[Progress in the treatment for nasolacrimal duct obstruction].
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Dacryocystorhinostomy↗
Biomedical subjects
Y Qian
Publications and source records attributed to Y Qian.
[Effect of raw radish on pharmacological action of ginseng].
Ginseng decoction can increase the memory-improvement, weary-resistance, antihypoxia, sedation in mice, and increase the weight of spleen and accessory sexual gland, but reduce the weight of thymus. Raw radish can decrease these pharmacological effects in varying degrees, except the sedation.
Animals↗
[Rat heart transplantation immune tolerance induced by blockade of B7-CD28 interaction].
OBJECTIVE: To study immune tolerance induced by blockade of B7-CD28 interaction after heart transplantation in rats. METHODS: A model of rat heterotopic heart allograft was established by using Ono's technique. CTLA-4-Ig was administered by intraperitoneal injection at dose of 100 micro. Survival times, histological changes, and the concentration of IL-2, IgG and IgM were measured after operation. RESULTS: Survival times were 31.60 +/- 1.82 days in experimental group and vs 7.25 +/- 0.71 days in control groups. In the experimental group, the graft survival time prolonged significantly (P < 0.01). Histological changes were characterized by mild scattered mononuclear infiltration and patchy myocyte injury in the experimental group (grade I - II). In the control group, the changes were characterized by severe lymphocyte infiltration, myocyte injury and necrosis, interstitial edema, and hemorrhage (grade IV). IL-2 was significantly decreased after operation (P < 0.01). IgG and IgM were not changed significantly in comparison with those in the control group (P > 0.05). CONCLUSION: The blockade B7-CD28 interaction can induce rat heterotopic heart allograft immune tolerance. It may provide a new way to treat immune rejection after transplantation.
Abatacept↗
Correlation between PAP-dependent steroid binding activity and substrate specificity of mouse and human estrogen sulfotransferases.
Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as an activated sulfate donor. A finding of undetermined significance in the study of EST has been that the guinea pig EST is able to bind pregnenolone and estradiol with high affinity in the presence of PAP, the reaction by-product of the sulfate donor PAPS. This finding has raised the possibility that EST may have other physiological functions independent of its enzymatic activity as a sulfotransferase. To determine if the PAP-dependent steroid binding activity is a common property shared by other estrogen sulfotransferases, we have expressed the mouse and human EST in bacteria and used the purified protein to address this question. We found that, in the presence of PAP, both recombinant mouse and human EST were able to bind estradiol with high affinity but only the human EST was able to bind pregnenolone. In addition, we show that human but not the mouse EST was also able to bind dehydroepiandrosterone, a property that was not described for the guinea pig EST. Furthermore, we demonstrate that the promiscuity of human EST in steroid binding is mirrored by a correspondingly low substrate specificity in its enzymatic activity as a sulfotransferase. Reversely, the lack of stable binding of pregnenolone and dehydroepiandrosterone by the mouse EST is paralleled by a lack of sulfotransferase activity of this enzyme toward these two steroids. Mutagenesis of mouse EST within a domain critical for PAPS binding abolished both its sulfotransferase and PAP-dependent estrogen binding activity. These data suggest that stable binding of steroids such as pregnenolone or estrogen is not an independent property of estrogen sulfotransferases but rather is related to their catalytic activity.
Animals↗
Studies of the DXS7 polymorphism at the MAO loci in unipolar depression.
From the fact that DXS7 polymorphism is closely related to monoamine oxidase (MAO) genes and MAO inhibitors are widely used in the treatment of unipolar depression, it is of particular interest to study the relationship between the DXS7 polymorphism and unipolar depression. Thus, this study examined the possible association between DXS7 polymorphism and unipolar depression in 66 cases versus 85 controls from Shanghai. Polymerase chain reaction and amplification fragment length polymorphism techniques were used for genotyping of the DXS7 locus in this study. Four alleles at the DXS7 locus were detected with length generated by polymerase chain reaction amplification ranging from 157 to 167 bp. Comparison of allele frequency in the DXS7 locus showed no difference between unipolar depression cases and normal controls in the total population set. When subclassified by age, a significant difference of allele frequency distribution was observed between early onset (before age 40) and late onset (after age 40) patients. The frequency of the 157-bp allele was decreased, whereas the frequency of the 165 allele was increased in late onset patients (0.3810 for the 157-bp allele and 0.5238 for the 165-bp allele) compared with that of early onset patients (0.6304 for the 157-bp allele and 0. 3261 for the 165-bp allele). There was also a difference of allele frequency between patients and normal controls with age over 40 years. The frequency of 165-bp allele increased significantly in late onset patients (0.5238) compared with that of controls within the same age range (0.3454). Association studies suggested that in the population with age over 40 years, presence of the 165-bp allele of DXS7 locus was significantly associated with unipolar depression (relative risk = 2.08, P < 0.05), whereas in the total population set, this association did not exist. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:598-600, 1999.
