Ontogeny of a novel pituitary protein (7B2) in the human fetal intestine

The developmental profile of the concentration of a novel pituitary protein (7B2) was studied immunochemically in the human gastrointestinal tract from 12 weeks of gestation to 4 months after birth and was compared to the distribution in the adult. 7B2-like immunoreactivity (IR-7B2) was detected in all segments studied, but no gross changes were seen through fetal life. At term higher concentrations of IR-7B2 were found in the duodenum and the antrum, which is similar to the distribution of adult man. Gel permeation chromatography revealed that the main peak of 7B2 immunoreactivity in the fetal intestinal extract eluted with a Kav of 0.3. Similar elution profiles were also observed in extracts of human adult intestine.     

Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain

The immunocytochemical localization of neurons containing the 41 amino acid peptide corticotropin-releasing factor (CRF) in the rat brain is described. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the rats and by silver intensification of the diaminobenzidine end-product. The presence of immunoreactive CRF in perikarya, neuronal processes, and terminals in all major subdivisions of the rat brain is demonstrated. Aggregates of CRF-immunoreactive perikarya are found in the paraventricular, supraoptic, medial and periventricular preoptic, and premammillary nuclei of the hypothalamus, the bed nuclei of the stria terminalis and of the anterior commissure, the medial septal nucleus, the nucleus accumbens, the central amygdaloid nucleus, the olfactory bulb, the locus ceruleus, the parabrachial nucleus, the superior and inferior colliculus, and the medial vestibular nucleus. A few scattered perikarya with CRF-like immunoreactivity are present along the paraventriculo-infundibular pathway, in the anterior hypothalamus, the cerebral cortex, the hippocampus, and the periaqueductal gray of the mesencephalon and pons. Processes with CRF-like immunoreactivity are present in all of the above areas as well as in the cerebellum. The densest accumulation of CRF-immunoreactive terminals is seen in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The widespread but selective distribution of neurons containing CRF-like immunoreactivity supports the neuroendocrine role of this peptide and suggests that CRF, similarly to other neuropeptides, may also function as a neuromodulator throughout the brain.     

Plasma 7B2 (a novel pituitary protein) immunoreactivity concentrations in patients with various endocrine disorders

We have measured plasma 7B2 (a novel pituitary protein)-immunoreactivity (IR) concentrations in patients with various endocrine disorders. Mean (+/- SEM) basal plasma 7B2-IR concentrations (ng/L) in patients with acromegaly (81 +/- 14.6), Cushing's disease (57.2 +/- 8.5), prolactinoma (71.4 +/- 9.5), panhypopituitarism (50.6 +/- 7.6), isolated ACTH deficiency (47.9 +/- 11.6), hyperthyroidism (57.9 +/- 6.7) and hypothyroidism (60.8 +/- 9.4) were on the same levels as those in age-matched normal subjects. However, basal plasma 7B2-IR concentrations were increased to more than 100 ng/L in 5 out of 25 patients with acromegaly (20%). Mean basal plasma 7B2-IR concentrations in patients with medullary carcinoma of the thyroid and pheochromocytoma were 293 +/- 38.1 ng/L (range: 225.7-357.4 ng/L, n = 3) and 221 +/- 82.8 ng/L (range: 48.5-527.8 ng/L, n = 5), respectively, and significantly higher than those in age-matched normal subjects (P less than 0.001). These results suggest that plasma 7B2-IR may have some diagnostic value for acromegaly and may be useful as a marker for medullary carcinoma of the thyroid and pheochromocytoma.     

Immunocytochemical localization of S-100 protein in the brain of adult rat

Summary The cellular and subcellular distribution of the nervous system-specific S-100 protein has been investigated in the brain of adult rat at the ultrastructural level by the pre-embedding unlabelled antibody PAP method. The protein is found in both fibrous and protoplasmic astrocytes and in the ependymal cells. The neurons, the oligodendrocytes as well as the microglial cells are lacking S-100. The labelled cells show a reaction product diffusely distributed in the cytoplasmic matrix and on specialized membranes, namely plasma membranes, outer mitochondrial membranes and membranes of the endoplasmic reticulum and Golgi apparatus. The astrocytic filaments and the axonemes of the ependymal cilia exhibit a strong immunoreactivity. The reaction product is also present in the nucleoplasm of the astrocytes and ependymal cells but it is absent from the nucleolus and nuclear envelope. This immunocytochemical data on tissue with satisfactory ultrastructural preservation, provides new information on the localization of the S-100 protein, and could contribute to the understanding of the biological role of the protein.     

