Induction of cells with phenotypic features of neuronal cells by treatment with dibutyryl cyclic adenosine 3',5'-monophosphate in a human parotid gland adenocarcinoma cell line in culture.

A human parotid gland adenocarcinoma cell line, with an intercalated duct cell phenotype of the salivary gland and expression of vasoactive intestinal polypeptide and amylase, was cultivated in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP). Morphological changes occurred; cells formed long cytoplasmic processes densely packed with ample microfibrils, as well as microtubules, and grew in a netlike appearance. In addition, it has been found by the immunofluorescence staining technique, immunoblotting, or immunoelectron microscopy that the cells treated with dB-cAMP express neurofilaments, neuron-specific enolase, synaptophysin, and HNK-1 antigen, as well as the alpha- and beta-chains of tubulin, whereas these antigens are not detected in untreated cells. The expression of vasoactive intestinal polypeptide detected diffusely in the cytoplasm of untreated cells was restricted to the cell membranes during the cultivation of cells in the presence of dB-cAMP, while expression of amylase persisted in the treated cells in a fashion similar to that in untreated cells. Moreover, both anchorage-independent and anchorage-dependent growth of the cells was markedly suppressed in the presence of dB-cAMP. After removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype and growth rate of the untreated cells. These findings indicate that reversible conversion into cells with phenotypic features of neuronal cells of a human parotid adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Uroplakin I: a 27-kD protein associated with the asymmetric unit membrane of mammalian urothelium.

The luminal surface of mammalian urothelium is covered with numerous plaques (also known as the asymmetric unit membrane or AUM) composed of semi-crystalline, hexagonal arrays of 12-nm protein particles. Despite the presumed importance of these plaques in stabilizing the urothelial surface during bladder distention, relatively little is known about their protein composition. Using a mouse mAb, AE31, we have identified a 27-kD protein that is urothelium-specific and is differentially expressed in superficial umbrella cells. This protein (pI approximately 5.8) partitions into the detergent phase during Triton X-114 phase separation. Pulse-chase experiments using cultured bovine urothelial cells showed that this protein is synthesized as a 32-kD precursor that is processed through a 30-kD intermediate, to the mature 27-kD form. In cytoplasmic vesicles containing immature AUM, the AE31 epitope is detected in patches on the cytoplasmic side, but in mature, apical AUM it is detected exclusively on the luminal side. This suggests an unusual translocation of the AE31 epitope during AUM maturation; more data are required, however, to substantiate this interpretation. Immunoaffinity purification of the 27-kD protein results in the copurification in approximately molar ratio of a 15-kD protein, as well as a small and variable amount of a 47-kD protein. Immunoblotting data indicate that these three proteins are immunologically distinguishable. This copurified 15-kD protein is relative basic (pI approximately 8.0). Like the 27-kD protein, it is urothelium-specific and is present mainly in the umbrella cells. Together, our data indicate that a 27-kD protein is urothelial plaque-associated (uroplakin I). Based on complex formation data, we provisionally name the 15-kD protein uroplakin II; additional data will be required to determine whether this and the 47-kD protein are integral parts of AUM. The identification of these AUM-associated and -related proteins, plus the availability of a culture system capable of synthesizing and processing some of these molecules, offer new opportunities for studying the detailed structure, assembly, and function of asymmetrical unit membrane.

Alteration of the erythrocyte membrane skeletal ultrastructure in hereditary spherocytosis, hereditary elliptocytosis, and pyropoikilocytosis.

The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.

Immunohistologic abnormalities of the microfibrillar-fiber system in the Marfan syndrome.

BACKGROUND: Indirect-immunofluorescence studies of skin and cultured dermal fibroblasts from patients with the Marfan syndrome demonstrate apparent deficiency of one element of connective tissue--the microfibrillar-fiber system--in assays using specific antibodies against fibrillin, a major microfibrillar protein. This study was designed to test whether these immunostaining abnormalities are consistent and diagnostic features of the disease. METHODS: We studied patients with either the Marfan syndrome or various other inherited connective-tissue disorders and normal subjects according to a single-blind protocol in which coded samples of skin, fibroblast cultures, or both were analyzed without knowledge of the clinical diagnosis and classified as "Marfan" or "non-Marfan" before the sample codes were broken. RESULTS: Of the 27 patients with the Marfan syndrome, 24 were correctly identified by the decreased content of microfibrillar fibers in their skin, cultured fibroblasts, or both; in contrast, 19 of 25 patients with other heritable disorders of connective tissue and all 13 normal subjects were correctly classified as "non-Marfan" by these assays (P less than 0.001). CONCLUSIONS: These results document consistent, relatively specific abnormalities of microfibrillar fibers in the Marfan syndrome. The biomechanical incompetence of these structural elements, due to quantitative or qualitative abnormalities, may account for the pleiotropic clinical manifestations of the disease. Therefore, various defects in the expression, structure, assembly, or degradation of the constituent structural glycoprotein (or glycoproteins) of microfibrils may be implicated in the causation of the Marfan syndrome.

