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G M Lee

Publications and source records attributed to G M Lee.

At least 19 recordsLinked to original sources
PubMed1994

Does the solid-state structure of endothelin-1 provide insights concerning the solution-state conformational equilibrium?

Additional NMR data (local NOE ratios and chemical shifts) for endothelin-1 supporting the existence of a relatively regular helix initiated abruptly at Lys9 (with Asp8 as an N-cap) and extending in all cases to Cys15 (and in a frayed form to Asp18 in some analogs) is presented. The recent solids-state structure [Janes et al. (1994), Nature Struct. Biol. 1, 311-319], in contrast, places the helix in the extreme C-terminal section of structure and the Lys9-Tyr13 segment is not helical. The X-ray structure does not predict the NOEs or chemical shifts observed for endothelins in aqueous media containing polar organic co-solvents. An analysis of the chemical shift data for reporter groups indicates that the helical conformational preference of endothelins is not significantly altered by the addition of acetonitrile, acetic acid, or ethylene glycol. The validity of the analytic strategy is supported by results for both more rigid and less helical analogs. We conclude that the structure observed in crystals obtained from purely aqueous media is influenced by intermolecular interactions in the solid state and is not a significant contributor to the conformational equilibrium observed for monomeric ET-1.

Amino Acid Sequence↗
PubMed1994

Enhanced specific antibody productivity of calcium alginate-entrapped hybridoma is cell line-specific.

In order to determine whether the enhanced specific antibody productivity (qMAb) of calcium alginate-entrapped hybridoma is cell line-specific, calcium alginate-entrapped hybridomas (4A2 and DB9G8) were cultivated under the condition where we had previously observed significantly enhanced qMAb of calcium alginate-entrapped S3H5/gamma 2bA2 hybridoma. Unlike S3H5/gamma 2bA2 hybridoma, neither 4A2 nor DB9G8 hybridomas showed persistently enhanced qMAb when they were entrapped in calcium alginate beads. The enhanced qMAb of entrapped 4A2 and DB9G8 hybridomas, which was 2-3 times higher than the qMAb of free-suspended cells in a control experiment, was observed only during the early stage of the culture. During the early stage of the culture, the viable cell concentration decreased probably due to cell damage during the entrapment process. As cell growth resumed, the qMAb decreased to the similar level of qMAb of free-suspended cells within 5-7 days. Thus, we conclude that the enhanced qMAb of calcium alginate-entrapped hybridomas is cell line-specific.

Alginates↗
PubMed1993

Protein lateral mobility as a reflection of membrane microstructure.

The lateral mobility of membrane lipids and proteins is presumed to play an important functional role in biomembranes. Photobleaching studies have shown that many proteins in the plasma membrane have diffusion coefficients at least an order of magnitude lower than those obtained when the same proteins are reconstituted in artificial bilayer membranes. Depending on the protein, it has been shown that either the cytoplasmic domain or the ectodomain is the key determinant of its lateral mobility. Single particle tracking microscopy, which allows the motions of single or small groups of membrane molecules to be followed, promises not only to reveal new features of membrane dynamics, but also to help explain longstanding puzzles presented by the photobleaching studies, particularly the so-called immobile fraction. The combination of the two complementary technologies should measurably enhance our understanding of membrane microstructure.

Cell Membrane↗
PubMed1993

Expansion of human bone marrow progenitor cells in a high cell density continuous perfusion system.

We describe here a continuous perfusion bioreactor system that enables a population of unselected human mononuclear bone marrow cells obtained from adult donors to expand up to 20 to 25-fold over a two-week period. Colony-forming units of granulocyte-macrophage (CFU-GM) progenitor cells expand 10 to 30-fold. These expansions depend on the gas phase oxygen concentration, the seeding density and time of cell harvest. Under operating conditions that allow for good cell proliferation, 3 to 4 million mononuclear cells can be obtained per square centimeter, with 0.5 to 0.8% being progenitor cells. Autologous human sera supported cell expansion as efficiently as animal sera. Increasing the size of the perfusion system to produce a clinically meaningful number of CFU-GMs could have important applications in bone marrow transplantation therapies.

Blood↗
PubMed1993

Increased neurite outgrowth induced by inhibition of protein tyrosine kinase activity in PC12 pheochromocytoma cells.

Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.

Adenosine Triphosphate↗
PubMed1991

Direct observation of brownian motion of lipids in a membrane.

Nanovid microscopy, which uses 30- to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual molecules in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid molecules were followed in a supported, planar bilayer containing fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepared by conjugating only anti-Fl to the gold. Paucivalent probes were prepared by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an average lateral diffusion coefficient (D) of 0.26 x 10(-8) cm2/sec, and paucivalent probes had an average D of 0.73 x 10(-8) cm2/sec. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 x 10(-8) cm2/sec, which, after allowance for stochastic variation, are consistent with the D of 1.3 x 10(-8) cm2/sec measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diameter, resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results. Nanovid microscopy is a valid method for analyzing the movements of single or small groups of molecules within membranes.

Antibodies, Monoclonal↗
PubMed1990

The effect of surgical handwashing routines on the microbial counts of operating room nurses.

Many factors may affect the efficiency of handwashing techniques. This study examined two interdependent factors: the time taken to wash the hands and the type of antiseptic solution used. A 3-minute initial scrub and 30-second consecutive scrub regimen was compared with a current standard regimen of a 5-minute initial scrub and a 3-minute consecutive scrub. Chlorhexidine gluconate 4% and povidone-iodine 7.5% were the antiseptics used in the two regimens. The sample (n = 34) was drawn from nurses employed in the operating room suite of a 950-bed hospital. Chlorhexidine gluconate was found to be responsible for lower numbers of colony-forming units of bacteria than povidone-iodine. The duration of the scrub had no significant effect on the numbers of bacteria when povidone-iodine was used. The optimal regimen was found to be the 5-minute initial and 3-minute consecutive scrubs with chlorhexidine gluconate.

Chlorhexidine↗
PubMed1989

Effect of serum concentration on hybridoma cell growth and monoclonal antibody production at various initial cell densities.

Hybridoma cells (S3H5/gamma 2bA2) were cultivated using RPMI 1640 media with different serum concentrations at various initial cell densities. It was found that the serum requirement for cell growth was population-dependent. At an initial cell density of 1.10(4) cells/ml, the minimum serum concentration for cell growth was 5% (v/v) and reduced to 1% at the initial cell density of 1.10(5) cells/ml. When the initial density was increased to 1.10(6) cells/ml, serum was not needed for cell growth. The maximum cell density and the integral of viable cells over cultivation time increased as serum concentration or initial cell density was increased. However, specific antibody production rate was almost constant at 0.16 pg/cell.hr irrespective of serum concentration or initial cell density.

Animals↗
PubMed1989

Serum can act as a shear protecting agent in agitated hybridoma cell cultures.

Hybridoma cells, S3H5/gamma 2bA2, were grown in spinner flasks containing RPMI 1640 media at different levels of serum. Cells in low serum media (1% fetal bovine serum) were more sensitive to shear induced by mechanical agitation than cells in high serum media (10%), indicating that serum can protect cells from shear induced by mechanical agitation. The data suggest that serum alters the physiological properties of the cells to make them more shear-resistant, rather than by changing the physico-chemical properties of medium.

Animals↗
PubMed1989

Mechanically cut mitotic spindles: clean cuts and stable microtubules.

We have discovered an easy way to cut through the mitotic spindle at any desired place. Spindles of demembranated cricket or grasshopper spermatocytes were severed with a microneedle between the chromosomes and one pole, and the cut-off polar piece was swept away. Spindle structure and microtubule dynamics in cut spindles were studied by anti-tubulin immunostaining and electron microscopy. The cut is clean: all microtubules are severed and only a few extend beyond the others. This provides the basis for a clear test of whether traction fibers pull chromosomes to the pole in anaphase, because the putative traction fiber is cleanly severed. Cutting creates new plus ends on microtubules in the cut-off polar piece and new minus ends on microtubules in the main spindle body. The microtubules with new plus ends are unstable, as expected from the dynamic instability of microtubules. However, the microtubules with new minus ends are as stable as uncut microtubules in the same spindle. Our mechanical method of cutting microtubules very likely creates native, reactive ends, and therefore the surprising stability of new minus ends is genuinely interesting, not an artifact of cutting.

Animals↗
PubMed1989

Characterization of mitotic motors by their relative sensitivity to AMP-PNP.

