Biomedical subjects

H Fu

Publications and source records attributed to H Fu.

At least 73 records · Page 4Linked to original sources

PubMed1999

Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa.

The 14-3-3 proteins are a family of conserved, dimeric proteins that interact with a diverse set of ligands, including molecules involved in cell cycle regulation and apoptosis. It is well-established that 14-3-3 binds to many ligands through phosphoserine motifs. Here we characterize the interaction of 14-3-3 with a nonphosphorylated protein ligand, the ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa. By using affinity chromatography and surface plasmon resonance, we show that the zeta isoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro. The interaction between ExoS and 14-3-3zeta is of high affinity, with an equilibrium dissociation constant of 7 nM. ExoS lacks any known 14-3-3 binding motif, but to address the possibility that 14-3-3 binds a noncanonical phosphoserine site, we assayed ExoS for protein-bound phosphate by using mass spectrometry. No detectable phosphoproteins were found. A phosphopeptide ligand of 14-3-3, pS-Raf-259, was capable of inhibiting the binding of 14-3-3 to ExoS, suggesting that phosphorylated and nonphosphorylated ligands may share a common binding site, the conserved amphipathic groove. It is conceivable that 14-3-3 proteins may bind both phosphoserine and nonphosphoserine ligands in cells, possibly allowing kinase-dependent as well as kinase-independent regulation of 14-3-3 binding.

14-3-3 Proteins↗

PubMed1999

Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel "unnatural" natural products.

The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds-"unnatural" natural products-by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.

Catalytic Domain↗

PubMed1999

Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzyme complexes that are organized into modules, where each module carries the domains needed to catalyze the condensation of an extender unit onto a growing polyketide chain. Each module also dictates the stereochemistry of the chiral centers introduced into the backbone during the chain elongation process. Here we used domain mutagenesis to investigate the role of the acyl transferase (AT) domains of individual modules in the choice and stereochemical fate of extender units. Our results indicate that the AT domains of DEBS do not influence epimerization of the (2S)-methylmalonyl-CoA extender units. Hence, stereochemical control of the methyl-branched centers generated by DEBS most likely resides in the ketosynthase (KS) domains of the individual modules. In contrast, several recent studies have demonstrated that extender unit specificity can be altered by AT domain substitution. In some of these examples, the resulting polyketide was produced at considerably lower titers than the corresponding natural product. We analyzed one such attenuated mutant of DEBS, in which the methylmalonyl transferase domain of module 2 was replaced with a malonyl transferase domain. As reported earlier, the resulting PKS produced only small quantities of the expected desmethyl analogue of 6-deoxyerythronolide B. However, when the same hybrid module was placed as the terminal module in a truncated 2-module PKS, it produced nearly normal quantities of the expected desmethyl triketide lactone. These results illustrate the limits to modularity of these multifunctional enzymes. To dissect the role of specific amino acids in controlling AT substrate specificity, we exchanged several segments of amino acids between selected malonyl and methylmalonyl transferases, and found that a short (23-35 amino acid) C-terminal segment present in all AT domains is the principal determinant of their substrate specificity. Interestingly, its length and amino acid sequence vary considerably among the known AT domains. We therefore suggest that the choice of extender units by the PKS modules is influenced by a "hypervariable region", which could be manipulated via combinatorial mutagenesis to generate novel AT domains possessing relaxed or altered substrate specificity.

Acyl Coenzyme A↗

PubMed1999

Mass spectra of aminoacyl adenylate pentacoordinated phosphorus compounds

Aminoacyl adenylate pentacoordinated phosphorus compounds were analyzed by field desorption (FD) and fast atom bombardment (FAB) mass spectrometries, together with the B/E linked scan technique, and their mass spectral fragmentation pathways were investigated. For the five bonds (one P-N bond, three P-O bonds and one mixed anhydride bond P-O-CO), the cleavage usually occurred more on the P-N bond, the mixed anhydride bond and the O-C bond adjacent to the P-O bond, and less on the P-O bond. Ion YH(+), corresponding to water loss from protonated 2',3'-O-isopropylidene-adenosine, was the base peak. The results reflect the structural characteristics of aminoacyl adenylate pentacoordinated phosphorus compounds. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗

PubMed1999

The barriers to bluetongue virus infection, dissemination and transmission in the vector, Culicoides variipennis (Diptera: Ceratopogonidae).

