Biomedical subjects

H Fu

Publications and source records attributed to H Fu.

At least 91 records · Page 5Linked to original sources

PubMed1998

Mutations in the hydrophobic surface of an amphipathic groove of 14-3-3zeta disrupt its interaction with Raf-1 kinase.

14-3-3 proteins bind to a diverse group of regulatory molecules such as Raf-1, Cbl, and c-Bcr that are involved in signal transduction pathways. The crystal structure of 14-3-3zeta reveals a conserved amphipathic groove that may mediate the association of 14-3-3 with diverse ligands. Consistently, mutations on the charged surface of the groove (Lys-49, Arg-56, and Arg-60) decrease the binding of 14-3-3zeta to the ligands tested (Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H. (1997) J. Biol. Chem. 272, 13717-13724). Here we report that mutations that altered the hydrophobic property of the groove, V176D, L216D, L220D, and L227D, disrupted the interaction of 14-3-3zeta with Raf-1 kinase. The reduced binding of the 14-3-3zeta mutants to Raf-1 was apparently not because of gross structural changes in the mutants as judged by their ability to form dimers, by partial proteolysis profiles, and by circular dichroism analysis. These hydrophobic residues appeared to be required for the binding of 14-3-3zeta to distinct activation states of Raf-1 because mutations V176D, L216D, L220D, and L227D reduced the interaction of 14-3-3zeta with Raf-1 from both phorbol 12-myristate 13-acetate-stimulated and unstimulated Jurkat T cells. These same mutations also disrupted the association of 14-3-3zeta with other regulatory molecules such as Cbl and c-Bcr, suggesting that the hydrophobic surface of the amphipathic groove represents part of a binding site shared by a number of 14-3-3-associated proteins. The conservation of the hydrophobic residues Val-176, Leu-216, Leu-220, and Leu-227 among known 14-3-3 family members implies their general importance in ligand binding.

14-3-3 Proteins↗

PubMed1998

14-3-3zeta binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove.

14-3-3 proteins bind a variety of molecules involved in signal transduction, cell cycle regulation and apoptosis. 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus motif, RSXpSXP, but must bind unphosphorylated ligands, such as glycoprotein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif. Here we report the crystal structures of the zeta isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS-Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived from phage display (R18, PHCVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1. The two peptides bind within a conserved amphipathic groove on the surface of 14-3-3 at overlapping but distinct sites. The phosphoserine of pS-Raf-259 engages a cluster of basic residues (Lys49, Arg56, Arg60, and Arg127), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating the same basic cluster. 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 A apart. The ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins.

14-3-3 Proteins↗

PubMed1998

Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1.

The 26 S proteasome is a multisubunit proteolytic complex responsible for degrading eukaryotic proteins targeted by ubiquitin modification. Substrate recognition by the complex is presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through lysine 48. We have identified previously a candidate for one such receptor from diverse species, designated here as Mcb1 for Multiubiquitin chain-binding protein, based on its ability to bind Lys48-linked multiubiquitin chains and its location within the 26 S proteasome complex. Even though Mcb1 is likely not the only receptor in yeast, it is necessary for conferring resistance to amino acid analogs and for degrading a subset of ubiquitin pathway substrates such as ubiquitin-Pro-beta-galactosidase (Ub-Pro-beta-gal) (van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R. D. (1996) Mol. Cell. Biol. 16, 6020-28). To further define the role of Mcb1 in substrate recognition by the 26 S proteasome, a structure/function analysis of various deletion and site-directed mutants of yeast and Arabidopsis Mcb1 was performed. From these studies, we identified a single stretch of conserved hydrophobic amino acids (LAM/LALRL/V (ScMcb1 228-234 and At-Mcb1 226-232)) within the C-terminal half of each polypeptide that is necessary for interaction with Lys48-linked multiubiquitin chains. Unexpectedly, this domain was not essential for either Ub-Pro-beta-gal degradation or conferring resistance to amino acid analogs. The domain responsible for these two activities was mapped to a conserved region near the N terminus. Yeast and Arabidopsis Mcb1 derivatives containing an intact multiubiquitin-binding site but missing the N-terminal region failed to promote Ub-Pro-beta-gal degradation and even accentuated the sensitivity of the yeast delta mcb1 strain to amino acid analogs. This hypersensitivity was not caused by a gross defect in 26 S proteasome assembly as mutants missing either the N-terminal domain or the multiubiquitin chain-binding site could still associate with 26 S proteasome and generate a complex indistinguishable in size from that present in wild-type yeast. Together, these data indicate that residues near the N terminus, and not the multiubiquitin chain-binding site, are most critical for Mcb1 function in vivo.

