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J Sun

Publications and source records attributed to J Sun.

At least 91 records · Page 5Linked to original sources
PubMed2001

Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO.

We have shown previously that at physiologically relevant oxygen tension (pO(2) approximately 10 mmHg), NO S-nitrosylates 1 of approximately 50 free cysteines per ryanodine receptor 1 (RyR1) subunit and transduces a calcium-sensitizing effect on the channel by means of calmodulin (CaM). It has been suggested that cysteine-3635 is part of a CaM-binding domain, and its reactivity is attenuated by CaM [Porter Moore, C., Zhang, J. Z., Hamilton, S. L. (1999) J. Biol. Chem. 274, 36831-36834]. Therefore, we tested the hypothesis that the effect of NO was mediated by C3635. The full-length RyR1 single-site C3635A mutant was generated and expressed in HEK293 cells. The mutation resulted in the loss of CaM-dependent NO modulation of channel activity and reduced S-nitrosylation by NO to background levels but did not affect NO-independent channel modulation by CaM or the redox sensitivity of the channel to O(2) and glutathione. Our results reveal that different cysteines within the channel have been adapted to serve in nitrosative and oxidative responses, and that S-nitrosylation of the cysteine-containing CaM-binding domain underlies the mechanism of CaM-dependent regulation of RyR1 by NO.

Amino Acid Substitution↗
PubMed2001

Antibody repertoire development in fetal and neonatal piglets. IV. Switch recombination, primarily in fetal thymus, occurs independent of environmental antigen and is only weakly associated with repertoire diversification.

The epitheliochorial placenta of swine is considered a barrier to Ag and selective transport of IgG, so this species should be an excellent model with which to determine whether switch recombination is Ag dependent. Analysis of Ig levels and Ig isotype profiles in >150 normal and virus-infected fetuses from 38-110 days of gestation (DG) suggested that IgG, IgA, and IgM were most likely the result of de novo fetal synthesis. Although transcripts for IgM could be recovered at DG 50 (114 DG is full gestation) in all major fetal lymphoid tissues, those for IgG and IgA first became prominent at 60 DG in thymus, and transcription and spontaneous secretion became especially pronounced in this organ in older fetuses. Data on transcription, secretion, and serum isotype profiles suggest that although all fetal IgA and IgM may result from de novo synthesis, some IgG may result from low-level selective transport. The complementarity-determining region 3 spectratypes of thymic IgA and IgG transcripts at 70 and 90 days, respectively, were as polyclonal as that of IgM, indicating a broad repertoire of switched B cells although the VDJs transcribed with these switched isotypes in normal fetuses were not diversified in comparison to those from animals exposed to environmental Ags such as age-matched, virus-infected fetuses, colonized isolator piglets, and conventional adults. However, VDJs expressed with switched isotypes were more diversified than those expressed with IgM. Thus, switch recombination in fetal life does not appear to be driven by environmental Ag and is only weakly coupled to VDJ diversification. These findings, and the fact that the oligoclonal IgA and IgM repertoires in a noninductive site of the mucosal immune system (parotid gland) become polyclonal in piglets reared germfree, suggest that initial expansion of the switched cells in the B cell compartment of fetal and neonatal piglets is not driven by environmental Ag.

Animals↗
PubMed2001

Synthesis of epothilone analogues by antibody-catalyzed resolution of thiazole aldol synthons on a multigram scale. Biological consequences of C-13 alkylation of epothilones.

