[Suppressive effect of lansoprazole on anti-Candida activity of murine macrophages].

This study investigated the influences of lansoprazole (AG1749), a proton pump inhibitor (PPI), and its active derivative (AG2000) on Candida albicans growth and the anti-Candida activity of macrophages. Under concentration of 100 microM, AG1749 and AG2000 had no effect on Candida growth. Murine peritoneal macrophages inhibited the growth of C. albicans in vitro. AG2000 suppressed the anti-Candida activity of macrophages dose-dependently, but AG1749 didn't. The suppressing activity of AG2000 for macrophages was neutralized by adding a SH-compound (L-cysteine) in the medium. This suggests that AG2000 may suppress macrophage function in a similar manner with inhibition of proton pump through binding to SH-molecules. When macrophages were preincubated with AG2000 for 1 hr and washed, their anti-Candida activity remained to be partially inhibited for 14 hrs. These results were discussed in relation to the pathogenesis of esophageal candidiasis.

Conservation of a tRNA core for aminoacylation.

The core region of Escherichia coli tRNA(Cys)is important for aminoacylation of the tRNA. This core contains an unusual G15:G48 base pair, and three adenosine nucleotides A13, A22 and A46 that are likely to form a 46:[13:22] adenosine base triple. We recently observed that the 15:48 base pair and the proposed 46:[13:22] triple are structurally and functionally coupled to contribute to aminoacylation. Inspection of a database of tRNA sequences shows that these elements are only found in one other tRNA, the Haemophilus influenzae tRNA(Cys). Because of the complexity of the core, conservation of sequence does not mean conservation of function. We here tested whether the conserved elements in H. influenzae tRNA(Cys)were also important for aminoacylation of H. influenzae tRNA(Cys). We cloned and purified a recombinant H. influenzae cysteine-tRNA synthe-tase and showed that it depends on 15:48 and 13, 22 and 46 in a relationship analogous to that of E. coli cysteine-tRNA synthetase. The functional conservation of the tRNA core is correlated with sequence conservation between E.coli and H.influenzae cysteine-tRNA synthetases. As the genome of H. influenzae is one of the smallest and may approximate a small autonomous entity in the development of life, the dependence of this genome on G15:G48 and its coupling with the proposed A46:[A13:A22] triple for aminoacylation with cysteine suggests an early role of these motifs in the evolution of decoding genetic information.

Precursor of pro-apoptotic cytokine modulates aminoacylation activity of tRNA synthetase.

Endothelial monocyte activating polypeptide II (EMAPII) is a cytokine that is specifically induced by apoptosis. Its precursor (pro-EMAPII) has been suggested to be identical to p43, which is associated with the multi-tRNA synthetase complex. Herein, we have demonstrated that the N-terminal domain of pro-EMAPII interacts with the N-terminal extension of human cytoplasmic arginyl-tRNA synthetase (RRS) using genetic and immunoprecipitation analyses. Aminoacylation activity of RRS was enhanced about 2.5-fold by the interaction with pro-EMAPII but not with its N- or C-terminal domains alone. The N-terminal extension of RRS was not required for enzyme activity but did mediate activity stimulation by pro-EMAPII. Pro-EMAPII reduced the apparent Km of RRS to tRNA, whereas the kcat value remained unchanged. Therefore, the precursor of EMAPII is a multi-functional protein that assists aminoacylation in normal cells and releases the functional cytokine upon apoptosis.

Genetic dissection of protein-protein interactions in multi-tRNA synthetase complex.

