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PubMed1998

Comparison of glycerol, other polyols, trehalose, and raffinose to provide a defined cryoprotectant medium for mouse sperm cryopreservation.

Most procedures for mouse sperm cryopreservation have utilized raffinose to provide hypertonicity for cell desiccation prior to freezing and glycerol to block intracellular ice formation. Trehalose has been shown in other cell systems to provide positive protection to the plasma membrane and so was examined as a replacement for raffinose. Comparison of 3 and 6% glycerol and 7.5 and 20% sugar showed that 6% glycerol and 7.5% sugar gave maximal protection consistently and so were adopted as standard. Comparison of raffinose and trehalose at this concentration showed trehalose to give significantly better recovery of intact cells: 48 +/- 6% for trehalose, 36 +/- 9% for raffinose (+/- SE, n = 5; arc sine transformed data; P < 0.01). Less hydrophilic polyols should prove more permeant to the membrane than glycerol, enter the cell rapidly, and so possibly inhibit lethal intracellular ice formation effectively. We hypothesized that one of these polyols plus glycerol would be a more effective cryoprotectant than glycerol alone. The polyols tested as supplements to 6% glycerol were propane-1,2-diol, propane-1,3-diol, 1,1,1-tris-(hydroxymethyl)ethane (THME), and 2-ethyl-2-(hydroxymethyl)-propane-1,3-diol (EHMP). With 6% glycerol and 7.5% raffinose or trehalose, the two diols and THME gave less cryoprotection than with glycerol alone, and EHMP reduced postthaw membrane integrity to nil, thus invalidating the hypothesis. Comparison of bicarbonate-containing medium MJB to bicarbonate-free medium NTP, both with 6% glycerol/7.5% trehalose, showed no difference in recovery of membrane-intact cells. For ease of pH maintenance, NTP was chosen for studies of addition prefreeze and removal postthaw of 6% glycerol/7.5% trehalose cryoprotectant with in vitro fertilization as endpoint. Three protocols for cryoprotectant handling were tested: serial addition/dilution; dialysis addition and removal; and dialysis addition and direct insemination without cryoprotectant removal. The last proved significantly superior (P < 0.01), giving 62% fertilized eggs, normalized to controls, compared to 21% for dialysis addition and removal and 32% for serial addition and dilution. The glycerol/trehalose combination thus provides a defined cryoprotectant which, when used with addition by dialysis prefreeze and direct insemination postthaw, yields a satisfactory yield of fertilized eggs in an in vitro fertilization system.

Animals↗

PubMed1997

Towards a molecular understanding of phase separation in the lens: a comparison of the X-ray structures of two high Tc gamma-crystallins, gammaE and gammaF, with two low Tc gamma-crystallins, gammaB and gammaD.

gamma-Crystallins, although closely related in sequence, show intriguing differences in their temperature-dependent interactions: those that have a high or intermediate Tc for phase separation are cryoproteins whereas low Tc gamma-crystallins are not. To address the molecular basis of phase separation, X-ray crystallography has been used to define the structural differences between high and low Tc gamma-crystallins. A pre-requisite for this study was to clarify the assignment of bovine gene sequences to bovine gamma-crystallin proteins used for biophysical measurements. Based on nucleotide sequence analyses of gamma E and gamma F bovine crystallin genes, gamma F corresponds to the previously crystallised high Tc protein bovine gamma IVa and gamma E corresponds to the high Tc bovine protein fraction previously known as gamma IIIa. The gamma F sequence has enabled the completion of the refinement of the bovine gamma F crystal structure which shows that the molecule has an additional surface tryptophan explaining why gamma F has different spectroscopic properties from gamma B. A high Tc protein from rat lens, gamma E crystallin, has been crystallised and the X-ray structure solved at 2.3 A resolution. Comparison of the X-ray structures of two high Tc proteins, rat gamma E and bovine gamma F, with the structures of two low Tc proteins, bovine gamma B and bovine gamma D, shows that the main conformational change between high and low Tc proteins is in the cd surface loop of motif 3. All four structures have numerous ion pairs on their surfaces leading to a high surface charge density, yet with low overall charge. Comparison of the lattice contacts of the two high Tc proteins with the two low Tc gamma-crystallins indicates that these high Tc proteins utilise more amino-aromatic interactions such as between histidine and arginine. Comparison of the sequences of all the gamma-crystallins which have been characterised for phase separation temperature indicates that only residue Arg/Lys 163 uniquely distinguishes cryo from non-cryo gamma-crystallins and it is close to the altered surface loop. Although this region probably contributes to phase separation, Tc is likely to be a function of an overall global property that is responsive to overall charge distribution. Calculated dipole moments of native gamma-crystallins, low Tc gamma-crystallin sequences threaded into high Tc gamma-crystallin structures, and vice versa, show how both sequence and 3D structure contribute to this overall property. High Tc gamma-crystallins have on average higher Arg/Lys ratios and higher histidine content. It is hypothesised that this increases the proportion of surface static paired charged networks which thus reduces the repulsive hydration force and so increases the attractive interactions of the protein-rich phase in binary liquid phase separation.

