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Biomedical subjects

L Tong

Publications and source records attributed to L Tong.

At least 19 recordsLinked to original sources
PubMed1998

Structural basis for inhibition of the cyclin-dependent kinase Cdk6 by the tumour suppressor p16INK4a.

The cyclin-dependent kinases 4 and 6 (Cdk4/6) that control the G1 phase of the cell cycle and their inhibitor, the p16INK4a tumour suppressor, have a central role in cell proliferation and in tumorigenesis. The structures of Cdk6 bound to p16INK4a and to the related p19INK4d reveal that the INK4 inhibitors bind next to the ATP-binding site of the catalytic cleft, opposite where the activating cyclin subunit binds. They prevent cyclin binding indirectly by causing structural changes that propagate to the cyclin-binding site. The INK4 inhibitors also distort the kinase catalytic cleft and interfere with ATP binding, which explains how they can inhibit the preassembled Cdk4/6-cyclin D complexes as well. Tumour-derived mutations in INK4a and Cdk4 map to interface contacts, solidifying the role of CDK binding and inhibition in the tumour suppressor activity of p16INK4a.

Adenosine Triphosphate↗
PubMed1998

Carboxymethyl-phenylalanine as a replacement for phosphotyrosine in SH2 domain binding.

The crystal structure of human p56(lck) SH2 domain in complex with an inhibitor containing the singly charged p-(carboxymethyl)phenylalanine residue (cmF) as a phosphotyrosine (Tyr(P) or pY) replacement has been determined at 1.8 A resolution. The binding mode of the acetyl-cmF-Glu-Glu-Ile (cmFEEI) inhibitor is very similar to that of the pYEEI inhibitor, confirming that the cmFEEI inhibitor has a similar mechanism of SH2 domain inhibition despite its significantly reduced potency. Observed conformational differences in the side chain of the cmF residue can be interpreted in terms of maintaining similar interactions with the SH2 domain as the Tyr(P) residue. The crystal structure of the free p56(lck) SH2 domain has been determined at 1.9 A resolution and shows an open conformation for the BC loop and an open phosphotyrosine binding pocket, in contrast to earlier studies on the src SH2 domain that showed mostly closed conformation. The structural information presented here suggests that the carboxymethyl-phenylalanine residue may be a viable Tyr(P) replacement and represents an attractive starting point for the design and development of SH2 domain inhibitors with better pharmaceutical profiles.

Binding Sites↗
PubMed1998

Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis.

In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage.

Animals↗
PubMed1998

Conserved mode of peptidomimetic inhibition and substrate recognition of human cytomegalovirus protease.

Human cytomegalovirus (HCMV) protease belongs to a new class of serine proteases, with a unique polypeptide backbone fold. The crystal structure of the protease in complex with a peptidomimetic inhibitor (based on the natural substrates and covering the P4 to P1' positions) has been determined at 2.7 A resolution. The inhibitor is bound in an extended conformation, forming an anti-parallel beta-sheet with the protease. The P3 and P1 side chains are less accessible to solvent, whereas the P4 and P2 side chains are more exposed. The inhibitor binding mode shows significant similarity to those observed for peptidomimetic inhibitors or substrates of other classes of serine proteases (chymotrypsin and subtilisin). HCMV protease therefore represents example of convergent evolution. In addition, large conformational differences relative to the structure of the free enzyme are observed, which may be important for inhibitor binding.

Amino Acid Sequence↗
PubMed1998

Signal transduction in neuronal death.

Apoptosis in the nervous system is a necessary event during the development of the nervous system and is also present after genotoxic events, be they chronic as in aging or more acute after trauma and ischemia. Apoptotic events reflect an interplay between intrinsic signaling events that rely on cytokines, neurotransmitters, and growth factors and responses to extrinsic events that increase levels of radical oxygen species. Both intrinsically and extrinsically driven signal-transduction pathways act via transcription factors that regulate the coordinated timely expression of stress-response genes as part of a decision-making process that can commit cells to apoptosis or survival. Here we discuss the role of two transcription factors that participate in apoptosis in the nervous system: the activator protein AP-1 and nuclear factor kappaB.

