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Biomedical subjects

X Q Wu

Publications and source records attributed to X Q Wu.

At least 19 recordsLinked to original sources
PubMed2000

Mouse testis brain ribonucleic acid-binding protein/translin colocalizes with microtubules and is immunoprecipitated with messenger ribonucleic acids encoding myelin basic protein, alpha calmodulin kinase II, and protamines 1 and 2.

Testis brain RNA-binding protein (TB-RBP) is a sequence-dependent RNA-binding protein that binds to conserved Y and H sequence elements present in many brain and testis mRNAs. Using recombinant TB-RBP and a highly enriched tubulin fraction, we demonstrate here that recombinant TB-RBP binds to microtubules assembled in vitro. The interaction between recombinant TB-RBP and microtubules was inhibited by high salt and by the microtubule disassembling agents colcemid and calcium, but not by the microfilament-disassembling agent cytochalasin D. Confocal microscopy confirmed colocalization of TB-RBP and tubulin in the cytoplasm of male germ cells. An affinity-purified antibody prepared against recombinant TB-RBP specifically precipitated mRNAs encoding myelin basic protein and alpha calmodulin-dependent kinase II-two transported mRNAs, and protamines 1 and 2-two translationally regulated testicular mRNAs. These data indicate an intracellular association between TB-RBP and specific target mRNAs and suggest an involvement of TB-RBP in microtubule-dependent mRNA transport in the cytoplasm of cells.

Amino Acid Sequence↗
PubMed1999

Protein-protein interactions between the testis brain RNA-binding protein and the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin and Trax in male germ cells and the brain.

Numerous functions have been proposed for the testis brain RNA-binding protein (TB-RBP) and its human homologue, Translin, ranging from mRNA transport and translational regulation to DNA rearrangement and repair. To gain insight into the likely functions of this 26 kDa protein, immunoprecipitation was used to identify proteins that interact with TB-RBP in mouse cytosolic extracts. Three proteins, the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin, and Trax, were specifically immunoprecipitated with an affinity-purified antibody to recombinant mouse TB-RBP. In vitro binding assays with recombinant proteins and EM immunocytochemistry confirm that TB-RBP interacts with the TER ATPase in vitro and in vivo. Confocal microscopy has demonstrated that TB-RBP colocalizes with actin in the cytoplasm of male germ cells. The immunoprecipitation of Trax with TB-RBP confirms a published report demonstrating protein interactions between the two proteins in a yeast two-hybrid assay. These data support the hypothesis that TB-RBP serves as a link in attaching specific mRNAs to cytoskeletal structures and suggests an involvement for the ubiquitously expressed TER ATPase in intracellular and/or intercellular mRNA transport.

Actins↗
PubMed1999

Testis-brain RNA-binding protein (Translin) is primarily expressed in neurons of the mouse brain.

The subcellular location(s) of the DNA- and RNA-binding protein, Testis-Brain RNA-Binding Protein (TB-RBP)/Translin in mouse brain has been determined in paraffin sections by immunocytochemistry with an affinity purified antibody to mouse recombinant TB-RBP. Nuclear staining was frequently seen in neurons throughout the brain, but no TB-RBP/Translin was detected in many of the neurons in superficial layers of the cerebral cortex and in some cells of the cerebellum. Cytoplasmic staining extending into the dendrites was seen in large neurons such as pyramidal neurons in Layer 5 of the cortex and magnocellular neurons of the hypothalamus or the brainstem raphe.

Animals↗
PubMed1999

Study on the direct electron transfer process of superoxide dismutase.

The electron transfer process of biological important species, superoxide dismutase (SOD), was studied using cyclic voltammetric method at different conditions. The rate of electron transfer process of SOD at conventional bare gold electrode was very low. Different methods were used to enhance the rate of this process. Gold microdisk electrode and gold minigrid electrode were used to replace the used conventional size gold electrode and the electron transfer rate was enhanced obviously. Different promoters, such as bis(4-pyridyl)disulfide, histidine and arginine, were used to promote the electron transfer process also. Promising results were observed with the help of those promoters. The rate enhancement through the participation of amino acids might be more interesting for the exploration of the real process of SOD-related reaction inside the human body.

Arginine↗
PubMed1998

The DNA/RNA-binding protein, TB-RBP, moves from the nucleus to the cytoplasm and through intercellular bridges in male germ cells.

The genetically haploid spermatids are functionally diploid as a result of the sharing of gene products through their intercellular bridges. This movement of molecules among haploid cells is crucial for the production of spermatozoa since numerous essential proteins are encoded on sex chromosomes. Testis-brain RNA-binding protein (TB-RBP) is a single-stranded DNA- and RNA-binding protein prominent in the nuclei and cytoplasm of specific stages of differentiating male germ cells. Here we present evidence that TB-RBP moves from the nucleus to the cytoplasm and through intercellular bridges of male germ cells. Based on its RNA-binding capabilities, we propose a role for TB-RBP in the distribution of equal amounts of mRNAs in haploid male germ cells.

