Search PubMed⌕ Search

Biomedical subjects

L Y Jan

Publications and source records attributed to L Y Jan.

At least 127 records · Page 7Linked to original sources
PubMed1994

Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn.

The N-methyl-D-aspartate (NMDA) receptor has been implicated in a variety of systems that undergo plastic changes in the central nervous system. We used electron microscopic immunocytochemistry with an antibody directed against an alternatively spliced exon near the C terminus of NMDAR1, the essential functional subunit of the NMDA receptor, to study the distribution of the NMDA receptor in the spinal cord and CA1 region of the hippocampus, two regions where NMDA-mediated long-term plasticity has been demonstrated. In CA1, we found that the NMDA receptor is exclusively expressed on postsynaptic structures. By contrast, in the spinal cord we found that in about one-third of labeled synapses, the receptor is located in the presynaptic terminal, immediately adjacent to the vesicle release site at the active zone. Using combined postembedding immunocytochemistry, we also showed that > 70% of the NMDA receptor immunoreactive terminals are glutamate positive, which suggests that the presynaptic NMDA receptor is an autoreceptor. Nerve ligation studies demonstrated that the receptor is transported in dorsal roots and sciatic nerve to the spinal cord and periphery, respectively. These data indicate that an NMDA autoreceptor is located in terminals of primary afferent fibers, where it could facilitate the transmission of inputs to the spinal cord by increasing the release of neurotransmitter from the primary afferent terminal.

Amino Acid Sequence↗
PubMed1994

Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion.

The small GTPases of the Rac/Rho/Cdc42 subfamily are implicated in actin cytoskeleton-membrane interaction in mammalian cells and budding yeast. The in vivo functions of these GTPases in multicellular organisms are not known. We have cloned Drosophila homologs of rac and CDC42, Drac1, and Dcdc42. They share 70% amino acid sequence identity with each other, and both are highly expressed in the nervous system and mesoderm during neuronal and muscle differentiation, respectively. We expressed putative constitutively active and dominant-negative Drac1 proteins in these tissues. When expressed in neurons, Drac1 mutant proteins cause axon outgrowth defects in peripheral neurons without affecting dendrites. When expressed in muscle precursors, they cause complete failure of, or abnormality in, myoblast fusion. Expressions of analogous mutant Dcdc42 proteins cause qualitatively distinct morphological defects, suggesting that similar GTPases in the same subfamily have unique roles in morphogenesis.

Actins↗
PubMed1994

Activation of the cloned muscarinic potassium channel by G protein beta gamma subunits.

Acetylcholine released during parasympathetic stimulation of the vagal nerve slows the heart rate through the activation of muscarinic receptors and subsequent opening of an inwardly rectifying potassium channel. The activation of these muscarinic potassium channels is mediated by a pertussis toxin-sensitive heterotrimeric GTP-binding protein (G protein). It has not been resolved whether exogenously applied G alpha or G beta gamma, or both, activate the channel. Using a heterologous expression system, we have tested the ability of different G protein subunits to activate the cloned muscarinic potassium channel, GIRK1. We report here that coexpression of GIRK1 with G beta gamma but not G alpha beta gamma in Xenopus oocytes results in channel activity that persists in the absence of cytoplasmic GTP. This activity is reduced by fusion proteins of the beta-adrenergic receptor kinase and of recombinant G alpha i-GDP, both of which are known to interact with G beta gamma. Moreover, application of recombinant G beta gamma, but not G alpha i-GTP-gamma S, activates GIRK1 channels. Thus G beta gamma appears to be sufficient for the activation of GIRK1 muscarinic potassium channels.

Amino Acid Sequence↗
PubMed1994

Atonal is the proneural gene for Drosophila photoreceptors.

