Presence of the betaII isotype of tubulin in the nuclei of cultured mesangial cells from rat kidney

Tubulin has generally been considered to be a cytosolic protein whose only function is to form microtubules. This assumption is supported by a great deal of evidence derived from immunohistochemical studies using antibodies directed against whole tubulin or its component polypeptides alpha- and beta-tubulin. We have re-examined the intracellular distribution of tubulin using monoclonal antibodies specific for the betaI, betaII, betaIII, and betaIV isotypes of beta-tubulin. Our test system is the cultured rat kidney mesangial cell. We have found that betaIII is absent from these cells and that beta1 and betaIV are present in microtubules throughout the cytosol. In contrast, betaII is present largely in the nuclei. Immunoblotting of purified nuclear extracts shows that the betaII-reactive antigen co-migrates with beta-tubulin. Extraction of the cytosol and chromatin suggests that betaII is concentrated in the nucleoli and also in a reticulated network in the rest of the nucleoplasm. An antibody to tyrosinated alpha-tubulin shows that alpha is also present in the nucleoli. Treatment of the cells with fluorescent colchicine shows an accumulation of colchicine in the nucleoli. Finally, fluorescently labeled alphabetaII-tubulin dimers, when microinjected into the cells, enter the nuclei and are concentrated in the nucleoli. These results suggest that the betaII isotype of tubulin is present as an alphabetaII dimer in the nuclei of cultured mesangial cells and suggest the possibility that different tubulin isotypes may have specific functions within the cell.     

Class II tubulin, the major brain beta tubulin isotype is polyglutamylated on glutamic acid residue 435.

Protein sequencing shows that porcine brain tubulin retains the N-terminal sequences of alpha and beta tubulin after a mild treatment with subtilisin. C-terminal peptides released by subtilisin were purified and characterized by automated Edman degradation and mass spectrometry. We confirm the polyglutamylation of alpha tubulin on glutamic acid residue 445 reported by others and show in addition that class II beta tubulin, the major beta tubulin isotype of adult brain, is also polyglutamylated. The substitution is restricted to glutamic acid residue 435. Thus all major tubulin isotypes of adult brain are subjected to polyglutamylation.     

A tubulin identified by a monoclonal antibody is not present in mitotic spindles

A monoclonal antibody, G8, which recognizes a form of tubulin (G8-tubulin) with a novel distribution in Rat-1 cells and Potorous tridactylis kidney (Ptk-2) cells was isolated. G8 labeled the interphase cytoskeleton of Rat-1 fibroblasts but not mitotic spindles or midbodies. G8 also stained a fiber network in some but not all Ptk-2 interphase cells but did not label mitotic spindles or midbodies in these cells. G8-tubulin is the only identified tubulin known to be absent from these structures. This distribution may indicate that G8-tubulin possesses functional specificity.     

The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei

The data presented here show for the first time that the protein known as "ribonuclease (RNase) inhibitor" (RI or RNH1) is present not only in the cell cytosol, but also in mitochondria, the central organelles in cell redox homeostasis. This finding directly correlates with the reported ability of RI to protect the cell from oxidative stress, with its sensitivity to oxidation and reactivity as a reactive oxygen species scavenger. While this study was carried out we also surprisingly discovered the presence of RI in the cell nucleus. We deem that these data open new views in the investigation on the cellular role(s) of the RI.     

Lacandonia granules are present in Ginkgo biloba cell nuclei

Lacandonia schismatica is a rare flowering plant with the sex organs spatially inverted. Several aspects of its cell biology are now known. Interestingly, within the cell nucleus, the chromatin is reticulated and it is associated to a novel structure named Lacandonia granules, a very abundant ribonucleoprotein particle showing similarities to perichromatin and Balbiani ring granules, which are involved in nuclear mRNA metabolism. To see whether these particles are present in other plants, we study the nucleus of Ginkgo biloba, a non-flowering plant. Light, electron and atomic force microscopy show that the cell nuclei of G. biloba are reticulated. Ultrastructural analysis showed that in the nucleoplasm, abundant intranuclear particles 32 nm in diameter are present. The EDTA regressive staining suggested that they contain RNA. Ultrastructural in situ hybridization confirmed the presence of RNA in these particles. Therefore, we conclude that the nuclei of G. biloba are reticulated and contain Lacandonia granules. We suggest that these particles may also be present in other plants.     

