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Biomedical subjects

Y M Li

Publications and source records attributed to Y M Li.

At least 91 records · Page 5Linked to original sources
PubMed1995

Mapping peptide-binding domains of the substance P (NK-1) receptor from P388D1 cells with photolabile agonists.

The tachykinin substance P (SP) is a peptide transmitter of primary afferents. Its actions on both central and peripheral targets are mediated by a G-protein-coupled receptor of known primary structure. To identify contact sites between the undecapeptide SP and its receptor, we prepared radiolabeled photoreactive analogs of SP (H-RPKPQQFFGLM-NH2) by replacing amino acids in the peptide with p-benzoyl-L-phenylalanine (BPA). SP, BPA3-SP, and BPA8-SP bind with high affinity (Kd < 3 nM) to SP receptors on the murine cell line P388D1, triggering intracellular calcium responses. Both binding and calcium responses are blocked by the specific SP receptor antagonist CP-96345. On photolysis, radioiodinated BPA3-SP, and BPA8-SP covalently label a heterogeneously glycosylated protein of about 75 kDa; labeling is abolished by excess unlabeled SP or CP-96345. The labeled receptors were digested with V8 protease and/or trypsin, and the resulting fragments were analyzed by electrophoresis, high pressure liquid chromatography, and chemical or enzymatic modification. BPA3-SP and BPA8-SP photo-incorporate into different regions of the murine SP receptor. The results establish that the third and the eighth positions of SP, respectively, interact with the NH2-terminal extracellular tail (residues 1-21) and second extracellular loop (residues 173-183) of the SP receptor. A model for the agonist peptide-binding sites of the SP receptor is proposed based on photoaffinity labeling and mutagenesis studies.

Affinity Labels↗
PubMed1995

Antibacterial activity of lysozyme and lactoferrin is inhibited by binding of advanced glycation-modified proteins to a conserved motif.

Why diabetes is associated with abnormally high susceptibility to infection remains unknown, although two major antibacterial proteins, lysozyme and lactoferrin, have now been shown to specifically bind glucose-modified proteins bearing advanced glycation end products (AGEs). Exposure to AGE-modified proteins inhibits the enzymatic and bactericidal activity of lysozyme, and blocks the bacterial agglutination and bacterial killing activities of lactoferrin. Peptide mapping revealed a single AGE binding domain in lysozyme and two AGE binding domains in lactoferrin; each domain contains a 17- to 18- amino acid cysteine-bounded loop motif (CX15-16C) that is markedly hydrophilic. Synthetic peptides corresponding to these motifs in lysozyme and lactoferrin exhibited AGE binding activity, and similar domains are also present in other antimicrobial proteins. These results suggest that elevated levels of AGEs in tissues and serum of diabetic patients may inhibit endogenous antibacterial proteins by binding to this conserved AGE-binding cysteine-bounded domain 'ABCD' motif, thereby increasing susceptibility to bacterial infections in the diabetic population.

Amino Acid Sequence↗
PubMed1995

Local anesthetics inhibit substance P binding and evoked increases in intracellular Ca2+.

BACKGROUND: During spinal and epidural anesthesia, local anesthetics reach concentrations in cerebrospinal fluid and spinal cord tissues at which their actions may extend beyond the classic blockade of sodium channels. This study examines the effects of several clinical and experimental local anesthetics on the binding and actions of a peptide neurotransmitter, substance P, known to be important in nociceptive transmission in the dorsal horn. METHODS: The binding of radiolabeled (Bolton-Hunter modified) substance P was studied in chick brain membranes in the presence of local anesthetics. The increase in intracellular calcium [Ca2+]in evoked by substance P was measured by the fluorescent indicator fura-2 loaded in a murine cell line expressing substance P (NK1) receptors. Cells were preincubated with bupivacaine before and during the transient addition of substance P. RESULTS: Both substance P binding and Ca2+ increase were inhibited half-maximally by approximately 1 mM bupivacaine at pH 7.5, whereas tetracaine, lidocaine, and benzocaine were slightly less potent at inhibiting binding. Concentration-dependent substance P-binding studies showed that bupivacaine's inhibition was not competitive. Inhibition of substance P binding by bupivacaine increased with increasing pH, but the protonated species appears to have some inhibitory activity, and quaternary lidocaine also inhibited binding. There was no stereoselectively to the binding inhibition. CONCLUSIONS: Because millimolar concentrations of local anesthetics are within the range measured in spinal cord during intrathecal and epidural procedures, these results are consistent with a direct action of local anesthetics on tachykinin-mediated neurotransmission during regional anesthesia.

