Product Of Immune Reaction Research Articles

Distribution of GABARAP and β-III-tubulin Proteins in the Rats Neocortex in the Neonatal Period after Exposure to Perinatal Hypoxia

The content of the intracellular transport protein GABARAP associated with the GABAA-receptor and the isoform of the cytoskeletal protein β-III-tubulin in the layers of the somatosensory area of the neocortex in the neonatal period in Wistar rats after exposure to hypoxia was studied. The model of human preterm pregnancy was used in the work. Immunohistochemical methods were used to detect GABARAP and β-III-tubulin. The exposure of hypoxia was carried out on the 2nd day after birth, in a special chamber with an oxygen content in the respiratory mixture of 7.8%. The study of the neocortex was carried out on days 5 and 10. It was shown that animals in control, by the end of the neonatal period (P10), the content of GABARAP (according to the staining density of the immune reaction product) significantly increased: in the upper layers II-III by 2, and in the deep layers IV-VI by 4 times compared with the earlier period development (P5). The content of β-III-tubulin in the layers of the cortex is distributed evenly. Animals that survived exposure to hypoxia in the neonatal period showed a significant increase in the content of GABARAP and β-III-tubulin in the upper layers of the neocortex. These changes occur against the background of a significant decrease in the content of the GABAA-receptor subtype (data obtained earlier). GABARAP and β-III-tubulin, as well as the GABAA-receptor, are part of a complex of elements mediating GABA transmission in the neocortex. Hypoxia causes a change in the content of these proteins in the cerebral cortex, which can lead to disruption of the inhibitory effects of GABA.

ALDEHYDE DEHYDROGENASE 1L1 IN RAT BRAIN CELLS

Aldehyde dehydrogenase belonging to family 1, member L1 (Aldh1L1) is one of the most important enzymes called astrocytes that determine such important functions of these cells as participation in folate metabolism and inactivation of formiate in the CNS. The aim of this study was immunocytochemical detection of cells synthetizing Aldh1L1 in rat brain, and determination of their topography and morphology features. The brain of adult male Wistar rats (n=10) was used at the work. The material was fixed in zinc-ethanol-formaldehyde, a special fixative providing high preservation of antigenic determinants. It was shown that A ldh1L1 is detected in all brain anatomic structures. Among brain cells expressing Aldh1L1 macroglia cells - astrocytes and ep-endymocytes - were identified. In astrocytes the most intensive immunohistochemical reaction was observed in the endfeets of processes of these cells and in superficial astrocytes forming the superficial glial border membrane. In ependymocytes, the product of immune reaction was detected in the cytoplasm as numerous granules of various shapes and size (from 0.1x0.2 to 1.3X3.7 pm). The predominant localization of Aldh1L1 in brain cells involved in transport of substances through blood-brain, CSF-brain, and CSF-blood barriers allows one to expect that this protein will prove to be useful functional marker during experimental investigations of the CNS barrier system and evaluating the reaction of brain to toxic metabolic products of alcohol surrogates.

Microstructure of immunoprecipitation patterns in gel: The mechanism of spatial self-organization of interacting protein populations in gel-like media

The description [1] utilized two models—the model of reaction–diffusion type and cluster aggregation model. Describing this system as a simple bimolecular reaction, the authors demonstrated that the immunoprecipitation structure was determined by spatial concentration of the immune reaction product. The concentrating region in the considered system has a shape of a regular ring. Study of the aggregation of AG and AB molecules using an imitation cluster model has demonstrated that the reaction product in this region is represented by rather large clusters, which allows for a visual recording of immunoprecipitation. At the first glance, the proposed description gives a comprehensive concept of the formation and structure of immunoprecipitation ring at both macroand microlevels. However, confocal microscopy examination of precipitation rings has demonstrated that they display a rather complex microstructure. The goal of this study was to explain the formation of this microstructure.

Immunocytochemical localization of annexin V (CaBP33), a Ca(2+)-dependent phospholipid- and membrane-binding protein, in the rat nervous system and skeletal muscles and in the porcine heart.

We investigated the ultrastructural localization of annexin V a Ca(2+)-dependent phospholipid- and membrane-binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca(2+)-dependent binding of the protein to brain and heart membranes, and on existence of both EGTA- and Triton X-100-extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells.

Detection of S-100b protein in Triton cytoskeletons: an immunocytochemical study on cultured Schwann cells.

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.

