Biomedical subjects

D X Liu

Publications and source records attributed to D X Liu.

At least 55 records · Page 3Linked to original sources

PubMed1999

Sequence analysis and in vitro expression of genes 6 and 11 of an ovine group B rotavirus isolate, KB63: evidence for a non-defective, C-terminally truncated NSP1 and a phosphorylated NSP5.

An ovine group B rotavirus (GBR) isolate, KB63, was isolated from faeces of a young goat with diarrhoea in Xinjiang, People's Republic of China. Sequence determination and comparison of genes 6 and 11 with the corresponding sequences of GBR strains ADRV and IDIR showed that they were the cognate genes encoding NSP1 and NSP5, respectively. While the overall identities of nucleotide sequences between these two genes and the corresponding genes of strains ADRV and IDIR were in the range 52.6-57.2%, the identities of deduced amino acid sequences were only 34.9-46.3%. These results demonstrate that the substantial diversity of NSP1 observed among group A rotaviruses (GAR) also exists within GBRs and that a high degree of diversity also exists among NSP5 of GBRs, in contrast to GAR NSP5. The NSP1 gene of KB63 contains three ORFs, whereas the NSP1 genes of other GBR strains contain only two. ORFs 2 and 3 of the KB63 gene may be derived from a single ORF corresponding to ORF2 of other GBR strains by the usage of a stop codon created by an upstream single base deletion and single point mutations. In vitro expression studies showed that ORFs 1 and 2, but not 3, of gene 6 can be translated, suggesting that ORF2 may encode a C-terminally truncated, potentially functional product. It may play a role, together with the product of ORF1, in virus replication, as the virus can be passaged further in kids. Similarly, gene 11 can be translated in vitro. Like its counterpart in GARs, the protein encoded by gene 11 was shown to be phosphorylated in vitro.

Amino Acid Sequence↗

PubMed1999

Molecular modeling on kappa opioid receptor and its interaction with nonpeptide kappa opioid agonists.

AIM: To study the interaction between kappa-opioid receptor and its nonpeptide agonists. METHODS: The "conservation patterns" for G-protein coupled receptors (GPCR) were used to determine 7 transmembrane (TM) regions. Taking the crystallographic coordinates of bacteriorhodopsin (BR) as the template, the 3D structural model was constructed for 7 TM of kappa-opioid subtype with molecular mechanics (MM) method. Five highly active nonpeptide kappa-opioid agonists were docked into the 7 helices of kappa-opioid receptor to study the ligand-receptor interaction. RESULTS: Four important interactions between U-50488-like agonists and kappa-opioid receptors were drawn according to our modeling study: (1) the protonated pyrrolidine nitrogen of the ligands formed a hydrogen-bond with the carboxyl of Asp138; (2) the carbonyl oxygen of ligands forms a hydrogen bond to the hydroxyl of Ser187; (3) the aryl groups connected to acylamide of the agonists inserted into a hydrophobic cavity enclosed by residues Val239, Val236, Phe235, Val232, Leu186, and Trp183; (4) the pyrrolidine of the ligands in the complexes was surrounded by Ile290, Asp138, Ile194, Ile135, and Cys131. CONCLUSION: The proposed interaction mechanism is helpful for further mutant experiments and designing novel potent kappa-opioid agonists.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1999

Dauricine inhibits redistribution of platelet membrane glycoprotein IV and release of intracellular alpha-granule thrombospondin induced by thrombin.

AIM: To study the possibility of dauricine (Dau) inhibiting redistribution of platelet membrane glycoprotein IV (GPIV) and release of intracellular alpha-granule thrombospondin (TSP) on platelet activation. METHODS: Using the flow cytometric assay of washed platelet to record expression of GPIV and release of TSP induced by thrombin. RESULTS: Dau did not affect GPIV and TSP on resting platelet membrane but inhibited redistribution of GPIV to the platelet surface and TSP release on activated platelet. There was a marked positive correlation between changes of GPIV and TSP (r = 0.511, P < 0.01). The inhibitory effect of Dau appeared not to be Ca2+ concentration-dependent. CONCLUSION: Dau inhibited redistribution of GPIV and release of intracellular alpha-granule thrombospondin induced by thrombin.

Adult↗

PubMed1998

Proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3C-like proteinase and identification of two novel cleavage products.