Anesthetics, Local↗
PubMed1995

Identification of galectin-3 as a high-affinity binding protein for advanced glycation end products (AGE): a new member of the AGE-receptor complex.

BACKGROUND: Advanced glycation end products (AGE), the reactive derivatives of nonenzymatic glucose-protein condensation reactions, are implicated in the multiorgan complications of diabetes and aging. An AGE-specific cellular receptor complex (AGE-R) mediating AGE removal as well as multiple biological responses has been identified. By screening an expression library using antibody against a previously identified component of the AGE-R complex p90, a known partial cDNA clone was isolated with homology to galectin-3, a protein of diverse identity, and member of the galectin family. MATERIALS AND METHODS: To explore this unexpected finding, the nature of the interactions between galectin-3 and AGE was studied using intact macrophage-like RAW 264.7 cells, membrane-associated and recombinant galectin-1 through -4, and model AGE-ligands (AGE-BSA, FFI-BSA). RESULTS: Among the members of this family (galectin-1 through 4), recombinant rat galectin-3 was found to exhibit high-affinity 125I-AGE-BSA binding with saturable kinetics (kD 3.5 x 10(7) M-1) that was fully blocked by excess unlabeled naturally formed AGE-BSA or synthetic FFI-BSA, but only weakly inhibited by several known galectin-3 ligands, such as lactose. In addition to the p90, immunoprecipitation with anti-galectin-3, followed by 125I-AGE-BSA ligand blot analysis of RAW 264.7 cell extracts, revealed galectin-3 (28 and 32 kD), as well as galectin-3-associated proteins (40 and 50 kD) with AGE-binding activity. Interaction of galectin-3 with AGE-BSA or FFI-BSA resulted in formation of SDS-, and beta-mercaptoethanol-insoluble, but hydroxylamine-sensitive high-molecular weight complexes between AGE-ligand, galectin-3, and other membrane components. CONCLUSIONS: The findings point toward a mechanism by which galectin-3 may serve in the assembly of AGE-R components and in the efficient cell surface attachment and endocytosis by macrophages of a heterogenous pool of AGE moieties with diverse affinities, thus contributing to the elimination of these pathogenic substances.

Animals↗
PubMed1995

[Health behavior in Hualien city high school students].

Chronic diseases and motor vehicle-related injuries are currently the major causes of death in Taiwan. Smoking, alcohol use and inappropriate driving behavior are important risk factors. Most health behavior develop during adolescence. Smoking and alcohol use always serve as "gateways" for other adverse health behavior. In this study, 1195 high school students from 27 classes were selected by a random systematic cluster sampling method, and a structured self-administered questionnaire was used to investigate their prevalent health behavior. The prevalence of current smoking was 9.9%, alcohol 18.7% and betel-nut chewing 5.4% among the academic high school students, whereas the prevalence among vocational school students was smoking 20.5%, alcohol 26.6% and betel-nut chewing 13.9%. Seventeen percent of vocational students reported that they often rode motorcycles after alcohol consumption. An aggregation of adverse health behavior, such as smoking, alcohol consumption and chewing betel-nuts was commonly observed. Designing comprehensive intervention programs to prevent and modify high school students adverse health behavior is an important task for school health-officials.

Adolescent↗
PubMed1994

Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats.

High levels of tissue advanced glycation end products (AGEs) that result from the spontaneous modification of proteins by glucose occur in diabetes and aging. To address the potential pathogenic role of AGEs in the glomerulosclerosis of diabetes or nephrosclerosis of aging, doses of AGE-modified rat albumin (25 mg per kg per day, i.v.) sufficient to elevate circulating AGE levels to the range of diabetic serum were administered daily to healthy rats alone or in combination with the AGE inhibitor aminoguanidine. After 5 months, the AGE content of renal tissues in AGE-treated rats rose to 50% above controls (P < 0.025), whereas serum contained 2.8-fold greater AGE levels (P < 0.025). Light and electron microscopy of kidneys from AGE-treated rats revealed a more than 50% increase in glomerular volume compared to controls (P < 0.001), significant periodic acid/Schiff reagent-positive deposits, basement membrane widening, and mesangial extracellular matrix increase and indicated significant glomerulosclerosis compared to untreated (P < 0.002) or albumin-treated controls (P < 0.002). These changes were associated with significant loss of protein (P < 0.005) and albumin (P < 0.002) in the urine of AGE-treated rats compared to controls. Cotreatment with aminoguanidine markedly limited both the structural and functional defects. These in vivo data demonstrate that AGEs influence glomerular structure and function in a manner leading to glomerulosclerosis. The effects are AGE-specific, as they are ameliorated by a pharmacological AGE inhibitor, aminoguanidine.