Animals↗
PubMed1998

Dimerization of the testis brain RNA-binding protein (translin) is mediated through its C-terminus and is required for DNA- and RNA-binding.

Testis brain-RNA-binding protein (TB-RBP) is a single-stranded DNA- and RNA-binding protein that is involved in chromosomal translocations, mRNA transport and translational regulation. Here we show from in vitro and in vivo protein binding studies that TB-RBP dimers are the minimum structural unit needed for DNA- and RNA-binding. Truncation studies demonstrate that the C-terminus of 55 amino acids of TB-RBP is essential, but not sufficient for DNA- or RNA-binding, and deletion of the leucine zipper motif in the C-terminus abolishes DNA- and RNA-binding. Changing cysteine 225 in the C-terminus to alanine does not significantly reduce DNA- or RNA-binding, but reduces the stability of the dimer. We conclude that the leucine zipper motif is required to maintain two molecules of TB-RBP as a dimer which is stabilized by a disulfide bond involving cysteine 225.

Amino Acid Sequence↗
PubMed1998

The RNA- and DNA-binding protein TB-RBP is spatially and developmentally regulated during spermatogenesis.

Testis brain RNA-binding protein (TB-RBP) suppresses translation in vitro and attaches mRNAs to microtubules by binding to conserved elements in the 3' untranslated regions (UTRs) of specific testis and brain mRNAs. Purification of TB-RBP from testicular and brain cytoplasmic extracts has revealed that mouse TB-RBP is 99% identical to the human protein translin, a recombination "hot spot" binding protein associated with chromosomal translocations. Using a cDNA encoding TB-RBP, the gene copy number and the developmental expression of TB-RBP have been analyzed by Southern blotting, Northern blotting, and in situ hybridization. In the mouse, TB-RBP is encoded by a single copy gene. In mouse testes, three TB-RBP mRNAs of about 1.2, 1.7, and 3.0 kb are developmentally regulated with high levels of expression in meiotic and postmeiotic germ cells. A fourth TB-RBP transcript of about 3.2 kb is seen in the brain. In situ hybridization confirms high levels of testicular TB-RBP mRNAs in meiotic and postmeiotic cells, with the highest levels of TB-RBP mRNAs in pachytene spermatocytes and round spermatids of the mouse and in round spermatids of the rat. RNase H digestion assays reveal that the three TB-RBP mRNAs of mouse testes result from processing differences in their 3' untranslated regions. These data demonstrate that multiple TB-RBP mRNAs are primarily expressed in meiotic and postmeiotic germ cells in the mammalian testis, and although the specific RNA-binding ability of TB-RBP appears limited to brain and testis, TB-RBP mRNAs are widely expressed.

Animals↗
PubMed1997

The RNA-binding protein, TB-RBP, is the mouse homologue of translin, a recombination protein associated with chromosomal translocations.

The mouse RNA-binding protein, TB-RBP, suppresses translation in vitro and attaches mRNAs to microtubules by binding to conserved elements in the 3' untranslated regions of specific mRNAs. We have now purified TB-RBP from testicular and brain cytoplasmic extracts and cloned its cDNA. We find that the mouse TB-RBP cDNAs contain an open reading frame of 228 amino acids with a leucine zipper domain within its C terminus, a transmembrane helix, and a group of putative phosphorylation sites. TB-RBP shows 99% identity to the human protein, translin, a recombination hotspot-binding protein associated with chromosomal translocations [Aoki, K., Suzuki, K., Sugano, T., Tasaka, T., Nakahara, K., Kuge, O., Omori, A. & Kasai, M. (1995) Nat. Genet. 10, 167-174]. As shown for translin, TB-RBP also binds to single-stranded DNAs containing a broad range of consensus sequences, many of which are similar to the Y and H RNA-binding sequences. Recombinant TB-RBP was synthesized and an antiserum was prepared against the recombinant protein. The identity between translin and TB-RBP was confirmed by demonstrating that immunoprecipitation of TB-RBP from testicular extracts abolished formation of the RNA-TB-RBP complex. Based upon its DNA binding to target sequences in clustered breakpoint regions, we propose that TB-RBP may be involved in DNA recombination or DNA repair in male germ cells.

Amino Acid Sequence↗
PubMed1997

Effect of the amino acid attached to Escherichia coli initiator tRNA on its affinity for the initiation factor IF2 and on the IF2 dependence of its binding to the ribosome.

