Biomedical subjects

L S Kan

Publications and source records attributed to L S Kan.

At least 37 records · Page 2Linked to original sources

PubMed1990

Molecular weight determination of underivatized oligodeoxyribonucleotides by positive-ion matrix-assisted ultraviolet laser-desorption mass spectrometry.

A Wiley-McLaren type time-of-flight mass spectrometer has been used for molecular weight measurements of several unprotected oligodeoxyribonucleotides using matrix-assisted UV laser desorption. Approximately 10 to 100 pmol of sample was required for recording their positive-ion mass spectra with a mass resolution in the range of 150 to 300 (Full width at half maximum) (FWHM). Little fragmentation was observed.

Mass Spectrometry↗

PubMed1990

300 MHz proton NMR study of the differentiation of diploid human epidermal keratinocytes.

The nuclear magnetic resonance spin-lattice (T1) and spin-spin (T2) relaxation times are closely related to the molecular motions of the molecules in a liquid sample. T1 and T2 of human epidermal cells were measured at 300 MHz as functions of harvesting methods (i.e., scraping vs trypsinization) and age in culture. It was found that T1 and T2 values have smaller variances when the cell is harvested by trypsinization rather than scraping. The correlation coefficients for both T1 and T2, obtained from cells harvested by scraping. More importantly, this is the first report to monitor in vitro aging through relaxation times measurement. There is a significant increase in the values of T1 and T2 from the third to seventh passages. Human keratinocytes slowed down and even ceased to grow the seventh passage. Therefore, the cellular water molecules of human keratinocytes have higher mobility in a more differentiated state. The factors contributing to the change in relaxation times as cells progress toward senescence are discussed.

Cell Differentiation↗

PubMed1989

Characterization of novel glycolipids from the giant cockroach (Blaberus colosseus).

A novel class of glycolipids, assigned the trivial name blaberosides, was isolated from whole head tissues of the giant cockroach (Blaberus colosseus). The class consists of two closely related families, blaberoside I and blaberoside II, each containing species differing by 26 atomic mass units. The structure of these gentiobiose-based glycoglycerolipids was elucidated by chromatographic behavior, nuclear magnetic resonance spectroscopy, mass spectrometry, and analysis of chemical degradation products and derivatives. Species in the blaberoside I family have been identified as 2-O-[6'-O-(6"-O-3-hydroxy-11-eicosenoyl-beta-D-glucopyranosyl)-bet a-D- glucopyranosyl]-3-(hexadecyloxy)-1-(3-hydroxy-11-eicosenoyl)-1,2-p ropanediol (blaberoside Ia) and 2-O-[6'-O-(6"-O-3-hydroxy-11-eicosenoyl-beta-D-glucopyranosyl)-bet a- D-glucopyranosyl]-3-(6-octadeceloxy)-1-(3-hydroxy-11-eicosenoyl )-1,2- propanediol (blaberoside Ib). Two smaller homologs of the blaberoside II family were discerned to be 2-O-[6'-O-(6"-O-3-hydroxy-11- eicosenoyl-beta-D-glucopyranosyl)-beta-D-glucopyranosyl]-3-(hex ade cyloxy)- 1,2-propanediol (blaberoside IIa), and 2-O-[6'-O-(6"-O-3-hydroxy-11-eicosenoyl-beta-D- glucopyranosyl)-beta-D-glucopyranosyl]-3-(4-octadeceloxy)-1,2-prop anediol (blaberoside IIb). These compounds are unique because they are animal origin glyceroglycolipids with a highly flexible gentiobiose backbone, and a beta-linkage of the carbohydrate to the glycerol ether at the 2 position rather than the usual 1 position.

Animals↗

PubMed1989

High-frequency 1H NMR studies of the effects of growth factors and phorbol esters on normal Syrian hamster diploid fibroblast cells.

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T1), and spin-spin (T2) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (less than or equal to 100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T1 values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T2 values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986), Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T1 and T2 were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T2 values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-alpha-phorbol-12,13-didecanoate (4 alpha PDD). Treatment with either growth factor resulted in smaller T2 values, but a statistically significant decrease was not observed for PDD or 4 alpha PDD. The observed reductions in T2 values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.

