Biomedical subjects

J Chen

Publications and source records attributed to J Chen.

At least 739 records · Page 41Linked to original sources

PubMed2000

A patient with type 2 diabetes mellitus associated with mutations in calcium sensing receptor gene and mitochondrial DNA.

A 44-year-old female with familial hypocalciuric hypercalcemia (FHH) due to a homozygous missense mutation (Pro40Ala) in calcium sensing receptor (CaSR) gene has type 2 diabetes mellitus. The identical heterozygous mutation of CaSR gene was observed in consanguineous parents and all other family members examined except her two sisters. Many subjects with abnormal glucose tolerance were observed in this family, which is compatible with maternal inheritance. Mitochondrial function of complex I (NADH-coenzyme Q reductase) activity in cybrid cells between mitochondrial DNA (mtDNA)-deleted (rho(0)) HeLa cells and mtDNA from the proband was decreased by 35%. The proband has eight substitutions and among these 4833 A/G is a missense substitution in NADH dehydrogenase 2 gene and may probably be a major pathogenic mutation of impaired complex I activity. These results suggest that coexistence of nuclear gene and mtDNA mutations may have caused or modified the development of abnormal glucose tolerance in this family.

Adult↗

PubMed2000

Memory of chirality in diastereoselective alpha-alkylation of isoleucine and allo-isoleucine derivatives.

[reaction: see text] alpha-Methylation of 3 gave 5 as a major product whereas 4 gave 6 predominantly, although both 3 and 4 have an (S)-chiral center at C(3). This indicates that chirality at C(2) in 3 and 4 was memorized in the corresponding intermediate enolates and the induced chirality made a major contribution in the stereochemical course of the reaction, while chirality at the adjacent chiral center C(3) had little effect.

Alkylation↗

PubMed2000

Identification of beta-glucosidase aggregating factor (BGAF) and mapping of BGAF binding regions on Maize beta -glucosidase.

In certain maize genotypes (nulls), beta-glucosidase does not enter the gel and therefore cannot be detected on zymograms. Such genotypes were initially thought to be homozygous for a null allele at the glu1 gene. We have shown that a beta-glucosidase aggregating factor (BGAF) is responsible for the null phenotype, and it specifically interacts with maize beta-glucosidases and forms large insoluble aggregates. To understand the mechanism of the beta-glucosidase-BGAF interaction, we constructed chimeric enzymes by domain swapping between the maize beta-glucosidase isozymes Glu1 and Gu2, to which BGAF binds, and the sorghum beta-glucosidase (dhurrinase) isozyme Dhr1, to which BGAF does not bind. The results of binding assays with 12 different chimeric enzymes showed that an N-terminal region (Glu(50)-Val(145)) and an extreme C-terminal region (Phe(466)-Ala(512)) together form the BGAF binding site on the enzyme surface. In addition, we purified BGAF, determined its N-terminal sequence, amplified the BGAF cDNA by reverse transcriptase-polymerase chain reaction, expressed it in Escherichia coli, and showed that it encodes a protein whose binding and immunological properties are identical to the native BGAF isolated from maize tissues. A data base search revealed that BGAF is a member of the jasmonite-induced protein family. Interestingly, the deduced BGAF sequence contained an octapeptide sequence (G(P/R)WGGSGG) repeated twice. Each of these repeat units is postulated to be involved in forming a site for binding to maize beta-glucosidases and thus provides a plausible explanation for the divalent function of BGAF predicted from binding assays.

Amino Acid Sequence↗

PubMed2000

Pituitary tumor transforming gene causes aneuploidy and p53-dependent and p53-independent apoptosis.

The pituitary tumor transforming gene, PTTG, is abundantly expressed in several neoplasms. We recently showed that PTTG overexpression is associated with apoptosis and therefore have now studied the role of p53 in this process. In MCF-7 breast cancer cells that express wild type p53, PTTG overexpression caused apoptosis. p53 was translocated to the nuclei in cells expressing PTTG. Overexpression of p53, along with PTTG, augmented apoptosis, whereas expression of the human papillomavirus E6 protein inhibited PTTG-induced apoptosis. In MG-63 osteosarcoma cells that are deficient in p53, PTTG caused cell cycle arrest and subsequent apoptosis that was inhibited by caspase inhibitors. A proteasome inhibitor augmented PTTG expression in stable PTTG transfectants, suggesting that down-regulated PTTG expression is required for cell survival. Finally, MG-63 cells expressing PTTG showed signs of aneuploidy including the presence of micronuclei and multiple nuclei. These results indicate that PTTG overexpression causes p53-dependent and p53-independent apoptosis. In the absence of p53, PTTG causes aneuploidy. These results may provide a mechanism for PTTG-induced tumorigenesis whereby PTTG mediates aneuploidy and subsequent cell transformation.

