Isolation of α and β Brain Tubulin Subunits after Alkaline Treatment of the Protein

We demonstrate here that brain purified tubulin can be dissociated into and subunits at pH > 10 and that the subunits can be separated by using the Triton X-114 phase separation system. After phase partition at pH > 10, tubulin but not tubulin behaves as a hydrophobic compound appearing in the detergent rich phase. After three extractions of the alkaline aqueous phase with Triton X-114, about 90% of the tubulin was recovered in the detergent rich phase. The hydrophobic behavior observed for tubulin after its dissociation at pH 11.5 was not due to an irreversible change of the protein, because when the detergent rich phase containing tubulin was diluted with a buffer solution at pH 7.3 and the solution allowed to partition again, -tubulin is recovered in the aqueous phase. The detergent in the aqueous phase of the and tubulin preparations can be removed up to 90% by 12 h dialysis. The and subunits of tubulin from kidney and liver behave, in this phase separation system, like those of brain tubulin.     

The relationship of hydrophobic tubulin with membranes in neural tissue

Brain membrane preparations contain tubulin that can be extracted with Triton X-114. After the extract is allowed to partition, 8% of the total brain tubulin is isolated as a hydrophobic compound in the detergent-rich phase. Cytosolic tubulin does not show this hydrophobic behaviour since it is recovered in the aqueous phase. Membrane tubulin can be released by 0.1 M Na₂CO₃ treatment at pH11.5 in such a way that the hydrophobic tubulin is converted into the hydrophilic form. These results suggest that tubulin exists associated with some membrane component that confers the hydrophobic behaviour to tubulin. If the tissue is homogenized in microtubule-stabilizing buffer containing Triton X-100, the hydrophobic tubulin is isolated from the microtubule fraction. This result indicates that the hydrophobic tubulin isolated from membrane preparations belongs to microtubules thatin vivo are associated to membranes. Therefore, hydrophobic tubulin (tubulin-membrane component complex) can be obtained from membranes or from microtubules depending on the conditions of brain homogenization.Abbreviations TBS Tris-buffered saline - Mes 2-(N-morpholine) ethane sulfonic acid     

Tyrosinated,detyrosinated and acetylated tubulin isotypes in rat brain membranes. Their proportions in comparison with those in cytosol

The heterogeneity of -tubulin and the relative proportions of the tubulin isotypes were investigated in brain membranes of rats of 1, 25 and 180 days of age by using four anti--tubulin antibodies: a) the monoclonal DM1A antibody, specific for -tubulin; b) the monoclonal 6-11B-1 antibody, specific for acetylated tubulin; c) a polyclonal antibody (Glu antibody), specific for detyrosinated tubulin; and d) a polyclonal antibody (Tyr antibody), specific for tyrosinated tubulin. We found that rat brain membranes contain the three tubulin isotypes mentioned above. The proportions of tyrosinated and detyrosinated tubulin relative to total -tubulin were somewhat lower in membrane than in cytosol in animals of 25 and 180 days of age. At day one of development, the proportions in membrane were similar to those found in cytosol. With respect to the acetylated form, it was about 20 times higher in membrane than in cytosol at the three ages studied. The proportion of acetylated tubulin was determined in different subcellular fractions: myelin, synaptic vesicles, mitochondria, microsomes, and plasma membrane. While the amount of total tubulin differed between the different subcellular fractions, the proportion of acetylated tubulin relative to total -tubulin was constant and similar to that found in total membranes. The proportion of acetylated tubulin was also investigated in non-neural tissues (kidney, liver and lung). Although values for cytosol were about 10-fold higher than that found in brain cytosol, no detectable values for membranes could be obtained in these organs.     

Brain plasmamembrane NA+:,K+-ATPase is inhibited by acetylated tubulin

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the -subunit of Na⁺,K⁺-ATPase. In the present work we show that the interaction of tubulin with Na⁺K⁺-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na⁺K⁺-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

Distribution of acetylated α-tubulin in brain

Summary We studied the solubility properties of brain acetylated -tubulin, as well as the localization of this tubulin in brain tissue. Endogenous unpolymerized tubulin and cytoskeletal tubulin were fractionated after brain Triton-solubilization. Using the immunoblotting technique, we found that acetylated -tubulin was recovered in the cytoskeletal fraction, and that most (92%) of the acetylated microtubules of this fraction were depolymerized by cold/Ca²⁺ treatment. In another set of experiments, axonal and soma-dendritic preparations were found to have equivalent amounts of acetylated -tubulin. By immunogold electron microscopy, we established that acetylated microtubules are widely distributed in dendrites of the central nervous system.     