Three monoclonal aldolase antibodies (84G3, 85H6, and 93F3), generated against a beta-diketone hapten (II) by the reactive immunization technique, catalyzed highly enantioselective retro-aldol reactions of the racemic thiazole aldols 13-20. Antibody 84G3 (0.0004-0.005 mol%) was used to resolve (+/-)-13-(+/-)-18 to afford compounds 13-18 in multigram quantities. Multiple 13-alkyl analogues of epothilone (7-12) and their trans isomers ((E)-7-(E)-12) were synthesized starting from thiazole aldols 13-18. Construction of the trisubstituted olefin moiety in compounds 7-12 and (E)-7-(E)-12 was catalyzed by Grubbs' catalyst (X). Initial biological testing with compounds 7-10 and their trans isomers showed that compounds 9, 10, and (E)-10 have appreciable tubulin polymerization and antiproliferative activities that approached those of epothilone C. The most active compound, (E)-9, even displayed potencies comparable to those observed for epothilones A and D. Interestingly, all trans analogues were more potent than their corresponding cis isomers. While introduction of an alkyl group at C-13 in the cis series led to an overall reduction in biological activity (compared to epothilone C), appropriate modification of the thiazole moiety (replacement of the 2-methyl substituent by a 2-methylthio group) was able to compensate for this loss. These results are encouraging in view of the expectation that epoxidations of these compounds should further increase their cellular activities. Thus, compounds 9, 10, and (E)-9 and (E)-10 represent highly promising candidates for further studies.

Aldehyde-Lyases↗
PubMed2001

CD98-mediated links between amino acid transport and beta 1 integrin distribution in polarized columnar epithelia.

In non-polarized cells, CD98 has been shown to both influence beta(1) integrins and heterodimerize with LAT-2, which confers amino acid transport capability on the LAT-2/CD98 heterodimer. Since LAT-2 is most heavily expressed in intestine and CD98 associates with the beta(1) integrin splice form selectively found in such epithelia, we investigated the relationship and polarity of these proteins using the intestinal epithelial model Caco2-BBE. CD98 was found to selectively coimmunoprecipitate with both LAT-2 and beta(1) integrin, and, logically, all three proteins were polarized to the same (basolateral) domain. Furthermore, expression of CD98 in polarized epithelia lacking human CD98 (MDCK cells) disrupted beta(1) integrin surface distribution and cytoskeletal architecture, suggesting that CD98 can influence integrin function. Expression of a CD98 mutant lacking the specific residues conferring LAT-2 binding similarly affected cells, confirming that the latter effect was not due to LAT-2 sequestration. Use of CD98 truncation mutants suggest that a 10-amino acid domain located at the putative cytoplasmic tail/transmembrane domain interface was necessary and sufficient to induce the phenotype change. We conclude that the CD98/LAT-2 amino acid transporter is polarized to the same domain on which beta(1) integrin resides. CD98 appears to associate with beta(1) integrin and, in doing so, may influence its function as revealed by disruption of the outside-in signaling that confers cytoskeletal organization. Furthermore, such findings suggest a link between classic transport events and a critical element of barrier function: integrin-mediated influences on cytoskeletal organization.

Amino Acid Transport System y+↗
PubMed2001

Three monoalkoxy-substituted nido-platinaboranes: [(PPh3)2PtB10H11-8-(OCH3)], [(PPh3)2PtB10H11-8-[OCH-(CH3)2]] and [(PPh3)2PtB10H10-9-[OCH(CH3)2]].

Each of the title compounds, 8-methoxy-7,7-bis(triphenylphosphine-P)-8,9:10,11-di-muH-7-platina-nido-undecaborane dichloromethane hemisolvate, [Pt(CH(14)B(10)O)(C(18)H(15)P)(2)].0.5CH(2)Cl(2), (I), 8-isopropoxy-7,7-bis(triphenylphosphine-P)-8,9:10,11-di-muH-7-platina-nido-undecaborane dichloromethane solvate, [Pt(C(3)H(18)B(10)O)(C(18)H(15)P)(2)].CH(2)Cl(2), (II), and 9-isopropoxy-7,7-bis(triphenylphosphine-P)-8,9:10,11-di-muH-7-platina-nido-undecaborane dichloromethane solvate, [Pt(C(3)H(18)B(10)O)(C(18)H(15)P)(2)].CH(2)Cl(2), (III), has an 11-vertex nido polyhedral skeleton, with the 7-platinum centre ligating to two exo-polyhedral PPh(3) groups and an alkoxy-substituted polyhedral borane ligand. Compounds (II) and (III) are isomers. The Pt-B distances are in the range 2.214 (7)-2.303 (7) A for (I), 2.178 (16)-2.326 (16) A for (II) and 2.205 (6)-2.327 (6) A for (III).

