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Biomedical subjects

C Mao

Publications and source records attributed to C Mao.

At least 91 records · Page 5Linked to original sources
PubMed1998

Crystallization and preliminary diffraction analysis of a hyperthermostable DNA polymerase from a Thermococcus archaeon.

The hyperthermostable DNA polymerase from a marine Thermococcus archaeon has been crystallized in space group P212121, with unit-cell dimensions a = 94.8, b = 98.2, c = 112.2 A with one molecule per asymmetric unit. Conditions for data collection at 98 K have been identified, and a complete data set was collected to 2.2 A resolution. Strategies employed here may facilitate crystallization of other hyperthermostable proteins. The structure of this enzyme will provide the first structural data on the archaeal and hyperthermostable classes of DNA polymerases. Sequence homology to human polymerase alpha (polymerase B family) may make it a model for studying eukaryotic and viral polymerases and for the development of anti-cancer and anti-viral therapeutics.

Crystallization↗
PubMed1998

Structure-based design of N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea and N-[2-(1-piperazinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea as potent non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A novel computer model of the HIV reverse transcriptase (RT) non-nucleoside inhibitor (NNI) binding pocket, which was generated using high resolution crystal structure information from 9 individual RT/NNI complexes, revealed previously unrecognized ligand derivatization sites for phenethylthiazolylthiourea (PETT) derivatives. Spatial gaps surrounding the pyridyl ring of the active PETT derivative trovirdine were discovered during modeling procedures. Docking studies using the computer-generated model of the binding pocket (composite binding pocket) suggested that the replacement of the planar pyridyl ring of trovirdine with a nonplanar piperidinyl or piperazinyl ring, which occupy larger volumes, would better fill the spacious Wing 2 region of the butterfly-shaped NNI binding pocket. The anti-HIV activity of the synthesized heterocyclic compounds N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea and N-[2-(1-piperazinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea was examined in HTLVIIIB-infected peripheral blood mononuclear cells. Both compounds were more potent than trovirdine and abrogated HIV replication at nanomolar concentrations without any evidence of cytotoxicity.

Antiviral Agents↗
PubMed1998

5-Alkyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-ones as potent non-nucleoside reverse transcriptase inhibitors of S-DABO series.

Novel dihydroalkoxybenzyloxopyrimidine (S-DABO) derivatives targeting the non-nucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been synthesized using a novel computer model for the NNI binding pocket and tested for their RT inhibitory activity in cell-free assays using purified recombinant HIV RT as well as for their anti-HIV activity in HTL VIIIB-infected peripheral blood mononuclear cells. Our computational approach allowed the identification of several ligand derivatization sites for the generation of more potent S-DABO derivatives. Our lead S-DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one (compound 3), elicited potent anti-HIV activity with an IC50 value of less than 1nM for inhibition of HIV replication without any evidence of cytotoxicity and an unprecedented selectivity index of > 100,000.

Anti-HIV Agents↗
PubMed1998

Mutations affecting cooperative DNA binding of phage HK022 CI repressor.

Cooperative protein-DNA interactions play critical roles in gene regulation in all organisms. Among the best-studied cooperative interactions is that of phage lambda repressor, which binds cooperatively to two adjacent operators. Similar cooperative interactions are also shown by several other lambdoid phage repressors, including HK022 CI repressor, which we study here. This protein has a much higher degree of cooperativity than seen with lambda repressor, and previous evidence has suggested that cooperativity may play roles in HK022 gene regulation that have no parallel in lambda. We have isolated several cooperativity or Coop- mutations in HK022 cI. These mutant proteins were partially defective in vivo for binding to two adjacent operators, but normal or nearly so for binding to a single operator. Two mutations showed mutual suppression, in that the double mutation had wild-type behavior. Analysis of several purified mutant proteins showed that they were also defective for cooperative binding in vitro. Unexpectedly, the mutant proteins showed an altered pattern of in vitro binding to DNA at non-operator sites. Several of them also increased the rate of specific repressor cleavage. We propose a conformational model in which the various functions of the wild-type protein are carried out by differing conformations; these conformations are normally in balance, and the mutations perturb this balance.

