Biomedical subjects
Publications and source records attributed to X Lu.
From the root of Salvia trijuga three triterpenoids were isolated. Based on the analysis of their spectra, they were identified as coleonolic acid with a contracted A-ring, euscaphic acid and 2 alpha-hydroxyursolic acid.
Therapeutic effect of Xiaopiling Granules on gastric precancerous changes was observed, using image analyzer and electronic microscope. The result indicated that the Granules could efficiently eliminate gastric mucositis, nourish gastric mucosa, soften or restrain dysplasia, ameliorate structure atypism and cell atypism of dysplasia, promote elimination of dysplasia and efficiently ameliorate the ultrastructure of gastric precancerous changes.
One way in which wild-type p53 is able to regulate cell cycle progression is thought to be via the induction of its downstream target gene Waf1/CIP1, thus indirectly regulating the transcriptional activity of E2F. The E2F transcription factors are known to be key effectors of the cell cycle. We report here that there is a physical and functional interaction between p53 and two of the components of the E2F transcription factors, E2F1 and DP1. The expression of wild-type p53 can inhibit the transcriptional activity of E2F, and the expression of both E2F1 and DP1 can also downregulate p53-dependent transcription. The transcriptional activity of p53 is known to be inhibited by the direct binding of mdm2, but we demonstrate here that both E2F1 and DP1 can inhibit p53 transcriptional activity independently of mdm2. Detailed studies of protein-protein interactions have provided evidence that E2F1 and its co-operating factor DP1 can complex with p53 both in vitro and in vivo.
We have previously demonstrated [Lu, Williams, Deadman, Salmon, Kakkar, Wilkinson, Baruch, Authi and Rahman (1994) Biochem. J. 304, 929-936] the preferential antagonism of the interactions of the integrin alpha IIb beta 3 on activated platelets with three immobilized glycoprotein ligands (fibrinogen, fibronectin and von Willebrand factor) by a selected panel of snake-venom RGD (Arg-Gly-Asp)-containing proteins including the disintegrins kistrin and elegantin, and the neurotoxin variant dendroaspin. Kistrin and dendroaspin, although structurally unrelated, contain similar amino acids flanking the tripeptide RGD and behaved as identical antagonists preferentially inhibiting platelet adhesion to immobilized fibrinogen as opposed to fibronectin. In contrast, elegantin, which shares extensive sequence similarity with kistrin but has different amino acids around the tripeptide RGD, preferentially inhibited platelet adhesion to immobilized fibronectin as opposed to fibrinogen. To develop further insights into the mechanisms underlying the preferential antagonism shown by the venom proteins in the adhesion studies, we, in the present study, sought to determine the binding properties of kistrin, elegantin and dendroaspin to the alpha IIb beta 3 complex by radioligand kinetic and competition studies. In direct binding experiments, both kistrin and dendroaspin were observed to bind to a single class of binding site on ADP-activated platelets with apparent equilibrium dissociation constant (Kdapp) values of 42 +/- 2 nM and 21 +/- 6 nM respectively. In competition studies, dendroaspin blocked the binding of 125I-labelled kistrin to ADP-activated platelets in a simple competitive manner, with an apparent equilibrium inhibition constant (Kiapp) of 143 +/- 14 nM, from which an indirect Kdapp = 22 nM for dendroaspin was determined. This result suggests that kistrin and dendroaspin bind to the same site on the integrin alpha IIb beta 3 consistent with their similar inhibitory properties. In contrast, elegantin recognized two classes of binding sites on the alpha IIb beta 3 complex with Kdapp values of 10.5 +/- 0.8 nM and 175 +/- 10 nM, and, unlike dendroaspin, did not inhibit the binding of 125I-labelled kistrin to ADP-activated platelets. However, in reciprocal experiments both kistrin and dendroaspin inhibited the binding of 125I-elegantin to ADP-activated platelets in a non-competitive manner, with Kiapp values of 34 +/- 3 nM and 21 +/- 2 nM respectively. Thus elegantin appears to interact with distinct but interacting sites on the alpha IIb beta 3 complex from the binding site of kistrin and dendroaspin, consistent with its distinctive inhibitory preferences as shown in platelet adhesion studies.(ABSTRACT TRUNCATED AT 400 WORDS)