Tumor-derived chaperone-rich cell lysates are effective therapeutic vaccines against a variety of cancers

With the clinical use of purified, tumor-derived chaperone proteins as anti-cancer vaccines already in clinical trial stages, we have focused our attention on the utility of chaperone-rich cell lysates (CRCL) in cancer immunotherapy. CRCL, as prepared from tumor lysates via a free solution-isoelectric focusing (FS-IEF) technique, is a high-yield vaccine enriched for numerous chaperone proteins. We have compared the efficacy of CRCL vaccines to that of individual chaperone protein vaccines in in vivo settings, including ELISPOT assays, tumor-growth assays and survival assays. In all experiments, CRCL vaccines were at least as effective, and in some settings perhaps even more effective, than either of the two most heavily studied components of CRCL, HSP70 and GRP94/gp96, in reduction in tumor growth and prolongation of survival in both prophylactic and pre-existing tumor settings against tumors of diverse origin and genetic background. Combining CRCL preparations with dendritic cells ex vivo resulted in a cellular vaccine that could eradicate pre-existing tumors in a high percentage of cases. The high yields of CRCL vaccines from small quantities of starting materials, the relative ease of its procurement and the functional data presented here suggest that CRCL vaccines are worthy of evaluation in pilot clinical trial cancer immunotherapy protocols.     

Interaction of tubulin with the plasma membrane: tubulin is present in purified plasmalemma and behaves as an integral membrane protein

Using monoclonal antibodies we have studied the interaction of tubulin with the plasma membrane of leaves of Nicotiana sylvestris (Speg. et Comes) and tobacco suspension-culture cells. The results show that isolated plasma membranes contain tightly bound -tubulins. Their association with the plasma membrane is resistent to non-ionic detergent and to low and high ionic strength. Only extraction with sodium dodecyl sulfate is capable of dissociating these cytoskeletal proteins. It is unlikely that this membrane-bound tubulin is present in its polymeric form because electron-microscopical analysis does not reveal the presence of filaments, whereas treatment of membranes with oryzalin (which has been shown to destabilize microtubules in vitro) does not remove the tubulins from isolated plasma membrane. When living cells are treated with oryzalin, the amount of membrane-associated tubulin is drastically reduced, which could mean that its presence is related to in-vivo microtubule dynamics.Abbreviations Mes 2 (N-morpholino) ethane sulfonic acid - NP40 Nonidet P40     

A developmentally regulated wound epithelial antigen of the newt limb regenerate is also present in a variety of secretory/transport cell types

The role of the wound epithelium in amphibian limb regeneration is not understood. We showed previously that monoclonal antibody (mAb) WE3 stains the wound epithelium but not skin epidermis, suggesting that the WE3 antigen may be a marker for, or be important in, the function of the wound epithelium. In the present study, we conducted an extensive immunohistochemical survey of adult newt tissues to define the distribution of the WE3 antigen. The results show that the antigen is most commonly found in tissues specialized in macromolecular secretion and/or ion transport. Since the enzyme, carbonic anhydrase, serves as a useful marker for a variety of specialized transporting cell types, we examined whether this enzyme was present in WE3-reactive cells. Of the tissues examined, a striking degree of colocalization of carbonic anhydrase and the WE3 antigen was observed, further strengthening the view that the WE3 antigen is an important constituent of specialized transporting cells. A preliminary biochemical characterization suggests that the antigen is probably a glycoprotein, which elutes during gel filtration as a species of over 660 kDa. Possible implications for the function of the wound epithelium are discussed.     

Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin

Tubulin, the major constituent protein of microtubules, is a heterodimer of alpha and beta subunits. Both alpha and beta exist in multiple isotypic forms. It is not clear if different isotypes perform different functions. In order to approach this question, we have made a monoclonal antibody specific for the beta III isotype of tubulin. This particular isotype is neuron-specific and appears to be phosphorylated near the C terminus. We have used immunoaffinity depletion chromatography to prepare tubulin lacking the beta III subunit. We find that removal of the beta III isotype results in a tubulin mixture able to assemble much more rapidly than is unfractionated tubulin when reconstituted with either of the two microtubule-associated proteins (MAPs), tau or MAP 2. Our results suggest that the different isotypes of tubulin differ from each other in their ability to polymerize into microtubules. We have also found that the anti-beta III antibody can stimulate microtubule assembly when reconstituted with tubulin and either tau or MAP 2. When reconstituted with tubulin lacking the beta III isotype, the antibody causes the tubulin to polymerize into a polymer that is a microtubule in the presence of MAP 2 and a ribbon in the presence of tau.     

The integrity of tubulin molecule is not required for the activity of tubulin carboxypeptidase

Tubulin dimer, alpha-tubulin subunit, and C-terminal peptides obtained from the alpha-tubulin subunit were compared in their capabilities to act as substrates of tubulin carboxypeptidase. The results obtained indicate that the enzyme does not require the beta-tubulin subunit to release tyrosine from alpha-tubulin. The 17-Kd C-terminal peptide of the alpha-tubulin subunit was obtained and it was detyrosinated at the same rate as tubulin dimer. A smaller C-terminal peptide of 2.8-3.7 Kd showed a lower capability to act as substrate. Similar results were obtained with pancreatic carboxypeptidase A. From the analysis of the results we consider that an optimal activity of the tubulin carboxypeptidase depends mainly on the accessibility of the C-terminal end of alpha-tubulin.     