Bacteriophage lambda↗
PubMed1998

Calf spleen purine nucleoside phosphorylase complexed with substrates and substrate analogues.

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway, which provides an alternative to the de novo pathway for the biosynthesis of purine nucleotides. PNP catalyzes the reversible phosphorolysis of 2'-deoxypurine ribonucleosides to the free bases and 2-deoxyribose 1-phosphate. Absence of PNP activity in humans is associated with specific T-cell immune suppression. Its key role in these two processes has made PNP an important drug design target. We have investigated the structural details of the PNP-catalyzed reaction by determining the structures of bovine PNP complexes with various substrates and substrate analogues. The preparation of phosphate-free crystals of PNP has allowed us to analyze several novel complexes, including the ternary complex of PNP, purine base, and ribose 1-phosphate and of the completely unbound PNP. These results provide an atomic view for the catalytic mechanism for PNP proposed by M. D. Erion et al. [(1997) Biochemistry 36, 11735-11748], in which an oxocarbenium intermediate is stabilized by phosphate and the negative charge on the purine base is stabilized by active site residues. The bovine PNP structure reveals several new details of substrate and inhibitor binding, including two phosphate-induced conformational changes involving residues 33-36 and 56-69 and a previously undetected role for His64 in phosphate binding. In addition, a well-ordered water molecule is found in the PNP active site when purine base or nucleoside is also present. In contrast to human PNP, only one phosphate binding site was observed. Although binary complexes were observed for nucleoside, purine base, or phosphate, ribose 1-phosphate binding occurs only in the presence of purine base.

Acyclovir↗
PubMed1998

Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal.

DNA polymerases copy DNA templates with remarkably high fidelity, checking for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps. Despite extensive biochemical, genetic and structural studies, the mechanism by which nucleotides are correctly incorporated is not known. Here we present high-resolution crystal structures of a thermostable bacterial (Bacillus stearothermophilus) DNA polymerase I large fragments with DNA primer templates bound productively at the polymerase active site. The active site retains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation. The polymerase also retains its ability to discriminate between correct and incorrectly paired nucleotides in the crystal. Comparison of the structures of successively translocated complexes allows the structural features for the sequence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions with the first four to five base pairs in the minor groove, location of the terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch from B-form to underwound A-form DNA at the polymerase active site.

Binding Sites↗
PubMed1998

Rational design and synthesis of phenethyl-5-bromopyridyl thiourea derivatives as potent non-nucleoside inhibitors of HIV reserve transcriptase.

A series of novel phenethylthiazolylthiourea (PETT) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of HIV reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket. The structure-based design and synthesis of these new PETT derivatives were complemented by biological assays of their anti-HIV activity. Modeling studies for rational drug design included the construction of a composite NNI binding pocket from nine RT-NNI crystal structures, the analyses of surface complementarity between NNI and RT, and application of Ki calculations combined with a docking procedure involving the novel PETT derivatives. The use of the composite NNI binding pocket allowed the identification and structure-based design of three promising PETT derivatives with ortho-F (2), ortho-Cl (3), and meta-F (5) substituents on the phenyl ring. These novel PETT derivatives were more active than AZT or trovirdine and showed potent anti-HIV activity with IC50[p24] values of < 1 nM and selectivity indices of > 100,000.

Anti-HIV Agents↗
PubMed1998

Isoform-specific regulation of nitric oxide synthase mRNA in the kidney by sodium and blood pressure.