Transmission of bluetongue virus (BTV) by a vector species of Culicoides was studied using immunohistochemistry, virus titration and in vitro transmission tests. Adult female C. variipennis were used from two colonies that are either "transmission competent" or "transmission refractory" after oral infection with BTV. Intrathoracic (i.t.) injection of BTV into the haemocoel always resulted in a fully disseminated infection and transmission of virus in saliva. However, after ingestion of an infectious blood meal, only 30% (approximately) of midges from either colony became persistently infected. Although none of the orally infected insects from the "refractory" colony were able to transmit virus, 12% of those from the "competent" colony (containing > or = 10(3.0)TCID50 of virus/midge) did transmit BTV in their saliva. The most important barriers to BTV transmission in Culicoides vector species appeared to be a mesenteron infection barrier (MIB), which controls initial establishment of persistent infection, a mesenteron escape barrier (MEB) which can restrict virus to gut cells and a dissemination barrier (DB) which can prevent virus which enters the haemocoel from infecting secondary target organs. Culicoides variipennis do not appear to present either a salivary gland infection barrier (SGIB), or a salivary gland escape barrier (SGEB) to BTV.

Animals↗

PubMed1999

Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase.

BACKGROUND: A single modular polyketide synthase (PKS) gene cluster is responsible for production of both the 14-membered macrolide antibiotic picromycin and the 12-membered macrolide antibiotic methymycin in Streptomyces venezuelae. Building on the success of the heterologous expression system engineered using the erythromycin PKS, we have constructed an analogous system for the picromycin/methymycin PKS. Through heterologous expression and construction of a hybrid PKS, we have examined the contributions that the PKS, its internal thioesterase domain (pikTE) and the Pik TEII thioesterase domain make in termination and cyclization of the two polyketide intermediates. RESULTS: The picromycin/methymycin PKS genes were functionally expressed in the heterologous host Streptomyces lividans, resulting in production of both narbonolide and 10-deoxymethynolide (the precursors of picromycin and methymycin, respectively). Co-expression with the Pik TEII thioesterase led to increased production levels, but did not change the ratio of the two compounds produced, leaving the function of this protein largely unknown. Fusion of the PKS thioesterase domain (pikTE) to 6-deoxyerythronolide B synthase (DEBS) resulted in formation of only 14-membered macrolactones. CONCLUSIONS: These experiments demonstrate that the PKS alone is capable of catalyzing the synthesis of both 14- and 12-membered macrolactones and favor a model by which different macrolactone rings result from a combination of the arrangement between the module 5 and module 6 subunits in the picromycin PKS complex and the selectivity of the pikTE domain.

Amino Acid Sequence↗

PubMed1999

Structure and functional analysis of the 26S proteasome subunits from plants.

As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN 10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10delta strains which grow normally, Physcomitrella rpn10delta strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.

Arabidopsis↗

PubMed1999

Functional analysis of the proteasome regulatory particle.

We have developed S. cerevisiae as a model system for mechanistic studies of the 26S proteasome. The subunits of the yeast 19S complex, or regulatory particle (RP), have been defined, and are closely related to those of mammalian proteasomes. The multiubiquitin chain binding subunit (S5a/Mcb1/Rpn10) was found, surprisingly, to be nonessential for the degradation of a variety of ubiquitin-protein conjugates in vivo. Biochemical studies of proteasomes from deltarpn10 mutants revealed the existence of two structural subassemblies within the RP, the lid and the base. The lid and the base are both composed of 8 subunits. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The base is sufficient to activate the 20S core particle for degradation of peptides, but the lid is required for ubiquitin-dependent degradation. The lid subunits share sequence motifs with components of the COP9/signalosome complex, suggesting that these functionally diverse particles have a common evolutionary ancestry. Analysis of equivalent point mutations in the six ATPases of the base indicate that they have well-differentiated functions. In particular, mutations in one ATPase gene, RPT2, result in an unexpected defect in peptide hydrolysis by the core particle. One interpretation of this result is that Rpt2 participates in gating of the channel through which substrates enter the core particle.

