Biomedical subjects

C L Wei

Publications and source records attributed to C L Wei.

14 recordsLinked to original sources

PubMed1999

RNase E is required for the maturation of ssrA RNA and normal ssrA RNA peptide-tagging activity.

During recent studies of ribonucleolytic "degradosome" complexes of Escherichia coli, we found that degradosomes contain certain RNAs as well as RNase E and other protein components. One of these RNAs is ssrA (for small stable RNA) RNA (also known as tm RNA or 10Sa RNA), which functions as both a tRNA and mRNA to tag the C-terminal ends of truncated proteins with a short peptide and target them for degradation. Here, we show that mature 363-nt ssrA RNA is generated by RNase E cleavage at the CCA-3' terminus of a 457-nt ssrA RNA precursor and that interference with this cleavage in vivo leads to accumulation of the precursor and blockage of SsrA-mediated proteolysis. These results demonstrate that RNase E is required to produce mature ssrA RNA and for normal ssrA RNA peptide-tagging activity. Our findings indicate that RNase E, an enzyme already known to have a central role in RNA processing and decay in E. coli, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3' end of ssrA RNA.

Base Sequence↗

PubMed1999

Corticotropin-releasing factor enhances brain-derived neurotrophic factor gene expression to facilitate memory retention in rats.

In the present study, we investigated the effects of corticotropin-releasing factor (CRF) injected into the dentate gyrus (DG) of the hippocampus on brain-derived neurotrophic factor (BDNF) mRNA expression and studied whether N-methyl-D-aspartate (NMDA) receptor mediates the effects of CRF on BDNF mRNA expression in the DG. Since both CRF and BDNF gene expressions are involved in memory processing in rats, we further investigated whether CRF facilitates memory retention through enhanced BDNF mRNA expression in the hippocampus. Effect of direct BDNF injection to the DG on retention performance in rats was also assessed. Results indicated that intra-DG CRF injection produced a dose-dependent (0.1 microg, 1.0 microg and 10 microg) increase in BDNF mRNA level, while intra-DG MK801 injection produced a dose-dependent (0.08 microg, 0.2 microg and 2.0 microg) decrease in BDNF mRNA expression in the DG. MK801, at a dose having no significant effect alone (0.08 microg), significantly antagonized the effect of CRF on BDNF mRNA expression. On the other hand, CRF (1.0 microg) consistently and markedly improved retention performance in rats in an inhibitory avoidance learning task. BDNF antisense oligonucleotide treatment, at a concentration which did not affect retention performance alone (0.5 mM), blocked the memory-enhancing effect of CRF. However, direct and chronic BDNF injection to the DG did not improve memory performance in rats. These results together suggest that at least one of the mechanisms responsible for the memory-facilitating effect of CRF is mediated through enhanced BDNF mRNA expression in the hippocampus. The lack of an effect of intra-DG BDNF injection on memory retention is also discussed.

Animals↗

PubMed1999

The proapoptotic function of Drosophila Hid is conserved in mammalian cells.

Three genes-reaper, grim, and hid-are crucial to the regulation of programmed cell death in Drosophila melanogaster. Mutations involving all three genes virtually abolish apoptosis during development, and homozygous hid mutants die as embryos with extensive defects in apoptosis. Although Hid is central to apoptosis in Drosophila, it has no mammalian homologue identified to date. We present evidence that expression of Drosophila Hid in mammalian cells induces apoptosis. This activity is subject to regulation by inhibitors of mammalian cell death. We show that the N terminus of Hid, which is a region of homology with Reaper and Grim, is essential for Hid's function in mammalian cells. We demonstrate that Hid is localized to the mitochondria via a hydrophobic region at its C terminus and functionally interacts with BclXL. This study shows that the function of Hid as a death inducer in Drosophila is conserved in mammalian cells and argues for the existence of a mammalian homologue of this critical regulator of apoptosis.

Animals↗

PubMed1998

RNA components of Escherichia coli degradosome: evidence for rRNA decay.