We have previously reported that the 3C-like proteinase of the coronavirus infectious bronchitis virus (IBV) is responsible for processing of the 1a and 1a/1b polyproteins to three mature products of 24, 10, and 100 kDa (Liu et al., 1994, 1997; Ng and Liu, 1998). The C-terminal cleavage site of the 100-kDa protein was defined to be the Q891(1b)-S892(1b) dipeptide bond encoded by nucleotides 15,129 to 15,134 (Liu and Brown, 1995). In this report, other cleavage sites of the 3C-like proteinase in the polyprotein encoded by the ORF 1b region were mapped by coexpression, deletion, and site-directed mutagenesis studies. Using two ORF 1b-specific antisera, V58 and V17, three more Q-S(G) dipeptide bonds, encoded by nucleotides 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, were demonstrated to be the cleavage sites of the 3C-like proteinase. Cleavage at these four positions would result in the release of four mature products with molecular masses of approximately 68, 58, 39, and 35 kDa. Among them, the 39- and 35-kDa proteins were specifically identified in IBV-infected cells. Taken together with the 100-kDa protein previously identified, these results suggest that the ORF 1b region of IBV mRNA1 may be able to encode five mature products.

Animals↗

PubMed1998

Characterization of the two overlapping papain-like proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination of the C-terminal cleavage site of an 87-kDa protein.

In a previous report, we showed that proteolytic processing of an 87-kDa mature viral protein from the coronavirus infectious bronchitis virus (IBV) 1a and 1a/1b polyproteins was mediated by two putative overlapping papain-like proteinase domains (PLPDs) encoded within the region from nucleotides 4243 to 5553 of ORF 1a (Liu et al., 1995). In this study, we demonstrate that only the first domain, PLPD-1, is responsible for this cleavage, as deletion of the second domain did not affect the formation of the 87-kDa protein. Site-directed mutagenesis studies further showed that a previously predicted nucleophilic cysteine residue (Cys1274) and a histidine residue (His1437) were essential for the proteinase activity, indicating that they may be important components of the catalytic center of the proteinase. Meanwhile, expression of a series of deletion mutants revealed that the 87-kDa protein was encoded by the 5'-most 2.6 kb of ORF 1a. Deletion and amino acid substitution mutation studies demonstrated that the Gly673-Gly674 dipeptide bond was most likely the cleavage site responsible for releasing the C-terminus of the 87-kDa protein from the 1a and 1a/1b polyproteins.

Coronavirus Papain-Like Proteases↗

PubMed1998

Identification of a 24-kDa polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3C-like proteinase and determination of its cleavage sites.

We report here the identification of a 24-kDa polypeptide in IBV-infected Vero cells by immunoprecipitation with a region-specific antiserum raised in rabbits against the IBV sequence encoded between nucleotides 10,928 and 11,493. Coexpression, deletion, and mutagenesis studies have demonstrated that this protein is encoded by ORF 1a from nucleotide 10,915 to 11,544 and is released from the 1a polyprotein by the 3C-like proteinase-mediated proteolysis. A previously predicted Q-S (Q3462S3463) dipeptide bond encoded by the IBV sequence from nucleotide 10,912 to 10,917 is identified as the N-terminal cleavage site, and a Q-N (Q3672N3673) dipeptide bond encoded by the IBV sequence between nucleotides 11,542 and 11,547 is identified as the C-terminal cleavage site of the 24-kDa polypeptide.

Animals↗

PubMed1998

Interpreting the effect of methyl group at the three carbon bridge of (-)-huperzine A on its anticholinesterase activity by molecular dynamics method.

Based on the recently resolved crystal structure of complex (-)-huperzine A-AChE, we simulated the interaction between (-)-huperzine A analogues and AChE using molecular dynamics method. It was revealed that the methyl group at the three carbon bridge of (-)-huperzine A can form a weak hydrogen bond with the phenol hydroxyl oxygen of Tyr121 and the main-chain oxygen of Gly118 of AChE, respectively.

Alkaloids↗

PubMed1998

Ubiquitin-dependent degradation of cyclin B is accelerated in polyploid megakaryocytes.

During the endomitotic cell cycle of megakaryocytic cell lines, the levels of cyclin B1 and the activity of cyclin B1-dependent Cdc2 kinase, although detectable, are reduced as compared with megakaryocytes undergoing a mitotic cell cycle. The levels of cyclin A, however, are comparable during both cell cycles. The expression of cyclin B1 mRNA is also equivalent in proliferating and polyploidizing cells. In the current study, we found that the rate of cyclin B1 protein degradation is enhanced in polyploidizing megakaryocytes. This finding has led us to further investigate whether the ubiquitin-proteosome pathway responsible for cyclin B degradation is accelerated in these cells. Our data indicate that polyploidizing megakaryocytic cell lines nad primary bone marrow cells treated with the megakaryocyte proliferation- and ploidy-promoting factor, the c-Mpl ligand, display increased activities of the ubiquitin-proteosome pathway, which degrades cyclin B, as compared with proliferating megakaryocytic cell lines or diploid bone marrow cells, respectively. This degradation has all the hallmarks of a ubiquitin pathway, including the dependence on ATP, the appearance of high molecular weight conjugated forms of cyclin B, and inhibition of the proteolytic process by a mutated form of the ubiquitin-conjugating enzyme Ubc4. Our studies also indicate that the ability to degrade cyclin A is equivalent in both the mitotic and endomitotic cell cycles. The increased potential of polyploid megakaryocytes to degrade cyclin B may be part of the cellular programming that leads to aborted mitosis.