Cytoplasmic aminoacyl-tRNA synthetases of higher eukaryotes acquired extra peptides in the course of their evolution. It has been thought that these appendices are related to the occurrence of the multiprotein complex consisting of at least eight different tRNA synthetase polypeptides. This complex is believed to be a signature feature of metazoans. In this study, we used multiple sequence alignments to infer the locations of the peptide appendices from human cytoplasmic tRNA synthetases found in the multisynthetase complex. The selected peptide appendices ranged from 22 aa of aspartyl-tRNA synthetase to 267 aa of methionyl-tRNA synthetase. We then made genetic constructions to investigate interactions between all 64 combinations of these peptides that were individually fused to nonsynthetase test proteins. The analyses identified 11 (10 heterologous and 1 homologous) interactions. The six peptide-dependent interactions paralleled what had been detected by crosslinking methods applied to the isolated multisynthetase complex. Thus, small peptide appendices seem to link together different synthetases into a complex. In addition, five interacting pairs that had not been detected previously were suggested from the observed peptide-dependent complexes.

Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family.

We have cloned and sequenced a cDNA encoding human cytoplasmic asparaginyl-tRNA synthetase (AsnRS). The N-terminal appended domain of 112 amino acid represents the signature sequence for the eukaryotic AsnRS and is absent from archaebacterial or eubacterial enzymes. The canonical ortholog for AsnRS is absent from most archaebacterial and some eubacterial genomes, indicating that in those organisms, formation of asparaginyl-tRNA is independent of the enzyme. The high degree of sequence conservation among asparaginyl- and aspartyl-tRNA synthetases (AsxRS) made it possible to infer the evolutionary paths of the two enzymes. The data show the neighbor relationship between AsnRS and eubacterial aspartyl-tRNA synthetase, and support the occurrence of AsnRS early in the course of evolution, which is in contrast to the proposed late occurrence of glutaminyl-tRNA synthetase.

Biochemical and phylogenetic analyses of methionyl-tRNA synthetase isolated from a pathogenic microorganism, Mycobacterium tuberculosis.

Mycobacterium tuberculosis methionyl-tRNA synthetase (MetRS) has been cloned and characterized. The protein contains class I signature sequences but lacks the Zn2+ binding motif and the C-terminal dimerization appendix that are found in MetRSs from several organisms including E. coli MetRS. Consistent with these features, the enzyme behaved as a monomer in a gel filtration chromatography and did not contain the bound Zn2+. Nonetheless, it was active to the tRNAMet of E. coli as determined by in vivo genetic complementation and in vitro reaction. Phylogenetic analysis separated the M. tuberculosis and E. coli MetRSs into prokaryote and eukaryote-archaea group, respectively. This result is consistent with the taxonomic locations of the organism but is an interesting contrast to the case of its paralogous protein, isoleucyl-tRNA synthetase, and suggests that the two enzymes evolved in separate idiosyncratic pathways.

Proliferating cell nuclear antigen in hypertrophied spinal ligaments. Immunohistochemical localization of proliferating cell nuclear antigen in hypertrophied posterior longitudinal ligament of the cervical spine.