The relative sensitivities of the motors for mitotic chromosome movements and saltatory motion were compared using a nonhydrolyzable analog of ATP, AMP-PNP. K+AMP-PNP was microinjected into PtKl cells at the time of nuclear envelope disassembly or at anaphase onset. To produce a dose-response curve for the effect of AMP-PNP on the rate of movement, the intracellular concentration of AMP-PNP in individual cells was measured. The volume injected into each cell was determined by adding dextrans labeled with Lucifer Yellow to the injection buffer, measuring the injected cell's fluorescence intensity, and then comparing the value with the fluorescence intensity of known volumes of Lucifer Yellow dextran solution. AMP-PNP produced a 50% inhibition of spindle elongation at 0.2 mM, of saltatory motion at 0.8 mM, and of chromosome movement at 8.6 mM. Prometaphase chromosome movement and anaphase chromosome-to-pole movement were similarly inhibited by AMP-PNP. Equivalent volumes of injection buffer containing 1% Lucifer Yellow dextran had no effect on chromosome movement, spindle elongation or saltatory motion. Although AMP-PNP occasionally produced shorter anaphase spindles, tubulin immunofluorescence revealed the presence of abundant spindle microtubules. Metaphase cells treated with very high cell concentrations of AMP-PNP had spindles with unusually long astral microtubules; thus microtubules are stabilized rather than broken down by AMP-PNP. In conclusion, spindle elongation is four times more sensitive than saltatory motion to AMP-PNP and 40 times more sensitive than chromosome movement. When these sensitivities to AMP-PNP are considered with the results from other studies, it can be concluded that the molecular motors for spindle elongation, chromosome movement and saltatory motion are different.

Adenosine Triphosphate↗
PubMed1989

Measurement of volume injected into individual cells by quantitative fluorescence microscopy.

Pressure microinjection is frequently used to introduce substances into mammalian cells, but precise quantitation of the volume injected into individual cells has been difficult. A simple and reliable procedure for determining the volume injected was developed in order to determine what intracellular concentration of AMP-PNP was necessary to inhibit specific cellular processes. The technique uses fluorescent Lucifer Yellow-labeled dextrans in the microinjection buffer and quantitative fluorescence microscopy to measure the fluorescence intensity of the injected cell. The volume injected is computed from a standard curve derived from the volume and fluorescence of spherical, microscopic droplets of Lucifer Yellow dextran solution. The droplets are ejected from a micropipet into immersion oil where they sink to rest on a siliconized coverslip. For the measurement of fluorescence, an inexpensive photomultiplier system that is attached to a fluorescence microscope is described. The potential uses of this method for other microassays are discussed.

Adenylyl Imidodiphosphate↗
PubMed1987

Chloral hydrate disrupts mitosis by increasing intracellular free calcium.

In examining how chloral hydrate affects mitosis, we found that extracellular application of 0.1% chloral hydrate produced an abrupt rise in cytosolic free Ca2+. Digitized fluorescence microscopy of Fura-2-loaded, mitotic and interphase PtK cells revealed that Ca2+ rose 15 s after chloral hydrate application, peaked within 1 min at a concentration two- to sevenfold above the basal level and then slowly dropped. Bathing cells in 0.1% chloral hydrate caused metaphase spindles to shorten, starting in 1-2 min, and inhibited spindle elongation without affecting chromosome-to-pole movement during anaphase, as determined by phase-contrast observation of living cells. Spindle elongation and chromosome movement were unaffected by intracellular injection of 7.5% chloral hydrate. Extensive mitotic microtubule breakdown occurred after cells were bathed for 7 min in 0.1% chloral hydrate, while interphase microtubules were unaffected as determined by immunofluorescence. The chloral hydrate-induced microtubule breakdown and metaphase spindle shortening were prevented by 10 mM-CoCl2, which has previously been shown to block Ca2+ influx and to stabilize microtubules in vitro. These results imply that disruption of mitotic spindle function and structure by chloral hydrate is due to a rise in cytosolic free Ca2+, and also indicate that mitotic microtubules are more Ca2+-labile than interphase microtubules.

Anaphase↗
PubMed1986

A misleading flow cytometric analysis of DNA in an adenocarcinoma: a comparative flow cytometric and cytogenetic study.

