Amino acid sequence of horseshoe crab, Tachypleus tridentatus, striated muscle troponin C

The amino acid sequence of troponin C obtained from horseshoe crab, Tachypleus tridentatus, striated muscle was determined by sequence analysis and alignments of chemically and enzymatically cleaved peptides. Troponin C is composed of 153 amino acid residues with a blocked N-terminus and contains no tryptophan or cysteine residue. The site I, one of the four Ca2+-binding sites, is considered to have lost its ability to bind Ca2+ owing to the replacements of certain amino acid residues.     

An ATP-driven Cl- pump in the brain

EDTA-treated microsomes prepared from rat brain mainly consisted of sealed membrane vesicles 200-500 nm in diameter and were rich in both Cl- -ATPase and Na+,K+-ATPase activities. Such Cl- -ATPase-rich membrane vesicles accumulated Cl- in an ATP-dependent and osmotically reactive manner in the presence of 1 nM ouabain. The Cl- uptake was maximally stimulated by ATP with a Km value of 1.5 mM; GTP, ITP, and UTP partially stimulated Cl- uptake, but CTP, beta, gamma-methylene ATP, ADP, and AMP did not. The ATP-dependent Cl- uptake was accelerated by an increase in the medium Cl- concentration with a Km value of 7.4 mM. Such stimulation of Cl- uptake by ATP was dependent on the pH of the medium, with an optimal pH of 7.4, and also on the temperature of the medium, with an optimal range of 37-42 degrees C. Ethacrynic acid dose dependently inhibited the ATP-dependent Cl- uptake with a concentration for half-maximal inhibition at 57 microM. N-ethylmaleimide (0.1 mM) completely inhibited and sodium vanadate (1 mM) partially inhibited the ATP-dependent Cl- uptake. The membrane vesicles did not accumulate H+ in the Cl- uptake assay medium. The ATP-dependent Cl- uptake profile agreed with that of Cl- -ATPase activity reported previously (Inagaki, C., Tanaka, T., Hara, M., and Ishiko, J. (1985) Biochem. Pharmacol. 34, 1705-1712), and this strongly supports the idea that Cl- -ATPase in the brain actively transports Cl-.     

Cloning and sequencing of the gene for Tetrahymena calcium-binding 25-kDa protein (TCBP-25)

With the intention of studying calcium-dependent ciliary reversal in Tetrahymena, we isolated a Tetrahymena calcium-binding protein of 10 kDa (TCBP-10) which was not calmodulin and reported its properties (Ohnishi, K., and Watanabe, Y. (1983) J. Biol. Chem. 258, 13978-13985). However, immunoblotting with an antiserum against TCBP-10 and sequencing of the cDNAs and partial genomic DNAs for this calcium-binding protein prove that this previously reported TCBP-10 is the degraded product of a 25-kDa calcium-binding protein. Thus, we correct the name of the protein from TCBP-10 to TCBP-25. From the analysis of the cDNA for TCBP-25, it is shown to be composed of 218 amino acid residues and its molecular weight is estimated to be 24,702. This protein is predicted to contain four EF-hand-type calcium binding domains and to be a member of the calmodulin family. Little sequence homology with other proteins was shown by a computer search, except in the EF-hand regions. The special feature of TCBP-25 is that the distance between calcium-binding domains II and III is extraordinarily long for a calmodulin family protein having four calcium-binding domains. The genomic DNA for TCBP-25 contains two introns situated at short distances before calcium-binding domains I and III, implying gene duplication in genealogy.     

Reversion of the apical hook of pea in the dark and its promotion by a red light pulse.

At the developmental stage at which the apical hook passed the 3rd and 4th nodes, dark-grown seedlings of pea ( Pisum sativum L. cv. Progress No.9) opened the hook upright and then formed a new hook above the node nearly in the opposite direction to the previous one. In cv. Alaska, in contrast, many (about 84%) seedlings closed the hook in the original direction after they partially (up to about 110°) opened it at the 3rd node, thus doing a wagging movement, while a small percentage (about 16%) of the seedlings reversed the hook direction. Exposure to red light of cv. Alaska seedlings for 10 min increased the percentage of the hook reversion up to 71% or more. The hook reversion was never observed except when the hook part passed the nodes, suggesting the involvement of the nodes in the phenomenon.     

Expression and transport into mitochondria of bovine cytochrome P-450(SCC) in insect cells using the baculovirus expression system.