The Drosophila peripheral nervous system comprises four major types of sensory element: external sense organs (such as mechano-sensory bristles), chordotonal organs (internal stretch receptors), multiple dendritic neurons, and photoreceptors. During development, the selection of neural precursors for external sense organs requires the proneural genes of the achaete-scute complex, which encode basic-helix-loop-helix transcription factors. These genes do not, however, control precursor selection for chordotonal organs or photoreceptors, raising the question of whether other proneural genes exist or a different mechanism of neurogenesis operates. Here we show that atonal (ato), originally isolated as a proneural gene for chordotonal organs, is also the proneural gene for photoreceptors. Pattern formation in the Drosophila eye involves a succession of cell fate specifications. Of the eight photoreceptors within each ommatidium of the compound eye, the photoreceptor R8 is the first to appear in the eye imaginal disc, right behind the morphogenetic furrow. The appearance of other photoreceptors (R1-7) follows in a defined sequence that is thought to arise by induction from R8 (refs 8, 9, 11, 12). We find that photoreceptor formation requires the function of atonal at the morphogenetic furrow and that atonal is specifically required for R8 selection. Formation of other photoreceptors does not directly require atonal function, but does depend on R8 selection by atonal. Thus, photoreceptors are selected by two mechanisms: R8 by a proneural mechanism, and R1-7 by local recruitment.

Animals↗
PubMed1994

Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte.

BACKGROUND: During oogenesis in Drosophila, determinants that will dictate abdomen and germline formation are localized to the 'polar plasm' in the posterior of the oocyte. Assembly of the polar plasm involves the sequential localization of several messenger RNAs and proteins to the posterior of the oocyte, beginning with the localization of oskar mRNA and Staufen protein during stages 8 and 9 of oogenesis. The mechanism by which these two early components accumulate at the posterior is not known. We have investigated whether directed transport along microtubules could be used to accomplish this localization. RESULTS: We have made a fusion protein composed of the bacterial beta-galactosidase enzyme as a reporter, joined to part of the plus-end-directed microtubule motor, kinesin, and have found that the fusion protein transiently localizes to the posterior of the oocyte during stages 8 and 9 of oogenesis. Treatment with the microtubule-depolymerizing agent colchicine prevents both the localization of the fusion protein and the posterior transport of oskar mRNA and Staufen protein. Furthermore, the fusion protein localizes normally in oocytes mutant for either oskar and staufen, but not in other mutants in which oskar mRNA and Staufen protein are mislocalized. CONCLUSIONS: Association with a plus-end-directed microtubule motor can promote posterior localization of a reporter protein during oogenesis. The genetic requirements for this localization and its sensitivity to colchicine, both of which are shared with the posterior transport of oskar mRNA and Staufen protein, suggest that similar mechanism may function in both processes.

Adenosine Triphosphate↗
PubMed1994

Changing subunit composition of heteromeric NMDA receptors during development of rat cortex.

Activation of the N-methyl-D-aspartate (NMDA) receptor is important for certain forms of activity-dependent synaptic plasticity, such as long-term potentiation (reviewed in ref. 1), and the patterning of connections during development of the visual system (reviewed in refs 2, 3). Several subunits of the NMDA receptor have been cloned: these are NMDAR1 (NR1), and NMDAR2A, 2B, 2C and 2D (NR2A-D). Based on heterologous co-expression studies, it is inferred that NR1 encodes an essential subunit of NMDA receptors and that functional diversity of NMDA receptors in vivo is effected by differential incorporation of subunits NR2A-NR2D. Little is known, however, about the actual subunit composition or heterogeneity of NMDA receptors in the brain. By co-immunoprecipitation with subunit-specific antibodies, we present here direct evidence that NMDA receptors exist in rat neocortex as heteromeric complexes of considerable heterogeneity, some containing both NR2A and NR2B subunits. A progressive alteration in subunit composition seen postnatally could contribute to NMDA-receptor variation and changing synaptic plasticity during cortical development.

Animals↗
PubMed1994

Genomic cloning and chromosomal localization of HRY, the human homolog to the Drosophila segmentation gene, hairy.

The Drosophila hairy gene encodes a basic helix-loop-helix protein that functions in at least two steps during Drosophila development: (1) during embryogenesis, when it partakes in the establishment of segments, and (2) during the larval stage, when it functions negatively in determining the pattern of sensory bristles on the adult fly. In the rat, a structurally homologous gene (RHL) behaves as an immediate-early gene in its response to growth factors and can, like that in Drosophila, suppress neuronal differentiation events. Here, we report the genomic cloning of the human hairy gene homolog (HRY). The coding region of the gene is contained within four exons. The predicted amino acid sequence reveals only four amino acid differences between the human and rat genes. Analysis of the DNA sequence 5' to the coding region reveals a putative untranslated exon. To increase the value of the HRY gene as a genetic marker and to assess its potential involvement in genetic disorders, we sublocalized the locus to chromosome 3q28-q29 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1994

Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells.

