Exposure of lumenal microtubule sites after mild fixation.

High-resolution analysis of tubulin structure and docking the structure of tubulin dimer into a map of microtubules led to a prediction that sites for tubulin acetylation are in the interior of microtubules. This is somehow difficult to reconcile with their susceptibility to proteases and acetylation in assembled microtubules. To assess the availability of acetylated alpha-tubulin for antibodies, immunofluorescence on detergent-extracted cells, on cells fixed under various conditions and in microinjected cells was performed with monoclonal antibodies of known epitope locations. The presented data indicate that acetylated alpha:Lys40 is not exposed on unfixed microtubules but that this region of lumenal microtubule surface becomes easily exposed under mild fixation conditions.

Exposure of tubulin structural domains in Nicotiana tabacum microtubules probed by monoclonal antibodies.

A panel of nine antibodies, specific to antigenic determinants located on N- or C-terminal structural domains of alpha and beta subunits of animal tubulin, and antibodies against acetylated, tyrosinated and polyglutamylated tubulins were utilized for probing the Nicotiana tabacum microtubules. The specificity of antibodies was confirmed by immunoblotting on whole cell lysates and on tubulin isoforms separated by high-resolution isoelectric focusing. Whereas antibodies TU-01 and TU-09 reacted with all alpha-tubulin isoforms and TU-06 reacted with all beta-tubulin isoforms, the other antibodies reacted with a limited number of tubulin isoforms. Antibody TU-14 reacted only with two beta-tubulin charge variants. In fixed cells, each of the antibodies stained microtubules of preprophase band, mitotic spindle and phragmoplast. Cortical microtubules were stained by all antibodies except TU-02 and TU-03, which did not decorate microtubules in interphase cells. Immunostaining of unfixed detergent-extracted cells revealed that antibodies against determinants on the C-terminal domains of both subunits decorated microtubules, but these were not stained with antibodies to determinants on the N-terminal domains. These data indicate that in plant microtubules at least several parts of the N-terminal domains of both subunits are either not exposed on the microtubule surface or are masked by the other proteins. In contrast, parts of the C-terminal domains are exposed on the exterior of microtubules. As for animal tubulins the majority of posttranslational modifications as well as binding sites for microtubule-associated proteins (MAPs) have been located to these regions, it is possible also in higher plants that the C-terminal structural domains of both tubulin subunits participate in the modulation of tubulin interactions with associated proteins.

Heterogeneity of microtubules recognized by monoclonal antibodies to alpha-tubulin.

A set of four monoclonal antibodies against tubulin (TU-01, TU-02, TU-03, and TU-04) were produced using pig brain microtubule protein as antigen. Their characterization shows that all recognize antigenic determinants located on the tubulin alpha-subunit. However, peptide mapping of isolated alpha-tubulin, followed by immunoblotting with the monoclonal antibodies, shows that the antigenic determinants are located on different peptide fragments in at least three cases. The immunoreactivity with tubulins from different cells and tissues, ranging from eukaryotic microorganisms to man, was studied by immunoblotting and immunofluorescence. The antigenic determinants recognized by the antibodies are not uniformly distributed but, in some instances, are absent from tubulins of lower eukaryotic cells. These antibodies also make it possible to distinguish between different sets of microtubules within individual cells. Antigenically different microtubules are particularly evident in mouse spermatozoa and in some protozoa (T. vaginalis, H. muscarum, L. tropica, N. gruberi) possessing different sets of microtubules with different functions. These monoclonal antibodies can clearly identify the heterogeneity of tubulin or microtubules both from different organisms and within the same cell.