Aneuploidy↗

PubMed2000

A mutant allele of essential, general translation initiation factor DED1 selectively inhibits translation of a viral mRNA.

Positive-strand RNA virus genomes are substrates for translation, RNA replication, and encapsidation. To identify host factors involved in these functions, we used the ability of brome mosaic virus (BMV) RNA to replicate in yeast. We report herein identification of a mutation in the essential yeast gene DED1 that inhibited BMV RNA replication but not yeast growth. DED1 encodes a DEAD (Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation of all yeast mRNAs. Inhibition of BMV RNA replication by the mutant DED1 allele (ded1-18) resulted from inhibited expression of viral polymerase-like protein 2a, encoded by BMV RNA2. Inhibition of RNA2 translation was selective, with no effect on general cellular translation or translation of BMV RNA1-encoded replication factor 1a, and was independent of p20, a cellular antagonist of DED1 function in translation. Inhibition of RNA2 translation in ded1-18 yeast required the RNA2 5' noncoding region (NCR), which also conferred a ded1-18-specific reduction in expression on a reporter gene mRNA. Comparison of the similar RNA1 and RNA2 5' NCRs identified a 31-nucleotide RNA2-specific region that was required for the ded1-18-specific RNA2 translation block and attenuated RNA2 translation in wild-type yeast. Further comparisons and RNA structure predictions suggest a modular arrangement of replication and translation signals in RNA1 and RNA2 5' NCRs that appears conserved among bromoviruses. The 5' attenuator and DED1 dependence of RNA2 suggest that, despite its divided genome, BMV regulates polymerase translation relative to other replication factors, just as many single-component RNA viruses use translational read-through and frameshift mechanisms to down-regulate polymerase. The results show that a DEAD-box helicase can selectively activate translation of a specific mRNA and may provide a paradigm for translational regulation by other members of the ubiquitous DEAD-box RNA helicase family.

Base Sequence↗

PubMed2000

Shape complementarity, binding-site dynamics, and transition state stabilization: a theoretical study of Diels-Alder catalysis by antibody 1E9.

Antibody 1E9 is a protein catalyst for the Diels-Alder reaction between tetrachlorothiophene dioxide and N-ethylmaleimide. Quantum mechanical calculations have been employed to study the 1E9-catalyzed Diels-Alder reaction in the gas phase. The transition states and intermediates were all determined at the B3LYP/6-31G*//HF/6-31G* level. The cycloaddition step is predicted to be rate-determining, and the endo reaction pathway is strongly favored. Binding of the reactants and the transition states to antibody 1E9 was investigated by docking and molecular dynamics simulations. The linear interaction energy (LIE) method was adopted to estimate the free energy barrier of the 1E9-catalyzed Diels-Alder reaction. The catalytic efficiency of antibody 1E9 is achieved by enthalpic stabilization of the transition state, near-perfect shape complementarity of the hydrophobic binding site for the transition state, and a strategically placed hydrogen-bonding interaction.

Antibodies, Catalytic↗

PubMed2000

Interferon-inducible Myc/STAT-interacting protein Nmi associates with IFP 35 into a high molecular mass complex and inhibits proteasome-mediated degradation of IFP 35.