Recently, we found that a multicomponent ribonucleolytic degradosome complex formed around RNase E, a key mRNA-degrading and 9S RNA-processing enzyme, contains RNA in addition to its protein components. Herein we show that the RNA found in the degradosome consists primarily of rRNA fragments that have a range of distinctive sizes. We further show that rRNA degradation is carried out in the degradosome by RNase E cleavage of A+U-rich single-stranded regions of mature 16S and 23S rRNAs. The 5S rRNA, which is known to be generated by RNase E processing of the 9S precursor, was also identified in the degradosome, but tRNAs, which are not cleaved by RNase E in vitro, were absent. Our results, which provide evidence that decay of mature rRNAs occurs in growing Escherichia coli cells in the RNA degradosome, implicate RNase E in degradosome-mediated decay.

Chromatography, Affinity↗

PubMed1998

Brain-derived neurotrophic factor antisense oligonucleotide impairs memory retention and inhibits long-term potentiation in rats.

We have examined the relationship between brain-derived neurotrophic factor gene expression in the hippocampus and memory retention as well as long-term potentiation of rats. One-way inhibitory avoidance learning was adopted as the behavioural paradigm. Results revealed that brain-derived neurotrophic factor messenger RNA levels in the dentate gyrus of the hippocampus were markedly increased at 1 h, 3 h and 6 h post-training in rats showing good retention performance when compared with the poor retention controls. Direct injection of brain-derived neurotrophic factor antisense oligonucleotide into the dentate gyrus of the hippocampus before memory consolidation takes place markedly impaired retention performance in rats. It also significantly decreased brain-derived neurotrophic factor messenger RNA level in the dentate gyrus. The same antisense treatment also markedly reduced the amplitude and slope of excitatory postsynaptic potential as well as the brain-derived neurotrophic factor messenger RNA level in the dentate gyrus. These results suggest that hippocampal brain-derived neurotrophic factor gene expression plays an important role in the memory consolidation process and in the expression of long-term potentiation in rats. These results provide the first evidence to relate brain-derived neurotrophic factor gene expression and memory function in vertebrates. It further suggests that brain-derived neurotrophic factor gene expression is involved in behavioural plasticity.

Animals↗

PubMed1996

The gene encoding UDP-glucose pyrophosphorylase is required for the synthesis of xanthan gum in Xanthomonas campestris.

Xanthomonas campestris pv. campestris produces a large quantity of exopolysaccharide, xanthan gum, rendering the colonies mucoid. G76E was a non-mucoid mutant isolated from Xc17 by Tn5 mutagenesis. A 3.0-kb KpnI-EcoRI fragment from the Xc17 chromosome was able to restore mucoid phenotype to G76E. Sequence analysis of the region responsible for the restoration revealed an open reading frame, ORF324, able to encode a polypeptide of 35,232 Da which shows striking similarity to the UDP-glucose pyrophosphorylases from bacteria. The activity of UDP-glucose pyrophosphorylase reduced drastically in G76E was found to be regained in the presence of the cloned 3.0-kb KpnI-EcoRI fragment. In vitro expression of the gene in the S30 transcription/translation system produced a protein of ca. 35 kDa.

Amino Acid Sequence↗

PubMed1996

Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.

Amino Acid Sequence↗

PubMed1995

Characterization of yeast translation initiation factor 1A and cloning of its essential gene.

Translation initiation factor eIF1A is required in vitro for maximal rates of protein synthesis in mammalian systems. It functions primarily by dissociating ribosomes and stabilizing 40 S preinitiation complexes. To better elucidate its precise role in promoting the translation initiation process, the yeast form of eIF1A has been identified in Saccharomyces cerevisiae and purified to homogeneity on the basis of its cross-reaction with antibodies prepared against mammalian eIF1A. The apparent mass of yeast eIF1A (22 kDa) resembles that of the mammalian homolog (20 kDa), and the yeast factor is active in stimulating methionyl-puromycin synthesis in an assay composed of mammalian components. The gene encoding yeast eIF1A, named TIF11, was cloned and shown to be single copy. TIF11 encodes a protein comprising 153 amino acids (17.4 kDa); the deduced amino acid sequence exhibits 65% identity with the sequence of human eIF1A. Both human and yeast eIF1A contain clusters of positive residues at the N terminus and negative residues at the C terminus. Deletion/disruption of TIF11 demonstrates that eIF1A is essential for cell growth. Expression of human eIF1A cDNA rescues the growth defect of TIF11-disrupted cells, indicating that the structure/function of yeast and mammalian eIF1A is highly conserved.