Journal Article↗
PubMed2001

The preparation and crystallization of Fab fragments of a family of mouse esterolytic catalytic antibodies and their complexes with a transition-state analogue.

The Fab fragments of a family of mouse esterolytic monoclonal antibodies MS6-12, MS6-126 and MS6-164 have been obtained by digestion of whole antibodies with papain, purified and crystallized in a range of different forms either alone or in complex with a transition-state analogue. The crystals diffract X-rays to resolutions between 2.1 and 1.2 A and are suitable for structural studies. The determination of these structures could be important in understanding the different catalytic power of each of these related catalytic antibodies.

Animals↗
PubMed2001

Cancer treatment by photodynamic therapy combined with adoptive immunotherapy using genetically altered natural killer cell line.

Adoptive immunotherapy mediated by human natural killer (NK) cell line genetically altered to produce interleukin-2 (NK92MI) was evaluated as adjuvant to photodynamic therapy (PDT) of subcutaneous tumors. The combined effect of these two modalities was first examined with SiHa tumors (human cervical squamous cell carcinoma) growing in NOD-scid mice. The most effective protocol for NK92MI cell transfer in conjunction with PDT mediated by photosensitizer mTHPC was the injection of 5 x 10(7) cells (peritumoral or intravenous) given immediately after PDT, which produced a marked improvement in the therapeutic outcome compared with the effect of PDT alone. The same protocol was tested with HT-29 tumor model (human colorectal adenocarcinoma) xenografted in NOD-scid mice. The results demonstrate that the adoptive immunotherapy with NK92MI cells (which when used alone were not effective in controlling tumor growth) significantly improved the cures of PDT-treated HT-29 tumors, whereas such benefit was not observed with the parental cell line NK92 (not producing interleukin-2). Flow cytometry-based analysis revealed a higher percentage of p.t. injected NK92MI cells in PDT-treated than in non-treated HT-29 tumors. Further investigation showed that the NK92MI cell-based adoptive immunotherapy is also a highly effective adjuvant for PDT treatment of murine EMT6 tumors growing in immunocompetent syngeneic BALB/c mice. This result diminishes the concern that adoptively transferred NK92MI cells may be rendered ineffective by an allogenic reaction of the host. The findings of this study suggest that advanced protocols of NK cell-based adoptive immunotherapy can be developed as efficient adjuvants to PDT used for the treatment of solid malignant tumors.

Animals↗
PubMed2001

A short course of methylprednisolone immunosuppression inhibits both rejection and spontaneous acceptance of rat liver allografts.

BACKGROUND: The effects of immunosuppressive drugs on transplant tolerance have not been extensively studied, although their effect on rejection is well established. METHODS: We examined the effects of a short course of treatment with the immunosuppressive drug methylprednisolone (MP) on the survival of PVG liver allografts in Dark Agouti (DA) recipients that accepted the livers and in Lewis recipients that rejected the livers. Infiltration of liver allografts was examined by immunohistochemical staining of liver sections, and apoptosis was measured by terminal deoxynucleotide transferase-mediated dUTP nick end labeling. RESULTS: A 5-day course of MP (days 0 to 4) led to rejection of four of six livers (mean survival time [MST] 99 days) in DA recipients compared with long-term survival (MST >100 days) in untreated animals. Delayed administration of MP (days 3 to 7) exacerbated rejection in DA recipients, and all eight animals rejected the graft (MST 68.5 days). Treatment of Lewis recipients with MP did not significantly prolong survival when administered from days 0 to 4 (MST 13 days), although delay of administration improved the outcome. Treatment from days 3 to 7 resulted in an MST of 21 days, whereas treatment from days 7 to 11 resulted in an MST of 41.5 days. MP treatment from day 3 to day 7 reduced T cells and interleukin 2 receptor-expressing cells but increased the numbers of apoptotic cells infiltrating both DA and Lewis strain allografts. CONCLUSIONS: These results show that immunosuppression with MP inhibits both spontaneous tolerance and rejection of liver allografts in a rat model and question the efficacy of administering MP to all liver allograft recipients from the time of transplantation.

