High levels of a heat-labile calmodulin-binding protein (CaM-BP80) in bovine neostriatum

Bovine brain contains a heat-labile, 80,000-dalton calmodulin-binding protein (CaM-BP80) which inhibits the calmodulin-dependent activities of cyclic 3',5'-nucleotide phosphodiesterase, adenylate cyclase, and Ca2+-ATPase in vitro. CaM-BP80 is composed of two polypeptides (60,000 and 18,500 daltons) present in a 1:1 ratio. An antibody directed against CaM-BP80 was raised in rabbits, and a radioimmunoassay was developed, having a sensitivity of 60 fmol of CaM-BP80. Using the radioimmunoassay, we determined the levels of CaM-BP80 in various bovine tissues. The protein was found primarily in the brain, present in particularly high levels in the neostriatum. These results, together with immunohistochemical localization of CaM-BP80 at the postsynaptic densities and the microtubules of postsynaptic dendrites [Wood, J.G., Wallace, R., Whitaker, J., & Cheung, W.Y. (1980) J. Cell Biol. 84, 66-76], suggest that the protein may have a role in the cerebrum at the site of neurotransmitter action and at the level of microtubular function.     

Immunohistochemical localization of calmodulin in mouse brain

Calmodulin is a well-known calcium-binding protein which is ubiquitous in the plant and animal kingdoms and regulates many cellular processes. In this paper, the distribution of calmodulin in mouse brain was studied immunohistochemically using specific anti-calmodulin IgG which was raised in the rabbit by immunization with native calmodulin. Immunoreactive staining was observed in the cells in almost all areas of the brain, but its intensity varied. Some areas were stained heavily even in the presence of high concentration of NaCl. These differed from those stained immunohistochemically with antibody against other calcium-binding proteins, parvalbumin or vitamin D-dependent calcium-binding protein.     

Immunocytochemical localization of protein kinases and calmodulin in dog thyroid cells

By immunocytochemical methods, the protein effectors of known intracellular signal molecules were demonstrated in the thyroid follicular cell and their localization investigated. Cyclic AMP protein kinase subunit RII was clearly located in the nucleus. Protein kinase subunits RI and C were in the cytosol and on the apical membrane. Cyclic GMP kinase and calmodulin were mostly found in the cytoplasm and at the apical membrane; they were poorly represented in the nucleus. The only membrane underlined by several markers was the apical membrane, i.e. the site of iodination, oxido reduction and H2O2 generation.     

Immuno-electron microscopic localization of calmodulin and calmodulin-binding proteins in the mouse germ cells during spermatogenesis and maturation

When extracts of mouse testis were Western-blotted against a monoclonal antibody which reacts with calmodulin in the presence of Ca²⁺, all calmodulin was associated with the macromolecules of molecular weight above 50 kDa. Immuno-electron microscopy of testes using this antibody indicated that calmodulin is localized at higher density in the nucleus and cytoplasm of germ cells during the developmental phase between pachytene and round spermatid, showing the highest level just before meiotic divisions. There was no special association of calmodulin to any organelles in these cells. Extremely low levels of calmodulin occurred in spermatogonia and other testicular tissue cells. Calmodulin decreased dramatically as spermatids underwent metamorphosis, becoming detectable only at the perinuclear space of sperm heads. Further relocation to the postacrosomal region occurred during sperm transit to the cauda epididymis. Immunodetection after the calmodulin overlay on ultrathin sections revealed a sharp increase of calmodulin immunogold deposits in the nuclei of spermatids accompanying their condensation. The results indicate that some calmodulin-binding proteins, but not calmodulin itself, accumulate in the nuclei during the final steps of spermiogenesis.     