The four cells of an external sense organ in the Drosophila peripheral nervous system, the neuron, its sheath cell, and two "outer support cells" that form the hair and socket, are derived from a common precursor, the sensory organ precursor (SOP), after two rounds of division. We determined by immunocytochemistry that numb is a membrane-associated protein which localizes asymmetrically to one-half of the predivisional SOP cell. Upon division, numb segregates differentially to one daughter. Loss of numb function causes the descendants of the SOP to differentiate inappropriately, producing four outer support cells and no neuron or sheath. Ectopic expression of numb during the time of SOP division results in a transformation that is opposite to the null mutant transformation. Thus, numb functions to determine the fates of the secondary precursors; the differential distribution of numb as the SOP divides generates an asymmetric division in which the daughter cells acquire distinct identities.

Animals↗
PubMed1994

Images of purified Shaker potassium channels.

BACKGROUND: Voltage-gated K+ channels play an important role in the control of neuronal excitability and synaptic plasticity. Their low abundance and extraordinary heterogeneity have rendered their purification from natural sources difficult. We have previously cloned a voltage-gated K(+)-channel gene, Shaker, from Drosophila. The Shaker K(+)-channel polypeptide resembles one of the four internal repeats of a Na(+)- or Ca(2+)-channel alpha subunit, suggesting that this example of a K+ channel contains four identical or homologous subunits. Similar K(+)-channel polypeptides have been characterized from mammals, other vertebrate and invertebrate species, and from plants. Electrophysiological studies of K+ channels expressed in Xenopus oocytes suggest that they are indeed tetramers, and heteromultimeric K+ channels have been found in the mammalian brain. Until now, however, no K+ channel, nor any other member of the superfamily of voltage-gated ion channels, has been characterized by electron microscopy or other structural analysis. RESULTS: We have purified Shaker K+ channels, expressed in insect Sf9 cells, to apparent homogeneity, and imaged them using the electron microscope. The physical dimensions of these molecules, as well as their biochemical characteristics, are consistent with a tetrameric subunit composition. Moreover, the Shaker channel revealed by negative staining has the appearance of a four-fold symmetric tetramer, with a large, central vestibule that presumably constitutes part of the pathway for ions. CONCLUSION: These first clear images of a voltage-gated ion channel reveal a marked four-fold symmetry. The integrity of the purified tetrameric complex indicates that the purification scheme used in this study may be further developed for future structural analysis of voltage-gated K+ channels.

Animals↗
PubMed1994

Evidence that the S6 segment of the Shaker voltage-gated K+ channel comprises part of the pore.

Potassium channels are highly selective and allow the rapid flux of potassium ions through their pore. Several studies have implicated the H5 (P or SS1-SS2) segment as part of the pore in voltage-gated ion channels. The proposal that H5 spans at least 80% of the electric potential drop across the K+ channel pore is based on altered internal tetraethylammonium sensitivity arising from mutations of H5 residues that are 100% conserved among K+ channels having differing sensitivity to tetraethylammonium. Here we report that the S6 segment is also involved in K+ ion permeation and in governing the sensitivity to internal tetraethylammonium and barium. Transplanting the S6 segment of NGK2 into Shaker causes this S6 chimaera to adopt the single-channel conductance and sensitivity to internal tetraethylammonium and barium ions from the NGK2 channel. The differences between NGK2 and Shaker in external tetraethylammonium sensitivity, but not single-channel conductance, can be attributed to the differences in their H5 sequences. Three nonconserved S6 residues have been found to affect either single-channel conductance or internal tetraethylammonium sensitivity.

Amino Acid Sequence↗
PubMed1994

The role of gene cassettes in axis formation during Drosophila oogenesis.

Establishment of the anteroposterior and dorsoventral axes of the fly originates during oogenesis and relies on signaling between the oocyte and the surrounding somatic follicle cells. Some genes originally identified as playing a role in signaling during embryonic development also mediate cell-cell communication during oogenesis. These genes have previously been grouped on the basis of their functions during embryogenesis, and this classification is largely maintained in oogenesis. The EGF receptor, the transmembrane protein rhomboid and proteins in the ras signal transduction pathway are required to initiate dorsoventral polarity, whereas the products of the neurogenic genes Notch and Delta are necessary for formation of the anteroposterior axis in the oocyte.

Animals↗
PubMed1994

Neuronal cell fate specification in Drosophila.