STUDY DESIGN: An experimental immunohistochemical investigation using an antibody for proliferating cell nuclear antigen. Surgically-extirpated specimens of posterior longitudinal ligament tissues from patients with hypertrophy of the posterior longitudinal ligament and other disorders of the cervical spine were analyzed. OBJECTIVE: To analyze the developmental mechanism of hypertrophy of the posterior longitudinal ligament, the authors evaluated the growth activity of cells in the posterior longitudinal ligament tissues by examining the immunolocalization of the proliferating cell nuclear antigen. SUMMARY OF BACKGROUND DATA: Although a number of cases of hypertrophy of the posterior longitudinal ligament have been reported, the pathophysiology of ligament hypertrophy is still unclear. It is well established that the proliferating cell nuclear antigen is a cell proliferation marker, and immunohistochemical analysis using an anti-proliferating cell nuclear antigen antibody is of value in assessing the cell growth activity of several tissues. METHODS: During anterior decompression surgery in the cervical spine, the authors extirpated posterior longitudinal ligament tissues in one piece from patients with hypertrophy of the posterior longitudinal ligament, ossification of the posterior longitudinal ligament, cervical disc herniation, and cervical spondylotic myelopathy. Midsagittal sections of the specimens were stained with an antibody against the proliferating cell nuclear antigen. RESULTS: In cases of hypertrophy of the posterior longitudinal ligament, immunostaining with the proliferating cell nuclear antigen was detected in cells in the posterior longitudinal ligament, not only at the vertebral endplate level, but also at the midvertebral level. A similar distribution of proliferating cell nuclear antigen-positive cells was observed in cases of ossification of the posterior longitudinal ligament. In cases of cervical disc herniation, however, proliferating cell nuclear antigen-positive cells in posterior longitudinal ligament tissues were restricted to the vertebral endplate level. No immunostaining with the proliferating cell nuclear antigen was seen in posterior longitudinal ligament tissues in cases of cervical spondylotic myelopathy. CONCLUSIONS: Cell growth activity was accelerated in posterior longitudinal ligament tissues in cases of hypertrophy of the posterior longitudinal ligament; such an unusual phenotype of posterior longitudinal ligament cells was also expressed in cases of ossification of cervical disc herniation and cervical spondylotic myelopathy. Therefore, up-regulation of the growth of posterior longitudinal ligament cells may contribute to the development of hypertrophy of the posterior longitudinal ligament, and some common regulatory mechanism(s) on the proliferation of posterior longitudinal ligament cells seem to underlie the development of hypertrophy of the posterior longitudinal ligament and ossification of the posterior longitudinal ligament.

Human lysyl-tRNA synthetase accepts nucleotide 73 variants and rescues Escherichia coli double-defective mutant.

The nucleotide 73 (N73) "discriminator" base in the acceptor stem is a key element for efficient and specific aminoacylation of tRNAs and of microhelix substrates derived from tRNA acceptor stems. This nucleotide was possibly one of the first to be used for differentiating among groups of early RNA substrates by tRNA synthetases. In contrast to many other synthetases, we report here that the class II human lysyl-tRNA synthetase is relatively insensitive to the nature of N73. We cloned, sequenced, and expressed the enzyme, which is a close homologue of the class II yeast aspartyl-tRNA synthetase whose co-crystal structure (with tRNAAsp) is known. The latter enzyme has a strong requirement for G73, which interacts with 4 of the 14 residues within the "motif 2" loop of the enzyme. Even though eukaryotic lysine tRNAs also encode G73, the motif 2 loop sequence of lysyl-tRNA synthetase differs at multiple positions from that of the aspartate enzyme. Indeed, the recombinant human lysine enzyme shows little preference for G, and even charges human tRNA transcripts encoding the A73 found in E. coli lysine tRNAs. Moreover, while the lysine enzyme is the only one in E. coli to be encoded by two separate genes, a double mutant that disables both genes is complemented by a cDNA expressing the human protein. Thus, the sequence of the loop of motif 2 of human lysyl-tRNA synthetase specifies a structural variation that accommodates nucleotide degeneracy at position 73. This sequence might be used as a starting point for obtaining highly specific interactions with any given N73 by simple amino acid replacements.

Juvenile amyotrophy of the distal upper extremity: pathologic findings of the dura mater and surgical management.