Immunocytochemical localization of S-100 beta beta protein in olfactory and supporting cells of lamb olfactory epithelium.

By immunocytochemistry, we have identified two novel cell types, olfactory and supporting cells of lamb olfactory epithelium, expressing S-100 beta beta protein. S-100 immune reaction product was observed on ciliary and plasma membranes, on axonemes and in the cytoplasm adjacent to plasma membranes and to basal bodies of olfactory vesicles. A brief treatment of olfactory mucosae with Triton X-100 before fixation is necessary for detection of S-100 beta beta protein within olfactory vesicles. In the absence of such a treatment, the immune reaction product is restricted to ciliary and plasma membranes. On the other hand, irrespective of pre-treatment of olfactory mucosae, S-100 beta immune reaction product in supporting cells is restricted to microvillar and plasma membranes. The anti-S-100 beta antiserum used in these studies does not bind to basal cells of the olfactory epithelium or to cells of the olfactory glands, whereas it binds to Schwann cells of the olfactory nerve. An anti-S-100 alpha antiserum does not bind to cellular elements of the olfactory mucosa, Schwann cells, or axons of the olfactory nerve. The present data provide, for the first time, evidence for the presence of S-100 beta beta protein in mammalian neurons (olfactory cells).

Immunocytochemical localization of S-100b protein in degenerating and regenerating rat sciatic nerves.

We studied the cellular and subcellular distribution of S-100b protein in normal, crushed, and transected rat sciatic nerves by an immunocytochemical procedure. In uninjured nerves, S-100b protein was restricted to the cytoplasm and membranes of Schwann cells, with no reaction product present in the nucleus or in axons. Similar images were seen from the first to the thirtieth day after the crush in activated Schwann cells during the degeneration period, i.e., up to the seventh post-lesion day, and in normal Schwann cells reappearing during the regeneration period, i.e., after the seventh post-lesion day, in the zone of the crush and proximal and distal to it. By the technique employed, there seemed to be no differences in the intensity of the immune reaction product in normal and activated Schwann cells. Also, similar images were seen in the proximal stump of transected nerves. Only a slight S-100b protein immune reaction product could be observed in the rare activated Schwann cells present in the distal stump around the seventh post-lesion day, the majority of cell types being represented by fibroblasts and elongated cells at this stage and thereafter. By immunochemical assays, similar results as those presented here have been reported and interpreted as indicative of the presence of S-100 protein in axons or, alternatively, of axonal control over expression of S-100 protein in Schwann cells. Our immunocytochemical data clearly show that the strong reduction in the S-100 protein content of the distal stump of transected nerves is owing to the paucity of Schwann cells and to the decrease in the S-100 protein content of these cells, rather than to degeneration of axons.

Resolving GABAA/benzodiazepine receptors: cellular and subcellular localization in the CNS with monoclonal antibodies

Monoclonal antibodies, raised against a purified GABAA/benzodiazepine receptor complex from bovine cerebral cortex, have been used to visualize the cellular and subcellular distribution of receptorlike immunoreactivity in the rat CNS, cat spinal cord, and bovine and postmortem human brain. Two different antibodies have been used for these studies; bd-17 recognizes the beta-subunit (Mr 55 kDa) in all the species tested, whereas bd-24 recognizes the alpha-subunit (Mr 50 kDa) of bovine and human but not rat and cat tissues. In bovine and human brain, both antibodies produced very similar staining patterns, indicating a homogeneous receptor composition, at least in the brain areas investigated. The general distribution and density of receptor antigenic sites in all tissues studied were very similar to that of benzodiazepine binding sites radiolabeled with 3H-Ro 15-1788 and of glutamate decarboxylase (GAD)-stained nerve terminals. The results demonstrate a very high receptor density (around neuronal cell bodies and processes or less discretely distributed) in the rat olfactory bulbs, cerebral cortex, ventral pallidum, islands of Calleja, globus pallidus, hippocampus, dentate gyrus, substantia nigra, geniculate nuclei, inferior colliculus, cerebellum, reticular formation, spinal cord, and retina. In contrast, no receptors could be detected in white matter, pineal, pituitary, adrenals, and superior cervical ganglia. Only among the cerebellar layers did we observe a conspicuous difference between the staining intensity and the radiolabeling. In bovine and postmortem human brain, e.g., hippocampus, dentate gyrus, cerebral cortex, and substantia nigra, the same close correlation between the immunohistochemical and radiohistochemical findings was observed. At the electron microscopic level, the immune reaction product in the rat substantia nigra and globus pallidus, for example, was localized to pre- and postsynaptic membranes of axodendritic and axosomatic synapses. Whether the presynaptic labeling represents GABA autoreceptors is discussed. In the near future, the monoclonal antibodies will be used in double-labeling experiments with GAD to identify those GABAergic projections that are modulated by benzodiazepine minor tranquillizers. Furthermore, they could also be used, in studies of postmortem human brain, to diagnose receptor dysfunction possibly associated with CNS disorders such as epilepsy.

