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Z Deng

Publications and source records attributed to Z Deng.

At least 19 recordsLinked to original sources

Identification and characterization of a zinc finger gene (ZNF213) from 16p13.3.

During our search for the familial Mediterranean fever (FMF) gene, we identified by cDNA selection a 1.2 kb cDNA fragment representing a novel human gene that is expressed in a wide variety of tissues. This gene spans approx. 8.0 kb genomic DNA and has seven exons. Its 3' untranslated region contains a long tandem repeat that gives rise to a polymorphism with two alleles of approx. 1.1 kb and 1.0 kb, with the 1.1 kb allele in strong linkage disequilibrium with FMF in patients of different ethnic backgrounds. However, both genetic and mutational analyses have excluded this gene as the one responsible for FMF. The predicted 424 amino acid protein, designated ZNF213, contains three C2H2 zinc fingers, a Kruppel associated A box and a leucine rich motif (LeR domain/SCAN box), strongly suggestive of a transcription factor.

Amino Acid Sequence

A productive NADP+ binding mode of ferredoxin-NADP + reductase revealed by protein engineering and crystallographic studies.

The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin-nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of approximately 30 degrees, with the C4 atom 3 A from the flavin N5 atom.

Amino Acid Sequence

Probing the function of the invariant glutamyl residue 312 in spinach ferredoxin-NADP+ reductase.

Ferredoxin-NADP+ reductase, the prototype of a large family of structurally related flavoenzymes, pairs single electrons carried by ferredoxin I and transfers them as a hydride to NADP+. Four mutants of the enzyme, in which Glu-312 was replaced with Asp, Gln, Leu, and Ala to probe the role of the residue charge, size, and polarity in the enzyme activity, have been heterologously expressed, purified, and characterized through steady-state, rapid kinetic studies, ligand-binding experiments, and three-dimensional structure determination by x-ray crystallography. The E312L mutant was the only one that was almost inactive (approximately 1%), whereas unexpectedly the E312A reductase was 10-100% active with the various acceptors tested. Rapid kinetic absorption spectroscopy studies demonstrated that flavin reduction by NADPH was impaired in the mutants. Furthermore, NADP(H) binding was partially perturbed. These functional and structural studies lead us to conclude that Glu-312 does not fulfil the role of proton donor during catalysis, but it is required for proper binding of the nicotinamide ring of NADP(H). In addition, its charge modulates the two one-electron redox potentials of the flavin to stabilize the semiquinone form.

Amino Acid Sequence

On the active site of Old Yellow Enzyme. Role of histidine 191 and asparagine 194.

Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500-800 nm) charge-transfer complexes. The enzyme is reduced by NADPH, and oxygen, quinones, and alpha,beta-unsaturated aldehydes and ketones can act as electron acceptors to complete catalytic turnover. Solution of the crystal structure of OYE1 from brewer's bottom yeast (Fox, K. M., and Karplus, P. A. (1994) Structure 2, 1089-1105) made it possible to identify histidine 191 and asparagine 194 as amino acid residues that hydrogen-bond with the phenolic ligands, stabilizing the anionic form involved in charge-transfer interaction with the FMN prosthetic group. His-191 and Asn-194 are also predicted to interact with the nicotinamide ring of NADPH in the active site. Mutations of His-191 to Asn, Asn-194 to His, and a double mutation, H191N/N194H, were made of OYE1. It was not possible to isolate the N191H mutant enzyme, but the other two mutant forms had the expected effect on phenolic ligand binding, i.e. decreased binding affinity and decreased charge-transfer absorbance. Reduction of the H191N mutant enzyme by NADPH was similar to that of OYE1, but the reduction rate constant for NADH was greatly decreased. The double mutant enzyme had an increased rate constant for reduction by NADPH, but the reduction rate constant with NADH was lower by a factor of 15. The reactivity of OYE1 and the mutant enzymes with oxygen was similar, but the reactivity of 2-cyclohexenone was greatly decreased by the mutations. The crystal structures of the two mutant forms showed only minor changes from that of the wild type enzyme.

Amino Acid Sequence

Identification of two Krüppel-related zinc finger genes (ZNF200 and ZNF210) from human chromosome 16p13.3.

