Biomedical subjects

Y B Shi

Publications and source records attributed to Y B Shi.

At least 73 records · Page 4Linked to original sources

PubMed1995

Xenopus sonic hedgehog as a potential morphogen during embryogenesis and thyroid hormone-dependent metamorphosis.

The hedgehog family of proteins have been implicated as important signaling molecules in establishing cell positional information and tissue patterning. Here we present the cloning and characterization of a hedgehog homologue from Xenopus laevis similar to the sonic class of vertebrate hedgehog genes. We isolated Xenopus hedgehog (Xhh) from a subtractive hybridization screen designed to identify genes induced by thyroid hormone during metamorphosis of the X.laevis gastrointestinal tract. In the intestine, Xhh mRNA expression was up-regulated at the climax of metamorphosis (stage 62) when intestinal epithelium underwent morphogenesis. Treatment of pre-metamorphic tadpoles with exogenous thyroid hormone (TH) resulted in a similar pattern of Xhh induction. Furthermore, TH induction was resistant to inhibitors of protein synthesis suggesting that Xhh is a direct thyroid hormone response gene. The expression and TH regulation of Xhh was not limited to the intestine, but was also observed in the limb and a mixture of pancreas and stomach. Throughout development, Xhh mRNA was present at varying levels with the earliest expression being detected at neurula stage. The highest levels of Xhh were observed between stages 33 and 40 shortly before tadpole feeding begins. Whole mount in situ hybridization analysis of Xhh expression in pre-hatching, stage 32 tadpoles demonstrated staining in the notochord and floor plate similar to that observed for other vertebrate hedgehog genes. Together, these data suggest a putative role for Xhh in organ development during both amphibian embryogenesis and metamorphosis.

Aging↗

PubMed1995

Transcriptional activation of the matrix metalloproteinase gene stromelysin-3 coincides with thyroid hormone-induced cell death during frog metamorphosis.

A full-length cDNA was isolated for a thyroid hormone response gene in the metamorphosing frog intestine and shown by sequence analysis to be the frog homolog of the mammalian extracellular matrix metalloproteinase stromelysin-3 (ST3). Northern hybridization indicated that ST3 gene expression is differentially activated in tadpole tissues during metamorphosis. In the small intestine, in situ hybridization localized high levels of ST3 mRNA to fibroblast-like cells during thyroid hormone-induced metamorphosis. ST3mRNA was undetectable in the intestine prior to metamorphosis, while high levels were present at the metamorphic climax. At this time, primary intestinal epithelial cells are known to undergo cell death and replacement by secondary epithelial cells, arguing that ST3 is involved in the modification of the extracellular matrix during apoptosis. ST3mRNA was also expressed at high levels during tadpole tail resorption, but not in premetamorphic tail or developing hindlimb, further supporting a role for ST3 when tissue remodeling is accompanied by large-scale cell death. Premetamorphic tadpoles treated with thyroid hormone showed a similar but compressed time course of ST3 gene regulation, suggesting that thyroid hormone controls ST3 gene expression during metamorphosis. In contrast, during embryogenesis, ST3 was expressed before endogenous thyroid hormone is detectable, indicating that ST3 can also be regulated independently of thyroid hormone. These findings implicate that ST3 participates in the modification of the extracellular matrix during matamorphic apoptosis, but Northern analyses using heterologous probes raise the possibility that additional matrix metalloproteinases may also be involved.

Amino Acid Sequence↗

PubMed1995

Cell-cell and cell-ECM interactions in epithelial apoptosis and cell renewal during frog intestinal development.

