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.

Math6, a bHLH gene expressed in the developing nervous system, regulates neuronal versus glial differentiation.

BACKGROUND: Whereas multiple basic helix-loop-helix (bHLH) genes are expressed in the developing nervous system, they account for the differentiation of only subsets of neurones, suggesting that there may be as-yet unidentified bHLH genes. RESULTS: We have isolated a novel bHLH gene, designated Math6, a distant mammalian homologue of the Drosophila proneural gene atonal. Structural analysis of the Math6 gene demonstrated that the coding region is divided into three exons, whereas that of other atonal homologues is present in a single exon, indicating that the genomic structure of Math6 is unique among the atonal homologues. Math6 is initially expressed by neural precursor cells in the ventricular zone, but later by subsets of differentiating and mature neurones such as hippocampal neurones and cerebellar Purkinje cells. Mis-expression of Math6 with retrovirus in the developing retina induced neurogenesis, while inhibiting gliogenesis, without affecting cell proliferation and death. CONCLUSIONS: These results show that cells which would normally differentiate into glia adopted the neuronal fate by mis-expression of Math6, indicating that Math6 promotes neuronal vs. glial fate determination in the nervous system.

Dynamic expression and essential functions of Hes7 in somite segmentation.

The basic helix-loop-helix (bHLH) gene Hes7, a putative Notch effector, encodes a transcriptional repressor. Here, we found that Hes7 expression oscillates in 2-h cycles in the presomitic mesoderm (PSM). In Hes7-null mice, somites are not properly segmented and their anterior-posterior polarity is disrupted. As a result, the somite derivatives such as vertebrae and ribs are severely disorganized. Although expression of Notch and its ligands is not affected significantly, the oscillator and Notch modulator lunatic fringe is expressed continuously throughout the mutant PSM. These results indicate that Hes7 controls the cyclic expression of lunatic fringe and is essential for coordinated somite segmentation.

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.

Hes7: a bHLH-type repressor gene regulated by Notch and expressed in the presomitic mesoderm.

BACKGROUND: Whereas Notch signalling is essential for somitogenesis, mice deficient for the basic helix-loop-helix (bHLH) genes Hes1 and Hes5, downstream Notch effectors, display normal somite formation, indicating that there may be an as-yet unidentified Hes1-related bHLH gene. RESULTS: We identified a novel bHLH gene, designated Hes7, from mouse embryos. Hes7 has a conserved bHLH domain in the amino-terminal region and the WRPW domain at the carboxy-terminal end, like Hes1. The mouse Hes7 gene is located next to Aloxe3, which is mapped to a position 37.0 cM from the centromere on chromosome 11. In a transfection analysis, Hes7 represses transcription from the N box- and E box-containing promoters. In addition, Hes7 suppresses the E47-induced transcriptional activation. Promoter analysis indicated that Hes7 expression is controlled by Notch signalling. Strikingly, Hes7 is specifically expressed in the presomitic mesoderm in a dynamic manner. We also identified two related bHLH genes from human: one is closely related to mouse Hes7 and therefore designated hHes7 and the other designated hHes4. CONCLUSION: The structure, transcriptional activity and expression pattern in the presomitic mesoderm of Hes7 are very similar to those of Hes1, suggesting that Hes7, together with Hes1, may play a role in somite formation under the control of Notch signalling.

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.

Generation of structurally and functionally distinct factors from the basic helix-loop-helix gene Hes3 by alternative first exons.

The basic helix-loop-helix gene Hes3 is expressed by differentiating and mature Purkinje cells in the cerebellum and by neural precursor cells in the embryonal nervous system. We here found that the transcript of cerebellar Hes3, designated Hes3a, has a distinct 5'-terminal structure from that of embryonal Hes3, designated Hes3b, and that the two types of Hes3 transcripts are generated from different first exons. Hes3a lacks the amino-terminal half of the basic region and thus does not bind to the DNA, whereas Hes3b contains a complete basic region and binds to the N box sequence with a high affinity like Hes1, another member of the Hes family. Both types of Hes3 proteins functionally antagonize the neuronal determination factor Mash1, but only Hes3b represses transcription from the N box-containing promoter like Hes1. Furthermore, misexpression of Hes3b with retrovirus in neural precursor cells inhibits neuronal differentiation like Hes1, whereas Hes3a does not. Thus, alternative promoters and first exons that are differentially utilized during neural development generate structurally and functionally distinct proteins from a single Hes3 gene locus.

