Biomedical subjects
Publications and source records attributed to L Deng.
L-770,644 (9c) is a potent and selective agonist of the human beta3 adrenergic receptor (EC50 = 13 nM). It shows good oral bioavailability in both dogs and rats (%F = 27), and is a full agonist for glycerolemia in the rhesus monkey (ED50 = 0.21 mg/kg). Based on its desirable in vitro and in vivo properties, L-770,644 was chosen for further preclinical evaluation.
OBJECTIVE: To develope the method of two-color fluorescence in situ hybridization (FISH) assay and use it for detecting the aneuploidy frequency of human sperm chromosome. METHODS: Sperm sample was washed three times and the slides were prepared. The sperm heads were decondensed with dithiothreitol (DTT) and lithium diiodosalicylate (LIS). Then, the sperm nuclei were hybridized with biotin labeled alpha satellite X chromosome DNA probe (DXZ1) and digoxigenin labeled alpha satellite Y chromosome DNA probe (DYZ3). The hybridization signals were detected with CY3-Streptavidin, goat antistrepavidin for biotin labeled probe and with mouse antidigoxigenin, rabbit antimouse-FITC for digoxigenin labeled probe. RESULTS: Under the Nikon fluorescence microscope, the hybridization signals in the sperm heads were clearly visible. The sperm with one red hybridization signal was X chromosome sperm (X sperm), and the sperm with one green hybridization signal in the sperm head was Y chromosome sperm (Y sperm). In the case of two hybridization signals in one sperm head, the sperm should be a numerical abnormal one. By using two color FISH with one euchromosome probe and one sex chromosome probe, the sperm with same color of two hybridization signals in one sperm head could be identified as aneuploidy sperm or diploid sperm. CONCLUSION: The two color FISH assay may be used to detect the aneuploidy frequency of human sperms that were exposed to mutagents and environmental potential aneuoploidogenic agents.
Extraction of sodium atoms from a trapped Bose-Einstein condensate (BEC) by a coherent, stimulated Raman process is demonstrated. Optical Raman pulses drive transitions between trapped and untrapped magnetic sublevels, giving the output-coupled BEC fraction a well-defined momentum. The pulsed output coupling can be run at such a rate that the extracted atomic wave packets strongly overlap, forming a highly directional, quasi-continuous matter wave.
The cyclopentylpropylimidazolidinone L-766,892 is a potent beta3 AR agonist (EC50 5.7 nM, 64% activation) with 420- and 130-fold selectivity over binding to the beta1 and beta2 ARs, respectively. In anesthetized rhesus monkeys, L-766,892 elicited dose-dependent hyperglycerolemia (ED50 0.1 mg/kg) with minimal effects on heart rate.
Human beta3 adrenergic receptor agonists containing 5-membered ring ureas were shown to be potent partial agonists with excellent selectivity over beta1 and beta2 binding. L-760,087 (4a) and L-764,646 (5a) (beta3 EC50 = 18 and 14 nM, respectively) stimulate lipolysis in rhesus monkeys (ED50 = 0.2 and 0.1 mg/kg, respectively) with minimal effects on heart rate. Oral absorption in dogs is improved over other urea analogs.
VP39 is a bifunctional mRNA-modifying protein that acts as both an mRNA cap-specific 2'-O-methyltransferase and a processivity factor for VP55, the vaccinia poly(A) polymerase catalytic subunit. Although regions of the protein surface required for methyltransferase function are well defined, it has been unclear whether the protein polyadenylylation function requires direct RNA contact and, if so, where the contact site(s) might be located on the protein surface. Here, we show that the VP55-VP39 heterodimer forms a stable complex with a 50mer oligonucleotide bearing a U2-N25-U motif, as opposed to the U2-N15-U motif that is optimal for stable complex formation with VP55 alone. An oligonucleotide bearing a U2-N25-U motif in which the downstream U residue is replaced with 4thioU can be efficiently photocrosslinked to VP39, but only in the context of the VP55-VP39 heterodimer. By partial proteolysis of end-labeled VP39, the site of oligonucleotide photocrosslinking was localized to the region of VP39 between residues Lys90 and Arg122. Peptide microsequencing and confirmatory mutagenesis identified the side-chain of Arg107 as the photocrosslinking site. Substitution of this residue with lysine abolished photocrosslinking entirely, consistent with the established RNA binding role of arginine in other RNA-binding proteins. This study provides clear evidence for a polyadenylylation-specific RNA-contact site on the surface of VP39, which is distinct from the RNA-binding methyltransferase "cleft" characterized in recent crystallographic and biochemical studies.