Amino Acid Sequence↗

PubMed1998

Epitope mapping of mouse monoclonal antibody EP-5C7 which neutralizes both human E- and P-selectin.

The epitope of mouse monoclonal antibody (mAb) EP-5C7, which binds to and blocks both human E- and P-selectin, was mapped onto the protein structure of E-selectin. Analyses with E- and L-selectin chimeric proteins and randomly mutagenized E-selectins demonstrated that the EP-5C7 epitope consists of the amino acid residues at positions 21, 22, 23, 119 and 120 of E-selectin. The binding of three neutralizing anti-E-selectin mAb's (E-1E4, H18/7 and CL2), whose epitopes were found to overlap with the E-selectin binding site for carbohydrate ligands, was not affected by the amino acid substitutions at these five positions. Inspection of the three-dimensional structure of E-selectin indicated that the EP-5C7 epitope is located near the junction between the lectin and EGF-like domains. The ligand binding site was distant from the EP-5C7 epitope, suggesting that the amino acid residues in the EP-5C7 epitope play an important role other than ligand binding in selectin-mediated cell adhesion.

Animals↗

PubMed1998

Phylogenetic analysis of African horse sickness virus segment 10: sequence variation, virulence characteristics and cell exit.

African horse sickness virus (AHSV) genome segment 10 encodes the non-structural proteins NS3/NS3a, which is involved in release of virus from cells. Full length segment 10 cDNAs were amplified by reverse transcription-polymerase chain reaction, from isolates of AHSV serotypes 2, 3, 4, 5, 7, 8 and 9. These cDNAs were cloned, sequenced and their phylogenetic relationships analysed. High levels of sequence homology were detected in segment 10 from some isolates of different serotypes, confirming that they could be grouped on this basis (serotypes 4, 5, 6 and 9 (group alpha); serotypes 3 and 7 (group beta); serotypes 1, 2, and 8 (group gamma). However, data from bluetongue virus (the prototype orbivirus) indicate that the AHSV serotype is determined exclusively by the structural outer coat proteins VP2 and VP5, encoded by genome segments 2 and 5 respectively. Therefore, as a direct consequence of genome segment reassortment between AHSV strains from different serotypes, the differences observed in segment 10 do not give a reliable indication of virus serotype. Segment 10 of AHSV 3 (virulent) and AHSV 3att (attenuated) were also analysed. These strains, together with AHSV 8, have been used to study of the genetic basis of virulence using reassortment (O'Hara et al., this publication). Virus release studies, using Culicoides cell cultures, indicate that differences in segment 10 of AHSV 3att and 8 can influence the timing of virus release from the infected cell.

African Horse Sickness Virus↗

PubMed1998

Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties.

The rice Waxy gene encodes a granule-bound starch synthase (GBSS) necessary for the synthesis of amylose in endosperm tissue. We have previously shown that a CT microsatellite near the transcriptional start site of the GBSS gene can distinguish 7 alleles that accounted for more than 80% of the variation in apparent amylose content in an extended pedigree of 89 US rice cultivars (Oryza sativa L.). Furthermore, all the cultivars with 18% or less amylose were shown to have the sequence AGTTATA at the putative leader intron 5' splice site, while all cultivars with a higher proportion of amylose had AGGTATA. Here we demonstrate that this single-base mutation reduces the efficiency of GBSS pre-mRNA processing and results in alternate splicing at three cryptic sites. The predominant 5' splice site in CT18 low-amylose varieties is 93 bp upstream of the splice site used in intermediate and high amylose varieties and is immediately 5' to the CT microsatellite that we previously demonstrated to be tightly correlated with amylose content. Use of the leader intron 5' splice site at either -93 or -1 in conjunction with the predominant 3' splice site results in formation of a small open reading frame 38 bp upstream of the normal ATG and out of frame with it. This open reading frame is not produced when any of the 5' leader intron splice sites are used in conjunction with an alternate 3' splice site five bases further downstream which was observed in all rice varieties tested.