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.

Ribozyme-catalyzed tRNA aminoacylation.

The RNA world hypothesis implies that coded protein synthesis evolved from a set of ribozyme catalyzed acyl-transfer reactions, including those of aminoacyl-tRNA synthetase ribozymes. We report here that a bifunctional ribozyme generated by directed in vitro evolution can specifically recognize an activated glutaminyl ester and aminoacylate a targeted tRNA, via a covalent aminoacyl-ribozyme intermediate. The ribozyme consists of two distinct catalytic domains; one domain recognizes the glutamine substrate and self-aminoacylates its own 5'-hydroxyl group, and the other recognizes the tRNA and transfers the aminoacyl group to the 3'-end. The interaction of these domains results in a unique pseudoknotted structure, and the ribozyme requires a change in conformation to perform the sequential aminoacylation reactions. Our result supports the idea that aminoacyl-tRNA synthetase ribozymes could have played a key role in the evolution of the genetic code and RNA-directed translation.

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.

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.

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.

The tyrosinase gene from medakafish: transgenic expression rescues albino mutation.

We have determined the 9.8 kb genomic nucleotide sequence of the tyrosinase gene and its 5 upstream region from a teleost, medakafish (Oryzias latipes), and shown that the coding region is composed of five exons and four introns, spanning 4.7 kb. While the number and sizes of the exons were found to be similar to those of mammalian tyrosinase genes, however, the total size of the coding region (4.7 kb) was demonstrated to be less than one tenth those of mouse (ca. 70 kb) and human (> 70 kb) genes. Primer extension analysis revealed that the transcription initiation site starts with a long untranslated leader sequence (340 nucleotide long) from the AUG start codon. A characteristic CATGTG sequence known as a putative regulatory motif in melanocyte-specific genes was present in the 131st base upstream from the initiation site, while other typical regulatory elements such as the TATA-box or M-box common to terrestrial vertebrates were lacking. Transgenic experiments were carried out by microinjecting two kinds of plasmid clones into fertilized eggs of the albino i(l) mutant: one consisting of the genomic tyrosinase gene with the 10 kb 5 upstream region and the other the tyrosinase cDNA with the 3 kb 5 upstream region. The results showed that 53 and 45 of 114 and 118 transgenic eggs, respectively, developed normally beyond hatching and 15 and 10 exhibited a mosaic pattern of pigmentation. Despite the absence of typical regulatory elements like a TATA-box in both cases correct melanin pigmentation was obtained without ectopic expression. Thus, transgenic expression rescued from the albino-i(l) mutation, and the i locus of the medaka genome can be concluded to encode the tyrosinase gene.

The whole nucleotide sequence and chromosomal localization of the gene for human metabotropic glutamate receptor subtype 6.

Metabotropic glutamate receptor subtype 6 (mGluR6) is restrictedly expressed in the retinal ON bipolar cells and ablation of mouse mGluR6 by gene targeting results in a loss of ON responses to light stimulus and impairs the detection of visual contrasts. We have isolated genomic clones containing the human mGluR6 gene and determined the whole nucleotide sequence of the mGluR6 gene. The transcription initiation site of the human mGluR6 gene has been identified using primer extension analysis in combination with reverse transcriptase-mediated polymerase chain reaction analysis of human retinal RNA, while the termination of the mGluR6 mRNA has been assigned by the analysis of rapid amplification of 3'-cDNA ends. The human mGluR6 gene consists of 16,742 base pairs with 10 exons separated by nine introns. The human mGluR6 is composed of 877 amino acid residues with a signal peptide of 24 amino acid residues and the mature protein shows a 94.6% homology with the rat counterpart. A CpG-rich island is present at exon 1 and its preceding putative promoter region and this unusual sequence, like several tissue-specific genes, may be important for a specific expression of the mGluR6 gene in the retinal bipolar cells. The human mGluR6 gene has been mapped to chromosome 5q35 by the analyses of blot hybridization of a DNA panel of human/mouse/hamster somatic cell hybrids and fluorescence in situ hybridization of human chromosomes. This study should provide the genetic basis for not only better understanding the molecular mechanism underlying a tissue-specific expression of the mGluR6 gene but also exploring a potential defect in human mGluR6 in a certain inherited eye disease.

