Punctate Appearance Research Articles

Intracellular staining of angiotensin receptors in the PVN and SON of the rat

Our immunocytochemical results demonstrate the presence of angiotensin AT 1 receptors in the cytoplasm and at the cell membrane in paraventricular and supraoptic neurons of the rat hypothalamus. The reaction product had a punctate appearance. We found no staining in the cell nucleus. Similar results were obtained with an anti-idiotypic antibody to angiotensin II.

Epitope location in the Cryptococcus neoformans capsule is a determinant of antibody efficacy.

Monoclonal antibodies (mAbs) to the polysaccharide capsule of Cryptococcus neoformans can prolong survival in mice. However, the properties of antibodies that mediate protection are not fully understood. The IgM mAbs 12A1 and 13F1 originated from the same B cell and differ only by somatic mutations in their variable regions; yet mAb 12A1 protects against serotype D infection, while mAb 13F1 does not. Phage peptide display libraries were used to analyze the fine specificity of these two mAbs. The selection of distinct peptide motifs from identical libraries confirmed that mAbs 12A1 and 13F1 bound to two distinct epitopes. Immunofluorescence and immunoelectron microscopy studies revealed differences in antibody localization within the capsule of serotype D strain; mAb 12A1 bound to the outer rim of the capsule resulting in an annular pattern, whereas mAb 13F1 bound throughout the capsule and had a punctate appearance. The difference in the binding pattern of mAb 12A1 and 13F1 was not observed on serotype A organisms, where both mAbs bound to the capsule with an annular fluorescence pattern. The fluorescence pattern of binding correlated with protective efficacy; mAb 13F1 prolonged survival of mice infected with the J11 serotype A strain (annular fluorescence), but not serotype D strains (punctate pattern). Annular binding, but not punctate binding, was associated with increased opsonic efficacy for phagocytosis of C. neoformans by J774.16 macrophage-like cells. The correlation between capsular binding pattern, opsonic activity, and ability to prolong survival suggests that the efficacy of anticryptococcal antibodies is dependent upon where they bind in the polysaccharide capsule.

Cysteine String Proteins Are Associated with Chromaffin Granules

In this work, we have examined the subcellular distribution of cysteine string proteins (Csps) in bovine adrenal medullary chromaffin cells. Csps did not leak from digitonin-permeabilized chromaffin cells, suggesting that there is no cytosolic pool of the protein in these cells. Subcellular fractionation studies confirmed that there was essentially no Csp immunoreactivity in the cytosolic fraction. However, immunoreactivity was detected in the membrane fractions of these cells. Csp immunoreactivity codistributed with dopamine beta-hydroxylase, a granule marker protein, in sucrose gradient-separated granule fractions. Immunofluorescence studies showed that all chromaffin cells in culture were stained with a punctate appearance consistent with a granular localization. These results were confirmed by immunogold labeling, which demonstrated specific labeling of chromaffin granule membranes. In addition to its presence on synaptic vesicles, cysteine string protein is therefore a bona fide chromaffin granule membrane protein.

Metabotropic glutamate receptor mGluR5 subcellular distribution and developmental expression in hypothalamus.