Distribution of non-class-III beta-tubulin isoforms in neuronal and non-neuronal cells.

beta-Tubulin isoforms in brain tissues and in cell lines were analyzed by high-resolution isoelectric focusing in combination with monoclonal antibodies. Post-translational modifications of brain non-class-III beta-tubulin isoforms were found in phylogenetically distant species ranging from pig to carp. Less extensive modifications were also observed in Neuro-2a, HeLa and 3T3 cells, where most acidic isoforms were glutamylated, while the basic, most abundant isoforms were not. The data suggest post-translational modification of non-class-III beta-tubulin isoforms in neuronal as well as in non-neuronal cells. Such modification might modulate interaction of tubulin with microtubule-associated proteins.

Changes of MAP2 phosphorylation during brain development.

The microtubule-associated protein MAP2 is essential for development of early neuronal morphology and maintenance of adult neuronal morphology. Several splice variants exist, MAP2a-d, with a lack of MAP2a in cat brain. MAP2 is widely used as a neuronal marker. In this study we compared five monoclonal antibodies (MAbs) against MAP2. They show differences in the immunocytochemical distribution of MAP2 isoforms during development of the visual cortex and cerebellum of the cat. Local and temporal differences were seen with MAb AP18, an antibody directed against a phosphorylation-dependent epitope near the N-terminal end. In large pyramidal dendrites in visual cortex, the AP18 epitope remained in parts immunoreactive after treatment with alkaline phosphatase. Three MAbs, AP14, MT-01, and MT-02, recognized the central region of the MAP2b molecule, which is not present in MAP2c and 2d, and reacted with phosphorylation-independent epitopes. During the first postnatal week the immunostaining in cerebellum differed between antibodies in that some cellular elements in external and internal granular layers and Purkinje cells were stained to various degrees, whereas at later stages staining patterns were similar. At early stages, antibody MT-02 stained cell bodies and dendrites in cerebral cortex and cerebellum. With progressing maturation, immunoreactivity became restricted to distal parts of apical dendrites of pyramidal cells and was absent from perikarya and finer proximal dendrites in cortex. MT-02 did not stain MAP2 in cerebellum of adult animals. This study demonstrates that the immunocytochemical detection of MAP2 depends on modifications such as phosphorylation and conformational changes of the molecule, and that MAP2 staining patterns differ between MAbs. Phosphorylation and specific conformations in the molecule may be essential for modulating function and molecular stability of MAP2, and monoclonal antibodies against such sites may provide tools for studying the functional role of modifications.

Maintenance of allogeneic cell recognition in allograft tolerant mice.

The organ distribution of 51Cr-labelled lymph node cells following transfer to syngeneic, allogeneic and specifically tolerant allogeneic recipients were compared. Neonatal induction of transplantation tolerance in several donor-recipient combinations involving different H-2 haplotypes had no effect on the reduction of homing into lymph nodes and the values of the homing did not differ from those of the untreated allogeneic recipients. In both cases, they amounted to 45-55% of the syngeneic controls. Even those neonatally treated animals, which had not been rendered tolerant at birth and rejected test skin grafts, were not capable of removing the transferred allogeneic cells by a typical immune elimination. The results indicate that animals recognize allogeneic cells, irrespective of whether or not they reject test skin grafts.

Monoclonal antibodies against transferrin. Precipitating mixtures and lack of inter-species cross-reactivity.

Five stable hybridoma lines were prepared using the myeloma cell line P3-X63-Ag.653 and spleen cells of mice hyperimmunized by pig transferrin. All hybridomas grew well in mouse peritoneal cavity and produced antibodies of the IgG1 subclass. Antibody preparations obtained from ascitic fluids tested for their capacity of antigen precipitation. No precipitation was obtained with single antibodies and with pairs of antibodies. Three out of 10 possible triads gave clear and sharp precipitation zones and rings in immunodiffusion tests performed in agar gel. All 5 antibodies were shown by quantitative enzyme-immunoassay to be specific for pig transferrin: no cross-reaction was obtained with mouse, human, horse and sheep transferrins.

Purification of mRNA for immunoglobulin kappa-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA.