Adenosine Triphosphatases↗

PubMed1999

Structural and functional analysis of the six regulatory particle triple-A ATPase subunits from the Arabidopsis 26S proteasome.

The 26S proteasome is a multi-subunit ATP-dependent protease responsible for degrading most short-lived intracellular proteins targeted for breakdown by ubiquitin conjugation. The complex is composed of two relatively stable subparticles, the 20S proteasome, a hollow cylindrical structure which contains the proteolytic active sites in its lumen, and the 19S regulatory particle (RP) which binds to either end of the cylinder and provides the ATP-dependence and the specificity for ubiquitinated proteins. Among the approximately 18 subunits of the RP from yeast and animals are a set of six proteins, designated RPT1-6 for regulatory particle triple-A ATPase, that form a distinct family within the AAA superfamily. Presumably, these subunits use ATP hydrolysis to help assemble the 26S holocomplex, recognize and unfold appropriate substrates, and/or translocate the substrates to the 20S complex for degradation. Here, we describe the RPT gene family from Arabidopsis thaliana. From a collection of cDNAs and genomic sequences, a family of genes encoding all six of the RPT subunits was identified with significant amino acid sequence similarity to their yeast and animal counterparts. Five of the six RPT sub- units are encoded by two genes; the exception being RPT3 which is encoded by a single gene. mRNA for each of the six proteins is present in all tissue types examined. Five of the subunits (RPT1 and 3-6) complemented yeast mutants missing their respective orthologs, indicating that the yeast and Arabidopsis proteins are functionally equivalent. Taken together, these results demonstrate that the RP, like the 20S proteasome, is functionally and structurally conserved among eukaryotes and indicate that the plant RPT subunits, like their yeast counterparts, have non-redundant functions.

Adenosine Triphosphatases↗

PubMed1999

Agonist-like activity of antibodies to angiotensin II receptor subtype 1 (AT1) from rats immunized with AT1 receptor peptide.

In the present study, rats were immunized with angiotensin II receptor subtype 1 (AT1) receptor peptides for 3 months to see if the immunization produced specific anti-AT1 receptor antibodies and if continuous stimulation for 3 months affected blood pressure or induced morphological changes in the organs containing AT1 receptors. Our results showed that there were constant high levels of circulating antibodies throughout the study period in all rats of the immunized group, but not in the control rats, and that there were almost no significant cross-reactions of antisera with AT2 receptor peptide and alpha1 adrenoceptor peptide, except in four rats, which showed low cross-reactions with alpha1 adrenoceptor and AT2 receptor peptides. When an affinity-purified anti-AT1 receptor antibody was used, it specifically displayed the AT1-stimulatory positive chronotropic effect and also localized AT1 receptors. However, in the immunized group, saturation binding of AT1 in homogenates from kidneys showed no difference either in maximal binding sites (Bmax) or in antagonist affinity (Kd). No difference in mRNA of AT1a was found in either kidney or heart, and no morphological changes in the organs were observed, as compared with the control group. Furthermore, immunization did not cause hypertension. In conclusion, the synthetic peptide corresponding to the second extra-cellular loop of the human AT1 receptor was able to produce highly specific and functionally active anti-AT1 receptor antibodies, but unable to induce pathological structural changes or hypertension.

Amino Acid Sequence↗

PubMed1999

Multiubiquitin chain binding subunit MCB1 (RPN10) of the 26S proteasome is essential for developmental progression in Physcomitrella patens.

