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H Yan

Publications and source records attributed to H Yan.

At least 73 records · Page 4Linked to original sources
PubMed2000

Catalytic center assembly of HPPK as revealed by the crystal structure of a ternary complex at 1.25 A resolution.

BACKGROUND: Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7, 8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate pathway, and therefore is an ideal target for developing novel antimicrobial agents. HPPK from Escherichia coli is a 158-residue thermostable protein that provides a convenient model system for mechanistic studies. Crystal structures have been reported for HPPK without bound ligand, containing an HP analog, and complexed with an HP analog, two Mg(2+) ions, and ATP. RESULTS: We present the 1.25 A crystal structure of HPPK in complex with HP, two Mg(2+) ions, and AMPCPP (an ATP analog that inhibits the enzymatic reaction). This structure demonstrates that the enzyme seals the active center where the reaction occurs. The comparison with unligated HPPK reveals dramatic conformational changes of three flexible loops and many sidechains. The coordination of Mg(2+) ions has been defined and the roles of 26 residues have been derived. CONCLUSIONS: HPPK-HP-MgAMPCPP mimics most closely the natural ternary complex of HPPK and provides details of protein-substrate interactions. The coordination of the two Mg(2+) ions helps create the correct geometry for the one-step reaction of pyrophosphoryl transfer, for which we suggest an in-line single displacement mechanism with some associative character in the transition state. The rigidity of the adenine-binding pocket and hydrogen bonds are responsible for adenosine specificity. The nonconserved residues that interact with the substrate might be responsible for the species-dependent properties of an isozyme.

Adenosine Triphosphate↗
PubMed2000

The function of Xenopus Bloom's syndrome protein homolog (xBLM) in DNA replication.

The Bloom's syndrome gene (BLM) plays a pivotal role in the maintenance of genomic stability in somatic cells. It encodes a DNA helicase (BLM) of the RecQ family, but the exact function of BLM remains elusive. To study this question, we have cloned the BLM homolog of the frog Xenopus laevis (xBLM) and have raised antibodies to it. Immunodepletion of xBLM from a Xenopus egg extract severely inhibits the replication of DNA in reconstituted nuclei. Moreover, the inhibition can be rescued by the addition of the recombinant xBLM protein. These results provide the first direct evidence that BLM plays an important role in DNA replication, suggesting that Bloom's syndrome may be the consequence of defective DNA replication.

Adenosine Triphosphatases↗
PubMed2000

Co-expression of the Bordetella pertussis leader peptidase I results in enhanced processing and expression of the pertussis toxin S1 subunit in Escherichia coli.

Bordetella pertussis is the causative agent of whooping cough. Traditional vaccines against this disease are inherently reactogenic, thus research is currently focussed on the production of less reactive, acellular vaccines. Expression of candidate antigens for these vaccines in Escherichia coli would be preferable, however, several B. pertussis antigens undergo incorrect post-translational processing in E. coli. The leader peptidase gene (lep) of B. pertussis encodes a protein of 294 amino acid residues that shares homology with other prokaryote leader peptidase I sequences. Hydrophilicity analysis based on the predicted amino acid sequence has demonstrated a similar membrane topology to that of E. coli and Salmonella typhimurium leader peptidase I. Co-expression of the B. pertussis lep gene in E. coli strain TOPP2 expressing the pertussis toxin S1 subunit was found to markedly increase the expression and post-translational processing of the S1 protein.

Amino Acid Sequence↗
PubMed2000

Combinatorial signaling in the specification of unique cell fates.

How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Here, we demonstrate that together with the transcription factor Lozenge, the nuclear effectors of the EGFR and Notch signaling pathways directly regulate D-Pax2 transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of D-Pax2 expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. We expect that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication.

Animals↗
PubMed2000

Early localized peritoneal mesothelioma as an incidental finding at laparoscopy. Report of a case and implications regarding natural history of the disease.

BACKGROUND: Peritoneal mesothelioma is regarded as a fatal disease that presents with progressive ascites in a relatively late stage of its natural history. To the authors' knowledge, prior published articles have not described the early manifestations of this cancer. METHODS: A 30-year-old asymptomatic woman underwent laparoscopy for an infertility workup. Nodules noted in the pelvis were biopsied and determined to be mesothelioma. Standard immunohistochemical studies were performed. Cytoreductive surgery and heated intraoperative intraperitoneal chemotherapy were used for treatment. RESULTS: Multiple (approximately 30) tumor nodules up to 2 mm in dimension and limited to the pelvis were observed and resected. No primary tumor focus was evident. These tumor nodules stained positive for Calretinin and negative for carcinoembryonic antigen immunohistochemically. CONCLUSIONS: In this patient, no incidence for transcoelomic dissemination of mesothelioma from a single primary site was observed. Rather, this patient's clinical presentation suggested that mesothelioma may be multifocal in origin within a limited region of the peritoneal cavity. This hypothesis may support a rationale for aggressive local-regional management of selected patients in whom peritoneal mesothelioma is of limited distribution and mass.