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Human hepatitis B virus (HBV) envelopes contain three distinct glycoproteins called L, M, and S HBsAg. Each is posttranslationally modified to contain N-linked oligosaccharides. N-linked oligosaccharides, after attachment to a polypeptide backbone, are processed by enzymes within the endoplasmic reticulum (ER). There is uncertainty about what role, if any, these N glycans and their modification in the ER play in the function of the HBV envelope proteins. By treating hepatoblastoma cultures which secrete HBV (HepG 2.2.15 cells) with inhibitors of different steps of the glycosylation and glycan modifying pathway, we provide evidence that glycosylation and the first step in the processing pathway are necessary for virion, but not subviral particle, secretion. That is, using a highly sensitive immunoprecipitation/polymerase chain reaction system, enveloped HBV could not be detected in the medium of HepG2.2.15 cells incubated with tunicamycin. However, HBV subviral particle secretion was not prevented by tunicamycin. Moreover, inhibitors of alpha-glucosidase I (the first step in the glycan processing pathway) also prevented virion secretion. Inhibitors of mannose trimming (a later step) and glycolipid synthesis, did not prevent virion secretion, defining the limits of the glycosylation requirements in secretion. These results demonstrate a requirement for N-glycosylation and glucosidase processing in the secretion of virions and further distinguish between the requirements for virion and subviral particle secretion.
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The crystal structure of the tandem SH2 domains of human ZAP-70 in complex with a peptide derived from the zeta-subunit of the T-cell receptor reveals an unanticipated interaction between the two domains. A coiled coil of alpha-helices connects the two SH2 domains, producing an interface that constitutes one of the two critical phosphotyrosine binding sites. These and other unique features provide the molecular basis for highly selective association of ZAP-70 with the T-cell receptor.
Programmed cell death (apoptosis) is an intrinsic part of organismal development and aging. Here we report that many nonsteroidal antiinflammatory drugs (NSAIDs) cause apoptosis when applied to v-src-transformed chicken embryo fibroblasts (CEFs). Cell death was characterized by morphological changes, the induction of tissue transglutaminase, and autodigestion of DNA. Dexamethasone, a repressor of cyclooxygenase (COX) 2, neither induced apoptosis nor altered the NSAID effect. Prostaglandin E2, the primary eicosanoid made by CEFs, also failed to inhibit apoptosis. Expression of the protooncogene bcl-2 is very low in CEFs and is not altered by NSAID treatment. In contrast, p20, a protein that may protect against apoptosis when fibroblasts enter G0 phase, was strongly repressed. The NSAID concentrations used here transiently inhibit COXs. Nevertheless, COX-1 and COX-2 mRNAs and COX-2 protein were induced. In some cell types, then, chronic NSAID treatment may lead to increased, rather than decreased, COX activity and, thus, exacerbate prostaglandin-mediated inflammatory effects. The COX-2 transcript is a partially spliced and nonfunctional form previously described. Thus, these findings suggest that COXs and their products play key roles in preventing apoptosis in CEFs and perhaps other cell types.
In vertebrate skeletal muscle, excitation-contraction coupling may occur by a mechanical coupling mechanism involving protein-protein interactions between the dihydropyridine receptor (DHPR) of the transverse tubule membrane and the ryanodine receptor (RYR)/Ca2+ release channel of the sarcoplasmic reticulum membrane. We have previously shown that the cytoplasmic II-III loop peptides of the skeletal and cardiac muscle DHPR alpha 1 subunits (SDCL and CDCL, respectively) activate the skeletal muscle RYR. We now report that cyclic AMP-dependent protein kinase-mediated phosphorylation of Ser687 of SDCL yields a peptide that fails to activate the RYR, as determined in [3H]ryanodine binding and single channel measurements. The phosphorylated SDCL bound to the skeletal muscle but not cardiac muscle RYR, and the binding could be displaced by the unphosphorylated SDCL. A mutant SDCL with a Ser687-->Ala substitution failed to activate the RYR, but was still able to bind. Similarly, a Ser813-->Ala substitution in CDCL yielded a peptide that failed to activate the skeletal RYR. Use of three smaller overlapping peptides within the SDCL region identified an amino acid region from 666 to 726 including Ser687, which bound to and activated the skeletal muscle RYR. These results suggest that cyclic AMP-dependent protein kinase-mediated phosphorylation of the DHPR alpha 1 subunit may play a role in the functional interaction of the DHPR and RYR in skeletal muscle.
Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato, ripening-inhibitor (rin), and non-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning the rin and nor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning the rin locus is estimated to be 200-300 kb/cM, while the nor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked to rin and nor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains the nor locus.
The process of body prepatterning during Drosophila blastoderm formation relies on the localized activities of zygotic segmentation genes, which are controlled by asymmetrically distributed maternal determinants. The anterior determinant bicoid, a homeodomain transcription factor, forms an anterior-to-posterior concentration gradient. It interacts with the maternal transcription factor hunchback to activate the anterior zygotic patterning genes, including the central gap gene Krüppel (Kr). In contrast, the posterior maternal system does not provide such a decisive transcription factor, but rather prevents the repressor hunchback from acting in the posterior half so that the gap genes giant (gt) and knirps (kni) are activated by an as yet unknown transcription factor. Here we show that caudal, a conserved homeodomain protein that forms a posterior-to-anterior concentration gradient, and the anterior determinant bicoid cooperate to form a partly redundant activator system in the posterior region of the embryo.
Injection of Corynebacterium parvum into the rat dorsal root ganglion has previously been shown to cause an inflammatory reaction dominated by macrophages and to enhance regeneration of the central axons of primary sensory neurons. Here, neuronal mRNAs that are modified by nerve transection were analyzed by in situ hybridization following injection of C. parvum into the dorsal root ganglion. Neuronal concentrations of mRNAs for the growth-associated protein (GAP-43) and the immediate early gene c-jun were increased by a local inflammatory response just as after axotomy. The concentration of mRNA for calcitonin gene-related peptide (CGRP) was also increased in a constant subpopulation of sensory neurons after injection of C. parvum in contrast to its decrease following axotomy. The results are consistent with the hypothesis that some of the responses to sensory neurons to axotomy are sustained by macrophages which accumulate within the dorsal root ganglion after nerve injury.
BACKGROUND: HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that copies the RNA genome of HIV-1 into DNA. It is a heterodimer composed of a 66 kDa (p66) and a 51 kDa (p51) subunit. HIV-1 RT is a crucial target for structure-based drug design, and potent inhibitors have been identified, whose efficacy, however, is limited by drug resistance. RESULTS: The crystal structure of HIV-1 RT in complex with the non-nucleoside inhibitor alpha-anilinophenyl-acetamide (alpha-APA) R95845 has been determined at 2.8 A resolution. The inhibitor binds in a hydrophobic pocket near the polymerase active site. The pocket contains five aromatic amino acid residues and the interactions of the side chains of these residues with the aromatic rings of non-nucleoside inhibitors appear to be important for inhibitor binding. Most of the amino acid residues where mutations have been correlated with high levels of resistance to non-nucleoside inhibitors of HIV-1 RT are located close to alpha-APA. The overall fold of HIV-1 RT in complex with alpha-APA is similar to that found when in complex with nevirapine, another non-nucleoside inhibitor, but there are significant conformational changes relative to an HIV-1 RT/DNA/Fab complex. CONCLUSIONS: The non-nucleoside inhibitor-binding pocket has a flexible structure whose mobility may be required for effective polymerization, and may be part of a hinge that permits relative movements of two subdomains of the p66 subunit denoted the 'palm' and 'thumb'. An understanding of the structure of the inhibitor-binding pocket, of the interactions between HIV-1 RT and alpha-APA, and of the locations of mutations that confer resistance to inhibitors provides a basis for structure-based design of chemotherapeutic agents for the treatment of AIDS.
