Biomedical subjects

G A Griess

Publications and source records attributed to G A Griess.

At least 19 recordsLinked to original sources

PubMed2001

Application of the concept of an electrophoretic ratchet.

Fractionation via a gel electrophoretic ratchet has previously succeeded for comparatively large (radius R > or = 95 nm) spheres (Serwer, P, Griess, G.A., Anal. Chim. Acta 1998, 372, 299-306). The electrical oscillations are the following electrical field pulses: high field --> low field --> high field, etc. The field is inverted after each pulse; the time-integral of the field can be zero. Response to the ratchet is caused by steric trapping in the high field-direction, but not in the low field-direction. Trapping and, therefore, response to the ratchet decrease as R decreases. The smaller spheres do not respond to the ratchet. In the present study, spheres with R values smaller than 95 nm are made, for the first time, to respond to a similar gel electrophoretic ratchet. To achieve this objective, the heterogeneity of pore size is increased for the gel used. The heterogeneity of pore size is increased by (i) forming the gel with degraded hydroxyethyl agarose, and (ii) gelling at comparatively high temperature. If a particle still does not respond to the ratchet (because the particle is too small), this particle has a net migration in the high field-direction, when the above-described pulsed field is biased in the high field-direction. If a particle does respond to the improved ratchet, the particle has a net migration in the low field-direction. Here, the R of ratchet-responding spheres is reduced to 30-50 nm. These ratchet-responding spheres include both intact bacteriophage particles (R = 30 nm) and latex spheres. The smaller ratchet-responding spheres have an electrophoretic mobility that decreases in magnitude as the electrical field increases in magnitude. A ratchet-based procedure is developed here to achieve continuous preparative gel electrophoresis.

Bacteriophage T3↗

PubMed2001

Improving the length-fractionation of DNA during capillary electrophoresis.

The present study develops a path-lengthening strategy for capillary electrophoresis of short double-stranded DNA molecules, in an aqueous solution of neutral polymer (hydroxypropylmethylcellulose). Tests of the dependence of fractionations on pulse times reveal the operation of at least one mechanism in addition to increase in effective path length. Electrophoresis is performed in the following two-stage cycles (cyclic electrophoresis): The first analysis-stage of each cycle is a constant field (forward) capillary electrophoresis. This analysis-stage reveals the length distribution of the shortest DNA molecules not previously analyzed. The second, enhancement-stage of each cycle is zero-integrated field electrophoresis (ZIFE). The enhancement-stage improves the DNA length-fractionation for the next DNA molecules to be analyzed. A slight reverse migration occurs in the enhancement-stage. Increase in both peak separation and peak sharpness contribute to improvement in the length-fractionation of DNA molecules.

DNA↗

PubMed2000

Unlimited increase in the resolution of DNA ladders.

Fractionation of DNA ladders by gel electrophoresis is limited by the progressive compressing of the long DNA end of a ladder. Improvement in the resolution of this DNA is achieved by use of the following two-step electrophoresis. Initially, the DNA ladder is fractionated by conventional constant field agarose gel electrophoresis. Subsequently, gel electrophoresis is performed in the reverse direction by pulsing the electrical field (PFGE). A newly developed type of pulsing is used, which causes inversion of a double-stranded DNA ladder: the distance migrated increases as the length of the DNA molecule increases. Thus, the resolution of DNA bands continues to increase during the PFGE. These two stages of electrophoresis are serially repeated. Eventually, both the short and the long DNA ends of the ladder migrate out of the gel while a selected region of the ladder undergoes progressive increase in resolution during back-and-forth migration. Improved resolution of DNA bands is achieved, without a known limit.

Chemical Fractionation↗

PubMed1999

Advances in the separation of bacteriophages and related particles.

Nondenaturing gel electrophoresis is used to both characterize multimolecular particles and determine the assembly pathways of these particles. Characterization of bacteriophage-related particles has yielded strategies for characterizing multimolecular particles in general. Previous studies have revealed means for using nondenaturing gel electrophoresis to determine both the effective radius and the average electrical surface charge density of any particle. The response of electrophoretic mobility to increasing the magnitude of the electrical field is used to detect rod-shaped particles. To increase the capacity of nondenaturing gel electrophoresis to characterize comparatively large particles, some current research is directed towards either determining the structure of gels used for electrophoresis or inducing steric trapping of particles in dead-end regions within the fibrous network that forms a gel. A trapping-dependent technique of pulsed-field gel electrophoresis is presented with which a DNA-protein complex can be made to electrophoretically migrate in a direction opposite to the direction of migration of protein-free DNA.