Immunocytochemical localization of a growth-associated protein (GAP-43) in rat adrenal gland

We have localized at light and electron-microscopic level the growth-associated protein GAP-43 in adrenal gland using single and double labelling immunocytochemistry. Clusters of GAP-43-immunofluorescent chromaffin cells and many immunofluorescent fibres were observed in the medulla. GAP-43-immunoreactive fibres also formed a plexus under the capsule, crossed the cortex and ramified in the zona reticulata. Double labelled sections showed the coexpression of GAP-43 with a subpopulation of tyrosine hydroxylase-and of dopamine--hydroxylase-immunoreactive chromaffin cells. Dual colour immunofluorescence for GAP-43 and calcitonin gene-related peptide (CGRP) revealed that some of the GAP-43-immunoreactive fibres also express CGRP. Pre-embedding electron microscopy showed GAP-43 immunoreactivity associated with the plasma membranes and cytoplasm of noradrenaline-producing chromaffin cells, and with processes of nonmyelin-forming Schwann cells. Immunoreactive unmyelinated axons and terminals were also observed. The immunostained terminals made symmetrical synaptic contacts with chromaffin cells. Immunoreactive unmyelinated fibres and small terminals were present in the cortex. Our results show that GAP-43 is expressed in noradrenergic chromaffin cells and in various types of nerve fibres that innervate the adrenal. Likely origins for these fibres include preganglionic sympathetic fibres which innervate chromaffin cells, postganglionic sympathetic fibres in the cortex, and CGRP containing sensory fibres.     

Evidence for a novel pituitary protein (7B2) in human brain, cerebrospinal fluid and plasma: brain concentrations in controls and patients with Alzheimer's disease

A novel pituitary protein, designated as 7B2, recently purified in our laboratory was measured using a specific radioimmunoassay in conjunction with immuno-affinity extraction, in cerebrospinal fluid (CSF) and in plasma obtained from normal volunteers. The mean concentrations of immunoreactive (IR)-7B2 were 2154 pg/ml in CSF and 29 pg/ml in plasma. Studies by SDS-poly-acrylamide gel electrophoresis revealed that both CSF IR-7B2 and plasma IR-7B2 have an apparent molecular weight of around 20,000-21,000 as previously observed in various rat tissues. IR-7B2 was also measured in various brain regions obtained from control subjects and patients with Alzheimer's disease. IR-7B2 was widely distributed in the human brain, with the highest concentrations in substantia nigra and caudate. IR-7B2 brain concentrations were found to be similar between control subjects and patients with Alzheimer's disease. Gel permeation chromatography of extracts of various brain regions revealed two major peaks with apparent molecular weights of 45,000-50,000 and 11,000-16,000 in hypothalamus, caudate, frontal cortex, hippocampus, putamen and locus coeruleus, and only one peak with an apparent molecular weight of 14,000-16,000 in substantia nigra and globus pallidus. These data suggest that this novel pituitary protein may play a role of consequence perhaps as a neurotransmitter or as a neuromodulator in the human central nervous system.     

The novel pituitary polypeptide 7B2 is a highly-conserved protein coexpressed with proopiomelanocortin

