Coexistence of the genes for putrescine transport protein and ornithine decarboxylase at 16 min on Escherichia coli chromosome

The nucleotide sequence of one of the putrescine transport operons (pPT71), located at 16 min of the Escherichia coli chromosome, was determined. It contained the genes for an induced ornithine decarboxylase and a putrescine transport protein. The gene for the ornithine decarboxylase contained a 2,196-nucleotide open reading frame encoding a 732-amino acid protein whose calculated Mr was 82,414, and the predicted amino acid sequence from the open reading frame had 65% homology with that of a constitutive ornithine decarboxylase encoded by the gene at 64 min. The ornithine decarboxylase activity was observed in the cells carrying pPT71 cultured at pH 5.2, but not in the cells cultured at pH 7.0. The gene for the putrescine transport protein contained a 1,317-nucleotide open reading frame encoding a 439-amino acid protein whose calculated Mr was 46,494. The hydropathy profile of the putrescine transport protein revealed that it consisted of 12 putative transmembrane spanning segments linked by hydrophilic segments of variable length. The transport protein was in fact found in the membrane fraction. When the gene for the putrescine transport protein was linked to the tet promoter of the vector instead of its own promoter, the putrescine transport activity increased greatly. The results suggest that the gene expression of the operon is repressed strongly under standard conditions.     

A novel L-glutamate transporter inhibitor reveals endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

We previously reported for the first time that D-aspartate (D-Asp) is biosynthesized by cultured mammalian cells such as pheochromocytoma (PC)12 cells and its subclone MPT1 (FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92). We speculated that D-Asp levels in the intra- and extracellular spaces of the cultured cells are maintained in a dynamic state of homeostasis. To test this here, we utilized a novel and potent L-Glu transporter inhibitor, TFB-TBOA. This inhibitor proved to be a genuine nontransportable blocker of the transporter even during long periods of culture. Use of this inhibitor with MPT1 cells confirmed that D-Asp levels are in a dynamic steady state where it is constantly released into the extracellular space by a yet undefined mechanism as well as being constantly and intensively taken up by the cells via the L-Glu transporter. We estimated the rate with which D-Asp is constitutively released from MPT1 cells is approx. 3.8 pmol/h/1x10(5) cells.     

Overexpression of Bop3 confers resistance to methylmercury in Saccharomyces cerevisiae through interaction with other proteins such as Fkh1, Rts1, and Msn2

We found that overexpression of Bop3, a protein of unknown function, confers resistance to methylmercury in Saccharomyces cerevisiae. Bmh2, Fkh1, and Rts1 are proteins that have been previously shown to bind Bop3 by the two-hybrid method. Overexpression of Bmh2 and the homologous protein Bmh1 confers resistance to methylmercury in yeast, but overexpression of either Fkh1 or Rts1 has a minimal effect. However, the increased level of resistance to methylmercury produced by overexpression of Bop3 was smaller in Fhk1-deleted yeast as compared with that of the wild-type strain. In contrast, the degree of resistance was significantly elevated in Rts1-deleted yeast. Msn2 and Msn4 were previously reported as proteins that bind to Bmh1 and Bmh2. Overexpression of Msn2 conferred a much greater sensitivity to methylmercury in yeast, while deletion of the corresponding gene lowered the degree of resistance to methylmercury induced by overexpression of Bop3. These results suggest that multiple proteins are involved in minimizing the toxicity of methylmercury induced by overexpression of Bop3.     

D-Glutamic acid-induced muscle contraction in the silkworm, Bombyx mori

Agonists for muscle contraction in silkworms were screened by injecting test solutions into the hemolymph of decapitated silkworm larvae. Kainic acid, a glutamate receptor agonist, and D-glutamic acid induced muscle contractions, and D-aspartic acid was partially effective, whereas NMDA and AMPA, representative mammalian glutamate receptor agonists, did not induce contraction. L-Glutamic acid inhibited the kainic acid or D-glutamic acid-induced contraction. Amino acid analysis revealed that 3% of the total glutamic acid in the silkworm hemolymph is D-glutamic acid. These results suggest that d-glutamic acid acts physiologically as an agonist for muscle contraction in silkworms, and that L-glutamic acid functions as an inhibitor.     