Amphibian intestinal remodeling during metamorphosis is a developmental system that is entirely controlled by thyroid hormone. It transforms a simple tubular organ into a complex multiply folded frog intestine similar to that in higher vertebrates. This process involves the degeneration of the larval epithelium through programmed cell death (apoptosis) and concurrent proliferation and differentiation of adult cell types. Earlier morphological and cellular studies have provided strong evidence implicating the importance of cell-cell and cell-ECM (extracellular matrix) interactions in this process. The recent molecular characterization of the genes that are regulated by thyroid hormone has begun to reveal some molecular clues underlying such interactions. In particular, the Xenopus putative morphogen hedgehog appears to be involved in regulating/mediating cell-cell interactions during adult epithelial proliferation, differentiation, and/or intestinal morphogenesis. On the other hand, several matrix metalloproteinases (MMPs) may be involved in remodeling the ECM. Of special interest is stromelysin-3, whose spatial and temporal expression profile during intestinal metamorphosis implicates a role in ECM remodeling, which in turn facilitates cell fate determination, i.e., apoptosis vs proliferation and differentiation. Understanding the mechanisms of action for those extracellular molecules will present a future challenge in developmental research.

Animals↗

PubMed1994

Tissue-dependent developmental expression of a cytosolic thyroid hormone protein gene in Xenopus: its role in the regulation of amphibian metamorphosis.

We have cloned the cDNA encoding the Xenopus laevis homolog of mammalian cytosolic thyroid hormone binding protein (CTHBP). We found that while its mRNA level varies little in whole animals during development, the expression of CTHBP is inversely correlated with tissue-specific transformations during metamorphosis. A high level of its mRNA was observed in the tail of premetamorphic tadpoles. However, the expression is dramatically repressed with the onset of rapid tail resorption. In the hindlimb, the expression of CTHBP is very low during morphogenesis. Subsequently, its expression continuously increases during the period of limb growth. In contrast, a low level of CTHBP expression was detected in the intestine throughout metamorphosis. These results suggest that CTHBP could function to modulate the metamorphic process by regulating the level of intracellular thyroid hormones.

Amino Acid Sequence↗

PubMed1994

Thyroid hormone-dependent differential regulation of multiple arginase genes during amphibian metamorphosis.

We have cloned three nonhepatic arginase genes in Xenopus laevis. The deduced amino acid sequences of the three arginases are almost identical and share about 60% identity with mammalian as well as Xenopus liver arginase. Both the liver and nonhepatic arginase genes are activated early during embryogenesis. The liver arginase gene is strongly expressed in tadpole liver, but weakly in other tissues. In contrast, the nonhepatic arginase genes have the strongest expression in the tadpole tail. During metamorphosis, the liver and nonhepatic arginase genes show distinct regulation patterns. In the intestine, both types of arginase genes are activated during the remodeling period. In the tail, the liver arginase gene is activated during tail resorption, whereas the nonhepatic ones are highly expressed throughout all stages examined. Finally, in the hindlimb, the liver arginase is up-regulated slightly during development, whereas the nonhepatic ones have low levels of expression until the end of metamorphosis. During 3,5,3'-L-triiodothyronine (T3)-induced metamorphosis, the nonhepatic arginase genes are activated very quickly, whereas the liver arginase gene is a late T3 response gene. These differential regulation patterns during normal and T3-induced metamorphosis suggest potential functions for the arginases during tissue remodeling.

Amino Acid Sequence↗

PubMed1994

Transcriptional repression of Xenopus TR beta gene is mediated by a thyroid hormone response element located near the start site.

We report here the detailed analysis of the promoter of a thyroid hormone receptor (TR) gene that is regulated by the hormone itself. The receptor gene, TR beta A, is one of the two TR beta genes in Xenopus laevis. It has two transcription start sites. The mRNAs derived from one of them are up-regulated by thyroid hormone, whereas those derived from the other are independent of the hormone. We have characterized the hormone-inducible promoter using a transient transfection assay in a Xenopus tissue culture cell line (A6). Deletion and mutational analysis identifies the first amphibian thyroid hormone response element (TRE). This TRE consists of near perfect direct repeats of AGGTCA with a 4-base pair spacing similar to mammalian TREs. The TRE forms specific complexes with extracts of A6 cells that have similar sequence specificities as those found for the complexes between mammalian TRs and TREs. However, unlike TREs found in other thyroid hormone-inducible promoters, this TRE is located at the putative transcription start site and mediates transcriptional repression by unliganded TRs. The addition of thyroid hormone at physiological concentrations overcomes the repression and induces further transcriptional activation at higher concentrations. These results suggest a potential mechanism for the regulation of amphibian metamorphosis, a process that is entirely controlled by thyroid hormone.

