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W J Song

Publications and source records attributed to W J Song.

At least 37 records · Page 2Linked to original sources
PubMed1997

The murine Dyrk protein maps to chromosome 16, localizes to the nucleus, and can form multimers.

We mapped the murine copy of the Dyrk gene and examined its subcellular localization and self-interaction. We found that: (1) Dyrk maps to the distal portion of MMU chromosome 16, consistent with previous mapping of the human DYRK gene to the Down syndrome critical region on HSA 21q22.2. (2) The Dyrk protein localizes to the cell nucleus, affording the potential of controlling the expression of other gene(s). (3) The Dyrk protein can self-associate in a two-hybrid system, in accord with the presence of a leucine zipper motif noted in the original sequence. In particular, its expression pattern in frontal brain nuclei during murine embryogenesis, its subcellular localization and its ability to interact with other proteins all suggest that this protein remains a good candidate to mediate some of the pleiotropic effects of Down syndrome.

3T3 Cells↗
PubMed1997

D2 dopamine receptors reduce N-type Ca2+ currents in rat neostriatal cholinergic interneurons through a membrane-delimited, protein-kinase-C-insensitive pathway.

Dopamine has long been known to regulate the activity of striatal cholinergic interneurons and the release of acetylcholine. Yet, the cellular mechanisms by which this regulation occurs have not been elucidated. One way in which dopamine might act is by modulating voltage-dependent Ca2+ channels. To test this hypothesis, the impact of dopaminergic agonists on Ca2+ channels in neostriatal cholinergic interneurons was studied by combined whole cell voltage-clamp recording and single-cell reverse transcription-polymerase chain reactions. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. Nearly, all interneurons tested (90%, n = 17) coexpressed D2 (short and long isoforms) and D1b (D5) dopamine receptor mRNAs. D1a receptor mRNA was found in only a small subset (20%) of the sample and D3 and D4 receptor mRNAs were undetectable. D2 receptor agonists rapidly and reversibly reduced N-type Ca2+ currents. D1b/D1a receptor activation had little or no effect on Ca2+ currents. The D2 receptor antagonist sulpiride blocked the effect of D2 agonists. Dialysis with guanosine-5'-O-(2-thiodiphosphate) or brief exposure to the G protein (Gi/o) alkylating agent N-ethylmaleimide also blocked the D2 modulation. The reduction in N-type currents was neither accompanied by kinetic slowing nor significantly reversed by depolarizing prepulses. The D2 receptor effects were mediated by a membrane-delimited pathway, because the modulation was not seen in cell-attached patches when agonist was applied to the bath and was not disrupted by perturbations in cytosolic signaling pathways known to be linked to D2 receptors. Activation of M2 muscarinic receptors occluded the D2 modulation, suggesting a shared signaling element. However, activation of protein kinase C attenuated the M2 modulation without significantly affecting the D2 modulation. Taken together, our results suggest that activation of D2 dopamine receptors in cholinergic interneurons reduces N-type Ca2+ currents via a membrane-delimited, Gi/o class G protein pathway that is not regulated by protein kinase C. This signaling pathway may underlie the ability of D2 receptors to reduce striatal acetylcholine release.

Animals↗
PubMed1996

Isolation of human and murine homologues of the Drosophila minibrain gene: human homologue maps to 21q22.2 in the Down syndrome "critical region".

The presence of an extra copy of human chromosome 21 (trisomy 21), especially region 21q22.2, causes many phenotypes in Down syndrome, including mental retardation. To study genes potentially responsible for some of these phenotypes, we cloned a human candidate gene (DYRK) from 21q22.2 and its murine counterpart (Dyrk) that are homologous to the Drosophila minibrain (mnb) gene required for neurogenesis and to the rat Dyrk gene (dual specificity tyrosine phosphorylation regulated kinase). The three mammalian genes are highly conserved, >99% identical at the protein level over their 763-amino-acid (aa) open reading frame; in addition, the mammalian genes are 83% identical over 414 aa to the smaller 542-aa mnb protein. The predicted human DYRK and murine Dyrk proteins both contain a nuclear targeting signal sequence, a protein kinase domain, a putative leucine zipper motif, and a highly conserved 13-consecutive-histidine repeat. Fluorescence in situ hybridization and regional mapping data localize DYRK between markers D21S336 and D21S337 in the 21q22.2 region. Northern blot analysis indicated that both human and murine genes encode approximately 6-kb transcripts. PCR screening of cDNA libraries derived from various human and murine tissues indicated that DYRK and Dyrk are expressed both during development and in the adult. In situ hybridization of Dyrk to mouse embryos (13, 15, and 17 days postcoitus) indicates a differential spatial and temporal pattern of expression, with the most abundant signal localized in brain gray matter, spinal cord, and retina. The observed expression pattern is coincident with many of the clinical findings in trisomy 21. Its chromosomal locus (21q22. 2), its homology to the mnb gene, and the in situ hybridization expression patterns of the murine Dyrk combined with the fact that transgenic mice for a YAC to which DYRK maps are mentally deficient suggest that DYRK may be involved in the abnormal neurogenesis found in Down syndrome.

