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Biomedical subjects

G Li

Publications and source records attributed to G Li.

At least 775 records · Page 43Linked to original sources
PubMed1995

Synthesis of N,N'-substituted piperazine and homopiperazine derivatives with polyamine-like actions at N-methyl-D-aspartate receptors.

A series of N,N'-substituted piperazine and homopiperazine derivatives have been synthesized with the objective of producing compounds that interact with polyamine modulatory sites on N-methyl-D-aspartate (NMDA) receptors. These novel compounds exhibited polyamine-like actions, enhancing [3H]MK-801 binding to NMDA receptors in rat forebrain membranes. The potencies of N,N'-bis(2-aminoacetyl)homopiperazine (15), N,N'-bis(N-methyl-4-aminobutyl)-piperazine (7), and N,N'-bis(3-aminopropyl)homopiperazine (11) (EC50 18.0, 21.3, and 24.4 microM, respectively) to enhance [3H]MK-801 binding were comparable to that of spermine (EC50 5.2 microM). However, the efficacies of 15, 7, and 11 in this measure were lower (by approximately 40%, 32%, and 24%, respectively) than spermine, which may be indicative of partial agonist actions. Like spermine, the ability of these piperazine and homopiperazine derivatives to enhance [3H]MK-801 binding could be inhibited by both a competitive polyamine antagonist (arcaine) and a specific, noncompetitive polyamine antagonist (conantokin-G). However, unlike endogenous polyamines, high concentrations (up to 1 mM) of these novel polyamine-like compounds did not inhibit [3H]MK-801 binding. N,N'-Aminoalkylated and aminoacylated piperazine and homopiperazine derivatives may prove useful for studying polyamine recognition sites associated with NMDA receptors.

Animals↗
PubMed1995

Hybridization of fluorescein-labeled DNA oligomers detected by fluorescence anisotropy with protein binding enhancement.

Fundamental aspects of the application of fluorescence anisotropy to detect the hybridization of fluorescein-labeled DNA oligomers were explored. The oligomers included a binding site for the EcoRI restriction enzyme, which binds to double-stranded DNA and is used in this work to enhance the difference between the anisotropies of the single-stranded and double-stranded oligomers by increasing the effective volume of the latter. The fluorescence anisotropy increases upon hybridization and further upon binding of EcoRI to the double strand. By varying the length of the tether used to attach the fluorescein to the 5' end of the oligonucleotide, it was found that a 6-carbon tether was optimal, providing the most dramatic increases in anisotropy in the presence of EcoRI. Dynamic fluorescence anisotropy (DFA) provided insight into the increases in steady-state anisotropy. In most cases, the best fits to the DFA data were obtained using a biexponential decay model, which describes an anisotropic rotator. Upon hybridization, the faster rotational motion is more hindered, and the contribution of the slower rotational component is increased. This effect is enhanced by binding of EcoRI to the double strand, especially when the EcoRI binding site is near the fluorescein at the 5' end and the tether length is in the optimal range. Because the rotational correlation time of the slower anisotropy decay component is much longer than the fluorescence lifetime, it is possible in some cases to reduce the anisotropic rotator model to the special limiting case of a hindered rotator.

Base Sequence↗
PubMed1995

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

Androstadienes↗
PubMed1995

bcl-2 suppresses expression of p21WAF1/CIP1 in breast epithelial cells.

The tumor suppressor gene p53 regulates G1 checkpoint prior to the initiation of DNA synthesis, which can either induce G1 arrest or signal apoptosis. The involvement of p53 in apoptosis may also be related to its ability to down-regulate transcription of the bcl-2 gene. The bcl-2 gene product prevents most types of apoptotic cell death, suggesting that bcl-2 interferes with an essential signaling molecule involved in the apoptotic cell death pathway. Although the bcl-2 protein is shown to be overexpressed in many types of human tumor including breast cancer, its biochemical or pathological consequences are poorly understood. To determine the effects of bcl-2 overexpression on apoptosis and transformation of breast epithelial cells and to investigate whether bcl-2 interferes with the p53 pathway, we introduced the bcl-2 expression vector into MCF10A cells, which were derived from diploid human breast epithelial cells containing the wild-type p53 gene. Overexpression of bcl-2 prevented free radical-induced apoptosis and induced a partially transformed phenotype in MCF10A cells. Although overexpression of bcl-2 did not affect the expression of the p53 gene, p53-dependent gene transcription such as p21WAF1/CIP1 was suppressed. These results suggest that bcl-2 may inhibit p53 functional activity and is involved in the regulation of an early commitment step either to proliferate or suicide.

Apoptosis↗
PubMed1995

Platelet-derived growth factor induces apoptosis in growth-arrested murine fibroblasts.