Nmi is an interferon (IFN)-inducible protein homologous to IFN-inducible protein IFP 35. The homology consists of a novel Nmi/IFP 35 domain (NID) of 90-92 amino acids that is repeated in tandem in each protein and mediates Nmi-Nmi protein interactions and subcellular localization. In a yeast two-hybrid screen with a fragment of Nmi protein containing both NIDs, we identified an interaction between Nmi and IFP 35. Deletion derivatives of the proteins indicate that both NIDs are required for the interaction between Nmi and IFP 35. In mammalian cells, Nmi and IFP 35 co-immunoprecipitate and co-localize in large cytoplasmic speckles. Nmi and IFP 35 proteins associate into a high molecular mass complex of 300-400 kDa as determined by native gel electrophoresis and gel filtration. The association of Nmi and IFP 35 into a complex can be demonstrated in multiple cell lines and is not dependent on treatment with IFN. Short term and long term cultures of transfected HEK293 cells suggest that Nmi and IFP 35 proteins stabilize each other through complex formation. IFP 35 appears to be more labile because Nmi was stable in the absence of IFP 35, whereas IFP 35 was degraded in the absence of Nmi. A deletion analysis revealed that Nmi must interact with IFP 35 to prevent its degradation and that the amino terminus of Nmi is required, but not sufficient, for this function. Inhibition of the proteasome, but not other proteases, led to increased levels of IFP 35. Thus, we have shown that Nmi and IFP 35 associate into a protein complex, that IFP 35 is degraded in a proteasome-mediated process, and that a novel function of Nmi is to prevent IFP 35 degradation. The stabilization of IFP 35 by Nmi may serve to amplify the physiologic effects of IFNs.

Carrier Proteins↗

PubMed2000

Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to pacemaking activity in specialized neurons and cardiac myocytes. HCN channels have a structure similar to voltage-gated K(+) channels but have a much larger putative S4 transmembrane domain and open in response to membrane hyperpolarization instead of depolarization. As an initial attempt to define the structural basis of HCN channel gating, we have characterized the functional roles of the charged residues in the S2, S3, and S4 transmembrane domains. The nine basic residues and a single Ser in S4 were mutated individually to Gln, and the function of mutant channels was analyzed in Xenopus oocytes using two-microelectrode voltage clamp techniques. Surface membrane expression of hemagglutinin-epitope-tagged channel proteins was examined by chemiluminescence. Our results suggest that 1) Lys-291, Arg-294, Arg-297, and Arg-300 contribute to the voltage dependence of gating but not to channel folding or trafficking to the surface membrane; 2) Lys-303 and Ser-306 are essential for gating, but not for channel folding/trafficking; 3) Arg-312 is important for folding but not gating; and 4) Arg-309, Arg-315, and Arg-318 are crucial for normal protein folding/trafficking and may charge-pair with Asp residues located in the S2 and S3 domains.

Amino Acid Sequence↗

PubMed2000

Tau epitopes in spinal cord neurofibrillary lesions in Chamorros of Guam.

We examined spinal cord sections from Guamanian Chamorros with or without amyotrophic lateral sclerosis or parkinsonism-dementia complex using immunohistochemistry and antibodies to epitopes that span the length of tau to characterize the tau epitope profile of neurofibrillary tangles in these spinal cords. Most (16/20) spinal cords, including some from Chamorros without documented clinical disease, contained tangles with a tau epitope profile similar to the tangles found in the forebrain and brain stem of these patients.

Aged↗

PubMed2000

Selective estrogen receptor modulator idoxifene inhibits smooth muscle cell proliferation, enhances reendothelialization, and inhibits neointimal formation in vivo after vascular injury.

BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.

Adult↗

PubMed2000

Protein kinase C-zeta phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin.

Protein kinase C-zeta (PKC-zeta) is a serine/threonine kinase downstream from phosphatidylinositol 3-kinase in insulin signaling pathways. However, specific substrates for PKC-zeta that participate in the biological actions of insulin have not been reported. In the present study, we identified insulin receptor substrate-1 (IRS-1) as a novel substrate for PKC-zeta. Under in vitro conditions, wild-type PKC-zeta (but not kinase-deficient mutant PKC-zeta) significantly phosphorylated IRS-1. This phosphorylation was reversed by treatment with the serine-specific phosphatase, protein phosphatase 2A. In addition, the overexpression of PKC-zeta in NIH-3T3(IR) cells caused significant phosphorylation of cotransfected IRS-1 as demonstrated by [(32)P]orthophosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimmunoprecipitated with endogenous PKC-zeta, and this association was increased 2-fold upon insulin stimulation. Furthermore, the overexpression of PKC-zeta in NIH-3T3(IR) cells significantly impaired insulin-stimulated tyrosine phosphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-1-associated phosphatidylinositol 3-kinase activity. Taken together, our results raise the possibility that IRS-1 is a novel physiological substrate for PKC-zeta. Because PKC-zeta is located downstream from IRS-1 and phosphatidylinositol 3-kinase in established insulin signaling pathways, PKC-zeta may participate in negative feedback pathways to IRS-1 similar to those described previously for Akt and GSK-3.

