Quantitation of Chill-Induced Release of a Tubulin-Like Factor and Its Prevention by Abscisic Acid in Gossypium hirsutum L

The degree of tubulin polymerization in cotton (Gossypium hirsutum L. cv Acala) cotyledonary tissue was estimated by radioimmunoassay which measured the amount of a tubulin-like factor. It was assumed that the release of this tubulin-like factor indicated depolymerization of microtubules. Exposure to chilling resulted in complete release of the tubulin-like factor. Pretreatment with abscisic acid in the light almost completely prevented the chill-induced release of the tubulin-like factor. Addition of colchicine during the chilling period accelerated the release of the tubulin-like factor. Pretreatment with abscisic acid greatly reduced this effect of colchicine. It is concluded that the destruction of the microtubular network is involved in the development of chilling injury in cotton. Abscisic acid apparently decreased chilling injury by stabilization of the microtubular network.     

Isolation of human nerve growth factor from placental tissue

Nerve growth factor (NGF) has been isolated from human placental tissue. Using the chicken embryo dorsal root ganglia assay, we determined levels of NGF activity for the amnion, placental cotyledons, cord serum, fetal serum, and maternal serum. The highest levels of NGF activity were measured in placental cotyledons. After homogenization and centrifugation of the placental cotyledons, the supernatant was sequentially chromatographed, at neutral pH, on Sephadex G-100, DEAE-11, and Sephadex G-150. A high-molecular-weight protein fraction (150,000), which contained all the biological activity, was isolated in this fashion. Analytical isoelectric focusing of this fraction revealed a basic protein component (pI 9.5) of the high-molecular-weight species. Assays for NGF activity of all protein components separated by analytical isoelectric focusing showed that NGF activity was associated only with the basic protein component. Correspondingly, preparative isoelectric focusing of the high-molecular-weight species yielded a basic protein with very high biological activity (1–3 ng per biological unit) that was immunochemically active against rabbit IgG made against mouse -NGF.To whom reprint requests should be addressed.     

Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.     

Localization of microfilaments and a tubulin-like protein in crustacean (Rhynchocinetes typus) spermatozoon

Sperm from the decapod crustacean Rhynchocinetes typus undergo dramatic shape changes as they pass from the vas deferens to seawater and interact with the oocyte envelopes. Using FITC-phalloidin and antitubulin antibodies, we were able to localize microfilaments and a tubulin-like protein in R. typus spermatozoon. Microfilaments and the tubulin-like protein were associated with the sperm rays and spines, but were absent at the spike and at its base. Folded and unfolded spermatozoa display similar fluorescence patterns. SDS-PAGE of whole spermatozoa and electrotransfer to nitrocellulose confirmed the presence of actin and two proteins at 97 kd and 120 kd that bind to tubulin antibodies (tubulin-like proteins). These results demonstrate the presence of actin, but not tubulin, and localize microfilaments in these sperm. It is proposed that this cytoskeletal component is active in sperm during crustacean fertilization.     

Production of monoclonal antibodies to estriol and their application in the development of a sensitive nonisotopic immunoassay

The development of highly specific monoclonal antibodies to estriol and a nonisotopic immunoassay (EIA) for unconjugated estriol based on the use of these monoclonal antibodies have been described. The monoclonal antibodies show little cross reactivity with other steroids and steroid conjugates and can be used directly in immunoassays without any purification. The EIA described here can be performed in 96-well microtiter plates or polystyrene tubes that have been coated with estriol-bovine serum albumin conjugate. In this assay, estriol in the standard or clinical samples (serum or saliva) competes with the immobilized steroid on the plate or the tube for binding with the antibody. The assay shows good agreement with radioimmunoassay (RIA) and is highly sensitive and reliable. Since no prior processing or extraction of the clinical samples is necessary, the method is potentially applicable for routine use in fetal monitoring as well as in a steroid laboratory.     