Patterns of tubulin isotype synthesis and usage during mitotic spindle morphogenesis in Physarum

Tubulin synthesis in the naturally synchronous plasmodium of Physarum polycephalum is a markedly periodic event restricted to the late G2 period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle. We have shown that all four tubulin isotypes identified in the plasmodium (alpha 1, alpha 2, beta 1 and beta 2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: beta 2 greater than alpha 1 greater than alpha 2 greater than beta 1. We have also shown that tubulins synthesized in the G2 period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin-containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucleokinesis.     

Sphingosylphosphorylcholine is a remarkably potent mitogen for a variety of cell lines

The effect of spingosylphosphorylcholine on cellular proliferation was investigated in a variety of cell types. Spingosylphosphorylcholine at low concentrations greatly stimulated DNA synthesis and cell division in quiescent Swiss 3T3 fibroblasts. The increased DNA synthesis was also accompanied by pronounced morphological alterations. Spingosylphosphorylcholine was remarkably more potent than other known growth factors and also acted synergistically with insulin, epidermal growth factor, fibroblast growth factor, and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to induce cellular proliferation. Sphingosylphosphorylcholine was less effective in stimulating DNA synthesis in rapidly growing normal and transformed cells. Spingosylphosphorylcholine appears to be a new type of potent, wide-spectrum growth promoting agent.     

Composition and organization of tubulin isoforms reveals a variety of axonemal models

In the flagellum of mammalian spermatozoa, glutamylated and glycylated tubulin isoforms are detected according to longitudinal gradients and preferentially in axonemal doublets 1-5-6 and 3-8, respectively. This suggested a role for these tubulin isoforms in the regulation of flagellar beating. In the present work, using antibodies directed against various tubulin isoforms and quantitative immunogold analysis, we aimed at investigating whether the particular accessibility of tubulin isoforms in the mammalian sperm flagellum is restricted to this model of axoneme surrounded with periaxonemal structures or is also displayed in naked axonemes. In rodent lung ciliated cells, all studied tubulin isoforms are uniformly distributed in all axonemal microtubules with a unique deficiency of glutamylated tubulin in the transitional region. A similar distribution of tubulin isoforms is observed in cilia of Paramecium, except for a decreasing gradient of glutamylated tubulin labeling in the proximal part of axonemal microtubules. In the sea urchin sperm flagellum, predominant labeling of tyrosinated and detyrosinated tubulin in 1-5-6 and 3-8 doublets, respectively, were observed together with decreasing proximo-distal gradients of glutamylated and polyglycylated tubulin labeling and an increasing gradient of monoglycylated tubulin labeling. In flagella of Chlamydomonas, the glutamylated and glycylated tubulin isoforms are detected at low levels. Our results show a specific composition and organization of tubulin isoforms in different models of cilia and flagella, suggesting various models of functional organization and beating regulation of the axoneme.     

Enolase is present in the cell wall of Saccharomyces cerevisiae

We have reported that the macrophage-like cell line J774.1, when infected with the periodontopathic bacterium Actinobacillus actinomycetemcomitans, undergoes apoptosis. In this study, we examined whether stimulation of J774.1 cells with lipopolysaccharide (LPS) before the infection affects the subsequent apoptosis. Cytotoxicity on the LPS-stimulated cells was about half of the unstimulated control cells. DNA fragmentation in the LPS-stimulated cells was also significantly lower than in the control cells, whereas it was increased to a level similar to that of the control cells by addition of a nitric oxide (NO) inhibitor. In addition, significantly smaller numbers of live A. actinomycetemcomitans were recovered from the LPS-stimulated macrophages at 8 h after the infection as compared with the control cells. These findings suggest that the inhibitory effect of LPS on apoptosis results from an enhanced NO-mediated bactericidal activity.     

Distinct colchicine binding kinetics of bovine brain tubulin lacking the type III isotype of beta-tubulin

In mammalian brain, beta-tubulin occurs as a mixture of four isotypes designated as types I, II, III, and IV. It has been speculated in recent years that the different tubulin isotypes may confer functional diversity to microtubules. In an effort to investigate whether different tubulin isotypes differ in their functional properties we have studied the colchicine binding kinetics of bovine brain tubulin upon removal of the beta III isotype. We found that the removal of the beta III isotype alters the binding kinetics from biphasic to monophasic with the disappearance of the slow phase. The kinetics become biphasic with the reappearance of the slow phase when the beta III-depleted tubulin was mixed with the beta III fraction eluted from the affinity column with 0.5 M NaCl. The analysis of the kinetic data reveals that the tubulin dimers containing beta III bind colchicine at an on-rate constant of 35 M-1 s-1 while those lacking beta III bind at 182 M-1 s-1. Our results strongly suggest that the beta-subunit plays a very important role in the interaction of tubulin with colchicine.     