The platelet-derived growth factor (PDGF) is a potent mitogen for murine fibroblasts. PDGF-stimulated cells express a set of immediate-early-response genes but require additional (progression) factors in serum to progress through the cell cycle. Serum-deprived cells are reversibly arrested in G0 phase and fail to fully traverse the G1 phase of the cell cycle when stimulated by PDGF alone. We now report that serum-deprived normal rat kidney fibroblast (NRK) cells stimulated by either PDGF AA or PDGF BB homodimers undergo apoptotic cell death. Furthermore, we show that epidermal growth factor also induces apoptotic cell death in serum-deprived NRK cells, epidermal growth factor enhances the rate of apoptosis in PDGF-treated cells, and a progression factor (insulin) but not endogenously expressed Bc1-2 fully protects NRK cells from PDGF-stimulated apoptosis. The results indicate that PDGF induces apoptosis in growth-arrested NRK cells and that the inability of NRK cells to transit the G1/S checkpoint is the critical determinant in establishing the genetic program(s) to direct the PDGF signal to apoptosis. The results suggest that polypeptide growth factors in vivo may signal cell fate positively or negatively in settings that limit the potential of cells to completely transit the cell cycle.

Animals↗
PubMed1995

Endogenous ligands of imidazoline receptors: classic and immunoreactive clonidine-displacing substance and agmatine.

1. There are several endogenous ligands that bind to I-receptors of both the I1 and I2 subclass. These include: (a) classic CDS, a partially purified entity isolated by the criteria that it displaces binding ligands to alpha 2- and I-receptors; (b) immunoreactive (ir)-CDS, a moiety that binds to antibodies raised against clonidine, para-amino-clonidine, or idazoxan; and (c) agmatine. 2. Classic-CDS, not yet defined structurally, binds to I1, I2, and alpha 2-adrenergic receptors, is neither a peptide nor a catecholamine, and has purportedly a molecular weight of 588 Da. By ligand binding assays, it was found in brain, serum, CSF, and placenta and in a neural-glial cell line. Partially purified classic CDS is bioactive. Like clonidine, it contracts aorta and vas deferens and inhibits platelet aggregation, effects largely attributable to agonism at alpha 2-adrenergic receptors. Unlike clonidine, it contracts rat gastric fundus and releases catecholamines from chromaffin cells, effects attributable to actions at I-receptors. Injected into the RVL, classic CDS alters arterial pressure, but the direction of change of pressure has differed between groups of investigators. However, in the absence of structure, it is possible that ligand binding and bioactivity may be attributable to different molecules. 3. Ir-CDS, also of unknown structure, is a material(s) that binds to antibodies raised against clonidine, PAC, or idazoxan. Ir-CDS, measured by radioimmunoassay, is unevenly distributed in brain with highest concentrations in the hypothalamus, midbrain, and dorsal medulla. It is contained in the gastric fundus, adrenal gland, heart, kidney, and serum in amounts substantially higher than found in brain. Ir-CDS may be elevated in the serum of some patients with hypertension and in the CSF of patients with structural brain disease. The concentration of ir-CDS and bioactivity on gastric fundus directly correlates, suggesting that it may share similarities with classic-CDS. However, until the structure of classic and ir-CDS is determined, the possibility that ligand binding and antibody recognition are properties of different molecules must be considered. 4. Agmatine (decarboxylated arginine) is the only endogenous molecule that, like CDS, binds to alpha 2- and I-receptors of both classes. It and its biosynthetic enzyme arginine decarboxylase are present in brain, and agmatine is widely distributed throughout the body. However, the distribution of agmatine and ir-CDS differs, whereas the biological actions of agmatine do not mimic those of classic CDS. Its presence raises the possibility of an alternative pathway for polyamine biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Agmatine↗
PubMed1995

Separation of the eleven priority pollutant phenols by capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) provides highly efficient separation of the eleven US EPA priority pollutant phenols. All of these phenols are completely resolved in fewer than 15 min in a 100 cm x 75 microns I.D. uncoated fused-silica capillary at 22.5 kV using a pH 9.8 phosphate-borate buffer. Buffer pH is the most critical parameter controlling resolution and separation time. A simple theoretical treatment greatly simplifies the pH optimization procedure. The effects on the separation of buffer concentration, applied voltage, and sample quantity injected were studied. Good calibration data were obtained for phenol concentrations up to 50 mg/l. Limits of detection for all phenols were less than 1 ppm.

Buffers↗
PubMed1995

Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease.

Some cases of Alzheimer's disease are inherited as an autosomal dominant trait. Genetic linkage studies have mapped a locus (AD3) associated with susceptibility to a very aggressive form of Alzheimer's disease to chromosome 14q24.3. We have defined a minimal cosegregating region containing the AD3 gene, and isolated at least 19 different transcripts encoded within this region. One of these transcripts (S182) corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. Five different missense mutations have been found that cosegregate with early-onset familial Alzheimer's disease. Because these changes occurred in conserved domains of this gene, and are not present in normal controls, they are likely to be causative of AD3.

Alzheimer Disease↗
PubMed1995

Catalytic cleavage of an RNA target by 2-5A antisense and RNase L.

