Selective up-regulation of an NMDA receptor subunit mRNA in cultured cerebellar granule cells by K(+)-induced depolarization and NMDA treatment.

High KCI or NMDA treatment promotes the survival of cultured neonatal cerebellar granule cells, and these cells become sensitive to NMDA toxicity after prolonged K+ depolarization. Following both treatments, the NMDA receptor increases, as assessed by fura-2 fluorescence analysis of NMDA receptor-mediated intracellular Ca2+ increase. Northern analysis indicates that both treatments specifically up-regulate NMDAR2A subunit mRNA through an increase in resting intracellular Ca2+ concentration. Antisense oligonucleotide analysis further indicates that NMDAR2A mRNA up-regulation is responsible for NMDA receptor induction. Our results demonstrate that regulation of a specific NMDA receptor subunit mRNA governs NMDA receptor induction, which is thought to play an important role in granule cell survival and death.

The basic helix-loop-helix gene hesr2 promotes gliogenesis in mouse retina.

Members of a subclass of hairy/Enhancer of split [E(spl)] homologs, called hesr genes, are structurally related to another subclass of hairy/E(spl) homologs, Hes genes, which play an important role in neural development. To characterize the roles of hesr genes in neural development, we used the retina as a model system. In situ hybridization analysis indicated that all hesr genes are expressed in the developing retina, but only hesr2 expression is associated spatially with gliogenesis. Each member was then misexpressed with retrovirus in the retinal explant cultures prepared from mouse embryos or neonates, which well mimic in vivo retinal development. Interestingly, hesr2 but not hesr1 or hesr3 promoted gliogenesis while inhibiting rod genesis without affecting cell proliferation or death, suggesting that the cells that normally differentiate into rods adopted the glial fate by misexpression of hesr2. The gliogenic activity of hesr2 was more profound when it was misexpressed postnatally than prenatally. In addition, double mutation of the neuronal determination genes Mash1 and Math3, which increases Müller glia at the expense of bipolar cells, upregulated hesr2 expression. These results indicate that, among structurally related hesr genes, only hesr2 promotes glial versus neuronal cell fate specification in the retina and that antagonistic regulation between hesr2 and Mash1-Math3 may determine the ratios of neurons and glia.

Mitochondrial genes are found on minicircle DNA molecules in the mesozoan animal Dicyema.

Animal mitochondrial DNA genomes are generally single circular molecules, 14-20 kb in size, containing a number of functional RNAs and 13 protein-coding genes. Among these, the COI, COII and COIII genes encode three subunits of cytochrome c oxidase. We have isolated and characterized these three mitochondrial genes from the mesozoan Dicyema, a primitive multicellular animal. Surprisingly, the COI, COII and COIII genes are encoded on three small, separate circular DNA molecules (minicircles) of length 1700, 1599 and 1697 bp, respectively. We estimated the copy number of each minicircle at 100 to 1000 per cell, and have shown a mitochondrial localization of the minicircles by in situ hybridization. Furthermore, we could not detect a putative "maxicircle" DNA molecule containing any combination of the COI, COII and COIII genes using either PCR or genomic Southern hybridization. Thus, our results show a novel mitochondrial genome organization in the mesozoan animal Dicyema.

Translation of synonymous codons in family boxes by Mycoplasma capricolum tRNAs with unmodified uridine or adenosine at the first anticodon position.

In Myocoplasma capricolum, codon family boxes except for arginine and threonine (CUN leucine, GUN valine, UCN serine, CCN proline, GCN alanine, and GGN glycine) have only a single tRNA species with the anticodon sequence UNN (N is U, C, A or G), the first nucleoside U being unmodified. Incorporation of the [3H]amino acid into the peptide fraction was examined with the M. capricolum cell-free translation system. Synthetic mRNA containing each of the respective amino acid codons in the coding frame was subjected to translation. The tRNAUNN translated all the family box codons with similar, if not equal, efficiencies. In M. capricolum, there are two species of threonine tRNA, tRNA(UGUThr) and tRNA(AGUThr), the first nucleoside U or A being unmodified. The tRNA(UGUThr) species translates codons ACA, ACG and ACU efficiently, and ACC only poorly. In contrast, the tRNA(AGUThr) species translates codons ACU, ACC and ACG efficiently and ACA poorly.