Animals↗
PubMed2001

Importance of planar chirality in chiral catalysts with three chiral elements: the role of planar chirality in 2'-substituted 1,1'-P,N-ferrocene ligands on the enantioselectivity in Pd-catalyzed allylic substitution.

A series of novel planar chiral 2'-substituted 1,1'-P,N-ferrocene ligands 9-11, 14, and 16 were prepared with diastereopurity >99:1 and found to be effective in asymmetric allylic alkylation and amination reactions. Ligand 14 furnished the highest enantiomeric excess, 98.5% and 96.5% ee in alkylation and amination reactions, respectively. The role of planar chirality in asymmetric reactions has been examined, and decisive effects on enantioselectivity as well as the control of absolute configuration in palladium-catalyzed allylic alkylation and amination reactions were observed. To clarify why and how the planar chirality governed the stereochemical outcome, X-ray crystallographic structures of eta(3)-diphenylallyl Pd complexes, (1)H NMR, (31)P NMR spectra of palladium dichloride complexes, and eta(3)-diphenylallyl Pd complexes of three 1,1'-P,N-ferrocene ligands were analyzed with the aid of COSY and 2D NOESY experiments. All results led to the conclusion that planar chirality influences the stereochemical outcome by changing or even inverting the ratio of two rotamers because of the steric interaction between a planar chiral group and the coordination site.

Journal Article↗
PubMed2001

Molecular characterization of the VLDL receptor homolog mediating binding of lipophorin in oocyte of the mosquito Aedes aegypti.

Lipophorin (Lp) functions as a yolk protein precursor in the mosquito Aedes aegypti and it is internalized via receptor-mediated endocytosis (Insect Biochem. Mol. Biol., 30 (2000) 1161). We cloned and molecularly characterized a putative mosquito ovarian lipophorin receptor (AaLpRov) cDNA. The cDNA has a length of 3468 bp coding for a 1156-residue protein with a predicted molecular mass of 128.9 kDa. The deduced amino acid sequence of the cDNA revealed that it encodes a protein homolog of the LDL receptor superfamily, and that it harbors eight cysteine-rich ligand binding repeats at the N-terminus like vertebrate VLDL receptors. The deduced amino acid sequence of this mosquito ovarian receptor is most similar to that of the locust lipophorin receptor (LmLpR) (64.3%), and is only distantly related to the mosquito vitellogenin receptor (VgR) (18.3%), another ovarian LDLR homolog with a different ligand. The AaLpRov cDNA was expressed in a TnT Coupled Reticulocyte Lysate system, and co-immunoprecipitation experiments confirmed that the receptor protein specifically binds Lp. Developmental expression profiles clearly showed that AaLpRov transcripts are present in the vitellogenic ovary, with peak expression at 24-36 h post blood meal. In situ hybridization indicated that AaLpRov transcripts are present only in female germ line cells. Distance-based phylogenetic analyses suggest that the insect LpR and vertebrate LDL/VLDL receptor lineages separated after divergence from the insect VgR lineage.

Aedes↗
PubMed2001

Role for p27(Kip1) in Vascular Smooth Muscle Cell Migration.

BACKGROUND: Rapamycin is a potent inhibitor of smooth muscle cell (SMC) proliferation and migration. Rapamycin-mediated inhibition of SMC proliferation is associated with upregulation of the cyclin-dependent kinase inhibitor p27(Kip1). Previously, we showed that mixed embryonic fibroblasts obtained from p27(Kip1)(-/-) mice were relatively rapamycin-resistant, suggesting that p27(Kip1) plays an integral role in modulating the antiproliferative effects of rapamycin. We hypothesized that the antimigratory effect of rapamycin may also be mediated by p27(Kip1). METHODS AND RESULTS: Rapamycin (1 to 10 nmol/L) inhibited basic fibroblast growth factor-induced migration of wild-type (WT) but not p27(Kip1)(-/-) SMCs in a dose-dependent manner (P<0.05) in a modified Boyden chamber. The effects of rapamycin on aortic SMC explant migration were also studied with WT, p27(+/-), and p27(-/-) mice. Rapamycin 4 mg. kg(-1). d(-1) IP for 5 days inhibited SMC migration by 90% in the WT and p27(Kip1)(+/-) (P<0.05) but not p27(Kip1)(-/-) animals. CONCLUSIONS: Lack of p27(Kip1) reduces rapamycin-mediated inhibition of SMC migration. These novel findings suggest a role for p27(Kip1) in the signaling pathway(s) that regulates SMC migration.