During the course of cloning the gene for familial Mediterranean fever (FMF), we identified a number of transcripts from a 275-kb genomic region on 16p13.3. Two of the transcripts were found to contain multiple C2H2-type zinc finger motifs in tandem arrays, indicating that they are members of the Krüppel-type family. One transcript was found to be an alternatively spliced form of a previously reported zinc finger gene, ZNF200. The other transcript, ZNF210, is 2017 bp and encodes an open reading frame of 504 aa. Northern blot analysis indicates that ZNF210 is expressed in all the tissues tested with the highest expression in heart, skeletal muscle, pancreas, prostate, ovary, and colon. On the other hand, the strongest expression of ZNF200 is in testis, with very low levels in all the other tissues tested. Sequence analysis reveals eight C2H2 zinc finger motifs at the C-terminus of ZNF210 and five in ZNF200. In addition, ZNF210 also possesses a Krüppel-associated box at its N-terminus, indicating that it might function as a transcription repressor. The intron-exon structures of both genes were determined and showed that ZNF210 has seven exons while the coding part of ZNF200 is distributed in four exons. The locations of ZNF200 and ZNF210 are 10 and 120 kb telomeric to the FMF gene, respectively.

Amino Acid Sequence

Diagnosis of familial Mediterranean fever by a molecular genetics method.

BACKGROUND: Familial Mediterranean fever is a recessively inherited disorder characterized by episodes of fever with abdominal pain, pleurisy, or arthritis. The familial Mediterranean fever gene, designated MEFV, was recently cloned, and at least three missense mutations (M6801, M694V, and V726A) that account for a large percentage of patients with this disease were identified. OBJECTIVE: To establish a diagnostic test for familial Mediterranean fever. DESIGN: Cross-sectional study of a convenience sample of patients attending familial Mediterranean fever clinics. SETTING: Tertiary referral hospitals. PATIENTS: 107 patients with familial Mediterranean fever, their family members, and controls. MEASUREMENTS: Mutations in the 107 samples were assessed by amplifying genomic DNA with use of primers that selectively amplify the normal or altered DNA sequence of the 3 MEFV mutations (amplification refractory mutation system [ARMS]). Mutations were independently assessed by automated sequencing of genomic DNA amplified by polymerase chain reaction to evaluate the sensitivity and specificity of the ARMS assay. RESULTS: The ARMS assay correctly identified M6801, M694V, and V726A mutations in 82 persons with mutations documented by DNA sequencing (21 homozygotes, 2 compound heterozygotes, and 59 simple heterozygotes). Of 7 persons known from family studies to be noncarriers and 18 unrelated persons who were negative for these mutations by sequencing, none had MEFV mutations according to ARMS. CONCLUSION: The ARMS assay is a rapid, cost-effective, and accurate method for detecting three common mutations in familial Mediterranean fever.

Cross-Sectional Studies

An essential methionine residue involved in substrate binding by phosphofructokinases.

An alignment of all PPi-dependent phosphofructokinases and all allosteric ATP-dependent PFKs shows relatively few residues that are fully conserved. One residue that is conserved is a methionine residue that appears from the crystal structure of Escherichia coli PFK to be interacting with fructose 6-P. Very conservative substitutions for this methionine with leucine or isoleucine by site-directed mutagenesis of E. coli ATP-PFK and Entamoeba histolytica PPi-PFK produced profound decreases either in the apparent affinity for fructose 6-P or in maximal velocity, or both. Methionine provides a highly specific interaction with fructose 6-P for binding and for transition state stabilization.

Allosteric Regulation

An intercalated and thermally stable FAPY adduct of aflatoxin B1 in a DNA duplex: structural refinement from 1H NMR.