Interaction of Phomopsin A with Normal and Subtilisin-Treated Bovine Brain Tubulin

Tubulin, the major component of microtubules, has a tendency to lose its ability to assemble or to bind to ligands in a time-dependent process known as decay. The decay process also causes tubulin to expose sulfhydryl groups and hydrophobic areas. The antimitotic drug phomopsin A strongly protects the tubulin molecule from decay. Here we have studied the interaction of phomopsin A with tubulin and tubulin which has been treated with subtilisin to remove selectively the C-termini of the and chains (_ss). The binding of phomopsin A to tubulin decreases the sulfhydryl titer by approximately 1.0 mol/mol. Selective removal of the peptides from the C-terminal ends does not affect phomopsin A's interaction with tubulin. Moreover, the _ss tubulin–phomopsin A complex appears to be more stable than the tubulin–phomopsin A complex as determined by the time-dependent increase in exposure of sulfhydryl groups and hydrophobic areas on tubulin. In fact, phomopsin A inhibits the decay process of _ss tubulin completely. This observation raises the possibility of determining the conformtion of this configuration of tubulin.     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.     

The incorporation and characterization of powdery mildew resistance from Aegilops longissima in common wheat (T. aestivum L.)

Summary In the progeny of a hybrid between monotelosomic line 3B of Chinese Spring wheat and Chinese Spring — Aegilops longissima ditelosomic addition line G a cytologically stable strain was selected consisting of 20 wheat chromosome pairs, one pair of telosomic chromosome 3BL and one pair of telosomic longissima chromosome G. Inoculating Chinese Spring — Aegilops longissima addition and substitution lines with ten different powdery mildew isolates, partial resistance was observed. The infection grade as well as the number of spores/cm² leaf area were significantly reduced.     

Structures of the N-linked sugar chains in the PAS-6 glycoprotein from the bovine milk fat globule membrane

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man16 (Man13) Man14GlcNAc14GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal13Gal14GlcNAc12Man16 (Gal13Gal14GlcNAc12Man13) Man14GlcNAc14 (Fuc16)GlcNAc-PA;6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively. © 1998 Rapid Science Ltd     

Variation of seed α-amylase inhibitors in the common bean

Variation of seed -amylase inhibitors was investigated in 1 154 cultivated and 726 non-cultivated (wild and weedy) accessions of the common bean, Phaseolus vulgaris L. Four -amylase inhibitor types were recognized based on the inhibtion by seed extracts of the activities of porcine pancreatic -amylase and larval -amylase and larval -amylase of the Mexican bean weevil, Zabrotes subfasciatus Boheman. Of the 1 880 accessions examined most (1 734) were able to inhibit porcine pancreatic -amylase activity, but were inactive against the Z. subfasciatus larval -amylase; 41 inhibited only the larval -amylase activity, 52 inhibited the activities of the two -amylases, and 53 did not inhibit the activity of either of the -amylases. The four different inhibitor types were designated as AI-1, AI2, AI-3, and AI-0, respectively. These four inhibitor types were identified by the banding patterns of seed glycoproteins in the range of 14–20 kDa by using SDSpolyacrylamide gel electrophoresis. Additionally, four different banding patterns were recognized in accessions with AI-1, and were designated as AI-1a, 1b, 1c, and 1d. Two different patterns of the accessions lacking an -amylase inhibitory activity were identified and designated as AI-0a and AI-0b. The largest diversity for seed -amylase inhibitors was observed in non-cultivated accessions collected from Mexico where all eight inhibitor types were detected. The possible relationships between the variation of seed -amylase inhibitors and bruchid resistance are discussed.     

Mammalian Sperm Tubulin: An Exceptionally Large Number of Variants Based on Several Posttranslational Modifications

Extraction of demembranated bull sperm flagella by SDS was used to maximize tubulin solubilization. The - and -tubulin separated by SDS-PAGE were treated with endoproteinases LysC and AspN, respectively. Carboxy-terminal fragments were isolated by Mono Q chromatography and reversed-phase HPLC. Automated sequencing and mass spectrometry revealed an astonishingly high number of tubulin variants. Many variants were due to polyglutamylation and in particular to polyglycylation. The number of side-chain glycyl residues ranged from 0 to 28 in and 0 to 15 in . Corresponding values for side-chain glutamyl residues were 0–6 in and 0–3 in . Additional variability was based on carboxy-terminal detyrosination and partial loss of the penultimate glutamate. A major glycylation site in - and -tubulin was mapped. Some variants seem to display both glycyl and glutamyl side chains.     

