gene that maps to the same chromosomal location. The expression of the genes follows four different patterns: firstly, four closely related genes, LysB, C, D and E, are all strongly expressed in the midgut of larvae and adults; secondly, LysP is expressed in the adult salivary gland; thirdly, LysS is expressed mainly in the gastric caecae of larvae; and finally, LysX is primarily expressed in the metamorphosing midgut of late larvae and early pupae. The LysD-like genes and LysS are strongly repressed in artificially infected animals, possibly reflecting a malaise reaction in the digestive tract. None of the genes is expressed in the fat body or haemocytes. Thus rather than being a component of the haemolymph, the Drosophila lysozymes are found mainly in the digestive tract where they are expressed at a high level. Furthermore all genes, except LysP, encode acidic proteins, in contrast to the strongly basic typical lysozymes. This is highly reminiscent of the situation in ruminants, where the lysozymes have been recruited for the digestion of symbiotic bacteria in the stomach.     

中华蟾蜍消化道6种酶的组织化学定位

目的研究中华蟾蜍消化道酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、腺苷三磷酸酶(ATPase)、非特异性酯酶(NSE)、过氧化物酶(POX)和琥珀酸脱氢酶(SDH)等6种酶的分布。方法在消化道的8个部位取材,采用冰冻切片技术、石蜡切片技术、酶的组织化学方法和光密度定量分析。结果 ACP主要分布于胃贲门中贲门腺部,十二指肠和回肠中酶反应呈弱阳性。ALP主要分布于食管、十二指肠至回肠的粘膜上皮,十二指肠酶活性最高。ATPase在消化道各部位均有分布,胃中胃腺部和回肠粘膜上皮酶活性显著较高(P0.05)。NSE和POX在整个消化道粘膜上皮和粘膜固有层均有分布,胃各部位酶活性显著较低(P0.05)。SDH除在食管和直肠酶活性显著较低外,其它部位均有大量分布,十二指肠和回肠酶活性显著较高(P0.05)。结论中华蟾蜍消化道粘膜6种酶的分布同其它动物有相似之处,也有其自身特点。6种酶在消化道中的分布与消化道各部位的生理机能密切相关。     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.