Animals↗
PubMed1997

Determination of Association Constants for Cyclodextrin-Surfactant Inclusion Complexes: A Numerical Method Based on Surface Tension Measurements

Inclusion complexes of beta-cyclodextrin (beta-CD) with sodium octyl sulfonate (C8As), sodium dodecyl sulfonate (C12As), and sodium hexadecyl sulfonate (C16As) in aqueous solutions are studied by surface tension measurement at the air/water interface at different temperatures. At fixed concentrations of the surfactants, the surface tension increases with an increase in beta-CD concentration to a maximum value, at which it holds. The surface tension curves of the surfactants in the presence of beta-CD are higher than those in the absence of beta-CD. The curves rise higher with the increase in beta-CD concentration for each surfactant. The apparent critical micelle concentrations (CMC*) of the surfactants vary linearly with beta-CD concentration. The CMC* and surface tension values (including those after the CMC*) for the same system decrease with increase in temperature. A numerical method based on surface tension measurements is developed to determine the association constants for 1:1 inclusion complexes. This method is very reliable and easy to perform. The results demonstrate that the longer the hydrophobic tail of the surfactant, the greater the association constant with beta-CD, and that for the same surfactant the association constant is higher at lower temperatures.

Journal Article↗
PubMed1997

Potent HIV protease inhibitors containing a novel (hydroxyethyl)amide isostere.

A series of HIV protease inhibitors containing a novel (hydroxyethyl)amidosuccinoyl core has been synthesized. These peptidomimetic structures inhibit viral protease activity at low nanomolar concentrations (IC50 < 10 nM for HIV-1 protease). The inhibition constant (Ki) for inhibitor 19 was determined to be 7.5 pM against HIV-1 and 1.2 nM against HIV-2 proteases, respectively. Several compounds (19-24) inhibited HIV-1 replication in cell culture assays with 50% effective concentrations (EC50) = 3.7-35 nM. This series of inhibitors was found to exhibit poor bioavailability (< 10%) in the rat, following oral administration. The synthesis and biological properties of these compounds are discussed. In addition, an X-ray structure of one of these inhibitors (23) in complex with HIV-2 protease provides insight into the binding mode of this novel class of HIV protease inhibitors.

Administration, Oral↗
PubMed1997

Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase.

The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms.

Animals↗
PubMed1997

Cytokine levels and inflammatory responses in developing late-phase allergic reactions in the skin.

BACKGROUND: Cytokines could play roles in the attraction of leukocytes into sites of IgE-mediated late-phase reactions (LPR) or in the activation of such cells at the sites. Previous studies found increased release of IL-6, IL-1, and granulocyte-macrophage colony-stimulating factor into skin chambers overlying allergen-challenged sites, mainly after 6 hours when LPR are already well-developed. OBJECTIVE: To compare levels of several cytokines with inflammatory responses in skin chambers overlying developing LPRs. METHODS: Skin chambers were appended to denuded blister bases in 15 sensitive subjects, then challenged over a 5-hour period with pollen allergens (Ag) or buffer control (B). Levels of several chemotactic cytokines, eosinophil cationic product (released from eosinophils), and lactoferrin (released from neutrophils) were measured and leukocyte accumulation was assessed. RESULTS: Levels of the chemokines IL-8, RANTES, and monocyte chemotactic protein-1 (but not IL-1, IL-6, or granulocyte-macrophage colony-stimulating factor) were significantly higher at Ag-challenge sites than at B-challenge sites. IL-5 was not detected. In individual chamber fluids at Ag-challenge sites, (1) IL-8 levels correlated strongly with levels of lactoferrin but not with eosinophil cationic protein levels; (2) RANTES levels correlated with numbers of eosinophils but not with eosinophil cationic protein levels; and (3) levels of monocyte chemotactic protein-1 correlated weakly with histamine released after the first hour. CONCLUSIONS: During LPRs developing during the first 5 hours of Ag challenge, there is increased local release of several chemokines, which could play roles in the observed attraction and subsequent activation of leukocytes and continued histamine release observed at such sites.

Blister↗
PubMed1997

A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket.

The crystal structure of human p38 mitogen-activated protein (MAP) kinase in complex with a potent and highly specific pyridinyl-imidazole inhibitor has been determined at 2.0 A resolution. The structure of the kinase, which is in its unphosphorylated state, is similar to that of the closely-related ERK2. The inhibitor molecule is bound in the ATP pocket. A hydrogen bond is made between the pyridyl nitrogen of the inhibitor and the main chain amido nitrogen of residue 109, analogous to the interaction from the N1 atom of ATP. The crystal structure provides possible explanations for the specificity of this class of inhibitors. Other protein kinase inhibitors may achieve their specificity through a similar mechanism. The structure also reveals a possible second binding site for this inhibitor, with currently unknown function.

Adenosine Triphosphate↗
PubMed1997

Retinoic acid induces apoptosis in PC12 cells independent of neurotrophic factors.