Bacteriophages↗

PubMed1998

The formation of small-pore gels by an electrically charged agarose derivative.

Previous studies have shown that, during the formation of an underivatized agarose gel, agarose molecules laterally aggregate to form thicker fibers called suprafibers; the suprafibers branch to form a gelled network. In the present study, electron microscopy of thin sections is used to investigate both the thickness and the spacing of the fibers of gels formed by agarose chemically derivatized with carboxymethyl (negatively charged) groups. For carboxymethyl agarose, electron microscopy reveals that gels cast in water consist of both fibers narrower and pores smaller than those observed for water-cast underivatized agarose gels at the same concentration. This result is confirmed by using the electrophoretic sieving of spheres to determine the radius (PE) of the effective pore of the gel. At a given concentration of gel less than 1%, the PE for a water-cast carboxymethyl agarose gel is 0.25-0.30x the PE for a water-cast underivatized agarose gel. The value of PE predicts the extent of the electrophoretic sieving that is observed when double-stranded DNA is subjected to electrophoresis through a water-cast carboxymethyl agarose gel; DNA bands formed in a water-cast carboxymethyl agarose gel are comparable in quality to DNA bands formed in a water-cast underivatized agarose gel of equal PE. The following observation supports the hypothesis that electrical charge-charge repulsion among carboxymethyl agarose molecules inhibits the formation of suprafibers in water-cast carboxymethyl agarose gels: Increased content of suprafibers in carboxymethyl agarose gels is observed when the ionic strength is raised by the presence of NaCl, MgCl2, or any of several buffers during gelation of carboxymethyl agarose.

DNA↗

PubMed1998

Intracellular Ca2+ thresholds that determine survival or death of energy-deprived cells.

Increase of intracellular ionized or free Ca2+ is thought to play a central role in cell death due to ATP depletion. However, concurrently operative mechanisms of injury that do not require intracellular Ca2+ increases have made it difficult to test this hypothesis or to determine the concentrations at which intracellular Ca2+ becomes lethal. The predominant Ca2+-independent mechanism of injury during ATP depletion involves the loss of cellular glycine. This type of damage can be fully inhibited by adding the amino acid exogenously. Using glycine to suppress Ca2+-independent plasma membrane damage, we have examined the effect of intracellular Ca2+ elevations on cell viability during ATP depletion. Madin-Darby canine kidney (MDCK) cells were depleted of ATP by incubation with a mitochondrial uncoupler in glucose-free medium. Free Ca2+ concentration in the medium was varied between 26 nmol/L and 1.25 mmol/L in the presence of a Ca2+ ionophore. Measurements with the Ca2+ probes fura-2, furaptra, and fura-2FF showed that intracellular Ca2+ was clamped at extracellular levels under these conditions. Cell survival during ATP depletion was indicated by viable cells recovered 24 hours later. The results show that ATP-depleted cells can sustain high levels of intracellular Ca2+ (100 micromol/L) for prolonged periods and remain viable if plasma membrane damage is prevented by glycine. Cell death was observed only when intracellular free Ca2+ was allowed to increase beyond 100 micromol/L, and this was associated with dramatic nuclear alterations: chromatin condensation, loss of nuclear lamins, and breakdown of DNA into large 50- to 150-kb fragments. Our studies demonstrate unexpectedly high resistance of cells to calcium cytotoxicity if glycine that is lost during ATP depletion is restored. In addition, they provide insights into novel mechanisms of nuclear disintegration and DNA damage that are triggered when the high thresholds of intracellular Ca2+ required for cell death are exceeded.

Adenosine Triphosphate↗

PubMed1997

The conformation of packaged bacteriophage T7 DNA: informative images of negatively stained T7.