ADP Ribose Transferases↗
PubMed2001

c-Abl tyrosine kinase binds and phosphorylates phospholipid scramblase 1.

Phospholipid scramblase 1 (PLSCR1) is a plasma membrane protein that has been proposed to play a role in the transbilayer movement of plasma membrane phospholipids. PLSCR1 contains multiple proline-rich motifs resembling Src homology 3 (SH3) domain-binding sites. An initial screen against 13 different SH3 domains revealed a marked specificity of PLSCR1 for binding to the Abl SH3 domain. Binding between intracellular PLSCR1 and c-Abl was demonstrated by co-immunoprecipitation of both proteins from several cell lines. Deletion of the proline-rich segment in PLSCR1 (residues 1--118) abolished its binding to the Abl SH3 domain. PLSCR1 was Tyr-phosphorylated by c-Abl in vitro. Phosphorylation was abolished by mutation of Tyr residues Tyr(69)/Tyr(74) within the tandem repeat sequence (68)VYNQPVYNQP(77) of PLSCR1, implying that these residues are the likely sites of phosphorylation. Cellular PLSCR1 was found to be constitutively Tyr-phosphorylated in several cell lines. The Tyr phosphorylation of PLSCR1 was increased upon overexpression of c-Abl and significantly reduced either upon cell treatment with the Abl kinase inhibitor STI571, or in Abl-/- mouse fibroblasts, suggesting that cellular PLSCR1 is a normal substrate of c-Abl. Cell treatment with the DNA-damaging agent cisplatin activated c-Abl kinase and increased Tyr phosphorylation of PLSCR1. The cisplatin-induced phosphorylation of PLSCR1 was inhibited by STI571 and was not observed in Abl-/- fibroblasts. These findings indicate that c-Abl binds and phosphorylates PLSCR1, and raise the possibility that an interaction between c-Abl and plasma membrane PLSCR1 might contribute to the cellular response to genotoxic stress.

Amino Acid Sequence↗
PubMed2001

Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1.

Heme controls expression of genes involved in the synthesis of globins and heme. The mammalian transcription factor Bach1 functions as a repressor of the Maf recognition element (MARE) by forming antagonizing hetero-oligomers with the small Maf family proteins. We show here that heme binds specifically to Bach1 and regulates its DNA-binding activity. Deletion studies demonstrated that a heme-binding region of Bach1 is confined within its C-terminal region that possesses four dipeptide cysteine-proline (CP) motifs. Mutations in all of the CP motifs of Bach1 abolished its interaction with heme. The DNA-binding activity of Bach1 as a MafK hetero-oligomer was markedly inhibited by heme in gel mobility shift assays. The repressor activity of Bach1 was lost upon addition of hemin in transfected cells. These results suggest that increased levels of heme inactivate the repressor Bach1, resulting in induction of a host of genes with MARES:

Amino Acid Sequence↗
PubMed2001

Neuraminidase from a bacterial source enhances both HIV-1-mediated syncytium formation and the virus binding/entry process.