Animals↗

PubMed1989

Epitopic discrimination by monoclonal antibodies directed against the same alkylated nucleoside.

Epitopic specificity of three monoclonal antibodies (mAb's) (coded as ER-6, ER-3, and EM-1) was examined through the utilization of haptenic structural analogs. The binding affinity expressed by the microscopic equilibrium constant (Ki) (Yuhasz, et al., Biochemistry 26, 2334-2342 (1987] of the immunizing hapten, O6-ethyl-2'-deoxy-guanosine (*G) and eight structural analogs, were analyzed by a nitrocellulose affinity filter assay (NAFA) and radioimmunoassay (RIA) for each mAb to determine the protein-hapten interaction between the epitope and the binding cavity. Several components of the *G hapten were determined to be critical for each mAb recognition, while all three mAb's were found to require the O6-ethyl moiety, conjugated guanine base ring, the glycosyl bond and the sugar ring C [1'] and C [2'] position. This investigation further probes and categorizes the binding specificity of the monoclonal antibodies after incorporation of the *G monomer into three short deoxyribooligomeric haptens: O6-ethyl-2'-deoxyguanylyl 3',5' deoxyadenosine (*GA), 2'-deoxyadenylyl 3',5' O6-ethyl-2'-deoxyguanylyl 3',5' 2'-deoxyadenosine (A*GA), and O6-ethyl-2'-deoxyguanylyl 3',5' 2'-deoxyadenylyl 3',5'-2'-deoxyadenylyl 3',5' 2'-deoxycytosine (*GAAC). Unlike the similar binding profiles for the monoclonal antibodies and the haptenic structural analogs, the binding profiles for the deoxyribooligomeric haptens were found to differ in their modes of recognition. These results will be compared to ascertain the key components of monomer and oligomer interaction of the binding cavity. It is important for investigations where monoclonal antibodies derived from small haptens are utilized in recognition of larger antigens containing those haptens.

Alkylating Agents↗

PubMed1988

Phosphorus-31 nuclear magnetic resonance spectroscopy of human retinoblastoma cells: correlation with metabolic indices.

The 31P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by 31P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1, 2 and 3 days post feeding showed a 40% decrease in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.

Cell Line↗

PubMed1988

Hairpin and duplex formation in DNA fragments CCAATTTTGG, CCAATTTTTTGG, and CCATTTTTGG: a proton NMR study.

Three DNA fragments, CCAATTTTGG (1), CCAATTTTTTGG (2), and CCATTTTTGG (3), were studied by proton NMR spectroscopy in aqueous solution. All these oligodeoxyribonucleotides contain common sequences at the 5' and 3' ends (5'-CCA and TGG-3'). 2 as well as 3 forms only hairpin structures with four unpaired thymidylyl units, four and three base pair stems, respectively, in neutral solution under low and high NaCl concentrations. At high salt concentration the oligomer 1 forms a duplex structure with -TT- internal loop. On the other hand, the same oligomer forms a stable hairpin structure at low salt and low strand concentrations at pH 7. The hairpin structure of 1 has a stem containing only three base pairs (CCA.TGG) and a loop containing four nucleotides (-ATTT-) that includes a dissociated A.T base pair. The two secondary structures of 1 coexist in an aqueous solution containing 0.1 M NaCl, at pH 7. The equilibrium shifts to the hairpin side when the temperature is raised. The stabilities and base-stacking modes of all three oligonucleotides in two different structures are reported.

Base Sequence↗

PubMed1988

2,4-Dinitrophenylhydrazides of polysialogangliosides.