Polydnaviruses are the only known group of mutualistic viruses. They are required for successful parasitization in many braconid and ichneumonid parasitoids. The intimacy of this mutualistic association is indicated by the integration and vertical transmission of polydnaviruses in wasp genomes and by their asymptomatic, developmentally regulated replication. The evolution of this mutualism raises several interesting issues that require a better understanding of the viral genome and viral replication. To develop probes for virus replication and morphogenesis, we have begun to characterize several viral structural proteins. A 699 bp cDNA encoding the p12 viral structural protein was cloned and sequenced. The p12 gene localizes to viral segment Y and encodes a predicted protein of 92 amino acids that does not encode a signal peptide and is unrelated to known peptide or nucleic acid sequences. The p12 mRNA is detected at the onset of virus replication. mRNA titers increase with increasing rates of virus replication. Polyclonal antisera raised against histidine-tagged p12 protein expressed in bacteria reacted specifically with the p12 polypeptide in Western blots of CsPDV virions. The p12 polypeptide was not detected in non-replicative wasp or lepidopteran tissues by Western blot analyses but was readily detected in protein extracts of wasp ovaries. The data indicate that the p12 gene is a viral gene encoding a virion protein and provides a specific probe for virus replication that will be useful for studying the evolution of this group of mutualistic viruses.
OBJECTIVE: To develop the method of two-color fluorescence in situ hybridization (FISH) assay and use it for detecting the aneuploidy frequency of human sperm chromosome. METHODS: Sperm sample was washed three times and the slides were prepared. The sperm heads were decondensed with dithiothreitol (DTT) and lithium diiodosalicylate (LIS). Then, the sperm nuclei were hybridized with biotin labeled alpha satellite X chromosome DNA probe (DXZ1) and digoxigenin labeled alpha satellite Y chromosome DNA probe (DYZ3). The hybridization signals were detected with CY3-Streptavidin, goat antistrepavidin for biotin labeled probe and with mouse antidigoxigenin, rabbit antimouse-FITC for digoxigenin labeled probe. RESULTS: Under the Nikon fluorescence microscope, the hybridization signals in the sperm heads were clearly visible. The sperm with one red hybridization signal was X chromosome sperm (X sperm), and the sperm with one green hybridization signal in the sperm head was Y chromosome sperm (Y sperm). In the case of two hybridization signals in one sperm head, the sperm should be a numerical abnormal one. By using two color FISH with one euchromosome probe and one sex chromosome probe, the sperm with same color of two hybridization signals in one sperm head could be identified as aneuploidy sperm or diploid sperm. CONCLUSION: The two color FISH assay may be used to detect the aneuploidy frequency of human sperms that were exposed to mutagents and environmental potential aneuoploidogenic agents.
OBJECTIVE: To search differentially expressed sequences correlated with pathogenesis of human nasopharyngeal carcinoma (NPC), including the candidates of tumor suppressor genes. METHODS: cDNA representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from normal human primary cultures of nasopharyngeal epithelial cells and cDNA from NPC cell line HNE1. The sources of differentially expressed products were proved by Southern blot and Northern blot. The fragments were cloned with pGEM-T easy kit and sequenced by the chain termination reaction. RESULTS: Four differentially expressed cDNA fragments were isolated in the fourth subtractive hybridization using cDNA from normal human primary cultures of nasopharyngeal epithelial cells as tester amplicon and cDNA from NPC cell line HNE1 as driver amplicon by cDNA RDA. These differential cDNA fragments revealed that they really came from the tester amplicon and were not expressed or down-regulated in the NPC HNE1 cells. Of these obtained clones, some are the fragments of the human known genes including house-keeping genes, the others are novel genes. CONCLUSION: NPC involves alteration of multiple genes. Some of known genes matched with the differentially expressed sequences have an effective suppressive ability on the carcinoma.
The 3-pyridylethanolamine L-757,793 is a potent beta 3 AR agonist (EC50 6.3 nM, 70% activation) with 1,300- and 500-fold selectivity over binding to the beta 1 and beta 2 ARs, respectively. L-757,793 stimulated lipolysis in rhesus monkeys (ED50 0.2 mg/kg) with a maximum response equivalent to that elicited by isoproterenol.
OBJECTIVE: To establish partial gene expression map of 7q32 in nasopharyngeal carcinoma (NPC) cell line, tissues and primary culture normal nasopharyngeal epithelial cells. METHODS: We detected the expression of 20 ESTs at 7q32 in NPC cell line HNE1,13 NPC biopsies and primary culture normal nasopharyngeal epithelial cells using differential RT PCR and Northern hybridization. RESULTS: 8 ESTs (AA188181, AA13079,N27556, AA031919, N22721, H20825, T91284, AA001936) expressed equally in both of HNE1 and primary culture normal nasopharyngeal epithelial cells; 7 ESTs (T64215, AA025822, R60014,R80002,H06688, R60192,R95096) expressed in neither of them; 3 ESTs (H19830,W72688,AA130630) overexpressed in HNE1 ; and 2 ESTs (AA070437, H90882) overexpressed in primary culture normal nasopharyngeal epithelial cells. W72688 and H19830 each overexpressed in 77%(10/13) of NPC biopsies; AA070437 down-expressed in 30.7% of NPC biopsies. CONCLUSION: Partial gene expression map of 7q32 in nasopharyngeal carcinoma cell line ,tissues and primary culture normal nasopharyngeal epithelial cells has been established. The up-regulation of W72688, H19830 and down-regulation of AA070437 may be related to the occurrence of NPC.
