S100-like protein in the goldfish (Carassius auratus) kidney

The presence and distribution of S100-like protein in the goldfish (Carassius auratus L.) kidney has been studied by the use of immunohistochemical and histochemical methods. Simple immunohistochemistry (peroxidase anti-peroxidase method) was carried out with a polyclonal antibody against a mixture of both S100alpha and S100beta proteins. In order to confirm the cell-type containing S-100-like immunoreactivity, the colocalization of S-100-like protein immunoreactivity with periodic acid-Schiff (PAS) reaction was investigated by using double staining with indirect immunofluorescence and PAS histochemistry. S100-like immunoreactivity was detected only in juxtaglomerular cells located in the renal arterial branch and never on afferent arterioles. No immunoreactivity was observed in other tracts of the nephron or in the interstitial cells. Double staining confirmed that S-100-like immunoreactivity and PAS reactivity were colocalized in juxtaglomerular cells. These findings are the first regarding the presence and distribution of S100-like protein in the teleost kidney; they add a new member to the list of extra-neural S100-like-containing cell types and confirm that the antigen cannot be regarded as nervous-system-specific. In addition, a concentration of S100-like immunoreactivity in juxtaglomerular cells suggests the presence of S100-like calcium-binding protein-mediated activities in these cell types.     

Differential changes in calcitonin, somatostatin and gastrin/cholecystokinin-like immunoreactivities in rat thyroid parafollicular cells during ontogeny

Immunocytochemical quantitative studies on the development of rat thyroid calcitonin (C) cells have been performed. In neonatal rat pups up to day 6 nearly 90% of all calcitonin cells are also somatostatin immunoreactive and 45% of these cells also show immunoreactivity to a C-terminal gastrin/cholecystokinin antiserum (CCK-4-like immunoreactivity). Already at day 8 the frequency of somatostatin immunoreactive calcitonin cells has dropped to 25%, whereas half of the calcitonin cells still display CCK-4-like immunoreactivity. In adult rats, less than 1% of the calcitonin cells are somatostatin immunoreactive, whereas 90% of the calcitonin cells display CCK-4-like immunoreactivity. These data show that between day 6-8 a pronounced change in the peptide repertoire of rat thyroid C cells occur and that these cells, prior to this time, mainly contain calcitonin and somatostatin immunoreactivity and after this time mainly contain calcitonin and CCK-4-like immunoreactivity. The time course of the change in the C cell peptides is similar to that observed with changes in transitory peptides of neonatal rat pancreas and duodenum, suggesting that possible hormonal mechanisms during this period act to change the peptide repertoire of several endocrine cell types simultaneously. It is possible that many of the transitory peptides exert actions in the developing individual that are necessary for growth and differentiation. Interestingly, many of these transitory peptides reappear in tumours, where theoretically they could exert similar actions.     

Differential changes in calcitonin,somatostatin and gastrin/cholecystokinin-like immunoreactivities in rat thyroid parafollicular cells during ontogeny

Summary Immunocytochemical quantitative studies on the development of rat thyroid calcitonin (C) cells have been performed. In neonatal rat pups up to day 6 nearly 90% of all calcitonin cells are also somatostatin immunoreactive and 45% of these cells also show immunoreactivity to a C-terminal gastrin/cholecystokinin antiserum (CCK-4-like immunoreactivity). Already at day 8 the frequency of somatostatin immunoreactive calcitonin cells has dropped to 25%, whereas half of the calcitonin cells still display CCK-4-like immunoreactivity. In adult rats, less than 1% of the calcitonin cells are somatostatin immunoreactive, whereas 90% of the calcitonin cells display CCK-4-like immunoreactivity. These data show that between day 6–8 a pronounced change in the peptide repertoire of rat thyroid C cells occur and that these cells, prior to this time, mainly contain calcitonin and somatostatin immunoreactivity and after this time mainly contain calcitonin and CCK-4-like immunoreactivity. The time course of the change in the C cell peptides is similar to that observed with changes in transitory peptides of neonatal rat pancreas and duodenum, suggesting that possible hormonal mechanisms during this period act to change the peptide repertoire of several endocrine cell types simultaneously. It is possible that many of the transitory peptides exert actions in the developing individual that are necessary for growth and differentiation. interestingly, many of these transitory peptides reappear in tumours, where theoretically they could exert similar actions.     