The structure of a formamidopyrimidine (FAPY) adduct arising from imidazole ring opening of the initially formed trans-8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct under basic conditions and positioned in the 5'-d(CTATFAPYGATTCA)-3'*5'-d(TGAATCATAG)-3' oligodeoxynucleotide was determined. The FAPY adduct may be a major progenitor of aflatoxin B1-induced mutations in DNA. The freshly prepared sample showed biphasic melting, with transitions at 28 and 56 degreesC. NMR initially showed multiple subspectra. Over a period of several days at 4 degreesC, the sample converted to a single species with a Tm of 56 degreesC, 15 degrees C greater than the unmodified duplex. The deoxyribose was in the beta configuration about the anomeric carbon, evidenced by NOEs between FAPYG5 H3', H2', H2", and H1'. FAPY formation resulted in the loss of the guanine H8 proton, and the introduction of the formyl proton, which showed NOEs to FAPYG5 H1' and A6 N6Ha. A total of 31 NOEs from AFB1 to DNA protons were observed, mostly to the 5'-neighboring base, T4 in the modified strand. Sequential NOEs were interrupted between T4 and FAPYG5 in the modified strand, between C16 and A17 in the complementary strand, and between T4 N3H and FAPYG5 N1H. An NOE between FAPYG5 N1H and C16 N4H showed intact hydrogen bonding at FAPYG5*C16. Upfield chemical shifts were observed for T4 H6 and A17 H8. Molecular dynamics calculations converged with pairwise rmsd differences of <0.9 A. The sixth root residual was 8.7 x 10(-2). The AFB1 moiety intercalated from the major groove between FAPYG5 and T4*A17, and stacked with T4 and FAPYG5 and partially stacked with A17. The base step between T4*A17 and FAPYG5*C16 was increased from 3.4 to 7 A. The duplex unwound by about 15 degrees. The FAPY formyl group was positioned to form a hydrogen bond with A6 N6Ha. Strong stacking involving the AFB1 moiety, and this hydrogen bond explains the thermal stabilization of four base pairs by this adduct, and may be a significant factor in its processing.

Aflatoxin B1

Cloning and expression of the gene for the active PPi-dependent phosphofructokinase of Entamoeba histolytica.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) from Entamoeba histolytica (HM-1) was purified from trophozoites. Oligonucleotide probes based on partial amino acid sequence were used to clone and sequence the gene and the cDNA of the enzyme. The molecular mass of the subunit was greater than, and the derived sequence significantly different from, that of the product of the PPi-PFK gene previously cloned from E. histolytica [Huang, Albach, Chang, Tripathi and Kemp (1995) Biochim. Biophys. Acta 1260, 215-217; Bruchhaus, Jacobs, Denart and Tannich (1996) Biochem. J. 316, 57-63]. The sequence identity between the two proteins was 17%. The sequence bore greater identity with the more phylogenetically advanced plant PPi-PFKs than with bacterial PPi-PFKs. The cloned cDNA was expressed and the protein purified. The kinetic properties were identical with those of the enzyme isolated from the organism. Furthermore, the specific activity was more than three orders of magnitude higher than that described for the product of the previously cloned E. histolytica PFK gene [Bruchhaus et al. (1996)]. The pH-dependence and apparent substrate affinities of the cloned enzyme were identical with those of the PPi-PFK in trophozoite extracts, indicating that the product of the cloned gene accounts for most if not all of the PFK activity in E. histolytica trophozoites.

Amino Acid Sequence

Effect of oral aluminum and aluminum citrate on blood level and short-term tissue distribution of aluminum in the rat.

Aluminum (Al) absorption seems to be very low, but many factors can enhance it in animals and humans. In the present study, we investigated the acute effect of Na citrate on Al absorption by monitoring Al levels in blood and several tissues. For this purpose, 18 Wistar male rats were divided into 3 groups: control, Al, and Al + Na citrate. After a 14-h fasting period, animals were dosed orally with deionized water, or 2 mmol Al chloride, or 2 mmol Al chloride plus 2 mmol Na citrate. Blood samples were taken before and 1, 2, 4, and 6 h after the gavage. Al concentrations in blood, liver, tibia, kidney, and intestinal wall were determined by ICP-OES. In the Al and Al + citrate groups, Al blood concentrations peaked at 1 h and 2 h with higher levels in the Al + citrate group. Al gavage resulted in an increase in Al level in intestinal wall, but not in the other investigated tissues. Simultaneous gavage of citrate with Al significantly increased its tissue levels in tibia, kidney, and in intestinal wall. Our data show clearly that Al as chloride can be absorbed, but not well retained by the organism tissues. Furthermore, the model used in the present study is appropriate for acute studies to investigate the effect of various compounds on Al absorption in the rat.

Administration, Oral

Familial Mediterranean fever at the millennium. Clinical spectrum, ancient mutations, and a survey of 100 American referrals to the National Institutes of Health.