PC12 cells are known to undergo programmed cell death (apoptosis) when they are deprived of serum. Nerve growth factor (NGF) rescues PC12 cells from serum deprivation-induced apoptosis. In the present study, we examined the effects of retinoic acid (RA), a classic morphogen, on apoptosis in PC12 cells after serum deprivation and NGF-mediated rescue. In naive PC12 cells, all trans-RA treatment induced cell death in the presence of NGF. RA also abolished the protective effects of dibutyryl cyclic AMP or insulin under serum-free conditions. The death process was accompanied by nuclear condensation and DNA fragmentation, typical of apoptosis. In addition, RA also increased the extent of apoptosis in PC12 cells after serum deprivation. Cycloheximide, an inhibitor of protein synthesis, did not abolish the effects of RA on serum-deprived PC12 cells. RA also decreased thymidine incorporation and proliferation in NGF-treated PC12 cells. Furthermore, although the total DNA binding activity of the AP-1 transcription factor was not changed after RA treatment, RA decreased a specific AP-1 transcriptional activity. It is surprising that differentiated PC12 cells resisted the toxic effects of RA. These data suggest that RA might function as an endogenous inducer of apoptosis during neural differentiation by a mechanism distinct from that of serum deprivation.

Animals↗
PubMed1996

Canine prosomatostatin: isolation of a cDNA, regulation of gene expression, and characterization of post-translational processing intermediates.

Somatostatin is a tetradecapeptide (SS-14) initially isolated from the hypothalamus that is also found in D cells of the stomach and pancreas where it exerts an inhibitory action on a variety of gastrointestinal functions. Since many of concepts important to an understanding of gastrointestinal physiology are derived from experiments in the dog we examined somatostatin gene expression and post-translational processing in the canine fundus, antrum and pancreas. The canine somatostatin cDNA which is highly homologous to other known mammalian somatostatins was used to examine somatostatin expression in isolated canine fundic D-cells. Somatostatin expression induced by cholecystokinin (10(-8) M) was inhibited by the somatostatin analog, octreotide (10(-7) M). To examine somatostatin processing in the canine gut we noted that synthesis of SS-14 and somatostatin octacosapeptide (SS-28) involves endoproteolytic cleavage of prosomatostatin (proSS) at both paired and single basic amino-acid residues, respectively. Antisera capable of recognizing the amino-terminal residues of SS-28, SS-28(1-14) and SS-28(1-12) were characterized and identified concentrations of SS-28(1-12) but not SS-28(1-14) in the fundus, antrum and pancreas equivalent to those of SS-14. Since previous biosynthetic studies in canine fundic D-cells showed that SS-14 was synthesized without the appearance of a SS-28 intermediate, we hypothesize that proSS is sequentially cleaved at a dibasic site to produce SS-14 followed by monobasic cleavage that results in the formation of SS-28(1-12). Furthermore, equivalent amounts of SS-14 and SS-28(1-12) were co-released from canine fundic D-cells by CCK (10(-8) M) suggesting that the generation of these products occurs within the same regulated pathway of secretion.

Amino Acid Sequence↗
PubMed1996

A new serine-protease fold revealed by the crystal structure of human cytomegalovirus protease.

Human cytomegalovirus (hCMV), a herpesvirus, infects up to 70% of the general population in the United States and can cause morbidity and mortality in immunosuppressed individuals (organ-transplant recipients and AIDS patients) and congenitally infected newborns. hCMV protease is essential for the production of mature infectious virions, as it performs proteolytic processing near the carboxy terminus (M-site) of the viral assembly protein precursor. hCMV protease is a serine protease, although it has little homology to other clans of serine proteases. Here we report the crystal structure of hCMV protease at 2.0 angstroms resolution, and show that it possesses a new polypeptide backbone fold. Ser 132 and His 63 are found in close proximity in the active site, confirming earlier biochemical and mutagenesis studies. The structure suggests that the third member of the triad is probably His 157. A dimer of the protease with an extensive interface is found in the crystal structure. This structure information will help in the design and optimization of inhibitors against herpesvirus proteases.

Crystallography, X-Ray↗
PubMed1996

Lung carcinoma in former smokers.