Cysteamine and the endocrine pancreas: Immunocytochemical,immunochemical, and functional aspects

Summary To evaluate the previously reported depletion of pancreatic somatostatin by cysteamine (-mercaptoethylamine), mice were injected subcutaneously with the drug at 300 mg/kg. Immunocytochemical analysis performed on sections from tissue taken at 4 h after the injection revealed an elimination of somatostatin-14-like immunoreactivity without alterations in the somatostatin-28_{(1 – 12)}-like immunoreactivity. In sections from tissues taken at 24 h after injection, no differences between cysteamine-injected animals and controls were observed. Immunochemical analysis of somatostatin-14-like immunoreactivity in pancreatic extracts showed a significant reduction of the concentration (P< 0.001). In contrast, no change in the insulin concentration was observed. Functionally, cysteamine lowered the plasma glucose levels at l h after injection; this effect persisted for 6 h. Plasma insulin levels were likewise reduced transiently by cysteamine. Concomitant administration of somatostatin did not influence these effects of cysteamine. The plasma glucose-lowering effect of cysteamine was seen also in alloxan-diabetic mice. We conclude that cysteamine alters the immunoreactive characteristics of pancreatic somatostatin without affecting the immunoreactivity of insulin, and that cysteamine transiently reduces plasma glucose and insulin levels     

Protective effect of erythropoietin against ketamine-induced apoptosis in cultured rat cortical neurons: Involvement of PI3K/Akt and GSK-3 beta pathway

Erythropoietin (EPO) prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals in models of neurodegenerative diseases. Here we investigated the neuroprotective effect of EPO in ketamine-induced neurotoxicity in primary cortical neurons. EPO in combination with ketamine greatly increased the cell viability and reduced the number of TUNEL-positive cells. To elucidate a possible mechanism by which EPO exerts its neuroprotective effect, we investigated the phosphoinositide3-kinase pathway using LY294002. The neuroprotection of EPO was prevented by LY294002. Immunoblotting revealed that EPO induced the phosphorylation/activation of Akt and phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK-3β). Moreover, the caspase-3-like activity was increased by addition of ketamine, and decreased by administration of ketamine with EPO. Decreased caspase-3-like activity by administration of ketamine with EPO was restored by LY294002. Our results suggest that PI3K/Akt and GSK-3β pathway are involved in the neuroprotective effect of EPO. You Shang and Yan Wu have contributed equally to this work.     

Neurokinin A-like immunoreactivity in feline dental pulp: its distribution,origin and coexistence with substance P-like immunoreactivity

Summary The distribution and origin of neurokinin A (NKA)-like immunoreactivity were investigated in feline dental pulp by an indirect immunofluorescence method. NKA-containing nerve fibres with varicosities, which entered the dental pulp via apical foramen, were distributed throughout this tissue. Many NKA-containing nerve fibres were localized around blood vessels, but some were observed apart therefrom. At the odontoblastic layer, thin NKA-containing nerve fibres were observed running straight toward the pulp-predentinal border between odontoblasts. After inferior alveolar nerve section, all NKA-containing nerve fibres disappeared in the dental pulp, while the removal of the superior cervial ganglion resulted in no change in the distribution of these fibres. The correlation of NKA-like immunoreactivity and substance P (SP)-like immunoreactivity was also investigated by double-immunofluorescence technique. The distribution of NKA-containing nerve fibres was very similar to that of SP-containing nerve fibres; it appeared that all NKA-containing nerve fibres contained SP.     

Peptides related to insulin-like growth factor 1 in the gastro-entero-pancreatic system of bony and cartilaginous fish.