Adult↗
PubMed1996

Coordinated expression of dopamine receptors in neostriatal medium spiny neurons.

In recent years, the distribution of dopamine receptor subtypes among the principal neurons of the neostriatum has been the subject of debate. Conventional anatomical and physiological approaches have yielded starkly different estimates of the extent to which D1 and D2 class dopamine receptors are colocalized. One plausible explanation for the discrepancy is that some dopamine receptors are present in physiologically significant numbers, but the mRNA for these receptors is not detectable with conventional techniques. To test this hypothesis, we examined the expression of DA receptors in individual neostriatal neurons by patch-clamp and RT-PCR techniques. Because of the strong correlation between peptide expression and projection site, medium spiny neurons were divided into three groups on the basis of expression of mRNA for enkephalin (ENK) and substance P (SP). Neurons expressing detectable levels of SP but not ENK had abundant mRNA for the D1a receptor. A subset of these cells (approximately 50%) coexpressed D3 or D4 receptor mRNA. Neurons expressing detectable levels of ENK but not SP had abundant mRNA for D2 receptor isoforms (short and long). A subset (10-25%) of these neurons coexpressed D1a or D1b mRNAs. Neurons coexpressing ENK and SP mRNAs consistently coexpressed D1a and D2 mRNAs in relatively high abundance. Functional analysis of neurons expressing lower abundance mRNAs revealed clear physiological consequences that could be attributed to these receptors. These results suggest that, although colocalization of D1a and D2 receptors is limited, functional D1 and D2 class receptors are colocalized in nearly one-half of all medium spiny projection neurons.

Animals↗
PubMed1996

Identification and analysis of the human and murine putative chromatin structure regulator SUPT6H and Supt6h.

We have isolated and sequenced SUPT6H and Supt6h, the human and murine homologues of the Saccharomyces cerevisiae and Caenorhabditis elegans genes SPT6 (P using 1603 aa = 6.7 e-95) and emb-5 (P using 1603 aa = 7.0 e-288), respectively. The human and murine SPT6 homologues are virtually identical, as they share >98% identity and >99% similarity at the protein level. The derived amino acid sequences of these two genes predict a 1603-aa protein (human) and a 1726-bp protein (mouse), respectively. There were several known features, including a highly acidic 5'-region, a degenerate SH2 domain, and a leucine zipper. These features are consistent with a nuclear protein that regulates transcription, whose extreme conservation underscores the likely importance of this gene in mammalian development. Expression of human and murine SPT6 homologues was analyzed by Northern blotting, which revealed a 7. 0-kb transcript that was expressed constitutively. The SPT6 homologue was mapped to chromosome 17q11.2 in human by somatic cell hybrid analysis and in situ hybridization. These data indicate that SUPT6H and Supt6h are functionally analogous to SPT6 and emb-5 and may therefore regulate transcription through establishment or maintenance of chromatin structure.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of the human and mouse homologues (SUPT4H and Supt4h) of the yeast SPT4 gene.