Adolescent↗
Purification, characterization, and cDNA cloning of a new fibrinogenlytic venom protein, Agkisacutacin, from Agkistrodon acutus venom.
Agkisacutacin is a new fibrinogenlytic protein from Agkistrodon acutus venom. It consists of two heterologous subunits linked by an intersubunit disulfide bond. The cDNAs encoding the two chains of Agkisacutacin were cloned from a lambdagt11 cDNA library of the snake venom gland and sequenced, including the leader peptides (23/23 amino acid residues) and mature subunits (129/123 amino acid residues). It is structurally related to the family of IX/X-binding protein (IX/X-bp)-like proteins and shows high similarity (alpha-70%/beta-64%) to habu IX/X-bp from Trimeresurus flavoridis, but displays distinct biological activity with direct action on fibrinogen.
Agkistrodon↗
Crystal structure of avian carboxypeptidase D domain II: a prototype for the regulatory metallocarboxypeptidase subfamily.
The crystal structure of domain II of duck carboxypeptidase D, a prohormone/propeptide processing enzyme integrated in a three repeat tandem in the natural system, has been solved, constituting a prototype for members of the regulatory metallocarboxypeptidase subfamily. It displays a 300 residue N-terminal alpha/beta-hydrolase subdomain with overall topological similarity to and general coincidence of the key catalytic residues with the archetypal pancreatic carboxypeptidase A. However, numerous significant insertions/deletions in segments forming the funnel-like access to the active site explain differences in specificity towards larger protein substrates or inhibitors. This alpha/beta-hydrolase subdomain is followed by a C-terminal 80 residue beta-sandwich subdomain, unique for these regulatory metalloenzymes and topologically related to transthyretin and sugar-binding proteins. The structure described here establishes the fundamentals for a better understanding of the mechanism ruling events such as prohormone processing and will enable modelling of regulatory carboxypeptidases as well as a more rational design of inhibitors of carboxypeptidase D.
Amino Acid Sequence↗
Quinone reductase inhibitors block SAPK/JNK and NFkappaB pathways and potentiate apoptosis.
A variety of environmental stresses stimulate the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEKK) > stress-activated protein kinase (SAPK)-ERK kinase (SEK) > SAPK/c-Jun NH(2)-terminal kinase (JNK) stress-activated protein kinase cascade and coordinately activate the transcription factor NFkappaB. Mechanisms of stress activation upstream of MEKK1 have not been precisely determined. Redox mechanisms involving sulfhydryls are likely because N-acetyl-cysteine at millimolar concentrations blocks stress signals. Because intracellular sulfhydryl concentrations can be regulated through redox cycling involving reactive quinones (1), we tested the ability of quinone reductase inhibitors to alter stress signaling. Several quinone reductases are inhibited by dicoumarol, a coumarin derivative. Dicoumarol prevented SAPK activation in vivo by chemical cell stressors and also prevented SAPK activation induced by expression of the tumor necrosis factor alpha (TNFalpha) receptor-associated protein TRAF2 but not by expression of truncated active MEKK1. Other coumarin derivatives failed to block SAPK activation, but other inhibitors of quinone reductases, particularly menadione, similarly blocked SAPK activation. Cells deficient in a major quinone reductase, NQO1, displayed hypersensitivity to dicoumarol stress inhibition, whereas SAPK in cells reconstituted with the NQO1 gene displayed relative dicoumarol resistance. Consistent with the proposed role of overlapping upstream signaling cascades in activation of NFkappaB, dicoumarol also blocked NFkappaB activation in primary macrophages stimulated with either lipopolysaccharide or TNFalpha. In addition, dicoumarol strongly potentiated TNFalpha-induced apoptosis in HeLa cells, probably by blocking the anti-apoptotic effect of NFkappaB. The ability of dicoumarol to simultaneously inhibit SAPK and NFkappaB activation and to potentiate apoptotic cell death suggests that SAPK is not an obligate participant in apoptosis. Dicoumarol, currently in clinical use as an oral anticoagulant, represents a potential therapeutic inhibitor of the SAPK and NFkappaB response.