Effect of volar angulation of extra-articular distal radius fractures on distal radioulnar joint stability: a biomechanical study.

The relationship between increased volar tilt of the distal radius and distal radioulnar joint stability was examined. Distal radioulnar joint stiffness was recorded at 10° intervals from 10° dorsal angulation to 20° of volar angulation from the anatomical position of the radius. Tests were performed with the intact radioulnar ligament and repeated after partial and then complete sectioning of the radioulnar ligament at the ulnar fovea. With the intact radioulnar ligament, distal radioulnar joint stiffness increased significantly at 10° and 20° of volar angulation. Partial sectioning of the radioulnar ligament resulted in an approximate 10% decrease of distal radioulnar joint stiffness compared with the intact state, but distal radioulnar joint stiffness still increased significantly with greater volar tilt. Complete sectioning of the radioulnar ligament significantly decreased distal radioulnar joint stiffness, and increasing the volar tilt did not result in increased distal radioulnar joint stiffness. These results suggest that volar angulation deformities of the distal radius should be corrected to 10° of volar tilt when the triangular fibrocartilage complex is intact. Level of evidence: N/A.

Decreased phospholipase D (PLD) activity in ceramide-induced apoptosis of human keratinocyte cell line HaCaT.

Ceramide is recognized as an intracellular lipid second messenger, which induces various kinds of cell function including apoptosis. To evaluate the competence of ceramide on the keratinocyte apoptosis, we examined effects of a cell-permeable ceramide, N-acetylsphingosine (C2-ceramide), on a human keratinocyte cell line, HaCaT. C2-ceramide induced a distinct apoptosis in HaCaT cells in a time-dependent manner, as inferred by morphologic hallmarks of apoptosis such as bleb formation, cell body shrinkage, nuclear chromatin condensation, and internucleosomal DNA fragmentation. In sharp contrast, an inactive C2-ceramide, dihydroC2-ceramide, which lacks the 4-5trans double bond, failed to induce the apoptosis. The apoptotic HaCaT cells induced by C2-ceramide showed a significant suppression of phospholipase D (PLD) activity, regardless of the presence or absence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). This indicates that C2-ceramide inhibits both GTPgammaS dependent and GTPgammaS independent PLD. The membrane associated GTPgammaS dependent PLD activity was stimulated by recombinant adenosine diphosphate-ribosylation factor. The adenosine diphosphate-ribosylation factor dependent and independent PLD activities were inhibited by C2-ceramide in a concentration dependent manner, but not by the inactive C2-ceramide. The concentration of C2-ceramide to inhibit the membrane associated PLD activity was comparable with that required for apoptosis induction in HaCaT cells. It was thus suggested that downregulation of PLD activity may be involved in the mechanism underlying C2-ceramide induced keratinocyte apoptosis.

Expression of the tyrosinase-encoding gene in a colorless melanophore mutant of the medaka fish, Oryzias latipes.

In the medaka fish Oryzias latipes many mutants for body colors have been isolated. Among them, a colorless melanophore mutant b, carrying b alleles homozygously, has pigmented black eyes but orange-colored skin with amelanotic melanophores, suggesting the presence of a tissue-specific mechanism of melanin formation. To cast light on the molecular basis of the mechanism, we have cloned cDNAs for tyrosinase (Tyr), a key enzyme in melanin biosynthesis, from the wild-type (wt) fish. DNA sequence analysis revealed that all clones encode a protein of 540 amino acids, having five potential glycosylation sites and two copper-binding sites that are characteristic features of Tyr. Genomic DNA blot analysis disclosed that the Tyr gene is present as a single copy in the fish genome. Using a cDNA clone as a probe, RNA blot analysis was carried out. In the wt, the 2.2-kb Tyr mRNA was expressed in eyes and skin but not in liver, corresponding to tissue-specific melanin formation. In the b mutant, contrary to expectation, the mRNA was detected not only in eyes but also in amelanotic skin. Therefore, pigmentation of the skin controlled by b is not directly related to expression of the Tyr gene.