Bone Marrow Cells↗

PubMed1998

Proteolytic processing of the polyprotein encoded by ORF1b of the coronavirus infectious bronchitis virus (IBV).

We present here evidence demonstrating that four previously predicted Q-S(G) cleavage sites, encoded by the IBV sequences from nucleotide 15,129 to 15,134, 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, can be recognised and transcleaved by the 3C-like proteinase. Five mature products with sizes of approximately 100 kDa, 65 kDa, 63 kDa, 42 kDa and 35 kDa are released from the ORF1b polyprotein by the 3C-like proteinase-mediated cleavage at these positions. Meanwhile, expression of plasmids containing only the ORF1b region showed no autocleavage of the polyprotein encoded, suggesting that the 3C-like proteinase may be the sole proteinase involved in processing of the 1b polyprotein. These data may therefore represent a complete processing map of the polyprotein encoded by ORF1b of mRNA1.

Antibodies↗

PubMed1998

Further characterisation of the coronavirus IBV ORF 1a products encoded by the 3C-like proteinase domain and the flanking regions.

Coronavirus IBV encodes a piconarvirus 3C-like proteinase. In a previous report, this proteinase was shown to undergo rapid degradation in vitro in reticulocyte lysate due to a posttranslational event involving ubiquitination of the protein. Several lines of evidence presented here indicate that the proteinase itself is stable. Translation of the IBV sequence from nucleotide 8864 to 9787 resulted in the synthesis of a 33 kDa protein, representing the full-length 3C-like proteinase. Pulse-chase and time-course experiments showed that this protein was stable in reticulocyte lysate for up to 2 hours. However, a 45 kDa protein encoded by the IBV sequence from nucleotide 8693 to 9911 underwent rapid degradation in reticulocyte lysate, but was stable in wheat germ extract, suggesting that an ATP-dependent protein degradation pathway may be involved in the turnover of the 45 kDa protein. To identify the IBV sequence responsible for the instability of this 45 kDa protein species, the region from nucleotide 8693 to 9787 was translated both in vitro and in vivo, leading to the synthesis of a stable 43 kDa protein. These results suggest that a destabilising signal may be located in the IBV sequences between the nucleotides 9787 and 9911. Meanwhile, protein aggregation was observed when the product encoded by the IBV sequence from nucleotide 9911 to 10,510 was boiled for 5 minutes before being analysed in SDS-PAGE; when the same product was treated at 37 degrees C for 15 minutes, however, protein aggregation was not detected. Deletion studies indicate that the presence of a hydrophobic domain downstream of the 3C-like proteinase-encoding region may be the cause for the aggregation of the product encoded by this region of ORF 1a.

Animals↗

PubMed1998

Characterisation of a papain-like proteinase domain encoded by ORF1a of the coronavirus IBV and determination of the C-terminal cleavage site of an 87 kDa protein.

Our previous studies have shown that two overlapping papain-like proteinase domains (PLPDs) encoded by the IBV sequence from nucleotides 4155 to 5550 is responsible for cleavage of the ORF 1a polyprotein to an 87 kDa protein. In this study, we demonstrate that only the more 5' one of the two domains, PLPD-1 encoded between nucleotides 4155 and 5031, is required for processing to the 87 kDa protein. Site-directed mutagenesis studies have shown that the Cys1274 and His1435 residues are essential for the PLPD-1 activity, suggesting that they may be the components of the catalytic centre of this proteinase. Coexpression and immunoprecipitation studies have further revealed that PLPD can interact with the 87 kDa protein. Meanwhile, data obtained from the construction and expression of a series of deletion mutants have indicated that the 87 kDa protein is encoded by the 5'-most 2600 bp part of ORF1a. further deletion and mutagenesis studies are underway to determine precisely the C-terminal cleavage site of the 87 kDa protein.

