Brain transglutaminase: in vitro crosslinking of human neurofilament proteins into insoluble polymers.

The accumulation in aged human neurons of insoluble, high molecular weight filamentous polymers apparently linked by nondisulfide covalent bonds led us to examine human brain for the presence of transglutaminase (EC 2.3.2.13) and endogenous protein substrates for this crosslinking enzyme. We demonstrate the presence in brain of a transamidating enzyme that can covalently crosslink brain proteins into insoluble polymers in vitro by forming gamma-glutamyl-epsilon-lysine intermolecular bridges. Brain transglutaminase is Ca2+ dependent, has an electrophoretic mobility similar to that of erythrocyte transglutaminase, and is active in human postmortem brain from aged normal individuals and patients with Alzheimer disease (senile dementia). Brain neurofilament fractions incubated in the presence of transglutaminase, Ca2+, and the fluorescent amine dansylcadaverine form a fluorescent, nondisulfide-bonded insoluble polymer; this process is associated with a decrease in the amount of soluble neurofilament polypeptides in the preparation. Electron microscopy of the polymeric material reveals an extensive network of connecting filaments, which can be immunostained with various neurofilament antisera. Cystamine, an inhibitor of transglutaminase, prevents the neurofilament crosslinking. Glial filaments and myelin basic protein can also serve as substrates of brain transglutaminase in vitro. Although Alzheimer disease-type paired helical filaments are not formed under the specific in vitro conditions employed, the data suggest one possible mechanism for the covalent crosslinking of filaments into insoluble polymers during human neuronal aging.     

Neurofilament immunoreactivity in myenteric neurons differs from that found in the central nervous system

Neurofilaments are 10-nm diameter protein fibers found within neurons and are composed predominantly of a triplet of polypeptides usually referred to as low-, medium-, and high-molecular-weight subunits. We describe the results of a study of myenteric plexus neurons using a panel of neurofilament triplet protein antibodies and indirect immunofluorescence techniques. Polyclonal antibodies to each of the three neurofilament subunits reliably stained myenteric neurons and their processes, indicating the presence of all three proteins in these cells. However, several well-characterized monoclonal antibodies to epitopes on high- and medium-molecular-weight subunits showed immunoreactivity in brain tissue but not in myenteric neurons and their processes. Some of the antibodies that do not stain recognize only phosphorylated epitopes, indicating that the level of neurofilament phosphorylation is very low in enteric neurons. Other antibodies that are not thought to be sensitive to the level of neurofilament phosphorylation show reduced or no staining of enteric neurons, suggesting the presence of immunologically distinct neurofilaments in these cells. These results suggest the presence of modified neurofilament structures in enteric neurons, possibly reflecting their unique mechanical character within the moving intestinal wall.     

Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains

Neurofibrillary tangles (NFT) and neurites associated with senile plaques (SP) in Alzheimer disease-affected brain tissues were specifically immunostained with affinity-purified antibody preparations directed against ubiquitin. In addition, a class of neurites seen in brain regions containing NFT and SP were also specifically stained. Cross-reactivity of the ubiquitin antisera for tau protein, neurofilament proteins, and high molecular weight microtubule-associated proteins (MAPs) were ruled out by (i) the inability of the ubiquitin antisera to stain these proteins in immunoblotting experiments and (ii) the inability of tau, neurofilament, and MAP preparations, when preincubated with the ubiquitin antisera, to inhibit the selective neurofibrillar staining observed. Our results are consistent with the suggestion that ubiquitin is covalently associated with the insoluble neurofibrillary material of NFT and SP. We propose that the ubiquitin-mediated degradative pathway may be ineffective in removing these fibrillar structures in Alzheimer disease brain.     

日本血吸虫成虫培养细胞骨架系统的研究

目的：研究日本血吸虫成虫培养细胞的骨架系统。方法：将日本血吸虫成虫细胞接种于小盖玻片上,以RPMI－1640含20％小牛血清附加常量抗菌素的培养基培养,用考马斯亮蓝染色法和魁笔环肽荧光染色法显示培养细胞骨架。结果：考马斯亮蓝染色显示日本血吸虫成虫细胞骨架呈网络状结构,均钭分布于胞质中;用（NH4）2SO4抽提后考马斯亮蓝色显示细胞骨架无明显改变;鬼笔环肽荧光染色后可见细胞内有均匀荧光亮点。结论：日本血吸虫成虫培养细胞骨架呈致密网络状结构,以中间纤维为主,缺乏粗大的微丝束。     