Animals↗

PubMed1994

Cloning and characterization of the ribosomal protein L8 gene from Xenopus laevis.

A full-length cDNA clone encoding the Xenopus laevis ribosomal protein L8 has been isolated and sequenced. The cDNA has an open reading frame of 257 amino acids. The deduced amino acid sequence is rich in basic residues and extremely homologous to the rat L8 protein. The Xenopus gene is ubiquitously expressed at fairly constant levels during development, consistent with its role as a house-keeping gene.

Amino Acid Sequence↗

PubMed1994

Thyroid hormone-dependent regulation of the intestinal fatty acid-binding protein gene during amphibian metamorphosis.

To investigate, at the molecular level, the remodeling of small intestine during amphibian metamorphosis, a subtractive hybridization approach was used to identify genes that are differentially regulated by thyroid hormone. A frog cDNA was isolated from Xenopus laevis and determined to be the gene encoding the intestinal fatty acid-binding protein (IFABP) based on its high sequence homology to the previously cloned mammalian IFABP gene. Northern blot analyses and in situ hybridization histochemistry also showed that, like the mammalian IFABP genes, frog IFABP gene expression is restricted to the intestinal epithelium. Xenopus embryos express detectable IFABP mRNA at stage 33/34, suggesting that intestinal epithelial cells differentiate well before feeding begins at stage 45. Moreover, during metamorphosis, levels of IFABP mRNA were gradually down-regulated over a period of about 20 days between stages 54 and 62, reaching a minimum at metamorphic climax, after which they were reelevated as the secondary epithelium forms. This reduction in IFABP gene expression could be reproduced in only 3 days by treating premetamorphic tadpoles with thyroid hormone. Our findings also show that this effect, while likely to be indirect, takes place before overt morphological changes are evident in primary epithelial cells. Thus, the down-regulation of IFABP mRNA is one of the early molecular events preceding epithelial cell death during intestinal remodeling.

Amino Acid Sequence↗

PubMed1994

New techniques of hearing assessment.

This article reviews current literature in the areas of otoacoustic emissions and auditory brain stem responses (ABRs) and their application to the evaluation of peripheral auditory function in infants and children. The different types of otoacoustic emissions are described along with their incidence, development, clinical applications, and interpretation in the pediatric group. The development of the auditory system, reflected by ABRs, is presented in detail with previously unpublished results from three-channel Lissajous' trajectory studies performed in infants. Clinical application of frequency-specific ABRs, both air and bone conducted, are presented. Finally, a discussion of the need to develop objective estimators of signal quality and threshold detection for otoacoustic emissions and ABRs in infants and children is presented, which includes previously unpublished results evaluating a new technique.

Auditory Perception↗

PubMed1993

The earliest changes in gene expression in tadpole intestine induced by thyroid hormone.

Genes induced by thyroid hormone (TH) to change their expression in the Xenopus laevis gastrointestinal (GI) tract have been isolated using a subtractive hybridization method. An exhaustive search for down-regulated genes identified a single gene. Thirty-two different cDNA fragments derived from the up-regulated mRNA of tadpole intestine 18 h after addition of TH were cloned. They map to no more than 22 distinct genes. The isolation of multiple cDNA fragments derived from a single mRNA indicates that the complexity of up-regulated genes is limited. Both ubiquitous and intestine-specific up-regulated genes were found in this screen. The majority of these genes respond directly to TH induction as judged by the resistance of up-regulation to inhibitors of protein synthesis. The biological significance of these genes is supported by their dramatic regulation in the GI tract during spontaneous metamorphosis.