BACKGROUND: The roles of nitric oxide synthases (NOS) in kidney function are still controversial, principally due to the lack of isoform-specific inhibitors of NOS. OBJECTIVE: To investigate the relative roles of each isoform of NOS in regulation of sodium and volume homeostasis. DESIGN: We studied the effects of long-term modifications of sodium diet and blood pressure on expression of NOS mRNA in the renal cortex, where the three isoforms of NOS are present. METHODS: We used quantitative reverse-transcription-polymerase chain reaction assays specific to each isoform of NOS to determine amounts of their respective mRNA in control rats, deoxycorticosterone acetate (DOCA)-salt hypertensive rats, rats fed a high-salt diet, and furosemide-treated rats fed a low-sodium diet. Nicotinamide adenine nucleotide phosphate H (NADPH) diaphorase staining was performed on DOCA-salt and control rat kidneys. RESULTS: Levels of NOS I mRNA in DOCA-salt rats were decreased by treatment, those in low-salt-diet rats remained unaffected and those in high-salt diet rats tended to be intermediate between those of the other rat groups. Expression of NOS III mRNA was not significantly modified by either treatment Levels of NOS II mRNA in DOCA-salt rats were increased, those in high-salt-diet rats remained unaffected, and those in low-salt-diet were decreased by treatment, but these levels are more than 100-fold lower than those observed for the other isoforms of NOS. NADPH diaphorase staining in macula densa of DOCA-salt rats was markedly decreased compared with that in macula densa of control rats but staining in renal inflammatory and fibrous lesions became detectable, and staining in the vessels did not differ from that for control rats. CONCLUSIONS: Our results show that intake of sodium and extracellular fluid volume regulate levels of mRNA of the three NOS isoforms in the renal cortex differently, suggesting that each of them plays a specific role.

Animals↗
PubMed1998

Localization of leptin binding domain in the leptin receptor.

The leptin receptor is a member of the class I cytokine receptor family and is involved in the control of appetite and body weight. The predicted amino acid sequence of the extracellular region of the cloned leptin receptor differs from that of many other cytokine receptors in that it contains two homologous segments representing potential ligand binding sites. After the analysis of various deletion and substitution mutants of the leptin receptor, we found that the first potential binding motif is not required for leptin binding and receptor activation, whereas modification of the second potential binding motif can lead to inactive receptor mutants. Further deletion analysis generated a minimal binding domain that retains high affinity leptin binding. The leptin binding domain thus has been localized to residues 323-640, which contain the second segment of cytokine receptor domain/fibronectin type 3 domain (residues 428-635). Coexpression of the active isoform of leptin receptor (OB-Rb) with an inactive mutant lacking high affinity leptin binding site led to suppression of the activity mediated by OB-Rb, suggesting that the leptin receptor may exist as a multimeric complex in the absence of leptin.

Amino Acid Sequence↗
PubMed1998

Structure-based design of novel dihydroalkoxybenzyloxopyrimidine derivatives as potent nonnucleoside inhibitors of the human immunodeficiency virus reverse transcriptase.

Two highly potent dihydroalkoxybenzyloxopyrimidine (DABO) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket and tested for anti-HIV activity. Our lead DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-on e, elicited potent inhibitory activity against purified recombinant HIV RT and abrogated HIV replication in peripheral blood mononuclear cells at nanomolar concentrations (50% inhibitory concentration, <1 nM) but showed no detectable cytotoxicity at concentrations as high as 100 microM.

Binding Sites↗
PubMed1998

Carcinoid tumors of the pancreas. Status report based on two cases and review of the world's literature.

