Biomedical subjects

Xing Zhang

Publications and source records attributed to Xing Zhang.

At least 73 records · Page 4Linked to original sources

PubMed2003

Structure of the bacteriophage T4 DNA adenine methyltransferase.

DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a beta-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.

Amino Acid Sequence↗

PubMed2003

Structure of the predominant protein arginine methyltransferase PRMT1 and analysis of its binding to substrate peptides.

PRMT1 is the predominant type I protein arginine methyltransferase in mammals and highly conserved among all eukaryotes. It is essential for early postimplantation development in mouse. Here we describe the crystal structure of rat PRMT1 in complex with the reaction product AdoHcy and a 19 residue substrate peptide containing three arginines. The results reveal a two-domain structure-an AdoMet binding domain and a barrel-like domain-with the active site pocket located between the two domains. Mutagenesis studies confirmed that two active site glutamates are essential for enzymatic activity, and that dimerization of PRMT1 is essential for AdoMet binding. Three peptide binding channels are identified: two are between the two domains, and the third is on the surface perpendicular to the strands forming the beta barrel.

Amino Acid Sequence↗

PubMed2003

Structural basis for the product specificity of histone lysine methyltransferases.

DIM-5 is a SUV39-type histone H3 Lys9 methyltransferase that is essential for DNA methylation in N. crassa. We report the structure of a ternary complex including DIM-5, S-adenosyl-L-homocysteine, and a substrate H3 peptide. The histone tail inserts as a parallel strand between two DIM-5 strands, completing a hybrid sheet. Three post-SET cysteines coordinate a zinc atom together with Cys242 from the SET signature motif (NHXCXPN) near the active site. Consequently, a narrow channel is formed to accommodate the target Lys9 side chain. The sulfur atom of S-adenosyl-L-homocysteine, where the transferable methyl group is to be attached in S-adenosyl-L-methionine, lies at the opposite end of the channel, approximately 4 A away from the target Lys9 nitrogen. Structural comparison of the active sites of DIM-5, an H3 Lys9 trimethyltransferase, and SET7/9, an H3 Lys4 monomethyltransferase, allowed us to design substitutions in both enzymes that profoundly alter their product specificities without affecting their catalytic activities.

Catalytic Domain↗

PubMed2003

Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa.

Besides serving to package nuclear DNA, histones carry information in the form of a diverse array of post-translational modifications. Methylation of histones H3 and H4 has been implicated in long-term epigenetic 'memory'. Dimethylation or trimethylation of Lys4 of histone H3 (H3 Lys4) has been found in expressible euchromatin of yeasts and mammals. In contrast, methylation of Lys9 of histone H3 (H3 Lys9) has been implicated in establishing and maintaining the largely quiescent heterochromatin of mammals, yeasts, Drosophila melanogaster and plants. We have previously shown that a DNA methylation mutant of Neurospora crassa, dim-5 (defective in methylation), has a nonsense mutation in the SET domain of an H3-specific histone methyltransferase and that substitutions of H3 Lys9 cause gross hypomethylation of DNA. Similarly, the KRYPTONITE histone methyltransferase is required for full DNA methylation in Arabidopsis thaliana. We used biochemical, genetic and immunological methods to investigate the specific mark for DNA methylation in N. crassa. Here we show that trimethylated H3 Lys9, but not dimethylated H3 Lys9, marks chromatin regions for cytosine methylation and that DIM-5 specifically creates this mark.

DNA Methylation↗

PubMed2003

[Reproductive toxicity of metadoxine in rats].

OBJECTIVE: To study the reproductive toxicity of metadoxine. METHODS: Male and female rats were given metadoxine before pregnancy and early gestation, i.e. to feed metadoxine to male rats for 60 days before copulation and continue feeding during copulation, and feed metadoxine to female rats for 14 days before copulation. RESULTS: No significant toxic effect was observed in the 400 mg/kg group. A few rats showed paralysis of hind leg in the 800 mg/kg group. The dosage of 1 600 mg/kg caused significant paralysis of hind legs, emaciation, and reduced weight gain. In the 1600 mg/kg group, the mating rate of male rats was significantly affected (P < 0.01). In the 800 and 1 600 mg/kg group, fertility of male rats was markedly reduced (P < 0.01). In the 800 mg/kg group, the effect on sperm counts of epididymis of male rats was markedly reduced (P < 0.05). In the 1 600 mg/kg group, testicle weight and body weight ratio and sperm counts of epididymis rate were significantly (P < 0.001) reduced. In the 1 600 mg/kg group, the fertility rate of female rats was remarkably (P < 0.001) reduced. In the 800 mg/kg group, the weight gain of pregnant rats was significantly reduced (P < 0.001). In both the 800 and 1 600 mg/kg groups, the gestation rate was greatly reduced (P < 0.001). In the 800 mg/kg group, mortality rate before nidation (P < 0.001) and average live fetus number were significantly reduced (P < 0.05). In the 400 mg/kg group, the fetal weight was significantly reduced (P < 0.001). In the 800 mg/kg group, body length, tail length, body weight and sternum development of fetal rats were significantly affected (P < 0.001). CONCLUSION: Under the presented experimental conditions, metadoxine has no teratogenic effects on SD rats and the no effect dose is 400 mg/kg. And the no effect dose for the developmental toxicity is less than 400 mg/kg.

