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Biomedical subjects

H Fu

Publications and source records attributed to H Fu.

At least 109 records · Page 6Linked to original sources
PubMed1997

Raf-1 kinase and exoenzyme S interact with 14-3-3zeta through a common site involving lysine 49.

14-3-3 proteins are a family of conserved dimeric molecules that bind to a range of cellular proteins involved in signal transduction and oncogenesis. Our solution of the crystal structure of 14-3-3zeta revealed a conserved amphipathic groove that may allow the association of 14-3-3 with diverse ligands (Liu, D., Bienkowska, J., Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191-194). Here, the contributions of three positively charged residues (Lys-49, Arg-56, and Arg-60) that lie in this Raf-binding groove were investigated. Two of the charge-reversal mutations greatly (K49E) or partially (R56E) decreased the interaction of 14-3-3zeta with Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 14-3-3zeta with another target protein, exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3zeta required for ExoS activation increased by approximately 110-, 5-, and 2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic reduction of 14-3-3zeta/ligand affinity by the K49E mutation is due to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteolysis and circular dichroism analysis. This work identifies the first point mutation (K49E) that dramatically disrupts 14-3-3zeta/ligand interactions. The parallel effects of this single point mutation on both Raf-1 binding and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3zeta.

14-3-3 Proteins↗
PubMed1997

Selection of diphtheria toxin active-site mutants in yeast. Rediscovery of glutamic acid-148 as a key residue.

Saccharomyces cerevisiae was transformed with expression plasmids carrying the DTA gene under control of the GAL1 promoter; colonies that formed under inducing conditions were selected; and plasmids from these colonies were screened for mutations in DTA that failed to block expression of the protein. Substitutions at three sites were identified, all of which are in the active-site cleft; and each of the substitutions reduced ADP-ribosyltransferase activity by > 10(5). The substitutions include a charge reversal mutation of a catalytically important residue (Glu148Lys) and replacements of either of two glycines (Gly22 and Gly52) with bulky residues. The fact that multiple mutations were identified in these same residues implies that there are relatively few sites at which substitutions ablate ADP-ribosyltransferase activity without blocking expression of the full-length protein. Incorporation of a primary attenuating mutation into the DTA gene allowed S. cerevisiae also to be used to select complementary secondary mutations which altered activity less drastically. Besides elucidating structure-activity relationships, mutations identified by these approaches may be useful in designing new vaccines.

Binding Sites↗
PubMed1997

Fine mapping of the epitopes of humanized anti-L-selectin monoclonal antibodies HuDREG-55 and HuDREG-200.

Blocking the function of L-selectin with a monoclonal antibody (mAb) is a promising way to prevent neutrophils from causing tissue damage during inflammation. HuDREG-55 and HuDREG-200 are humanized mAb which bind to human L-selectin and block its function as an adhesion molecule. To understand the mechanism of the action of HuDREG-55 and HuDREG-200, we determined their epitopes on L-selectin at the amino acid level. The analysis of human E- and L-selectin chimeric proteins demonstrated that the lectin domain of L-selectin is necessary for the binding of HuDREG-55 and HuDREG-200. Mutational analysis of Escherichia coli-expressed L-selectin showed that HuDREG-55 binding is sensitive to amino acid changes at positions 11, 56, 87, 89, 105, 107 and 111 (counting from the amino-terminus of mature L-selectin) while HuDREG-200 binding is sensitive to amino acid changes at 45, 46 and 47. Both epitopes are located close to the predicted carbohydrate binding site, indicating that HuDREG-55 and HuDREG-200 block the function of L-selectin by directly inhibiting the binding to carbohydrate ligands.

Amino Acid Sequence↗
PubMed1997

ATPase and ubiquitin-binding proteins of the yeast proteasome.