Cytokeratin expression during AFB1-induced carcinogenesis.

The early stages of the carcinogenic process induced by aflatoxin B1 (AFB1) in rat liver during 24 weeks of feeding and the resulting tumours have been studied with respect to cytokeratin (CK) expression. A previously uncharacterized monoclonal antibody, MRCTU/J1, has been shown to recognize rat CK18 and together with antibodies against human CK8, 18 and 19, has been used to examine the possible lineage of tumour cells and also to identify the altered foci that might be most relevant to tumorigenesis. Results suggested that AFB1-induced transformation in liver may occur in more than one cell type, since tumours with the normal hepatocyte CK pattern and those with bile duct or oval cell CK phenotype were identified. Additionally, hepatocytes with a bile duct CK phenotype appeared during the early stages of carcinogenesis. The in vivo pattern of CK expression also appeared to be maintained in one normal and one hepatoma-derived cell line. Overexpression of CKs (particularly of CK19) was a much more selective marker for altered foci, compared to gamma-glutamyltranspeptidase, and was more consistently expressed at high levels in tumours, suggesting that it might be a more reliable way of identifying those cells involved in the transformation process.

Glycophorin C content of human erythrocyte membrane is regulated by protein 4.1.

Human erythrocyte transmembrane sialoglycoprotein, glycophorin C, plays a functionally important role in maintaining erythrocyte shape and regulating membrane material properties, possibly through its interaction with protein 4.1. Moreover, it has previously been shown that membranes deficient in protein 4.1 exhibit decreased content of glycophorin C. To further define the relationship between protein 4.1 and glycophorin C, a series of studies were performed using both protein 4.1- and glycophorin C-deficient erythrocytes. Quantitation by flow cytometry showed that the glycophorin C content of cells totally deficient in protein 4.1 was 9% of normal and that of cells partially deficient in protein 4.1 was 44% of normal. Interestingly, while homozygous glycophorin C-deficient cells had no detectable levels of this sialoglycoprotein, cells from obligate heterozygotes had normal levels. Protein 4.1 content of membranes of these glycophorin C-deficient cells was also normal. These data suggest that glycophorin C may be synthesized in excess by erythroid cells and its membrane content regulated by protein 4.1. To investigate if this regulation is due to association between protein 4.1 and glycophorin C, we examined the retention of glycophorin C in membrane skeletons (Triton shells) prepared from normal membranes, protein 4.1-deficient membranes, and protein 4.1-deficient membranes reconstituted with exogenous protein 4.1. Glycophorin C is retained by Triton shells prepared from normal membranes, whereas Triton shells prepared from protein 4.1-deficient membranes are totally devoid of this sialoglycoprotein. However, reconstitution of protein 4.1-deficient membranes with purified protein 4.1 resulted in retention of glycophorin C with the Triton shells. This finding suggests that protein 4.1 is necessary for association of glycophorin C with the membrane skeleton. Furthermore, these data suggest that through its interaction with glycophorin C, protein 4.1 may play a role in regulating the membrane content of this sialoglycoprotein in mature human erythrocytes.

A polypeptide of 59 kDa is associated with bundles of cytoplasmic filaments in Neurospora crassa.

Complex arrangements of filamentous structures have been isolated from vegetative cells of the fungus Neurospora crassa. They were enriched by differential centrifugation and purified by permeation chromatography. The filamentous structures are made up of units of 8-10 nm diameter and were isolated in bundles of up to six to nine units. The main constituent of these structures is a polypeptide with an apparent molecular mass of 59 kDa (P59Nc), which represents 4-5% of the total N. crassa proteins. The filamentous structures are cold-stable and are not affected by high-ionic-strength solutions or by the presence of 10 mM-EDTA or 1% (w/v) Triton X-100; they were disassembled by raising the pH of the solution or by using Tris-based buffers. The disassembled form assembled into structures sedimentable at 105,000 g after dialysis against the isolation buffer. The sedimentable structures were organized in the form of regular aggregates of 42-45 nm polypeptides and reacted weakly with anti-IFA, a monoclonal antibody which recognizes an epitope common to many of the higher-eukaryote intermediate-filament polypeptides. Immunofluorescence examination of wall-digested hyphae of N. crassa using affinity-purified antibodies prepared against P59Nc showed immunostaining of abundant filamentous and dot-shaped structures distributed in the cytoplasm.