Animals↗

PubMed2003

[Clinical significance of CEA mRNA detection in pleural and peritoneal effusions by real-time fluorescent quantitative RT-PCR].

OBJECTIVES: To detect CEA mRNA levels in benign and malignant pleural and peritoneal effusions and evaluate their clinical significance. METHODS: Samples of pleural and peritoneal effusions from 58 patients with malignant diseases and 76 patients with benign diseases were collected and total RNAs were prepared and subjected to real-time fluorescent quantitative RT-PCR to determine the CEA mRNA levels in these samples. The positive rate of this examination was compared with that of shed cell pathological examination. RESULTS: Nineteen samples (32.8%) of pleural and peritoneal effusions from the 58 patients with malignant diseases showed positive results in shed cell examination, while the number of CEA mRNA >1 CN was 46 (79.3%) (chi(2) = 21.81, P = 0.000). Nineteen samples of pleural and peritoneal effusions from the 76 patients with benign diseases showed CEA mRNA > 1 CN (25.0%), which was significantly different from that of the patients with malignant diseases (chi(2) = 38.85, P = 0.000). CONCLUSION: CEA mRNA levels in pleural and peritoneal effusions can be quantified by real-time fluorescent quantitative PCR, which is more sensitive than shed cell pathological examination. This technique is helpful in discrimination of benign and malignant pleural and peritoneal effusions.

Ascitic Fluid↗

PubMed2003

[Study on the pulmonary fibrogenetic effect induced by rush-mat dust in rats].

OBJECTIVE: To investigate the fibrogenetic effects induced by rush-mat dust in rats. METHODS: SD rats were treated with 50 mg of rush-mat dust per rat by intra-tracheal instillation, sacrificed 3, 6, and 12 months respectively after exposure. The lung tissue and lung lymph-node were taken out for pathological and electron microscopic examination. The content of collagen and ceruloplasmin (CP) in lung tissues were also determined. RESULTS: After treatment for 12 months, fresh wet lung weight in rush-mat dust group [(2.69 +/- 0.22) g] was higher than those in saline group [(1.87 +/- 0.25) g], TiO(2) group [(2.25 +/- 0.26) g], but lower than that in SiO(2) group [(11.41 +/- 1.63) g]; dry lung weight in rush-mat dust group [(0.47 +/- 0.03) g] was higher than those in saline group [(0.32 +/- 0.03) g], TiO(2) group [(0.41 +/- 0.08) g], but lower than that in SiO(2) group [(2.06 +/- 0.28) g]; lung collagen content in rush-mat dust group [(103.08 +/- 14.79) mg] was higher than those in saline group [(75.96 +/- 13.91) mg, TiO(2) group [(85.84 +/- 17.62) mg], but lower than that in SiO(2) group [(497.50 +/- 100.80) mg]; CP content in rush-mat dust group [(18.03 +/- 1.87) U/L] was higher than those in saline group [(15.05 +/- 2.24) U/L], TiO(2) group [(16.92 +/- 1.67) U/L], but lower than that in SiO(2) group [(25.37 +/- 3.58) U/L], P < 0.05 or P < 0.01. Pathological examination showed lung macrophage alveolitis, broadening of alveolar interval, one to two grade of silicotic nodes and increased amount of type II epithelial cell in alveolar as well as slight collagenous fibrosis in lung tissue of rush-mat dust group. Under electron microscope, primary and secondary lysosome and medullary sheath-like phagocytic residual body were found in lung tissue of rush-mat dust group, meanwhile the amount of type II alveolar epithelial cell and collagen fiber were slightly increased but these changes were less than those of quartz group. CONCLUSION: The rush-mat dusts have slight pulmonary fibrogenetic effect on rat.