The 26S proteasome is a 2-Megadalton proteolytic complex with over 30 distinct subunits. The 19S particle, a subcomplex of the 26S proteasome, is thought to confer ATP-dependence and ubiquitin-dependence on the proteolytic core particle of the proteasome. Given the complexity of the 19S particle, genetic approaches are likely to play an important role in its analysis. We have initiated biochemical and genetic studies of the 19S particle in Saccharomyces cerevisiae. Here we describe the localization to the proteasome of several ATPases that were previously proposed to be involved in transcription. Independent studies indicate that the mammalian 26S proteasome contains closely related ATPases. We have also found that the multiubiquitin chain binding protein Mcb1, a homolog of the mammalian S5a protein, is a subunit of the yeast proteasome. However, contrary to expectation, MCB1 is not an essential gene in yeast. The mcb1 mutant grows at a nearly wild-type rate, and the breakdown of most ubiquitin-protein conjugates is unaffected in this strain. One substrate, Ub-Proline-beta gal, was found to require MCB1 for its breakdown, but it remains unclear whether Mcb1 serves as a ubiquitin receptor in this process. Our data suggest that the recognition of ubiquitin conjugates by the proteasome is a complex process which must involve proteins other than Mcb1.

Adenosine Triphosphatases↗
PubMed1997

[Comparison of changes of bone mass, parathyroid hormone and calcitonin between two animal models of bone loss].

The changes in bone mass, blood parathyroid hormone (PTH) and calcitonin (CT) between rats suspended for 14 and 21 days, simulated weightlessness, and rats ovariectomized (OVX) for 30 and 60 days were observed. The results revealed that mineral density of T6 and L3 was significantly increased in rats suspended for 14 days. T6 mineral density was also significantly increased in rats suspended for 21 days, but L3 mineral density was significantly decreased. T6 mineral density had no change in OVX rats, but L3 mineral density was significantly decreased in them both for 30 and 60 days. Mineral content at proximal 1/3 of femur shaft was significantly decreased in rats suspended for 14 and 21 days, and in rats OVX for 60 days. Bone biomechanical properties of suspended rats were more seriously deteriorated than that in OVX rats. There was no change in blood PTH, but CT was significantly increased in rats suspended for 14 and 21 days. Blood PTH and CT were significantly decreased in rats OVX for 30 and 60 days. The results indicate that the mineral redistribution and bone loss are presented in trabecular bone of suspended rats, but not in OVX rats. The deterioration of bone quality was more seriously in suspended rats than that in OVX rats. Both the cortical and trabecular bone in the two models were affected.

Animals↗
PubMed1997

[Study on tumor necrosis factor receptor of human gastric cancer cells].

OBJECTIVE: To explore the relationship between the number of tumor necrosis factor receptors (TNFR) and the degree of differentiation of gastric cancer cells, and the relationship between the number of TNFR and the cytotoxicity of tumor necrosis factor mutant (TNF-m). METHODS: With 125I-TNF-m, the radio-ligand binding assay was used to detect the TNFR on three gastric cancer cell lines (MKN28, SGC7901 MKN45) with from high to low degrees of differentiation. MTT colorimetric method was used to study the cytotoxicity of TNF-m on the three gastric cancer cell lines in vitro. RESULTS: The number of TNFR of the three gestric cancer cells was 9.8 x 10(-12) nmol, 5.6 x 10(-12) nmol and 3.2 x 10(-12) nmol per cell respectively it differed significent from each other (P < 0.05) but the dissociation constant was basically the same. The rate of TNF-m internalization of the three gastric cancer cells was nearly the same and temperature dependent. The half-life of TNFR was about 90 mins. The ratio of membrane receptors to cytoplasm receptors was about 0.5. The maximum killing rate of TNF-m was 86%, 60% and 34% for MKN28, SGC7901, MKN45 cells, respectively, which were significantly different (P < 0.05). Killing rate of TNF-m to these cells at 39 degrees C was higher than at 37 degrees C. CONCLUSIONS: The number of TNFR on the surface of gastric cancer cells was associated with the degree of differentiation of gastric cancer cells. The cytotoxicity of TNF-m was related to the number of TNFR and the rate of internalization of TNF-m.

Adenocarcinoma↗
PubMed1997

[Comparative studies on pharmacological and toxic actions of raw fructus Psoraleae and its salt-baked and improved salt-baked products].

The pharmacological and toxic actions of raw Fructus Psoraleae and its salt-baked, and improved salt-baked products were comparatively studied. The leucogenic action of the improved salt-baked product was more effective than that of the original salt-baked one. But no significant difference in antidiarrheal or antiandrogenoid effect was found between the two salt-baked products. The toxicity(LD50 and the injury on kidney) of the improved salt-baked product was lower than that of the salt-baked. one. The results indicate that this improved processing method would contribute to increasing the efficacy and decreasing the toxicity of Fructus Psoraleae.

