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C Yu

Publications and source records attributed to C Yu.

At least 379 records · Page 21Linked to original sources
PubMed1993

Calculating three-dimensional molecular structure of paliurine B from atom-atom distance and restrained energy minimization.

The conformation of paliurine B, a 13-membered cyclopeptide alkaloid isolated from Paliurus ramosissimus, has been determined from 2D NMR and distance geometry, followed by the restrained energy minimization calculation. The conformation of the 13-membered ring is well-defined but that of the acyclic dipeptide tail region is relatively disordered. In addition, the cavity in the 13-membered ring is just large enough to insert a magnesium ion but is a little small for calcium or sodium ions.

Alkaloids↗
PubMed1993

A new mammalian DNA topoisomerase I poison Hoechst 33342: cytotoxicity and drug resistance in human cell cultures.

Hoechst dye 33342 (Ho33342), like many other DNA minor groove binding ligands and its parent compound Hoechst dye 33258 (Ho33258), nonspecifically inhibits the catalytic activities of many DNA enzymes. However, both Ho33258 and Ho33342 also specifically interrupt the breakage/reunion reaction of mammalian DNA topoisomerase I by trapping reversible topoisomerase I cleavable complexes. The enhanced membrane permeability of Ho33342 over its parent compound Ho33258 has allowed studies of the cellular action of Ho33342. Our results suggest that Ho33342 also traps topoisomerase I but not topoisomerase II into reversible cleavable complexes in human KB cells. Although Ho33342 shares a similar mechanism of action with camptothecin, a prototypic topoisomerase I poison, in trapping topoisomerase I cleavable complexes, Ho33342 differs from camptothecin in its effect on drug-resistant cells. Different from camptothecin, Ho33342 was shown to be about 200-fold less cytotoxic in MDR1-overexpressing human KB V1 cells relative to parental human KB 3-1 cells. Ho33342 is only 5-fold less cytotoxic for camptothecin-resistant CPT-K5 cells, which expresses a highly camptothecin-resistant from of topoisomerase I, than for the wild type human lymphoblast RPMI 8402 cells. Our studies suggest a potential use of Hoechst 33342 as a new topoisomerase I poison in antitumor chemotherapy.

Benzimidazoles↗
PubMed1993

Solution conformation of cobrotoxin: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study.

The solution conformation of cobrotoxin has been determined by using proton nuclear magnetic resonance spectroscopy. With the combination of various two-dimensional NMR techniques, the 1H-NMR spectrum of cobrotoxin was completely assigned (Yu et al., 1990). A set of 435 approximate interproton distance restraints was derived from nuclear Overhauser enhancement (NOE) measurements. These NOE constraints, in addition to the 29 dihedral angle constraints (from coupling constant measurements) and 26 hydrogen bonding restraints (from the pattern of short-range NOEs), form the basis of 3-D structure determination by the hybrid distance geometry-dynamical simulated annealing method. The 23 structures that were obtained satisfy the experimental restraints, display small deviation from idealized covalent geometry, and possess good nonbonded contacts. Analysis of converged structures indicated that there are two antiparallel beta sheets (double and triple stranded), duly confirming our earlier observations. These are well defined in terms of both atomic root mean square (RMS) differences and backbone torsional angles. The average backbone RMS deviation between the calculated structures and the mean structure, for the beta-sheet regions, is 0.92 A. The mean solution structure was compared with the X-ray crystal structure of erabutoxin b, the homologous protein. This yielded information that both structures resemble each other except at the exposed loop/surface regions, where the solution structure seems to possess more flexibility.

Cobra Neurotoxin Proteins↗
PubMed1993

Uptake of antisense oligonucleotides and functional block of acetylcholine receptor subunit gene expression in primary embryonic neurons.

