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H H Yeh

Publications and source records attributed to H H Yeh.

At least 19 recordsLinked to original sources
PubMed2001

Green fluorescent protein expression from recombinant lettuce infectious yellows virus-defective RNAs originating from RNA 2.

Lettuce infectious yellows virus (LIYV) RNA 2 defective RNAs (D RNAs) were compared in protoplasts for their ability to replicate and to express the green fluorescent protein (GFP) from recombinant D RNA constructs. Initially four LIYV D RNAs of different genetic composition were compared, but only two (LIYV D RNA M5 and M18) replicated to high levels. Both of these contained at least two complete ORFs, one being the 3'-terminal ORF encoding P26. Northern hybridization analysis using probes corresponding to 3' regions of LIYV RNA 2 detected the P26 subgenomic RNA from protoplasts infected with LIYV RNAs 1 and 2 or protoplasts inoculated only with RNA 1 plus either the LIYV D RNA M5 or M18, suggesting that these LIYV D RNAs served as templates to generate the P26 subgenomic RNA. The GFP coding region was inserted as an in-frame insertion into the P26 coding region of the LIYV M5 and M18 D RNAs, yielding M5gfp and M18gfp. When transcripts of M5gfp and M18gfp were used to inoculate protoplasts, bright fluorescence was seen only when they were co-inoculated with LIYV RNA 1. The percentage of fluorescent protoplasts ranged from experiment to experiment, but was as high as 5.8%. Time course analyses showed that fluorescence was not detected before 48 h pi, and this correlated with the timing of LIYV RNA 2 and RNA 2 D RNA accumulation, but not with that of LIYV RNA 1.

Blotting, Northern↗
PubMed2001

Distinct NMDA and AMPA receptor-mediated responses in mouse and human Cajal-Retzius cells.

This study examined glutamate-activated current responses of mouse and human Cajal-Retzius (C-R) cells. Thin cortical slices were prepared from the brains of mice 4-6 days after birth and from those of midgestational human fetuses. Both human and mouse C-R cells displayed glutamate-induced whole-cell current responses that were voltage-dependent and included an N-methyl-D-aspartate (NMDA) receptor-mediated component that was differentially sensitive to blockade by the NMDA receptor antagonists 2-amino-5-phosphonovaleric acid and ifenprodil. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a non-NMDA glutamate receptor agonist, induced current responses in human but not in mouse C-R cells. These results, taken together, lead us to conclude that human C-R cells express both NMDA and AMPA types of glutamate receptors very early during development of the cortex. In contrast, mouse C-R cells express only the NMDA type of glutamate receptor. Thus we demonstrate a species-dependent sensitivity of C-R cells to glutamate and postulate that this differential sensitivity may account in part for a species-dependent difference in the persistence of C-R cells during cortical development.

2-Amino-5-phosphonovalerate↗
PubMed2000

A heterogeneous population of defective RNAs is associated with lettuce infectious yellows virus.

Preparations of dsRNAs and virion RNAs extracted from Nicotiana clevelandii plants infected with the bipartite Lettuce infectious yellows virus (LIYV) were found to contain multiple LIYV RNA species. In addition to the two LIYV genomic RNAs, three types of RNAs were observed: (a) 3' coterminal subgenomic RNAs; (b) RNAs containing LIYV RNA 1 or RNA 2 5' terminus but lacking the 3' terminus; and (c) RNAs with both LIYV RNA 2 3' and 5' termini but each with a central extensive deletion, a structure typical of defective RNAs (D RNAs). No D RNA-like RNAs were detected for LIYV RNA 1. A reverse transcription followed by polymerase chain reaction (RT-PCR) strategy was used to clone from virion RNAs several LIYV RNA 2 D RNAs as cDNAs. Nucleotide sequence analysis of 43 cloned cDNAs showed in some D RNAs the presence of a stretch of 1-5 nt in the junction site that is repeated in the genomic RNA 2 in the two positions flanking the junction site or in close proximity. Some D RNAs contained in the junction site one or several extra nucleotides not present in the LIYV genomic RNA 2. Two of the cloned cDNAs were used to generate in vitro transcripts, and infectivity studies showed that both D RNAs were replication competent in protoplasts when coinoculated with LIYV RNAs 1 and 2 or with only LIYV RNA1. Neither D RNA showed obvious effects upon LIYV RNA 1 and RNA 2 accumulation in coinfected protoplasts. These data suggest that LIYV infections contain a heterogeneous population of LIYV RNA 2 D RNAs, and some are encapsidated into virions.