Androgen Antagonists↗
PubMed1997

[Toxic studies on various processed products of Fructus Psoraleae].

Toxic studies on Fructus Psoraleae (FP) and its processed products are carried on. LD50 is 37.7 +/- 0.54 g crude drug/kg in oral administration of raw FP in comparison with 43.25 +/- 6.1/kg in that of Salt-bake one in mice. When 5.0 g/kg is given by oral for 14 days, the wet weight of testicles, preputial galnds, prostate and semianl vesicle is decreased in raw FP and its stir-bake tested groups. The wet weight of preputial glends, prostate, seminal vesicle in Lei Gong's Procession group, or prostate, and seminal vesicle in salt-steam group is also decreased to a certain extent. But no significant change on the weight of those sex organs is found in liquor-bake or salt-bake procession group, The glomerulal cell hyperplasia and cloudy swelling of renal tubules are induced by administration of raw FP in pathological examination.

Animals↗
PubMed1997

[Comparision on the effects of ALI aeute liver injury of three species of concha haliotidis in mice].

The liver injury mice model with Haliotis ruber (Leach), Haliotis disus hannai Ino and Haliotis lacvigata (Donovan) was studied. The results indicated three species Haliotidis could versus carbon tetrachloride-induced ALI aeute liver injury in mice. Comparision of reduced serum glutamic-pyruvic transaminass (SGPT) activity showed Halitis disacus hannai Ino > Haliotis Lacvigata (Donovan) > Haliotis ruber (Leach). The Liver starch was determined. It is suggested protection liver effect of Haliotis discus hannai Ino and Haliotis lacvigata (Donovan) are most powerful.

Alanine Transaminase↗
PubMed1996

PCR cloning of the cDNA encoding baboon L-selectin.

The cDNA encoding baboon L-selectin was isolated from baboon peripheral blood lymphocytes-derived cDNA and its nucleotide (nt) sequence was determined. The deduced 372-amino acid (aa) sequence of baboon L-selectin is 95% identical to that of human L-selectin.

Amino Acid Sequence↗
PubMed1996

Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries.

New phage display vectors for in vivo recombination of immunoglobulin (Ig) heavy (VH) and light (VL) chain variable genes, to make single-chain Fv fragments (scFv), were constructed. The VH and VL genes of monoclonal antibody (mAb) EP-5C7, which binds to both human E- and P-selectin, were cloned into a pUC19-derived plasmid vector, pCW93, and a pACYC184-derived phagemid vector, pCW99, respectively. Upon induction of Cre recombinase (phage P1 recombinase), the VH and VL genes were efficiently recombined into the same plasmid via the two loxP sites (phage P1 recombination sites), one located downstream from a VH gene in pCW93 and another upstream from a VL gene in pCW99. In the resulting phagemid, the loxP sequence also encodes a polypeptide linker connecting the VH and VL domains to form a scFv of EP-5C7. Whether expressed on the phage surface or as a soluble form, the EP-5C7 scFv showed specific binding to human E- and P-selectin. This phagemid vector system provides a way to recombine VH and VL gene libraries efficiently in vivo to make extremely large Ig combinatorial libraries.

Amino Acid Sequence↗
PubMed1996

Engineered biosynthesis of novel polyketides: regiospecific methylation of an unnatural substrate by the tcmO O-methyltransferase.

TcmO is an O-methyltransferase that methylates the C-8 hydroxyl to Tcm B3, a four-ring aromatic intermediate in the tetracenomycin biosynthetic pathway of Streptomyces glaucescens. The gene encoding this enzyme was expressed in Streptomyces coelicolor CH999 together with the actinorhodin polyketide synthase (PKS) gene cluster, which is responsible for the biosynthesis of 3,8-dihydroxy-methylanthraquinone carboxylic acid (DMAC) and its decarboxylated analog, aloesaponarin. The resulting recombinant strain produced approximately equal quantities of aloesaponarin and a new product but no DMAC. Spectroscopic analysis revealed that the novel polyketide was the 3-O-methylated analog of DMAC. An in vitro radioisotopic assay was developed for tcmO. The enzyme requires S-adenosylmethionine as a co-substrate. It has a Km of 3 microM and a kcat of 2.7 min-1 for DMAC. A series of monocyclic, bicyclic, and tricyclic aromatic compounds were also tested as candidate substrates in vitro. Remarkably, none was modified by tcmO within detectable limits of the assay. Together, these results highlight the interesting molecular recognition features of this enzyme. On one hand, there appears to be some flexibility in the number and structures of unreactive rings, since both Tcm and B3 and DMAC are good substrates. However, 6-methylsalicylic acid, a monocyclic analog of the reactive ring, is not recognized by the enzyme. Likewise, neither aloesaponarin (which only differs from DMAC in the reactive ring) nor carminic acid (which only differs in the distal nonreactive ring) is modified. Thus, the binding energy for the tcmO-catalyzed methyl transfer appears to involve significant contributions from both the aromaticity and the functionality of polycyclic substrates.