Several recent studies have used antisense oligonucleotides in the nervous system to probe the functional role of particular gene products. Since antisense oligonucleotide-mediated block of gene expression typically involves uptake of the oligonucleotides, we have characterized the mechanism of this uptake into developing neurons from embryonic chickens. Antisense oligonucleotides (15 mers) added to the bathing media are taken up into the embryonic chicken sympathetic neurons maintained in vitro. A portion of the oligonucleotide uptake is temperature dependent and saturates at extracellular oligonucleotide concentrations > or = 20 microM. This temperature sensitive, saturable component is effectively completed by single nucleotides of ATP and AMP and is reminiscent of receptor-mediated endocytosis of oligonucleotides described in non-neuronal cells. The efficiency of the oligonucleotide uptake system is dependent on the developmental stage of the animal but independent of the number of days that the neurons are maintained in vitro. Following the uptake of antisense oligonucleotides directed against ion channel subunit genes expressed by these neurons (nicotinic acetylcholine receptor subunit alpha 3; nAChR alpha 3), biophysical assays reveal that the functional expression of the target gene is largely blocked. Thus the number of wild type nAChR channels expressed is decreased by approximately 80%-90%. Furthermore, following antisense deletion of alpha 3, "mutant" nAChRs with distinct functional characteristics are expressed. In sum, these studies characterize the uptake of antisense oligonucleotide and demonstrate the functional block of specific gene expression in primary developing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Synapse elimination from the mouse neuromuscular junction in vitro: a non-Hebbian activity-dependent process.

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three-compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2-7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p < 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i.e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p < 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development.

Action Potentials↗
PubMed1993

Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on fibroblast-like synoviocytes: paradoxical induction of IFN-gamma and TNF-alpha receptor expression.

We recently described mutual antagonism between IFN-gamma and TNF-alpha on human fibroblast-like synoviocytes (FLS). TNF-alpha inhibits IFN-gamma-induced HLA-DR expression and IFN-gamma blocks TNF-alpha-dependent synoviocyte proliferation, collagenase production, and GM-CSF secretion. To study the mechanism of antagonism we have analyzed the effect these factors on the expression of cytokine surface receptors. 125I-Labeled cytokine binding was measured on cultured FLS and the results were analyzed by Scatchard plots. Unstimulated synoviocytes expressed 9300 +/- 1560 IFN-gamma binding sites per cell. A single class of high-affinity receptor was observed (Kd = 4.5 +/- 2.5 x 10(-10) M). TNF-alpha did not competitively inhibit 125I-IFN-gamma binding. When FLS were incubated with TNF-alpha (100 ng/ml), there was a paradoxical 49.5 +/- 5.6% increase in the number of binding sites for IFN-gamma (P = 0.001), with no change in the Kd. Unstimulated FLS also expressed 2850 +/- 700 TNF-alpha receptors per cells, with a single Kd consistent with the lower-affinity TNF-alpha receptor (7.4 +/- 0.2 x 10(-10) M). IFN-gamma did not directly interfere with TNF-alpha binding. Preincubation of FLS with 100 U/ml of IFN-gamma resulted in a 28.9 +/- 9.0% increase in TNF-alpha receptor expression (P < 0.008), with no change in the Kd. Low levels of the soluble 55-kD TNF receptor were detected in FLS supernatants. IFN-gamma did not effect soluble TNF receptor production.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthritis, Rheumatoid↗
PubMed1993

A structure-activity relationship study of batracylin analogues.

A number of isoindolo[1,2-b]quinazolines and some benzo[4,5]isoquinolino[1,2-b]quinazolines as structural modification analogues of the antitumor compound batracylin were synthesized and evaluated against HL-60 cell growth and in topoisomerase II-mediated DNA cleavage assays. Of the compounds studied, 10,12-dihydro-7,8-methylenedioxyisoindolo[1,2-b]quinazolin-1 2(10H)-one (1d), 2-amino-10,12-dihydroisoindolo[1,2-b]quinazolin-12(10H)-one (1p), and 2-amino-7,8-methylenedioxy-10,12-dihydroisoindolo[1,2-b] quinazolin-12(10H)-one (1ab) exhibited good inhibitory activities against HL-60 cell lines as well as induction of topo II-mediated DNA cleavage activities.

Antineoplastic Agents↗
PubMed1993

The relevance of antibody concentration to the immunohistological quantification of cell proliferation-associated antigens.