STUDY DESIGN: Five cases of juvenile amyotrophy of the distal upper extremity were reviewed retrospectively to elucidate the pathophysiology of spinal cord dysfunction and the results of surgical management. OBJECTIVES: To clarify the pathogenesis of juvenile amyotrophy of the distal upper extremity and to present the results of a new surgical treatment. SUMMARY OF BACKGROUND DATA: Hirayama first reported this disorder in 1959. It is characterized by juvenile onset, slow progression, and involvement of the unilateral distal upper extremity. Recently, compression of the cervical spinal cord during neck flexion was implicated as a possible etiology of the disorder, but the exact etiology is still unknown. The value of surgical treatment for patients with juvenile amyotrophy of the distal upper extremity has not been established. METHODS: The clinical and radiographic characteristics of five patients with juvenile amyotrophy of the distal upper extremity were examined. All five patients were treated surgically with duraplasty in combination with posterior spinal fusion. Dynamic and computed tomographic myelography were performed before and after surgery. Intraoperative ultrasonography and conductive spinal cord evoked potentials were recorded before and after duraplasty. The surgical results and the histology of the resected dura were studied. RESULTS: Myelograms taken with the neck in a neutral position showed that the spinal cord was flattened in all five patients. When the neck was flexed, the dura and the spinal cord were compressed further. Intraoperative ultrasonography during neck flexion revealed an anterior shift of the spinal cord and decreased spinal cord pulsation. Amplitude of the conductive spinal cord evoked potentials decreased with neck flexion but increased after dural incision. Histologically, the dura appeared abnormal in that it contained few elastic fibers without the normal wavy structure. CONCLUSIONS: Juvenile amyotrophy of the distal upper extremity was characterized by inelastic dura that constricts and compresses the cervical spinal cord when the neck is in either a neutral or a flexed position. Abnormal dura appeared to be the cause of juvenile amyotrophy of the distal upper extremity. Duraplasty with spinal fusion are proposed as treatments.

Maintaining genetic code through adaptations of tRNA synthetases to taxonomic domains.

The universal genetic code is determined by the aminoacylation of tRNAs. In spite of the universality of the code, there are barriers to aminoacylation across taxonomic domains. These barriers are thought to correlate with the co-segregation of sequences of synthetases and tRNAs into distinct taxonomic domains. By contrast, we show here examples of eukaryote-like synthetases that are found in certain prokaryotes. The associated tRNAs have retained their prokaryote-like character in each instance. Thus, co-segregation of domain-specific synthetases and tRNAs does not always occur. Instead, synthetases make adaptations of tRNA-protein contacts to cross taxonomic domains.

Effects of nifedipine on physical dependence on barbital or diazepam in rats.

The effects of nifedipine on the development of physical dependence on barbital and diazepam in rats were examined using the drug-admixed food method. Rats were chronically treated with either barbital- or barbital in combination with nifedipine-admixed food for 28 days, and with either diazepam- or diazepam in combination with nifedipine-admixed food for 26 days, on schedules of gradually increasing doses of barbital or diazepam. Withdrawal was conducted by substituting normal food for drug-admixed food on the last day of the treatment. Co-administration of nifedipine with barbital potentiated weight loss and withdrawal scores after the termination of barbital treatment. However, the withdrawal signs after the termination of diazepam treatment were not affected by co-administration of nifedipine with diazepam. These results suggest that nifedipine potentiates the development of physical dependence on barbital but not diazepam. It is known that co-administration of dihydropyridine derivative nitrendipine suppresses the development of physical dependence on ethanol. Basing on the differences in sensitivity of central depressants, barbiturates, benzodiazepines and ethanol, to three types of voltage-dependent Ca2+ channels, such as L-, N- and T-types studied so far, the development of physical dependence on central depressants may be modified differently by L-type Ca2+ channel blockers, corresponding to respective depressants.

Human glycyl-tRNA synthetase. Wide divergence of primary structure from bacterial counterpart and species-specific aminoacylation.

Several class I and class II human tRNA synthetases are clearly related to their bacterial counterparts. We report here the cloning, cDNA sequence, deduced primary structure, and expression in bacteria of a class II human glycyl-tRNA synthetase. While the human sequence aligns well with a Bombyx mori and a Saccharomyces cerevisiae sequence for glycyl-tRNA synthetase, particularly in the region of the class II-defining sequence motifs, it diverges widely from that of the Escherichia coli enzyme. The divergence is so great that from the sequences alone we cannot conclude that the human and E. coli proteins are descended from homologous genes. Moreover, even though the human and E. coli class II alanyl-tRNA synthetases cross-acylate their respective tRNAs, aminoacylations by the recombinant human and E. coli glycyl-tRNA synthetases are restricted to their homologous tRNAs. The species-specific aminoacylations correlate with a nucleotide sequence difference at a location in the acceptor stem that is known to be critical for aminoacylations by the E. coli enzyme. Thus, glycyl-tRNA synthetase may have followed a path of historical development different in at least some respects from that of several other tRNA synthetases.