[Immunohistochemical analysis of the liver using monoclonal antibodies against intermediate filaments]. / Imunohistochemická analýza pecene pomocou monoklonálnych protilátok proti niektorým typom intermediárnych filamentov.

The expression of some types of cytokeratin and vimentin in the liver was studied by using monoclonal antibodies to these types of intermediary filaments. Necrotic samples from livers without pathological finding were examined. A panel of 5 monoclonal antibodies, prepared in Czechoslovakia, to cytokeratins Nos. 7,8,18, and 19, as well as to vimentin was used. The findings confirmed the characteristic expression of cytokeratins 8 and 18 in hepatocytes and of cytokeratins 7, 8, 18, and 19 in epithelial cells of intrahepatic bile canaliculi, that monoclonal antibodies prepared in Czechoslovakia (LAMO, Brno and VUKEO, Brno) can be successfully used in immunochemical studies of intermediary filaments in the liver.

Insulin induces cell adhesion and normal flattened morphology in Krebs II ascites tumour cells.

Insulin induced Krebs II ascites cells to attach to the substratum and to adopt a flattened morphology associated with normal adhesion and movement. The changes were associated with a reorganization of cellular actin. The results show that insulin has important effects on cell structure and morphology. Insulin may thus be involved in the nutritional control of normal and malignant growth.

Neuroimmunohistochemical analysis of peridiscal nociceptive neural elements.

Twenty-three perioperative tissue samples from lumbar disc operations on 11 patients were studied immunohistochemically using the sensitive avidin-biotin-peroxidase complex (ABC) method and specific heterologous antisera for the presence of neurofilament-positive neural elements containing nociceptive neuropeptides substance P (SP) and/or calcitonin gene-related peptide (CGRP). Histologically, neural elements were especially abundant in the posterior longitudinal ligament, there being also a few demonstrable nerves in the peripheral anulus fibrosus. These nerves often showed a co-localization of cytoskeletal neurofilaments together with SP and/or CGRP immunoreactivity. It is suggested that pressure and chemical irritation of nociceptive nerves dependent on degenerated discs excite sensory neural elements, especially in the posterior longitudinal ligament and possibly also in the peripheral parts of the anulus fibrosus, while the disc itself, at least if not penetrated by vascular granular tissue, is painless and neuroanatomically lacks a structural basis for pain perception.

Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells.

We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.

The analysis of glycoproteins in cells and tissues by two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to identify and analyse subsets of proteins in cells and tissues. The combination of 2-D PAGE and [125I] concanavalin A overlay revealed an extraordinary complexity and diversity in the glycoprotein profiles of different cell types. However, the glycoproteins are not expressed idiosyncratically. Rather, their expression is closely linked to the state of differentiation of a particular cell type. Such glycoproteins can therefore be used to generate antibodies specific for differentiated cells. 2-D PAGE analyses of cellular glycoproteins also revealed a major common glycoprotein of 100 kDa. This was localised to the lumen of the endoplasmic reticulum and is referred to as endoplasmin. The combination of 2-D PAGE with electroblotting and 45Ca overlay revealed that endoplasmin and several other luminal endoplasmic reticulum proteins (reticuloplasmins) are high capacity, low affinity calcium binding proteins which could function as calcium storage proteins in the endoplasmic reticulum. One of these called calreticulin is also found in the sarcoplasmic reticulum. 2-D PAGE and 45Ca overlay has been used to demonstrate the presence of a calcium-binding protein (CP22/sorcin) in the cytosol of rodent multidrug resistant cells. Analyses of murine serum by 2-D PAGE revealed the presence of a novel stress protein serum amyloid P component. These studies illustrate the value of 2-D PAGE when used in combination with detection methods which select specific subsets of proteins such as glycoproteins.

Isolation and characterization of endothelial cells from bovine cerebral arteries.

