Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190,000

A heat-stable microtubule-associated protein (MAP) with apparent molecular weight of 190,000 is a major non-neural MAP which distributes ubiquitously among bovine tissues (termed here MAP-U). Previously we reported that microtubule-binding chymotryptic fragments of MAP-U and tau contain a common assembly-promoting (AP) sequence of 22 amino acid residues (Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1989) J. Biol. Chem. 264, 5885-5890). We isolated cDNA clones for MAP-U containing the whole coding sequence. Northern blot analysis revealed that a major species of MAP-U mRNA is 5 kilobases in length and is expressed ubiquitously among bovine tissues. Nucleotide sequence analysis revealed the complete amino acid sequence of MAP-U which consists of 1,072 amino acid residues. Analysis of the deduced amino acid sequence of MAP-U indicated that this molecule is clearly divided into two domains in terms of electrostatic charge distribution: an amino-terminal acidic domain (residues 1-640) and a carboxyl-terminal basic domain (residues 641-1072). The amino-terminal domain of MAP-U shows no significant sequence homology with other known protein sequences including neural MAPs, tau, and MAP-2. The amino-terminal domain of MAP-U contains unique 18 1/2 repeats of 14-amino acid motif which have not been observed in other MAPs. The carboxyl-terminal domain of MAP-U is further divided into three regions: a Pro-rich region (residues 641-880), an AP sequence region (residues 881-1003), and a short hydrophobic tail (residues 1004-1072). The Pro-rich region is mainly composed of five species of amino acid residues, Pro, Ala, Lys, Ser, and Thr. The AP sequence region contains four tandem repeats of AP sequences, and thus, this region is considered to play a leading role in the interaction of MAP-U with microtubules.     

The beta subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230

Our studies, which are aimed at understanding the catalytic mechanism of the beta subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to clarify the functional roles of several putative active site residues. Although previous chemical modification studies have suggested that histidine 86, arginine 148, and cysteine 230 are essential residues in the beta subunit, our present findings that beta subunits with single amino acid replacements at these positions have partial activity show that these 3 residues are not essential for catalysis or substrate binding. These conclusions are consistent with the recently determined three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Amino acid substitution of lysine 87, which forms a Schiff base with pyridoxal phosphate in the wild type beta subunit, yields an inactive form of the beta subunit which binds alpha subunit, pyridoxal phosphate, and L-serine. We also report a rapid and efficient method for purifying wild type and mutant forms of the alpha 2 beta 2 complex from S. typhimurium from an improved enzyme source. The enzyme, which is produced by a multicopy plasmid encoding the trpA and trpB genes of S. typhimurium expressed in Escherichia coli, is crystallized from crude extracts by the addition of 6% poly(ethylene glycol) 8000 and 5 mM spermine. This new method is also used in the accompanying paper to purify nine alpha 2 beta 2 complexes containing mutant forms of the alpha subunit.     

Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication

Summary A subset of Escherichia coli heat shock proteins, DnaK, DnaJ and GrpE were shown to be required for replication of mini-F plasmid. Strains of E. coli K12 carrying a missense mutation or deletion in the dnaK, dnaJ, or grpE gene were virtually unable to be transformed by mini-F DNA at the temperature (30° C) that permits cell growth. When excess amounts of the replication initiator protein (repE gene product) of mini-F were provided by means of a multicopy plasmid carrying repE, these mutant bacteria became capable of supporting mini-F replication under the same conditions. However, the copy number of a high copy number mini-F plasmid was reduced in these mutant bacteria as compared with the wild type in the presence of excess RepE protein. Furthermore, mini-F plasmid mutants that produce altered initiator protein and exhibit a very high copy number were able to replicate in strains deficient in any of the above heat shock proteins. These results indicate that the subset of heat shock proteins (DnaK, DnaJ and GrpE) play essential roles that help the functioning of the RepE initiator protein in mini-F DNA replication.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Stereochemical Control in Microbial Reduction. Part 10. Asymmetric Reduction of Alkyl 3-Methyl-2-Oxobutanoate with Immobilized Bakers' Yeast in Hexane

Esters of 3-methyl-2-oxobutanoic acid are reduced with bakers' yeast by three methods: free bakers' yeast in water, immobilized bakers' yeast in water, and immobilized bakers' yeast in hexane. Although (R)-hydroxy esters are obtained in all cases, the enantiomeric excess varies from 3% (reduction of the methyl ester with free bakers' yeast in water) to 93% (reduction of the butyl ester with immobilized bakers' yeast in hexane) depending on the structure of substrate and on the reaction conditions. The mechanism of the present stereochemical control is discussed.     