In the amphibian intermediate pituitary gland the biosynthetic activity for production of the precursor protein proopiomelanocortin (POMC) can be physiologically manipulated; POMC synthesis is high in animals adapted to a black background and low in white-adapted animals. In order to study genes associated with POMC gene expression we applied a differential hybridization technique involving screening of a pituitary cDNA library with probes derived from RNA of inactive and physiologically activated intermediate pituitary cells of the amphibian Xenopus laevis. A differentially hybridizing Xenopus pituitary cDNA clone encoded the novel polypeptide 7B2. This Mr-21,000 secretory granule-associated protein of unknown function is shown to be highly conserved between Xenopus and human (83% amino acid sequence similarity). Conserved segments within the 7B2 structure included the N-terminal portion, three pairs of basic amino acids which are potential recognition sites for proteolytic enzymes, and three regions sharing similarity with putative GTP-binding domains. Levels of 7B2 mRNA were about 3% of POMC mRNA levels in Xenopus pituitary glands. In the intermediate pituitary the amount of both POMC and 7B2 mRNA was much higher in black-adapted toads than in white-adapted animals. These physiologically-induced changes in POMC and 7B2 mRNA levels were not found in the anterior pituitary. We conclude that the POMC and 7B2 genes are coexpressed and that modulation of the activity of these genes is tissue-specific.     

Immunocytochemical localization of a calcium-binding protein in the rat duodenum

Summary The cellular localization of the vitamin D-dependent calcium-binding protein (CaBP) in the duodenum of rat was studied using indirect immunofluorescence and immunoperoxidase staining methods. Specific positive reaction product, indicative of the presence of CaBP, was exclusively located within the villous part of the duodenal mucosa. Moreover, CaBP was detected mainly within the supranuclear region of the cytoplasm of absorptive cells and also at the level of their basal laminae. CaBP was not demonstrable either in the nuclei or associated with the brush border membrane of absorptive cells. Also, CaBP was neither detectable in goblet cells nor in sub-epithelial layers. When the specific anti-CaBP antiserum was replaced by nonimmune rabbit serum or when it was preabsorbed on a CaBP-Sepharose conjugate, no positive immunostaining was seen. Together with recent biochemical data our observations agree well with the view that CaBP may act as an intracellular buffer by protecting the cell against too high Ca²⁺ concentrations.     

Immunocytochemical localization of a developmentally regulated, isoproterenol-inducible protein (LM protein) in rat submandibular gland

Administration of the beta-adrenergic drug isoproterenol (IPR) produces hyperplastic and hypertrophic enlargements of the submandibular gland of the rat and induces the synthesis of specific proteins in this organ. One of these proteins, the LM (large mobile) protein, was demonstrated immunocytochemically in the submandibular glands of developing untreated and IPR-treated rats. Immunoreactive LM protein was absent in the glands of 20-day-old fetuses and 1- and 2-day-old rats. It was localized in the proacinar and immature acinar cells in the glands of 6- to 21-day-old animals, but it was undetectable at 28 days of age. In the glands of adult rats, secretory granules of the granular convoluted tubule cells showed immunostaining for the LM protein which was also present in trace amounts in the acinar cells. Daily administration of IPR for 5 days to newborn or 8- or 15-day-old rats caused an apparent acceleration of proacinar/acinar cell differentiation, and consequently it increased the frequency of cells immunostained for the LM protein as well as the amount of immunoreactive material in these cells. Thus, the expression of LM protein in the submandibular gland is developmentally regulated, and it is restricted to the stage of differentiation of proacinar cells from terminal tubule cells. IPR is capable of inducing this protein in fully differentiated acinar cells in 3-week-old or older animals.     

Immunocytochemical localization of angiotensin-(1–7) in the rat forebrain

Recent findings by this group have led us to reconsider the view that amino (N−) terminal fragments of angiotensin (Ang) II are inactive degradation products of renin-angiotensin system. To further examine this possibility, an antibody to Ang-(1–7), the N-terminal heptapeptide, was produced to demonstrate the neuroanatomical distribution of the rat brain. Ang-(1–7)-immunoreactivity was found in paraventricular, supraoptic, and suprachiasmatic nuclei, bed nucleus of the stria terminalis, substantia innominata, median eminence, and neurohypophysis. This distribution of Ang-(1–7) in the rat forebrain, together with our previous demonstrations of vasopressin secretion in response to this peptide, suggest that Ang-(1–7) functions as a neuromodulator.     

