Biomedical subjects

T F Hsieh

Publications and source records attributed to T F Hsieh.

8 recordsLinked to original sources

PubMed2001

Gene expression in the developing mouse retina by EST sequencing and microarray analysis.

Retinal development occurs in mice between embryonic day E11.5 and post-natal day P8 as uncommitted neuroblasts assume retinal cell fates. The genetic pathways regulating retinal development are being identified but little is understood about the global networks that link these pathways together or the complexity of the expressed gene set required to form the retina. At E14.5, the retina contains mostly uncommitted neuroblasts and newly differentiated neurons. Here we report a sequence analysis of an E14.5 retinal cDNA library. To date, we have archived 15 268 ESTs and have annotated 9035, which represent 5288 genes. The fraction of singly occurring ESTs as a function of total EST accrual suggests that the total number of expressed genes in the library could approach 27 000. The 9035 ESTs were categorized by their known or putative functions. Representation of the genes involved in eye development was significantly higher in the retinal clone set compared with the NIA mouse 15K cDNA clone set. Screening with a microarray containing 864 cDNA clones using wild-type and brn-3b (-/-) retinal cDNA probes revealed a potential regulatory linkage between the transcription factor Brn-3b and expression of GAP-43, a protein associated with axon growth. The retinal EST database will be a valuable platform for gene expression profiling and a new source for gene discovery.

Animals↗

PubMed1999

Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco.

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed1998

BDNF restores the expression of Jun and Fos inducible transcription factors in the rat brain following repetitive electroconvulsive seizures.

The expression of inducible transcription factors was studied following repetitive electroconvulsive seizures (ECS), c-Fos, c-Jun, JunB, and JunD immunoreactivities were investigated following a single (1 x ECS) or repetitive ECS evoked once per day for 4, 5, or 10 days (4 x ECS, 5 x ECS, or 10 x ECS). Animals were killed 3 or 12 h following the last ECS. Three hours after 1 x ECS, c-Fos was expressed throughout the cortex and hippocampus. After 5 x ECS and 10 x ECS, c-Fos was reexpressed in the CA4 area, but was completely absent in the other hippocampal areas and cortex. In these areas, c-Fos became only reinducible when the time lag between two ECS stimuli was 5 days. In contrast to c-Fos, intense JunB expression was inducible in the cortex and hippocampus, but not CA4 subfield, after 1 x ECS, 5 x ECS, and 10 x ECS. Repetitive ECS did not effect c-Jun and JunD expression. In a second model of systemic excitation of the brain, repetitive daily injection of kainic acid for 4 days completely failed to express c-Fos, c-Jun, and JunB after the last application whereas injection of kainic acid once per week did not alter the strong expressions compared to a single application of kainic acid. In order to study the maintenance of c-Fos expression during repetitive seizures, brain-derived neurotrophic factor (BDNF) was applied in parallel for 5 or 10 days via miniosmotic pumps and permanent cannula targeted at the hippocampus or the parietal cortex. Infusion of BDNF completely reinduced c-Fos expression during 5 x ECS or 10 x ECS in the cortex ipsilaterally to the cannula and, to a less extent, also increased the expression of c-Jun and JunB when compared to saline-treated controls. BDNF had no effect on the expression patterns in the hippocampus. ECS with or without BDNF infusion did not change the expression patterns of the constitutive transcription factors ATF-2, CREB, and SRF. These data demonstrate that various transcription factors substantially differ in their response to acute and chronic neural stimulation. Repetitive pathophysiological excitation decreases the transcriptional actions of neurons over days in the adult brain, and this decrement can be prevented by BDNF restoring the neuroplasticity at the level of gene transcription.

Animals↗

PubMed1998

Peripheral nerve insult induces NMDA receptor-mediated, delayed degeneration in spinal neurons.

Injury of a peripheral nerve gives rise to adaptive functional and structural alterations in spinal neurons. We report that the rearrangement of the spinal circuitry in response to sciatic nerve transection in adult rats involves a delayed mode of degeneration of lumbar spinal cord neurons. Nuclear fragmentation was detected by the TUNEL technique 7 days after sciatic neurectomy but not after 3 or 14 days. Dying cells were preferentially located in the ipsilateral superficial dorsal horn and expressed the neuronal cytoskeletal marker SMI-31. Degeneration was prevented by continuous systemic treatment with the NMDA receptor-antagonist MK-801. These data are supportive that apoptosis is induced in spinal neurons in a transsynaptic manner by an early signal from injured afferent fibres via activation of spinal NMDA receptors.

Animals↗

PubMed1997

Expression of activating transcription factor-2, serum response factor and cAMP/Ca response element binding protein in the adult rat brain following generalized seizures, nerve fibre lesion and ultraviolet irradiation.