The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin kappa-chains of mouse myeloma MOPC 21 and mouse hybridoma PTF-02. The DNA fragment comprising the 3'-untranslated region and a part of the constant region of the kappa-chain gene was covalently attached to diazobenzyloxymethyl-cellulose and used as an affinity adsorbent. A homogeneous 14S mRNA species was obtained by hybridization of total mRNA to the affinity adsorbent at 52 degrees C and by elution at 60 degrees C. Addition of the purified mRNA to a fractionated cell-free translation system resulted in a significant increase in the radioactivity immunoprecipitated by pig anti-mouse immunoglobulin antibodies. A single radioactive polypeptide of apparent Mr of 25,000, corresponding obviously to the kappa-chain, was identified as the only translation product.

Limited mobility of membrane receptors of adhered or proteose peptone-stimulated macrophages.

The mobility of the membrane receptors of macrophages, obtained from normal and proteose peptone-stimulated mice, was studied by counting the cells which form patches or caps after incubation with anti-H-2a antibody or soybean agglutinin (SBA). The test was carried out with macrophage suspension by the indirect fluorescence method. When macrophages from unstimulated donors were used, the antibodies against H-2a antigens bind at 4 degrees C to the surface of the macrophages in the form of small aggregates; only with SBA lectin a larger percentage of cells (20%) does form caps even at this low temperature. After heating the cells to 37 degrees C, a significant increase in the number of cells with receptors in caps was observed in the control mice. When macrophages from stimulated donors were used, no cap formation was observed with any of the ligands, even after a 120-minute-incubation; the ligand-receptor complexes form patches predominantly on the membrane. The pretreatment of macrophages with colchicine did not lead to a cap formation. A similar inhibition of the capping phenomenon also occurred with macrophages obtained from unstimulated mice, but tested in a monolayer.

Expression of class III beta-tubulin in neuroendocrine tumours of gastrointestinal tract.

Class III b-tubulin is presented as a specific marker for the cells of neuronal origin as well as for the tumours originating from these cells. Its expression is considered one of the earliest events that appear in the cells revealing neuronal differentiation. Using monoclonal antibody TU-20 in an immunohistochemical analysis, we studied the expression of class III b-tubulin in gastrointestinal carcinoid tumours. Paraffin-embedded, formalin-fixed tissue sections from 49 tumour samples obtained from following locations: stomach (4 cases), small intestine (8 cases), appendix (18 cases), rectum (3 cases), pancreas (5 cases), liver metastases (7 cases) and lymph node metastases (4 cases) were used in the study. In 41 of the 49 tumour samples (83.7%), positive staining for class III b-tubulin was detected, while 8 tumour samples (16.3%) were negative. Expression of class III b-tubulin was prominent in all three rectal carcinoids and in three atypical carcinoids located in small intestine. Pancreatic neuroendocrine tumours revealed either weak immunostaining (2 cases), or were negative for this marker (3 cases). The intensity of class III b-tubulin immunolabelling was not related to the degree of tumour differentiation. The results of this study suggest that class III b-tubulin could be a perspective marker for gastrointestinal neuroendocrine tumours. Moreover, the differences in its expression could also elucidate some aspects of histogenetic relationships of neuroendocrine tumours of gastrointestinal tract.

Adherence of lymphocytes to Leydig cell surface as an effect of the functional activity of Leydig cells in vitro.

Formation of lymphocyte rosettes around unstimulated and/or LH/hCG-treated Leydig cells in culture as well as in suspension was investigated. The ability of Leydig cells to interact with lymphocytes was positively correlated with their steroidogenic activity, as measured by means of a histochemical test for delta (5)3 beta-HSD activity and by radioimmunoassay for androgen level in culture media. In order to rule out nonspecific binding of lymphocytes, rosette specificity tests were performed. Time-dependent dynamics of cell-cell interaction was also followed. A phenomenon resembling phagocytosis of the adhered lymphocytes was observed after 4 h of co-culture. Morphology of this interaction was examined under light microscope and transmission electron microscope.

