Heterogeneity of tubulin epitopes in mouse fetal tissues

A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves. Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.     

Differential distribution of tubulin epitopes in human spermatozoa

Two monoclonal antibodies (16 D3 and 24 E3) were used to map tubulin domains in human spermatozoa by indirect immunofluorescence. Their specificity to tubulin in these cells was established by Western blotting. Whereas 16 D3 uniformly stained the principal piece of the flagellum, the staining provided by 24 E3 decreased along the tail to become very weak 30 micron further away from the midpiece. This latter antibody also reacted with the proximal centriole as well as the midpiece, but not all spermatozoa stained identically at this level indicating heterogeneity within the population of sperm cells from a given donor. 16 D3 reacted weakly with the head, and the staining was interrupted after a bright spot in the neck. The study of a pathological case (the short tail spermatozoon) with an abnormal arrangement of dense fibers was consistent with a correlation between the distribution of the epitope defined by 24 E3 and that of peri-axenomal structures. The existence of tubulin domains interacting with these structures is postulated.     

Physiological regulation of total tubulin and polymerized tubulin in tissues

Polymerized and depolymerized forms of tubulin were measured in rat and mouse liver, rat islets, human lymphocytes, and platelets. The percent of the total tubulin present in the polymerized form varied from 30.3 +/- 1.5% in the liver of the fed rat to 89.2 +/- 0.2% in human platelets. Fasting decreased the total tubulin and to a greater extent the polymerized form of tubulin in both rat and mouse liver. Glucose feeding increased the polymerized tubulin without affecting the total tubulin content in rat liver. Phytohemagglutinin-stimulated lymphocytes exhibited at least a three-fold increase in total tubulin (expressed in terms of DNA content), which during the initial 48 h of incubation was accounted for in toto by an increase in polymerized tubulin. It is suggested that the lectin not only accelerates tubulin synthesis but also stimulated the polymerization process. Storage of platelets at 4 degrees C for 6 days resulted in a marked decrease in total tubulin and an even greater reduction in the polymerized form. It is concluded that both the total tubulin content and its degree of polymerization can be modulated independently by a wide variety of physiological factors.     

Hydroxyurea (HU)-induced apoptosis in the mouse fetal tissues

Hydroxyurea (HU), a ribonucleotide reductase inhibitor, induces morphological anomalies in the central nervous system (CNS), craniofacial tissues and limb buds in animals, and neonatal respiratory distress in humans. In the present study, pregnant mice were treated with 400 mg/kg of HU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue to clarify the mechanisms of HU-induced fetotoxicity and teratogenecity. At 6 and 12 HAT, a moderate to marked increase in the number of pyknotic cells was detected in the CNS and lung. A mild increase in the number of pyknotic cells was also found in the craniofacial mesenchymal tissues, limb buds and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results suggest that the HU-induced fetotoxicity is characterized by excess apoptotic cell death in the fetal tissues, and that such excess cell death in the fetal CNS, lung, craniofacial tissue and limb bud may have a certain relation to the later occurrence of morphological or functional anomalies reported in these tissues following HU-administration.     

Heterogeneity of the alpha subunit of tubulin and the variability of tubulin within a single organism

When tubulins obtained from particular microtubules of the sea urchin (ciliary doublet A tubules, flagellar doublet microtubules, and mitotic microtubules) are analyzed by electrophoresis in a polyacrylamide gel system containing sodium dodecyl sulfate and urea, heterogeneity of the alpha subunit, and differences between the tubulins are revealed. The alpha subunit of tubulin from mitotic apparatus and from A microtubules of ciliary doublets is resolved into two bands, while the alpha subunit of flagellar doublet tubulin gives a single band. The mitotic and ciliary tubulins differ in the mobilities of their two alpha species, or in the relative amounts present, or both. The existence of differences between the tubulins has been confirmed by a preliminary analysis of their cyanogen bromide peptides.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

The origin and role of confronting cisternae in selected fetal mouse and rat tissues

Confronting cisternae (CC) are described for the first time in normal fetal rat and mouse liver and intestinal epithelial cells. In these cells, CC characteristically consist of 2 parallel cisternae which are devoid of ribosomes on their juxtaposed surfaces. The intracisternal spacing is consistently 20 nm. While CC occur in rapidly proliferating tissues such as fetal liver and intestinal epithelium, they do not occur in hepatocytes following partial hepatectomy. Although it has been postulated that the intact CC profiles represent a mechanism of assuring the presence of pre-formed nuclear envelope (NE) or rough endoplasmic reticulum (RER) in cells, it is more likely that the subunits which result from CC degradation serve as a pool of membrane precursors for new NE or RER.     

Tubulin evolution: Ciliate-specific epitopes are conserved in the ciliary tubulin of metazoa

Summary In spite of their overall evolutionary conservation, the tubulins of ciliates display electrophoretic and structural particularities. We show here that antibodies raised againstParamecium andTetrahymena ciliary tubulins fail to recognize the cytoplasmic tubulins of all the metazoans tested. Immunoblotting of peptide maps of ciliate tubulins reveals that these antibodies react with one or very few ciliate-specific epitopes, in contrast to polyclonal antibodies against vertebrate tubulins, which are equivalent to autoantibodies and recognize several epitopes in both ciliate and vertebrate tubulins. Furthermore, we show that the anti-ciliate antibodies recognize ciliary and flagellar tubulins of metazoans ranging from sea urchin to mammals (with the exception of humans). The results support the conclusion that although duplication and specialization of tubulin genes in metazoans may have led to distinct types of tubulins, the axonemal one has remained highly conserved.     