Cloning of the Mycoplasma capricolum gene encoding peptide-chain release factor.

In Mycoplasma capricolum (Mc), a relative of Gram+ eubacteria with a high genomic A + T-content, the UGA codon is assigned to Trp instead of being a stop codon. We previously showed the lack of peptide-chain release factor (RF) activity in vitro responding to the UGA codon in this bacterium [Inagaki et al., Nucleic Acids Res. 21 (1993) 1335-1338]. To obtain more information on the translation termination mechanism of Mc, we isolated and sequenced the gene encoding RF. The deduced amino-acid sequence has no RF-2-specific + 1 frameshift site and shows 50 and 36% identity to Escherichia coli RF-1 and RF-2, respectively. We conclude that this gene encodes the putative RF-1 which would possess the conserved 'five-domain' structure of RF family found in various organisms.

Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats.

Five N-methyl-D-aspartate (NMDA) receptor subunits have been identified thus far: NR1, NR2A, NR2B, NR2C, and NR2D. Here, we have analyzed the expression patterns of mRNAs for the NMDA receptor subunits in the developing and adult rats by in situ hybridization. The developmental changes of the expression patterns were most salient in the cerebellum. In the external granular layer, hybridization signals of mRNAs for NR1, NR2A, NR2B, and NR2C appeared by postnatal day 3, but no NR2D mRNA was expressed at any developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stages examined. The signals for the NR2A mRNA appeared in Purkinje cells and granule cells during the second postnatal week. The signals for the NR2B mRNA in granule cells were seen transiently during the first 2 weeks after birth. The signals for NR2C mRNA appeared in granule cells and glial cells during the second postnatal week. The signals for NR2D mRNA appeared transiently in Purkinje cells during the first 8 postnatal days; in adult rats, these were seen in stellate and Golgi cells. In the cerebellar nuclei, mRNAs for NR1, NR2A, NR2B, and NR2D were more or less expressed on postnatal day 0, while expression signals for the NR2C mRNA were first detected in postnatal day 14. Thus, the most conspicuous changes of expression patterns were observed in the cerebellar cortex during the first 2 weeks after birth, when development and maturation of the cerebellum proceed most rapidly.

Molecular diversity of glutamate receptors and their physiological functions.