A number of different factors can profoundly influence the quantification of immunostained cells. Given the characteristics of immunohistological detection systems with non-linearity of signal and antigen concentration, we investigated the relationship of signal (number of stained cells) to the dilution of antibody employed. Three antibodies were studied which have been advocated as being effective in fixed material as markers of cell proliferation: PC10 (anti-proliferating cell nuclear antigen (PCNA)), Ki-S1 and MIB1 (a novel anti-Ki-67). Serial sections of tonsil were immunostained with a range of antibody dilutions using a fixed detection system and the number of stained cells quantified. Similar experiments were performed on tumour xenografts with known growth fraction and, in vitro, on human diploid fibroblasts in logarithmic growth phase. With both PC10 and Ki-S1 the number of stained cells increased with decreasing antibody dilution with no plateau being identified. In contrast, MIB1 showed a clear plateau. Immunocytological data indicate that PCNA and Ki-S1 antigen are present at low (but detectable) levels in at least some non-cycling cells and thus an artificial 'cut-off' has to be employed in assessing the number of proliferating cells with these antibodies. The superiority of MIB1 probably reflects the rapidity of catabolism of the Ki-67 antigen at the end of M phase. Taken together, these data point to the importance of carefully considering fundamental immunochemical properties such as antibody concentration (as well as antibody affinity and sensitivity of detection system) when employing immunological markers of cell proliferation in quantitative procedures.

Animals↗
PubMed1993

Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 (teniposide).

Inhibition of DNA topoisomerase II in simian virus 40 (SV40)-infected BSC-1 cells with a topoisomerase II poison, VM-26 (teniposide), resulted in rapid conversion of a population of the SV40 DNA into a high-molecular-weight form. Characterization of this high-molecular-weight form of SV40 DNA suggests that it is linear, double stranded, and a recombinant with SV40 DNA sequences covalently joined to cellular DNA. The majority of the integrants contain fewer than two tandem copies of SV40 DNA. Neither DNA-damaging agents, such as mitomycin and UV, nor the topoisomerase I inhibitor camptothecin induced detectable integration in this system. In addition, the recombination junctions within the SV40 portion of the integrants correlate with VM-26-induced, topoisomerase II cleavage hot spots on SV40 DNA. These results suggest a direct and specific role for topoisomerase II and possibly the enzyme-inhibitor-DNA ternary cleavable complex in integration. The propensity of poisoned topoisomerase II to induce viral integration also suggests a role for topoisomerase II in a pathway of chromosomal DNA rearrangements.

Animals↗
PubMed1993

An analysis of the correlation between angiographic and clinical findings in cerebral arteriovenous malformations.

One hundred and sixty-seven cases of cerebral arteriovenous malformations (AVMs) confirmed by digital subtract angiography (DSA) and surgery were analyzed to find the correlation between clinical and angiographic findings with regard to sex, age, size of angioma, and number of drainage veins and feeding arteries. Statistical analysis showed that the risk of hemorrhage was highest in small AVMs with only one drainage vein, and decreased with increases of volume and number of AVMs. Statistics also demonstrated that small AVMs usually have one drainage vein and so have a high risk of hemorrhage.

Adolescent↗
PubMed1992

NMR study of the solution conformation of actinomycin D.

The solution conformation of actinomycin D, the Gram-positive antibiotic and DNA-binding drug, has been determined by 1H-NMR in deuterated dimethyl sulfoxide. The structure determination is based on the experimental data set of NOE restraints. Four structures were obtained from the distance geometry and restrained molecular dynamics calculation. The resultant structures satisfy the experimental restraints very well. These structures are found to be compatible with the X-ray crystal structures.

Chemical Phenomena↗
PubMed1992

IL-1 receptor antagonist protein production and gene expression in rheumatoid arthritis and osteoarthritis synovium.