Animals↗

PubMed1993

[Evoked otoacoustic emission behavior in clinical sensorineural hearing loss].

The present study revealed a significantly higher EOAE incidence in subjects with sensorineural hearing loss of unknown causes (81.82%) than in those with tumor of the cerebellopontine angle (42.86%), sudden deafness (41.38%) and equilibrium disorders (50%). EOAEs had been detected in some ears with mean pure-tone hearing threshold across 0.5, 1 and 2 kHz above 40 dB HL. For subjects with sensorineural hearing loss of unknown causes, the correlation coefficient between the mean hearing threshold and EOAE detection threshold amounted to only 0.40. It seems that EOAEs are related primarily to the nature of hearing loss rather than simply to pure-tone audiometric behavior.

Adolescent↗

PubMed1992

Genomic organization and alternative promoter usage of the two thyroid hormone receptor beta genes in Xenopus laevis.

Each of the two Xenopus laevis thyroid hormone receptor beta genes is at least 70 kilobases in length with similar intron-exon organization. There are up to eight alternatively spliced exons in the 5'-untranslated region. Excluding the extreme amino terminus, each receptor is encoded by six exons spanning about 6 kilobases of the genome, in which each of the two zinc fingers that comprise the DNA-binding domain is encoded by a separate exon and the hormone-binding domain is split into three exons. The last exon of the coding region also contains at least 600 base pairs of the 3'-untranslated region, which is about 8 kilobases. Each of the receptor genes has two promoters and just one of them is up-regulated in tadpoles by the administration of thyroid hormone.

Amino Acid Sequence↗

PubMed1990

Xenopus laevis alpha and beta thyroid hormone receptors.

The Xenopus laevis genome encodes two genes for the alpha (TR alpha) and two genes for the beta (TR beta) thyroid hormone receptors. The two TR alpha genes closely resemble their rat, human, and chicken counterparts. No alternatively spliced TR alpha cDNA clones were found in the 5' untranslated region (5' UTR). In contrast, complex alternative splicing of TR beta mRNA occurs within the 5' UTR as well as possible alternative transcriptional start sites. As many as eight exons encoding mainly the 5' UTR are alternatively spliced, giving rise to at least two amino termini for each of the two TR beta proteins. The 5' UTR of transcripts from both TR alpha and TR beta genes contain multiple AUG sequences with short open reading frames suggesting translational control mechanisms might play a role in expression of TR genes.

Amino Acid Sequence↗

PubMed1990

Developmental and thyroid hormone-dependent regulation of pancreatic genes in Xenopus laevis.

We have isolated and sequenced a cDNA encoding Xenopus laevis pancreatic trypsin, which has approximately 70% amino acid sequence identity to mammalian trypsinogen. Northern blotting analysis shows that the trypsin gene is activated just before the tadpole starts to feed, reaches peak activity in the swimming tadpole (premetamorphosis), and is then repressed during prometamorphosis, attaining its lowest activity at the climax of metamorphosis. The same gene is then activated again in frogs but to a much lower level. The pattern of the changes in trypsin gene expression is followed by at least two other pancreas-specific genes and marks the remodeling of the pancreas of the animal at metamorphosis. Thyroid hormone, which is the causative agent of metamorphosis, can down-regulate trypsin gene expression prematurely.

Amino Acid Sequence↗

PubMed1988

Evidence for structural deformation of the DNA helix by a psoralen diadduct but not by a monoadduct.