3T3 Cells↗

PubMed2000

Amperometric ultramicrosensors for peroxynitrite detection and its application toward single myocardial cells.

The research studied the concentration variation of peroxynitrite anion (O=N-O-O-) released from cultured neonatal myocardial cells induced by ischemia/reperfusion and studied the protective effect of melatonin on the injury. For this purpose, amperometry peroxynitrite ultramicrosensors (UMS) were fabricated and constructed by electropolymerizing inorganic macromolecular film of tetraaminophthalocyanine manganese(II) and coating chemically with poly(4-vinylpyridine). Under optimum conditions, the UMS showed high selectivity and sensitivity to peroxynitrite determination with a calculated detection limit of 1.8 x 10(-8) mol/L (S/N of 3). The detection of peroxynitrite was based on electrocatalytic reduction of peroxynitrite. The mechanism of catalysis was also discussed. The UMS should be promising for in vivo measurement of peroxynitrite without interference or fouling. Peroxynitrite released from myocardial cells both in the ischemic period and in the reperfusion period was measured directly. This approach may lead to important information for myocardial cells on the mechanism of injury and prospective treatments of medicine such as melatonin.

Animals↗

PubMed2000

Characterization of tumor-associated Chk2 mutations.

The integrity of the DNA damage response pathway is essential for prevention of neoplastic transformation. Several proteins involved in this pathway including p53, BRCA1, and ATM are frequently mutated in human cancer. Checkpoint kinase 2 (Chk2) is a DNA damage-activated protein kinase that lies downstream of ATM in this pathway. Recently, heterozygous germline mutations in Chk2 have been identified in a subset of patients with Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype, suggesting that Chk2 is a tumor suppressor gene. In this study, we have reported the biochemical characterization of the four tumor-associated Chk2 mutants. Two of the reported Chk2 mutations identified in Li-Fraumeni syndrome result in loss of Chk2 kinase activity. Whereas one mutation within the Chk2 forkhead homology-associated (FHA) domain, R145W, retains some basal kinase activity, this mutant cannot be phosphorylated at an ATM-dependent phosphorylation site (Thr-68) and cannot be activated following gamma radiation. Wild-type Chk2 exists mainly in a protein complex of M(r) approximately 200,000 whereas the R145W mutant forms a larger, presumably inactive complex in the cell. The other FHA domain mutant, I157T, behaves as wild-type Chk2 in all the assays used here. Because the FHA domain is involved in protein-protein interactions, this mutation may affect associations of Chk2 with other proteins. Additionally, we have shown that Chk2 can also be inactivated by down-regulation of its expression in cancer cells. Thus, Chk2 may be inactivated by multiple mechanisms in the cell.

Cell Transformation, Neoplastic↗

PubMed2000

Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding.

Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding.

Adenosine Triphosphate↗

PubMed2000

A structural basis for drug-induced long QT syndrome.

Mutations in the HERG K(+) channel gene cause inherited long QT syndrome (LQT), a disorder of cardiac repolarization that predisposes affected individuals to lethal arrhythmias [Curran, M. E. , Splawski, I., Timothy, K. W., Vincent, G. M., Green, E. D. & Keating, M. T. (1995) Cell 80, 795-804]. Acquired LQT is far more common and is most often caused by block of cardiac HERG K(+) channels by commonly used medications [Roden, D. M., Lazzara, R., Rosen, M., Schwartz, P. J., Towbin, J. & Vincent, G. M. (1996) Circulation 94, 1996-2012]. It is unclear why so many structurally diverse compounds block HERG channels, but this undesirable side effect now is recognized as a major hurdle in the development of new and safe drugs. Here we use alanine-scanning mutagenesis to determine the structural basis for high-affinity drug block of HERG channels by MK-499, a methanesulfonanilide antiarrhythmic drug. The binding site, corroborated with homology modeling, is comprised of amino acids located on the S6 transmembrane domain (G648, Y652, and F656) and pore helix (T623 and V625) of the HERG channel subunit that face the cavity of the channel. Other compounds that are structurally unrelated to MK-499, but cause LQT, also were studied. The antihistamine terfenadine and a gastrointestinal prokinetic drug, cisapride, interact with Y652 and F656, but not with V625. The aromatic residues of the S6 domain that interact with these drugs (Y652 and F656) are unique to eag/erg K(+) channels. Other voltage-gated K(+) (Kv) channels have Ile and Val (Ile) in the equivalent positions. These findings suggest a possible structural explanation for how so many commonly used medications block HERG but not other Kv channels and should facilitate the rational design of drugs devoid of HERG channel binding activity.