Apoptosis↗
Characterization of the enzymatic properties of the first and second domains of metallocarboxypeptidase D.
Carboxypeptidase D (CPD) contains three domains with homology to other metallocarboxypeptidases. To further characterize the various domains, we constructed a series of point mutants with a critical active site Glu of duck CPD converted to Gln. The proteins were expressed in the baculovirus system, purified to homogeneity, and characterized. Point mutations within both the first and second domains eliminated enzyme activity, indicating that the third domain is inactive toward dansyl-Phe-Ala-Arg. CPD removed only the C-terminal Lys or Arg from peptides, with the first domain more efficient toward Arg and the second domain more efficient toward Lys. Peptides containing Pro in the penultimate position were poorly cleaved by either domain. Cleavage of a peptide with Ala in the penultimate position was most efficient, with the relative order Ala >/= Met > Ser, Phe > Tyr > Trp > Thr >/= Gln, Asp, Leu, Gly >> Pro for CPD with both domains active. There were only minor differences between the first and the second domains regarding the influence of the penultimate amino acid. The first domain was optimally active at pH 6.3-7.5, whereas the second domain was optimally active at pH 5. 0-6.5. Thus, the first and second carboxypeptidase domains have complementary enzyme activities. Furthermore, the finding that CPD with both domains active shows a broad activity to a wide range of substrates is consistent with a role for this enzyme in the processing of many proteins that transit the secretory pathway.
Animals↗
Antitumor efficacy of a novel class of non-thiol-containing peptidomimetic inhibitors of farnesyltransferase and geranylgeranyltransferase I: combination therapy with the cytotoxic agents cisplatin, Taxol, and gemcitabine.
Ras malignant transformation requires posttranslational modification by farnesyltransferase (FTase). Here we report on the design and antitumor activity, in monotherapy as well as in combination therapy with cytotoxic agents, of a novel class of non-thiol-containing peptidomimetic inhibitors of FTase and the closely related family member geranylgeranyltransferase I (GGTase I). The non-thiol-containing FTI-2148 is highly selective for FTase (IC50, 1.4 nM) over GGTase I (IC50, 1700 nM), whereas GGTI-2154 is highly selective for GGTase I (21 nM) over FTase (IC50, 5600 nM). In whole cells, the corresponding methylester prodrug FTI-2153 is >3000-fold more potent at inhibiting H-Ras (IC50, 10 nM) than Rap1A processing, whereas GGTI-2166 is over 100-fold more selective at inhibiting Rap1A (IC50, 300 nM) over H-Ras processing. Furthermore, FTI-2153 was highly effective at suppressing oncogenic H-Ras constitutive activation of mitogen-activated protein kinase and human tumor growth in soft agar. FTI-2148 suppressed the growth of the human lung adenocarcinoma A-549 cells in nude mice by 33, 67, and 91% in a dose-dependent manner. Combination therapy of FTI-2148 with either cisplatin, gemcitabine, or Taxol resulted in a greater antitumor efficacy than monotherapy. GGTI-2154 in similar antitumor efficacy experiments is less potent than FTI-2148 and inhibits tumor growth by 9, 27, and 46%. Combination therapy of GGTI-2154 with cisplatin, gemcitabine, or Taxol is also more effective. Finally, FTI-2148 and GGTI-2154 are 30- and 33-fold more selective and 30- and 16-fold more potent in whole cells than our previously reported thiol-containing FTI-276 and GGTI-297, respectively. Thus, our results demonstrate that this highly potent and selective novel class of non-thiol-containing peptidomimetics inhibits human tumor growth in whole animals and that combination therapy with cytotoxic agents is more beneficial than monotherapy.