Attenuation of anticonvulsant effects of diazepam after chronic treatment with bicuculline.

Changes in the GABAergic system after chronic treatment with bicuculline were examined in two strains of inbred rats, Fischer 344 (F344) and Lewis (LEW). Rats received an IP injection of either bicuculline (2 mg/kg) or vehicle once a day for 12 days. After this chronic treatment, the effects of diazepam (1 mg/kg, IP) and pentobarbital (20 mg/kg, IP) on bicuculline-induced convulsions were measured. Bicuculline was acutely infused into a tail vein at 0.0415 mg/min, and the infusion was terminated when rats showed seizure. Following the chronic bicuculline treatment, the anticonvulsant effect of diazepam, but not of pentobarbital, was significantly reduced as compared to its effect following chronic vehicle treatment in both strains. Both diazepam and pentobarbital showed a significant difference in anticonvulsant effects between strains (F344 > LEW). The hypnotic effects of muscimol, barbital, pentobarbital, and ethanol following chronic bicuculline treatment were examined. There was no significant difference in sleep time induced by these drugs between bicuculline- and vehicle-treated rats. These results suggest that the attenuation of diazepam's anticonvulsant effect after chronic bicuculline treatment may result from functional changes in benzodiazepine receptors and that the anticonvulsant effects of diazepam and pentobarbital may be influenced by genetic factors. Moreover, the hypnotic effects of several drugs tested are apparently not affected by chronic bicuculline treatment.

Genetic differences in the development of physical dependence upon diazepam in Lewis and Fischer 344 inbred rat strains.

The purpose of the present study was to investigate physical dependence upon diazepam systematically in two inbred strains of rats, Lewis (LEW) and Fischer 344 (F344). Rats were chronically fed food containing diazepam on an escalating drug dosage schedule, from 1 and 2 to 12 mg/g of food, over a period of 30 days. During treatment, the growth curve in LEW and F344 rats was suppressed compared with the respective controls. Motor incoordination was evaluated by a rotarod performance test. The ranking of the motor incoordination during the final concentration of diazepam was as follows: F344 greater than LEW. After substitution of normal food for the diazepam-admixed food, various signs of diazepam withdrawal occurred 16-120 h later. These signs included vocalization, irritability, muscle rigidity, ear-twitching, Straub's tail, piloerection, fascicular twitch, tremor, convulsion, and death. The incidences of vocalization, ear-twitching, piloerection, and tremor in F344 were significantly higher than those in LEW rats. Furthermore, two of six F344 rats showed spontaneous convulsions and one rat died of convulsions. Overall withdrawal scores were significantly greater in F344 (16.0) than in LEW (6.3) rats. These results suggest that diazepam withdrawal severity is strongly influenced by genetic factors, and F344 rats are highly susceptible to dependence upon benzodiazepines.

Susceptibility to, tolerance to, and physical dependence on ethanol and barbital in two inbred strains of rats.

1. Ethanol-induced sleep time was significantly longer in F344 than LEW rats. However, there is no difference in barbital-induced sleep time between F344 and LEW. 2. Development of tolerance to ethanol-induced motor impairment was slightly faster in F344 than in LEW rats. While, LEW rats more easily developed tolerance to the impairment by barbital in comparison with F344 rats. 3. F344 and LEW rats were chronically treated with liquid diet containing ethanol or with barbital-admixed food. After the termination of ethanol and barbital treatments, various withdrawal signs occurred in F344 rats, including tremor and convulsions, whereas LEW rats showed no convulsions. Withdrawal scores of ethanol and barbital were significantly higher in F344 than in LEW rats. 4. These results suggest that strain differences in physical dependence on ethanol and barbital may be mainly influenced by the susceptibility to ethanol and the development of tolerance to barbital, respectively.