Amino Acid Sequence↗

PubMed2000

Disruption of aldose reductase gene (Akr1b1) causes defect in urinary concentrating ability and divalent cation homeostasis.

Aldose reductase (AKR1B1) is the first enzyme in the polyol pathway through which glucose is converted to sorbitol, and has been implicated in the etiology of diabetic complications. However, its physiological role is still not well understood. In the kidney, AKR1B1 is quite abundant in the collecting tubule cells and thought to provide protection against hypertonic environment. We report here that the mice lacking AKR1B1 showed hypercalciuria, hypercalcemia, hypermagnesemia, and reduced ability to concentrate urine, suggesting a new physiological role of AKR1B1 in divalent cation homeostasis.

Aldehyde Reductase↗

PubMed2000

Increasing the thermostability of staphylococcal nuclease: implications for the origin of protein thermostability.

Seven hyper-stable multiple mutants have been constructed in staphylococcal nuclease by various combinations of eight different stabilizing single mutants. The stabilities of these multiple mutants determined by guanidine hydrochloride denaturation were 3.4 to 5.6 kcal/mol higher than that of the wild-type. Their thermal denaturation midpoint temperatures were 12.6 to 22.9 deg. C higher than that of the wild-type. These are among the greatest increases in protein stability and thermal denaturation midpoint temperature relative to the wild-type yet attained. There has been great interest in understanding how proteins found in thermophilic organisms are stabilized. One frequently cited theory is that the packing of hydrophobic side-chains is improved in the cores of proteins isolated from thermophiles when compared to proteins from mesophiles. The crystal structures of four single and five multiple stabilizing mutants of staphylococcal nuclease were solved to high resolution. No large overall structural change was found, with most changes localized around the sites of mutation. Rearrangements were observed in the packing of side-chains in the major hydrophobic core, although none of the mutations was in the core. It is surprising that detailed structural analysis showed that packing had improved, with the volume of the mutant protein's hydrophobic cores decreasing as protein stability increased. Further, the number of van der Waals interactions in the entire protein showed an experimentally significant increase correlated with increasing stability. These results indicate that optimization of packing follows as a natural consequence of increased protein thermostability and that good packing is not necessarily the proximate cause of high stability. Another popular theory is that thermostable proteins have more electrostatic and hydrogen bonding interactions and these are responsible for the high stabilities. The mutants here show that increased numbers of electrostatic and hydrogen bonding interactions are not obligatory for large increases in protein stability.

Crystallography, X-Ray↗

PubMed2000

Pseudouridine synthase 3 from mouse modifies the anticodon loop of tRNA.

A cDNA encoding mouse pseudouridine synthase 3 (mPus3p) has been cloned. The predicted protein has 34% identity with yeast pseudouridine synthase 3 (Pus3), an enzyme known to form pseudouridine at positions 38 and 39 in yeast tRNA. The cDNA is 1.7 kb, and when used as a probe on a Northern blot of total RNA from mouse tissues or cells in culture, a band at 1.8 kb was observed. The open reading frame codes for a protein of 481 amino acids with a predicted molecular mass of 55 552 Da. When mPus3p was in vitro translated and used in reactions with tRNA substrates from both yeast and humans, uridines at position 39 were modified to pseudouridine. In a tRNA substrate with a uridine at position 38 (human tRNA(Leu)), there was very slight formation of pseudouridine at that position after incubation with mPus3p.

Amino Acid Sequence↗