A case is presented in which flow cytometric and cytogenetic analysis was done on a biopsy from a highly anaplastic metastatic adenocarcinoma. Flow cytometric analysis of DNA content failed to show a significant population of aneuploid cells. However, histologic examination revealed a substantial number of tumor cells, and cytogenetic analysis produced chromosome counts ranging from 20 to 144.

Adenocarcinoma↗
PubMed1986

Genetic analysis of cystic fibrosis: linkage of DNA and classical markers in multiplex families.

Linkage of cystic fibrosis (CF) to DNA and classical markers was studied in 36 families of two or three generations with at least two living affected children. Among the 79 affected children, no recombinants were detected between the disease and the markers MET and pJ3.11, previously shown to be linked to CF. No linkage between the human trypsin gene family (which appears to include at least 10 members) and CF was found, although not all genes of the trypsin family have been screened yet. In one of the CF families, recombination between MET and pJ3.11 was detected in an unaffected sib. Data from our families suggest that the gene order of markers among chromosome 7q is: (7cen;p8.33)collagen(COL1A2);DOCR1-917;paraoxonase+ ++(PON);(MET-cf-J3.11);T-cell receptor beta chain (TCRB);qter. There was no evidence for (or against) either postzygotic selection or meiotic drive to explain the high frequency of CF in Caucasian populations.

Cystic Fibrosis↗
PubMed1985

Improved in situ hybridization and G-banding by pretreatment with Denhardt's solution and gelatin-chrome alum.

Various pretreatments of metaphase spreads were examined to obtain optimal DNA labelling patterns while maintaining chromosome integrity during in situ hybridization procedures. Preparations of African green monkey (AGM) chromosomes fixed in methanol-acetic acid (CV-1 cell line) were treated by coating with Denhardt's solution, dilute gelatin-chrome alum, nonfat instant dry milk dissolved in saline-citrate solution (SSC) and/or acetylation prior to denaturation of chromosomal DNA in 70% formamide-2 X SSC for 2 min at 70 degrees C. A 3H-labelled, cloned DNA fragment of the highly repetitive AGM component alpha DNA was hybridized to the chromosomes by incubation at 45 degrees C for 16 h. Treatment with gelatin-chrome alum prior to denaturation greatly improved chromosome morphology and decreased background, but reduced pericentromeric labelling. Sequential treatment with 5 X Denhardt's solution followed by gelatin-chrome alum resulted in enhanced specificity of labelling and excellent chromosome morphology, as well as reduced levels of background. Acetylation had little effect after pretreatment with gelatin-chrome alum, but reduced background levels after pretreatment with Denhardt's solution. Chromosomes treated with Denhardt's solution plus gelatin-chrome alum can be routinely G-banded using trypsin after in situ hybridization.

Animals↗
PubMed1984

Cytophotometric comparisons of DNA levels in neuronal and glial cells of the cerebellum: a comparative study.

Several cytochemical studies of the DNA content and ploidy status of neuronal cell nuclei in the central nervous system have reported the occurrence of hyperdiploid amounts of DNA in Purkinje cells and suggest the existence of some type of 'extra' DNA, the biological significance of which is, as yet, unknown. To explore this phenomenon further, the DNA content of glial and Purkinje cell nuclei was determined in several vertebrate species, using the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) to stain isolated cerebellar nuclei for analysis with a single parameter flow cytometer. The Feulgen reaction for DNA was used to stain liver and cerebellar tissue imprints for the measurement of individual nuclei with a Vickers M86 integrating microdensitometer. In both types of analyses, chicken erythrocyte nuclei served as an internal reference standard of 2.5 pg DNA per cell. The mean DNA content of Purkinje cells and glial or granule cells was essentially the same as that found for diploid (2C) non-neuronal cells, such as hepatocytes, in rainbow trout, Amazon molly fish, salamander (Plethodon), mouse, rat, rabbit, cat, dog, monkey and human. Although Purkinje cell nuclei with 4C DNA levels were found in all of these species, except salamander and rabbit, the frequency of such cells was low (1-7%) and varied with the species. There was a low incidence of Purkinje cell nuclei with interclass DNA amounts in all species examined. Our data show that most neuronal cell nuclei in the cerebellum contain 2C levels of DNA.

Animals↗