Blotting, Northern↗
PubMed2000

Heterogeneity of GABA(A) receptor-mediated responses in the human IMR-32 neuroblastoma cell line.

The gamma-aminobutyric acid (GABA) response profiles of IMR-32 human neuroblastoma cells were examined using whole-cell patch clamp and RT-PCR techniques. GABA activated a concentration-dependent and bicuculline-sensitive current, and RT-PCR revealed the expression of multiple GABA(A) receptor subunit mRNAs (alpha(1), alpha(3), alpha(4), beta(1), beta(3), gamma(2), and delta). A pharmacological profile of the GABA-induced current was derived using several subunit-selective agents. Diazepam, which requires the presence of a gamma subunit in order to modulate GABA(A) receptor-mediated responses, potentiated GABA-induced currents in a subset of IMR-32 cells. Two populations of GABA-activated currents were also evident based on sensitivity to modulation by zinc. Comparison of zinc- and diazepam-induced modulation of GABA-induced current responses in the same cells revealed an inverse correlation between these two modulators. No differences, however, were observed with the GABA(A) receptor modulators loreclezole, allopregnanolone, and pentobarbital. Thus, IMR-32 cells maintained in culture are heterogeneous in terms of expression of GABA(A) receptor isoforms.

Anticonvulsants↗
PubMed2000

Asynchronous accumulation of lettuce infectious yellows virus RNAs 1 and 2 and identification of an RNA 1 trans enhancer of RNA 2 accumulation.

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation.

Base Sequence↗
PubMed2000

Chronic exposure to ethanol alters GABA(A) receptor-mediated responses of layer II pyramidal cells in adult rat piriform cortex.

This study examined the effect of chronic exposure to ethanol on gamma-aminobutyric acid type-A (GABA(A)) receptor-mediated responses of layer II pyramidal neurons of the piriform cortex. Slices containing the piriform cortex were derived from pair-fed adult rats maintained on ethanol-supplemented or control liquid diet for 30 days. Responses of identified layer II pyramidal neurons to exogenously applied GABA were monitored by whole-cell patch-clamp recording. Chronic exposure to ethanol resulted in a rightward shift in the EC(50) of GABA and a decrease in the amplitude of maximal GABA response. GABA-induced responses were modulated by acutely applied ethanol (10-100 mM) in both chronic ethanol-treated and control groups. No significant difference was found in the average change in GABA response, suggesting that tolerance to acute ethanol exposure did not develop. When the modulatory responses of individual cells were classified and grouped as either being attenuating, potentiating, or having no effect, the incidence of potentiation in the ethanol-treated group was significantly higher. Consistent with the absence of tolerance to acute ethanol, cross-tolerance to diazepam was not observed following 30 days of treatment with ethanol. These results are discussed in light of regionally specific effects of chronic ethanol treatment on GABA(A) receptor-mediated responses of layer II piriform cortical neurons.

Age Factors↗
PubMed1999

Neuregulin induces GABA(A) receptor subunit expression and neurite outgrowth in cerebellar granule cells.