Anthraquinones↗
PubMed1996

Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector species.

Previous studies (Mertens et al., Virology 157, 375-386, 1987) have shown that removal of the outer capsid layer from bluetongue virus (BTV) significantly reduces (approximately x 10(-4)) the infectivity of the resultant core particle for mammalian cells (BHK 21 cells). In contrast, the studies reported here, using a cell line (KC cells) derived from a species of Culicoides that can act as a vector for BTV (Culicoides variipennis), demonstrated a much higher infectivity of core particles than that in mammalian cells (approximately x 10(3)). This increase resulted in a specific infectivity for cores that was only 20-fold less than that of purified disaggregated virus particles (stored in the presence of 0.1% sodium-N-lauroylsarcosine (NLS)). Removal of this detergent caused intact virus particle aggregation and (as previously reported) resulted in an approximately 1 log10 drop in the specific infectivity of those virus particles which remained in suspension. In consequence the specific infectivity of core particles for the KC cells was directly comparable to that of the intact but aggregated virus. These data are compared with the results from oral infectivity studies using two vector species (C. variipennis and Culicoides nubeculosus), which showed similar infection rates at comparable concentrations of purified cores, or of the intact but aggregated virus particles (NLS was toxic to adult flies). The role of the outer core proteins (VP7) in cell attachment and penetration, as an alternative route of initiation of infection, is discussed. Previous studies (Mertens et al., Virology 157, 375-386, 1987) also showed that the outer capsid layer of BTV can be modified by proteases (including trypsin or chymotrypsin), thereby generating infectious subviral particles (ISVP). The specific infectivity of ISVP for mammalian cells (BHK21 cells) was shown to be similar to that of disaggregated virus particles. In contrast, we report a significantly higher specific infectivity of ISVP but not of the intact virus (approximately x 100) for two insect cell lines (KC cells and C6/36 mosquito cells (derived from Aedes albopictus)). In oral infection studies with adults of the two vector species, ISVP produced the same infection rate at approximately 100-fold lower concentrations than either core particles or the intact but aggregated virus particles. The importance of mammalian host serum proteases, or insect gut proteases, in modification of the intact virus particle to form ISVP and their role in initiation of infection and the vector status of the insect is discussed.

Animals↗
PubMed1996

Magnetic stimulation of muscle evokes cerebral potentials by direct activation of nerve afferents: a study during muscle paralysis.

We tested the hypothesis that magnetic stimulation of muscle evokes cerebral potentials by causing a muscle contraction that then activates muscle receptors. We measured cerebral evoked potentials accompanying magnetic stimulation of muscle in 3 patients during surgery both before and after muscle paralysis with succinylcholine, a depolarizing agent. The magnetic stimulation was at low intensity (30%) and at a 2/s rate. The administration of succinylcholine sufficient to produce muscle paralysis did not alter cerebral potentials evoked by either low-intensity magnetic stimulation of muscle (gastrocnemius/soleus) or electrical stimulation of peripheral nerve (tibial nerve). In 1 normal subject, the S1 nerve root action potentials conducting at rapid velocity (> 60 m/s) were detected at the S1 foramen with a needle electrode using electrical stimulation of the tibial nerve. However, no S1 nerve root potentials could be identified to magnetic stimulation of muscle that evoked a cerebral potential. We conclude that magnetic stimulation of muscle activates terminal afferents in the muscle to provide the afferent drive for the cerebral potentials independent of muscle contraction. The failure to detect the afferent volley in S1 nerve root to magnetic stimulation suggests that only a few afferents are activated or that the activation of afferents is temporally dispersed.