Evidence for the presence of peptides, related to insulin-like growth factor 1 (IGF-1) has been obtained in serum and various organs of representatives of osteichthyes and chondrichthyes, i.e., the bony fish Myoxocephalus (Cottus) scorpius and the cartilaginous fish Raja clavata. The peptides were identified by means of gel chromatography and an IGF-1 radioimmunoassay. IGF-1-like immunoreactivity was detected in three different apparent molecular mass forms, i.e., 17 kDa, 6 kDa and 4 kDa, the occurrence of which seemed to depend on the species. When the same antiserum was used immunohistochemically, IGF-1-like immunoreactivity was observed in endocrine cells of the open type in the intestinal mucosal epithelium. These cells exhibited distinct and species-specific distribution patterns. Endocrine cells of the pancreas as well as epithelial cells of the pancreatic duct also showed IGF-1-like immunoreactivity. Occasionally, IGF-1-like immunoreactivity was observed also in interstitial cells. The distribution patterns and densities of the IGF-like immunoreactive cells correlated with the results obtained by radioimmunoassay of the crude extracts. Absorption studies indicated that the IGF-1-like peptides observed differ from mammalian and submammalian insulins as well as from mammalian IGF-1.     

Co-localization of peptides in the Brockmann bodies of the cod (Gadus morhua) and the rainbow trout (Oncorhynchus mykiss)

Endocrine cells exhibiting immunoreactivity to FMRFamide-like, LPLRFamide-like, neuropeptide Y(NPY)-like and peptide YY(PYY)-like peptides were found in the periphery of the Brockmann bodies of the cod, Gadus morhua, and rainbow trout, Oncorhynchus mykiss. No immunoreactivity or very weak labelling was found with antisera to pancreatic polypeptide (PP). Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was found in nerve fibres, whereas labelling with VIP antiserum in endocrine cells disappeared after preincubation with nonimmune serum. There were always more immunoreactive cells in the rainbow trout than in the cod. No immunoreactivity could be seen with antisera to gastrin/cholecystokinin (CCK) or enkephalin. Double-labelling studies were performed to study the colocalization of the peptides in peripheral endocrine cells. Cells immunoreactive to NPY were also labelled with antisera to FMRFamide, LPLRFamide and PYY. The co-localization pattern of NPY varied; in some Brockmann bodies, a population of the immunoreactive cells showed co-localization and others contained NPY-like immunoreactivity only, whereas in other Brockmann bodies, all NPY-labelled cells also contained FMRFamide-like, LPLRFamide-like and PYY-like immunoreactivity. Cells immunoreactive to PYY similarly contained FMRFamide-like, LPLRFamide-like and NPY-like immunoreactivity, comparable to the patterns observed with NPY. Glucagon-like immunoreactivity was found at the periphery of the Brockmann bodies. A subpopulation of the glucagon-containing cells contained NPY-like immunoreactivity. PYY-like immunoreactivity was also found co-localized with glucagon-like immunoreactivity, as were FMRFamide-like and LPLRFamide-like immunoreactivity. Therefore, either NPY-like and PYY-like immunoreactivity together with FMRFamide-like and LPLRFamide-like immunoreactivity occur in the same endocrine cells of the Brockmann body of the cod and rainbow trout, or a hybrid NPY/PYY-like peptide recognized by both NPY and PYY antisera is present in the Brockmann body.     

S-100-like immunoreactivity in a planarian

Summary The presence of an S-100-like immunoreactivity was investigated in the planarian Dugesia gonocephala. By microcomplement fixation assay, measurable amounts of S-100-like immunoreactive material (0.11g/mg soluble protein) were detected in planarian high-speed supernatants. The index of immunological dissimilarity between ox S-100 and planarian S-100-like immunoreactive material was higher than that previously calculated between ox S-100 and all the vertebrates tested. By the immunohistochemical PAP method, S-100-like immunoreactivity was only detectable in the cilia of the epidermal cells. Although the biological meaning of S-100-like immunoreactivity in planarian remains to be clarified, the present data introduce new perspectives into the investigation of S-100.     