The metabotropic glutamate receptor mGluR5 is a G-protein coupled receptor that plays a key role in release of Ca2+ from internal stores via inositol triphosphate mobilization. Western and Northern blot analyses revealed a greatly enhanced expression of mGluR5 in rats during early stages of hypothalamic development compared with the adult. This enhanced developmental expression provides an explanation for the dramatic physiological response of developing neurons to metabotropic glutamate receptor activation and supports the argument that metabotropic glutamate receptors may play an important role in hypothalamic development. During development, expression of the mGluR5 gene was reduced, not only in the hypothalamus but also in other regions of the brain. A differential decrease in mGluR5 protein was found in different brain regions with Western blot analysis. The hypothalamus showed a sixfold decrease in mGluR5 with development, whereas the cortex showed only a threefold decrease. Immunocytochemistry with an affinity-purified antibody against a peptide deduced from the cloned mGluR5 gene revealed selective expression in some regions in the adult hypothalamus. In the adult and developing (postnatal day 10) brain, immunoreactive neurons were found in the suprachiasmatic nucleus, preoptic area, lateral hypothalamus, and mammillary region, areas where the related metabotropic glutamate receptor mGluR1 is also found. In contrast, the ventromedial nucleus, an area critically involved in the regulation of food intake and metabolic balances, showed strong mGluR5 immunoreactivity but no mGluR1 immunoreactivity. Little or no mGluR5 staining was found in the neurosecretory neurons of the paraventricular, supraoptic, and arcuate nuclei. Ultrastructurally, mGluR5 was associated with the cytoplasmic face of the plasmalemma on hypothalamic dendrites, dendritic spines, and neuronal perikarya in the adult. The strongest immunoreactivity was found in patches on the membrane, sometimes associated with the postsynaptic side of synapses and sometimes associated with nonsynaptic dendritic or perikaryal membrane. Intense immunostaining was found on some astrocyte processes surrounding synaptic complexes containing asymmetrical synapses. These astrocytes would be in an ideal position to receive excitatory signals from glutamatergic axons. Unlike the punctate appearance of immunolabeling on neuronal membranes, astrocytes showed continuous staining along the plasma membrane.

Cysteine string protein immunoreactivity in the nervous system and adrenal gland of rat

Cysteine string proteins (csps) are a recently discovered class of cysteine-rich proteins. They have been shown to associate preferentially with synaptic vesicle fractions of Torpedo electric organ or rat brain where they have been implicated in events associated with transmitter secretion. However, to date there has been no information concerning the distribution of csps in rat tissues. We investigated the localization of csps in the rat retina and CNS using immunohistochemistry with affinity purified anti-csp antibodies. Specific csp immunoreactivity having a punctate appearance is present throughout the neuraxis. Csp immunoreactivity is particularly abundant in synapse-rich regions including those of the retina, main olfactory bulb, hippocampal formation, and cerebellum. White matter tracts are devoid of csp immunoreactivity. Neuromuscular junctions show strong csp immunoreactivity. This localization of csp immunoreactivity is compatible with a role for csps in presynaptic events at a wide variety of synapses. Immunohistochemical analysis of a non-neuronal, secretory tissue, the adrenal gland, reveals prominent csp immunoreactivity in the chromaffin cells of the adrenal medulla. However, csp immunoreactivity is not detected in adrenal cortical regions. These findings are confirmed and extended by immunoblot and Northern analyses which identify a 35 kDa and a 5 kb product, respectively, in extracts of adrenal. The presence of csps in the adrenal medulla suggests that these proteins may also participate in secretion-related events in certain non-neuronal cells.

Immunocytochemical localization of glycine receptors in the mammalian retina

The distribution of glycinergic synapses in the mammalian retina was studied with monoclonal antibodies against glycine receptors and a glycine receptor-related protein (gephyrin). Monoclonal antibody 2b is specific for the alpha 1 subunit of the glycine receptor; monoclonal antibody 4a is specific for all known alpha subunits and the beta subunit, and monoclonal antibody 7a is specific for gephyrin. The three antibodies were applied to the retina of cat, macaque monkey, rat, and rabbit. The general staining pattern is comparable in all these species and it is similar but distinct with all of the three antibodies. Labeling is characterized by a punctate appearance indicating that it occurs at synapses. In the inner plexiform layer, labeling is concentrated in two bands. One band is located close to the inner nuclear layer; the other band is located in the middle of the inner plexiform layer. In the outer plexiform layer, sparse punctate labeling is seen. The distribution of gephyrin was also studied at the ultrastructural level in cat and monkey retina. Gephyrin is present on the postsynaptic membrane of amacrine cells and ganglion cells. The presynaptic profile to gephyrin immunoreactivity is always of an amacrine cell. The AII amacrine cell, the crucial glycinergic interneuron of the rod pathway, is presynaptic to gephyrin immunoreactivity in the OFF-sublamina and is itself gephyrin-positive at an input synapse from another (possibly GABAergic) amacrine cell in the ON-sublamina.