Preparation of hydrophobic cotton cloth

Summary Hydrophobic cotton cloths were prepared by heating cotton flannel in a mixture of alcohols or phenols, epichlorohydrin and 4 M NaOH. These cloths adsorbed as much bovine serum albumin as did a commercial preparation of phenyl agarose. -Galactosidase and -glucosidase adsorbed on the cloths were about 50% as active as free enzymes. Glucoamylase immobilized on naphthyl cloth in a packed bed column efficiently hydrolyzed soluble starch to glucose. These inexpensive media would be useful for commercial-scale hydrophobic chromatography and enzyme immobilization.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis,cotyledons and endosperm

The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), -galactosidase and -mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo--mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - EDTA ethylenediamine tetraacetic acid disodium salt - TCA trichloroacetic acid Part 2 of a series, of which the first was published in Planta 139, 1–8 (1978)     

Immunostaining of human spermatozoa with tubulin domain-specific monoclonal antibodies. Recognition of a unique beta-tubulin epitope in the sperm head

Summary Four monoclonal antibodies that discriminate between structural domains of alpha-(TU-01, TU-04) or beta-(TU-06, TU-12) tubulin and a polyclonal anti-tubulin antibody were used for immunostaining of human spermatozoa using immunofluorescence microscopy. Specificity of antibodies was confirmed by immunoblotting experiments. Antibodies TU-01 and TU-06 uniformly stained the whole tail and the neck, whereas antibodies TU-04, TU-12 showed differential distribution of corresponding epitopes in the stable arrays of flagellar microtubules. Of the monoclonal antibodies used, only TU-12 against the antigenic determinant on C-terminal domain of -tubulin showed strong reactivity with the equatorial segment of the head. The results document a differential exposure of tubulin epitopes at the single-cell level and suggest the existence of distinct tubulin populations in various structural compartments of the human spermatozoon.     

Immobilization of glucoamylase on naphthyl cotton cloth

Summary Aspergillus niger glucoamylase was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme was cross-linked with glutaraldehyde. The immobilized glucoamylase exhibited greater pH dependence though the optimal pH did not change. The immobilized glucoamylase in a packed bed column completely hydrolysed 5% soluble starch at a specific velocity of about 4. Used naphthyl cloth could be regenerated by heating in 2 N NaOH at 100°C for 1 hour.     

Tubulin evolution: An electrophoretic and immunological analysis

This paper summarizes a survey of the electrophoretic behavior of the tubulins of 23 species (mostly protists) as well as their reactivity towards 4 anti-tubulin antibodies (raised against two ciliate tubulins and two vertebrate ones). Some generalizations concerning the relative migration rates of VS tubulin could be made, in particular the / inversion, first described inPhysarum was extended to several ciliates. Antivertebrate tubulin antibodies displayed a very broad spectrum of reactions, reacting with virtually all the species tested. They appear to correspond to auto-antibodies no exclusively directed against species specific determinants. In contrast, the two anti-ciliate tubulin antibodies displayed a narrow species specificity reacting only with a limited subset of protists. They were shown to be specific for a small number of immunological determinants present on ciliate tubulins. This allowed a rough evaluation of evolutionary relatedness between the various groups of protists analyzed. The results are discussed within the framework of a number of published phyllogenies and shown to be in striking agreement with some of the schemes.     

Generation of chicken polyclonal antibodies against distinct maize isotubulins

Summary Antibodies specific to five different maize isotubulins were made. From predicted amino acid sequences established from previously sequenced maize tubulin genes, peptide antigens were synthesized matching the carboxyl-terminal 11–13 amino acids of each of three maize -tubulins and two maize -tubulins. Antibodies were generated by injecting conjugated antigens into hens, collecting their eggs, and extracting immunoglobulin Y from the egg yolk. Specificity of each antibody was tested by immunoblotting of fusion proteins containing the antigenic sequence of the specific - and -tubulin isoforms. For all five isotubulins, antibodies were affinity-purified with fusion proteins corresponding to their respective antigens, to remove nonspecific binding found in the antibody preparations. Further preparation of anti--tubulins was required to eliminate cross-reactivity of antibodies with members of other -tubulin subfamilies. For this, affinity-purified antibodies against a specific -tubulin were preadsorbed with peptides representing cross-reactive -tubulin antigens. Results indicated that virtually all cross-reactivity between members of different -tubulin subfamilies could be eliminated, resulting in labeling of only the fusion protein containing the specific antigen. All five isotubulin antibodies generated showed labeling of discrete spots on two-dimensional immunoblots of maize proteins, demonstrating the specificity of the antibodies in complex tubulin mixtures. These antibodies should prove valuable for analyzing the developmental distribution, and possible functional significance, of several maize isotubulins.Abbreviations BSA bovine serum albumin - 2-D two-dimensional - GCG Genetics Computer Group - Ig Immunoglobulin - KLH keyhole limpet hemocyanin - PBS phosphate-buffered saline - PVDF polyvinylidene-difluoride - SDS-PAGE sodium dodecyl sulfate-poly-acrylamide gel electrophoresis - TBS Tris-buffered saline - TE Tris EDTA buffer     