Animals↗

PubMed2003

[A 40-year cohort study on cancer mortality among female workers with manual spinning of chrysotile asbestos].

The mortality of malignant tumors among female workers with manual spinning of chrysotile alone was investigated by a retrospective cohort study and was calculated based on the person-year at risk of the observed group by the life-table method. The standardized mortality ratio (SMR), the relative risk (RR), attributive risk (AR) and their corresponding 95% confidence intervals (CIs) were calculated based on the mean age-specific mortality rate of local female population. 5,681 female workers with manual spinning of chrysotile for at least one year between 1960 and 1980 were traced to the end of the year of 2000. A total number of 858 death cases by all causes were found. The results showed that the percentage of specific causes of death, cancer (24.83%) was the first cause of death, and the lung cancer (40.85%) was the most prevalent. SMRs for all cancers (1.35) and for lung cancer (3.88) were significantly elevated than those of the control group. The results indicated that the excess of lung cancer among female workers with manual spinning of chrysotile did not likely to be dependent on the amphibole hypothesis, the hypothesis of mineral oils exposure and cigarette smoking, but was likely related to manual spinning of asbestos operation produced airborne fibers that were generally longer than 10 microns in length than other operations using chrysotile.

Adult↗

PubMed2002

Mismatch repair in methylated DNA. Structure and activity of the mismatch-specific thymine glycosylase domain of methyl-CpG-binding protein MBD4.

MBD4 is a member of the methyl-CpG-binding protein family. It contains two DNA binding domains, an amino-proximal methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific glycosylase domain. Limited in vitro proteolysis of mouse MBD4 yields two stable fragments: a 139-residue fragment including the MBD, and the other 155-residue fragment including the glycosylase domain. Here we show that the latter fragment is active as a glycosylase on a DNA duplex containing a G:T mismatch within a CpG sequence context. The crystal structure confirmed the C-terminal domain is a member of the helix-hairpin-helix DNA glycosylase superfamily. The MBD4 active site is situated in a cleft that likely orients and binds DNA. Modeling studies suggest the mismatched target nucleotide will be flipped out into the active site where candidate residues for catalysis and substrate specificity are present.

Amino Acid Sequence↗

PubMed2002

Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase.

AdoMet-dependent methylation of histones is part of the "histone code" that can profoundly influence gene expression. We describe the crystal structure of Neurospora DIM-5, a histone H3 lysine 9 methyltranferase (HKMT), determined at 1.98 A resolution, as well as results of biochemical characterization and site-directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for methyl transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the HKMT family and the SET domain.

Amino Acid Sequence↗

PubMed2002

Determinants of the interaction of the spinal muscular atrophy disease protein SMN with the dimethylarginine-modified box H/ACA small nucleolar ribonucleoprotein GAR1.

Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the common, fatal neuromuscular disease spinal muscular atrophy. The SMN protein is important in small nuclear ribonucleoprotein (snRNP) assembly and interacts with snRNP proteins via arginine/glycine-rich domains. Recently, SMN was also found to interact with core protein components of the two major families of small nucleolar RNPs, fibrillarin and GAR1, suggesting that SMN may also function in the assembly of small nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudor domain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN, but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.

Amino Acid Substitution↗

PubMed2002

Loss of activities for mRNA synthesis accompanies loss of lambda2 spikes from reovirus cores: an effect of lambda2 on lambda1 shell structure.

The 144-kDa lambda2 protein, a component of the transcriptionally active reovirus core particle, catalyzes the last three enzymatic activities for formation of the 5' cap 1 structure on the viral plus-strand transcripts. Limited evidence suggests it may also play a role in transcription per se. Particle-associated lambda2 forms pentameric turrets ("spikes") around the fivefold axes of the icosahedral core. To address the requirements for lambda2 in core functions other than the known functions in RNA capping, particles depleted of lambda2 were generated from cores in vitro by a series of treatments involving heat, protease, and ionic detergent. The resulting particles contained less than 5% of pretreatment levels of lambda2 but showed negligible loss of the other four core proteins or the 10 double-stranded RNA genome segments. Transmission cryo-electron microscopy (cryo-TEM) and scanning cryo-electron microscopy demonstrated loss of the lambda2 spikes from these otherwise intact particles. In functional analyses, the "spikeless cores" showed greatly reduced activities not only for RNA capping but also for transcription and nucleoside triphosphate hydrolysis, suggesting enzymatic or structural roles for lambda2 in all these activities. Comparison of the core and spikeless core structures obtained by cryo-TEM and three-dimensional image reconstruction revealed changes in the lambda1 core shell that accompany lambda2 loss, most notably the elimination of small pores that span the shell near the icosahedral fivefold axes. Changes in the shell may explain the reductions in transcriptase-related activities by spikeless cores.