To study gene regulation mediated by chromatin in mammals, we isolated the human (SUPT4H) and murine (Supt4h) counterparts of the yeast gene encoding SPT4; the product of this gene presumably interacts with the products of the mammalian homologues (which we have also cloned) of yeast SPT5 and SPT6, thereby modulating chromatin formation and activity. We isolated two different sized human SUPT4H cDNA clones (1464 and 728 nt) and one murine Supt4h (688 nt) cDNA clone; all three encode the same 117-amino-acid protein with conservation of the zinc finger motif found in SPT4. Conservation of this zinc finger motif from yeast to mouse and human implies functional importance. Although the overall sequence homology at the DNA level between the human 728-nt transcript and the murine 688-nt transcript is only 78.4%, the DNA sequence homology is 97.7% within the coding region. At the protein level, the amino acid sequences of the translated murine Supt4h and the human SUPT4H gene products are identical. The likely functional copy of SUPT4H, which has at least two introns, maps to human chromosome 17, with candidate intronless pseudogenes on chromosomes 2, 12, and 20. Buttressing the hypothesis that this is a gene required constitutively, both the human SUPT4H transcripts and the murine Supt4h transcript are expressed widely, although not at equal levels (e.g., such as most histones), in all fetal and adult tissues that we examined.

Adult↗
PubMed1996

Use of a fluorescent-PCR reaction to detect genomic sequence copy number and transcriptional abundance.

We present a fluorescent-PCR-based technique to assay genomic sequence copy number and transcriptional abundance. This technique relies on the ability to follow fluorescent PCR progressively in real time during the exponential phase of the reaction so that quantitative PCR is accomplished. We demonstrated the ability of this technique to quantitate both known deletions and amplifications of loci that have been measured previously by other methods, and to measure transcriptional abundance. Using an efficient variant of the fluorescent-PCR technology, we can monitor transcription semiquantitatively. The ability to detect all amplifications and deletions at any single copy locus by PCR makes this the technique of choice to assay genomic sequence copy number anomalies in birth defects and cancers. The ability to detect variations in transcript abundance enables this technique to fashion a time and tissue analysis of transcription.

Chromosome Aberrations↗
PubMed1996

Voltage-dependent facilitation of calcium channels in rat neostriatal neurons.

1. Voltage-dependent facilitation of Ca2+ channels was studied in acutely isolated adult rat neostriatal neurons. Particular attention was paid to the facilitation of L-type channels. 2. In the absence of neuromodulators, the current-voltage relationship for whole cell Ba2+ currents was enhanced by a prepulse to +100 mV. The median enhancement at -20 mV was nearly 60%. The voltage dependence and kinetics of the processes underlying the facilitation were similar to those reported in other neurons. N-, P-, Q-, and L-type currents contributed to the observed facilitation. 3. Voltage-dependent facilitation of L-type currents was studied by subtracting nifedipine-insensitive currents from control currents. Although the kinetics were similar to those of the whole cell currents, the half-activation voltage for facilitation of L-type currents [half-activation voltage (Vh) = -0.6 mV, slope factors (Vc) = 11.8 mV, [n = 5] was significantly less depolarized than that of the pooled currents (Vh = 47.3 mV, Vc = 12.3 mV, n = 7). 4. Repetitive depolarization with spikelike waveforms was also able to induce facilitation of L-type currents, suggesting that facilitation was not simply a consequence of a modal shift in gating like that induced by Bay K 8644. 6. Combined whole cell recording and single-cell reverse transcription-polymerase chain reaction amplification revealed that neostriatal medium spiny neurons expressed detectable levels of either class C or class D L-type channel alpha 1, subunit mRNA. Both neurons expressing class C L-type channels and neurons expressing class D L-type channels exhibited voltage-dependent facilitation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Morphology of individual axons in crossed corticorubral projections in developing cats and effects of partial denervation.

Ordered neuronal connections in mature brains are thought to be sculpted from initially diffuse projections by elimination of inappropriate projections and strengthening of appropriate ones. Although evidence suggests that neuronal activity plays a role in these processes, the mechanism behind the modification of neuronal connections remains obscure. To gain insight into the mechanisms of axonal elimination and projection strengthening, we examined the morphology of individual axons that were to be eliminated as well as the consequences of partial denervation. While corticorubral projections in adult cats are thought to be uncrossed, early in postnatal development and after early unilateral lesions to the sensorimotor cortex, however, a significant amount of crossed corticorubral projections occurs. We examined the morphology of individual corticorubral axons in fetal cats and kittens from embryonic day 59 to postnatal day 48 and those that had received early unilateral lesions to the cortex, by serial reconstruction of Phaseolus-vulgaris-leucoagglutinin- or biocytin-labeled axons. For about 2 weeks during pre- and postnatal development, crossed axons remained simple in morphology, with few branches. Thereafter, they showed an increase in branch number, but then began to show fewer branches again. Axons and their collaterals were found in nonrestricted areas of the red nucleus (RN) throughout the period of observation, indicating that axons can sit at an inappropriate target for weeks but fail to ramify. In contrast, crossed corticorubral axons in kittens with cortical lesions showed terminal-arbor-like structures in the RN region that are in mirror symmetry to topographically appropriate areas in the ipsilateral RN, although some showed simple morphology without arbors. These complicated forms of morphology of individual axons during development and after partial denervation may not be explained by a simple activity-dependent mechanism.