Treatment with 2,4-dinitrophenylhydrazine HCl in the presence of dicyclohexylcarbodiimide, converts gangliosides to their dinitrophenylhydrazides. This derivatization is the basis of a useful method for HPLC determination of gangliosides (K. Miyazaki, N. Okamura, Y. Kishimoto and Y. C. Lee (1986) Biochem. J. 235, 755-761). This procedure, however, yields two different GT1b products. By characterizing these two products using plasma desorption mass spectrometry, proton magnetic resonance and other chemical and physical techniques, we found that either one or two of the three sialic acid carboxyl groups in GT1b, were converted to dinitrophenylhydrazides. The remaining underivatized carboxyl groups formed lactones with hydroxyl groups from other carbohydrate residues. Also, while sialic acid residues of GD1a were fully derivatized, only one sialic acid in GD1b, two sialic acids in GT1a and two in GQ1b were converted to dinitrophenylhydrazides, the remaining carboxyl groups probably forming lactones. Sialic acid residues between galactose of the gangliotetraose chain and another sialic acid in polysialogangliosides appear to be underivatized possibly because of steric hindrance.

Chemical Phenomena↗

PubMed1988

Phosphate catalyzed exchange rates of NH-N protons of DNA.

We have studied the catalysis of the exchange of the hydrogen-bonded NH-N protons of the short DNA helix (d-CCAAGCTTGG)2 by phosphate addition. The NH exchange rates were monitored by the line widths of the corresponding NH resonances in the 1H nmr spectra. The exchange catalyst phosphate is most effective on the exchange rate of the terminal CG1 base pairs. However, all internal base pairs are also moderately affected by phosphate which suggests an exchange mechanism governed by a fast equilibrium between opened and closed states of the duplex. Within the limits of error the same effectiveness of phosphate on the exchange rate of all internal NH-N protons has been observed. With the exception of the terminal base pairs, no sequence and/or position specificity of the exchange rates of the NH-N protons of the base pairs has been found.

Base Sequence↗

PubMed1987

Helix geometry, hydration, and G.A mismatch in a B-DNA decamer.

The DNA double helix is not a regular, featureless barberpole molecule. Different base sequences have their own special signature, in the way that they influence groove width, helical twist, bending, and mechanical rigidity or resistance to bending. These special features probably help other molecules such as repressors to read and recognize one base sequence in preference to another. Single crystal x-ray structure analysis is beginning to show us the various structures possible in the B-DNA family. The DNA decamer C-C-A-A-G-A-T-T-G-G appears to be a better model for mixed-sequence B-DNA than was the earlier C-G-C-G-A-A-T-T-C-G-C-G, which is more akin to regions of poly(dA).poly(dT). The G.A mismatch base pairs at the center of the decamer are in the anti-anti conformation about their bonds from base to sugar, in agreement with nuclear magnetic resonance evidence on this and other sequences, and in contrast to the anti-syn geometry reported for G.A pairs in C-G-C-G-A-A-T-T-A-G-C-G. The ordered spine of hydration seen earlier in the narrow-grooved dodecamer has its counterpart, in this wide-grooved decamer, in two strings of water molecules lining the walls of the minor groove, bridging from purine N3 or pyrimidine O2, to the following sugar O4'. The same strings of hydration are present in the phosphorothioate analog of G-C-G-C-G-C. Unlike the spine, which is broken up by the intrusion of amine groups at guanines, these water strings are found in general, mixed-sequence DNA because they can pass by unimpeded to either side of a guanine N2 amine. The spine and strings are perceived as two extremes of a general pattern of hydration of the minor groove, which probably is the dominant factor in making B-DNA the preferred form at high hydration.

Base Composition↗

PubMed1987

NMR study of the effect of sugar-phosphate backbone ethylation on the stability and conformation of DNA double helix.

Temperature variation studies of the imido proton NMR and 31P NMR resonances of the self-associated d(C-C-A-A-G-A-T-T-G-G) and d[C-C-A-A-G-p(Et)-A-T-T-G-G] duplexes (both the R and S diastereoisomers) and the heteroduplexes formed with their complementary strand d(C-C-A-A-T-C-T-T-G-G) were carried out in aqueous solution. Results demonstrate that phosphate backbone ethylation did not disrupt the interstrand hydrogen bonding involved in double-helix formation but perturbed the helix. The S isomer perturbed the duplex more than the R isomer. The line broadening patterns and faster fraying motion in the alkylated duplexes compared to those in the nonalkylated duplexes indicate that the perturbation introduced in the middle propagates along the backbone to the end of the duplex.