Glutamate receptors play an important role in many integrative brain functions and in neuronal development. We report the molecular diversity of NMDA receptors and metabotropic glutamate receptors on the basis of our studies of molecular cloning and characterization of the diverse members of these receptors. The NMDA receptors consist of two distinct types of subunits. NMDAR1 possesses all properties characteristic of the NMDA receptor-channel complex, whereas the four NMDAR2 subunits, termed NMDAR2A-2D, show no channel activity but potentiate the NMDAR1 activity and confer functional variability by different heteromeric formations. The NMDA receptor subunits are considerably divergent from the other ligand-gated ion channels, and the structural architecture of these subunits remains elusive. The mGluRs form a family of at least seven different subtypes termed mGluR1-mGluR7. These receptor subtypes have, seven transmembrane segments and possess a large extracellular domain at their N-terminal regions. The seven mGluR subtypes are classified into three subgroups according to their sequence similarities, signal transduction mechanisms and agonist selectivities: mGluR1/mGluR5, mGluR2/mGluR3 and mGluR4/mGluR6/mGluR7. On the basis of our knowledge of the molecular diversity of the NMDA receptors and mGluRs, we have studied the physiological roles of individual receptor subunits or subtypes. We have shown that K(+)-induced depolarization or NMDA treatment in primary cultures of neonatal cerebellar granule cells induces the functional NMDA receptor and specifically up-regulates NMDAR2A mRNA among the multiple NMDA receptor subunits through the increase in resting intracellular Ca2+ concentrations. Our study demonstrates that the regulation of the specific NMDA receptor subunit mRNA governs the NMDA receptor induction that is thought to play an important role in granule cell survival and death. Analysis of an agonist selectivity and an expression pattern of mGluR6 has indicated that mGluR6 is responsible for synaptic neurotransmission from photoreceptor cells to ON-bipolar cells in the visual system. We have also investigated the function of mGluR2 in granule cells of the accessory olfactory bulb by combining immunoelectron-microscopic analysis with slice-patch recordings on the basis of the identification of a new agonist selective for this receptor subtype. Our results demonstrate that mGluR2 is present at the presynaptic site of granule cells and modulates inhibitory GABA transmission from granule cells to mitral cells. This finding indicates that the mGluR2 activation relieves excited mitral cells from GABA inhibition but maintains the lateral inhibition of unexcited mitral cells, thus resulting in enhancement of the signal-to-noise ratio between the excited mitral cells and their neighboring unexcited mitral cells.

Glutamate receptor agonists enhance the expression of BDNF mRNA in cultured cerebellar granule cells.

The influence of glutamate and its analogues on the expression of BDNF mRNA was studied in cultured cerebellar granule cells. Four-hour exposure of the neurons to the glutamate receptor agonists, quisqualate, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA), increased levels of BDNF mRNA. Glutamate in combination with antagonists of the ionotropic glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), D-2-amino-5-phosphonovalerate (AP-5) and/or (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine hydrogen maleate (MK-801), also increased levels of BDNF mRNA. However, the addition of glutamate itself to the cultures produced severe neuronal death and failed to increase the mRNA level. The onset of the increase in BDNF mRNA by kainate and NMDA lagged behind that by quisqualate. These results indicate that the non-ionotropic glutamate receptor might be involved in the induction of BDNF mRNA. Quisqualate is known to be a potent agonist of both the AMPA/kainate receptor and the metabotropic glutamate receptor. The specific antagonists of the AMPA/kainate receptor, CNQX and 6,7-dinitroquinoxaline-2,3-dione (DNQX) failed to block the increase of BDNF mRNA by quisqualate. Moreover, the desensitization of the metabotropic glutamate receptor by phorbol ester abolished the increase of BDNF mRNA by quisqualate. These results suggest that stimulation of the metabotropic glutamate receptor may be the most predominant component to increase BDNF mRNA in cerebellar granule cell culture.

Phosphatidylcholine-specific phospholipase C, but not phospholipase D, is involved in pemphigus IgG-induced signal transduction.

The precise mechanism of the acantholysis after pemphigus IgGs bind to desmoglein (Dsg) 3 and/or Dsg 1 on the cell surface is as yet unknown. We have previously reported that pemphigus IgG (P-IgG) causes a transient increase in intracellular calcium and inositol 1,4,5-trisphosphate concentration, and subsequent activation of protein kinase C (PKC) in DJM-1 cells, a squamous cell carcinoma line. In order to see whether phosphatidylcholine (PC)-specific phospholipase C (PLC) or phospholipase D (PLD) is involved in the P-IgG-induced signaling process, the production of 1,2-diacylglycerol (DAG) and phosphatidylbutanol (PBut), a potential marker for the determination of PLD activity in the presence of butanol, was determined in DJM-1 cells. A biphasic accumulation of DAG, which consisted of a first transient phase and a second sustained phase, was observed. The second phase of DAG accumulation was profoundly inhibited by pretreatment with D609, a selective inhibitor of PC-PLC, but not by propranolol, an inhibitor of phosphatidate phosphohydrolase. Pemphigus serum after preadsortion of antibodies to Dsg 3 and Dsg 1 with recombinant Dsg 3 and Dsg 1 did not show formation of DAG. PBut was not generated following the addition of P-IgG. In addition, the levels of [3H]phosphocholine, a direct metabolite of PC-PLC, were elevated after the addition of P-IgG. These results suggest that the PC-PLC pathway plays a major role in P-IgG-induced transmembrane signaling by causing prolonged generation of DAG, which may lead to long-term activation of PKC.