Ultrastructural immunohistochemical localization of poliovirus during virulent infection of mice.

Ultrastructural immunohistochemistry was used to localize type 2 human poliovirus (HPV 2) during virulent infection of mice caused by the Lansing strain. In the spinal cord, immune-reaction product was exclusively localized within neurons and their processes. The absence of viral antigen in glial, endothelial and inflammatory cells further supports the strict neuronotropicity of HPV. In addition, viral antigen and virus-like particles were localized in synaptic complexes and axons, including preterminal axons. This clear demonstration of HPV in neuronal cell bodies, their axons, and synaptic elements strongly supports the hypothesis of HPV dissemination in the central nervous system via axonal transport.

Immunocytochemical localization of the myelin-associated glycoprotein Fact or Artifact?

Immune staining for the myelin-associated glycoprotein (MAG) by the peroxidase-antiperoxidase technique in vibratome, paraffin and Epon sections of peripheral nerve resulted in a periaxonal ring of immune reaction product and an absence of staining over compact myelin. The thicknesses of the periaxonal rings of staining in Epon sections were similar when axons were surrounded by single Schwann cell processes and when they were surrounded by compact myelin sheats. Thicker rings of periaxonal staining were present when axons were surrounded by multiple layers of uncompacted Schwann cell membranes. When swelling of the Schwann cell periaxonal cytoplasm separated the periaxonal Schwann cell membrane from compact myelin by distances that could be readily resolved in the light microscope, immune staining for MAG was found over the periaxonal Schwann cell membrane but not over compact myelin. Biochemical experiments in which myelin or MAG was treated with sodium ethoxide and hydrogen peroxide, under conditions similar to those used for etching Epon sections prior to immune staining, showed that the immune reactivity of MAG was stable to these treatments. Finally, a pre-embedding technique for immune staining at the electron-microscopic level showed MAG reaction product on the cytoplasmic side of Schwann cell periaxonal membranes and the membranes of Schmidt-Lanterman incisures and paranodal loops. These results confirm and strengthen the evidence from previous reports indicating that MAG is confined to periaxonal membranes, Schmidt-Lanterman incisures, paranodal loops, and the outer mesaxon of myelinating Schwann cells and is absent from compact myelin. These results are discussed in reference to a recent report apparently showing the presence of MAG in compact CNS myelin. Based on the data presented and discussed in this paper, it is concluded that MAG is located in specific non-compact regions of central and peripheral myelin sheaths and not in compact myelin lamellae.

Estrogen-responsive creatine-kinase in the reticulo-epithelial cells of rat thymus.

We have evaluated the presence of the so-called "estrogen-induced protein" (IP) in the thymus of immature rat. IP is one of the earliest proteins synthesized in response to estrogen in rat uterus and has recently been identified as the BB-isozyme of creatine-kinase (J. Biol. Chem. 256 (1981) 5741). Estrogen-exposed tissues were labelled with [35S]methionine and the soluble proteins separated by SDS-polyacrylamide gel electrophoresis followed by fluorography. It was observed that estrogen stimulated the labelling of a thymic protein with electrophoretic mobility identical to that of uterine IP. The effect was dose-related and specific for estrogen receptor ligands. Thymic IP, like that of the uterus, was immunoprecipitated equally well by either anti-IP antiserum from rat uterus or anti-BB-creatine-kinase antiserum from rat brain. After purification by ion-exchange and affinity chromatography, there was a parallel increase in the extent of recovery of IP and BB-creatine-kinase activity in both the thymus and the uterus. Using the immunohistochemical peroxidase-antiperoxidase method with anti-IP serum, the immune-reaction product was localized in a few cells identified as reticulo-epithelial cells. The thymocytes were always free of any reaction product. These data demonstrate that the reticulo-epithelial cells of rat thymus are a target for estrogens.