Neuregulin (NRG), a growth and differentiation factor that signals via erbB receptor tyrosine kinases, has been shown to have biological effects in both the CNS and the peripheral nervous system. We report here that erbB4 is expressed in mature cerebellar granule cells, where it appears to be concentrated at the granule cell postsynaptic terminals. We also show that one form of NRG, Ig-NRG, plays a crucial role in aspects of cerebellar granule cell development in vitro. First, Ig-NRG treatment of granule cells in culture selectively induces the expression of the GABA(A) receptor beta2 subunit. This increase in subunit expression is paralleled by an increase in functional GABA(A) receptors. In contrast to its effects on GABA(A) receptor subunit expression, Ig-NRG does not upregulate NMDA receptor N2B and N2C subunit expression. Second, we demonstrate that Ig-NRG also enhances neurite outgrowth from cultured granule cells. Ig-NRG does not, however, act as a survival factor for the granule cells. We have compared the effect of Ig-NRG with the effects of brain-derived neurotrophic factor (BDNF), a neurotrophin that exerts specific effects on granule cells in culture, and found that BDNF does not mimic the effects of Ig-NRG on GABA(A) receptor subunit expression. Our results show that Ig-NRG has specific effects on granule cell development and maturation and may regulate these processes in vivo.

Aging↗
PubMed1999

Immunohistochemical localization of the INsP4 receptor GTPase-activating protein GAP1IP4BP in the rat brain.

The distribution of GAP1(IP4BP), a GTPase-activating protein showing high affinity and stereospecificity for inositol 1,3,4,5-tetrakisphosphate (InsP4), was investigated by Western blot and immunohistochemistry of rodent brain with polyclonal antibodies generated against the carboxy-terminus of the cloned protein. GAP1(IP4BP)-like immunoreactivity was found throughout the brain, most notably in the pyriform cortex, neocortex, hippocampus, striatum, and cerebellar cortex. However, the most striking immunolabeling was consistently localized to area CA1 of the hippocampus and the central, medial, and intercalated nuclei of the amygdala. Western blot analysis of the corresponding brain regions corroborated these immunohistochemical observations. The regionally specific expression of GAP1(IP4BP) provides the prerequisite neuroanatomical substrate toward elucidating the functional role of InsP4 and GAP1(IP4BP) in the central nervous system.

Age Factors↗
PubMed1999

Ethanol modulates AMPA-induced current responses of primary somatosensory cortical neurons.

This study examined the effect of ethanol on responses of primary somatosensory cortical neurons to AMPA. Thin (200-250 microns) brain slices were sectioned to include the primary somatosensory cortex of rats 6-15 days after birth. Visually-identified neurons were selected for whole-cell patch clamp recording and an eight-barrel drug pipet assembly was used to deliver test agents. Ethanol (5-100 mM) either positively or negatively modulated AMPA (100 microM)-induced current to varying degrees in approximately 70% of primary somatosensory cortical neurons. As revealed in layer V large pyramidal neurons, the outcome of an ethanol-induced modulation appeared to be age-dependent, the trend being one of potentiation in slices derived from younger rats (postnatal days 6-9) but one of attenuation in those derived from older animals (postnatal days 13-15). These findings indicate that ethanol at physiologically relevant concentrations modulates non-NMDA receptor-mediated responses of neurons in the rat primary somatosensory cortex.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1999

Fetal human cortical neurons grown in culture: morphological differentiation, biochemical correlates and development of electrical activity.

Cultured fetal human cortical neurons derived from second trimester human fetal cortex were analyzed with regard to their morphological differentiation and expression of cell-specific markers. The culture method was adapted from standardized protocols originally developed for the isolation and culture of rodent oligodendrocytes and astrocytes. This technique takes advantage of the different adhesive properties and stratification of central nervous system cells in vitro. Under these culture conditions fetal human cortical neurons underwent morphological differentiation, expressed neuron-specific markers and voltage- and ligand-gated ion channels. Highly enriched cultures of microglia and astrocytes generated from the same starting material also expressed cell-type specific markers. These cultures serve as a valuable tool for the establishment of normative data and as experimental models for neurodevelopmental and neurodegenerative studies.