Postoperative lumbar spinal instability occurring or progressing secondary to laminectomy.

The manifestations and pathomechanism of postoperative lumbar spinal instability, occurring or progressing secondary to laminectomy, was clarified by means of functional radiographic analysis in a series of 46 patients over 40 years of age. The relation between instability and the clinical symptoms also is discussed. In patients under 60 years of age, instability at the operated level tended to appear in cases of wide laminectomy more often than in cases of partial laminectomy. Occurrence or progress of instability seems to be promoted by resection of the posterior spinal elements rather than the disc. It is further considered that the postoperative aggravation of clinical symptoms may be influenced not only by instability, but also by the other factors.

Glutamate and quisqualate regulate expression of metabotropic glutamate receptor mRNA in cultured cerebellar granule cells.

Metabotropic glutamate receptor (type 1; mGluR1) is expressed predominantly in the hippocampus and the cerebellum. Using cultured cerebellar granule cells, we investigated the regulation of the mGluR1 mRNA expression. Levels of mGluR1 mRNA were decreased to less than half by high potassium stimulation and by glutamate and quisqualate. Although these glutamate receptor agonists tested are also known to cause neuronal cell death in culture, the effect of cell death cannot explain the observed reduction in mGluR1 mRNA because of the following reasons: (a) antagonists of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors inhibited cell death, but not the reduction of the level of mGluR1 mRNA; (b) mGluR1 mRNA returned to its initial level 48 h after the agonist application; and (c) the mRNA level of one of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptors (GluR1) was not altered by these conditions. Therefore, we conclude that the glutamate or quisqualate stimulation can specifically inhibit the expression of mGluR1 mRNA. The dose response of quisqualate for the reduction in mGluR1 mRNA is consistent with that for inositol phosphate formation stimulated through the cloned mGluR1. The mRNA reduction did not require extracellular calcium. Desensitization of mGluR1 with phorbol ester abolished the mRNA reduction. These results suggest that the reduction in mGluR1 mRNA is mediated by the activation of the metabotropic receptor itself.

Lack of peptide-release activity responding to codon UGA in Mycoplasma capricolum.

In Mycoplasma capricolum, a relative of Gram-positive eubacteria with a high genomic AT-content (75%), codon UGA is assigned to tryptophan instead of termination signal. Thus, in this bacterium the release factor 2 (RF-2), that recognizes UAA and UGA termination codons in eubacteria such as Escherichia coli and Bacillus subtilis, would be either specific to UAA or deleted. To test this, we have constructed a cell-free translation system using synthetic mRNA including codon UAA [mRNA(UAA)], UAG [mRNA(UAG)] and UGA [mRNA(UGA)] in-frame. In the absence of tryptophan, the translation of mRNA(UGA) ceased at UGA sites without appreciable release of the synthesized peptides from the ribosomes, whereas with mRNA(UAA) or mRNA(UAG) the bulk of the peptides was released. Upon addition of the E.coli S-100 fraction or B.subtilis S-100 fraction to the translation system, the synthesized peptides with mRNA(UGA) were almost completely released from the ribosomes, presumably because of the presence of RF-2 active to UGA in the added S-100 fraction. These data suggest that RF-2 is deleted or its activity to UGA is strongly weakened in M.capricolum.

Planarian mitochondria. I. Heterogeneity of cytochrome c oxidase subunit I gene sequences in the freshwater planarian, Dugesia japonica.