2-5A antisense (2-5A-AS) molecules are chimeric oligonucleotides that cause 2-5A-dependent RNase (RNase L) to catalyze the selective cleavage of RNA in human cells. These composite nucleic acids consist of a 5'-monophosphorylated, 2',5'-linked oligoadenylate known as 2-5A (an activator of RNase L) covalently attached to antisense 3',5'-oligodeoxyribonucleotides. Here, we characterize the targeted cleavage of the double-stranded RNA-dependent protein kinase (PKR) mRNA by purified, recombinant human RNase L. A 2-5A-AS chimera, which contains complementary sequence to PKR mRNA, and unmodified 2-5A, which causes general RNA decay, were about 20- and 40-fold more active, respectively, than 2-5A-AS chimeras in which the DNA domains are not complementary to sequences in PKR mRNA. Directed cleavage was efficient because each 2-5A-AS chimera targeted many RNA molecules. Moreover, RNase L caused the catalytic cleavage of the RNA target (kcat of approximately 7 s-1). The precise sites of PKR mRNA cleavage caused by 2-5A-AS were mapped, using a primer extension assay, to phosphodiester bonds adjacent to the 3' terminus of the chimera binding site (5' on the RNA target) as well as within the chimera's oligonucleotide binding site itself. The selectivity of this approach is shown to be provided by the antisense arm of the chimera, which places the RNA target in close proximity to the RNase.

Base Sequence↗
PubMed1995

A paradigm for drug discovery employing encoded combinatorial libraries.

Very large combinatorial libraries of small molecules on solid supports can now be synthesized and each library element can be identified after synthesis by using chemical tags. These tag-encoded libraries are potentially useful in drug discovery, and, to test this utility directly, we have targeted carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) as a model. Two libraries consisting of a total of 7870 members were synthesized, and structure-activity relationships based on the structures predicted by the tags were derived. Subsequently, an active representative of each library was resynthesized (2-[N-(4-sulfamoylbenzoyl)-4'-aminocyclohexanespiro]-4-oxo-7 -hydroxy- 2,3-dihydrobenzopyran and [N-(4-sulfamoylbenzoyl)-L-leucyl]piperidine-3-carboxylic acid) and these compounds were shown to have nanomolar dissociation constants (15 and 4 nM, respectively). In addition, a focused sublibrary of 217 sulfamoylbenzamides was synthesized and revealed a clear, testable structure-activity relationship describing isozyme-selective carbonic anhydrase inhibitors.

Benzopyrans↗
PubMed1995

Ultraviolet radiation induction of squamous cell carcinomas in p53 transgenic mice.

Mutations of the p53 gene have been implicated in the pathogenesis of cutaneous squamous cell carcinoma (SCC). To examine the role of p53 in skin carcinogenesis, we observed the development of skin cancers in p53 transgenic mice which carry multiple copies of a mutant allele of the p53 gene with or without chronic UVB radiation. Thirty-one tumors developed in 19 UV-irradiated p53 transgenic mice versus 14 of 19 in the control group; 9 p53 transgenic mice but none of the control mice developed multiple tumors. Histologically. 14 of 14 tumors in the CD-1 mice were SCCs. In the p53 transgenic mice, 25 of 31 tumors were SCCs, and 6 were benign tumors. The mean time to appearance of tumors did not differ between CD-1 mice (26.3 weeks) and the p53 transgenic mice (25.7 weeks; P = 0.512). The p53 protein, which was undetectable by immunohistochemistry in the keratinocytes of CD-1 mice, was elevated in 93% (13 of 14) of tumors from CD-1 mice. These data indicate that mutation of the p53 gene is an important step in the development of SCC. p53 mutations do not alter the latent period of UV-induced SCC but significantly increase the number of tumors and the propensity for multiple tumor development.

Animals↗
PubMed1995

Enzymatic and metabolic studies on retrograde regulation mutants of yeast.

Two nuclear genes, RTG1 and RTG2, which sense the functional state of yeast mitochondria, have been described recently. Yeast strains with null alleles of either of these two genes (delta rtg1, delta rtg2) cannot grow on acetate as the sole carbon source and are auxotrophic for glutamate and aspartate. We report here a series of metabolic experiments and enzyme activity measurements that were made in an attempt to determine the reason for the acetate- phenotype and the glutamate/aspartate auxotrophy. Decreases in the activities (approximately 50%) in mitochondrial citrate synthase (CS1), acetyl-CoA synthetase, NAD isocitrate dehydrogenase, and pyruvate carboxylase were noted. When CS1 was overexpressed in the delta rtg1 and delta rtg2 mutants, these strains could grow on acetate but were still auxotrophic for glutamate/aspartate. We propose that, in the mutant strain, CS1 activity becomes limiting for efficient acetate utilization, but that other complex metabolic interactions are affected, limiting production of intermediates that would allow synthesis of glutamic and aspartic acids.

Acetate-CoA Ligase↗