IL-1 can participate in the perpetuation of arthritis through direct stimulation of synoviocytes and augmentation of matrix degradation. Hence, local production of the IL-1R antagonist protein (IRAP) might be an important negative feedback signal that regulates synovitis. We assessed synovial IRAP production in synovia from 30 individuals, by using a specific mAb and the immunoperoxidase staining method. IRAP was detected in 11 of 12 rheumatoid arthritis (RA) synovial tissues (ST) and was located primarily in the sublining, particularly in perivascular regions enriched for macrophages. Some staining was observed in the intimal lining of the synovium, although this was significantly less than in the sublining (p less than 0.05). Nine of 12 osteoarthritis (OA) tissues were positive for IRAP. In contrast to RA, the staining was observed primarily in the synovial lining in OA, with only minimal sublining IRAP being detected. Synovia from four patients without arthritis were negative (three autopsy specimens and one post-traumatic sample). Of the other two patients with miscellaneous diagnoses, one sample was negative (tenosynovitis) and one was positive (seronegative inflammatory arthritis) (sublining). Studies of serial sections and double-immunostaining experiments indicated that macrophages are the major cells containing immunoreactive IRAP. IRAP gene expression in vivo was determined by performing in situ hybridization on ST from 17 arthritis patients. RNA sense IRAP probes did not hybridize to any tissues. Anti-sense IRAP probes bound to two of nine RA tissues, two of six OA tissues, one of one seronegative inflammatory arthropathy tissue, and none of one flexor tenosynovitis tissue. As with immunoreactive protein, IRAP mRNA was primarily localized to cells in the synovial lining in OA but was more prominent in perivascular lymphoid aggregates in RA and seronegative inflammatory arthropathy. Northern blot analysis was performed on RNA isolated from nine ST. The appropriately sized IRAP band was identified in six of nine samples (five of six RA and one of three OA). Supernatants from cultured RA and OA ST cells contained immunoreactive and biologically active IRAP. Hence, IRAP gene expression and protein production occur in RA and OA synovium, albeit in different distributions.

Amino Acid Sequence↗
PubMed1992

Conformational properties of oxytocin in dimethyl sulfoxide solution: NMR and restrained molecular dynamics studies.

The conformation of oxytocin, the neurohypophyseal nonapeptide hormone, in solution in deuterated dimethyl sulfoxide has been determined by 1H-nmr. The structural determination is based on the experimental data set of nuclear Overhauser effect restraints. Obtained after the restrained molecular dynamics simulation on an initial structure of extended conformation, five resultant structures satisfy the experimental restraints well. These structures resemble that of the crystal structure of deamino-oxytocin, an analogue of oxytocin, in terms of a close correlation observed both at two beta-turn regions of the 20-membered tocin ring and at the tripeptide tail end. Based on this comparison and analysis of restrained molecular dynamics trajectories, we found that, although the turns are stabilized by the formation of hydrogen bonds, the oxytocin molecule possesses a slight twist in DMSO solution relative to the orientation of deamino-oxytocin in the crystalline state. Analyses of oxytocin conformation indicate that the tripeptide tail is more flexible than the tocin ring.

Amino Acid Sequence↗
PubMed1992

In search of the central respiratory neurons: I. Dissociated cell cultures of respiratory areas from the upper medulla.

Dissociated cells from the areas of the nucleus ambiguus and the nucleus tractus solitarius obtained by tissue punch or block dissection from coronal slices of the medulla at the level of the obex were cultured from fetal rats at 18 to 21 days gestation. The dissociated neurons were plated either directly in vitrogen-coated 35 mm tissue culture dishes or in such dishes which had been seeded with subcultures of cortex- or medulla-derived astrocytes. After the astrocytes reached confluency and were treated with an antimitotic agent, dissociated nucleus ambiguus or nucleus tractus solitarius was plated at 0.5-1.0 x 10(6) cells per dish. Neurons grew well on monolayers of medullary or cortical astrocytes, but survived poorly on vitrogen-coated dishes without a cellular substrate. Rat medulla was preferred as the source of astrocytes. Tissue dissociation with papain rather than trypsin produced less cellular debris, and the neuronal yield from the tissue was higher. The neuronal population was heterogenous in morphology including small and large bipolar, pyramidal, and multipolar cells. Neurons sensitive to CO2 and/or low pH (Rigatto et al., J Neurosci Res 33:590-597, 1992) did not appear to have any definitive morphologic characteristics, but most were multipolar. These neurons stained well with antibodies to neuron-specific enolase and Fragment C of tetanus toxin, but not to choline acetyltransferase (ChAT). These findings suggest that neurons possibly responsible for the central regulation of respiration can be maintained for several weeks in dissociated cell culture, providing a system for neurotransmitter, electrophysiological, and morphological studies.