Bovine cytochrome P-450(SCC) introduced with the baculovirus host vector system was found to be expressed in Spodoptera frugiperda cells. Cell fractionation analysis indicated that the P-450(SCC) expressed as the precursor form was transported into mitochondria and converted to a mature form. However, this form did not exhibit definite activity for cholesterol side chain cleavage. These findings suggest that most of the P-450(SCC) expressed by this system is an inactive protein within mitochondria that is not folded to the conformation of the active enzyme and/or does not incorporate heme appropriately.     

Neurotransmitter-Mediated Regulation of Brain Aromatase: Protein Kinase C- and G-Dependent Induction

Abstract: Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by α₁-adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the α₁-agonist phenylephrine than in control neurons, whereas prazosin, an α₁-antagonist, suppressed this increase, and ligands for α₂- or β-adrenergic receptors did not exert any influence. The profile of α₁-adrenergic receptor subtypes during actual development in vivo suggested that the α_1B subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12-myristate 13-acetate and dibutyryl-cyclic GMP, whereas forskolin and dibutyryl-cyclic AMP caused a decrease. These data indicate that stimulation via α₁ (possibly α_1B)-adrenergic receptors, as well as receptors of specific neuropeptides, controls the expression of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. β-Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.     

Cyclization reaction catalyzed by branching enzyme.

The action of branching enzyme (EC 2.4.l.l8) from Bacillus stearothermophilus on amylose was analyzed. The enzyme reduced the molecular size of amylose without increasing the reducing power. This result could not be explained by the normal branching reaction model. When the product was treated with glucoamylase (an exo++-type amylase), a resistant component remained. The glucoamylase-resistant component was easily digested by an endo-type alpha-amylase or by isoamylase plus glucoamylase. These results suggested that the glucoamylase-resistant component was a cyclic glucan composed of alpha-1,4- and alpha-l,6-glucosidic linkages. In other words, it was suggested that branching enzyme catalyzed cyclization of the alpha-l,4-glucan chain of the amylose molecule to form an alpha-l,6-glucosidic linkage, thereby forming two smaller molecules. Mass spectrometry also supported the cyclic nature of the product.     

A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae

During periods of water deficit, plants accumulate late embryogenesis-abundant (LEA) proteins which are thought to protect cells from stresses associated with dehydration. One of these genes, le25, is expressed in tomato leaves and roots in response to water deficit and abscisic acid accumulation. To study the function of this protein and to test the effect of overproduction of the LE25 protein in Saccharomyces cerevisiae (Sc), a recombinant plasmid in which le25 is expressed under the control of the GAL1 promoter was constructed. The content of LE25 was high in Sc cells transformed with the recombinant plasmid. The transformant exhibited several stress-tolerant phenotypes. Growth of the transformant in a medium with 1.2 M NaCl was improved, as compared to a control strain. While the control strain showed a long lag phase of 40 h, le25-expressing cells showed a shortened lag phase of 10 h. However, no growth improvement was observed in a medium with 2 M sorbitol. In addition, the transformant had an increased survival rate after freezing stress, but not after high-temperature stress. These results, together with its predicted secondary structure, may indicate that LE25 functions as an ion scavenger.     

Production of (R)-1-phenylethanol through enantioselective oxidation of (S)-isomer from their racemic mixture by Pachysolen tannophilus IFO 1007

Summary Stereoselective oxidation of (S)-isomer of rac-1-phenylethanol (1-PA) by the yeast Pachysolen tannophilus IFO 1007 immobilized into calcium-alginate gels was investigated to produce (R)-isomer. Continuous production of (R)-isomer was accomplished for more than 80 h with an enantiomeric excess of > 90% using a bioreactor of a fluidized-bed type.     

Cloning and functional analysis of molecular chaperone genes from Bacillus stearothermophilus SIC1

Genes homologous to groES and groEL, which are recognized as molecular chaperone genes, from Bacillus stearothermophilus SIC1 were cloned and sequenced. By addition of GroES, GroEL and ATP in vitro, remaning activity of the alcohol dehydrogenase from Saccharomyces cerevisiae after heat treatment at 50°C for 6 min was improved from 55% to 90%. Furthermore, even though inclusion bodies were formed when a single chain Fv(sFv) was expressed in E. coli cells, during in vivo coexpression with molecular chaperone, a significant amount of the antibody protein could be recovered from the soluble fraction.