Cryoelectron Microscopy↗

PubMed2002

Nab2p is required for poly(A) RNA export in Saccharomyces cerevisiae and is regulated by arginine methylation via Hmt1p.

From transcription to translation, mRNA is complexed with heterogeneous nuclear ribonucleoproteins (hnRNP proteins) that mediate mRNA processing, export from the nucleus, and delivery into the cytoplasm. Although the mechanism is unknown, export of mature mRNA from the nucleus is a critical regulatory step in gene expression. Analyses of hnRNP proteins have shown that many of these proteins are required for this essential cellular process. In this study, we characterize the Saccharomyces cerevisiae Nab2 protein, which was first identified as a poly(A) RNA-binding protein (Anderson, J. T., Wilson, S. M., Datar, K. V., and Swanson, M. S. (1993) Mol. Cell. Biol. 13, 2730-2741). Our work indicates that poly(A) RNA export from the nucleus is dependent upon a functional Nab2 protein; correspondingly, export of Nab2p from the nucleus is dependent upon ongoing RNA polymerase II transcription. Furthermore, we show that Nab2p is modified within its RGG domain by the type I protein-arginine methyltransferase, Hmt1p. Our experiments demonstrate that arginine methylation is required for the export of Nab2p from the nucleus and therefore establish an in vivo effect of this modification. Overall, these experiments provide evidence that Nab2p is an hnRNP protein that is required for poly(A) RNA export and whose export from the nucleus is regulated by Hmt1p.

ATP-Binding Cassette Transporters↗

PubMed2002

The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds.

The PWWP domain is a weakly conserved sequence motif found in > 60 eukaryotic proteins, including the mammalian DNA methyltransferases Dnmt3a and Dnmt3b. These proteins often contain other chromatin-association domains. A 135-residue PWWP domain from mouse Dnmt3b (amino acids 223--357) has been structurally characterized at 1.8 A resolution. The N-terminal half of this domain resembles a barrel-like five-stranded structure, whereas the C-terminal half contains a five-helix bundle. The two halves are packed against each other to form a single structural module that exhibits a prominent positive electrostatic potential. The PWWP domain alone binds DNA in vitro, probably through its basic surface. We also show that recombinant Dnmt3b2 protein (a splice variant of Dnmt3b) and two N-terminal deletion mutants (Delta218 and Delta369) have approximately equal methyl transfer activity on unmethylated and hemimethylated CpG-containing oligonucleotides. The Delta218 protein, which includes the PWWP domain, binds DNA more strongly than Delta369, which lacks the PWWP domain.

Amino Acid Motifs↗

PubMed2002

Structure of the SET domain histone lysine methyltransferase Clr4.

Methylation of histone H3 lysine 9 is an important component of the 'histone code' for heterochromatic gene silencing. The SET domain-containing Clr4 protein, a close relative of Su(var)3-9 proteins in higher eukaryotes, specifically methylates lysine 9 of histone H3 and is essential for silencing in Schizosaccharomyces pombe. Here we report the 2.3 A resolution crystal structure of the catalytic domain of Clr4. The structure reveals an overall fold rich in beta-strands, a potential active site consisting of a SAM-binding pocket, and a connected groove that could accommodate the binding of the N-terminal tail of histone H3. The pre-SET motif contains a triangular zinc cluster coordinated by nine cysteines distant from the active site, whereas the post-SET region is largely flexible but proximal to the active site. The structure provides insights into the architecture of SET domain histone methyltransferases and establishes a paradigm for further characterization of the Clr4 family of epigenetic regulators.

Amino Acid Sequence↗

PubMed2002

A case-control study on risk of changing food consumption for colorectal cancer.