IGF-2-like peptides are present in insulin cells of the elasmobranchian endocrine pancreas: an immunohistochemical and chromatographic study

Evidence for the presence of peptides, related to insulin-like growth factor 2 (IGF-2), has been obtained in the endocrine pancreas of the elasmobranchian species Raja clavata, the sting ray. By radioimmunoassay, IGF-2-like immunoreactivity was detected in Raja pancreas extract. Further characterization of this activity by acid gel chromatography revealed two distinct peaks of IGF-2-like immunoreactivity with apparent molecular weights of approximately 8.2 kDa and 4.5 kDa. Using the same IGF-2 antibody as well as antisera specific for mammalian IGF-1, insulin, glucagon, somatostatin and pancreatic polypeptide in double immunofluorescence studies, IGF-2-like immunoreactivity was located exclusively in insulin-immunoreactive cells. In contrast, IGF-1-like immunoreactivity was mainly observed in somatostatin-and glucagon-immunoreactive cells. A varying proportion (0–70%) of insulin-immunoreactive cells, however, displayed both IGF-1- and IGF-2-like immunoreactivity. Absorption studies indicated that the IGF-2-like peptides in Raja are different from mammalian and submammalian insulin and mammalian IGF-1, but similar to mammalian IGF-2. Thus, IGF-2-like peptides seem to occur during evolution as early as the phylogenetic development of the elasmobranchians. Furthermore, the results indicate a particularly conservative evolution of the islet IGF-2 system.     

Localization of S1- and S2-like immunoreactivity in the nervous system of the brittle star Amphipholis squamata (Delle Chiaje 1828).

The recent isolation and characterization of the SALMFanide neuropeptides S1 GFNSALMFamide; and S2 (SGPYSFNSGLTFamide) from the sea stars. Asterias rubens and Asterias forbesi have initiated numerous studies on their morphological localization and distribution within the phylum Echinodermata. It has been shown by immunocytochemistry and radioimmunoassay that these peptides are widely distributed in the nervous system of some asteroids, echinoids and ophiuroids. A physiological approach has also shown that S1 and S2 potentiate the luminescence of the small ophiuroid Amphipholis squamata. In the present study. S1- and S2-like immunoreactivity have been localized in A. squamata by immunocytochemistry on both wholemount preparation and histological sections. The results reveal a widespread neuronal distribution of S1-like immunoreactivity in the circumoral ring, radial nerve cord, and tube feet. S1-like immunoreactivity was found to be associated with axons and cell bodies in both the ectoneural and hyponeural components of the nervous. S2-like immunoreactivity was detected only in the ectoneural plenus of the circumoral ring and radial nerve cord.     

神经肽Y和β—内啡肽样免疫阳性物质在文昌鱼神经系统和哈氏窝的分布

用免疫组织化学方法首次发现,神经肽Y（NPY）和β－内啡肽（β－Ep)样免疫阳性物质分布在文昌鱼神经系统和哈氏窝。NPY样免疫阳性神经元出现在端脑前部和中部、中脑前部和中部以及后脑,NPY样免疫阳性神经纤维在文昌鱼脑的各部分与神经元交错呈网状密集分布。神经管的背面与中部均可观察到NPY样免疫阳性神经元及其阳性性纤维。β－Ep样免疫阳性神经元及其神经纤维定位在中脑前部和中部以及神经管,且分布范围明显小于NPY。文昌鱼哈氏窝也有NPY和β－Ep样免疫阳性物质分布。这些结果表明,NPY和β－Ep可能作为脑内的一种神经递质,像鱼类那样,参与调节文昌鱼哈氏窝促性腺激素分泌细胞的分泌活动,这为文昌鱼脑－哈氏窝复合体的密切关系提供新的形态学证据。     

Distribution and characterization of cyclo (His-Pro)-like immunoreactivity in anuran (frog) skins

The distribution of cyclo (His-Pro)-like immunoreactivity in frog skins from seven frog species was examined. The chromatographic elution profile of cyclo (His-Pro)-like immunoreactivity in amphibian skins measured by radioimmunoassay corresponded precisely to that of [³H-Pro]-cyclo (His-Pro) after DEAE-Cellulose, Sephadex G-25 and high-pressure liquid chromatography. The concentrations of cyclo (His-Pro) in frog skins were much higher than the concentrations of TRH previously observed in skin and the concentrations of cyclo (His-Pro) in both brain and gastrointestinal tract.     