The Extracellular Matrix is an Integrated Unit: Ultrastructural Localization of Collagen Types I,III, IV,V, VI,Fibronectin, and Laminin in Human Term Placenta

The human term placenta is used extensively as a source of extracellular matrix components. To elucidate the tissue distribution and interrelationships of seven of these components, monospecific antibodies directed against collagen types I, III, IV, V, VI, fibronectin, and laminin were reacted with human term placenta and studied by light and electron immunohistochemistry.Type I collagen was the basic structural unit of human term placenta, present as 30\2-35 nm, cross-banded fibers, often in the form of large fiber bundles. Type III collagen was present as thin 10\2-15 nm, beaded fibers often forming a meshwork which encased type I collagen fibers. Types V and VI collagen were present as 6\2-10 nm filaments, often closely associated with types I and III collagen. Type VI collagen also coated collagen fibers of all diameters, enhancing their periodicity, providing a staining pattern often similar to that observed with anti-fibronectin antibodies. Fibronectin was present in both maternal and fetal plasma and throughout the stroma of the chorionic villus, as both free filaments and coating collagen fibers. Basement membranes contained laminin and type IV collagen, but no fibronectin. In summary, the non-basement membrane proteins studied often codistributed with type I collagen, between and apparently attached to fibers, suggesting that they may act as binding proteins, linking type I fibers and bundles, to themselves and to other structures.     

Pineal parenchymal tumors: Cell differentiation and prognosis

Eleven pineal parenchymal tumors were studied using various antibodies specific to the central nervous system and cell-proliferation-related antigen MIB-1 in order to examine the divergent types of cell differentiation and also evaluate prognosis. Electron microscopy was also performed. All tumors were immunohistochemically positive to chromogranin A and B crystallin and were also highly positive to retinal S protein. Pineocytoma cells contained microtubules, intermediate filaments, glial bundles, clear-centered vesicles and synaptic apparati. Pineoblastoma cells also had microtubules and neurofilaments, but glial filaments and definite synapses were not identifiable. Pineal parenchymal tumors were considered to be of pinealocyte origin, and there was a continuous spectrum of divergent cell differentiation between pineocytoma and pineoblastoma cells. The MIB-1 labeling index correlated well with histological malignancy, neuronal differentiation evaluated immunohistochemically by both neurofilament protein and synaptophysin, and cases with seeding potentials. Although histopathological features of neuronal development were, until recently, seen as the hallmark of benign prognosis in pineal parenchymal tumors, they are now thought to be only one of the pieces of evidence that may be used for purposes of prognosis.Abbreviations PC pineocytoma - PB pineoblastoma - NFP neurofilament protein - GFAP ghal fibrillary acidic protein     

Dynorphin immunocytochemical localization in brain and peripheral nervous system: preliminary studies.

Using antisera specific for the opioid peptide dynorphin, we have carried out immunocytochemical studies of the distribution in rat brain and periphery. In the central nervous system, cells that stain positively for dynorphin are found in the supraoptic nucleus, with less-well-stained cells in the paraventricular nucleus of the hypothalamus. Few positive fibers were detected in brain, suggesting problems with fixation and preservation of antigenicity. In pituitary no staining was seen in the anterior and intermediate lobes but heavy staining was detected in the posterior lobe. In the guinea pig, adrenal chromaffin cells stained with dynorphin antisera. Staining of these cells could be blocked with excess of dynorphin-(1-13) or either enkephalin. Radioimmunoassays revealed a great excess of the enkephalins in the adrenal, suggesting cross competition between dynorphin antiserum and adrenal medullary enkephalin. Finally, the dynorphin antiserum stained a complex of fibers in guinea pig ileum. Staining of these fibers could be blocked by moderate amounts of enkephalin as well as by smaller amounts of dynorphin-(1-13). We conclude that in some places (brain and pituitary) dynorphin exists separately from leucine-enkephalin. In other parts of brain and in the periphery the relationship between dynorphin and the enkephalins is very complex and requires further study and improved antisera.     

Histologic and ultrastructural features of normal human parietal pericardium

Morphologic studies of normal anterior parietal pericardium from seven patients revealed this tissue to be composed of three layers: (1) the serosa, consisting of a surface layer of mesothelial cells and a narrow submesothellal space, (2) the fibrosa, containing variously oriented layers of collagen fibrils and small elastic fibers, and (3) the epipericardial connective tissue layer, containing mainly large coarse bundles of collagen and forming part of the pericardiosternal ligament. Scanning electron microscopic examination is most useful for study of the surface features of pericardial mesothelial cells, which have single cilia and are covered with microvilli. The latter bear friction and increase the surface area for fluid transport. Junctional complexes between adjacent mesothelial cells consist of desmosomes, which reinforce intercellular adhesion and zonulae occludentes, which form permeability barriers. Actin-like filaments (50 Å in diameter) are present in microvilli and in immediately subjacent regions of the cells; these filaments mediate changes in cell shape. Intermediate filaments (100 Å in diameter) are associated with desmosomes and form bundles in the perinuclear regions; these filaments provide structural support to the cytoplasm.     