We have detected sequence heterogeneity in the cytochrome c oxidase subunit I (COI) gene of freshwater planarian, Dugesia japonica, collected in one locality. A part of the COI gene was amplified via the polymerase chain reaction (PCR) using template DNA prepared from a mixture of 500 individuals or from each of 18 individuals. Analyses of DNA sequences by standard strategies for cloning and sequencing or by direct sequencing clearly show that (1) considerable sequence heterogeneity exists in DNA prepared from the mixed individuals, (2) 11 individuals have almost identical sequences (type A), and (3) 7 individuals have sequences different from one another (Seq-D1 to Seq-D7; collectively called type D). Each of the Seq-D1-D7 sequences except for Seq-D5 shows some heterogeneity even in a single individual (heteroplasmy). A possible cause of the sequence heterogeneities is discussed.

Planarian mitochondria. II. The unique genetic code as deduced from cytochrome c oxidase subunit I gene sequences.

The cytochrome c oxidase subunit I (COI) gene sequences from planarian (Dugesia japonica) DNA, most probably of mitochondrial origin, are heterogeneous. Taking advantage of the heterogeneity that occurs primarily in silent sites of the COI DNA sequences, amino acid assignments of several codons have been deduced as nonuniversal: UGA = Trp, AAA = Asp, and AGR (R: A or G) = Ser. In addition, UAA, a stop codon in the universal genetic code, is tentatively assumed to be a tyrosine codon, because three of the sequences examined have UAA at the well-conserved tyrosine site of UAY (Y: U or C) in other planarian sequences as well as in the mitochondria of human, Xenopus, sea urchin, Drosophila, Trypanosoma, and Saccharomyces cerevisiae. AUA would most probably be an isoleucine codon in these mitochondria, whereas it is a methionine codon in the majority of nonplant mitochondria.

Hes1 and Hes3 regulate maintenance of the isthmic organizer and development of the mid/hindbrain.

The isthmic organizer, which is located at the midbrain-hindbrain boundary, plays an essential role in development of the midbrain and anterior hindbrain. It has been shown that homeobox genes regulate establishment of the isthmic organizer, but the mechanism by which the organizer is maintained is not well understood. Here, we found that, in mice doubly mutant for the basic helix-loop-helix genes Hes1 and Hes3, the midbrain and anterior hindbrain structures are missing without any significant cell death. In these mutants, the isthmic organizer cells prematurely differentiate into neurons and terminate expression of secreting molecules such as Fgf8 and Wnt1 and the paired box genes Pax2/5, all of which are essential for the isthmic organizer function. These results indicate that Hes1 and Hes3 prevent premature differentiation and maintain the organizer activity of the isthmic cells, thereby regulating the development of the midbrain and anterior hindbrain.

The bHLH gene Hes6, an inhibitor of Hes1, promotes neuronal differentiation.

We have isolated the basic helix-loop-helix (bHLH) gene Hes6, a novel member of the family of mammalian homologues of Drosophila hairy and Enhancer of split. Hes6 is expressed by both undifferentiated and differentiated cells, unlike Hes1, which is expressed only by the former cells. Hes6 alone does not bind to the DNA but suppresses Hes1 from repressing transcription. In addition, Hes6 suppresses Hes1 from inhibiting Mash1-E47 heterodimer and thereby enables Mash1 and E47 to upregulate transcription in the presence of Hes1. Furthermore, misexpression of Hes6 with retrovirus in the developing retina promotes rod photoreceptor differentiation, like Mash1, in sharp contrast to Hes1, which inhibits cell differentiation. These results suggest that Hes6 is an inhibitor of Hes1, supports Mash1 activity and promotes cell differentiation. Mutation analysis revealed that Hes1- and Hes6-specific functions are, at least in part, interchangeable by alteration of the loop region, suggesting that the loop is not simply a nonfunctional spacer but plays an important role in the specific functions.