Mouse hepatitis virus strain A59 (MHV-A59) encodes within the 22-kb gene 1 a large polyprotein containing three proteinase domains with proven or predicted cysteine catalytic residues. E64d, a specific, irreversible inhibitor of cysteine (thiol) proteinases, inhibits the processing of the gene 1 polyprotein. Specifically, E64d blocks the carboxy-terminal cleavage of p65. E64d also inhibits replication of MHV-A59 in murine DBT cells in a dose-dependent manner, resulting in reduced virus titers and viral syncytia formation. This inhibition of replication is associated with a rapid shutoff of new viral RNA synthesis, in a manner similar to that seen in the presence of cycloheximide. The E64d-associated inhibition of RNA synthesis likely results from E64d-specific inhibition of processing of the gene 1 polyprotein, resulting in inactive proteinase or replicase proteins. These results indicate that processing of the MHV-A59 gene 1-encoded polyprotein is required throughout infection to sustain RNA synthesis and virus replication.
To investigate the effect of phosphorylation on the transcription activity of p53, ten phosphorylation mutants were constructed covering all the identified phosphorylation sites of rat p53. These included mutants of two casein kinase I sites (Ser6 and Ser9), two DNA-PK sites (Ser15 and Ser39), a p34cdc2 site (Ser313), the adjacent Ser312 and a casein kinase II site (Ser390). Two double phosphorylation mutants (Ser4, 6 and Ser15, 390) and one triple phosphorylation mutant (Ser4, 6 and 15) were also constructed. The transcription activity of all the p53 phosphorylation mutants was tested by transfection into two different types of cells, Saos-2 cells and p53(-/-) fibroblasts derived from p53 knock out mice, which both lack endogenouse p53. Surprisingly, all the p53 phosphorylation mutants retain transcription activity and the seven mutants tested can also suppress cell growth.
The p53 gene is required for the normal apoptotic response of mammalian cells to DNA damage caused by ionizing radiation and DNA damaging drugs. DNA damage results in the accumulation of biologically active p53. This response is potentially lethal and is therefore highly regulated. By using both biochemical and cell biological approaches a number of discrete control pathways have been identified. These include analysis of cellular and viral proteins that bind to p53 to inactivate its function, the discovery of cells with defects in the p53 activation pathway and the analysis of an allosteric regulation of p53 function controlled by phosphorylation.
This investigation was conducted to test whether topical nitroprusside (NP), a cytosolic guanylate cyclase activator, would increase the level of cyclic GMP and alter O2 consumption or blood flow in the cerebral cortex of rats. Male Long-Evans rats were used in a control (n = 9), low dose NP (n = 13, 10(-3) M) or high dose NP (n = 12, 10(-2) M) group. Nitroprusside or saline was topically applied to the right side of the cerebral cortex and the left side was used as a control. The cyclic GMP level was determined in five rats in each group using a radioimmunoassay. In the other rats in each group, regional cerebral blood flow was measured by [14C]iodoantipyrine and regional arterial and venous O2 saturations were determined microspectrophotometrically. Nitroprusside significantly increased the cyclic GMP level from 21.4 +/- 12.0 pmol/g (contralateral cortex) to 52.2 +/- 36.7 pmol/g (NP treated cortex) in low dose nitroprusside group and from 19.9 +/- 22.6 pmol/g (contralateral cortex) to 58.5 +/- 15.1 pmol/g (NP treated cortex) in high dose nitroprusside group. High dose nitroprusside significantly increased cerebral blood flow from 80 +/- 11 ml.min-1.100 g (contralateral cortex) to 114 +/- 11 ml.min-1.100 g (NP treated cortex). However, there was no significant difference in O2 extraction and O2 consumption between the NP treated cortex and contralateral cortex in either the low or the high dose NP groups.(ABSTRACT TRUNCATED AT 250 WORDS)