Spontaneous chlorophyll mutants of Pennisetum americanum: Genetics and chlorophyll quantities

Summary Thirteen spontaneously occurring chlorophyll deficient phenotypes have been described and their genetic basis was established. Ten of these — white, white tipped green, patchy white, white virescent, white striping 1, white striping 2, white striping 4, fine striping, chlorina and yellow virescent showed monogenic recessive inheritance and the remaining three — yellow striping, yellow green and light green seedling phenotypes showed digenic recessive inheritance. The genes for (i) white tipped green (wr) and yellow virescent (yv) and (ii) patchy white (pw) and white striping 1 (wst 1) showed independent assortment. Further, the genes for white (w), white tipped green (wr) and yellow virescent (yv) were inherited independently of the gene for hairy leaf margin (Hm).In the mutants — white tipped green, patchy white, white striping 1, white striping 2, fine striping, chlorina, yellow virescent, yellow striping, yellow green and light green phenotypes total quantity of chlorophyll was significantly less than that in the corresponding controls, while in white virescent there was no reduction in the mature stage. For nine of the mutants the quantity of chlorophyll was also estimated in F₁'s (mutant x control green). In F₁'s of six of the mutants — white tip, patchy white, chlorina, yellow virescent, fine striping and yellow striping the quantity of chlorophyll was almost equal to the wild type. In the F₁'s of three of the mutants — white striping 1, white striping 2 and light green an intermediate value between the mutant and wild types was observed. In yellow virescent retarded synthesis of chlorophyll, particularly chlorophyll a was observed in the juvenile stage. Reduced quantity of chlorophyll was associated with defective chloroplasts. In the mutants — white tipped green, white virescent, fine striping, chlorina, yellow striping, yellow green and light green defective plastids were also observed. In patchy white secondary destruction of chlorophylls and the presence of defective plastids were found to be associated with reduced chlorophyll quantity at maturity.Paper chromatographic studies of leaf flavonoids revealed some variation between the inbreds, but there were three common spots, 7, 8 and 9, except for PDP in which the spot 8 was absent. Chlorophyll deficient mutants differed from their respective controls in the absence of one or more of the spots present in the controls and in the presence of new spots in some of the mutants.Most of the chlorophyll mutants showed higher survival rate in the Kharif season than in Rabi season which was attributed to the higher mean day temperature and longer day light period in the Kharif season than in Rabi season.     

Magnetic orientation and magnetoreception in birds and other animals

Animals use the geomagnetic field in many ways: the magnetic vector provides a compass; magnetic intensity and/or inclination play a role as a component of the navigational map, and magnetic conditions of certain regions act as sign posts or triggers, eliciting specific responses. A magnetic compass is widespread among animals, magnetic navigation is indicated e.g. in birds, marine turtles and spiny lobsters and the use of magnetic sign posts has been described for birds and marine turtles. For magnetoreception, two hypotheses are currently discussed, one proposing a chemical compass based on a radical pair mechanism, the other postulating processes involving magnetite particles. The available evidence suggests that birds use both mechanisms, with the radical pair mechanism in the right eye providing directional information and a magnetite-based mechanism in the upper beak providing information on position as component of the map. Behavioral data from other animals indicate a light-dependent compass probably based on a radical pair mechanism in amphibians and a possibly magnetite-based mechanism in mammals. Histological and electrophysiological data suggest a magnetite-based mechanism in the nasal cavities of salmonid fish. Little is known about the parts of the brain where the respective information is processed.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Structural study of fucoidan from Cladosiphon okamuranus tokida

A structural study was carried out on a fucoidan isolated from the brown seaweed Cladosiphon okamuranus. The polysaccharide contained fucose, glucuronic acid and sulfate in a molar ratio of about 6.1 : 1.0 : 2.9. The results of Smith degradation showed that this polysaccharide has a linear backbone of 13-linked -fucopyranose with a half sulfate substitution at the 4-positions, and a portion of the fucose residues was O-acetylated. The data obtained from partial acid hydrolysis, a methylation analysis and NMR spectra indicated that the -glucuronic acid residue is linked to the 2-positions of the fucose residues, which were not substituted by a sulfate group. These results indicated that the average structure of this fucoidan is as follows: -[(3Fuc-4(±OSO₃-)1–)₅3[GlcA12]Fuc1–]_n–. (Half of each fucose residue was sulfated. One O-acetyl ester was present in every 6 fucose residues.)     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