Within the icosahedral protein outer shell of bacteriophage T7, a 40-kbp DNA genome occupies a cavity also occupied by a protein cylinder that projects into the DNA from the outer shell. However, neither the internal cylinder nor separately resolved DNA segments are revealed in the conventional negatively stained specimens of intact bacteriophage T7. In the present study, a procedure of negative staining is used that reveals both internal proteins and separately resolved segments of packaged DNA during electron microscopy of intact particles of a hybrid T7 bacteriophage; the hybrid is genetically T7, except for a tail fiber gene that has a segment from the T7-related bacteriophage, T3. The negatively stained packaged DNA segments of this hybrid bacteriophage are found to be wrapped around the axis of the internal cylinder. To obtain additional information about the conformation of packaged T7 DNA, electron microscopy is performed of negatively stained capsids that are incompletely filled with DNA (ipDNA-capsids); a procedure is described for improved isolation of ipDNA-capsids from lysates of hybrid bacteriophage T7-infected cells. The packaged DNA segments of ipDNA-capsids are found not to be wrapped around any axis. Images of ipDNA-capsids are explained by the hypothesis that DNA does not achieve its wrapped condition until the capsid is more than 40% full of DNA. Wrapping via folding is, therefore, proposed to explain the images of DNA packaged in bacteriophage T7.

Bacteriophage T3↗

PubMed1996

Use of excluded volume to increase the heterogeneity of pore size in agarose gels.

When testing theoretical models that quantitatively describe the sieving of macromolecules during gel electrophoresis, investigators have been limited by absence of control of the heterogeneity of the size of pores in the gel. In a recent study performed by electron microscopy of thin sections (G. A. Griess et al., J. Struct. Biol. 1993, III, 39-47), pore size heterogeneity has been increased for agarose gels by a combination of both derivatization and molecular weight reduction of the polysaccharide chains of agarose. In the present study, pore size heterogeneity is increased by a mechanism that appears to have an origin different from the origin of this previously observed increase in heterogeneity: Pore size heterogeneity is increased by addition of a polyethylene glycol (PEG) of high molecular weight (18,500) to molten agarose before gelation. In contrast, the use of a lower molecular weight PEG (either 4,000 or 7,500) causes the formation of micron-sized precipitates within a gelled network of agarose fibers. Thus far, the PEG-induced heterogeneity of pore size occurs primarily in 100-1,000 microns scale zones separated from each other by interzone regions of decreased agarose fiber density. More uniform gels are needed for the study of sieving.

Dose-Response Relationship, Drug↗

PubMed1996

Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii.

Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp.

Borrelia↗

PubMed1995

Non-denaturing gel electrophoresis of biological nanoparticles: viruses.

Although gel electrophoresis is usually used for the fractionation of monomolecular particles, it is also applicable to the fractionation of the multimolecular complexes produced during both cellular metabolism and assembly of viruses in virus-infected cells. Gel electrophoretic procedures have been developed for determining both the size of a spherical particle and some aspects of the shape of a non-spherical particle. Capsids bound to DNA outside of the capsid can also be both fractionated and characterized. The procedures developed will be used for screening viral mutants; they also can potentially be used for diagnostic virology. Sensitivity of detection, the major current limitation, is being improved by use of both improved stains and scanning fluorimetry. The gels used for fractionation sometimes approximate random straight fiber gels, but become increasingly biphasic as the gel concentration is decreased.

Animals↗

PubMed1995

A desktop, low-cost video fluorometer for quantitation of macromolecules after gel electrophoresis.

After gel electrophoresis, quantification of in-gel fluorescence is sometimes used to assay the macromolecules fractionated. Both procedures and equipment that have the following improvements are presented here for direct video fluorometry of gels used for electrophoresis: comparatively low cost, high ease of use and low consumption of space. This equipment has a linear response to the amount of ethidium-stained DNA that forms a band in an agarose gel.

DNA, Viral↗

PubMed1993

The relationship of agarose gel structure to the sieving of spheres during agarose gel electrophoresis.