Elevation of plasma 7B2 (a novel pituitary protein) in cord blood at obstetrical delivery and the possible correlation with GH

Plasma 7B2-immunoreactivity (7B2-IR) concentrations in umbilical artery (UA), umbilical vein (UV) and maternal vein (MV) were measured by RIA at the time of obstetrical delivery at term. Plasma 7B2-IR concentrations (Mean +/- SEM) in UA (N = 12), UV (N = 16) and MV (N = 16) were 725 +/- 69, 699 +/- 64 and 116 +/- 4.5 pg/ml, respectively. Plasma 7B2-IR concentrations in UA and UV were much higher than those in MV. There was no arterio-venous gradient between UA and UV. A trace amount of 7B2-IR (Mean +/- SEM, 226 +/- 16.8 pg/g tissue) was detected in the placental extracts. A statistically significant positive correlation (r = 0.7595, p less than 0.005) was found between plasma 7B2-IR and GH in the UV. Significant negative correlations between body weight of the neonates and plasma levels of GH (r = -0.6836, p less than 0.01) and 7B2-IR (r = -0.4939, p less than 0.05) were also apparent. When analyzing cord blood plasma using gel permeation chromatography and SDS-polyacrylamide gel electrophoresis, a major peak with an apparent molecular weight of 20,000 was observed. These findings suggest that 7B2-IR in UA and UV originates from the fetus and that 7B2-IR does not permeate through the placenta. The possibility of involvement of 7B2 in fetal growth warrants attention.     

Immunocytochemical localization of a galanin-like peptidergic system in the brain and pituitary of some teleost fish

Summary Immunostaining of brain and pituitary sections of teleost fishes (eels, salmonidae, cyprinidae, gourami, sculpin, mullet) with anti porcine galanin (GAL) revealed the presence of immunoreactive (ir) perikarya and a rich network of fibers. Ir-perikarya were located rostrodorsally to the recessus preopticus, and in the posterior tuberal hypothalamus. Ir-fibers were abundant in basal telencephalon and around diencephalic ventricular recesses but never contacted their lumen. Furthermore, they were observed in basal hypothalamus, brainstem and ventral medulla. Ir-fibers passed along corticotropic (ACTH), gonadotropic cells and somatotropes (GH cells) in eel and trout pars distalis, but rarely ended in caudal neurohypophysis. In goldsfish pituitary ir-fibers occurred in neural digitations and among different cell types which however did not contain a GAL-like peptide. The relation GAL fibers/GH cells appeared more evident in species with a high growth rate. The other species showed a similar distribution of brain GAL. In eels and trout, ir-perikarya were not observed in areas containing somatostatin, GH- and ACTH-releasing factor, and ACTH-like perikarya, suggesting that GAL did not coexist with these peptides. The widespread distribution of a GAL-like peptide in teleost brain suggests that it could play a role of neurotransmitter and/or neuromodulator and regulate the secretion of adenohypophysial hormone(s). Abbreviations used in the text: GAL galanin. In the brain: CSF cerebrospinal fluid; NLT nucleus lateralis tuberis; NPO nucleus preopticus; NPP nucleus preopticus periventricularis; NPVa nucleus periventricularis anterior; NRP nucleus recessus posterioris; RI recessus infundibularis; RL recessus lateralis; RPO recessus preopticus. In the pituitary: ACTH corticotropin; CRF corticotropin-releasing factor; GH growth hormone; GRF growth hormone-releasing factor; GTH gonadotropin; MSH melanotropin; NH neurohypophysis; NIL: neurointermediate lobe; PPD proximal pars distalis; RPD proximal pars distalis     

Age-related change in 7B2 (a novel pituitary polypeptide) concentrations in human cerebrospinal fluid

Concentrations of 7B2 (a novel pituitary polypeptide) immunoreactivity (7B2-IR) were measured using a specific 7B2 radioimmunoassay (RIA) in cerebrospinal fluids (CSFs) from 87 humans. The mean (+/- S.E.M.) concentration of 7B2-IR in CSF was 2022 +/- 68 pM and a statistically significant decrease with aging was observed in those concentrations (R = -0.28, t = 2.73, P less than 0.01), although it was not a strong relation based on the R-value. In the gel permeation chromatography of CSF on Sephadex G-100, a major peak with an apparent mol. wt. of 43 kDa (43K) and a minor peak with that of 11 kDa (11K) were observed.     