Three-Dimensional Labeling Program for Elucidation of the Geometric Properties of Biological Particles in Three-Dimensional Space

For all biological particles such as cells or cellular organelles, there are three-dimensional coordinates representing the centroid or center of gravity. These coordinates and other numerical parameters such as volume, fluorescence intensity, surface area, and shape are referred to in this paper as geometric properties, which may provide critical information for the clarification ofin situmechanisms of molecular and cellular functions in living organisms. We have established a method for the elucidation of these properties, designated the three-dimensional labeling program (3DLP). Algorithms of 3DLP are so simple that this method can be carried out through the use of software combinations in image analysis on a personal computer. To evaluate 3DLP, it was applied to a 32-cell-stage sea urchin embryo, double stained with FITC for cellular protein of blastomeres and propidium iodide for nuclear DNA. A stack of optical serial section images was obtained by confocal laser scanning microscopy. The method was found effective for determining geometric properties and should prove applicable to the study of many different kinds of biological particles in three-dimensional space.     

Protein Kinases Are Involved in Prolonged Acetylcholine Release from Rat Hippocampus Induced by Thyrotropin-Releasing Hormone Analogue NS-3

Abstract: The effects of various protein kinase inhibitors on acetylcholine release from the rat hippocampus induced by the local application of NS-3 (montirelin hydrate, CG-3703), a thyrotropin-releasing hormone analogue, into the medial septum-diagonal band were examined using in vivo microdialysis. Perfusion of NS-3 (1 µ M ) into the medial septum-diagonal band for 20 min produced a pronounced and prolonged increase in the hippocampal acetylcholine efflux. Pretreatment of the medial septum-diagonal band with either K-252a, a nonselective protein kinase inhibitor, or selective protein kinase A inhibitor H-89 almost completely blocked the acetylcholine efflux evoked by NS-3, and selective protein kinase C inhibitor calphostin C inhibited the action of NS-3. On the other hand, NS-3 (0.1–10 µ M ) or TRH (1–100 µ M ) increased the cyclic AMP efflux from the medial septum-diagonal band in a concentration-dependent manner, as measured by microdialysis. These findings suggest that protein kinases A and C in the neurons of the medial septum-diagonal band are involved in the mechanism of the prolonged stimulation of acetylcholine release from the hippocampus induced by thyrotropin-releasing hormone and its analogue, NS-3.     

Ras-Regulated Hypophosphorylation of the Retinoblastoma Protein Mediates Neuronal Differentiation in PC12 Cells

Abstract: To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12- O -tetradecanoylphorbol 13-acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin-6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa-Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras-dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells.     

Sensory cues for the gradual frequency fall responses of the gymnotiform electric fish, Rhamphichthys rostratus

The sensory cues for a less known form of frequency shifting behavior, gradual frequency falls, of electric organ discharges (EODs) in a pulse-type gymnotiform electric fish, Rhamphichthys rostratus, were identified. We found that the gradual frequency fall occurs independently of more commonly observed momentary phase shifting behavior, and is due to perturbation of sensory feedback of the fish's own EODs by EODs of neighboring fish. The following components were identified as essential features in the signal mixture of the fish's own and the neighbor's EOD pulses: (1) the neighbor's pulses must be placed within a few millisecond of the fish's own pulses, (2) the neighbor's pulses, presented singly at low frequencies (0.2–4 Hz), were sufficient, (3) the frequency of individual pulse presentation must be below 4 Hz, (4) amplitude modulation of the sensory feedback of the fish's own pulses induced by such insertions of the neighbor's pulses must contain a high frequency component: sinusoidal amplitude modulation of the fish's own EOD feedback at these low frequencies does not induce gradual frequency falls. Differential stimulation across body surfaces, which is required for the jamming avoidance response (JAR) of wave-type gymnotiform electric fish, was not necessary for this behavior. We propose a cascade of high-pass and low-pass frequency filters within the amplitude processing pathway in the central nervous system as the mechanism of the gradual frequency fall response.Abbreviations EOD electric organ discharge - f frequency of EOD or pacemaker command signal - JAR jamming avoidance response - S ₁ stimulus mimicking fish's own EOD - f ₁ frequency of S₁ - S ₂ stimulus mimicking neighbor's EOD - f ₂ frequency of S₂