3T3 Cells↗
Effect of lead exposure and accumulation on copper homeostasis in cultured C6 rat glioma cells.
C6 rat glioma cells resemble rat astroglia in culture in that both cell types accumulate lead (Pb) intracellularly from the medium. As such, C6 cells are a model for Pb accumulation by the brain. In this study, an increase in intracellular Pb accumulation induced by p-chloromercuribenzoate (PCMB) after exposure to 10 microM Pb acetate suggests a role for sulfhydryl groups in Pb retention. Stimulation of Pb accumulation by nifedipine suggests the entry of Pb into these cells by a novel path. Most of the intracellular Pb from exposure for 7 days to 1 microM Pb was associated with high-molecular weight components in cytosol. Pb exposure increased the abundance of three proteins with the following characteristics on two-dimensional gels: 81 kDa with pI of 5.6, 81 kDa with pI of 4. 9, and 71 kDa with pI of 5.6. The levels of five other proteins, ranging in size from 37-41 kDa with pIs of 6.0-6.8 declined. Exposed C6 cells accumulated copper (Cu) intracellularly, and Cu accumulation after Pb exposure was shown by kinetic analysis with 67Cu to result from an increased uptake and a decreased efflux for Cu. Pb-exposed cells also showed increased Cu binding to membranes, which is consistent with the increase of Cu uptake. These data indicate that intracellular Pb interacts with high molecular weight proteins in C6 cells, and exposure also alters membrane transport properties for copper.
Animals↗
Human exonuclease 1 functionally complements its yeast homologues in DNA recombination, RNA primer removal, and mutation avoidance.
Yeast exonuclease 1 (Exo1) is induced during meiosis and plays an important role in DNA homologous recombination and mismatch correction pathways. The human homolog, an 803-amino acid protein, shares 55% similarity to the yeast Exo1. In this report, we show that the enzyme functionally complements Saccharomyces cerevisiae Exo1 in recombination of direct repeat DNA fragments, UV resistance, and mutation avoidance by in vivo assays. Furthermore, the human enzyme suppresses the conditional lethality of a rad27Delta mutant, symptomatic of defective RNA primer removal. The purified recombinant enzyme not only displays 5'-3' double strand DNA exonuclease activity, but also shows an RNase H activity. This result indicates a back-up function of exonuclease 1 to flap endonuclease-1 in RNA primer removal during lagging strand DNA synthesis.
Cell Division↗
Wettability, Oil Recovery, and Interfacial Tension with an SDBS-Dodecane-Kaolin System.
The wettability of kaolin with SDBS (sodium dodecyl benzene sulfonate) aqueous solutions was measured by the Washburn equation expressed as contact angles. The contact angle changes for SDBS aqueous solutions on kaolin surface was studied. The interfacial tension between the SDBS solutions and n-dodecane was measured using both drop volume and spinning drop methods. Then the oil recovery of n-dodecane on the kaolin surface was tested. It was found that the minimum contact angle (the most hydrophilic condition) and the maximum oil recovery occurred near the critical micelle concentration (CMC) of SDBS while the interfacial tension between the SDBS solution and n-dodecane was far from ultra-low. Copyright 1999 Academic Press.
Journal Article↗
Residues 48 and 82 at the N-terminal hydrophobic pocket of rabbit skeletal muscle troponin-C photo-cross-link to Met121 of troponin-I.
It has been proposed [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644], and confirmed by structural studies [Gagne et al. (1995) Nat. Struct. Biol. 2, 784-789], that the binding of Ca2+ to the triggering sites in troponin-C (TnC) causes the opening of the N-terminal hydrophobic pocket bound by the B, C, and D helices. This conformational change is believed to provide an additional binding site for troponin-I (TnI) and to lead to further events in the Ca2+ regulation process. To answer the question of which part of TnI interacts with this hydrophobic patch of TnC, we constructed two TnC mutants, each with a single cysteine, one at residue 48 between helices B and C and the other at residue 82 on the D helix. Each mutant was labeled with the photoactivatable cross-linker benzophenone-4-iodoacetamide, followed by reconstitution and UV irradiation. Studies were made in the binary complex composed of TnC and TnI, the ternary complex composed of TnC, TnI, and troponin-T (TnT), and the synthetic thin filament composed of troponin, tropomyosin, and F-actin. TnC-TnI photo-cross-linking was observed for both mutants and for all three types of complexes. Although no Ca2+ dependence in the photo-cross-linking was observed on the binary and ternary complexes, the extent of cross-linking was reduced in the absence vs the presence of Ca2+ in the thin filament. TnI Met121, five residues from the C-terminus of the inhibitory region, was identified as the cross-linking site for both TnC mutants using microsequencing and mass spectrometry following proteolysis. These results, obtained with intact TnC.TnI complexes, indicate that the TnI segment containing Met121 is in close contact with the N-terminal hydrophobic patch of TnC, and that in the thin filament the segment containing this residue moves away slightly from the hydrophobic patch in the absence of Ca2+, possibly triggering the translocation of the actin-binding region(s) of TnI toward actin.