Aging↗
PubMed1994

Continuous beds for microchromatography: cation-exchange chromatography.

Microcolumns (i.d. 10-320 microns) for cation-exchange chromatography can be prepared simply by polymerization of an aqueous solution of appropriate monomers, including the desired ligand, directly in the chromatographic tube (fused-silica tubing) in the presence of salt. The beds thus prepared are in the form of rods traversed by channels through which the eluent can pass. The walls of the channels are composed of very small particles and are impermeable to peptides and proteins, which is important for rapid mass transfer and thus for high resolution at high flow rates. The bed becomes attached covalently to the tube wall during synthesis. A complicated column tube design with a supporting frit at the bottom is thus eliminated. The absence of a frit reduces the flow resistance and facilitates interfacing to mass spectrometers. The covalent linkage of the bed to the tube wall also serves to suppress the zone-broadening "wall effect." A homogeneous "packing" of a continuous bed column with an inner diameter as small as 10 microns is easily obtained. The resolution, binding capacity, and flow rate (i.e., run time at a given pressure) can be varied by changing the composition of the monomer solution. One can thus tailor the beds to each separation problem. The chromatographic properties of the microcolumns are demonstrated by separations of model proteins.

Chromatography, Ion Exchange↗
PubMed1994

Reduction in superoxide anion secretion and bactericidal activity of neutrophils from aged rats: reversal by the combination of gamma interferon and growth hormone.

Polymorphonuclear neutrophils (PMN) from bone marrow of 24-month-old rats kill Escherichia coli less efficiently than PMN from 3-month-old rats. Secretion of O2- and killing of E. coli by PMN from both young and old rats can be significantly augmented by preincubation with either 250 U of gamma interferon (IFN-gamma) or 250 ng of growth hormone (GH) per ml. This priming is specific, because neutralizing monoclonal antibodies against either IFN-gamma or GH completely abrogate the enhanced O2- secretion by PMN from young rats. However, in contrast to PMN from young rats, PMN from aged rats are not primed to kill E. coli by 10-fold-lower concentrations of either IFN-gamma (25 U/ml) or GH (25 ng/ml). To explore the mechanism for the reduction in bacterial killing by PMN from old rats, a syngeneic GH-secreting pituitary cell line (GH3) was implanted in vivo. PMN from GH3-treated aged rats, but not control aged rats, could now be primed in vitro for O2- secretion by IFN-gamma (25 U/ml). Although PMN from aged rats do not respond to the lower doses of either IFN-gamma or GH, the combination of both reagents totally restores the ability of PMN to secrete O2- and to kill E. coli. This synergistic priming is observed with PMN from aged rats, but not with those from young rats, and can be detected when both reagents are added simultaneously or when they are added in either sequence. Furthermore, addition of a monoclonal antibody against either IFN-gamma or GH abrogates the synergism of these two molecules. Collectively, these data identify an important alteration in myeloid cells from aged rodents by showing that their PMN are intrinsically unable to respond to low concentrations of IFN-gamma by secreting O2- and killing bacteria. The results also define a previously unrecognized synergism in PMN from aged animals by showing that GH synergizes with IFN-gamma both in vivo and in vitro to restore these suppressed responses.

Aging↗
PubMed1994

Agonist-induced photoincorporation of a p-benzoylphenylalanine derivative of substance P into membrane-spanning region 2 of the Torpedo nicotinic acetylcholine receptor delta subunit.

The neuropeptide substance P acts, at micromolar concentrations, as a noncompetitive antagonist of nicotinic acetylcholine receptors (AChRs) of both neuronal and muscle subtypes. The mechanism of this inhibition has been shown to be most consistent with stabilization of a nonconducting desensitized state of the AChR, via binding to a site distinct from both the agonist site and the high affinity noncompetitive antagonist site. We have used a radioiodinated photoreactive analogue of substance P, containing the amino acid p-benzoyl-L-phenylalanine in place of the Phe8 residue of substance P, to identify the sites of interaction of substance P within the Torpedo california AChR. AChR-rich membrane suspensions were photolabeled in the absence or presence of the agonist carbamylcholine and/or nonradioactive substance P, and incorporation into AChR subunits was assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of agonist 125I incorporation was detected in each subunit and was insensitive to substance P, whereas in the presence of carbamylcholine there was a 2-fold increase in photoincorporation into the AChR delta subunit that was inhibited by the addition of an excess of substance P. The sites of specific photoincorporation in the delta subunit were initially mapped by use of Staphylococcus aureus V8 protease to a 14-kDa fragment extending from delta Ile-192 to Glu-280. Further fragmentation of this 14-kDa fragment with trypsin and S. aureus V8 protease established that the sites of specific incorporation were restricted to the region delta Ser-253 to Glu-280, which contains the membrane-spanning region 2 that is known to form the lining of the ion channel. These results establish that in the presence of agonist at least a part of the undecapeptide substance P binds within the ion channel in the desensitized state of the AChR, and it is likely that the binding of substance P to this site is responsible for the action of substance P as a noncompetitive AChR antagonist.