The influence of lactation products on the temporal expression of histo-blood group antigens in the intestines of suckling pigs: lectin histochemical and immunohistochemical analysis

Summary Lectins and carbohydrate-specific monoclonal antibodies were used as cytochemical probes to investigate the possible influence of lactation products on the expression of intestinal membrane and secretory glycoconjugates in suckling piglets. Two different lactational regimes were compared; the first involved normal rearing of piglets for 8 weeks on a single dam and the second involved repeated cross-fostering of littermates onto recently farrowed sows, thereby restricting them to early milk. Five histo-blood group phenotypes were recognized within the piglet population: two immature phenotypes, O immature or Oi and A immature or Ai, and three mature forms, O, A and -. Under the normal suckling regime the transition from immature to mature OA phenotypes was evident at the fifth weekpost partum. However, in the repeatedly cross-fostered piglets this transition was evident much earlier at 3 weekspost partum. It is suggested that qualitative or quantitative variations in milk composition during the sow's lactation may significantly influence the expression of intestinal histo-blood group antigens in her suckling young.Abbreviations GalNac N-acetylgalactosamine - Gal Galactose - GlcNac N-acetylglucosamine     

Microtubular structures developed in response to a carbamate herbicide in plant mitosis

Summary Indirect immunodetection of tubulin showed that the herbicide carbetamide activated silent signals left by the preprophase band (PPB) and by old phragmoplasts. Thus, after half an hour of treatment, 5.3% of anaphases inAllium cepa L. meristems showed spindle microtubules pointing to sites of the longitudinal cell membranes which, under control conditions, would only start attracting microtubules from the growing phragmoplast at late telophase. After 2 h, 12.8% of the telophases showed not only the expected phragmoplast between the two sister nuclei, but one or two additional phragmoplasts, at one or both cell tips, the sites of the phragmoplasts from the telophases of previous cycles. A few binucleate cells, obtained by aborting phragmoplast formation by a short caffeine treatment, developed three phragmoplasts in their next mitosis (bimitosis) in the presence of carbetamide: one between each sister pair of telophasic nuclei plus an extra one. The latter also occupied the site of the phragmoplast of the telophase of the previous cycle.Abbreviations PPB preprophase band of microtubules - EGTA ethylene glycol-bis(-amino-ethyl-ether)-N,N,N,N-tetraacetic acid - PMSF phenylmethylsulfonyl-fluoride - PIPES piperazine-N,N-bis(2-ethane sulphonic acid) - PBS phosphate-buffered saline - DAPI 4,6-diamidino-2-phenylindole     

Analogues of NADP+ as inhibitors and coenzymes for NADP+ malic enzyme from maize leaves

Structural analogues of the NADP⁺ were studied as potential coenzymes and inhibitors for NADP⁺ dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate ( NADP⁺), 3-acetylpyridine-adenine dinucleotide phosphate (APADP⁺), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP⁺) and -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate (23NADPc⁺) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in V_max for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP⁺), 3-aminopyridine-adenine dinucleotide phosphate (AADP⁺), adenosine 2-monophosphate (2AMP) and adenosine 2: 3-cyclic monophosphate (23AMPc) were competitive inhibitors with respect to NADP⁺, while -nicotinamide adenine dinucleotide 3-phosphate (3NADP⁺), NAD⁺, adenosine 3-monophosphate (3AMP), adenosine 2: 5-cyclic monophosphate (25AMPc), 5AMP, 5ADP, 5ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C₄ atom of the pyridine ring is the major factor that governs the coenzyme activity.Abbreviations NADP⁺ 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate - NHDP⁺ nicotinamide-hypoxanthine dinucleotide phosphate - APADP⁺ 3-acetylpyridine-adenine dinucleotide phosphate - SNADP⁺ thionicotinamide-adenine dinucleotide phosphate - AADP⁺ 3-aminopyridine-adenine dinucleotide phosphate - 23NADPc⁺ -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate - 3NADP⁺ -nicotinamide adenine dinucleotide 3-phosphate - 2AMP adenosine 2-monophosphate - 3AMP adenosine 3-monophosphate - 23AMPc adenosine 2: 3 monophosphate cyclic - A adenosine - RuBP ribulose 1,5-bisphosphate - SCF-MO Self-Consistent Field-Molecular Orbitals (method)