Alternative Splicing↗

PubMed1998

Epstein-Barr-based episomal chromosomes shuttle 100 kb of self-replicating circular human DNA in mouse cells.

We describe the microcell fusion transfer of 100-200 kb self-replicating circular human minichromosomes from human into mouse cells. This experimental approach is illustrated through the shutting of the latent 170 kb double-stranded DNA genome from the human herpesvirus, Epstein-Barr virus, into nonpermissive rodent cells. Using this interspecies transfer strategy, circular episomes carrying 95-105 kb of human DNA were successfully established at low copy number in mouse A9 cells. Selected episomes were stably maintained for 6 months, and unselected episomes were characterized by a 95% episomal retention per cell division. The establishment of a mouse artificial episomal chromosome system should facilitate evolutionary and therapeutic studies of large human DNA in rodent genetic backgrounds.

Animals↗

PubMed1998

Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana.

The 20S proteasome is the proteolytic complex in eukaryotes responsible for degrading short-lived and abnormal intracellular proteins, especially those targeted by ubiquitin conjugation. The 700-kD complex exists as a hollow cylinder comprising four stacked rings with the catalytic sites located in the lumen. The two outer rings and the two inner rings are composed of seven different alpha and beta polypeptides, respectively, giving an alpha7/beta7/beta7/alpha7 symmetric organization. Here we describe the molecular organization of the 20S proteasome from the plant Arabidopsis thaliana. From an analysis of a collection of cDNA and genomic clones, we identified a superfamily of 23 genes encoding all 14 of the Arabidopsis proteasome subunits, designated PAA-PAG and PBA-PBG for Proteasome Alpha and Beta subunits A-G, respectively. Four of the subunits likely are encoded by single genes, and the remaining subunits are encoded by families of at least 2 genes. Expression of the alpha and beta subunit genes appears to be coordinately regulated. Three of the nine Arabidopsis proteasome subunit genes tested, PAC1 (alpha3), PAE1 (alpha5) and PBC2 (beta3), could functionally replace their yeast orthologs, providing the first evidence for cross-species complementation of 20S subunit genes. Taken together, these results demonstrate that the 20S proteasome is structurally and functionally conserved among eukaryotes and suggest that the subunit arrangement of the Arabidopsis 20S proteasome is similar if not identical to that recently determined for the yeast complex.

Amino Acid Sequence↗

PubMed1998

Long term follow-up of patients with systemic lupus erythematosus.

To assess the impact of demographic and clinical factors on prognosis in patients with systemic lupus erythematosus (SLE), we studied a cohort composed of 566 patients in Huashan Hospital between 1959 and 1992 who were followed up to June 30, 1993. The survivorship was examined through life table analysis. The results showed that the survival rate from the time of SLE onset was 93% at 1 year, 73% at 5 years, and 60% at 10 years. On univariate analysis, we found that the following factors worsened the probability of survival: male, neuropsychiatric manifestations, pleurisy-pericarditis, hematological disorders, renal involvement, hypocomplementemia, abnormal electrocardiograph, and high corticosteroid dose of treatment. The time that the C3 depression occurred in the course of SLE affected the survival more significantly than did its decreased levels. The earlier the occurrence of C3 depression, the lower the patient's survival probability. On multivariate analysis, the independent risk factors were male gender, abnormal electrocardiograph, hypocomplementemia, and high corticosteroid dose of treatment. All of these indicated that clinical features of SLE might have value as predictors for its prognosis and that the occurrence of the decreased C3 in the early course of SLE might be the most important factor.

Adolescent↗

PubMed1998

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

Fifty cases of atrophic cholecystitis were treated mainly by regulation of the function of the spleen. Of them, 21 cases were cured, 18 markedly effective, and 7 effective. The total effective rate was 92.0%. By comparison of results of ultrasonography B performed before and after treatment, it was shown that both the longitudinal and transverse inner diameters of gallbladder cross section increased evidently, and the condition of atrophy was improved remarkably after treatment.

Adolescent↗

PubMed1998

[Synthesis and vasodilative activities of benzamide derivatives].