OBJECTIVES: To investigate the relative risk factor of food items for colorectal cancer in four time periods through a case-control study in a Chinese rural area. METHODS: Colorectal cancer patients diagnosed at a county cancer center, Hebei Province, China, and non-cancer outpatients with similar age, sex, and place of residence were selected for cases and controls, respectively. There were 102 (93.6%) colorectal cancer patients and 99 (90.8%) outpatients being the cases and controls, respectively in the present investigation, who agreed to be interviewed about their food intake, during a 20-year period, through a food frequency questionnaire. The risks of intake of different food items and lifestyle for colorectal cancer were compared between cases and controls. RESULTS: During the 20-year period, diets of both cases and controls changed with increase in intake of animal foods and fruits, and alcohol consumption tended to increase. In the food items, milk intake showed a protective effect in both males and females, and the odds ratios were 0.38 (95% CI 0.16-0.90) and 0.28 (95% CI 0.10-0.81) for males and females, respectively. A reduced risk of fruit intake could be seen in males, while a reduced risk of vegetables could be observed in females. Meat intake and saturated fats were the prominent risk factors for colorectal cancer in males and females, respectively. A comparison of life habits, showed that tea drinking had a consistent protective effect in females, and the odds ratios were 0.21 (0.08-0.58), 0.23 (0.08-0.67), 0.25 (0.10-0.64), and 0.11 (0.04-0.30) for periods of 20-, 10-, 5-years ago, and current time, respectively. CONCLUSIONS: These findings indicate that change in food consumption is strongly associated with a change in risk of colorectal cancer, and dietary meat has increased the risk of colorectal cancer. Increase in the consumption of milk and fruits may be a significant measure for colorectal cancer prevention in low-incidence areas.

Adult↗

PubMed2002

The hydrophilic amino-terminal arm of reovirus core shell protein lambda1 is dispensable for particle assembly.

The reovirus core particle is a molecular machine that mediates synthesis, capping, and export of the viral plus strand RNA transcripts. Its assembly and structure-function relationships remain to be well understood. Following the lead of previous studies with other Reoviridae family members, most notably orbiviruses and rotaviruses, we used recombinant baculoviruses to coexpress reovirus core proteins lambda1, lambda2, and sigma2 in insect cells. The resulting core-like particles (CLPs) were purified and characterized. They were found to be similar to cores with regard to their sizes, morphologies, and protein compositions. Like cores, they could also be coated in vitro with the two major outer-capsid proteins, micro 1 and sigma3, to produce virion-like particles. Coexpression of core shell protein lambda1 and core nodule protein sigma2 was sufficient to yield CLPs that could withstand purification, whereas expression of lambda1 alone was not, indicating a required role for sigma2 as a previous study also suggested. In addition, CLPs that lacked lambda2 (formed from lambda1 and sigma2 only) could not be coated with micro 1 and sigma3, indicating a required role for lambda2 in the assembly of these outer-capsid proteins into particles. To extend the use of this system for understanding the core and its assembly, we addressed the hypothesis that the hydrophilic amino-terminal region of lambda1, which adopts an extended arm-like conformation around each threefold axis in the reovirus core crystal structure, plays an important role in assembling the core shell. Using a series of lambda1 deletion mutants, we showed that the amino-terminal 230 residues of lambda1, including its zinc finger, are dispensable for CLP assembly. Residues in the 231-to-259 region of lambda1, however, were required. The core crystal structure suggests that residues in the 231-to-259 region are necessary because they affect the interaction of lambda1 with the threefold and/or fivefold copies of sigma2. An effective system for studies of reovirus core structure, assembly, and functions is hereby established.

Capsid↗

PubMed2002

An antibody to the putative aphid recognition site on cucumber mosaic virus recognizes pentons but not hexons.

Cucumber mosaic virus (CMV), the type member of the genus Cucumovirus (family Bromoviridae), is transmitted by aphids in a nonpersistent manner. Mutagenesis experiments identified the betaH-betaI loop of the capsid subunit as a potential key motif responsible for interactions with the insect vector. To further examine the functional characteristics of this motif, we generated monoclonal antibodies that bound to native virions but not to betaH-betaI mutants. Fab fragments from these antibodies were complexed with wild-type CMV and the virus-Fab structure was determined to 12-A resolution by using electron cryomicroscopy and image reconstruction techniques. The electron density attributed to the bound antibody has a turret-like appearance and protrudes from each of the 12 fivefold axes of the icosahedral virus. Thus, the antibody binds only to the pentameric clusters (pentons) of A subunits of the T=3 quasisymmetric virus and does not appear to bind to any of the B and C subunits that occur as hexameric clusters (hexons) at the threefold (quasi-sixfold) axes. Modeling and electron density comparisons were used to analyze the paratope-epitope interface and demonstrated that the antibody binds to three betaH-betaI loops in three adjacent A subunits in each penton. This antibody can discriminate between A and B/C subunits even though the betaH-betaI loop adopts the same structure in all 180 capsid subunits and is therefore recognizing differences in subunit arrangements. Antibodies with such character have potential use as probes of viral assembly. Our results may provide an additional rationale for designing synthetic vaccines by using symmetrical viral particles.

Animals↗