To understand the organization of fibers in an agarose gel, digitized electron micrographs are used here to determine the frequency distribution of interfiber distance (2Pc) in thin sections of agarose gels. For a preparation of underivatized agarose, a 1.5% gel has a Pc distribution that is indistinguishable from the Pc distribution of a computer-generated, random-fiber gel; the log of the occurrence frequency (F) decreases linearly as a function of Pc. As the agarose concentration decreases below 1.5%, the semilogarithmic F versus Pc plot becomes progressively less linear. Two straight lines represent the data; the plot is steeper at the lower Pc values. As the percentage of agarose increases above 1.5%, the semilogarithmic F versus Pc plot becomes steeper at the higher Pc values. This change in the shape of semilogarithmic F versus Pc plots is possibly explained by the existence in agarose gels of two zones, one whose Pc distribution is more sensitive to the average agarose concentration than the other. To compare the structure of agarose gels to their sieving during electrophoresis, the root mean square value of Pc (Pc) is compared to the sieving-based radius of the effective pore (PE; Griess et. al. (16)) for both underivatized agarose and a derivatized agarose that has a smaller PE at any given agarose percentage. For 0.8-2.0% gels of either underivatized or derivatized agarose, PE/Pc is a constant within experimental error. Deviations from this constant are observed at lower gel percentages. This relationship of PE to Pc constrains theoretical descriptions of the motion of spheres in fibrous networks.

Biophysical Phenomena↗

PubMed1993

The trajectories of spheres during agarose gel electrophoresis.

To develop a physical description of the gel-induced retardation of spheres during gel electrophoresis, the microscopic motion of single electrically charged latex spheres is statistically quantified here, by digital image analysis. To obtain adequate resolution in space, comparatively large spheres, 240 nm in radius, are used. The following observations are made during electrophoresis in a 0.2% agarose gel at 22 degrees C: (a) When a comparatively high field (3.0 V cm-1) is used, inelastic collisions result in field-induced trapping of spheres; no elastic collisions are observed. (b) Reduction of the field from 3.0 to 0.0 V cm-1 results in reverse migration of previously trapped spheres. (c) In the absence of trapping, the electrical field does not cause an alteration in the tortuosity of motion (i.e. motion in a field-perpendicular direction). (d) When results are obtained for a constant time between images (0.2 s), gel-dependent deviations from a true random walk are not observed in the absence of trapping. (e) When results are obtained as a function of time between images, significant gel-dependent deviation from a random walk is observed. In the absence of trapping, the data presented here indicate that retardation is derived primarily from dissipative processes that are concentrated near gel fibers. However, steric effects have not yet been distinguished from hydrodynamic effects.

Buffers↗

PubMed1992

Assembly-associated structural changes of bacteriophage T7 capsids. Detection by use of a protein-specific probe.

To detect changes in capsid structure that occur when a preassembled bacteriophage T7 capsid both packages and cleaves to mature-size longer (concatameric) DNA, the kinetics and thermodynamics are determined here for the binding of the protein-specific probe, 1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid (bis-ANS), to bacteriophage T7, a T7 DNA deletion (8.4%) mutant, and a DNA-free T7 capsid (metrizamide low density capsid II) known to be a DNA packaging intermediate that has a permeability barrier not present in a related capsid (metrizamide high density capsid II). Initially, some binding to either bacteriophage or metrizamide low density capsid II occurs too rapidly to quantify (phase 1, duration < 10 s). Subsequent binding (phase 2) occurs with first-order kinetics. Only the phase 1 binding occurs for metrizamide high density capsid II. These observations, together with both the kinetics of the quenching by ethidium of bound bis-ANS fluorescence and the nature of bis-ANS-induced protein alterations, are explained by the hypothesis that the phase 2 binding occurs at internal sites. The number of these internal sites increases as the density of the packaged DNA decreases. The accompanying change in structure is potentially the signal for initiating cleavage of a concatemer. Evidence for the following was also obtained: (a) a previously undetected packaging-associated change in the conformation of the major protein of the outer capsid shell and (b) partitioning by a permeability barrier of the interior of the T7 capsid.

Anilino Naphthalenesulfonates↗

PubMed1991

Variation of the permeability of bacteriophage T4: analysis by use of a protein-specific probe for the T4 interior.