Twenty two benzamide derivatives were synthesized of which twenty one were not reported before. Vasodilative activity screening in vitro has shown that most of the compounds possess various activities, among which compound H1, H11, H17, E1, E3 demonstrated a superior pharmacological profile to the lead compound when inhibiting effect on the noradrenaline (10(-7) mol.L-1) induced contraction of rat aortic strip was chosen as the evaluation criterion, while H7,15, E7 exerted significant inhibiting action toward 85.7 mmol.L-1 KCl induced contraction of rat aortic strip. Further evaluation assays for their KCO potentials showed that E1 might be an ATP sensitive KCO. Preliminary structure-activity relationships of this kind of aromatic amides are discussed herein.

Animals↗

PubMed1998

[Study on tissue culture of Poligonum multiflorum].

In the present thesis, suitable medium for callus inducement and relationship between culture time and callus growth of Poligonum multiflorum were studied by mono-factorial and orthogonal design test. The experiments showed that the best medium for callus inducement was MS + 2,4-D 1 mg/L + 6-BA 1 mg/L + IBA 0.5 mg/L, dark culture was better than light culture, callus grew fast when it was cultured 12 days, whole plant could be induced by callus, the induce rate was 95%.

Culture Media↗

PubMed1998

[Comparison studies on pharmacodynamics of three kind of Carapax trionycis].

Three kinds of Carapax Trionycis were used in rat to prevent and treat syndrom-complex of dificiency of Yin. Compareing the improvement of symptoms of dificiency of Yin, it was found that all of them actted effectively in various degrees. The effect of ZhongHua Carapax Trionycis (ZHCT) was similar to those of Shan Rui Carapax Trionycis (SRCT), but the efficacy of Green Carapax Trionycis (GCT) was inferior to those of ZHCT and SRCT. All kinds of C. Trionycis could increase the function of responding to iriation in mice with deficiency of Yin. The intensity of ZHCT was similar to that of GCT, and superior to that of SRCT. They all could enhance phagocytic function of eticuloedothelial system obviously, promot the development of thymus in different degrees with no efect on spleen in young mice. The efficacy of increasing the immune function of ZHCT and GCT were more potent than that of SRCT. ZHCT and GCT significantly decreased the activity of SGPT in mice poisoned by CC14. ZHCT and GCT protected liver better than SRCT did.

Animals↗

PubMed1998

Reconstructing the size of the African American population by age and sex, 1930-1990.

We estimate the size of the African American population in five-year age groups at census dates from 1930 to 1990 using a three-part strategy. For cohorts born after 1935, we follow the U.S. Census Bureau in using classical demographic analysis. To estimate the size of cohorts born before 1895, we use extinct-generation estimates. For remaining cohorts, we implement an age/period/cohort model of census counts. All approaches are applied to a data set in which the age distribution of deaths has been corrected for age misreporting. Results provide strong confirmation of the basic validity of Census Bureau estimates of census undercounts for African Americans while extending estimates to new cohorts and periods. Our estimates are less consistent with an historical series prepared by Coale and Rives (1973).

Adolescent↗

PubMed1997

Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.

Growth factors can promote cell survival by activating the phosphatidylinositide-3'-OH kinase and its downstream target, the serine-threonine kinase Akt. However, the mechanism by which Akt functions to promote survival is not understood. We show that growth factor activation of the PI3'K/Akt signaling pathway culminates in the phosphorylation of the BCL-2 family member BAD, thereby suppressing apoptosis and promoting cell survival. Akt phosphorylates BAD in vitro and in vivo, and blocks the BAD-induced death of primary neurons in a site-specific manner. These findings define a mechanism by which growth factors directly inactivate a critical component of the cell-intrinsic death machinery.

3T3 Cells↗

PubMed1997

14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain.

Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.

14-3-3 Proteins↗

PubMed1997

Synthesis and in vitro activity of long-chain 5'-O-[(alkoxycarbonyl)phosphinyl]-3'-azido-3'-deoxythymidines against wild-type and AZT- and foscarnet-resistant strains of HIV-1.