Amino Acid Sequence↗

PubMed1994

Large-scale genomic comparison using two-dimensional DNA gels.

Two-dimensional electrophoresis (2DE) of DNA fragments, in which separation occurs first by size and then by sequence variation, is a method enabling large-scale comparison of complex genomes. Combining 2DE with probing for various classes of repetitive genomic elements allows rapid and efficient comparison of thousands of fragments and millions of base-pairs of DNA distributed across most genomic regions. This approach is demonstrated here by analyzing the extent of genomic relatedness of different inbred strains of mice. Such strains are shown to differ from each other by approximately 0.2-1% of their nucleotides, above which level reproductive speciation occurs. The 2DE method of assessing the overall relationship between two genomes represents an appropriate tool for analyzing members of a single species, but is too sensitive for use in interspecies comparisons.

Animals↗

PubMed1997

Comparison of 1997 Medicare relative value units for gender-specific procedures: is Adam still worth more than Eve?

BACKGROUND: On January 1, 1992, Congress implemented a Medicare payment system based on relative value units (RVUs). The total RVU (which is made up of work, practice, and malpractice RVUs) is multiplied by a dollar conversion factor to set the reimbursement for all procedures covered by Medicare. In a previous study, we found that significant gender bias exists in Medicare reimbursement for female-specific services. Recently, HCFA approved increases (beginning January 1997) in the work RVU for many gynecologic procedures. This study was undertaken to compare work and total RVUs for gender-specific procedures effective January 1, 1997. METHODS: Using the May 1996 Federal Register, we compared work and total RVUs for 24 pairs of gender-specific procedures. The groups were matched so that the amount of work and level of difficulty would be similar, if not identical. We validated our selection of procedures for comparison by also evaluating the average time required to perform these procedures. RESULTS: Comparison of work RVUs for the 24 paired procedures revealed that in 19 cases (80%), male-specific procedures had a higher RVU; in 3 cases (12%), female-specific procedures were higher; and in 2 cases, there was no difference. On average, work RVUs were 49% higher for urologic procedures than for gynecologic procedures. Comparison of total RVUs revealed that in 20 cases (83%), urologic procedures had a higher total RVU and in 3 cases (12%), gynecologic procedures were higher. On average, male-specific surgeries are reimbursed at an amount which is 37% higher than that for female-specific surgeries. CONCLUSION: Recent increases in work RVUs for many gynecologic procedures have resulted in improved reimbursement. However, even with these improvements, significant gender bias still exists in the Medicare reimbursement of female-specific procedures. This gender bias is further magnified as more private insurance carriers use the system to set reimbursement.

Female↗

PubMed1993

A structural basis for sequence comparisons. An evaluation of scoring methodologies.

A residue-exchange matrix has been derived that is suitable for comparison of amino acid sequences. This matrix is based on the tabulation of 207,795 amino acid replacements observed in 65 homologous sets of structurally aligned three-dimensional structures (235 proteins). The majority of the data is from structural comparisons where there is between 15 and 40% sequence identity. As a result, a scoring matrix such as the one devised here should provide a sensitive basis for the comparison of amino acid sequences and the search for homologous sequences in amino acid databases. In order to assess the value of this matrix we have made a comparative analysis with 12 other published scoring matrices that have been used for the alignment of protein amino acid sequences. We find that the matrix derived here is among the better performers in terms of alignment significance, detection of homologous sequences and the accuracy of alignments.

Amino Acid Sequence↗

PubMed1995

A comparison of neutron diffraction and molecular dynamics structures: hydroxyl group and water molecule orientations in trypsin.

A comparison is presented of experimentally observed hydroxyl and water hydrogen atoms in trypsin determined from neutron density maps with the results of a 140 ps molecular dynamics simulation. Experimental determination of hydrogen and deuterium atom positions in molecules as large as proteins is a unique capability of neutron diffraction. The comparison addresses the degree to which a standard force-field approach can adequately describe the local electrostatic and van der Waals forces that determine the orientations of these hydrogen atoms. The molecular dynamics simulation, based on the all-atom AMBER force-field, allowed free rotation of all hydroxyl groups and movement of water molecules making up a bath surrounding the protein. The neutron densities, derived from 2.1 A 2H2O-H2O difference Fourier maps, provide a database of 27 well-ordered hydroxyl hydrogen atoms. Virtually all of the simulated hydroxyl orientations are within a standard deviation of the experimentally observed positions, including several examples in which both the simulation and the neutron density indicate that a hydroxyl group is shifted from a "standard" rotamer. For the most highly ordered water molecules, the hydrogen distributions calculated from the trajectory were in good agreement with neutron density; simulated water molecules that displayed multiple hydrogen-bonding networks had correspondingly broadened neutron density profiles. This comparison was facilitated by development of a method to construct a pseudo 2 A density map based on the hydrogen atom distributions from the simulation. This method is particularly useful for statically disordered water molecules, in which the average location assigned from a trajectory may represent a site of relatively low occupancy. The degree of disorder of internal water molecules is shown to result primarily from the electrostatic environment surrounding that water molecule as opposed to the cavity size available to the molecule.