Function of Mannan Chains of Yeast Repressible Acid Phosphatase on Its Enzymatic Properties

To find the function of the mannan chains covalently attached to yeast repressible acid phosphatase, the N-glycosidic carbohydrate chains were removed by endo-β-N-acetyl-glucosaminidase H under native conditions. Almost all of the N-glycosidic mannan chains were cleaved off by the glycosidase. The deglycosylated enzyme was shown to be a dimer structure as is the native enzyme. The deglycosylated enzyme retained enzyme activity, the same Km, and the same circular dichroism spectra as the native enzyme. These results indicate that the carbohydrate chains are not essential for maintaining the active enzyme structure, but the deglycosylated enzyme was shown to be more sensitive to acidic pH and high temperature.     

Peripheral mononeuropathy induced by loose ligation of the sciatic nerve in the rat: behavioral, electrophysiological and histopathologic studies.

The relationship between clinical parameters and pathological changes was investigated in an animal model of mononeuropathy, by behavioral, electrophysiological and histopathological methods. Mononeuropathy was induced in rats by loosely tying ligatures around the sciatic nerve. Eighty-four rats were used, and these were divided into fourteen groups to determine chronological changes in the withdrawal reflex latency, nerve conduction velocity and ultrastructure of the nerve from 1 to 84 days after nerve ligation surgery. Pathological changes around the ligated nerves were divisible in three phases: the first week was an inflammatory phase, when axonal degeneration, phagocyte infiltration and interstitial edematous changes were observed. The second and third weeks were a nerve-sprouting phase, when numerous axonal sprouts and remyelination were seen. The fourth to twelfth weeks were a recovery phase in which maturing myelination and interstitial fibrosis were characteristic. In the inflammatory phase, withdrawal reflex latencies were shortened, and sensory nerve conduction velocities (SCV) and motor nerve conduction velocities (MCV) gradually decreased. In the nerve-sprouting phase, the latency values remained low, and SCV and MCV were minimal. The parameters examined gradually returned to control levels during the recovery phase. In conclusion, these findings increase the knowledge of disease progression in mononeuropathy with hyperalgesia in human and animal models.     

Characteristics of the gene for a spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome

The nucleotide sequence of the gene for the spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome was determined. It contained four open reading frames encoding A, B, C, and D proteins. By making several subclones, we showed that expression of all the four proteins was necessary for maximal spermidine and putrescine transport activity. A single transport system was involved in the transport of both spermidine and putrescine. The A protein (Mr 43K) was found to be associated with membranes, as shown by Western blot analysis of the cell fractions. In addition, it had consensus amino acid sequences for the nucleotide binding site. B (Mr 31K) and C (Mr 29K) proteins consisted of six putative transmembrane spanning segments linked by hydrophilic segments of variable length as shown by cell localization of the proteins synthesized in maxicells and by hydropathy profiles. D protein (Mr 39K) was inferred to be a polyamine binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein. These results indicate that the spermidine and putrescine transport system can be defined as a bacterial periplasmic transport system.     

Apoptotic inducers activate the release of d-aspartate through a hypotonic stimulus-triggered mechanism in PC12 cells

We have characterized release of d-aspartate (d-Asp), a regulator of hormone synthesis and secretion, via a volume-sensitive organic anion channel (VSOC) in PC12 cells by studying its response to apoptotic stimuli. PC12 cells have been demonstrated to endogenously synthesize d-Asp. Apoptotic inducers, including staurosporin (STS), tumor necrosis factor (TNF)-α, H₂O₂, and C2-ceramide, activate the release of d-Asp through a hypotonic stimulus-triggered mechanism. Putative blockers of the anion channel, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 4,4′-diisothiocyanostilbene-2,2′-sulphonic acid (DIDS), significantly inhibited stress-induced d-Asp release under hypotonic conditions following the application of apoptotic inducers. Hypotonic conditions are essential for activation by apoptotic inducers. Phorbol 12-mirystate 13-acetate and the Ca²⁺ ionophore A23187 increased d-Asp efflux via the VSOC, implying the involvement of intracellular Ca²⁺ in the activation of the d-Asp efflux. However, hypotonic stress and STS had no effect on the concentration of intracellular Ca²⁺ in PC12 cells. Furthermore, an unknown EGTA-sensitive factor(s), other than Ca²⁺, and peroxynitrite may play pivotal roles in STS-enhanced d-Asp release.