Planarian mitochondria sequence heterogeneity: relationships between the type of cytochrome c oxidase subunit I gene sequence, karyotype and genital organ.

Freshwater planarians Dugesia japonica from three localities were examined for cytochrome c oxidase subunit I (COI) gene sequence, karyotype and the presence of genital organ. The planarians from Mt Fujiwara in Japan were composed of two different groups; one revealed inter- and intraindividual COI gene heterogeneity, while another revealed no sequence heterogeneity. The sequence in planarians from Mt Alishan in Taiwan was homogeneous, while that from the Kenting National Park in Taiwan revealed a considerable heterogeneity. All the planarians having the homogeneous gene sequences carry the 2X karyotype and many of them had genital organs. These are assumed to belong to the sexual lineage. In contrast, almost all planarians having heterogeneous sequences carry the karyotype of either 3X plus 2X (mixoploid) or 3X, and all of them lack genital organs. These lineages are assumed to be asexual. The heterogeneity of COI gene sequences in the presumed asexual lineages would have resulted from an accumulation of mutations by repeated asexual reproduction.

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.

Characterization of excitatory amino acid neurotoxicity in N-methyl-D-aspartate receptor-deficient mouse cortical neuronal cells.

Roles and mechanisms of N-methyl-D-aspartate (NMDA) receptors in glutamate neurotoxicity were investigated in cultures of NMDA receptor-deficient cortical neuronal cells. Mutant mice lacking a functional NMDA receptor were generated by gene targeting of the NR1 NMDA receptor subunit. Cortical neuronal cells prepared from wild-type NR1+/+, heterozygous NR1+/- and homozygous mutant NR1-/- mice at 15-17 days of gestation grew indistinguishably from each other. Brief exposures (5 min) of both NR1+/+ and NR1+/- neuronal cells to glutamate or NMDA, but not kainate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), resulted in widespread neuronal degeneration by the following day. In contrast, neither glutamate nor NMDA treatment caused neuronal degeneration in NR1-/- cells, indicating that NMDA receptors are responsible for rapidly triggered glutamate neurotoxicity. The above four compounds were all effective in inducing the death of NR1+/+ and NR1+/- neuronal cells after prolonged exposure (20-24 h). However, NMDA had no neurotoxic effects on NR1-/- cells, although the other three compounds wer neurotoxic with potencies comparable to those for NR1+/+ and NR1+/- cells. The AMPA and kainate receptors are thus sufficient for inducing slowly triggered glutamate neurotoxicity. Brief exposure of a mixed population of NR1+/+ and NR1-/- neuronal cells to NMDA selectively killed the NMDA receptor-expressing cells without any appreciable effects on neighbouring NMDA receptor-deficient cells. This finding further supports a direct and indispensable role for NMDA receptors in NMDA-evoked neuronal cell death.

Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish, Oryzias latipes.

In the medaka fish (Oryzias latipes) many mutants for body color have been isolated. A typical example is the recessive oculocutaneous albino mutant i, which has amelanotic skin and red-colored eyes with no tyrosinase activity. To cast light on the molecular basis of the albino mechanism, we performed Southern blot analysis of genomic DNA from the mutant with an authentic tyrosinase gene probe; the results demonstrate that an extra 1.9 kb fragment is present inside the first exon. The insertion is responsible for the oculocutaneous albinism. About 80 copies of this fragment are present in the genomes of albino-i and wild-type fish; these repeated sequences are here designated Tol1 elements and the particular element found in the tyrosinase gene of albino-i is denoted Tol1-tyr. The nucleotide sequence of Tol1-tyr shows that the fragment (i) carries terminal inverted repeats of 14 bp, and (ii) is flanked by duplicated 8 bp segments of the host chromosome. These are properties of DNA-mediated transposable elements. Comparison of the nucleotide sequence of Tol1-tyr with other sequences in DNA databases, with special attention to sequences of transposable elements known to date, did not reveal any similarity. Thus, Tol1 constitutes a hitherto unknown family of DNA transposable elements.

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.