Regarded as the most common and best understood of the hereditary periodic fever syndromes, familial Mediterranean fever (FMF) is a recessively inherited disease of episodic fever with some combination of severe abdominal pain, pleurisy, arthritis, and a characteristic ankle rash. The flares typically last for up to 3 days at a time, and most patients are completely asymptomatic between attacks; if untreated with prophylactic colchicine, some patients later develop amyloidosis and renal failure. The recent cloning of the FMF gene on the short arm of chromosome 16p, and the subsequent finding that its tissue expression is limited to granulocytes, has helped to explain the dramatic accumulation of neutrophils at the symptomatic serosal sites; the wild-type gene likely acts as an upregulator of an anti-inflammatory molecule or as a downregulator of a pro-inflammatory molecule. For nearly half a century, FMF was thought to cluster primarily in non-Ashkenazi Jews, Arabs, Armenians, and Turks, although the screening of the 8 known mutations in an American cohort has identified substantial numbers of people from the Ashkenazi Jewish and Italian populations in the United States who also have this disease. Nevertheless, the symptoms often go unrecognized and patients remain undiagnosed for years, not receiving the highly efficacious colchicine therapy; their histories often include multiple laparotomies, laparoscopies, and psychiatric evaluations. The combinations of clinical manifestations among FMF patients are quite heterogeneous, but our American cohort did not establish any connections between individual mutations and specific clinical pictures--as is seen in other diseases like cystic fibrosis, in which distinct genotypes target certain organ systems. Specifically, the data from our American series are insufficient to evaluate the hypothesis that the M694V/M694V genotype confers a more severe phenotype, or increases the risk of amyloidosis; but both our data and the recent literature (160) indicate that amyloidosis can occur in FMF patients with only 1 copy, or no copies, of the M694V mutation. It appears that specific MEFV mutations are probably not the sole determinants of phenotype, and that unknown environmental factors or modifying genes act as accomplices in this disease. Although we hope the discovery of the FMF gene will allow the diagnosis of FMF to become genetically accurate, the reality is that both clinical and genetic tools must still be used together unless mutations are identified on both of a patient's chromosomes. Physicians should be careful not to rule out the diagnosis in patients of high-risk ethnic backgrounds just because of atypical clinical features, as our data indicate that MEFV mutations are sometimes demonstrable in such patients. At the same time, physicians cannot yet rely solely on a genetic diagnosis because we have not yet identified a sufficient spectrum of mutations, and it is not currently feasible to examine every patient's full DNA sequence for the entire gene; screening an ethnically consistent and clinically positive patient for the 8 known mutations frequently identifies a mutation on only 1 chromosome, and genetic analysis of other classic cases will often reveal none of the 8 mutations. Still, our data suggest that ethnic background is an important predictor of finding 1 of the presently known mutations, and the knowledge of ancestries atypical for FMF can suggest the diagnosis of other hereditary periodic fever syndromes. As the list of FMF-associated MEFV mutations is expanded, and/or new sequencing technologies permit more rapid screening, the value and interpretation of genetic testing for FMF will become more straightforward. Moreover, as the pathophysiology of this disorder becomes less of a hypothesis and more of an understood entity, it is likely that treatment options will broaden beyond the use of daily prophylactic colchicine. (ABSTRACT TRUNCATED)

Adult

Construction of an approximately 700-kb transcript map around the familial Mediterranean fever locus on human chromosome 16p13.3.

We used a combination of cDNA selection, exon amplification, and computational prediction from genomic sequence to isolate transcribed sequences from genomic DNA surrounding the familial Mediterranean fever (FMF) locus. Eighty-seven kb of genomic DNA around D16S3370, a marker showing a high degree of linkage disequilibrium with FMF, was sequenced to completion, and the sequence annotated. A transcript map reflecting the minimal number of genes encoded within the approximately 700 kb of genomic DNA surrounding the FMF locus was assembled. This map consists of 27 genes with discreet messages detectable on Northerns, in addition to three olfactory-receptor genes, a cluster of 18 tRNA genes, and two putative transcriptional units that have typical intron-exon splice junctions yet do not detect messages on Northerns. Four of the transcripts are identical to genes described previously, seven have been independently identified by the French FMF Consortium, and the others are novel. Six related zinc-finger genes, a cluster of tRNAs, and three olfactory receptors account for the majority of transcribed sequences isolated from a 315-kb FMF central region (between D16S468/D16S3070 and cosmid 377A12). Interspersed among them are several genes that may be important in inflammation. This transcript map not only has permitted the identification of the FMF gene (MEFV), but also has provided us an opportunity to probe the structural and functional features of this region of chromosome 16.

Amino Acid Sequence

Expression of delta-endotoxin cryIA(c) gene of Bacillus thuringiensis in Escherichia coli and Streptomyces lividans.