Carbohydrate Metabolism↗

PubMed1987

Thermodynamic investigation of monoclonal antibody alkylated nucleoside interaction as a model for epitope recognition on nucleic acids.

The objective of this investigation is examination of the dominant forces that govern complex formation between a series of monoclonal antibodies directed against O6-ethyl-2'-deoxyguanosine. These monoclonal antibodies (coded as ER-6, ER-3, and EM-1) provide the basis for a thermodynamic comparative evaluation of the potentially different forces that stabilize the various monoclonal antibody (mAb) alkylated nucleoside complexes. The binding affinities of ER-6, ER-3, and EM-1 are measured in terms of specific (O6-ethyl-2'-deoxyguanosine, or O6-EtdGuo) and nonspecific (O6-methyl-2'-deoxyguanosine, or O6-MedGuo) antigens, under a variety of experimental conditions, including pH, sodium chloride addition, 1-propanol addition, and temperature, via a nitrocellulose affinity filter assay. The binding isotherms were analyzed via a least-squares routine fit to a two independent binding sites model. The temperature dependence of the van't Hoff enthalpies for the specific O6-EtdGuo interaction ranges from -15.18 to -18.60 kcal mol-1, while for O6-MedGuo the range was extended from -2.72 to -20.66 kcal mol-1. The standard and unitary entropies were negative for those mAb interactions with O6-EtdGuo as well as for ER-6/O6-MedGuo complex formation. However, it was found that the interactions between ER-3 and EM-1 with O6-MedGuo led to decidedly positive entropic values. These results indicate two different dominant forces at work in complex stabilization. The interaction of the three mAb's with their specific antigen, as well as ER-6/O6-MedGuo interaction (nonspecific), may well be controlled by van der Waals type forces, while ER-3 and EM-1 interactions with nonspecific antigen imply formal charge neutralization electrostatics as the dominant force.

Alkylation↗

PubMed1987

Structural and conformational studies on deoxyguanosyl-3',5'-deoxyadenosine monophosphate and its ethyl phosphotriester analogs--left-handed dimers.

The mode of base-base stacking, the handedness and the sugar(dGpA)phosphate backbone conformation of deoxyguanosyl 3'-5' deoxyadenosine and its diastereomeric ethyl phosphotriester analogs were studied by 1H NMR, UV and CD spectroscopy. The results indicate the three dimers are left-handed, while the sugar phosphate backbone is comprised predominantly of C2-endo,gg(C4-C5) and g'g (C5-O) conformers. The two bases are extensively stacked and interact about 90 degrees along the dyad axes. The extent of base overlap in dGpA is slightly greater than in either ethyl phosphotriester analog. The absolute configurations of the two ethyl phosphotriester diastereoisomers of dGpA can be assigned by one-dimensional and two-dimensional 1H NMR nuclear Overhauser enhancement experiments.

Alkylation↗

PubMed1987

Phosphorus-31 NMR spectroscopy of cultured human retinal pigmented epithelial cells.

Cultured human retinal pigmented epithelial cells were studied using phosphorus-31 nuclear magnetic resonance spectroscopy (P-31 NMR). Retinal pigmented epithelial cells from normal human donors were isolated and expanded using roller-bottle culture. The P-31 NMR spectrum of the intact living cells was obtained at 121 MHz by casting the cells in agarose threads and perfusing the threads with culture medium. The cells remained viable at 37 degrees C in the NMR magnet for at least 24 hr, as determined by the stability of the phosphorus spectrum and by trypan blue dye exclusion at the end of the experiments. The intact cell spectrum showed resonances from phosphorylethanolamine, phosphorylcholine, inorganic phosphate, phosphodiesters, phosphocreatine, ATP, NAD+, and UDP-N-acetylglucosamine, with phosphorylethanolamine, phosphorylcholine, and ATP being the major metabolites present. The resonances were assigned by making a perchloric acid (PCA) extract of the intact cells and running under high-resolution conditions. The PCA extract spectrum also detected sugar phosphates, ADP and UTP, with the latter being approximately 20% of the nucleotide pool. These studies provide the basis for the study of normal and diseased human RPE cells by NMR spectroscopic methods.