We show that the nature of the amino acid in the formylaminoacyl-tRNA influences initiation factor (IF) 2 dependence of its ribosome binding and that this IF2 dependence reflects the relative affinity of the formylaminoacyl-tRNA for the initiation factor IF2. We compared the template-dependent ribosome binding activities, in the presence of initiation factors, of wild type and anticodon sequence mutants of Escherichia coli initiator tRNAs that carry formylmethionine (fMet), formylglutamine (fGln), or formylvaline (fVal). The fGln-tRNA bound less well than fMet-tRNA whereas the fVal-tRNA bound as well as fMet-tRNA. The rate and extent of binding of fGln-tRNA to the ribosome was significantly increased by further addition of purified initiation factor IF2. In contrast, the binding of fVal-tRNA or fMet-tRNA was not affected much by the addition of IF2. Using gel mobility shift assay, we have measured the apparent Kd values of the IF2.formylaminoacyl-tRNA binary complexes. These are 1.8, 3.5, and 10.5 microM for fMet-tRNA, fVal-tRNA, and fGln-tRNA, respectively.

Amino Acids↗
PubMed1996

Selenocysteine synthesis in mammalia: an identity switch from tRNA(Ser) to tRNA(Sec).

The mechanism of selenocysteine insertion into proteins is distinct from all other amino acids in all lines of descent in that it needs specific protein cofactors and a structurally unique tRNA(Sec). It is first aminoacylated with serine and further recognized among all other serylated serine isoacceptors by a selenocysteine synthase and is converted to selenocysteyl-tRNA(Sec). We present here the complete set of identity elements for selenylation of mammalian seryl-tRNA(Sec) and show that the transplantation of these elements into normal serine tRNA allows its selenylation. Four particular structural motifs differentiate eukaryotic tRNA(Sec) from normal tRNA(Ser): the orientation of the extra arm, the short 4 bp T psi C-stem, the extra long 9 bp acceptor-stem and the elongated 6 bp dihydrouridine-stem. Only the last two are essential and only together sufficient for selenocysteine synthesis, whereby the additional base-pairs of the acceptor-stem may be replaced by non-paired nucleotides. Each exchange of the first three structural motifs mentioned above between tRNA(Ser) and tRNA(Sec) resulted in a significant loss of serylation, indicating that the overall composition of particular structure elements is necessary to maintain normal functions of tRNA(Sec). Since we find that all seryl-tRNAs which are selenylated are also substrates for serine phosphorylation we propose that phosphoseryl-tRNA(Sec) is a storage form of seryl-tRNA(Sec).

Acylation↗
PubMed1996

Ribosome-initiator tRNA complex as an intermediate in translation initiation in Escherichia coli revealed by use of mutant initiator tRNAs and specialized ribosomes.

For functional studies of mutant Escherichia coli initiator tRNAs in vivo, we previously described a strategy based on the use of tRNA genes carrying an anticodon sequence change from CAU to CUA along with a mutant chloramphenicol acetyltransferase (CAT) gene carrying an initiation codon change from AUG to UAG. Surprisingly, under conditions where the mutant initiator tRNA is optimally active, the CAT gene with the UAG initiation codon produced more CAT protein (3- to 9-fold more depending on the conditions) than the wild-type CAT gene. Here we show that two new mutant CAT genes having GUC and AUC initiation codons also produce more of the CAT protein in the presence of the corresponding mutant initiator tRNAs. These results are most easily understood if assembly of the 30S ribosome-initiator tRNA-mRNA initiation complex in vivo proceeds with the 30S ribosome binding first to the initiator tRNA and then to the mRNA. In cells overproducing the mutant initiator tRNAs, most ribosomes would carry the mutant initiator tRNA and these ribosomes would select the mutant CAT mRNA over the other mRNAs.

Acylation↗
PubMed1995

Inactivation and conformation changes of the glycated and non-glycated D-glyceraldehyde-3-phosphate dehydrogenase during guanidine-HCl denaturation.

The glycated D-glyceraldehyde-3-phosphate dehydrogenases have been isolated from rabbit muscle and erythrocytes (He et al. (1995) Biochem. J. 309, 133-139). The circular dichroism spectrum in the near-ultraviolet of gGAPDH was different from that of GAPDH. Changes in intrinsic protein fluorescence and in the 410 nm fluorescence of the NAD derivatives introduced at the active sites of both the glycated and non-glycated GAPDH from rabbit were compared on inactivation during denaturation in GuHCl. Complete inactivation for the non-glycated enzyme occurred in 0.5 M GuHCl solution, however, that for the glycated enzyme occurred in the 0.35 M solution. The kinetic inactivation of gGAPDH was a biphasic process (the fast and slow phases). The fast phase for gGAPDH was faster than that of GAPDH. The kinetic exposure of the fluorescent NAD derivatives at the active sites of both enzymes was also biphasic with fast phase rates which approach those of the inactivation. It appears that glycation of the enzyme may disturb the spatial geometry of the functional groups responsible for the catalytic mechanism and affect the activity.