For our laboratory's investigation into the role of the endothelial cells in vasospasm following subarachnoid hemorrhage and in inflammatory diseases, we found it necessary to devise a modified method of cell culture, which would be appropriate for studying human endothelial cells from lobectomized brain. We report our techniques to increase cell harvest and ensure reproducibility, our method of culturing endothelial cells from bovine major cerebral arteries, and our morphologic and immunocytochemical characterization of these cells. To increase the harvest of endothelial cells, the blood cells were washed from the lumen of the major cerebral arteries at the slaughterhouse and a modified reversed vessel technique was employed. The monolayer of cultured endothelial cells displayed a cobblestone appearance when it reached confluency and transmission electron microscopy revealed junctional complexes and interdigitation of cytoplasm at Passages 10 and 17. The cells stained positively for Factor VIII-related antigen at Passages 3, 5, 7, 10, and 15. Also the cells metabolized acetylated low-density lipoprotein at Passage 8. To determine the purify of the cultured endothelial cells, an immunocytochemical study of the cytoskeleton was performed on Passage 5 cells using either rhodamine-phalloidin or antibodies against smooth muscle myosin, desmin, and vimentin.

The role of membrane skeletal-associated alpha-globin in the pathophysiology of beta-thalassemia.

The beta-thalassemic mouse provides a useful model for testing hypotheses about the pathophysiology in human beta-thalassemia. The clinical picture of these mice and their red blood cell deformability characteristics are quite similar to those observed in human beta-thalassemia intermedia. The creation of transgenic mice that express human beta-globin (beta s) has provided an opportunity to study the effect of increasing the non-alpha-globin chain production on the thalassemic phenotype. A small increase in beta-globin production produces transgenic mice that are healthier, have almost normal hemoglobin values, and whose red blood cell deformability is increased. We quantified and characterized the membrane skeletal-associated globin in normal, transgenic thal/sickle, and thalassemic mice and showed that only alpha-globin was associated with the membrane skeleton in the pathologic red blood cells, and that the degree of rigidity as measured in the rheoscope correlated directly and closely with the amount of membrane skeletal-associated globin in these abnormal red blood cells.

Neurofilament and chromogranin expression in normal and neoplastic neuroendocrine cells of the human gastrointestinal tract and pancreas.

To differentiate neuroendocrine (NE) neoplasms arising at different levels of the gut and pancreas, the authors studied the expression of neurofilament (NF) proteins and chromogranin (CR) in normal and neoplastic NE cells of the human gastrointestinal tract (GIT) (14 ileal/jejunal carcinoids, six appendiceal carcinoids, 11 rectal carcinoids) and pancreas (23 islet cell tumors). Among pancreatic islet cell tumors, those with middle molecular weight (NF-M)-positive cells were more abundant than those with high molecular weight (NF-H)-positive cells; nearly all of these tumors expressed CR. Although NF-M was abundantly expressed in greater than 50% of tumor cells in a subset of these tumors, only one of these tumors exhibited diffuse immunoreactivity with NF-H. Among rectal carcinoid tumors, NF-M and NF-H-positive cells were present in approximately the same number of tumors, yet only diffuse immunoreactivity to NF-H could be detected. Chromogranin immunoreactivity in greater than 50% of tumor cells was present in 74% of islet cell tumors, 93% of ileojejunal carcinoids, and 83% of appendiceal carcinoids, but only in a minority of rectal carcinoids (36%). Although ileojejunal carcinoid tumors rarely expressed NF-M and did not express NF-H, diffuse immunoreactivity with CR was present in nearly all of these tumors. None of the appendiceal carcinoid tumors expressed NF-M or NF-H, yet all of these tumors demonstrated immunoreactivity with CR. Neurofilament immunoreactivity was not detected in normal GIT and pancreatic NE cells, whereas CR immunoreactivity was always present. These results suggest that for NE neoplasms of the GIT and pancreas the differential expression of NF subtypes appears to be related to tumor site; and CR is a marker of most GIT and pancreatic NE neoplasms although NF may discriminate subtypes of GIT and pancreatic NE tumors. Neurofilament subtyping may be useful in the evaluation of the origin of NE tumors presenting as metastatic lesions.

[Correlations of intracellular and cellular regeneration of the epithelium of the kidney tubules in necronephrosis]. / Sootnoshenie vnutrikletochnoi i kletochnoi regeneratsii épiteliia mochevykh kanal'tsev pochki pri nekronefroze.

The study of radioautographs in ultrathin and semithin sections extracted from animal tissues and following the impulsive as well as long-term injection of 3H-thymidine was made. Intracellular regeneration processes (external cell membrane synthesis, DNA reparation, restoration of the lost albumin-synthesizing complex and cytoskeleton) turned to develop in proximal canals of epithelial cells. The processes were aimed at the liquidation of distortions preventing cell from entering mitosis.