The specificity of human autoantibodies that react with both cell nuclei and plasma membranes: the nuclear antigen is present on core mononucleosomes.

We have examined the nature of the nuclear antigen recognized by certain natural human antibodies that react specifically with both cell nuclei and plasma membranes from many species. Partial purification of these antibodies, called X-ANA, is achieved by binding to and rapid elution from the surface of viable human leukocytes. Chicken erythrocyte chromatin was solubilized by digestion with staphylococcal nuclease and fractionated into a 0.15 M NaCl soluble fraction that consisted of core mononucleosomes lacking H1/H5, and a 0.15 M NaCl insoluble fraction composed of polynucleosomes with H1/H5 present. No proteins other than histones were detected. Native and reconstituted mononucleosomes displaced IgG of the leukocyte eluates from nuclei of frozen mouse kidney sections and from the walls of plastic tubes coated with polynucleosomes. The reconstituted core mononucleosomes were 4- 10-fold less efficient inhibitors than native mononucleosomes. Trypsin digested mononucleosomes, free high m.w. DNA, and free histones displayed no or very weak inhibitory activity. The data indicate that X-ANA recognize a complex consisting of the core histones H2A, H2B, H3, H4, and DNA of 140 to 200 base pairs in length.     

A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin

Mammalian brain tubulin consists of several isotypes of alpha and beta subunits that separate on polyacrylamide gels into three electrophoretic classes, designated alpha, beta 1, and beta 2. It has not been possible hitherto to resolve the different isotypes in a functional form. To this end, we have now isolated a monoclonal antibody, using as an immunogen a chemically synthesized peptide corresponding to the carboxyl-terminal sequence of the major tubulin isotype (type II) found in the beta 1-tubulin electrophoretic fraction. The antibody binds to beta 1 but not to alpha or beta 2. When pure tubulin from bovine brain is passed through an immunoaffinity column made from the anti-type II antibody, the tubulin that elutes in the unbound fraction is enriched greatly for the beta 2 electrophoretic variant. The tubulin that binds to the column appears to contain only alpha and beta 1, not beta 2. When these tubulin fractions are characterized by immunoblotting using the anti-type II antibody, the antibody binds only to the beta 1 band in the bound fraction, not to the beta 1 band in the unbound fraction. Using polyclonal antibodies generated against the carboxyl-termini of types I, III, and IV, we demonstrate that the beta 1 electrophoretic species is comprised of isotypes I, II, and IV, whereas the beta 2 variant is comprised exclusively of type III beta-tubulin. Further, we calculate that beta-tubulin in purified bovine brain tubulin is comprised of 3% type I, 58% type II, 25% type III, and 13% type IV tubulins.     

Genetic characterization of a reciprocal translocation present in a widely grown barley variety

Artificially induced translocation stocks have been used to physically map the barley genome; however, natural translocations are extremely uncommon in cultivated genotypes. Albacete is a barley variety widely grown in recent decades in Spain and carrying a reciprocal translocation which obviously does not affect its agronomical fitness. This translocation has been characterized by a combination of cytological and molecular genetic approaches. Firstly, recombination frequencies between markers on chromosomes 1H and 3H were estimated to determine the boundaries of the reciprocal interchange. Secondly, 1H-3H wheat barley telosome addition lines were used to assign selected markers to chromosome arms. Thirdly, fluorescence in situ hybridization (FISH) with rDNA probes (5S and 18S-5.8S-26S) and microsatellite probes [(ACT)(5), (AAG)(5) and (CAG)(5)] was used to determine the locations of the translocation breakpoints more precisely. Fourthly, fine-mapping of the regions around the translocation breakpoints was used to increase the marker density for comparative genomics. The results obtained in this study indicate that the translocation is quite large with breakpoints located on the long arms of chromosomes 1H and 3H, between the pericentromeric (AAG)(5) bands and above the (ACT)(5) interstitial distal bands, resulting in the reciprocal translocation 1HS.1HL-3HL and 3HS.3HL-1HL. The gene content around the translocation breakpoints could be inferred from syntenic relationships observed among different species from the grass family Poaceae (rice, Sorghum and Brachypodium) and was estimated at approximately 1,100 and 710 gene models for 1H and 3H, respectively. Duplicated segments between chromosomes Os01 and Os05 in rice derived from ancestral duplications within the grass family overlap with the translocation breakpoints on chromosomes 1H and 3H in the barley variety Albacete.