Animals↗
PubMed1995

Prenatal development of cerebrorubral and cerebellorubral projections in cats.

Cerebrorubral (CR) and cerebellorubral (CBR) afferents are localized to distal dendrites and somata of red nucleus (RN) neurons, respectively, in adult cat. To test if this synaptic site segregation is established by a sequential arrival of the two afferents, the development of CR and CBR projections was studied. CR axons arrived in the RN at embryonic day (E) 50 or 51, while CBR axons entered the RN before E35. These results suggest the possibility that arrival of CBR axons before CR axons leads to the segregation of synaptic sites.

Afferent Pathways↗
PubMed1995

Perinatal development of action potential propagation in cat rubrospinal axons.

1. The development of action potential conduction was studied by intracellular recording of antidromic spikes in cat rubrospinal cells. 2. The distance between the C1 and L1 spinal segments increased linearly from 5.6 cm at embryonic day (E) 59 to 9.8 cm at postnatal day (P) 30. 3. The conduction time from the C1 segment to the rubrospinal neuron soma, estimated from antidromic spike latency evoked by stimulation of the C1 segment, decreased rapidly prior to birth and then slowly thereafter. This coincided with a reduction in conduction time variation between cells. 4. The conduction time from the red nucleus to the L1 segment followed a similar time course during development. The conduction time reached the adult value by P30, at which time the spinal cord is only half the adult length. 5. The conduction velocity between the C1 and L1 segments increased monotonically between E59 and P30, from a low of 1 m s-1 to a maximum of 34 m s-1. 6. The rise time of rubrospinal neuron somadendritic spikes followed a developmental time course similar to that for conduction time. 7. Myelination of rubrospinal axons, as judged by the presence of myelinated segment spikes, began to occur prior to E59. 8. These findings suggest that development of action potential propagation in rubrospinal cells can be divided into an early and a late stage: conduction time reaches the adult value during the early stage, i.e. by the first postnatal month, and is maintained during the late stage. We propose that myelination, axon diameter increase and maturation of membrane properties act to reduce conduction time to adult values during the early stage, while a proportional increase in fibre diameter with axonal length results in a constant conduction time during the late stage.

Action Potentials↗
PubMed1995

Expressed sequence tags from the long arm of human chromosome 21.

We isolated expressed sequence tags (ESTs) on the long arm of chromosome 21. The ESTs were mapped by PCR using a monochromosomal somatic-cell mapping panel. Of a total of 55 cDNAs, 30 mapped back uniquely to chromosome 21, 7 mapped back to other chromosomes including chromosome 21, 8 mapped back to chromosomes other than 21, and 10 could not be assigned using this methodology. The 30 chromosome 21-specific markers so isolated represent useful EST markers. A rapid PCR-based method was used to delineate the expression pattern of these 30 pairs in different tissues.

Animals↗
PubMed1994

Kinetics and regulation of pantothenate kinase from Escherichia coli.

Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis and is regulated by feedback inhibition by CoA. Pantothenate kinase was purified to homogeneity from Escherichia coli and was shown to exist as a homodimer. Kinetic analysis indicated the presence of two ATP binding sites that exhibited positive cooperativity with a Hill coefficient of 1.46. Site-directed mutagenesis of lysine 101 to methionine (K101M) resulted in the inactivation of the enzyme, although dimer formation was not altered. The K101M mutant was unable to bind either adenosine 5'-O-(3-thiotriphosphate) or CoA, supporting the conclusion from kinetic analysis that both the substrate and inhibitor bind to the same site on the enzyme. CoA binding was not cooperative. Coexpression of the K101M mutant gene on a high copy number plasmid in the presence of a chromosomal copy of the wild-type gene resulted in the production of heterodimers between active and inactive subunits. Kinetic analysis of the chimeric heterodimers showed the absence of cooperative ATP interactions and indicated a sequential kinetic mechanism for pantothenate kinase with ATP binding first and pantothenate second. Thus, pantothenate kinase regulation involves the competitive binding of CoA to the ATP site, which blocks ATP binding at one site and prevents positive cooperative ATP binding to the second site on the dimer.

