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G M Lee

Publications and source records attributed to G M Lee.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1984

Picogram per cell determination of DNA by flow cytofluorometry.

Using nuclei isolated from less than 0.2 g tissue or 10(7) cells, a method is presented for the quantitative determination of amounts of DNA per cell at the picogram level. This technique is based on the enhanced fluorescence of 4',6-diamidino-2-phenylindole (DAPI) when it binds to DNA. A rapid, one-step nuclear isolation and DNA staining procedure is used to prepare tissue samples for flow cytometric analysis. Frozen tissues give results comparable to those for fresh tissue. Both chicken and trout erythrocyte nuclei were used as reference standards in the determination of amounts of DNA per diploid cell for several mammals and Amazon molly fish. The consistent values obtained for different tissues from the same organism show the accuracy of this method for DNA measurement.

Animals↗
PubMed1984

Proctolin in the lobster: the distribution, release, and chemical characterization of a likely neurohormone.

A radioimmunoassay, immunohistochemical techniques, and high pressure liquid chromatography (HPLC) methods have been developed for the study of the pentapeptide proctolin in the lobster Homarus americanus. Proctolin-like immunoreactivity is present in nearly every portion of the lobster nervous system; immunoreactivity is found in the brain, in each of the ganglia and connectives of the ventral nerve cord, and in many of the nerve roots that emerge from the cord. The greatest amounts are found in the pericardial organs, which are well known neurosecretory structures, and these structures have been selected for more detailed study. The immunoreactive material in the pericardial organs appears to be authentic proctolin. This material co-migrates with synthetic proctolin in two HPLC systems. Furthermore, a peptide that is purified from pericardial organs by HPLC is indistinguishable from synthetic proctolin in high resolution fast atom bombardment mass spectrometry. Cytochemistry reveals that the surface of the pericardial organs is densely covered with immunoreactive varicosities. No cell bodies that stain for proctolin are found in the pericardial organs, and the cells that give rise to the varicosities have not yet been located. The nerve endings in pericardial organs are capable of releasing proctolin-like material when depolarized in the presence of Ca++. These findings suggest that proctolin is a neurohormone in the lobster.

Animals↗
PubMed1982

In vitro injury of porcine aortic endothelial cells by very-low-density lipoproteins from diabetic rat serum.

Primary cultures of porcine aortic endothelial cells were used to detect injurious agents in diabetic rat serum. Media containing normal or diabetic serum (streptozotocin-induced) were incubated with subconfluent primary cultures of endothelial cells. Cells exposed to 17% diabetic serum were severely contracted and permeable to trypan blue after 18 h. Cultures in normal serum contained significantly more protein (P less than 0.005) than cultures in diabetic serum after 1 day. When normal and diabetic serum were mixed in different proportions and added to cells at 17% total serum concentration, a 50% reduction from growth in normal serum occurred at a ratio of 1 part diabetic serum to 9 parts normal serum. The toxicity of diabetic serum was not altered by heat inactivation. Administration of insulin to diabetic rats reversed both cytotoxicity and elevated triglyceride levels. Serum triglyceride levels were inversely correlated with endothelial cell growth (r = -0.878, P less than 0.001). In vitro addition of up to 1 U/ml insulin to cultures in diabetic serum did not alter toxicity. Ultracentrifugal flotation of diabetic serum demonstrated the toxic substance to be entirely localized in the very-low-density lipoprotein fraction (d less than 1.006 g/ml). A pool of d greater than 1.006 fractions from diabetic serum supported growth equivalent to normal serum. Diabetic serum contains a substance localized in the very-low-density lipoprotein fraction that is severely toxic to aortic endothelial cells in vitro. Very-low-density lipoprotein injury of endothelial cells may play an important role in the development of arterial vascular disease in insulin-dependent diabetes.

Animals↗
PubMed1981

The role of serum and biliary antibodies and cell-mediated immunity in the clearance of S-typhimurium from chickens.

The development of three parameters of immunity in response to a non-lethal infection of Salmonella typhimurium in adult chickens has been examined. Intravenous inoculation of 1 X 10(6) organisms established infection in the liver, spleen and intestinal tract of all birds; the organism persisted in these sites until day 9 of the infection, after which it was cleared rapidly from all sites. High levels of agglutinating and haemagglutinating antibodies were found in serum and bile 5 days after infection; they peaked at days 7 to 10, and detectable antibody was still present in both fluids 6 weeks after infection. The presence of this antibody did not appear to cause a significant reduction in organism numbers in any of the sites examined. Cell mediated immunity was detected at day 14. It is suggested that cells mediated immunity is responsible for clearance of the organisms from the tissues.

Animals↗
PubMed1977

Adriamycin interactions with T4 DNA polymerase. Two modes of template-mediated inhibition.

We examined the effect of adriamycin on kinetics of DNA synthesis catalyzed by DNA polymerase purified from bacteriophage T4-infected Escherichia coli. Two distinct modes of enzyme inhibition occur: uncompetitive and competitive at "low" and "high" drug:DNA nucleotide molar ratios, respectively. Competitive inhibition is not observed unless an unblocked amino group is present on the sugar (daunosamine) moiety. A model is proposed to relate the enzyme inhibition kinetics to intercalative and ionic binding of adriamycin to DNA.

Coliphages↗