Affinity Labels↗
PubMed1994

[The nuclear morphometric analysis and its clinical value in esophageal cancer].

The technique of computer-assisted morphometric analysis had been utilized to assess the prognosis for many cancers. However, there has not been the report about application of this method in esophageal cancer. The nuclear area (A) and nuclear roundness factor (NRF) of epithelial cells of 42 esophageal cancers and ten normal esophagus were measured by morphometry and the results were compared with the extent of the tumor (length, depth and lymph node metastasis), pathologic grades of the cancer, survival time of the patients, and so on. A and NRF of epithelial cells of esophageal cancer were significantly larger than those of normal esophagus (P < 0.001 and P < 0.05 respectively). In the esophageal cancer group, NRF of epithelial cells of patients with short survival time were significantly larger than those of long survival time (P < 0.05). NRF of epithelial cells of patients with lymphatic node metastasis were significantly larger than those of non-metastasis (P < 0.05). A and NRF of epithelial cells were not related with tumor length, invasive depth, and position, also not related with patient sex and age. The results confirmed that nuclear morphometry can play an important role in the diagnosis, differential diagnosis and prognosis of esophageal cancer.

Adult↗
PubMed1993

Protein phosphatase 2A and its [3H]cantharidin/[3H]endothall thioanhydride binding site. Inhibitor specificity of cantharidin and ATP analogues.

The target site for cantharidin (CA) and its analogues was isolated recently from mouse liver and identified as protein phosphatase (PP2A) in the heterodimeric form known as PP2A2. The most toxic CA analogue, endothall thioanhydride (ETA) (mouse i.p. LD50 0.3 mg/kg), appears to have the same binding site in mouse liver and brain based on studies comparing [3H]ETA and [3H]CA. ATP and its nonhydrolyzable analogues and pyrophosphate and related compounds including phosphonoformic acid inhibited both [3H]CA and [3H]ETA binding with IC50 values ranging from 2 to 81 microM. As with CA itself, the most potent inhibitors have two negatively charged groups in close proximity to each other. Inhibition of [3H]CA binding by 5,5'-dithiobis(2-nitrobenzoic acid) and stimulation by N-ethylmaleimide indicated the involvement of a thiol site in the CA-binding domain. CA and three analogues (cantharidic acid, palasonin and endothall) inhibited PP2A and protein phosphatase 1 (PP1) but not PP2B or PP2C. The catalytic subunit of PP2A was 5- to 12-fold more sensitive to these CA analogues than the catalytic subunit of PP1. CA and the herbicide endothall also inhibited spinach leaf PP1 and PP2A and, at 50 microM, decreased the PP2A-mediated light-induced activation of nitrate reductase in intact spinach leaves by 62 and 56%, respectively. This is consistent with PP2A as their site of action in plants, and indicates the potential use of CA analogues as pharmacological probes to investigate cellular processes that are regulated by reversible protein phosphorylation in vivo.

Adenosine Triphosphate↗
PubMed1993

Macrophages suppress lectin-induced proliferation of lymphocytes from aged rats.

Splenocytes from old rats exhibit a significant reduction in proliferative responses to Con A. This defect can be overcome by passing the cells over nylon wool, which suggests that nylon wool adherent cells contribute to the reduction in proliferation of mixed cell populations from aged rats. To examine the potential role of macrophages in this process, thioglycollate-elicited peritoneal macrophages were added to Con A-activated spleen cells from young and old rats. Addition of as few as 3% macrophages suppressed proliferation of Con-A-activated splenocytes and this inhibition was significantly greater if either the macrophages or spleen cells were derived from aged rats. Although prostaglandin E2 (PG-E2) inhibited the proliferation of splenocytes and nylon-wool enriched splenocytes from 3-month-old animals, its suppressive effects were much greater on Con A-activated lymphocytes derived from 24-month-old rats. Addition of catalase to deplete macrophage-derived H2O2 resulted in greater augmentation of Con-A-induced proliferative responses of splenocytes from old rats than those from young rats. TGF-beta 2, but not TNF-alpha, also significantly suppressed the proliferation of Con A-activated splenocytes to a greater degree in aged than in young rats. Since macrophages are known to release PG-E2, H2O2 and TGF-beta 2 after stimulation with Con A, these data support the idea that T cells within the splenocyte population become increasingly susceptible during aging to inhibitory products released by macrophages.