Cells, Cultured↗

PubMed1986

NMR relaxation study of water protons in Syrian hamster fetal cells at 300 MHz.

The T1 and T2 relaxation times of water protons in two cell types in culture derived from Syrian hamster fetuses (normal primary or secondary fetal cells vs BP6T tumor cells derived from the normal cells transformed by carcinogens) were measured at 7.05 Tesla magnetic field (proton frequency = 300 MHz). The T1/T2 ratios and the correlation time, tau c, calculated from the T1/T2 ratio of cellular water protons, are significantly different in these two fibroblastic cell types of the same biological origin and with similar morphologies and growth rates in culture.

Animals↗

PubMed1985

Isolation and purification of deoxyribonucleosides from 90% 13C-enriched DNA of algal cells and their characterization by 1H and 13C NMR.

13C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The 1H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive 1H-13C couplings. Similarly, the 13C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz 13C NMR spectrum, due to 13C-13C couplings. The 1H-13C and 13C-13C coupling constants were also measured and tabulated. The isotopic enrichment of 13C these deoxyribonucleosides was obtained by integration of the 1H and/or 13C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% 13CO2 during metabolism pathways. This protocol provides experimentally useful quantities of 13C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis.

Carbon Isotopes↗

PubMed1983

Nuclear Overhauser effect as a tool for the complete assignment of nonexchangeable proton resonances in short deoxyribonucleic acid helices.

A new strategy for the assignment of nonexchangeable proton resonances in oligonucleotide duplexes is presented and used to interpret the spectra of the oligonucleotide helix d(CpGpCpGpCpG) X d(CpGpCpGpCpG) in low salt (B form). This procedure is based on the use of sequential homodecoupling (1D) or COSY (2D) for the interconnection of the sugar resonances pertaining to the same residue and on the measurement of nuclear Overhauser effect (NOE) (1D or 2D) between critically located protons in order to establish the connectivity between the base protons and the sugar protons, as well as between consecutive base-sugar residues. The assignment performed by this method was found in perfect agreement with the one made previously by the incremental procedure [Cheng, D. M., Kan, L.-S., Frechet, D., Ts'o, P. O. P., Uesugi, S., Shida, T., & Ikehara, M. (1983) Biopolymers (in press)]. Also, this method is demonstrated to be applicable to DNA short helices containing A X T base pairs.

Base Sequence↗

PubMed1983

Nuclear magnetic resonance study on the exchange behavior of the NH-N protons of a ribonucleic acid miniduplex.

The exchange behavior of the guanine N(1) and uracil N(3) protons in the self-complementary hexanucleotide r(ApApGpCpUpU) has been studied at 5 degrees C in 80% H2O/20% D2O by proton NMR. Under these conditions, the hexanucleotide forms a stable miniduplex. The exchange rate of all Watson-Crick NH protons is unaffected by addition of trifluoroethylamine up to 0.07 M. On the other hand, addition of phosphate buffer, pH 6.9, enhances the exchange rate of the uracil N(3) protons of both terminal and internal A X U base pairs but does not influence the exchange rate of the guanine N(1) protons of the central G X C base pairs. Catalysis by increased phosphate concentrations results in an open-limited rate of the internal A X U base pairs with kex = 233 s-1, equivalent to a lifetime of 4.3 ms. The proton exchange of the central G X C is regulated by the opening rate of the central core of the miniduplex. On the other hand, the sensitivity of the exchange rate of internal as well as of terminal A X U base pairs can be explained by their reduced lifetime due to end "fraying" and a subsequent catalysis of the exchange process from the opened state. These results suggest that it may be possible to probe labilized parts of RNAs such as tRNA by gradual addition of the exchange catalyst phosphate and to monitor their exchange rates by proton NMR.

Base Composition↗