Unusual distribution of tubulin isoforms in the snail Lymnaea stagnalis

Immunocytochemistry and Western blotting techniques demonstrated that the nervous system and foot of the pond snail Lymnaea stagnalis are rich sources of tubulin, which can be extracted and assembled in vitro in the presence of taxol. Various broad-spectrum antibodies raised against -tubulin and -tubulin yielded qualitatively similar results. One monoclonal antibody to trypanosome -tubulin, however, labelled -tubulin more strongly on both probed sections and Western blots. Cytochemistry and immunoblotting revealed that tyrosinated tubulin constitutes a large proportion of total -tubulin in locomotor cilia of the foot and in axons of the nervous system. Detyrosinated tubulin also appeared to be abundant in the foot cilia but only a very faint band of detyrosinated tubulin was found on protein blots extracted from the central ganglia, and staining was barely detectable in central ganglia or peripheral nerves. Similarly, acetylated tubulin appeared to be abundant in foot cilia, but Western blotting indicated only low levels of acetylated tubulin in the nervous system. Immunocytochemistry indicated that, while most neurons possessed little or no acetylated tubulin, a small number of axons contained significant amounts of this isoform. Thus, while a large amount of tubulin was expected in the nervous system and locomotor cilia of L. stagnalis, the observed distribution of isoforms was unanticipated. Specifically, neurons of other organisms have generally been reported to contain substantial amounts of both detyrosinated -tubulin and acetylated -tubulin. Our results indicate that such findings cannot be generalized across all species. L. stagnalis, with its well studied nervous system and unusual distribution of tubulin isoforms, may prove to be particularly useful for studying the roles of tubulin isoforms in microtubule function and cell activity.     

Molecular cloning,expression and characterization of human mini antibodies against enterotoxin C1 of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We describe here the cloning, expression, and production of specific single-chain antibodies (scFv) against the recombinant enterotoxin C1 of Staphylococcus aureus. High-affinity scFv were selected from the phage library of human mini antibodies; afterwards, the cells of E. coli trxA gor double mutant were infected with a product obtained by fusion of DNA encoding of these mini antibodies with the trxA gene to induce soluble scFv synthesis in cell cytoplasm. The scFv obtained displayed high enterotoxin C1 affinity. Analysis for cross reactivity showed that mini antibodies interacted also with SEA- SEB-, SED-, SEE-, SEG-, and SEI-type enterotoxins, but they failed to interact with ricin, diphtheritic, and cholera toxins, or with both lethal and protective factors of the anthrax toxin. This property may be helpful in screening for staphylococcus enterotoxins.     

Production of monoclonal antibodies and enzyme immunoassay for typical adenylate cyclase activator,Forskolin

The ratio of hapten to bovine serum albumin in an antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against forskolin was produced by fusing splenocytes immunized with a forskolin-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.57%. A very small cross-reaction appeared with 1-deoxy, 9-deoxy and 1,9-dideoxy forskolin derivatives. The full measuring range of the assay extends from 6 ng to 200 ng of forskolin. The competitive ELISA assay used for this analysis was found to be more sensitive than TLC (10 g), GLC (30 ng) and HPLC (1 g) methods.     

Changes in glycerol solubility and amino acid incorporation of a protein possessing the characteristics of tubulin during first cleavage in the sea urchin embryo

A method is described for the quantitation of the pool size of a tubulin like protein during synchronized cell division in the sea urchin Lytechinus variegatus. The method involves the use of a thin SDS slab polyacrylamide gel system in which tubulin can be quantitated in the submicrogram range. Employing a microtubule stabilization buffer, the intact tubule fraction was removed and the soluble tubulin pool was quantitated with this gel system. Amino acid incorporation into this protein was also quantitated. The resulting specific activity values and values for the amount of tubulin-like protein present in the pool fraction suggested that the tubulin pool decreases at fertilization and then remains constant through the first cell cycle. Tubulin synthesis, however, steadily increased after fertilization and then decreased dramatically just prior to mitotic apparatus formation. No change in tubulin pool size was observed at the time of peak mitotic apparatus formation. These results are discussed in terms of the regulation of microtubule function.     