Lipophilic esters of 3'-azido-3'-deoxy-5'-O-(carboxyphosphinyl)thymidine (PFA-AZT) were synthesized and tested for antiretroviral activity in CD4+ HT4-6C cells infected with either wild-type HIV-1LAI, a PFA-resistant strain encoding a single-point mutation in reverse transcriptase (E89K), or an AZT-resistant clinical isolate (A018-post). Arbuzov condensation of 1-octadecyl, 1-eicosanyl, and 1-docosanyl chloroformate with trimethyl phosphite yielded the corresponding dimethyl long-chain alkyl triesters of PFA. Selective removal of one methyl group from the triesters with sodium iodide yielded monosodium salts, whereas treatment with bromotrimethylsilane cleaved both methyl groups while leaving the long-chain alkyl group intact. Neutralization of the resulting [(alkyloxy)carbonyl]phosphonic acids with 2 equiv of sodium methoxide afforded disodium salts of the phosphonic acid moiety. Similar chemistry was used to obtain the mono- and disodium salts of the cholesterol ester of PFA. Reaction of the triesters with phosphorous pentachloride, followed by coupling with AZT and O-demethylation with sodium iodide, afforded 3'-azido-3'-deoxy-5'-O-[[(1-octadecyloxy)carbonyl]phosphinyl ]thymidine (9a), 3'-azido-3'-deoxy-5'-O-[[(1-eicosanyloxy)carbonyl]phosphinyl ]thymidine (9b), 3'-azido-3'-deoxy-5'-O-[[(1-docosanyloxy)carbonyl]phosphinyl ]thymidine (9c), and 3'-azido-3'-deoxy-5'-O-[[(3 beta-cholest-5-enyloxy)carbonyl]phosphinyl]thymidine (9d). Concentrations of 9a-d found to inhibit replication of wild-type HIV-1LAI by 50% (EC50 values) as measured in a plaque reduction assay were in the 0.1-0.3 microM range as compared with 0.013 microM for AZT and 133 microM for PFA. The concentration at which toxicity was observed in 50% of the host cells (TC50 values) as measured by a visual grading scale of cellular morphology was 10 microM for 9a and 9d, 32 microM for 9b, and 320 microM for 9c. Thus, the TC50/EC50 ratio or selectivity index (SI) was 100 for 9a, 230 for 9b, and 1000 for 9c but only 33 for 9d, suggesting that the straight-chained fatty alcohol esters were more therapeutically selective. Similar TC50 and SI values were obtained for rapidly dividing CEM lymphoblasts as for HT4-6C cells. In assays against E89K, 9a-c had mean EC50 values of 0.13, 0.009, and 0.17 microM, whereas the EC50 of PFA was > 1000 microM and that of AZT was 0.009 microM; thus, E89K was highly resistant to PFA but not cross-resistant to either AZT or the lipophilic PFA-AZT conjugates. In viral replication assays against the A018C-post isolate, the mean EC50 values of 9a-c were 0.30, 0.53, and 0.77 microM as compared with 2.9 microM for AZT and 65 microM for PFA; thus, the virus recovered from a patient pretreated with AZT was not cross-resistant to either PFA or 9a-c. A notable feature of these results was that, in addition to being > 1000-fold more potent than PFA against the PFA-resistant mutant, the lipophilic PFA-AZT conjugates were more potent than PFA, as well as AZT, against AZT-resistant HIV-1.

Anti-HIV Agents↗

PubMed1997

Misclassification bias in estimates of bereavement effects.

Prospective studies that examine marital status differences in health and mortality frequently fail to update information on marital status in statistical models. The authors illustrate how the resulting misclassification of marital status can produce substantial bias in estimates of bereavement effects associated with widowhood. They use as their main source of data the Longitudinal Study of Aging (LSOA), 1984-1990, a national survey of persons aged 70 years and older. The estimates are based primarily on 3,192 respondents who were married and cohabiting with their spouses at the time of the baseline survey and who could be matched to their spouses' records. Comparisons of widowhood status derived from matched spouse records with reported marital status recorded in LSOA interviews demonstrate that reliance on interview information leads to substantial misclassification of marital status. Results from survival models indicate that estimates of marital status effects are sensitive to whether and how marital status is updated after baseline interviews. For example, updating marital status in hazard models from interview information alone indicates a protective effect of widowhood, whereas classifying widowhood on the basis of spouses' death records reveals a significant bereavement effect (relative mortality risks between 1.3 and 1.4).

Aged↗