Actins↗
Long-Range Electrostatic Interaction between a Charged Wall and Two Similarly Charged Colloidal Spheres at Low Surface Potentials.
The long-range electrostatic interaction between a pair of similarly charged colloidal spheres and a charged planar wall at low surface potentials is theoretically investigated. The linear Poisson-Boltzmann equation (PBE) and the point charge approximation of the charged sphere are used. The electrical potential distribution in the electrolyte solution is found from the PBE at the constant surface potentials using the image charge method. The electrostatic forces acting on the spheres are then calculated. The results show that the repulsive interaction between a pair of similarly charged colloidal spheres clearly decreases when a charged wall appears nearby, but it is impossible for an attractive force to emerge at the scaled surface potentials less than 1. There is, however, an attractive force between the charged wall and the similarly charged colloidal spheres, when the surface potential zetap on the wall is sufficiently higher than the surface potential zetas on the spheres to make zetap > zetasexp(kappah) (h is the distance from the wall to the sphere center). In this case, there are negative surface charges on the spheres at positive surface potential zetas. It is these negative charges that produce the above attraction. Copyright 1999 Academic Press.
Journal Article↗
The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts.
Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309-319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165-176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.
Actins↗
Glu300 of rat carboxypeptidase E is essential for enzymatic activity but not substrate binding or routing to the regulated secretory pathway.
Several recently discovered members of the carboxypeptidase E (CPE) gene family lack critical active site residues that are conserved in other family members. For example, three CPE-like proteins contain a Tyr in place of Glu300 (equivalent to Glu270 of carboxypeptidase A and B). To investigate the importance of this position, Glu300 of rat CPE was converted into Gln, Lys, or Tyr, and the proteins expressed in Sf9 cells using the baculovirus system. All three mutants were secreted from the cells, but the media showed no enzyme activity above background levels. Wild-type CPE and the Gln300 point mutant bound to a p-aminobenzoyl-Arg-Sepharose affinity resin, and this binding was competed by an active site-directed inhibitor, guanidinoethylmercaptosuccinic acid. The affinity purified mutant CPE protein showed no detectable enzyme activity (<0.004% of wild-type CPE) toward dansyl-Phe-Ala-Arg. Expression of the Gln300 and Lys300 mutant CPE proteins in the NIT3 mouse pancreatic beta-cell line showed that these mutants are routed into secretory vesicles and secreted via the regulated pathway. Taken together, these results indicate that Glu300 of CPE is essential for enzyme activity, but not required for substrate binding or for routing into the regulated secretory pathway.
Amino Acid Sequence↗
Potent, highly selective, and non-thiol inhibitors of protein geranylgeranyltransferase-I.
The design, synthesis, and biological evaluation of a family of peptidomimetic inhibitors of protein geranylgeranyltransferase-I (PGGTase-I) are reported. The inhibitors are based on the C-terminal CAAL sequence of many geranylgeranylated proteins. Using 2-aryl-4-aminobenzoic acid derivatives as mimetics for the central dipeptide (AA), we have attached a series of imidazole and pyridine derivatives to the N-terminus as cysteine replacements. These non-thiol-containing peptidomimetics show exceptional selectivity for PGGTase-I over the closely related enzyme protein farnesyltransferase (PFTase). This selectivity is retained in whole cells where the inhibitors were shown to block the geranylgeranylation of Rap-1A without affecting the farnesylation of small GTP-binding proteins such as Ras.
3T3 Cells↗