Visualization of G oα subtype guanine nucleotide-binding regulatory protein in the medulla oblongata and pons of the rat

This study reports on the visualization of G o protein in the medulla oblongata and pons of Sprague-Dawley rats, using a commercially available selective polyclonal antiserum against a 39-kDa protein corresponding to the α subunit of G o. Specific immunofluorescent-staining was widely distributed in neuropils and neuronal cell bodies but displayed regional heterogeneity. The immunoreactive product exhibited a punctate appearance and was most pronounced in the cell membrane and cytoplasm of the perikarya and proximal dendrites whereas the nucleus remained unstained. Positive immunoreactivity of G oα was present in nucleus reticularis gigantocellularis, nucleus reticularis paramedialis, nucleus tractus solitarii, nucleus nervi hypoglossi, nucleus raphé pallidus, locus coeruleus, nucleus tractus mesencephali nervi trigemini and nucleus cuneatus. These results provided anatomic evidence to support the participation of G o protein in both sensory and motor functions that originate from the medulla oblongata and pons of the rat.

Cytoskeletal structure of skeletal muscle: identification of an intricate exosarcomeric microtubule lattice in slow- and fast-twitch muscle fibers.

We used immunochemical quantification and indirect immunofluorescence to investigate the cell content, distribution, and organization of microtubules in adult rat slow-twitch soleus and fast-twitch vastus lateralis muscles. An immunoblotting assay demonstrated that the soleus muscle (primarily Type I fibers) was found to have a 1.7-fold higher relative content of alpha-tubulin compared with the superficial portion of the vastus lateralis muscle (primarily Type IIb fibers). Both physiological muscle types revealed a complex arrangement of microtubules which displayed oblique, longitudinal, and transverse orientations within the sarcoplasmic space. The predominance of any one particular orientation varied significantly from one muscle tissue section to another. Nuclei were completely surrounded by a dense net-like structure of microtubules. Both muscle fiber types were found to possess a higher density of microtubules in the subsarcolemmal region. These microtubules followed the contour of the sarcolemma in slightly contracted fibers and showed a fine punctate appearance indicative of a restricted distribution. The immunofluorescence results indicate that microtubules are associated with the sarcolemma and therefore may form a part of the membrane cytoskeletal domain of the muscle fiber. We conclude that the microtubule network of the adult mammalian skeletal muscle fiber constitutes a bone fide component of the exosarcomeric cytoskeletal lattice domain along with the intermediate filaments, and as such could therefore participate in the mechanical integration of the various organelles of the myofibers during the contraction-relaxation cycle.

Disorganization of microfilaments and intermediate filaments interferes with the assembly and stability of desmosomes in MDCK epithelial cells