We have investigated the structural change in a double-stranded DNA helix caused by covalent addition of a psoralen. A synthetic double-stranded DNA was constructed to contain either a psoralen furan-side monoadduct or an interstrand diadduct at a specific site. When the unmodified and psoralen modified DNAs were examined by electron microscopy in the presence of distamycin, which stiffens the DNA helix, the DNA containing the psoralen interstrand diadduct appeared bent (or kinked), whereas the furan-side monoadducted DNA appeared similar to the unmodified DNA. RecA protein from E. coli has been shown to preferentially bind UV (ultra violet) irradiated DNA presumably due to alterations in the normal DNA helical structure. Using a nitrocellulose filter binding assay, we have found that the psoralen interstrand diadduct enhances the binding of recA protein to the double-stranded DNA, whereas a furan-side monoadduct has little effect. Thus both the recA protein binding and the electron microscopic data suggest that a psoralen diadduct causes deformation of a DNA helix, most likely by kinking the helix, and that a monoadduct has little effect on the DNA helix structure.

Base Sequence↗

PubMed1988

Base-catalyzed reversal of a psoralen-DNA cross-link.

Base-catalyzed reversal of a psoralen-DNA cross-link has been observed under denaturing alkaline conditions at elevated temperatures. The cross-link was formed between 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen and the two thymidine residues (T) on opposite strands of the double-stranded DNA formed from the self-complementary oligonucleotide 5'-GGGTACCC-3'. In contrast to the photoreversal of the cross-link, which yields mostly the furan-side monoadducted oligonucleotide [Cimino, G. D., Shi, Y., & Hearst, J. E. (1986) Biochemistry 25, 3013-3020], base-catalyzed reversal of the cross-link yields only pyrone-side monoadducted oligonucleotides as identified on the basis of their mobilities on a 20% polyacrylamide-7 M urea gel and their chemical and photochemical properties. A mechanism has been proposed to explain the base-catalyzed reversal reaction. This observation suggests a way to make pyrone-side monoadducted DNA. It also suggests that caution must be taken when psoralen-adducted DNA is treated under denaturing alkaline conditions.

Chromatography, High Pressure Liquid↗

PubMed1988

Interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested at a specific psoralen crosslink site.

We have probed the interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested by a site-specifically placed psoralen crosslink using DNase I footprinting techniques. The psoralen derivative 4'-hydroxymethyl-4,5',8-trimethylpsoralen was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing an E. coli RNA polymerase promoter at one end. The psoralen molecule was photochemically attached to two adjacent thymidine residues on opposite strands as a diadduct. Using this crosslinked DNA as the template for transcription, we found that the E. coli RNA polymerase was blocked at the psoralen diadduct, yielding a transcript 29 nucleotides long. The arrested elongation complex inhibited DNase I digestion of both the coding strand and the non-coding strand from about 22 nucleotides upstream to 15 nucleotides downstream from the diadduct. These results, which suggest that the unwindase and the catalytic sites of the polymerase are very close to each other, have been incorporated into a model of the transcription elongation complex.

Base Sequence↗

PubMed1988

Interaction of T7 RNA polymerase with DNA in an elongation complex arrested at a specific psoralen adduct site.

We have probed the interaction of T7 RNA polymerase with DNA in an elongation complex arrested by a site specifically placed psoralen diadduct or furanside monoadduct using DNase I footprinting techniques. The psoralen derivative, HMT (4'-hydroxy-methyl-4,5',8-trimethylpsoralen), was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing a T7 RNA polymerase promoter at one end. The psoralen molecule was photochemically attached either to 2 adjacent thymidine residues on opposite strands as a diadduct or to only 1 thymidine residue on the coding strand as a furan-side monoadduct. Using these psoralen-modified DNAs as templates for transcription, we found that T7 RNA polymerase was blocked at the psoralen adduct site and that the arrested elongation complex protected about 15 nucleotides upstream from the adduct on the coding strand and 20 nucleotides around the adduct on the noncoding strand from DNase I digestion. The two psoralen-modified DNA templates yielded identical RNA transcripts and DNase I footprints. In contrast, T7 polymerase protected only the coding strand from -20 to +8 in the initiation complex. These results suggest that the RNA polymerase undergoes a marked conformational change upon converting from an initiation complex to an elongation complex.

Base Sequence↗