Astrocytes↗
PubMed1999

Lettuce infectious yellows virus: in vitro acquisition analysis using partially purified virions and the whitefly Bemisia tabaci.

Virions of lettuce infectious yellows virus (LIYV; genus Crinivirus) were purified from LIYV-infected plants and their protein composition was analysed by SDS-PAGE and immunoblotting. Virion preparations contained the major capsid protein (CP), but the minor capsid protein (CPm), p59 and the HSP70 homologue were also identified by immunoblot analysis. Immunogold labelling analysis showed that CP constituted the majority of the LIYV virion capsid, but CPm was also part of the capsid and localized to one end of the virion, similar to the polar morphology seen for viruses in the genus Closterovirus. p59 and the HSP70 homologue were not detected on virions by immunogold labelling, but were always detected in virion preparations by immunoblot analysis. Purified LIYV virions were used for in vitro acquisition analysis with Bemisia tabaci whiteflies and were efficiently transmitted to plants. Infectivity neutralization analyses were done using antisera to the LIYV-encoded CP, CPm, p59 and HSP70 homologue. Only antiserum to the CPm effectively neutralized LIYV transmission by B. tabaci. These data suggest that the LIYV-B. tabaci transmission determinants are associated with purified virions, and that the LIYV virion structural protein CPm is involved in transmission by B. tobaci.

Animals↗
PubMed1998

Ethanol-GABAA receptor interactions: a comparison between cell lines and cerebellar Purkinje cells.

This study compared the interaction between ethanol and gamma-aminobutyric acid (GABA)-mediated current responses elicited in several immortalized cell lines and stably transfected cells, as well as in cultured and acutely dissociated rat cerebellar Purkinje cells. Only cell lines that were found previously to possess functional GABAA receptors were examined in this study. Under identical recording conditions, ethanol (10-200 mM) exerted no effect on GABA-induced currents in any of the cell lines or stably transfected cells tested in this study. However, GABA responses monitored in both primary culture and acutely dissociated Purkinje cells were significantly potentiated by ethanol (25 and 50 mM). Mouse pancreatic cells (RINm5F) were insensitive to both diazepam and ethanol suggesting the expression of a GABAA receptor isoform lacking a gamma subunit. Immortalized neuroblastoma IMR-32 cells displayed GABA responses that could be distinguished based on differential sensitivity to diazepam. However, none of the IMR-32 cells displayed GABA responses that were sensitive to modulation by ethanol. GABA responses in the stably transfected cell lines, PA3 (alpha1beta1gamma2L) and WSS-1 (alpha1beta2gamma2), were also unaffected by exposure to ethanol. In Purkinje cells acutely dissociated from the neonatal cerebellum, the ethanol-induced potentiation of GABA-induced current response could be observed before postnatal day 7, when only the gamma2S but not the gamma2L splice variant is expressed. This indicates that the gamma2L subunit is not necessary for an ethanol-induced potentiation of GABAA receptor-mediated response to become manifest. In addition, the results point to inherent differences that should be taken into account in interpreting comparative data between native and recombinant GABAA receptors.

Animals↗
PubMed1997

Ethanol modulation of GABA-activated current responses in acutely dissociated retinal bipolar cells and ganglion cells.

This study examined the effect of acute ethanol exposure on GABA-activated whole-cell current responses elicited in bipolar cells and ganglion cells of the rat retina. Acute exposure to ethanol potentiated GABA responses in 86% of the bipolar cells and in 52% of the ganglion cells tested. As determined in bipolar cells, ethanol was maximally effective at a concentration of 50 mM. In bipolar cells, a GABAC receptor-mediated component of the whole-cell response to GABA could be uncovered which was also potentiated by ethanol. However, ethanol was ineffective in enhancing bipolar cell responses to glycine. GABA-activated current responses monitored in ganglion cells that were insensitive to modulation by ethanol were sensitive to potentiation by diazepam. At higher concentrations (100-175 mM), ethanol by itself occasionally induced a chloride-mediated current but this occurred independent of an ethanol-induced potentiation of GABA responses. These data establish that ethanol can modulate the sensitivity of retinal neurons to GABA. Overall, the results presented in this study set the stage for future studies to examine the cellular and molecular bases for a differential neuronal sensitivity to an ethanol-induced modulation of GABA responses.