Aging↗
PubMed1993

Comparative SPECT study of stroke using Tc-99m ECD, I-123 IMP, and Tc-99m HMPAO.

Brain perfusion SPECT imaging using Tc-99m ethyl cysteinate dimer (ECD) was carried out in 12 patients with chronic cerebrovascular disease. The sensitivity of lesion detection and lesion-to-normal tissue contrast was compared with those of I-123 IMP and Tc-99m HMPAO. Eight patients underwent all three studies and the remaining four patients had Tc-99m ECD and I-123 IMP scans. The sensitivity of lesion detection and lesion contrast was analyzed quantitatively using an asymmetric index determined by the equation: 100 x [right - left[/(right + left). In the cerebral cortex, cerebellum, and white matter, I-123 IMP showed higher lesion sensitivity than Tc-99m ECD and Tc-99m HMPAO. In the striatum and thalamus, Tc-99m ECD and I-123 IMP showed higher lesion sensitivity than Tc-99m HMPAO. I-123 IMP showed the highest lesion contrast in cerebral cortex and cerebellum, whereas Tc-99m ECD showed the highest contrast in thalamus and striatum. In all regions, Tc-99m ECD showed higher lesion contrast than Tc-99m HMPAO. These results suggest regional variation in the sensitivity of lesion detection and lesion contrast of Tc-99m ECD compared to I-123 IMP, and the superiority of Tc-99m ECD to Tc-99m HMPAO.

Adult↗
PubMed1993

[Studies on the phenolic derivatives from Galeola faberi Rolfe].

Eight phenolic derivatives, p-hydroxybenzaldehyde (I), 4,4'-dihydroxy-diphenyl methane(II), 2,4-bis(4-hydroxybenzyl) phenol (III), 5-methoxy-3-(2-phenyl-E-ethenyl)-2,4-bis (4-hydroxybenzyl) phenol (IV), p-hydroxybenzyl alcohol (V), 4-(beta-D-glucopyranosyloxy) benzyl alcohol (gastrodin) (VI), bis[4-(beta-D-glucopyranosyloxy) benzyl] (S)-2-isopropylmalate (VII), bis [4-(beta-D-glucopyranosyloxy) benzyl] (S)-2-sec-butylmalate (VIII) were isolated from the rhizome of Galeola faberi Rolfe for the first time. IV and VIII are new compounds. III is a new natural compound.

Benzhydryl Compounds↗
PubMed1992

Cantharidin-binding protein: identification as protein phosphatase 2A.

The toxic effects of cantharidin from blister beetles and its analogs, including the herbicide endothall, are attributable to their high affinity and specificity for a cantharidin-binding protein (CBP). An ammonium sulfate precipitate of mouse liver cytosol was purified by five chromatographic steps to isolate CBP in 14% yield and > 99% purity as monitored by [3H]cantharidin-binding activity. The purification factor of 2230-fold corresponds to a CBP content of 0.045% of the liver cytosolic protein. CBP is a heterodimer consisting of a 61-kDa alpha subunit and a 39-kDa beta subunit. Amino acid sequences of four peptides from CBP-alpha and three peptides from CBP-beta are identical with deduced amino acid sequences for the A alpha regulatory and C beta catalytic subunits, respectively, of protein phosphatase 2A (PP2A). This assignment of CBP as PP2A-AC from structural evidence is supported by biochemical studies with selective substrates and inhibitors. CBP dephosphorylation of phosphorylase alpha is sensitive not only to okadaic acid, as with PP2A, but also to cantharidin and its analogs, consistent with their potency in blocking the radioligand binding site of CBP. Okadaic acid is a potent inhibitor of [3H]cantharidin binding to CBP. PP2A is present in many mammalian tissues and in plants and is involved in regulatory phosphorylation-dephosphorylation events which modulate multiple cellular functions. Inhibition of PP2A activity may account for the diverse effects and toxicity of cantharidin and its analogs, including the herbicide endothall, in mammals and possibly plants.

Amino Acid Sequence↗