The expression of the constitutive transcription factors activating transcription factor-2 (ATF-2), serum response factor (SRF) and cAMP/Ca response element binding factor (CREB), and the phosphorylation of SRF and CREB were studied in the untreated adult rat nervous system and following seizure activities and neurodegenerative stimuli. In the untreated rat, intense nuclear SRF immunoreactivity was present in the vast majority of neurons in the forebrain, cortex, striatum, amygdala and hippocampus, and in some scattered neurons in the medulla and spinal cord. In contrast, SRF immunoreactivity was absent in the midline areas of the forebrain, e.g., the globus pallidum and septum, and in the hypothalamus, thalamus, mesencephalon and motoneurons. Nuclear ATF-2 was expressed at high levels in apparently all neurons, but not glial cells, throughout the neuraxis except for those neuronal populations which exhibit a high basal level of c-Jun, i.e. dentate gyrus and the motoneurons of cranial and somatosensory neurons. CREB immunoreactivity was present at a rather uniform intensity in all neuronal and glial cells throughout the neuraxis. Two hours, but not 5 h or 24 h, following systemic application of kainic acid, an increase in SRF was detectable by western blot analysis in hippocampal and cortical homogenates whereas the expression of ATF-2 and CREB did not change. Phosphorylation of CREB at serine 133 and of SRF at serine 103 were studied with specific antisera. In untreated rats, intense phosphoCREB and phosphoSRF immunoreactivities labelled many glial cells and/or neurons with the highest levels in the dentate gyrus, the entorhinal cortex and the retrosplenial cortex. Following kainate-induced seizures, phosphoSRF-IR but not phosphoCREB-IR transiently increased between 0.5 h and 2 h. Following transection of peripheral or central nerve fibres such as optic nerve, medial forebrain bundle, vagal and facial nerve fibres, ATF-2 rapidly decreased in the axotomized neurons during that period when c-Jun was rapidly expressed. SRF remained unchanged and CREB disappeared in some axotomized subpopulations. Similar to axotomy, c-Jun increased and ATF-2 decreased in cultured adult dorsal root ganglion neurons following ultraviolet irradiation. The distribution of SRF and ATF-2 suggests that their putative target genes c-fos, junB, krox-24 and c-jun can be independently regulated from SRF and ATF-2. The suppression of ATF-2 and the expression of c-Jun following axotomy and ultraviolet irradiation might be part of a novel neuronal stress response in the brain that strongly resembles the stress response characterized in non-neuronal cells.

Activating Transcription Factor 2↗

PubMed1997

Molecular approaches to identify novel genes expressed in Arabidopsis thaliana.

Our laboratory is engaged in an effort to identify genes expressed primarily during plant embryogenesis. Genes which exhibit unique expression profiles in the plant are also being sought. To this end, several methods to identify and clone novel genes based on specific expression patterns have been developed. These methods include virtual subtraction, differential display and other PCR based technologies. In addition to this, a yeast one-hybrid approach has been established to identify transcription factors which regulate these genes. To date, this work has identified several novel genes.

Arabidopsis↗

PubMed1996

Identification of novel mRNAs in immature seeds of Arabidopsis thaliana.

Although most of the major discernible morphogenetic events in plants occur after germination, the overall architectural pattern of the mature plant is established during early events of embryogenesis. So far, few genes that are expressed specifically during embryogenesis have been identified. This is due primarily to technical difficulties associated with the mass ratios of the embryo and the surrounding maternal tissue and to the lack of molecular and cellular markers to direct screening efforts. We have developed a series of molecular approaches to study the early events of embryogenesis. These include 'virtual subtraction' of a cDNA library with high specific-activity cDNA probes generated from both seed and non-seed tissue, PCR amplification of gene family members from an immature seed cDNA library using primers specific to conserved domains, differential display analysis of mRNA populations and high throughput expressed sequence tag (EST) analysis. These techniques have led to the identification and isolation of several novel seed-specific cDNAs.

Amino Acid Sequence↗

PubMed1990

Extracellular single-unit studies of suprachiasmatic neurons in brain slices. Effects of serotonin, dopamine, carbachol and LHRH.

Suprachiasmatic nucleus (SCN) is the major rhythm generating center in the central nervous system. Extracellular single-unit activities of the SCN neurons were recorded in brain tissue slices either from ovariectomized (OVX) rats or from OVX rats treated with estrogen (polyestradiol phosphate, PEP, 0.1 mg/rat, sc). Neuronal responses to luteinizing hormone-releasing hormone (LHRH) and various neurotransmitters, i.e., serotonin (5-HT), dopamine (DA) and carbachol (Carb, an acetylcholine agonist) were tested. A total of 137 units were recorded from OVX and 81 from OVX + PEP rats. The steady state firing rates of SCN neurons were between 0-9 spikes/sec in OVX and 0-23 in OVX + PEP group. Among them, most fired irregularly (86.9% in OVX and 71.6% in OVX + PEP rats), and a few fired regularly (10.2% and 25.9%, respectively). Few silent units were detected (2.9% and 2.5%, respectively). It seems that estrogen treatment somewhat increased the percentage of regular firing units. As for the agents tested in this study, LHRH by itself had no significant effect on the SCN neuronal activity; 5-HT affected more than 40% of the units, with about equal number of excitation (16.8%) and inhibition (24.4%), while Carb inhibited nearly half of the units tested (49.5%), excited only a few (10.3%). All the actions of these three agents were not significantly affected by in vivo estrogen pretreatment. Dopamine, however, excited more units (50%) in OVX than in OVX + PEP group (27.1%). Although LHRH had no direct effect by itself, it exerted a significant modulatory role on the actions of the other three agents. It either potentiated (42.9%) or inhibited (53.6%) the action of 5-HT, and it significantly potentiated the action of Carb (57.1%). For the action of DA, LHRH had a more significant potentiation effect on OVX + PEP than in OVX rats. (40.9% vs. 12.5%), while the inhibitory effect was the same. In conclusion, 5-HT, DA and Carb all exert significant effects on SCN neuronal activities. LHRH can have a modulatory role upon the action of 5-HT, DA and Carb, and estrogen pretreatment specifically affects that of DA in the SCN.

Animals↗