Animals↗
PubMed1996

Noradrenergic potentiation of cerebellar Purkinje cell responses to GABA: cyclic AMP as intracellular intermediary.

Norepinephrine and the beta-adrenergic receptor agonist, isoproterenol, have been shown to potentiate the amplitude of GABAA receptor-mediated whole-cell current responses in Purkinje cells acutely dissociated from the rat cerebellum. However, the steps leading from the activation of beta-adrenergic receptors to the modulation of GABAA receptor remain to be delineated. This study tested the hypothesis that a sequelae of intracellular intermediaries involving the cyclic AMP second messenger system serves as the subcellular link to promote this heteroreceptor interaction. Exposure to cholera toxin, but not to pertussis toxin, increased the amplitude of GABA-activated current responses in acutely dissociated Purkinje cells. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate) also resulted in a time- and dose-dependent augmentation of the response to GABA. while guanosine 5'-O-(2-thiodiphosphate) blocked the norepinephrine-mediated facilitation. A positive modulation of the current response to GABA was observed following intracellular delivery of cyclic AMP or the catalytic subunit of the cyclic AMP-dependent protein kinase. Furthermore, the norepinephrine-induced potentiation of the GABA-activated current response was prevented in the presence of the Rp isomer of cyclic AMP, the regulatory subunit of cyclic AMP-dependent protein kinase and an inhibitor of cyclic AMP-dependent protein kinase. These findings led to the formulation of a working model in which activation of the beta-adrenergic receptor triggers a Gs-protein-mediated transduction cascade in cerebellar Purkinje cells which activates adenylate cyclase, resulting in a rise in intracellular levels of cyclic AMP, increased phosphorylating activity by cyclic AMP-dependent protein kinase and, ultimately, a potentiation of GABAA receptor function.

Adenylate Cyclase Toxin↗
PubMed1996

Molecular and functional identification of m1 muscarinic acetylcholine receptors in rat ventricular myocytes.

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m1 and m2 but not for the m3 to m5 mAChRs. The identity of the m1 and m2 bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m1 and m2, but not m3, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m1 mAChR was responsible for the stimulatory effects on Ca2+ transients by high concentrations of carbachol ( > 10 mumol/L) known to occur in these cells. In pertussis toxin-treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 mumol/L) increased the basal Ca2+ level from 96 +/- 7 to 118 +/- 8 nmol/L and the peak Ca2+ transient level from 519 +/- 32 to 640 +/- 36 nmol/L (mean +/- SEM P < .05 for both, n = 8). These effects of carbachol on Ca2+ transients were antagonized by 10 nmol/L pirenzepine, an m1 mAChR-selective antagonist. In contrast, the m2 mAChR-selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m1 mAChRs are expressed on adult rat ventricular myocytes in addition to m2 mAChRs. The results further suggest that m1 mAChRs mediate the stimulatory responses on Ca2+ transients to high concentrations of cholinergic agonists seen in these cells.

Animals↗
PubMed1995

Deciphering the native GABAA receptor: is there hope?

The bewildering number of GABAA receptor subunits, their regionally dependent expression in the brain, and their supernumerary expression in single cells present major challenges in studying the function of native GABAA receptors. Which subunit combinations actually exist in native neurons? In this mini-review, GABAA receptor subunit diversity is considered in light of using the wealth of "structure-function" information gained from studying recombinant receptor to predict the subunit composition and functional properties of native GABAA receptors.

Animals↗