Adult↗
PubMed2000

Metal-induced B-H activation: addition of methyl acetylene carboxylates to Cp*Rh-, Cp*Ir-, (p-cymene)Ru-, and (p-cymene)Os half-sandwich complexes containing the chelating 1,2-dicarba-closo-dodecaborane-1,2-dithiolate ligand

The reactions of the 16e half-sandwich complexes [Cp*M[S2C2(B10)H10)]] (1: M=Rh; 2: M = Ir) and [eta6-(4-isopropyltoluene)M[S2C2(B10H10)] (3: M=Ru; 4: M=Os) with both methyl acetylene monocarboxylate and dimethyl acetylene dicarboxylate were studied in order to obtain more evidence for B-H activation, ortho-metalation, and B(3,6)-substitution of the carborane cluster. In the case of rhodium, the reaction of 1 with methyl acetylene monocarboxylate led to new complexes after twofold insertion into one of the Rh-S bonds (7), and twofold insertion together with B-substitution at the carborane cage (8). In the case of iridium, the reactions of 2 with methyl acetylene monocarboxylate gave two geometrical isomers 10 and 11, in which the alkyne is inserted into one of the Ir-S bonds, followed by hydrogen transfer from the carborane via the metal to the former alkyne and formation of an Ir-B bond. Only one type each (12 and 13) of these isomers was obtained from the reactions of the ruthenium and osmium half-sandwich complexes 3 and 4. The 16e starting materials 1-4 reacted with dimethyl acetylene dicarboxylate at room temperature to give the complexes 14-17, respectively, which are formed by addition of the C=C bond to the metal center and insertion into one of the metal-sulfur bonds. The proposed structures in solution were deduced from NMR data (1H, 11B, 13C, 103Rh NMR), and X-ray structural analyses were carried out for the rhodium complexes 7 and 8.

Journal Article↗
PubMed2000

CD4+ T cells acting independently of antibody contribute to protective immunity to Plasmodium chabaudi infection after apical membrane antigen 1 immunization.

Apical membrane Ag 1 (AMA1) is a leading malaria vaccine candidate. Homologues of AMA1 can induce protection in mice and monkeys, but the mechanism of immunity is not understood. Mice immunized with a refolded, recombinant, Plasmodium chabaudi AMA1 fragment (AMA1B) can withstand subsequent challenge with P. chabaudi adami. Here we show that CD4+ T cell depletion, but not gammadelta T cell depletion, can cause a significant drop in antiparasite immunity in either immunized normal or immunized B cell KO mice. In normal mice, this loss of immunity is not accompanied by a decline in Ab levels. These observations indicate a role for AMA1-specific Ab-independent T cell-mediated immunity. However, the loss of immunity in normal CD4+ T cell-depleted mice is temporary. Furthermore, immunized B cell KO mice cannot survive infection, demonstrating the absolute importance of B cells, and presumably Ab, in AMA1-induced immunity. CD4+ T cells specific for a cryptic conserved epitope on AMA1 can adoptively transfer protection to athymic (nu/nu) mice, the level of which is enhanced by cotransfer of rabbit anti-AMA1-specific antisera. Recipients of rabbit antisera alone do not survive. Some protected recipients of T cells plus antisera do not develop their own AMA 1-specific Ab response, suggesting that AMA 1-specific CMI alone can protect mice. These data are the first to demonstrate the specificity of any protective CMI response in malaria and have important implications for developing a malaria vaccine.

Amino Acid Sequence↗
PubMed2000

Dissecting the nucleotide binding properties of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase with fluorescent 3'(2)'-o-anthraniloyladenosine 5'-triphosphate.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7, 8-dihydropterin, the first reaction in the folate biosynthetic pathway. Like other enzymes in the folate pathway, HPPK is an ideal target for development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from humans and animals. Using 3'(2')-o-anthraniloyladenosine 5'-triphosphate as a fluorescent probe, a fluorometric competitive binding assay has been developed for measuring the dissociation constants of various compounds that bind to the ATP site of HPPK. The fluorometric assay has been used to determine the nucleotide specificity and dissect the energetics of the binding of MgATP. The order of affinity of various nucleoside triphosphates for HPPK is MgATP>MgGTP>MgITP>MgXTP approximately MgUTP approximately MgCTP. The affinity of MgATP for HPPK (K(d)=2.6+/-0.06 microM) is 260-fold higher than that of MgGTP and more than 1000-fold higher than those of the other nucleoside triphosphates, indicating that HPPK is highly specific with respect to the base moiety of the nucleotide. The affinity of ATP for HPPK in the presence of Mg(2+) is 15 times that in the absence of Mg(2+), indicating that the metal ion is important for the binding of the nucleotide. Removal of the gamma-phosphate from MgATP reduces its affinity for HPPK by a factor of approximately 21. The affinity of AMP for HPPK is about one third that of ADP and almost the same as that of adenosine. The result suggests that among the three phosphoryl groups of MgATP, the gamma-phosphoryl group is most critical for binding to HPPK and the alpha-phosphoryl group contributes little to the binding of the nucleotide. The affinity of MgATP is 18 times that of MgdATP, indicating that the 2'-hydroxyl group of MgATP is also important for binding. van't Hoff analysis suggests that binding of MgATP is mainly driven by enthalpy at 25 degrees C and the entropy of binding is also in favor of the formation of the HPPK.MgATP complex.