Immunocytochemical localization of a novel pituitary protein (7B2) within the rat brain and hypophysis

A novel pituitary protein called 7B2 was localized in rat pituitary and brain by immunocytochemistry (unlabeled antibody technique). Immunoreactive material was present in the secretory cells of anterior and intermediate lobes and in neural structures of the posterior lobe of the hypophysis. 7B2-immunoreactive neurons were evident within the hypothalamus in the supraoptic nucleus, paraventricular nucleus (magnocellular and parvocellular parts), and lateral hypothalamus. Immunoreactive nerve fibers were seen within the internal and external zone of the median eminence. Among extrahypothalamic regions, the substantia nigra, dorsal tegmental nucleus, cuneiform nucleus, dorsal parabrachial nucleus, spinal tract trigeminal nerve, interior olive, solitary nucleus, and layers I and II of the spinal cord contained 7B2-immunoreactive material. This anatomical distribution suggests a role for 7B2 in endocrine and autonomic functions.     

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.     

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.     

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.     

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.     

Immunocytochemical localization of progesterone-binding protein (PBP) in guinea-pig placental tissue

Summary The cellular localization of progesterone-binding protein (PBP) in the guinea-pig placenta was studied by use of immunocytochemical procedures. Within the chorioallantoic placenta, a strong positive reaction was observed in the interlobar and marginal trophoblast from the third week of gestation to term. PBP was localized in the cytoplasm of the syncytiotrophoblast, and the nuclei were never stained. At the ultrastructural level, the immunoreaction was associated with the rough endoplasmic reticulum, the Golgi apparatus and the perinuclear space. No deposits were seen in any other cell organelles. This localization strongly suggests that the interlobar syncytium is related to PBP synthesis. In the labyrinth, a weak immunoreaction was observed by light microscopy around some blood lacunae. At the ultrastructural level the dense deposits were localized in vesicles located near the maternal lacunae.The distribution of PBP was also studied by light microscopy in other tissues from pregnant guinea-pig. No PBP, or PBP-like material, was detected inside cells from liver, muscle, heart, lung, kidney, ovary, and uterus. A weak immunoreaction for PBP was detected in vascularized zones of these organs.These observations strongly suggest that PBP, a protein related to gestation in the guinea-pig, is elaborated by the placental tissue of this hystricomorph rodent. PBP is the first steroid-binding plasma protein shown to be of extrahepatic origin.     

Immunocytochemical localization of renin in mouse kidney

Summary The distribution of renin in mouse kidney was examined in immunohistochemical studies by using an antiserum against pure mouse submaxillary renin and the peroxidase-antiperoxidase (PAP) technique. At antibody dilutions from 1:10⁴ to 1:10⁶, renin was found in high concentrations in the epitheloid cells of the vasa afferentia and, in lower concentrations, in the wall of some of the vasa efferentia. Renin was also detected in most of the interlobular arteries. Mesangial cells and Goormaghtigh cells were always free of specific staining. At high antiserum concentrations (i.e., dilutions from 1:10² to 1:10⁴) specific reaction product was also observed in the apical part of proximal tubule cells. This staining may represent filtered and pinocytozed renin.     

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 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 somatostatin in human brain

The distribution of somatostatin (SRIF) was examined in normal human forebrain, using thick vibratome cut sections. The unlabeled antibody enzyme method of immunocytochemistry revealed a widespread distribution of SRIF immunoreactive neurons and fibers throughout the septum, diencephalon and corpus striatum. Within the septum SRIF neurons and fibers were observed in the medial and lateral septal nuclei, the nucleus of the diagonal band, the nucleus accumbens and the bed nucleus of the stria terminalis. SRIF neurons and fibers were found in several hypothalamic and anterior thalamic nuclei as well as all regions of the corpus striatum. An interesting collection of SRIF immunoreactive neurons and processes were observed forming a wide band extending anteriorly from the lateral preoptic area through the lateral hypothalamus and substantia innominata posteriorly. This report on the localization of immunoreactive SRIF in the human forebrain extends previous anatomical findings and lends morphological support to recent biochemical studies.     