The permeability of bacteriophage T4 and the change in T4 permeability caused by mutation to osmotic shock resistance are investigated here by quantification of the kinetics with which both a DNA-specific probe (ethidium) and a protein-specific probe [1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid, or bis-ANS)] bind to T4. In the case of an osmotic shock-resistant mutant, T40s41, both ethidium and bis-ANS bind with first order kinetics. The first-order rate constant (k*) for both bis-ANS and ethidium is a function of anion type and concentration. Adenosine triphosphate, phosphate, bisulfite, sulfate, and acetate anions all reduce k* below the k* observed when chloride is the only anion. When chloride is the only anion at 25 degrees C, k* values for binding to T40s41 are orders of magnitude above k* values for binding to wild-type T4 (T4wt). At 25 degrees C, k* for T4wt is too small to measure but k* for T4wt increases at 50-55 degrees C to values approaching those measured for T40s41, without inactivating T4wt, when chloride is the only anion; during heating, T4wt is stabilized by both ethidium and bis-ANS. Binding to T4wt is reversible at 50-55 degrees C, but not at 25 degrees C. Equilibrium binding of bis-ANS to T40s41 reveals 112 +/- 24 sites per T4 capsid. Equilibrium binding of ethidium to T40s41 reveals both high- and low-affinity sites previously observed in the packaged DNA of other bacteriophages. The ATP-induced decrease in k* is not accompanied by a decrease in equilibrium binding. The following hypotheses are presented to explain the above data: (a) All detected bis-ANS binding sites on T4 are interior to the outer surface of T4. (b) The value of k* for both bis-ANS and ethidium is controlled at the port(s) of passage through the outer shell of the T4 capsid. (c) The anions present control k* values at the port(s) of entry, probably by controlling the size of this port. The effects on k* of phosphate explain the otherwise paradoxical observation [P. J. McCall and V. A. Bloomfield (1976) Biopolymers 15, 2323-2336] that in a phosphate buffer the permeabilities of T4wt and T40s41 are the same.

Anilino Naphthalenesulfonates↗

PubMed1990

Gel electrophoresis of micron-sized particles: a problem and a solution.

The gel electrophoresis of spherical particles with a radius above 0.2 micron has not been reported yet. In the present study, video phase-contrast light microscopy is used to observe the motion of individual latex spheres, 0.52 micron in radius, during electrophoresis in 0.1% agarose gels. At 2 V/cm, the spheres initially migrate in the direction of the electrical field. However, each sphere eventually undergoes a cessation of all motion. Brownian motion is restored when the electrical potential gradient is reduced to zero. Arrest can be prevented by periodically inverting the direction of the electrical field. These observations are explained by electrical field-induced steric trapping of the spheres by gel fibers. Inversion of the electrical field should assist the application of agarose gel electrophoresis to micron-sized cellular organelles and cells.

Bacteria↗

PubMed1989

The sieving of spheres during agarose gel electrophoresis: quantitation and modeling.

By use of agarose gel electrophoresis, the sieving of spherical particles in agarose gels has been quantitated and modeled for spheres with a radius (R) between 13.3 and 149 nm. For quantitation, the electrophoretic mobility has been determined as a function of agarose percentage (A). Because a previously used model of sieving [D. Rodbard and A. Chrambach (1970) Proc. Natl. Acad. Sci. USA 65, 970-977] was found incompatible with some of these data, alternative models have been tested. By use of an underivatized agarose, two models, both based on the assumption of a single effective pore radius (PE) for each A, were found to yield PE values that were independent of R and that were in agreement with values of PE obtained independently (PE = 118 nm X A-0.74): sieving by altered hydrodynamics in a cylindrical tube of radius, PE, and sieving by steric exclusion from a circular hole of radius, PE. The same analysis applied to a 6.5% hydroxyethylated commercial agarose yielded a steeper PE vs A plot and also agreement of the above two models with the data. The PE vs A plot was significantly altered by both further hydroxyethylation and factors that cause variation in the electro-osmosis found in commercial agarose.

Biopolymers↗

PubMed1986

Determination of a particle's radius by two-dimensional agarose gel electrophoresis.

Electrophoresis in an agarose gel dilute enough to be almost nonretarding, followed by electrophoresis in an orthogonal direction into a more concentrated agarose gel, has been developed as a procedure to determine the radius of spherical particles. Unlike procedures of unidirectional electrophoresis in a single gel, the above procedure can be used to compare the radii of particles that differ in solid-support-free electrophoretic mobility. Accuracy of 0.3 nm has been achieved with particles 30 nm in radius. It was found that the apparent radius of the spherical capsid of bacteriophage P22 decreased by 3% during elevated temperature-induced ejection of DNA from the capsid. Though originally designed for use with multimolecular particles, the procedure described here should also be useful with monomolecular particles.

Bacteriophages↗