Pyridyloxypropanolamines L-749,372 (8, beta 3 EC50 = 3.6 nM) and L-750,355 (29, beta 3 EC50 = 13 nM) are selective partial agonists of the human receptor, with 33% and 49% activation, respectively. Both stimulate lipolysis in rhesus monkeys (ED50 = 2 and 0.8 mg/kg, respectively), with minimal effects on heart rate. Oral bioavailability in dogs, 41% for L-749,372 and 47% for L-750,355, is improved relative to phenol analogs.
Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.
A study of 4-acylaminobenzenesulfonamides in a cloned human beta 3 adrenergic receptor assay resulted in the discovery of n-hexylurea, L-755,507 (22). This 0.43 nM beta 3 agonist, which is > 440-fold selective over both beta 1 and beta 2 binding, is among the most potent human beta 3 agonists reported to date.
Protein phosphatase 2A (PP2A) is one of the most important and abundant serine/threonine phosphatases in mammalian tissues and plays a role in gene expression, cell division, and signal transduction. PP2A is activated by ceramide, which is produced by the hydrolysis of membrane sphingomyelin in response to a variety of stress-related stimuli. To further study the role of ceramide-mediated signal transduction in cellular processes such as senescence and apoptosis, we designed and synthesized a series of low molecular weight aromatic compounds, mainly of the isoquinolone and tetralone classes, and evaluated their ability to inhibit enzymes known to be activated by ceramide. Those enzymes studied were ceramide-activated protein kinase, protein kinase C zeta and PP2A. Of these, only PP2A was found to be inhibited. A few of the compounds inhibited both ceramide-activated as well as basal PP2A activity. In addition, several of the compounds activated PP2A by up to 300% above basal enzyme activity, but only in the presence of ceramide. Thus, modulation (both inhibition and activation) of the catatylic activity of ceramide-activated PP2A is demonstrated by certain low molecular weight aromatic compounds.
Metabolic stability of synthetic dynorphins [N-terminal fragments of dynorphin A (Dyn A)] were evaluated in vitro and in vivo. These peptides were applied at concentrations 100-1000 times higher than those of the endogenous dynorphins. Degradation kinetics of these peptides were studied in rat brain homogenate by using microbore gradient RP-LC assay, and limited information on their metabolism was obtained by electrospray ionization mass spectrometry (ESI-MS) of the isolated metabolites. In vivo cerebral microdialysis, in which the peptides were introduced via the probe placed in striatum region of the brain of the experimental animals, was used to circumvent contamination arising from autoproteolysis of brain during incubation of the samples in vitro. Metabolites of Dyn A (1-13) and Dyn A (1-11) were identified from electrospray ionization mass spectra of the microdialysates without chromatographic separation; the identification of peptides in the mixtures were supported by medium resolution ESI Fourier-transform ion cyclotron resonance MS. LC-MS was used to fully characterize the complex peptide mixture obtained after the striatal perfusion of Dyn A (1-12).
Vaccinia virus RNA polymerase terminates transcription in response to a specific signal UUUUUNU in the nascent RNA. Transduction of this signal to the elongating polymerase requires a trans-acting viral termination factor (VTF/capping enzyme), and is coupled to the hydrolysis of ATP. Recent studies suggest that ATP hydrolysis is catalyzed by a novel termination protein (factor X), which is tightly associated with the elongation complex. Here, we identify factor X as NPH-I (nucleoside triphosphate phosphohydrolase-I), a virus-encoded DNA-dependent ATPase of the DExH-box family. We report that NPH-I serves two roles in transcription (1) it acts in concert with VTF/CE to catalyze release of UUUUUNU-containing nascent RNA from the elongation complex, and (2) it acts by itself as a polymerase elongation factor to facilitate readthrough of intrinsic pause sites. A mutation (K61A) in the GxGKT motif of NPH-I abolishes ATP hydrolysis and eliminates the termination and elongation factor activities. Related DExH proteins may have similar roles at postinitiation steps during cellular mRNA synthesis.
Suramin, an organic polyanion with six sulphonic groups, is under clinical trials as an agent against hormone-refractory prostate cancer. The drug binds strongly to serum albumin. The objectives were to use electrospray to measure the molecular masses of the intact complexes of albumin and suramin to determine the number of suramin molecules bound under different molar ratios, and to investigate the binding of suramin in human serum. With albumin in excess (2:1 to 25:1 ratio), only 1 and 2 bound suramins were found; with suramin excess (2:1 to 1000:1) up to 20 bound suramin molecules/albumin were found. Up to 5 bound suramins were found in human serum with 4:1 suramin:albumin ratio, which corresponds to recommended therapeutic doses (200-300 micrograms/mL). At 8:1 ratio, which would be toxic, complexes with up to ten bound suramin molecules were found, and unreacted albumin diminished.