BACKGROUND: A reduction in the risk of lung carcinoma and a lower death rate among former smokers (FS) compared with current smokers (CS) have been documented in numerous U.S. and international studies. The main objective of our study was to compare the differences in demographic and clinical characteristics in groups stratified by smoking status and gender to evaluate the effect of smoking history and cessation on age at lung carcinoma diagnosis and on specific histologic type. METHODS: We conducted a cross-sectional study of lung cancer at The University of Texas M.D. Anderson Cancer Center from January 1986 to December 1990 and from January 1992 to December 1993. This study included 1039 patients age 19-88 with confirmed primary lung carcinoma who responded to self-administered risk factor questionnaires. Among them, 497 patients (47.83%) were CS, 444 patients (42.73%) were FS, 98 patients (9.43%) had never smoked (NS), and 840 patients (80.8%) were heavy smokers (more than 20 pack-years). RESULTS: The median age at lung carcinoma diagnosis for FS was slightly later than that for CS. The histologic type of lung carcinoma for those who had quit smoking more than 20 years previously was not significantly different from that of NS, but was significantly different from that of CS (P < 0.05) and from those who quit smoking fewer than 10 years previously (P < 0.10). CS was a positive predictor for both small cell carcinoma (odds ratio [OR] = 8.79) and squamous cell carcinoma (OR = 2.11) and negatively associated with adenocarcinoma (OR = 0.50), whereas FS was a positive predictor only for small cell carcinoma (OR = 5.50). The variable of pack-years was negatively associated with adenocarcinoma and positively associated with small cell carcinoma in all patients combined and in women, and was also positively associated with squamous carcinoma in all patients after adjustment by smoking status. CONCLUSIONS: These results indicate that smoking cessation or less life-time smoking exposure affects the distribution of specific histologic subtypes of lung cancer, especially for women, and that smoking cessation may postpone the age at which lung cancer occurs.

Adenocarcinoma↗
PubMed1996

Effect of nerve growth factor on AP-1, NF-kappa B, and Oct DNA binding activity in apoptotic PC12 cells: extrinsic and intrinsic elements.

Both intrinsic signals, such as serum and neurotrophic factor deprivation, and extrinsic events or agents, such as oxidative stress and glucose deprivation, can induce cell death in pheochromocytoma (PC12) cells. Also, treatment with nerve growth factor (NGF) reduces cell death due to the treatments mentioned. Serumless-induced cell death, as a model of apoptosis, has been intensively investigated in PC12 cells. In the present study, we investigated the molecular components of H2O2-induced cell death and compared it with serumless-induced cell death. Exposure of PC12 cells to intermediate concentrations of H2O2 (100 microM) induced nuclear condensation and DNA fragmentation, indicating that there is an apoptotic component in H2O2-induced cell death. Since transcription factors have been shown to play an essential role in the control of cellular proliferation, differentiation, and survival, we measured changes in the DNA binding activities of the transcription factors activator protein-1 (AP-1), nuclear factor kappa B (NF-kappa B), and octamer-binding protein (Oct) by electrophoretic mobility shift assay (EMSA) after H2O2 treatment and serum deprivation, both in the absence and presence of exogenous NGF in PC12 cells. AP-1 DNA binding activity transiently increased during apoptosis due to serum deprivation, and NGF treatment further stimulated AP-1 DNA binding activity in a more persistent fashion. NF-kappa B DNA binding activity only increased slightly after serum deprivation, and NGF treatment of PC12 cells decreased NF-kappa B binding activity in the late stages of serum deprivation. Oct DNA binding activity decreased after serum deprivation, while NGF had an opposite effect. AP-1 DNA binding activity also transiently increased after H2O2 treatment, as did NF-kappa B DNA binding activity. Our results suggest that AP-1 is likely to be a common component of signaling pathways associated with both the induction or suppression of apoptosis induced by intrinsic or extrinsic stimuli.

Animals↗
PubMed1996

Crystal structures of the human p56lck SH2 domain in complex with two short phosphotyrosyl peptides at 1.0 A and 1.8 A resolution.

src homology 2 (SH2) domains are modules of about 100 amino acid residues and bind to phosphotyrosine-containing motifs in a sequence-specific manner. They play important roles in intracellular signal transduction and represent potential targets for pharmacological intervention. The protein tyrosine kinase p56lck is a member of the src family and is involved in T-cell activation. The crystal structure of its SH2 domain with an 11-residue peptide showed that the phosphotyrosine and the Ile residue at the pY + 3 position are recognized by the SH2 domain. We present here the crystal structure of the SH2 domain of human p56lck in complex with the short phosphotyrosyl peptide Ac-pTyr-Glu-Glu-Ile (pYEEI peptide) at 1.0 A resolution. The structural analysis at atomic resolution reveals that residue Arg134 (alphaA2), which interacts with the phosphotyrosine side-chain, is present in two conformations in the complex. The structure at 1.8 A resolution of the complex with the phosphotyrosyl peptide Ac-pTyr-Glu-Glu-Gly (pYEEG peptide), which is 11 fold less potent, shows another binding mode for the pY + 3 residue as well as rearrangements of the side-chain of Arg196 (EF3) and one of the water molecules at the base of the pY + 3 pocket. The structure of the complex with the short pYEEI peptide at atomic resolution represents a good starting point for the design and optimization of new inhibitors. Comparative structural analysis of many different inhibitor complexes will be an important component of this drug discovery process.

Amino Acid Sequence↗