Immunocytochemical localization of d-amino acid oxidase in rat brain

d-amino acid oxidase (d-AAO) is a peroxisomal flavoenzyme, the physiological substrate and the precise function of which are still unclear. We have investigated D-AAO distribution in rat brain, by immunocytochemistry, with an affinity-purified polyclonal antibody. Immunoreactivity occurred in both neuronal and glial cells, albeit at different densities. Glial immunostaning was strongest in the caudal brainstem and cerebellar cortex, particularly in astrocytes, Golgi-Bergmann glia, and tanycytes. Hindbrain neurons were generally more immunoreactive than those in the forebrain. Immunopositive forebrain cell populations included mitral cells in the olfactory bulb, cortical and hippocampal neurons, ventral pallidum, and septal, reticular thalamic, and paraventricular hypothalamic nuclei. Within the positive regions, not all the neuronal populations were equally immunoreactive; for example, in the thalamus, only the reticular and anterodorsal nuclei showed intense labelling. In the hindbrain, immunopositivity was virtually ubiquitous, and was especially strong in the reticular formation, pontine, ventral and dorsal cochlear, vestibular, cranial motor nuclei, deep cerebellar nuclei, and the cerebellar cortex, especially in Golgi and Purkinje cells.     

Immunocytochemical localization of the glucose-transport protein in mammalian brain capillaries

Summary The endothelial cells of mammalian brain capillaries, which form the anatomical basis of the blood-brain barrier, have been investigated by immunocytochemical methods to determine the distribution of the glucose-transport protein. A monoclonal antibody raised against the intact human erythrocyte glucose-transport protein and polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal sequence of the human erythrocyte glucose-transport protein were used for immunofluorescent staining of isolated human and bovine cerebral cortex microvessels. The pattern of fluorescence with both antibodies demonstrated the antigen to the distributed throughout the plasma membrane of the capillary endothelial cells. These results provide further evidence for the homology between the human erythrocyte and brain capillary glucose-transport protein, and confirm its abundance in brain capillaries.     

Age-related change in plasma concentration of 7B2 (a novel pituitary polypeptide) in normal humans

Using a specific radioimmunoassay, we measured concentrations of plasma 7B2 (a novel pituitary polypeptide) immunoreactivity (7B2-IR) in normal human subjects, patients with chronic renal failure and those with liver cirrhosis. Mean (+/- SEM) values of plasma 7B2-IR in normal healthy men and women were 55.8 +/- 1.2 pg/ml (n = 266) and 56.1 +/- 0.9 pg/ml (n = 408), respectively. The elevation of plasma 7B2-IR showed a relationship with age of the subjects, in both men (r = 0.39, t = 6.86, p less than 0.001) and women (r = 0.35, t = 7.44, p less than 0.001). Plasma 7B2-IR concentrations were elevated in patients with chronic renal failure (536 +/- 45 pg/ml, Mean +/- SEM, n = 10) as well as those in liver cirrhosis (95 +/- 10 pg/ml, Mean +/- SEM, n = 15) compared to values in normal subjects, suggesting that 7B2 is mainly eliminated through the kidney and is partly metabolized in the liver.     

Immunocytochemical localization of cathepsins B and H in rat liver

Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Immunocytochemical localization of cathepsins B and H in rat liver

Summary Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultrathin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Immunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord.

Specific antisera directed against substance P and neuromedin K (neurokinin B) have been used in double-label immunofluorescence studies to unambiguously localize these two neuropeptides of the tachykinin family in single tissue sections of rat spinal cord and dorsal root ganglia. Substance P-like immunoreactivity (SPLI) is present but neuromedin K-like immunoreactivity (NMKLI) is undetectable in dorsal root ganglia. Both peptides are present in the spinal cord, but NMKLI is largely restricted to the dorsal gray while SPLI shows a broader distribution. In the spinal gray, NMKLI coexists with SPLI in some, but not all, fibers. While substance P in the dorsal spinal cord is largely of primary afferent origin, neuromedin K appears to originate largely from intrinsic spinal neurons.