The 26S proteasome, a multisubunit complex, is the primary protease of the ubiquitin-mediated proteolytic system in eukaryotes. We have recently characterized MCB1 (RPN10), a subunit of the 26S complex that has affinity for multiubiquitin chains in vitro and as a result may function as a receptor for ubiquitinated substrates. To define the role of MCB1 further, we analyzed its function in Physcomitrella patens by generating MCB1 gene disruptions using homologous recombination. PpMCB1, which is 50 to 75% similar to orthologs from other eukaryotes, is present in the 26S proteasome complex and has a similar affinity for multiubiquitin chains, using a conserved hydrophobic domain within the C-terminal half of the polypeptide. Unlike yeast Deltamcb1 strains, which grow normally, P. patens Deltamcb1 strains are viable but are under developmental arrest, generating abnormal caulonema that are unable to form buds and gametophores. Treatment with auxin and cytokinin restored bud formation and subsequent partial development of gametophores. Complementation of a Deltamcb1 strain with mutated versions of PpMCB1 revealed that the multiubiquitin chain binding site is not essential for the wild-type phenotype. These results show that MCB1 has an important function in the 26S proteasome of higher order eukaryotes in addition to its ability to bind multiubiquitin chains, and they provide further support for a role of the ubiquitin/26S proteasome proteolytic pathway in plant developmental processes triggered by hormones.

Amino Acid Sequence↗

PubMed1999

[Relationships of angiotensinogen gene M235T variant with diabetic nephropathy in Chinese type 2 diabetes mellitus].

OBJECTIVE: To investigate whether angiotensinogen(AGT) gene M235T variant is associated with type 2 diabetes mellitus without nephropathy (DM) and/or diabetic nephropathy (DN) in Chinese type 2 diabetes. METHODS: AGT genotypes of 84 cases with DM, 96 with DN and 98 controls were determined by polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism analysis of the region of the variant. RESULTS: Increased frequencies of T allele (0.82) and TT genotype (0.70) were observed in 96 subjects with DN, compared with those observed in 98 control subjects (0.63 and 0. 43 respectively, P = 0.003, P = 0.0004). The odds ratio associated with TT genotype was 3.47 (95% CI 1.51-7.94, P = 0.0033) for DN in analysis adjusted for several DN risk factors. There was no difference in allele distribution between 84 DM patients and the controls. CONCLUSION: TT genotype of the AGT gene might be an independent risk factor of diabetic nephropathy in Chinese patients with type 2 diabetes mellitus.

Adult↗

PubMed1999

Xylene-induced effects on brain neurotransmitters, behavior and fos protein in rats.

Male Sprague-Dawley rats administered xylene intraperitoneally on alternate days at a dose of 125 or 250 mg/kg for 30 days exhibited no marked changes in locomotor activity, learning and memory capacity. However in rats given xylene on alternate day at a dose of 500 mg/kg for 30 days, a significant decrease in locomotor activity, deficits in learning ability and memory loss were detected. These xylene-induced behavioral changes were associated with a decrease in beta-endorphin and leuenkaphlin concentrations in the pons-medulla. On the contrary, xylene at a dose of 500 mg/kg increased the beta-endorphin level in caudate and c-fos expression in hippocampus. These data suggest that the xylene-induced behavioral alterations might be associated with the expression of Fos protein in the hippocampus.

Animals↗

PubMed1999

Treatment of atrophic cholecystitis by regulating the spleen--a report of 50 cases.

Fifty cases of atrophic cholecystitis were treated by regulating of the spleen. Of them, 21 were cured, 18 remarkably effective, and 7 effective. The overall effective rate was 92.0%. As compared with the results of ultrasonography B performed before and after treatment, it was shown that both the longitudinal and transverse inner diameters of the gallbladder increased evidently, and the condition of atrophy improved remarkably after treatment.

Adolescent↗

PubMed1999

The change of insulin-like growth factor-1 in diabetic patients with neuropathy.