Binding, Competitive↗
PubMed2000

Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry.

Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.

Amino Acid Sequence↗
PubMed2000

Affinity adsorbents for the vancomycin group of antibiotics.

The vancomycin group of antibiotics kill Gram-positive bacteria by binding to nascent bacterial cell-wall peptidoglycan bearing the C-terminal sequence-D-Ala-D-Ala. In this paper, affinity adsorbents for the vancomycin group of antibiotics were prepared by immobilizing the peptidoglycan analogues -D-Ala-D-Ala, -succinyl-D-Ala and -succinyl-Gly on to crosslinked poly(N, N-dimethylacrylamide). The adsorption capacities of the three adsorbents for demethylvancomycin were 0.59, 0.35 and 0.29 mmol/g, respectively. The adsorption capacity of the adsorbent with-D-Ala-D-Ala for vancomycin was 0.53 mmol/g. In contrast, the adsorbent bearing -succinyl-L-Ala hardly adsorbed demethylvancomycin. Aqueous sodium carbonate (0.4 M)/acetonitrile (7/3, v/v) completely desorbed demethylvancomycin adsorbed on the adsorbents.

Adsorption↗
PubMed2000

Results of treatment of 33 patients with peritoneal mesothelioma.

BACKGROUND: Peritoneal mesothelioma is a rare peritoneal malignancy, representing approximately one-third of all mesotheliomas. It is regarded as a universally fatal cancer with few treatment options. METHODS: Records of 33 patients with peritoneal mesothelioma were reviewed retrospectively. Demographic, clinical and quantitative prognostic indicators were evaluated and analysed statistically using survival as endpoint. Patients were treated by a uniform strategy involving cytoreductive surgery with peritonectomy procedures and perioperative intraperitoneal chemotherapy (cisplatin, doxorubicin). RESULTS: There were ten women and 23 men; mean age was 53.0 years. Asbestos exposure was recorded in five patients and a family history of cancer in 13. Presentation was mainly abdominal distension and pain. Median survival was 31.0 months; overall projected survival at 3 years was 56 per cent. The most significant positive predictive factors of survival were: female sex (P= 0.003), low prior surgical score (P=0.002), completeness of cytoreduction (P=0.0002) and second-look surgery (P=0.019). The morbidity rate for this combined treatment was 33 per cent and the perioperative mortality rate was 3 per cent. CONCLUSION: Although peritoneal mesothelioma is rare, progress in its management has occurred. Survival has been extended and selection factors by which patients may be allocated to aggressive management strategies have been defined.

Adolescent↗
PubMed2000

Isolation and identification of the group B streptococcal toxin CM101 from infants with sepsis.

OBJECTIVE: To determine whether the group B streptococcal (GBS) polysaccharide exotoxin CM101, which induces a complement-activated cytokine-driven inflammatory response, is present in body fluids of infants with GBS disease. STUDY DESIGN: With a sandwich enzyme-linked immunosorbent assay, CM101 was measured in plasma, urine, and cerebrospinal fluid from newborn infants who were evaluated for possible infection and from older infants with culture-confirmed GBS disease. RESULTS: Urine from 11 newborn infants with culture-confirmed early-onset disease contained large amounts of CM101 (1.0 to 5.5 mg/48 h). Plasma concentrations were 62.6 +/- 10.5 microg/mL in these infants and were 69.0 +/- 21.2 microg/mL in 4 older infants with late-onset disease. Plasma CM101 concentrations did not correlate with indexes of illness severity, leukocyte counts, or interleukin-6 or interleukin-8 plasma concentrations. CM101 was present in cerebrospinal fluid of 5 infants with meningitis (8.4 +/- 1.6 microg/mL). CM101 was not found in control samples. CM101 isolated from urine had molecular weight and sugar composition similar to those obtained from GBS culture media, and they both elicited a comparable pathophysiologic response when infused intravenously in lambs. CONCLUSIONS: CM101 is present in infants with GBS disease, and it appears to be the same as CM101 obtained from GBS culture media.

Animals↗