Animals↗

PubMed1998

Regulation of mRNA 1 expression by the 5'-untranslated region (5'-UTR) of the coronavirus infectious bronchitis virus (IBV).

In this report, we show that expression of the coronavirus IBV mRNA1 is regulated by its 5'-UTR. Evidence presented demonstrates that the IBV sequence from nucleotide 1 to 1904 directs very inefficient synthesis of a product of approximately 43 kDa. Deletion of either the first 362 bp or the whole part of the 5'-UTR, however, dramatically increased the expression of the 43 kDa protein species. The mechanisms involved were investigated by two different approaches. Firstly, translation of the same construct in the presence of [3H]-leucine ruled out the possibility that initiation of small reading frames from non-AUG codons located in the 5'-UTR may compete with the authentic AUG initiation codon, and therefore inhibit the expression of ORF 1a. Secondly, expression and deletion analyses of a dicistronic construct showed that translation of the 43 kDa protein was initiated by ribosome internal entry mechanism. These studies suggest that a 'weak' ribosome internal entry signal is located in the 5'-UTR and is involved in the regulation of mRNA1 expression.

5' Untranslated Regions↗

PubMed1998

Establishment of kappa opioid receptor agonists pharmacophore with molecular modeling method.

AIM: To build up nonpeptide kappa-opioid receptor agonists pharmacophore. METHODS: Five structurally diverse, highly active nonpeptide kappa-opioid agonists were retrieved from MDL MDDR database. Molecular mechanics method were used to seek out 50 lower energy conformations for each compound. Taking nitrogen atom of pyrrolidine and acyl acetamide as overlay points, 5 agonists were superimposed to each other with their most favorite conformation. From the overlay map, the structure specificity of nonpeptide kappa-opioid receptor agonists were elucidated. RESULTS: According to this pharmacophore, the pyrrolidine ring, the carbonyl group of acyl acetamide, and the hydrophobic group attached to acyl acetamide were suggested to be the structure-specific moieties of kappa-opioid agonists. Moreover, by comparing kappa 1-opioid receptor sequence of mouse with other G-protein-coupled receptors, we determined those conserve residues existing on transmembrane regions which might interact with the suggested groups. The carboxyl of Asp138 might interact with N atom of pyrrolidine by forming a hydrogen bond. The hydroxyl of Ser187 and the carbonyl group of kappa-opioid agonists might form another hydrogen bond, which was critical for its kappa selective affinity. The hydrophobic group attached to acyl acetamide might have hydrophobic interaction with aromatic residues of kappa-opioid receptors. CONCLUSION: These kappa agonists pharmacophore were helpful to select specific positions in the lead compounds to be occupied by hydrophobic moieties to limit their ability to across the blood-brain barrier.

Amino Acid Sequence↗

PubMed1998

[Tweed-Merrifield analysis for Chinese Shangdong adults]

OBJECTIVE:This study was to establish cephalometric normals for Shangdong Chinese based on the Tweed-Merrifield analysis. MEHTODS: Lateral cephalograms of 80 young adults(40 female and 40 male) with normal occlusions were selected.The ages was 18-24. RESULTS: To establish cephalometric normals for Shangdong Chinese based on the Tweed-Merrifield analysis,and discover FMA female>male,AFH,PFH,IND(P/A) female>male. CONCLUSION: These normals will be used to orthodontic diagnosis and treatment planning for Shangdong adult patients.

Journal Article↗

PubMed1997

Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites.

Proteolytic processing of the polyprotein encoded by mRNA 1 is an essential step in coronavirus RNA replication and gene expression. We have previously reported that an open reading frame (ORF) 1a-specific proteinase of the picornavirus 3C proteinase group is involved in processing of the coronavirus infectious bronchitis virus (IBV) 1a/1b polyprotein, leading to the formation of a mature viral protein of 100 kDa. We report here the identification of a novel 10-kDa polypeptide and the involvement of the 3C-like proteinase in processing of the ORF 1a polyprotein to produce the 10-kDa protein species. By using a region-specific antiserum, V47, raised against a bacterial-viral fusion protein containing IBV sequence encoded between nucleotides 11488 and 12600, the 10-kDa polypeptide was detected in lysates from both IBV-infected and plasmid DNA-transfected Vero cells. Coexpression, deletion, and mutagenesis studies showed that this novel polypeptide was encoded by ORF 1a from nucleotide 11545 to 11878 and was cleaved from the 1a polyprotein by the 3C-like proteinase domain. Evidence presented suggested that a previously predicted Q-S (Q3783 S3784) dipeptide bond encoded by ORF 1a between nucleotides 11875 and 11880 was responsible for the release of the C terminus of the 10-kDa polypeptide and that a novel Q-N (Q3672 N3673) dipeptide bond encoded between nucleotides 11542 and 11547 was responsible for the release of the N terminus of the 10-kDa polypeptide.