The cryIA(c) gene of Bacillus thuringiensis was isolated from plasmid pOS1000. In order to obtain a proper cloning site and open reading frame, some DNA sequences preceding the initiating codon of the gene were replaced by synthetic oligonucleotide sequences. The isolated cryIA(c) was cloned into E. coli expression vector pKK223-3, and production of CryIA(c) protein was detected after induction by IPTG. A recombinant plasmid, pHZ1256, was constructed by insertion of the cryIA(c) gene into Streptomyces vector pHZ1272. pHZ1256 was introduced into Streptomyces lividans, and the production of CryIA(c) protein was confirmed by Western blotting after thiostrepton induction. A bioassy experiment showed that the CryIA(c) protein produced by E. coli and S. lividans caused 93% and 57% mortality to Plutella xylostella, respectively.

Animals

Construction of Escherichia coli-Streptomyces shuttle expression vectors for gene expression in Streptomyces.

pIJ4123 and pIJ6021 are high-copy-number vectors for gene expression in Streptomyces. They contain a strong, thiostrepton-inducible promoter, PtipA. Two E. coli-Streptomyces shuttle vectors, pHZ1271 and pHZ1272, were constructed by inserting a replicon and bla gene from E. coli into pIJ4123 and pIJ6021, respectively. Both vectors were structurally stable either in E. coli or in Streptomyces lividans. To demonstrate the utility of pHZ1272, the hemoglobin gene of Vitreoscillia (vhb) was cloned into pHZ1272 and expressed in Streptomyces lividans. The expression product (VHb) was detected by Western blotting and carbon monoxide binding activity analysis.

Amino Acid Sequence

A high-resolution genetic map of the familial Mediterranean fever candidate region allows identification of haplotype-sharing among ethnic groups.

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designated MEFV) on chromosome 16p13.3. We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placed MEFV in the approximately 285 kb between D16S468/D16S3070 and D16S3376. We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placed MEFV between D16S3082 and D16S3373 (approximately 200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only for D16S3370 and D16S2617 among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that some of these mutations arose before the affected Middle Eastern populations diverged from one another.

Alleles

Simultaneous expression of Borrelia OspA and OspC and IgM response in cerebrospinal fluid in early neurologic Lyme disease.

Lyme disease is the major tick-borne disease, caused by Borrelia burgdorferi (Bb). Neurological involvement is common in all stages. In vivo expression of Bb antigens (Ags) and the immune response to them has not been well investigated in the cerebrospinal fluid (CSF). Upregulation of outer surface protein (Osp) C and concomitant downregulation of OspA before tick inoculation of the spirochete has been reported in skin and blood in animals. CSF OspA Ag in early disease suggests otherwise in CSF. Early Ag expression and IgM response in human CSF was investigated here. Paired CSF and serum was collected from 16 early, predominantly erythema migrans Lyme disease patients with neurologic problems, 13 late Lyme disease patients, and 19 other neurologic disease (OND) controls. Samples were examined for IgM reactivity to recombinant Bb-specific Osps using ELISA and immunoblot. Of 12 early Lyme disease patients with neurologic involvement with both CSF and serum IgM against OspC, 7 (58%) had IgM to OspA (n = 5) or OspB (n = 2) that was restricted to the CSF, not serum. Overall, 12 of 16 (75%) of these early Lyme disease patients with neurologic involvement had CSF and serum IgM against OspC. Only 3 of 13 (23%) late Lyme disease patients and none of 19 OND controls had CSF IgM directed against OspC. In conclusion, in CSF, OspC and OspA can be coexpressed, and IgM response to them occurs in early Lyme disease patients with neurologic involvement. This biologic finding may also provide a discriminating marker for CNS infection in Lyme disease.

Adolescent

Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial Mediterranean fever locus (MEFV) on chromosome 16p 13.3.

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAC, and YAC clones, we have generated a contig of genomic clones spanning approximately 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAC, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the approximately 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene.

Base Sequence

Secondary and tertiary structural changes in gamma delta resolvase: comparison of the wild-type enzyme, the I110R mutant, and the C-terminal DNA binding domain in solution.

gamma delta Resolvase is a site-specific DNA recombinase (M(r) 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low-salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten tertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I110R resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I110R mutant contains a folded catalytic core of approximately 60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I110R resolvase. No increase in the helicity of the linker region of I110R resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.

Ammonium Sulfate