Detection of blood group A antigen expression in human colon cancer using monoclonal antibodies with different specificities.

Blood group antigen expression in human colon cancer was studied by means of two monoclonal antibodies of broad anti-A (HE-14) and anti-type 3 and type 4 chain-based A and H (HE-10) specificity. These antigens were proved to reappear in tumors of the distal colon, the HE-10 antibody reacting more frequently (9 out of 12 samples) than HE-14 (5 out of 12 samples) and frequently with supranuclear staining of the cytoplasm probably in those places of the Golgi apparatus where carbohydrate antigens are synthesized. This staining pattern is characteristic of HE-10 in normal colonic mucosa as well. With HE-14, staining was often absent in less differentiated tumors, while HE-10 did react in such tumors. In this connection, the possible expression of type 3 and type 4 chain H antigens in the tumor tissue is discussed. In some cases, these two antibodies gave different staining patterns in parallel sections from the same tissue sample, primarily at the cellular level. Three out of 12 cases showed blood group antigen expression in the mucosa of the distal colon adjacent to the tumor only when HE-10 antibody was used.

Rapid screening method for monoclonal antibodies against cytoskeletal proteins.

A horseradish peroxidase/anti-horseradish peroxidase monoclonal mouse antibody complex was prepared and used for the detection of mouse monoclonal antibodies against proteins of the whole cytoskeleton. A rapid qualitative assay of large number of antibody samples on the whole cytoskeleton and an early determination of their specificity by the immunoblot technique is described.

Fluorenone-azomethines, a novel class of microtubule inhibitors that specifically affect cell proliferation.

The effects of various azomethine derivatives on microtubule (MT) assembly in vitro as well as on cell proliferation, cell shape, and the cytoskeleton of some cultured murine cell lines were studied. 3 of them, the alpha-diphenylene-N-(p-[bis-(beta-hydroxyethyl)-amino]-phenyl)-nit rone (DHPN), alpha-diphenylene-N-(p-[N-(hydroxyethyl)-N-(gamma-hydroxypropyl)- amino]-phenyl)-nitrone, and alpha-diphenylene-N-(p-diethylaminophenyl)-nitrone, strongly inhibit the assembly of microtubules (MTs) in vitro (50% inhibition at 4 to 7 mumol/l). The same compounds are also able to disrupt preformed microtubules. Moreover, they were found to inhibit proliferation of leukaemia L 1210, melanoma B16 K, fibroblast L 929, and embryo fibroblast cells down to 1 to 10 mumol/l, completely. Immunofluorescence microscopy revealed that DHPN, used as a representative of the active azomethines, causes a reversible destruction of the microtubule part of the cytoskeleton. Apparently resulting from microtubule disruption, the intermediate filament system collapsed whereas the microfilament system remained unaffected. The results indicate that the antiproliferative action of the azomethines is based, at least partially, on their ability to attack microtubules.

Effect of some steroid hormones on the homing affinity of lymph node cells and monocytes.

The selective homing of labelled cells from lymph nodes and peritoneal exudate to lymph nodes and spleens is strongly inhibited after an in vivo administration of steroid hormones, cyproterone acetate, hydrocortisone acetate and hydrocortisone succinate. At the same time the number of lymphocytes and monocytes is greatly reduced in the peripheral blood. This decrease and changes in recirculation of lymphocytes and monocytes are due not to the lysis of cells but to an altered distribution of them in the body and an increased trapping in the bone marrow. The values in the peripheral blood picture are very rapidly reconstituted after the level of the soluble, short-term acting steroid--hydrocortisone succinate--in the body had decreased, and the transferred labelled cells re-enter the lymph nodes while their number in bone marrow is reduced. The altered distribution of cells in the body that is obviously not advantageous for the immune reactions may be one of the causes of the immunosuppressive effect of the steroids.