Localization and Characterization of Tubulin-Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane-Specific Isoform

A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit.     

Regeneration of almond from immature seed cotyledons

Adventitious shoots were regenerated from immature cotyledons of four almond cultivars (`Ne Plus Ultra', `Nonpareil', `Carmel' and `Parkinson'). Open-pollinated fruit were collected from orchard-grown trees 100–115 days after full bloom. The proximal ends of the cotyledons were excised and the embryonic axes discarded. The effects of different concentrations of thidiazuron (TDZ) and indole-3-butyric acid (IBA) and the presence or absence of light for the first 7 days of culture were determined. Shoot regeneration rates were highest for cotyledons cultured for 8 weeks on Murashige and Skoog (MS) basal medium containing TDZ (10.0 M), followed by 4 weeks on medium without plant growth regulators. Regeneration levels were further improved if cotyledons were maintained in darkness for the first 7 days. IBA (0.5 M) significantly reduced the development of adventitious shoots. The frequency of cotyledons that developed adventitious shoots under the optimum tested conditions for `Ne Plus Ultra', `Nonpareil', `Carmel', and `Parkinson' were 80.0%, 73.3%, 100.0% and 86.7%, respectively.     

Immobilization of galactosyltransferase and continuous galactosylation of glycoproteins in a reactor

We immobilized human milk galactosyltransferase covalently to CNBr- and tresylchloride-activated Sepharose. The enzyme was also immobilized non-covalently to Concanavalin A-Sepharose and to monoclonal anti-galactosyltransferase antibodies which were boundvia their Fc-fragment to Protein G-Sepharose. With the covalent methods, up to 72% of the enzyme could be bound to the carrier, but more than 90% of the specific activity was lost. In contrast, non-covalent immobilization yielded only about 50% immobilization efficiency, but 21% and 25% of specific activity, respectively, could be recovered. The stability of immobilized galactosyltransferase as compared to native enzyme was considerably increased: at room temperature, 55% of initial immobilized activity was lost after 65 hours compared to 95% of loss of soluble enzyme activity. Immobilized galactosyltransferase was then used for continuous galactosylation of the glycoproteins ovalbumin, endo H-treated yeast invertase and bovine serum albumin-N-acetylglucosamine in a slurry reactor. 55%, 35% and 25%, respectively, of all acceptor sites on these glycoproteins could be galactosylated by this method.     

β-Tubulin accumulation and DNA synthesis are sequentially resumed in embryo organs of cucumber (Cucumis sativus L.) seeds during germination

Summary Resumption of DNA synthetic activities and -tubulin accumulation was studied in embryo organs of germinating cucumber (Cucumis sativus L.) seeds. Flow-cytometric analysis indicated the existence of 2C, 4C, and 8C nuclei in the radicle of mature embryos, whereas in cotyledons most of the cells contained nuclei with 2C DNA content. Upon imbibition of water, nuclear DNA replication was initiated in the radicle within 15 h, subsequently spreading towards the cotyledons. Bromodeoxyuridine incorporation preceded detectable changes in the relative amounts of DNA, implying the occurrence of putative DNA repair. Organellar DNA synthesis occurred independently of the nuclear DNA synthetic cycle. Western blotting and immunohistochemical localization demonstrated that the constitutive level of -tubulin originated from preserved -tubulin granules. During imbibition, disappearance of fluorescent tubulin granules, accumulation of -tubulin, and formation of microtubular cytoskeleton were found in the radicle, but not in the cotyledon areas. Mitosis only occurred after radicle protrusion at 21 h of imbibition. It is concluded that the differences in the initiation and progress of these cellular and molecular events are associated with the discrete behaviors of the radicle and the cotyledons upon imbibition. The formation of cortical microtubular cytoskeleton and the accumulation of tubulins are important features in preparation of radicle protrusion, whereas DNA synthesis may contribute to postgerminative growth.     

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