CONCLUSION: The diagnosis of a pancreatic carcinoid should be based on the measurement of serotonin in serum or its demonstration in the tumor and/or by the measurement of its derivative (5-HIAA) in urine. Carcinoid of the pancreas is a rare but definite entity; usually having metastasized by the time of diagnosis. The term "serotonin-producing tumor of the pancreas" has been suggested as an alternative designation for "pancreatic carcinoid." BACKGROUND: The literature on carcinoid tumors of the pancreas is confusing because much of it preceded the development of the more specific immunological, chemical and staining techniques currently available. METHODS: 43 case reports were collected from the world's literature, based on a demonstrable pancreatic neuroendocrine tumor plus a positive finding of at least one of the following without another dominant hormone being demonstrated: elevation of 5-Hydroxytryptamine (5-HT) (serotonin) in the serum or detected in tumor tissue, and/or elevation of 5-Hydroxyindole acetic acid (5-HIAA) in the urine. In addition to these two hormone-specific assays, information was collected on the silver-staining properties of the tumor; properties which have traditionally been associated with carcinoid tumors. Positive silver staining in tumor cells (argyrophilic and/or argentaffin reaction) is strongly indicative of the carcinoid tumor but the findings are less specific than the hormone assays and immunohistologic stains. RESULTS: In this review of 43 cases, including two current ones, the pancreatic carcinoid tumor has the following important features: 1. It is a rare tumor that is usually diagnosed late when the tumor is large and has metastasized. Thirty-eight (88.4%) have been malignant. They are, therefore, associated with a high incidence of the "carcinoid syndrome." 2. To date, prognosis in therapy is poor, based on delayed diagnosis, a resultant low incidence of resectability, and an uncertain duration of survival after resection. 3. Pancreatic carcinoid tumors remain difficult to differentiate from other endocrine tumors. The measurement of urinary 5-HIAA excretion or the demonstration of elevated serotonin level in the tumor or in serum is essential to its distinction. Silver staining of the tumor, although of historic importance, has been superceded by the hormone-specific studies. 4. To distinguish it from other endocrine tumors of the pancreas, the terms "pancreatic serotoninoma" or "serotonin-producing tumor of the pancreas" have been suggested as possible alternatives. Its growth characteristics may be related more to its cell of origin than to its extent of hormone secretion. Not all of the tumors result in recognizable hyperserotoninemia.

Adult↗
PubMed1998

Alpha-cyano-beta-hydroxy-beta-methyl-N-[4-(trifluoromethoxy)phenyl] propenamide: an inhibitor of the epidermal growth factor receptor tyrosine kinase with potent cytotoxic activity against breast cancer cells.

Epidermal growth factor receptor (EGF-R) tyrosine kinase is known to be overexpressed in several malignancies and is an important target for anticancer drug design. We constructed a homology model to represent the structure of EGF-R and propose that this model can be used to design potent inhibitors of EGF-R. We used our EGF-R model and a docking procedure to rationally design compounds predicted to bind favorably to EGF-R. This approach led to the successful design of a leflunomide metabolite analogue, which was found to have an IC50 value of 1.7 microM in EGF-R inhibition assays and killed >99% of human breast cancer cells in vitro by triggering apoptosis. The reported studies may provide the basis for the development of a new class of potent and clinically useful anti-breast cancer agents.

Aniline Compounds↗
PubMed1997

Involvement of yeast sphingolipids in the heat stress response of Saccharomyces cerevisiae.

A role for sphingolipids in the yeast heat stress response has been suggested by the isolation of suppressors of mutants lacking these lipids, which are unable to grow at elevated temperatures. The current study examines the possible role of sphingolipids in the heat adaptation of yeast cells as monitored by growth and viability studies. The suppressor of long chain base auxotrophy (SLC, strain 7R4) showed a heat-sensitive phenotype that was corrected by transformation with serine palmitoyltransferase. Thus, the deficiency in sphingolipids and not the suppressor mutation was the cause of the heat-sensitive phenotype of the SLC strain 7R4. The ability of sphingolipids to rescue the heat-sensitive phenotype was examined, and two endogenous yeast sphingoid backbones, phytosphingosine and dihydrosphingosine, were found to be most potent in this effect. Next, the effect of heat stress on the levels of the three major classes of sphingolipids was determined. The inositol phosphoceramides showed no change over a 1.5-h time course. However, the four detected species of sphingoid bases increased after 15 min of heat stress from 1.4- to 10.8-fold. The largest increases were seen in two sphingoid bases, C20 phytosphingosine and C20 dihydrosphingosine, which increased 6.4- and 10.8-fold over baseline, respectively. At 60 min of heat stress two species of yeast ceramide increased by 9.2- and 10.6-fold over baseline. The increase seen in the ceramides was partially decreased by Fumonisin B1, a ceramide synthase inhibitor. Therefore, heat stress induces accumulation of sphingoid bases and of ceramides, probably through de novo synthesis. Taken together, these results demonstrate that sphingolipids are involved in the yeast heat stress adaptation.