Localization of substance P-like,leucine-enkephalin-like,methionine-enkephalin-like,and FMRFamide-like immunoreactivity in the eyestalk of the fiddler crab,Uca pugilator

Summary The occurrence and distribution of substance P (SP)-like, methionine-(Met)- and leucine-(Leu)-enkephalinlike, and FMRFamide-like immunoreactivities were determined in the neuroendocrine complex of the eyestalk of the fiddler crab, Uca pugilator, by immunocytochemistry. SP-like immunoreactivity was found in the optic peduncle, sinus gland, medulla externa, medulla interna, lamina ganglionaris, and retinular cells. Met-enkephalin-like and Leuenkephalin-like immunoreactivity was observed in most of the retinular cells, optic peduncle, sinus gland, medulla terminalis, and lamina ganglionaris. However, Met-enkephalin-like, but no Leu-enkephalin-like, immunoreactivity was seen in the medulla terminalis X-organ. FMRFamide-like immunoreactivity could be seen in all parts of the eyestalk except in the sinus gland, lamina ganglionaris, and retinular cells. FMRF-amide-like activity was especially strong in the three chiasmatic regions connecting the optic ganglia. The possibility that these four peptides may function as neuroregulators in the fiddler crab is discussed.This investigation was supported in part by Grant No. PCM-8300064 from the National Science Foundation to MF and Biomedical Research Support Grant No. 2 SO7RRO5373 SUB from the University of Kansas Medical Center to LLV     

Release of slow-reacting substance of anaphylaxis and leukotriene C4-like immunoreactivity from guinea pig colonic tissue

Colonic mucosa, muscularis propria and subserosa from ovalbumin-sensitized guinea pigs were incubated and challenged with antigen in vitro. Slow-reacting substance of anaphylaxix (SRS-A) was determined biologically as well as radioimmunologically in terms of leukotriene (LT) C₄-like immunoreactivity. Before antigenic challenge release of immunoreactive LTC₄ by all tissues was below or close to the detection limit of the radioimmunoassay. After addition of antigen colonic mucosa released considerable amounts of LTC₄-like immunoreactivity, while muscularis propria and subserosa were less active. The biological activity of the SRS-A formed after challenge was antagonized by FPL 55712. Contrary to LTC₄-like immunoreactivity release of 6-keto-prostaglandin (PG) F1_α was predominant in the subsero and smaller amounts were released from the smooth muscular and mucosal layers. Synthesis of SRS-A and LTC₄-like immunoreactivity, respectively, as well as synthesis of 6-keto-PGF_1α was inhibited by the dual inhibitor of lipoxygenase and cyclooygenase BW755C. The results suggest a role for LTs as local mediators of inflammatory reactions in colonic disease states, particularly those with possible involvement of immunological processes.     

Mineral status of embryos of domestic fowl following exposure in vivo to the carbonic anhydrase inhibitor acetazolamide

Na+/H+ exchangers are integral membrane proteins that exchange Na+ and H+ across cell membranes. The Na+/H+ exchangers 2 and 3 are epithelial isoforms in mammals and contribute to acid-base homeostasis. The gills of fishes, including elasmobranchs, are also associated with acid/base balance, and are probably the primary acid/base regulatory organ. This study examines the presence of Na+/H+ exchangers 2 and 3 using immunohistochemistry and immunoblotting in the gills of four species of elasmobranchs, the banjo ray (Trygonorrhina fasciata), southern eagle ray (Myliobatis australis), the gummy shark (Mustelus antarcticus) and the Australian angel shark (Squatina australis) using heterologous antibodies. Na+/H+ exchanger 2-like immunoreactivity was observed in the gills of the banjo ray, eagle ray and angel shark. In the banjo and eagle rays, this Na+/H+ exchanger-like immunoreactivity co-localised with immunoreactivity to Na+ /K+ -ATPase, a marker for the mitochondrial-rich cells of fishes. Na+/H+ exchanger 3-like immunoreactivity was only observed in the gills of the angel and gummy sharks, some Na+/H+ exchanger 3-like cells also showed Na+ /K+ -ATPase immunoreactivity. However, immunoblotting of banjo and eagle ray gill membranes demonstrated Na+/H+ exchanger 3-like immunoreactivity, which was not consistent with the immunohistochemical results. These data demonstrate the presence of epithelial Na+/H+ exchangers 2 and 3 in the gills of elasmobranchs and a link with acid/base regulation is suggested.     