Amino Acid Sequence↗

PubMed1995

Protein synthesis initiation factor eIF-1A is a moderately abundant RNA-binding protein.

Eukaryotic initiation factor (eIF) 1A (formerly called eIF-4C) is a small protein that promotes dissociation of 80 S ribosomes into subunits, stabilizes methionyl-tRNA binding to 40 S ribosomal subunits, and is required for the binding of mRNA to ribosomes. The sequence of eIF-1A derived from its cloned cDNA possesses a high frequency of basic residues and acidic residues at its N and C termini, respectively. Northwestern blotting with a fragment of mRNA indicates that eIF-1A binds RNA. Overexpression of the human eIF-1A cDNA in Escherichia coli and subsequent purification enabled us to prepare large quantities of active factor. The level of eIF-1A in HeLa cells determined by Western immunoblotting is 0.01% of total protein, which corresponds to 0.2 molecules of eIF-1A/ribosome. The moderate abundance means that eIF-1A is equal to or in excess of native 40 S subunits and suggests that the factor may not be limiting for protein synthesis, a conclusion reinforced by the failure of overproduced eIF-1A to stimulate translation rates in transiently transfected COS-1 cells. S1 nuclease protection and primer extension analyses show that eIF-1A mRNA possesses an unusually long 5'-untranslated leader that is very G/C-rich (72%). Unexpectedly, the mRNA is efficiently translated in HeLa cells as judged by polysome profile analyses.

Animals↗

PubMed1995

The major protein of messenger ribonucleoprotein particles in somatic cells is a member of the Y-box binding transcription factor family.

A cDNA encoding the major core protein, p50, of cytoplasmic messenger ribonucleoprotein particles (mRNPs) of somatic cells was cloned from a rabbit reticulocyte cDNA library. From the derived 324-amino acid sequence, p50 is identified as a member of the Y-box binding transcription factor family. The protein was earlier described as a repressor of globin mRNA translation. These findings suggest that p50 may affect protein biosynthesis at two levels: mRNA transcription in the nucleus and mRNA translation in the cytoplasm. Together with recently published results showing that masked mRNA in germ cells also is associated with proteins of the Y-box binding protein family, the present finding indicates that these proteins are universal core proteins responsible for the formation of cytoplasmic mRNPs in eukaryotes. Highly purified p50 forms large 18 S homomultimeric complexes with a molecular mass of about 800 kilodaltons and melts RNA secondary structure. This suggests that p50 may affect translation by changing the overall structure of the mRNA.

Amino Acid Sequence↗

PubMed1994

Determination of the amino acid sequence of rabbit, human, and wheat germ protein synthesis factor eIF-4C by cloning and chemical sequencing.

The small eukaryotic initiation factor (eIF)-4C is implicated in the initiation pathway, where it enhances ribosome dissociation into subunits and stabilizes the binding of the initiator Met-tRNA(i) to 40 S ribosomal subunits. In order to elucidate the function of eIF-4C, its structure has been further characterized. The amino acid sequence of many peptides from rabbit reticulocyte and wheat germ eIF-4C have been determined chemically. From the chemical sequencing of the rabbit protein, it was noted that at least two different eIF-4C molecules were present which differed by conservative substitutions at three positions (2 aspartic acid for glutamic acid switches and 1 valine for isoleucine switch). By the use of unique sequences with low codon degeneracy, primers were used to obtain a polymerase chain reaction product of appropriate size and sequence. This product was then used to isolate full-length coding sequence cDNA clones for human eIF-4C. A similar strategy was used to design PCR primers and then isolate a wheat cDNA clone which lacked the coding region for the first 23 amino acids, but contained a complete 3'-untranslated region. The protein amino acid sequence of wheat germ eIF-4C is 68% identical with the mammalian protein, and, allowing for the most conservative substitutions, the proteins are 76% similar. Both the mammalian and wheat germ proteins are 143 amino acids in length and have molecular weights of about 16,400. A unique feature of eIF-4C is its apparent "polarity" as 9 of the first 15 amino acids are basic while 13 of the last 20 amino acids are acidic. This dipole nature may enable the protein to interact with both the ribosome (perhaps via the rRNA) and other translation initiation factors.