Adult↗
PubMed1996

Antibiotic activity of polyketide products derived from combinatorial biosynthesis: implications for directed evolution.

A library of over 100 polyketides, generated via combinatorial cloning of genes encoding subunits of aromatic polyketide synthases, was screened for molecules capable of inhibiting the growth of gram-positive bacteria. A total of 26 polyketides, with varying levels of antibiotic activity in filter-disk assays, were purified. Most bioactive polyketides were produced as relatively minor compounds (< 1 mg/l), although two major anthraquinones, with yields in the range of 10-100 mg/l, were also identified and structurally characterized. When tested against Bacillus subtilis 168 beta, they were found to cause a 50% reduction in colony-forming units at concentrations of 20 and 300 micrograms/ml, respectively. We speculate that many of the minor (and possibly more potent) bioactive polyketides are synthesized via nonspecific enzymatic modifications of shunt products derived from engineered polyketide synthase pathways. If so, then these 'fortuitous' pathways should be amenable to further rationally guided manipulation. Our results support the notion that combinatorial biosynthesis can be used to generate novel, biologically active molecules. They also point to the feasibility of designing mutagenesis selection experiments aimed at the directed evolution of organic molecules with desirable pharmaceutical properties.

Anti-Bacterial Agents↗
PubMed1996

Deciphering the biosynthetic origin of the aglycone of the aureolic acid group of anti-tumor agents.

BACKGROUND: Mithramycin, chromomycin, and olivomycin belong to the aureolic acid family of clinically important anti-tumor agents. These natural products share a common aromatic aglycone. Although isotope labeling studies have firmly established the polyketide origin of this aglycone, they do not distinguish between alternative biosynthetic models in which the aglycone is derived from one, two or three distinct polyketide moieties. We set out to determine the biosynthetic origin of this moiety using a recombinant approach in which the ketosynthase and chain-length factor proteins from the antibiotic-producer strain, which determine the chain length of a polyketide, are produced in a heterologous bacterial host. RESULTS: The ketosynthase and chain-length factor genes from the polyketide synthase gene cluster from the mithramycin producer, Streptomyces argillaceus ATCC 12956, and the acyl carrier protein and ketoreductase genes from the actinorhodin polyketide synthase were expressed in Streptomyces coelicolor CH999. The recombinant strain produced a 20-carbon polyketide, comprising the complete backbone of the aglycone of mithramycin. CONCLUSIONS: The aglycone moieties of mithramycin, chromomycin, and olivomycin are derived from a single polyketide backbone. The nascent polyketide backbone must undergo a series of regiospecific cyclizations to form a tetracenomycin-like tetracyclic intermediate. The final steps in the aglycone biosynthetic pathway presumably involve decarboxylation and oxidative cleavage between C-18 and C-19, followed by additional oxidation, reduction, and methylation reactions.

Antibiotics, Antineoplastic↗
PubMed1996

Engineered biosynthesis of novel polyketides: properties of the whiE aromatase/cyclase.

The ORFVI from the cluster of genes, which is responsible for the biosynthesis of the Streptomyces coelicolor spore pigment, the whiE cluster, has been described as a bifunctional aromatase/cyclase. In order to evaluate its potential use for generating novel polyketides, combinations of this gene with those encoding minimal polyketide synthase enzymes with or without a ketoreductase from S. coelicolor A3(2) were constructed and analyzed in vivo. Analysis of the polyketide products generated from these constructs indicates that the whiE-ORFVI enzyme has properties similar to those of TcmN, although the whiE aromatase/cyclase normally acts on a polyketide intermediate that is four carbons longer than the TcmN substrate. The whiE aromatase/cyclase can influence the regiospecificity of the first cyclization of unreduced, but not reduced, backbones and is also responsible for the second ring aromatization. An unusual new polyketide, EM18, was identified which is not seen in equivalent strains expressing the tcmN aromatase/cyclase or the act aromatase genes. The structure of EM18 suggests that the WhiE-ORFVI product might have some unique properties within this family of polyketide synthase subunits, and may therefore be useful in the design of combinatorial biosynthetic strategies.

Anti-Bacterial Agents↗