To investigate the possible role(s) of cytoskeletal elements in desmosome assembly we have studied the effects of cytostatic drugs on the assembly of desmosomes in MDCK epithelial cells. We showed previously [Pasdar et al.: Cell Motil. Cytoskeleton 23:201-213, 1992] that selective disruption of microtubules has no effect on desmosome assembly. Here, we have treated MDCK cells with cytochalasin B and a combination of cytochalasin B and nocodazole and analysed the effects of desmosome assembly. Immunofluorescence analysis of MDCK cultures following drug treatment indicated complete disruption of actin microfilaments and disorganization of cytokeratin intermediate filaments. Biochemical analysis of newly synthesized desmosomal membrane core glycoproteins as well as the cell adhesion protein E-cadherin revealed no effect of these drugs on the kinetics of synthesis, intracellular processing, or transport to the plasma membrane either in the presence or absence of cell-cell contact. However, morphological analyses revealed a significant disruption in the spatial organization of desmosomal proteins and E-cadherin. Drug treatment in the absence of cell-cell contact resulted in the disruption of the normally observed homogeneous punctate staining pattern and appearance of aggregate staining. Induction of cell-cell contact in these cultures resulted in redistribution of some of the aggregate staining to the plasma membrane. In contrast to control cultures, significant amount of intracellular staining was retained for all desmosomal proteins. Biochemical analyses of turnover rates of newly synthesized desmosomal proteins indicated a significant decrease in metabolic stability of these proteins while the turnover rate of E-cadherin was not significantly different among control and drug-treated cultures. Taken together, these results suggest that intact actin and cytokeratin filaments are necessary for the stability, efficient assembly, and spatial organization of the junctional components at the membrane. The regulatory role of cytokeratins and actin filaments in assembly and stability of desmosomes on the plasma membrane is discussed.

Biochemical and immunohistochemical demonstration of a tightly bound form of prostaglandin E 2 in the rat brain

Basal levels of prostaglandin E 2 in the rat brain were determined by radioimmunoassay to be 0.68–0.79 pmol/g brain. About one-third of the prostaglandin E 2 (0.23–0.28 pmol/g) was resistant to extraction with ethanol, but could be recovered with a mixture of ethanol and 1 N HC1 (9:1, v/v), indicating that a tightly bound form of prostaglandin E 2 exists in the brain. The amount of the bound form of prostaglandin E 2 was almost unchanged by pentylenetetrazole-induced convulsion or by transcardial perfusion with a formaldehyde solution, although these treatments resulted in 40- to 80-fold increases in prostaglandin E 2 content extracted with ethanol at neutral pH. A polyclonal antibody against prostaglandin E 2-albumin conjugates recognized the bound form of prostaglandin E 2, giving a punctate appearance in many neuronal cell bodies in the brain. Although almost all of the neuronal perikarya were immunoreactive for prostaglandin E 2, intense immunoreactivity was observed in the mitral cell layer of the olfactory bulb, layer V of the cerebral neocortex, anterodorsal and reticular nuclei of the thalamus, supraoptic, paraventricular, accessory neurosecretory and lateral mammillary nuclei of the hypothalamus, mesencephalic trigeminal nucleus, nucleus of the trapezoid body and deep cerebellar nuclei. When the cerebral neocortical regions were observed electron microscopically, immunoreaction products were seen as fine granules which were clustered into small patches in the cytoplasm of neuronal cell bodies and proximal dendrites. No immunoreaction products were seen in glial cells or endothelial cells. These results suggest that prostaglandin E 2 is involved in fundamental processes of neurons.

Novel antigenic determinant expressed in neurons of the dorsolateral hypothalamus in rat and human.

Previous studies have identified a group of cells in the dorsolateral hypothalamus that project to many different areas in the CNS, such as thalamus, diagonal band of Broca, basal ganglia, cerebral cortex, hippocampus, and olfactory bulb. Their role is presently unknown, but the cells have been reported to stain for an intriguing array of putative neurotransmitter-related substances, including alpha-melanocyte-stimulating hormone (alpha MSH), melanin-concentrating hormone (MCH), human growth-hormone-releasing factor 1-37 (hGRF 1-37), corticotropin-releasing factor (CRF), metorphamide, and acetylcholine esterase. A monoclonal antibody produced in the present study, alpha C11, stains both the cell bodies of this system in hypothalamus, with a punctate pattern, and varicose fibers in the various target areas. In double-label immunocytochemical experiments in rat DLH, alpha C11 and MCH staining exactly overlaps. Concentrations of alpha MSH and MCH high enough to completely block staining with the corresponding antisera had no effect on staining with alpha C11. Similarly, CRF, hGRF 1-37, and metorphamide were unable to block alpha C11 staining. The results suggest that the antigenic epitope for alpha C11 is not contained in alpha MSH, MCH, CRF, hGRF, or metorphamide, and thus, that alpha C11 is detecting another antigen uniquely expressed in these neurons. The punctate appearance of staining in the hypothalamus and the concentration of staining in fiber varicosities suggests that the alpha C11 epitope may be involved in synaptic function.