Neuraminidases, also termed sialidases, which catalyze the removal of sialic acid residues from various glycoconjugates, have been previously reported to modulate HIV-1 replication. Given that some of the known opportunistic microbes found in patients infected with HIV-1 harbor neuraminidase (NA) activity, we speculated that pathogen-derived NA might be envisaged as an important factor in the pathogenesis of this retroviral infection. In the present study, we have monitored the putative modulation of HIV-1-mediated syncytium formation and virus replication by highly purified bacterial-derived NA from Arthrobacter ureafaciens. Taking advantage of a luciferase-based syncytium quantitative assay, we demonstrate here that the level of HIV-1-mediated syncytium formation is enhanced in the presence of NA and that it necessitates interaction between gp120 and CD4/chemokine coreceptor. By using pseudotyped recombinant luciferase-encoding HIV-1 particles, we found that NA treatment of human CD4-positive target cells (i.e., T lymphoid, monocytoid, and peripheral blood mononuclear cells) significantly augmented single-round infection by T- and macrophage-tropic isolates of HIV-1. The observed increase in HIV-1 infection was linked with an enhancement in the initial steps of the virus replicative cycle as monitored by viral binding and entry assays. Interestingly, NA treatment also enhances infectivity of HIV-1 pseudotypes with envelope glycoprotein from the amphotropic murine leukemia virus or the vesicular stomatitis virus. Taken together, our results provide useful information regarding the possible contribution of microbial agents carrying NA activity to HIV-1 pathogenesis.

Arthrobacter↗
PubMed2001

Variance estimation of a survival function for interval-censored survival data.

Interval-censored survival data often occur in medical studies, especially in clinical trials. In this case, many authors have considered estimation of a survival function. There is, however, relatively little discussion on estimating the variance of estimated survival functions. For right-censored data, a special case of interval-censored data, the most commonly used method for variance estimation is to use the Greenwood formula. In this paper we propose a generalization of the Greenwood formula for variance estimation of a survival function based on interval-censored data. Also a simple bootstrap approach is presented. The two methods are evaluated and compared using simulation studies and a real data set. The simulation results suggest that the methods work well.

Analysis of Variance↗
PubMed2001

Catalytic antibody route to the naturally occurring epothilones: total synthesis of epothilones A-F.

Naturally occurring epothilones have been synthesized starting from enantiomerically pure aldol compounds 9-11, which were obtained by antibody catalysis. Aldolase antibody 38C2 catalyzed the resolution of (+/-)-9 by enantioselective retro-aldol reaction to afford 9 in 90% ee at 50 % conversion. Compounds 10 and 11 were obtained in more than 99% ee at 50% conversion by resolution of their racemic mixtures using newly developed aldolase antibodies 84G3, 85H6 or 93F3. Compounds 9, 10 and 11 were resolved in multigram quantities and then converted to the epothilones by metathesis processes, which were catalyzed by Grubbs' catalysts.

Anti-Bacterial Agents↗
PubMed2001

The importance of hinge sequence for loop function and catalytic activity in the reaction catalyzed by triosephosphate isomerase.

We have determined the sequence requirements for the N-terminal protein hinge of the active-site lid of triosephosphate isomerase. The codons for the hinge (PVW) were replaced with a genetic library of all possible 8000 amino acid combinations. The most active of these 8000 mutants were selected using in vivo complementation of a triosephosphate isomerase-deficient strain of Escherichia coli, DF502. Approximately 0.3 % of the mutants complement DF502 with an activity that is between 10 and 70 % of wild-type activity. They all contain Pro at the first position. Furthermore, the sequences of these hinge mutants reveal that hydrophobic packing is very important for efficient formation of the enediol intermediate. However, the reduced catalytic activities observed are not due to increased rates of loop opening. To explore the relationship between the N-terminal and C-terminal hinges, three semi-active mutants from the N-terminal hinge selection experiment (PLH, PHS and PTF), and six active C-terminal hinge mutants from previous work (NSS, LWA, YSL, KTK, NPN, KVA) were combined to form 18 "double-hinge" mutants. The activities of these mutants suggest that the N-terminal and C-terminal hinge structures affect one another. It appears that specific side-chain interactions are important for forming a catalytically active enzyme, but not for preventing release of the unstable enediol intermediate from the active site of the enzyme. The independence of intermediate release on amino acid sequence is consistent with the absence of a "universal" hinge sequence in structurally related enzymes.

Amino Acid Sequence↗