Animals↗

PubMed1997

[Antigen strategy: a new way for gene therapy].

Synthetic purine-rich or pyridine-rich oligonucleotides have been shown to form stable triplex by binding to specific homopurine-homopyridine sequences in double-stranded DNA. Several studies have suggested that triplex formation in the transcription-regulated regions of genes could inhibit their transcription in vitro or in vivo. This new technology was called antigene strategy, which would be of great value in the treatment of cancer and virus infection. However, the low stability of triplex and restricting target selection of homopurine-homopyridine tracts in selected genes greatly reduced its utility in antigen strategies. Further work is still needed.

DNA↗

PubMed1996

Definition of a human herpesvirus-6 betaherpesvirus-specific domain in glycoprotein gH that governs interaction with glycoprotein gL: substitution of human cytomegalovirus glycoproteins permits group-specific complex formation.

Formation of the glycoprotein gH/gL heterooligomer has important implications for understanding the pathology of human herpesvirus-6(HHV-6)-associated disease because this complex is essential for infectivity and fusogenic cell-to-cell spread. Definition of the HHV-6 gH domain involved in protein-protein interactions was addressed by targeting regions defined by conserved cysteines identified by alignment of gH amino acid sequences representative of all herpesvirus subfamilies. Studies using site-directed mutagenesis and transient cellular expression showed that the N-terminus of HHV-6 gH includes a 230-amino-acid domain required for interaction with HHV-6 gL encompassing residues conserved specifically amongst betaherpesviruses. Interestingly, the human cytomegalovirus (HCMV) homologues, UL75 (gH) or UL115 (gL), can substitute for HHV-6 glycoproteins and participate in heterologous complex formation. Furthermore, the region which governs this heterologous gL binding also maps to the N-terminal portion of HHV-6 gH. Although both proteins can functionally substitute for complex formation there are also specific differences. Surprisingly, further deletion of HHV-6 gH to 145-amino-acid-domain residues abolishes complex formation with HHV-6 gL but allows interaction with HCMV gL. This may be related to requirements in HHV-6 for homodimer formation before complex formation between gH and gL. Under nonreducing conditions HHV-6 gH and gL form multimeric complexes consistent with intra- and intermolecular dimer formation stabilised by disulphide bonds whereas for HCMV there is no evidence for dimer formation for gH and multimeric complexes have only been observed between gH and gL. In summary, both HHV-6 and HCMV glycoproteins can interact and the heterologous complex between HHV-6 gH and HCMV gL is possibly more stable. This may result in important biological consequences in vivo during cellular coinfections by facilitating spread of the viruses, with applications to altered cellular tropisms and effects on reactivation from the latently infected cell.

Base Sequence↗

PubMed1995

Characterisation and mutational analysis of an ORF 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein.

Coronavirus gene expression involves proteolytic processing of the mRNA 1-encoded polyproteins by viral and cellular proteinases. Recently, we have demonstrated that an ORF 1b-encoded 100-kDa protein is proteolytically cleaved from the 1a/1b fusion polyprotein by a viral-specific proteinase of the picornavirus 3C proteinase group (3C-like proteinase). In this report, the 3C-like proteinase has been further analysed by internal deletion of a 2.3-kb fragment between the 3C-like proteinase-encoding region and ORF 1b and by substitution mutations of its catalytic centre as well as the two predicted cleavage sites flanking the 100-kDa protein. The results show that internal deletion of ORF 1a sequences from nucleotide 9911 to 12227 does not influence the catalytic activity of the proteinase in processing of the 1a/1b polyprotein to the 100-kDa protein species. Site-directed mutagenesis studies have confirmed that the predicted nucleophilic cysteine residue (Cys2922) and a histidine residue encoded by ORF 1a from nucleotide 8985 to 8987 (His2820) are essential for the catalytic activity of the proteinase, and that the QS(G) dipeptide bonds are its target cleavage sites. Substitution mutations of the third component of the putative catalytic triad, the glutamic acid 2843 (Glu2843) residue, however, do not affect the processing to the 100-kDa protein. In addition, cotransfection experiment shows that the 3C-like proteinase is capable of trans-cleavage of the 1a/1b polyprotein. These studies have confirmed the involvement of the 3C-like proteinase domain in processing of the 1a/1b polyprotein, the predicted catalytic centre of the proteinase, and its cleavage sites.

Amino Acid Sequence↗