Heterogeneity of Salmonella-host interactions in infected host tissues

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Competitive interactions between corticosterone and 11-dehydrocorticosterone in binding to receptors in fetal mouse tissues

The binding of ³H-corticosterone and ³H-11-dehydrocorticosterone to receptors in cytosol and nucleus was examined in fetal mouse brain and placenta using Sephadex gel filtration or charcoal to separate bound and unbound steroid. In the cytosol, competitive displacement of each steroid by the other was observed. The binding was unaffected by RNase, DNase, dithiothreitol or N-ethyl maleimide but was diminished by Pronase. Nuclei were isolated by hypotonic shock using dilute MgCl₂ and the steroid receptor-complexes of both steroids were obtained from the nuclear sap. Receptor-complexes of both steroids were observed in brain and placental tissues. Competitive displacement of each steroid by the other was also observed in nuclear binding. Both 11-dehydrocorticosterone and 11-deoxycorticosterone bound to a chromatin fraction as did the hormone corticosterone. Identity of the steroids was established by using chromatography and co-crystallization techniques. This work raises the possibility that in the fetal mouse, 11-dehydrocorticosterone, previously considered biologically inactive and an abundant metabolite in fetal mouse tissues, may in fact play a more positive role in regulation.     

A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes

Microtubule-associated proteins (MAPs) interact with tubulinin vitro andin vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of -tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that the 205 kDa protein contained a tubulin binding motif which was recognized by site-directed antibodies that also tag tubulin binding epitopes on MAP-2 and tau. This characteristic led us to designate this protein as MBD-205, a component that shares binding domains with these MAPs, rather than as a marker of the MAPs family. On the other hand, immunofluorescence experiments using site-specific antibodies, i.e. MAP-reacting monoclonal anti-idiotypic reagent MTB6.22 and a polyclonal antibody to the second tau repeat, revealed a MBD-205 co-localization with membrane structures and microtubule-organizing centers in Vero cells. Microinjection studies along with studies on the cell distribution suggest that MBD-205 appears to play a structural role at the level of the microtubule interactions in these cells.     

The structure and amount of tubulin in cells and tissues.

The NH2-terminals and amino acid compositions of three carboxymethylcysteine-containing peptides produced from carboxymethylated calf brain tubulin by digestion with elastase have been determined. The 3H/14C ratio obtained when these peptides were generated from mixtures of [14C]carboxymethylated calf brain tubulin and the [3H]carboxymethylated proteins of mouse brain or 3T3 cells and subsequently purified indicated that both mouse brain and 3T3 cells contained protein giving rise to peptides homologous to these peptides. This indicates that the tubulins of calf brain, mouse brain, and 3T3 cells are homologous in the regions of amino acid sequence accounting for the peptides examined. From the 3H/14C ratio of the isolated peptides, 13.5% of the protein of mouse brain and 3.5% of the protein of 3T3 cells were estimated to be tubulin.     

Comparative immunogold analysis of tubulin isoforms in the mouse sperm flagellum: Unique distribution of glutamylated tubulin

The distribution of different tubulin isoforms in the mouse sperm flagellum was studied using four site-directed antibodies to tubulin: DM1A and DM1B general anti α and β-tubulin, 6-11B-1 anti-acetylated α-tubulin, and GT335 anti-glutamylated α and β-tubulin. Quantitative immunogold analyses were performed on five regions of the flagellum: the middle piece, three successive regions of the principal piece, and the terminal piece. A uniform labeling was observed with DM1A and DM1B along the entire flagellum both for peripheral doublets and the central pair. Similar results were obtained with 6-11B-1 directed to acetylated α-tubulin, an N-terminal-modified tubulin isoform. In contrast, the labeling for glutamylated α and β-tubulin, C-terminal modified isoforms, was not uniform. The highest intensity was found in the middle piece and the terminal piece. The labeling which decreased significantly both for peripheral doublets and central pair along the principal piece was considered as a loss of glutamylated tubulin accessibility. From the middle piece to the end of the principal piece, this labeling was predominant in doublets 1-5-6, corresponding to the plane of the flagellar wave. However, the labeling for doublets 2-3-4-7-8-9 was heterogeneous, showing an increasing asymmetry. These results suggest that in the mammalian sperm cell model, the glutamylated tubulin might be involved in a functional heterogeneity among peripheral doublets of the flagellum. © 1996 Wiley-Liss, Inc.     

Heterogeneity of purified mouse interferons.

A procedure for obtaining highly purified stable interferon from mouse L cells is described. Interferon with a specific activity of 2.5 times 10-8 reference units/mg of protein is composed of 10 to 11 polypeptides. They differ in their molecular size as determined by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The molecular weight range is estimated to be from the smallest at 20, 000 to the largest at 32, 000. At least six of the polypeptides are glycoproteins and each of the polypeptides can be obtained as an apparent homogeneous entity and each has interferon activity.