Recent work indicates that the Drosophila nervous system develops in a progressive process of cell fate specification. Expression of specific proneural genes in clusters of cells (the proneural clusters) in the cellular blastoderm endows these cells with the potential to form certain types of neural precursors. Intercellular interactions that involve both proneural genes and neurogenic genes then allow the neural precursors to be singled out from the proneural clusters. Expression of neural precursor genes in all neural precursors is likely to account for the universal aspects of neuronal differentiation, such as axonal outgrowth. Selective expression of certain neuronal-type selector genes further specifies the type of neuron(s) that a neural precursor will produce.

Animals↗
PubMed1994

daughterless is essential for neuronal precursor differentiation but not for initiation of neuronal precursor formation in Drosophila embryo.

The first steps of neuronal precursor formation require several genes that encode transcription regulators with the helix-loop-helix (HLH) motif, including the proneural genes of the achaete-scute complex AS-C (achaete (ac), scute (sc) and lethal of scute (l'sc)) and daughterless (da). The da protein dimerizes with AS-C products in vitro to form DNA-binding proteins. Previous studies have shown that the AS-C genes are expressed initially in discrete clusters of ectodermal cells (the proneural clusters) and then more strongly in the neuronal precursors that arise from these clusters and delaminate from the epidermal layer. In this paper, we studied the distribution of da protein with an antibody raised against Da. We found that Da is ubiquitously but non-uniformly distributed. Within the ectodermal layer, its level is neither elevated (as in the case of AS-C genes) nor reduced (as in the case of emc product) in the proneural cluster. It is, however, at higher levels in many neuronal precursors. We further studied the requirement of da in neuronal precursor development by using a variety of markers for neuronal precursors. Our results reveal the existence of at least two stages in neuronal precursor formation. da is not required for the initial appearance of nascent neuronal precursors but is required for these cells to express multiple neuronal precursor genes and to produce the normal number of neurons.

Animals↗
PubMed1994

lola encodes a putative transcription factor required for axon growth and guidance in Drosophila.

Mutations in the gene longitudinals lacking (lola) lead to defects in the development of axon tracts in the Drosophila embryonic central nervous system. We now show that lola mutations also cause defects of axon growth and guidance in the peripheral nervous system, and causes a particular cluster of embryonic sense organs (lch5) to be oriented improperly. Axonal aberrations caused by lola are similar to those caused by mutations of three other genes, logo, Notch and Delta, raising the possibility that lola works in the same genetic pathway as do these other molecules. The lola gene encodes at least two nuclear protein products, apparently by differential RNA splicing. The predicted proteins contain an amino-terminal motif similar to that recently described for a family of transcription factors, including the products of the Drosophila genes tramtrack and the Broad Complex. Like Ttk and BR-C, one of the two characterized products of the lola locus bears sequences similar to the zinc-finger motif, but the other (neuronal) form of the protein has no recognizable DNA-binding motif.

Amino Acid Sequence↗
PubMed1994

Contrasting subcellular localization of the Kv1.2 K+ channel subunit in different neurons of rat brain.

In the nervous system, a wide diversity of K+ channels are formed by the oligomeric assembly of subunits encoded by a large number of K+ channel genes. The physiological functions of a specific K+ channel subunit in vivo will be dictated in part by its subcellular location within neurons. We have used a combined in situ hybridization and immunocytochemical approach to determine the subcellular distribution of Kv1.2, a member of the Shaker subfamily of K+ channel genes. In contrast to other characterized K+ channel subunits, Kv1.2 protein shows a complex differential subcellular distribution in neurons of rat brain. In some of these neurons (e.g., hippocampal and cortical pyramidal cells, and Purkinje cells), Kv1.2 is concentrated in dendrites, while in others (e.g., cerebellar basket cells), Kv 1.2 is predominantly, if not exclusively, localized to nerve terminals. Furthermore, Kv1.2 immunoreactivity was also detected in certain axon tracts. We hypothesize that the differential sorting of Kv1.2 could result from association of Kv1.2 with varying heterologous K+ channel subunits in different cell types, with the implication that Kv1.2 may participate in distinct heteromultimeric K+ channels in different subcellular domains. The findings suggest that Kv1.2-containing K+ channels may play diverse functional roles in several neuronal compartments, regulating presynaptic or postsynaptic membrane excitability, depending on the neuronal cell type.

Amino Acid Sequence↗