Animals↗
PubMed1992

In search of the central respiratory neurons: II. Electrophysiologic studies of medullary fetal cells inherently sensitive to CO2 and low pH.

Although extensively pursued, the central respiratory neurons have remained elusive. We departed from the more conventional physiologic and morphologic methods of system and tissue examination and cultured dissociated fetal rat cells (Fitzgerald et al., J Neurosci Res 33:579-589, 1992) from the area of the nucleus ambiguus and the nucleus tractus solitarius located within the 2 mm rostral to the obex. Pacemaker-like cells, with a regular single or bursting activity, studied at 3-5 weeks of age, responded to very small pulses of CO2 (50 ms) and low pH with an increase in spike frequency and a decrease in spike amplitude. Other irregularly beating or silent cells did not respond or else required very large pulses (> 200 ms) to do so. The pacemaker cells also responded to hypoxia induced by administration of sodium hydrosulfite with an increase in spike frequency and amplitude; high oxygen (> 600 torr) and adenosine produced a decrease in electrical activity. Most of these cells were multipolar after staining with antibodies to neuron-specific enolase (NSE) and Fragment C of tetanus toxin. They did not stain for choline acetyltransferase (ChAT). The results suggest that these cultured cells, expressing a phenotype inherently responsive to CO2 and low pH, have the characteristics of central respiratory chemoreceptors, and may be involved in the generation of the respiratory rhythm.

Animals↗
PubMed1992

Structure of the oxidized long-chain flavodoxin from Anabaena 7120 at 2 A resolution.

The structure of the long-chain flavodoxin from the photosynthetic cyanobacterium Anabaena 7120 has been determined at 2 A resolution by the molecular replacement method using the atomic coordinates of the long-chain flavodoxin from Anacystis nidulans. The structure of a third long-chain flavodoxin from Chondrus crispus has recently been reported. Crystals of oxidized A. 7120 flavodoxin belong to the monoclinic space group P2(1) with a = 48.0, b = 32.0, c = 51.6 A, and beta = 92 degrees, and one molecule in the asymmetric unit. The 2 A intensity data were collected with oscillation films at the CHESS synchrotron source and processed to yield 9,795 independent intensities with Rmerg of 0.07. Of these, 8,493 reflections had I > 2 sigma and were used in the analysis. The model obtained by molecular replacement was initially refined by simulated annealing using the XPLOR program. Repeated refitting into omit maps and several rounds of conjugate gradient refinement led to an R-value of 0.185 for a model containing atoms for protein residues 2-169, flavin mononucleotide (FMN), and 104 solvent molecules. The FMN shows many interactions with the protein with the isoalloxazine ring, ribityl sugar, and the 5'-phosphate. The flavin ring has its pyrimidine end buried into the protein, and the functional dimethyl benzene edge is accessible to solvent. The FMN interactions in all three long-chain structures are similar except for the O4' of the ribityl chain, which interacts with the hydroxyl group of Thr 88 side chain in A. 7120, while with a water molecule in the other two. The phosphate group interacts with the atoms of the 9-15 loop as well as with NE1 of Trp 57. The N5 atom of flavin interacts with the amide NH of Ile 59 in A. 7120, whereas in A. nidulans it interacts with the amide NH of Val 59 in a similar manner. In C. crispus flavodoxin, N5 forms a hydrogen bond with the side chain hydroxyl group of the equivalent Thr 58. The hydrogen bond distances to the backbone NH groups in the first two flavodoxins are 3.6 A and 3.5 A, respectively, whereas in the third flavodoxin the distance is 3.1 A, close to the normal value. Even though the hydrogen bond distances are long in the first two cases, still they might have significant energy because their microenvironment in the protein is not accessible to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