Supportive effect of simultaneous immunization to other H-antigens on the female response to male-specific antigen in mice.

The response of A-strain females to the male-specific antigen is very weak; both first- and second-set test skin grafts are rejected protractedly and about 50% of the grafts are retained in spite of generally present morphologic signs of cellylar immunity induced by these grafts. When the first-set skin graft involved H-incompatibilities additional to the MSA (such as H-2 only or H-2 plus multiple non-H-2) all the second-set grafts, which were only MSA-incompatible, underwent a markedly accelerated rejection. The possible mechanism of this adjuvant-like effect is discussed.

The mechanism of rosette formation between sheep red blood cells and L-A9 fibroblasts.

More than 20% of mouse L-A9 fibroblasts from rosettes with sheep red blood cells. This relatively weak interaction is mediated by receptors of glycoprotein nature; species-specific antigens of SRBC are part of, or lie in close proximity to, this binding. SRBC receptors are present on most L-A9 fibroblasts, neither Con A receptors nor MHL products are part of the SRBC receptors. However, Con A- and H-2 antibody-induced alterations in the membrane and cytoskeletal system accompanying cap formation produce a strong inhibition of rosette formation. This inhibition may be due to an interference into the adhesion by involving the cytoskeletal system in the redistribution processes because the same effect was obtained with Colcemid and cytochalasin B.

Analysis of different survival of weakly incompatible allografts of skin from the ear and back.

A comparison of the survival of skin grafts across the H-barrier presented by the male-specific antigen in mice was made with grafts of skin from different sources, namely from the back or from the ear. Their survival was roughly similar provided the ear skin was depleted of the subepidermal connective tissue with the cartilage. With this layer intact, the ear skin grafts had a prolonged survival and some of them even took permanently. A local administration of a mucolytic agent to the normal grafts from the back accelerated their rejection. The factors of the subepidermal connective tissue which might be responsible for the effect on the survival of weakly incompatible skin grafts and in this connection the role of the source of the skin are discussed.

Are histocompatibility antigens in neonatal skin grafts truly tolerogenic?

Neonatal skin grafts across a weak histocompatibility barrier (MSA-incompatible) were used only for sensitization of the recipients whose responsiveness was then tested in two different assays. On the basis of 1. functional inactivation of PEC in a PEC transfer system and 2. inhibition of macrophage migration. The difference between neonatal and adult skin grafts turned out to be quantitative rather than qualitative, i.e., both induced sensitization whose demonstration with the neonatal grafts required more sensitive techniques and/or more favourable timing because it was weaker. The possible nature of the difference, which was occasionally interpreted as being due to a tolerogenic rather than immunogenic activity of neonatal grafts, is discussed.

Spermatogenesis and SRBC haemolysin formation in various inbred mouse strains treated with cyproterone acetate.

The synthetic steroid antiandrogen cyproterone acetate (CA) was administered subcutaneously to adult male mice of the inbred strains B10, B10.D2, B10.BR and C57BL/6 either as a single dose of 1 mg or as 10 daily doses of 0.5 mg. At 48 h after a single dose of CA, the thymus and spleen weights were markedly reduced and the morphology of the thymus was changed (depletion of cortex lymphocytes) in some strains. At 6 h and at 12 days after the 10th dose of CA, thymuses in males of all strains were markedly diminished with a histologically apparent involution affecting the cortex, as well as the medulla and reticuloepithelial tissue. Spleen weights in males of some strains were also significantly lower. SRBC haemolysin titres at 12 days after the 10th dose and spermatogenesis at 6 h and at 12 days after the 10th dose were significantly decreased in males of all strains; in none of the males was sterility produced by the CA doses used, however, Indications of the strain differences could only be seen in the onset and duration of the damage to the spleen and the corresponding decrease in haemolysin formation. The importance of the consequences of short-term administration of CA is discussed in terms of potential risks for long-term administration of CA in the regulation of male fertility.