Nerve growth factor receptor-like immunoreactivity in primary and permanent canine tooth pulps of the cat

Summary The distribution of nerve growth factor receptor (NGF receptor)-like immunoreactivity in pulps of developing primary and mature permanent cat canine teeth was examined, by use of a monoclonal antibody against NGF receptor detected by fluorescence immunohistochemistry and pre-embedding immunocytochemical light- and electron microscopy. Both primary and permanent pulps contained a vast number of NGF receptor-like immunoreactive nerves. Immunolabelling appeared to be localized both to axons and Schwann cells. In addition, many blood vessel walls in immature primary tooth pulps showed NGF receptor-like immunoreactivity, in contrast to permanent pulps where blood vessels rarely were NGF receptor-immunoreactive. Double-labelling immunofluorescence experiments revealed that in the permanent pulp a majority of the NGF receptor-positive nerves also showed calcitonin gene-related peptide (CGRP)-like immunoreactivity, and many showed substance P-like immunoreactivity. However, nerve fibers with neuropeptide Y-like immunoreactivity lacked NGF receptor-like immunoreactivity. In developing primary tooth pulps fewer NGF receptor-positive nerves were CGRP-like immunoreactive or substance P-like immunoreactive, as compared to the permanent pulp. Neuropeptide Y-like immunoreactive nerve fibers were not detected in the primary tooth pulp. The results suggest a role for nerve growth factor in both developing and mature sensory nerves of the tooth pulp.     

Existence of different forms of adrenocorticotropin in rat hypothalamus

Adrenocorticotropin (ACTH)-like immunoreactivity and bioactivity were extracted from rat hypothalamus and fractionated by high pressure liquid chromatography. Analysis of the fractions either by radioimmunoassay or bioassay (corticosteroid production from rat adrenal cells) revealed several peaks of immunoreactivity and bioactivity. Only 20-25% of total ACTH-like immunoreactivity and bioactivity eluted with the same retention time as authentic ACTH 1-39. The results suggest that different forms of ACTH exist in rat hypothalamus.     

Anatomical mapping of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in outer hair cell efferents in adult rats

Summary The distribution of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in the cochleae of 15 adult Wistar white rats was investigated using the peroxidase-antiperoxidase (PAP) technique. A monoclonal antibody to ChAT and a polyclonal antiserum to GAD were used. Immunoreaction was investigated quantitatively, in the electron microscope, on tangential sections of the tunnel of Corti and the rows of outer hair cells. ChAT-like and GAD-like immunoreactivity was found in all efferent nerve fibres in the tunnel of Corti and in all efferent synapses on the outer hair cells. A coexistence of ChAT and GAD in the efferent system to the outer hair cells of the rat is therefore assumed.     

Enkephalin-like immunoreactivity in the pedal ganglion of Mytilus edulis (Bivalvia) and its proximity to dopamine-containing structures

The presence of enkephalin-like immunoreactivity in the pedal ganglion of the bivalve mollusc Mytilus edulis has been demonstrated. Enkephalin-like immunoreactivity is highly localized in those intraganglionic regions rich in dopamine-containing structures. In addition, FMRF-NH2-like (Phe-Met-Arg-Phe-HN2) immunoreactivity was found to occur in intraganglionic regions that are devoid of enkephalin-like immunoreactivity. The differential topographic distribution of these peptidergic immunoreactivities suggests involvement in separate functions. By contrast, the closeness of enkephalin-like immunoreactivity to dopamine histofluorescence supports previous data demonstrating biochemical and physiological links between these two systems.