Invariance and heterogeneity in the major structural and regulatory proteins of chick muscle cells revealed by two-dimensional gel electrophoresis.

A two-dimensional gel electrophoresis system is used to investigate some of the properties of desmin, the major subunit of the 100-A filaments from chick muscle cells, and to compare these properties to those of the other major contractile and regulatory proteins of muscle. Desmin from embryonic and adult smooth, skeletal, and cardiac muscle cells is resolved into two isoelectric variants, alpha and beta, which possess slightly different electrophoretic mobilities in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both the alpha and the beta variants from all six preparations appear to be identical in isoelectric point and apparent molecular weight. The alpha and beta desmin are present in approximately equal amounts in all three types of muscle, suggesting that both isoelectric variants of desmin serve as the structural subunits of the 100-A filaments in chick muscle cells. Tropomyosin also can be resolved into two subunits, alpha and beta, in all three types of muscle. However, in each type of muscle both subunits differ from their counterparts in the other types of muscle, either by molecular weight or by isoelectric point. These results indicate that, with regard to apparent isoelectric point and molecular weight, desmin, a major muscle structural protein, is invariant, while tropomyosin, a major muscle regulatory protein, exhibits heterogeneity in the three types of muscle.     

Brain-specific hyaluronate-binding protein: an immunohistological study with monoclonal antibodies of human and bovine central nervous system.

Hyaluronectin is a protein isolated from acid extracts of human brain by affinity chromatography on immobilized hyaluronate. With polyclonal antibodies, it was immunohistologically localized in the rat at the nodes of Ranvier of central and peripheral myelinated fibers and in mesenchymal tissues. Compared to adult rat, hyaluronectin-immunoreactive material was more abundant in embryonal rat brain and mesenchyma. We report a different localization in human and bovine tissues with monoclonal antibodies reacting with human hyaluronectin by NaDodSO4/PAGE and immunoblotting but not staining rat tissues by immunohistology. In human and calf the antigen reacting with hyaluronectin monoclonal antibodies was brain specific, while several peripheral tissues were stained by the polyclonal antibodies. In human and bovine central nervous system monoclonal antibodies stained white matter and tissues formed predominantly by glial fibers (e.g., subependymal glia). In white matter hyaluronectin-immunoreactive material formed a delicate mesh surrounding individual myelinated fibers, a pattern compatible with the distribution of fine astroglial processes in this location. Gray matter did not stain with monoclonal antibodies, the granular layer of the cerebellum excepted. The findings suggest that human hyaluronectin is heterogeneous and comprises at least two fractions. The main fraction is a brain-specific protein, probably produced by white matter astrocytes. Another fraction cross-reacting with rat is more abundant in embryonal tissues, including mesenchyma and brain.     

Cellular localization and characterization of Glut 3 glucose transporter isoform in human brain.

In the present study we examined the expression and localization of Glut 3 in human brain using peptide-specific antisera. Glut 3 was expressed at 2-3 times higher levels in cerebral cortex from adult (n = 6) compared to that from neonatal infants (n = 4; P less than 0.05). However, similar levels of immunoreactive Glut 3 were present in cerebellum from adults (n = 6) and newborns (n = 4). Cellular localization of Glut 3 in adult (n = 5) and neonatal (n = 5) infant brains was undertaken by immunohistochemical analysis. Glut 3 was visible in the adult neuropil of the cerebral cortex; in certain cellular processes within the deeper cortical layers; in intravascular white cells, including monocytes, lymphocytes and granulocytes; and in microvascular endothelial cells. Neither the premature nor the mature newborn cerebral cortex exhibited Glut 3 labeling in the neuropil or microvasculature. In the cerebellum, given the stratified nature of the cellular arrangement, Glut 3 was more clearly and definitively noted in the cellular processes at all stages of development. Double labeling studies using neuronal (neurofilament) and astrocytic (glial fibrillary acidic protein) markers indicated that Glut 3 was primarily expressed in neurons. We conclude that Glut 3 is localized in many cellular components, including white blood cells in human brain. The prominent localization of Glut 3 to mature neuronal processes suggests an essential role for this transporter in regulating fuel requirements for dendritic and axonal traffic, thereby mediating neurotransmission. Further study is required to address the possibility that another as yet undefined glucose transporter isoform is expressed in other cell-specific regions of the brain.     

Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells.

Antibodies to myosin light chain kinase, purified from turkey gizzard smooth muscle, were developed in rabbits and purified by affinity chromatography on a myosin light chain kinase-Sepharose 4B column. The purified antibodies crossreact with purified smooth muscle myosin light chain kinase but not with a variety of contractile or cytoskeletal proteins. The antibodies inhibit the catalytic activity of smooth muscle myosin light chain kinase and there is an inverse relationship between the kinase activity and the amount of antibody present in an assay. Half-maximal inhibition of myosin kinase activity occurs at an antibody/myosin kinase molar ratio of 10:1. The affinity-purified antibodies to smooth muscle myosin kinase were used to study the location of myosin kinase in a variety of nonmuscle cells. Immunofluorescence studies indicate that myosin light chain kinase is localized on microfilament bundles (stress fibers) in cultured fibroblasts. The stress fiber staining pattern is abolished when the antibodies are incubated with purified smooth muscle myosin light chain kinase prior to staining cells, while the staining pattern is unaffected when the antibodies are incubated with actin, myosin, alpha-actinin, or tropomyosin prior to staining. Moreover, the stress fiber staining pattern is periodic in well-spread gerbil fibroma cells and experiments have demonstrated that myosin light chain kinase appears to have the same periodic distribution as myosin but an antiperiodic distribution relative to alpha-actinin. These data indicate that myosin light chain kinase and its substrate, myosin, are in close proximity and are consistent with the hypothesis that myosin light chain kinase regulates actin-myosin interactions in nonmuscle cells.     

Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease.

The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death.     

Localization of bovine brain filament antibody on intermediate (100 A) filaments in guinea pig vascular endothelial cells and chick cardiac muscle cells.

Guinea pig vascular endothelial cells contain naturally occurring rings of intermediate filaments that completely encircle the nucleus. Indirect immunofluorescence staining showed that these perinuclear rings bound antibody prepared against protein from bovine brain 9-nm filaments. In endothelial cells grown in the presence of 1 muM demecolcine (Colcemid) the perinuclear ring "coils" into a juxtanuclear "cap". Throughout this process we could demonstrate staining of the intermediate filaments. Chick cardiac muscle cells in culture stained diffusely with the antibody. After treatment for 24 hr with 1 muM demecolcine the cardiac cells accumulated large bands of intermediate filaments. These bands stained intensely with the antibody. Our findings suggest that intermediate filaments in guinea pig endothelial cells and those induced in chick cardiac muscle cells are antigenically similar to bovine brain filaments. The staining of these filaments is not affected by treatment with demecolcine.     

Three-dimensional immunogold localization of alpha-actinin within the cytoskeletal networks of cultured cardiac muscle and nonmuscle cells.

The ultrastructural distribution of alpha-actinin was studied in cultured hamster heart cells by immunogold replica electron microscopy. This technique enabled us to localize alpha-actinin within the cytoskeletal networks at high resolution and in three dimensions. Colloidal gold, indicating the presence of alpha-actinin, was localized on the Z bands of nascent myofibrils in myocytes and on stress fiber bundles in nonmuscle cells. alpha-Actinin staining was also seen on stellate foci, where cytoskeletal filaments converged along the inner myocyte cell membranes. Intermediate filaments were associated with Z bands of myofibrils, stress fibers, and subplasmalemmal actin networks at the specific points where alpha-actinin was localized on these structures. Heavy meromyosin treatment prior to immunostaining confirmed that the thin filaments contained actin. These results suggest that alpha-actinin serves to interlink these various cytoskeletal elements. In addition, this protein may be involved in the initial phases of filament organization during myofibrillogenesis along the inner surface of the myocyte plasma membrane.     

Peptidergic Innervation and Endocrine Cells in the Human Liver

El-Salhy M, Stenling R, Grimelius L. Peptidergic innervation and endocrine cells in the human liver. Scand J Gastroenterol 1993;28:809-815.