OBJECTIVE: To evaluate the possible relationship between insulin-like growth factor-1 (IGF-1) and diabetic neuropathy (DNP). METHODS: Sixty-nine patients with Type 2 non-insulin-dependent diabetes (54 with peripheral neuropathy and 15 without neuropathy) were observed. Normal controls were 34 non-diabetic persons. Diabetic peripheral neuropathy diagnosis was carried out taking into account results of NS, ND, NC and AF. After an overnight fast, blood was taken for IGF-1, glucose, hemoglobin Alc, C-peptide, and insulin. Plasma IGF-1 was measured by radioimmunoassay (RIA) method. RESULTS: The neuropathic group had significantly lower levels for IGF-1 (86.43 ng/ml +/- 45.18 ng/ml) compared to normal controls (119.68 ng/ml +/- 89.42 ng/ml) (P < 0.05), and to diabetic patients without neuropathy (113.75 ng/ml +/- 66.58 ng/ml) (P < 0.05). No significant difference was found between diabetic non-neuropathic group and normal control subjects (P < 0.05). In diabetic subjects there was a positive correlation (gamma = 0.27, P < 0.05) between IGF-1 and beat to variation in heart rate. There were negative correlation between IGF-1 and postprandial blood glucose (gamma = -0.3, P < 0.05), and aspartic acid translocase (gamma = -0.27, P < 0.05). CONCLUSION: In diabetic patients with peripheral neuropathy there are abnormalities of IGF-1 that may contribute to the pathogeneses of diabetic neuropathy.

Aged↗

PubMed1999

[Clinical analysis of percutaneous balloon mitral valvotomy in 1063 patients].

This article reports the clinical study of 1063 patients who underwent percutaneous balloon mitral valvotomy (PBMV) and 1043 patients among them who did successfully (98.2%). After the procedure, left atrial mena pressure fell from 3.19 +/- 1.06 kPa to 2.00 +/- 0.93 kPa (P < 0.001), mitral pressure gradient decreased from 2.79 +/- 1.20 kPa to 1.33 +/- 1.06 kPa (P < 0.001). Disappearance of diastolic murmur and significant improvement of cardiac function were achieved. Among the 20 failed cases, there were cardiac perforation and/or pericardial tamponade (8 cases), cerebral embolism (3 cases), severe mitral regurgitation (3 cases), atrial septal defect (2 cases) and discontinuance of the procedure due to various causes. The short-term outcome of PBMV is satisfying, so it can replace the surgical closed valvotomy as an effective treatment for selected patients with mitral stenosis.

Adolescent↗

PubMed1999

[Gas chromatography/mass spectrometry analysis for determination of methyl alpha-(6-methoxyl-2-naphthyl) propionate from carbonylation].

alpha-(6-Methoxyl-2-naphthyl) ethanol can be carbonylated to form methyl-alpha-(6-methoxyl-2-naphthyl)propionate catalyzed by a catalyst in the pressnce of methanol. High efficiency capillary gas chromatography and GC/MS technique were used for determining products which were obtained from the carbonylation. An OV-101 fused silica capillary column (25 m x 0.2 mm i.d.) and a flame ionization detector (FID) were employed in the GC analysis. The column temperature was kept at 250 degrees C. Mass spectra were obtained by electron impact at 70 eV. The structures of four compounds were identified. The major product is methyl-alpha-(6-methoxy-2-naphthyl) propionate, and the two by-products are 2-vinyl-6-methoxynaphthalene and 1-methoxy-(6'-methoxy-2'-naphthyl)ethanol.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1998

A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein.

Protein kinases activated by sphingosine or N,N'-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739-747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50-60 kDa that is strongly activated by N, N'-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the alpha and zeta isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (eta, beta, zeta) but not others (sigma, tau). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 beta. The sigma and tau isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N, N'-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent phosphorylation of specific 14-3-3 isoforms that regulate signal transduction. In view of the three-dimensional structure of 14-3-3, it is likely that phosphorylation by SDK1 would alter dimerization of 14-3-3, and/or induce conformational changes that alter 14-3-3 association with other kinases involved in signal transduction.

14-3-3 Proteins↗