Adenosine Triphosphate↗
PubMed1994

Detection of Schistosoma japonicum antigen (Sj31/32) in sera of Chinese patients using a sandwich ELISA based on monoclonal antibody.

A sandwich-ELISA was developed using IgG from rabbits infected with Schistosoma japonicum as capture antibody and a monoclonal antibody as detecting reagent for circulating Sj 31/32 in patient sera. The assay detected circulating antigen in 36 of 53 sera from Chinese schistosomiasis japonica patients, but not in sera from healthy persons or patients with paragonimiasis, trichinosis or cysticercosis. This is the first demonstration that Sj 31/32 proteins may be used for circulating antigen-based serodiagnosis of schistosomiasis.

Animals↗
PubMed1993

Segregation of cerebrorubral and cerebellorubral synaptic inputs on rubrospinal neurons of fetal cats as demonstrated by intracellular recording.

Cerebrorubral and cerebellorubral inputs are localized to distal dendrites and somata of red nucleus neurons in adult cats, respectively. To examine if this segregation is established early in development, we performed intracellular recording from rubrospinal neurons of fetal cats aged from embryonic day 58 to 65. Stimulation of the contralateral cerebellar nuclei evoked excitatory postsynaptic potentials (EPSPs). EPSPs were also induced by stimulation of the ipsilateral pericruciate cortext but they were much slower in time course and smaller in amplitude compared to cerebellar ones. We suggest that cerebrorubral and cerebellorubral synapses are segregated on soma-dendritic membrane of rubrospinal neurons early in development.

Animals↗
PubMed1993

An electrophysiological study of a transient ipsilateral interpositorubral projection in neonatal cats.

We examined whether transient projections in the developing central nervous system of Mammalia form functional synapses on their target neurons, using transient ipsilateral interpositorubral (iIR) projection in kittens as a model system. Intracellular recordings were made from red nucleus (RN) neurons in 26 kittens aged 6-26 postnatal days (PD6-26). RN neurons were identified by monosynaptic excitatory postsynaptic potentials (EPSPs) evoked by stimulation of contralateral nucleus interpositus (IN), and additionally by intracellular staining in a few cells. Sixty-nine out of 362 RN neurons responded to stimulation of the ipsilateral IN. Of the 69 cells, 25 showed depolarizing responses with relatively short latency (2.1-6.7 ms) in kittens up to PD20. Such responses were not observed in older animals. Varying stimulus strength revealed that the potentials were unitary. Paired-pulse facilitation of the potential was observed, suggesting that the depolarizations are EPSPs. Several lines of evidence were obtained suggesting that the EPSPs are evoked monosynaptically. They followed high-frequency stimulation up to 50 Hz, and their latencies remained constant with varying stimulus strength. The latencies of ipsilaterally induced EPSPs were always longer than those of contralateral ones, evidence consistent with the longer course of ipsilaterally projecting axons than that of contralateral ones (Song and Murakami 1990). The age of disappearance of the monosynaptic EPSPs, i.e., PD20, also corresponds roughly with that of the anatomically demonstrable iIR fibers (PD15-PD25; Song and Murakami 1990). It is thus concluded that the transient iIR fibers in kittens form functional synapses on RN neurons.

Animals↗
PubMed1992

Developing corticorubral axons of the cat form synapses on filopodial dendritic protrusions.

Developing neurons transiently grow numerous spine- or filopodium-like dendritic protrusions (SLDPs). Electron microscopy on identified input and intracellular staining of postsynaptic cells were performed to gain insight into their significance. Newborn kitten-corticorubral axons, labelled with biocytin, commonly made synapses on SLDP, often multiply invaginated by the SLDPs. Correspondingly, intracellularly labelled kitten rubrospinal cells had numerous SLDPs. Taking into account that corticorubral synapses are largely formed on dendritic shafts in adult cats, it is likely that the SLDPs play some important role in the development of corticorubral synapses. We hypothesize that rubrospinal cells elongate SLDPs searching for corticorubral axons to form synapses.

Aging↗