Histologically normal liver biopsy specimens from patients with Hodgkin's lymphoma were investigated with three immunohistochemical methods for the occurrence of peptidergic nerve fibers and endocrine cells. Numerous immunoreactive nerve fibers were seen with antisera against peripheral nerves markers (neuron-specific enolase, neurofilament protein, and S-100). These nerve fibers were localized in the tunica media of branches of both the hepatic artery and portal vein, around the bile ducts, and in the connective tissue of the interlobular septa. In the liver, 10 types of peptidergic nerve fibers were detected: glucagon-, glucagon-like peptide- (GLP), somatostatin-, neuropeptide Y- (NPY), vasoactive intestinal polypeptide-, neurotensin-, gastrin/cholecystokinin C-terminus-, substance P-, serotonin-, and galanin-immunoreactive nerve fibers. GLP-, somatostatin-, NPY-, neurotensin-, substance P-, and galanin-immunoreactive nerve fibers were abundant; the other nerve fibers were scarce. The nerve fibers showed two distinct patterns of distribution: they occurred in the blood vessel wall and in connective tissue of the interlobular septum. Pancreatic polypeptide- and NPY-immunoreactive cells were found among the lining epithelial cells of the bile ducts in the interlobular septum.     

GABA-immunoreactive intrinsic and -immunonegative secondary neurons in the cat pineal organ

The pineal organ of the cat was studied by postembedding gamma-aminobutyric acid (GABA) immunocytochemistry. Two polyclonal rabbit GABA antisera were used with light microscopic peroxidase and electron microscopic immunogold techniques. A considerable number of intrinsic neurons are scattered in the proximal portion of the pineal organ. Some of the nerve cells were GABA-immunoreactive; other neurons as well as pinealocytes and glial/ependymal cells were immunonegative. A few GABA-immunoreactive neurons behave like CSF-contacting neurons by penetrating the ependymal lining of the pineal recess. GABA-immunoreactive neurons were more frequently found in the subependymal region. Small bundles of thin immunoreactive unmyelinated and thick immunoreactive myelinated nerve fibers occurred in the proximal pineal, especially near the habenular commissure. There were synapses of various types between GABA-immunoreactive and -immunonegative fibers. Myelinated immunoreactive axons seemed to loose their sheaths after entering the organ. Axon-like processes of pinealocytes terminated on dendrites of immunonegative neurons present near the posterior and habenular commissures. The axons of these neurons were found to join the commissural fibers and may represent a pinealofugal pathway conducting information originating from pinealocytes. The pinealocytic axons forming ribbon-containing synapses on dendrites of secondary neurons speak in favor of the sensory-cell nature of the pinealocytes. The pinealopetal myelinated GABA-immunoreactive axons and the intrinsic "GABA-ergic" neurons are proposed to inhibit the action of intrapineal neurons on which the pinealocytic axons terminate.     

Tropomyosin synthesis accompanies formation of actin filaments in embryonal carcinoma cells induced to differentiate by hexamethylene bisacetamide.

Hexamethylene bisacetamide (HMBA) induces in vitro the cytodifferentiation of PCC3/A/1 mouse embryonal carcinoma (EC) cells. In EC cells, actin is associated with surface structures but microfilament bundles are not seen. After 2 days of HMBA treatment, rounded EC cells are converted to flat adhesive ones with a developed cytoskeleton containing actin and tropomyosin. The ratio of actin to total proteins is constant in EC cells and their HMBA derivatives; but a striking difference is observed for one of the newly synthesized proteins (Mr 34,000) identified as tropomyosin. Synthesis of tropomyosin is followed by its association with actin microfilament bundles, as revealed by indirect immunofluorescence microscopy with specific antibodies.     

Intracellular distributions of mechanochemical proteins in cultured fibroblasts

We have used methods that have allowed simultaneous fluorescent staining of intracellular actin together with either myosin, filamin, or tubulin in normal rat kidney fibroblasts in monolayer culture. In the main portions of the cell body, the actin, myosin, and filamin are all present in two structures: in one, the three proteins are present in the same fiber bundles (stress fibers); in the other, there is a diffuse distribution of the three proteins. On portions of the cell periphery however-in the basal regions of microspikes, in ruffles, and in regions of cell-cell contact-actin and filamin are present, but myosin is severely depleted or absent. Microtubules are present in the cell body in a distribution independent of the stress fibers and are mostly absent from the cell periphery. Microspikes and ruffles are highly dynamic structures on the cell surface, and regions of cell-cell contact generally result from the association of ruffles on the two contacting cells. Therefore, the presence of filamin and actin but not myosin in these specialized regions on the cell surface, together with the recent demonstration [Wang, K. & Singer, S. J. (1977) Proc. Natl. Acad. Sci. USA 74, 2021-2025)] that pure filamin interacts with individual F-actin filaments in solution to form fiber bundles and sheet-like structures, suggest that in vivo filamin-actin interactions play an important role in the control of actin filament structure, in cell motility, and in the stabilization of cell-cell contacts.