Animals↗
PubMed1994

Eukaryotic selenocysteine inserting tRNA species support selenoprotein synthesis in Escherichia coli.

Although the tRNA species directing selenocysteine insertion in prokaryotes differ greatly in their primary structure from that of their eukaryotic homologues they share very similar three-dimensional structures. To analyse whether this conservation of the overall shape of the molecules reflects a conservation of their functional interactions it was tested whether the selenocysteine inserting tRNA species from Homo sapiens supports selenoprotein synthesis in E. coli. It was found that the expression of the human tRNA(Sec) gene in E.coli can complement a lesion in the tRNA(Sec) gene of this organism. Transcripts of the Homo sapiens and Xenopus laevis tRNA(Sec) genes synthesised in vitro were amino-acylated by the E.coli seryl-tRNA ligase although at a very low rate and the resulting seryl-tRNA(Sec) was bound to and converted into selenocysteyl-tRNA(Sec) by the selenocysteine synthase of this organism. Selenocysteyl-tRNA(Sec) from both eukaryotes was able to form a complex with translation factor SELB from E.coli. Although the mechanism of selenocysteine incorporation into seleno-proteins appears to be rather different in E.coli and in vertebrates, we observe here a surprising conservation of functions over an enormous evolutionary distance.

Animals↗
PubMed1994

The length and the secondary structure of the D-stem of human selenocysteine tRNA are the major identity determinants for serine phosphorylation.

Selenocysteine tRNA [tRNA(Ser)Sec] has been shown to be serylated by tRNA(Ser) synthetase. The serine moiety of seryl-tRNA(Ser)Sec in vertebrates is further phosphorylated by a kinase, in addition to being converted into selenocysteine. Using site-directed mutagenesis we have introduced a number of mutations into T7 RNA polymerase transcripts of human tRNA(Ser)Sec. Our results show that most of the unique structural features of tRNA(Ser)(Sec), like the 5'-triphosphate, the 9 bp long acceptor stem and the anticodon, are not identity elements for phosphorylation of human seryl-tRNA(Ser)Sec. However, the length and secondary structure of the D-stem (6 bp in contrast with 4 bp in the canonical serine tRNA) of human tRNA(Ser)Sec, but not its sequence, are the major identity determinants which discriminate this tRNA from common tRNA(Ser) and identify it as the substrate for phosphorylation by seryl-tRNA(Ser)Sec kinase. This notion is confirmed by the fact that normal seryl-tRNA(Ser), which is not a substrate for serine phosphorylation, becomes a substrate if two additional base pairs are introduced into its D-stem.

Anticodon↗
PubMed1994

Sexing bovine embryos using PCR amplification of bovine SRY sequence.

This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.

Animals↗
PubMed1993

The long extra arms of human tRNA((Ser)Sec) and tRNA(Ser) function as major identify elements for serylation in an orientation-dependent, but not sequence-specific manner.

Selenocysteine tRNA [tRNA((Ser)Sec)] is charged with serine by the same seryl-tRNA synthetase (SerRS) as the canonical serine tRNAs. Using site-directed mutagenesis, we have introduced a series of mutations into human tRNA((Ser)Sec) and tRNA(Ser) in order to study the identity elements of tRNA((Ser)Sec) for serylation and the effect of the orientation of the extra arm. Our results show that the long extra arm is one of the major identity elements for both tRNA(Ser) and tRNA((Ser)Sec) and gel retardation assays reveal that it appears to be a prerequisite for binding to the cognate synthetase. The long extra arm functions in an orientation-dependent, but not in a sequence-specific manner. The discriminator base G73 is another important identity element of tRNA((Ser)Sec), whereas the T- and D-arms play a minor role for the serylation efficiency.

Base Sequence↗
PubMed1992

[Synthesis of O,O'-dialkyl-O''-(5-substituted-3-benzothienglyoxylonitrile oximino) phosphates and thiophosphates].

In order to search for potential molluscicidal synergists, eighteen O,O'-dialkyl-O''-(5-substituted-3-benzothienglyoxylonitrile oximino) phosphates and thiophosphates were synthesized. Preliminary biological screening showed that compounds I2,3,7,11,12 combined with sodium pentachlorophenate exhibited significant molluscicidal synergism against snails (Oncomelanis hupensis).

Animals↗