Chromatography, High Pressure Liquid↗
PubMed1997

Identification and characterization of Saccharomyces cerevisiae dihydrosphingosine-1-phosphate phosphatase.

We have identified the yeast sphingosine resistance gene (YSR2) of Saccharomyces cerevisiae as encoding a protein that specifically dephosphorylates dihydrosphingosine 1-phosphate (DHS-1-P), and we refer to this protein as dihydrosphingosine-1-phosphate phosphatase. Overexpression of YSR2 conferred sphingosine resistance to the dihydrosphingosine-1-P lyase-defective mutant (JS16) of S. cerevisiae, which is hypersensitive to sphingosine. The ysr2Delta deletion mutant of S. cerevisiae accumulated DHS-1-P compared with its wild type strain upon labeling with D-erythro-[4, 5-3H]dihydrosphingosine, whereas overexpression of YSR2 increased dephosphorylation of DHS-1-P. An epitope-tagged fusion protein (YSR2-Flag) was partially purified and found to specifically dephosphorylate DHS-1-P to yield dihydrosphingosine. YSR2 failed to dephosphorylate ceramide 1-phosphate or phosphatidic acid. Functionally, the mutant bearing the ysr2Delta deletion decreased labeling of sphingolipids and increased labeling of glycerolipids dramatically following in vivo labeling with D-erythro-[3H]dihydrosphingosine, but it slightly affected labeling of sphingolipids with inositol. Taken together, these results identify YSR2 as dihydrosphingosine-1-phosphate phosphatase. They also raise the intriguing possibility that phosphorylation followed by dephosphorylation is required for incorporation of exogenous long chain sphingoid bases into sphingolipids.

Drug Resistance, Microbial↗
PubMed1997

The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology.

BACKGROUND: Purine nucleoside phosphorylase (PNP) from Escherichia coli is a hexameric enzyme that catalyzes the reversible phosphorolysis of 6-amino and 6-oxopurine (2'-deoxy)ribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate. In contrast, human and bovine PNPs are trimeric and accept only 6-oxopurine nucleosides as substrates. The difference in the specificities of these two enzymes has been utilized in gene therapy treatments in which certain prodrugs are cleaved by E. coli PNP but not the human enzyme. The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze the same basic chemical reaction. Structural comparison of the active sites of mammalian and E. coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E. coli PNP. RESULTS: The crystal structure of E. coli PNP at 2.0 A resolution shows that the overall subunit topology and active-site location within the subunit are similar to those of the subunits from human PNP and E. coli uridine phosphorylase. Nevertheless, even though the overall geometry of the E. coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different. In E. coli PNP, the purine- and ribose-binding sites are generally hydrophobic, although a histidine residue from an adjacent subunit probably forms a hydrogen bond with a hydroxyl group of the sugar. The phosphate-binding site probably consists of two main-chain nitrogen atoms and three arginine residues. In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP. CONCLUSIONS: The structures of human and E. coli PNP define two possible classes of nucleoside phosphorylase, and help to explain the differences in specificity and efficiency between trimeric and hexameric PNPs. This structural data may be useful in designing prodrugs that can be activated by E. coli PNP but not the human enzyme.

Binding Sites↗
PubMed1997

ART (protein product of agouti-related transcript) as an antagonist of MC-3 and MC-4 receptors.

The mRNA encoding an agouti related protein (ART) of unknown biochemical function was previously reported to be up-regulated in the hypothalamus of two genetically obese mouse strains. We have expressed human ART as a secreted protein in COS-7 cells, and show that recombinant ART is functionally active in inhibiting the binding of a radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analog to the human melanocortin-3 (MC-3) and melanocortin-4 (MC-4) receptors, while it is not a potent inhibitor of the human melanocortin-5 (MC-5) receptor. ART is an antagonist of the human MC-3 and MC-4 receptors as determined in functional assay. ART appears to be approximately 100-fold more potent than agouti with reference to the MC-3R and MC-4R binding affinity. These data suggest that ART may be a physiological regulator of feeding behavior.

Agouti-Related Protein↗