Immunocytochemical localization of (Na + + K +)-ATPase in the rat hippocampus

The adult rat hippocampus was investigated by light microscopic immunocytochemistry for (Na + + K +)-ATPase. In the CA1, CA2 and CA3 hippocampal regions, dense immunostaining for (Na + + K +)-ATPase, exhibiting a punctate appearance, was demonstrated along the soma plasmalemma of hippocampal pyramidal cells in the stratum pyramidale, thus outlining these cells distinctly, and along dendrites extending into the stratum radiatum. (Na + + K +)-ATPase immunostaining was dense in the neuropil of the strata oriens and radiatum of the rat hippocampus, but much lighter in the corpus callosum. Immunostaining at the periphery of pyramidal cell soma may be associated with the plexus formed by axon terminals of hippocampal basket cells.

Fine structure of nerve endings in extraocular muscle.

Introduction Recent electron microscopic studies have demonstrated the presence of two distinct types of muscle fiber, Fibrillenstruktur and Felderstruktur, 1 in the extraocular muscles of guinea pig, 2 cat, 3 and man. 4 In the Fibrillenstruktur fibers, the myofibrils have a punctate appearance, and the sarcoplasmic reticulum surrounds clearly the individual fibrils. The Felderstruktur fibers, on the other hand, have larger myofibrils which are not regularly separated from each other and appear to be fused together. Physiologically, the Fibrillenstruktur fibers appear to be associated with relatively rapid twitch responses, whereas the Felderstruktur fibers respond to stimulation with prolonged contracture. 3 There are, in addition, two kinds of nerve endings in the extraocular muscles which can be histochemically differentiated by cholinesterase staining; these are the en plaque and en grappe endings. 2-7 The en plaque endings stain intensely and show the same configuration as the subneural apparatus of motor end-plates in

Muscle fibres and efferent nerves in a crustacean receptor muscle

ABSTRACT The accessory flexor muscle of the crab myochordotonal organ consists of two kinds of muscle-fibres. In one type the fibrillar material has a uniform punctate appearance in cross-section. The Z bands are often broken across the fibre width and are spaced 2 to 3μapart. These resemble the ‘Fibrillenstruktur’ muscle of vertebrates. In the second fibre type the fibrillar material appears as large clumps in cross-section and it is often difficult to distinguish the outlines of individual fibres. The sarcomere length is 10 to 12μ. These resemble the ‘Felderstruktur’ muscle of vertebrates. Two types of expanded structures are seen in conjunction with the two efferent neurones innervating this muscle. With fresh material under phase microscopy one type appears as a 20 by 40μrectangular plaque containing vacuoles as well as dark clumps and granules. The other kind of enlargement consists of a group of spheres giving the appearance of a grape cluster and measuring 40 μin its greatest dimension. It is suggested that these expanded structures may be associated with efferent nerve terminations in this muscle.

APPEARANCE OF THE TONGUE IN B. COLI INFECTION

To the Editor: —After observing for many years the characters of the tongue inB. coliinfection, I think these notes may have some practical interest. The tongue exhibits a strawberry punctate appearance. The more confluent and close the points or dots, the more serious the infection. On the contrary, they become more scattered and discrete as the infection subsides. They remain, however, while the infection continues. Case reports are not needed to prove this observation: it is enough to test it in a patient. If a bacteriologic examination of the centrifugalized urine is made,B. coliwill be usually found. This phenomenon suggests per se the diagnosis, and if present in other conditions, one may be sure that there is also an associated colibacillary infection. In a Spanish textbook they speak of this tongue as typical of influenza. It is not so. When this kind of tongue is seen