Amino Acid Sequence↗

PubMed1989

A liver-specific nuclear factor interacts with the promoter region of the large surface protein gene of human hepatitis B virus.

The outer envelope of the 42-nm virion of the human hepatitis B virus (HBV) is composed of the large, the middle, and the major surface proteins. Whereas the middle and the major surface proteins are transcribed from the SPII promoter of the pre-S/S gene, the large surface protein is transcribed from the SPI promoter located upstream of SPII. We have previously shown that transcription of SPI (comprising nucleotides [nt] -380 to +17) occurs preferentially in differentiated hepatoma cell lines (H.K. Chang and L.P. Ting, Virology 170:176-183, 1989). In this report, we further demonstrated that a sequence of 95 base pairs in the upstream region of SPI (nt -95 to +17) was necessary and sufficient for such preferential expression in differentiated hepatoma cells. By analysis of the expression of the chloramphenicol acetyltransferase gene in a series of mutants with deletions at the 5' end of SPI, we identified a positive transcriptional cis-acting element mapping at nt -95 to -72 which appears to play a key role in the regulation of the expression of the large surface protein. This region shared a high degree of sequence homology with regulatory sequences of several liver-specific genes from human, mouse, and rat, with a consensus sequence (G/A)GTTA(A/C)TNNT(C/T)NNC(A/C). We further identified a nuclear factor present in the nuclear extracts of differentiated human hepatoma cell lines which interacted specifically with this element of the SPI promoter. This nuclear factor was similar to the rat liver-specific factor HNF-1, since an oligonucleotide containing the recognition sequence of HNF-1 could efficiently compete for the human factor in a footprinting assay. The sequence at nt -93 to -68 which was bound by this factor in SPI was termed the HNF-1-binding element. Activation of the SPI promoter by human differentiated hepatocyte nuclear factor 1, described in this report, probably explains, first, the formation of the 42-nm virion specifically in liver but not in several other tissues despite the synthesis of the middle and the major surface proteins in those tissues, and second, why only differentiated hepatoma cell lines are able to produce 42-nm-like virion particles on transfection by HBV DNA.

Base Sequence↗

PubMed1986

[Level of cyclic AMP-dependent protein kinase isozyme in normal liver and hepatoma tissue and the effect of sodium selenite].

Sodium selenite in normal saline was administered intraperitoneally (1 mg/kg) into mice bearing ascitic hepatocarcinoma for 4 days. The cyclic AMP-dependent protein kinase isozymes (type I and type II) in normal liver and hepatocarcinoma cells were separated and assayed. The results show that the level of type I/II is markedly higher in hepatocarcinoma than in the normal liver cells. Sodium selenite is able to reduce it towards the normal level. Further analysis shows that the chief function of sodium selenite is to reduce the raised level of type I/II in hepatocarcinoma cells which, in fact, is due to the increase of total amount of type I cyclic AMP-dependent protein kinase. This paper presents the speculation that one of the mechanisms of the inhibitory effect of sodium selenite on carcinogenesis may be due to the selective action of this compound on the cyclic AMP-dependent protein kinase isozymes in tumor cells, thus inhibiting cancer cell division and facilitating differentiation and reversion.

Animals↗

PubMed1983

In vivo and in vitro release of macromolecules from polymeric drug delivery systems.

In vivo release rates of a macromolecule from an ethylene-vinyl acetate copolymer have been shown to be indistinguishable from those of identical implants tested in vitro. The studies were conducted for approximately 2 months, and two different techniques were used to assess release rates. One of these techniques, using [3H]inulin as a marker, may be particularly useful in future studies assessing in vivo release rates from drug delivery systems. The appearance of [3H]inulin in the urine of rats bearing implants allowed continuous monitoring of release. A histological evaluation of tissue sections surrounding polymer implanted for 7 months showed no inflammatory cell reaction.

Delayed-Action Preparations↗