Neuronal localization of the tyrosine-specific protein kinase p62c-yes in rat basal ganglia

The cellular localization of the tyrosine-specific protein kinase p62^c-yes , the product of the proto-oncogene c-yes, has been examined in the striatonigral neurons which interconnect the rat neostriatum and substantia nigra. Although p62^c-yes was more enriched in the neostriatum than in the substantia nigra, excitotoxin-induced necrosis of nerve cells in the neostriatum led to 50–60% decreases of p62^c-yes both in the lesioned neostriatum and in the ipsilateral substantia nigra. Hence, the p62^c-yes tyrosine kinase is present both in the cell body region and in the axonal and nerve terminal region of the striatonigral neurons. This localization indicates that the enzyme may be involved in both presynaptic and postsynaptic functions in mammalian forebrain neurons.Special issue dedicated to Dr. Paul Greengard.     

Isolation of a new calmodulin-binding protein from rat brain

A new calmodulin-binding protein was isolated from rat brain by chromatographies on DEAE-Sephadex and hydroxyapatite followed by affinity chromatography on calmodulin-Sepharose. This protein, which constituted over 10% of the total amount of calmodulin-binding proteins in the supernatant from rat brain, gave one band of molecular weight 50K on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although bound to calmodulin-Sepharose even in the presence of 5 M urea, the protein was quickly released on removal of calcium. Rapid postmortem decrease of the protein was observed.     

Immunocytochemical localization of renin in mouse kidney.

The distribution of renin in mouse kidney was examined in immunohistochemical studies by using an antiserum against pure mouse submaxillary renin and the peroxidase-antiperoxidase (PAP) technique. At antibody dilutions from 1:10(4) to 1:10(6), renin was found in high concentrations in the epitheloid cells of the vasa afferentia and, in lower concentrations, in the wall of some of the vasa efferentia. Renin was also detected in most of the interlobular arteries. Mesangial cells and Goormaghtigh cells were always free of specific staining. At high antiserum concentrations (i.e., dilutions from 1:10(2) to 1:10(4)) specific reaction product was also observed in the apical part of proximal tubule cells. This staining may represent filtered and pinocytozed renin.     

Immunocytochemical localization of protein kinase C in developing brain tissue and in primary neuronal cultures

Antisera to protein kinase C (PKC) have been used to examine the presence and distribution of the enzyme in developing cerebellar cortex of postnatal rat and in cultures of rat sympathetic ganglia. In the cerebellar cortex of 2-,4-, and 6-day old rats, immunostaining was observed in areas of early-forming presynaptic terminals and growth cones. No staining was evident in the cortical proliferative zone. Beginning at 10 days postnatal, nuclear staining, not apparent at earlier stages, was prominent in Purkinje cells. In neuronal cultures of dissociated rat sympathetic ganglia, PKC was immunolocalized in cell bodies and bundles of neuronal processes. Immunoreactivity was particularly striking in growth cones of extending neurites and in axonal varicosities. These results suggest a role for PKC in neuronal growth following cell proliferation and in synaptic function. The appearance of nuclear staining in later developmental stages suggests that the enzyme may be involved in the promotion and maintenance of the differentiated state of neurons.     

Immunocytochemical localization of calspectin (a non-erythroid spectrin-like protein) in thyroid glands of normal and TSH-treated rats

The localization of calspectin (fodrin, a non-erythroid spectrin-like protein), which is known to bind calmodulin and F-actin, was detected in the thyroid gland of normal and TSH-treated rats by means of light-microscopic immunocytochemistry. Calspectin was demonstrated in the cytoplasm of the follicle epithelial cells especially along the baso-lateral plasma membrane in normal rats. In TSH-treated animals, in addition to the baso-lateral plasma membrane region, the apical plasma membrane region of the follicle epithelial cells also showed positive reaction to the immunostaining. These results suggest that calspectin, in conjugation with calmodulin and actin, play a role in the secretory activities including reabsorption activity of colloid of the follicle epithelial cell.