Excimer fluorescence of pyrenyliodoacetamide-labeled tropomyosin: a probe of the state of tropomyosin in reconstituted muscle thin filaments.

Rabbit skeletal tropomyosin (Tm) specifically labeled at cysteine groups with N-(1-pyrenyl)-iodoacetamide (PIA) exhibits excimer fluorescence. The excimer fluorescence was sensitive to the local conformation of Tm, to actin binding, and, in reconstituted thin filaments, to the Tm state change induced by binding of myosin subfragment 1 (S1). The properties of PIATm were similar to previously studied pyrenylmaleimide-labeled Tm (PMTm) [Ishii, Y., & Lehrer, S.S. (1985) Biochemistry 24, 6631] except that S1 binding to actin-Tm increased the excimer fluorescence in contrast to the time-dependent decrease seen for PMTm. The fluorescence properties of PIATm are sensitive to the Tm chain-chain interaction via equilibria among pyrene configurations and nonfluorescent dimer as well as the monomer and excimer-forming configurations. The effect of bound troponin (Tn) on the excimer fluorescence of PIATm in the reconstituted systems was dependent on ionic strength with a slight Ca2+ dependence. S1 titrations in the absence and presence of Tn and Ca2+ indicated that the excimer fluorescence probes the state change of Tm from the weak S1 binding state to the strong S1 binding state which is facilitated by Ca2+ [Hill et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186]. Binding of MgADP-S1 and MgAMPPNP-S1 produced the same total excimer fluorescence change as for nucleotide-free S1, showing that the strong S1 binding state of Tm-actin is independent of nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Whole-mount preparations of mouse lens epithelium for the fluorescent cytological study of actin.

A technique is given for the preparation of a sheet of epithelial cells from the capsule of the crystalline lens. A new method is described for fixation and staining with fluorescent phalloidin or actin antibody in order to localize the actin cytoskeleton in this tissue. Optical section of the preparation resolves such actin features as apical polygonal arrays, sequestered actin bundles, perinuclear actin aggregates, observed here for the first time, and filamentous networks in the basal region of the cell. This method is superior to previous ones in its ability to preserve actin-abundant sectors distinctively.

Buccal mucosa fibroblasts and periodontal ligament cells perturbed by tensile stimuli in vitro.

Human buccal mucosa fibroblasts and periodontal ligament cells grown in tissue culture were subjected to tensile forces approximating those used for orthodontic bodily tooth movement. The cells were synchronized into pre S phase and positively tested for response to nonmechanical physical stimuli. Two-dimensional gel analysis and immunohistochemical analysis of the three cytoskeletal components showed a lack of response. Similar negative results were found when the cells were perturbed in the presence of substance P. We hypothesize that perhaps these cells respond more readily to injury, a secondary effect of the forces of tooth movement, than to tensile forces.

Prosomes, subcomplexes of untranslated mRNP.

PROSOMES are a novel class of small RNP particles of uniform morphology, but of variable RNA (pRNA) and protein composition (about 650,000 MW; 12 nm diameter in the EM). They were discovered as subcomplexes of free mRNP, tightly attached to inactive mRNA in the cytoplasm. The pRNAs hybridize stably to mRNA. Prosomes associate in vitro to mRNA and inhibit cell free protein synthesis inducing an mRNA structure unable to interact with ribosomes. Many types of prosomes were observed. The individual particle is made up by a variable combination of about 20 characteristic proteins and one or several pRNa. Some prosomal proteins are glycosylated, phosphorylated and, possibly, ADP-ribosylated and are highly conserved in evolution whilst others vary with the species and the mRNA population they are associated to. A protease activity was found associated to prosomes. The function(s) of the prosomes is(are) still unknown. The differential inhibition of in vitro protein synthesis points to a capacity to recognize mRNA and to keep it in an inactive state. The observation with the aid of monoclonal antibodies (pMABs) that prosomes and thus mRNP are attached to the intermediate filaments (IF) raises the question if one of the functions of the IF might be in the topological distribution of mRNA within the cell. Similar to the cytokeratin fibers, the prosome networks bridge neighboring cells at specific positions. The nucleus also contains some prosomal antigens, located on chromosomes and on the nuclear matrix. Their presence and distribution in the cell compartments varies with the cell type and the prosomal antigen probed. Oocytes contain large amounts of prosomes.(ABSTRACT TRUNCATED AT 250 WORDS)