Crystallography, X-Ray↗

PubMed1996

A dataset of protein-protein interfaces generated with a sequence-order-independent comparison technique.

While there are a number of structurally non-redundant datasets of protein monomers, there is none of protein-protein interfaces. Yet, the availability of such a dataset is expected to provide an added insight into a number of investigations. First and foremost among these is analyzing the interfaces to obtain their prevailing architectures, the forces that account for the protein-protein associations and their packing considerations. Their comparisons with those of the monomers are likely to shed additional light on protein-protein recognition on the one hand and on the folding of the polypeptide chain on the other. Docking simulations are also expected to benefit from the existence of such a dataset. A major stumbling block to the generation of a dataset of interfaces has been that the interface is composed of at least two chains. Furthermore, in the interfaces, each of the chains might be represented by non-contiguous pieces. Their order in the interfaces being compared might be different as well. This discontinuity stems from the definition of an interface. An interface consists of interacting residues between the chains, and those that are in their vicinity in the supporting scaffold, within a certain distance threshold. This necessarily yields unordered fragments, as well as isolated residues. Our novel, efficient, sequence-order-independent structural comparison technique is ideally suited to handle the task of the generation of a library of structurally non-redundant protein-protein interfaces. As it is computer-vision based, it views atoms as collections of points in space, disregarding their chain connectivity. In this work, 351 interface-families are created. Comparisons of the interfaces, and separately, of the chains which contribute to them, yield some interesting cases. In one of the cases, while two interfaces are similar, the structure of only one of the two chains is similar between the two complexes. The structure of the second chain of the first complex differs from that of the second chain of the second complex. Here the structure of the cleft in the first chain dictates the specific binding interactions. In another case, while the interfaces in the two complexes are similar, both chains composing them differ between the complexes. Lastly, the chains composing the complexes are similar, but the interfaces are dissimilar, providing a set of data for investigations of the favorable orientations of protein-protein associations.

Algorithms↗

PubMed1999

Comparison of scalar measures used in magnetic resonance diffusion tensor imaging.

The tensors derived from diffusion tensor imaging describe complex diffusion in tissues. However, it is difficult to compare tensors directly or to produce images that contain all of the information of the tensor. Therefore, it is convenient to produce scalar measures that extract desired aspects of the tensor. These measures map the three-dimensional eigenvalues of the diffusion tensor into scalar values. The measures impose an order on eigenvalue space. Many invariant scalar measures have been introduced in the literature. In the present manuscript, a general approach for producing invariant scalar measures is introduced. Because it is often difficult to determine in clinical practice which of the many measures is best to apply to a given situation, two formalisms are introduced for the presentation, definition, and comparison of measures applied to eigenvalues: (1) normalized eigenvalue space, and (2) parametric eigenvalue transformation plots. All of the anisotropy information contained in the three eigenvalues can be retained and displayed in a two-dimensional plot, the normalized eigenvalue plot. An example is given of how to determine the best measure to use for a given situation by superimposing isometric contour lines from various anisotropy measures on plots of actual measured eigenvalue data points. Parametric eigenvalue transformation plots allow comparison of how different measures impose order on normalized eigenvalue space to determine whether the measures are equivalent and how the measures differ. These formalisms facilitate the comparison of scalar invariant measures for diffusion tensor imaging. Normalized eigenvalue space allows presentation of eigenvalue anisotropy information.

Anisotropy↗

PubMed1997

A mathematical model to determine the optimal number of fragments for comparison of bacterial chromosomic macrorestriction patterns.

To our knowledge, although comparison of chromosomic macrorestriction patterns has become one of the most feasible molecular tools of the current microbial taxonomy, a mathematical approach to optimize the choice of a restriction enzyme among the endonucleases tested for such comparison has not been previously described. The coincidence of restriction patterns for two tested bacterial strains with this chosen endonuclease will ensure a high genetic relatedness between them. We report a mathematical model to determine the probability of hazardously obtaining a particular chromosomic macrorestriction pattern by PFGE and to calculate the optimal number of fragments for its comparison. The model presented allows us to determine the optimal number of fragments in order to compare chromosomic restriction patterns. The model calculates this values as a function of the chromosome size and the restriction site length. The model is not useful for choosing a restriction enzyme previous to experimental steps, but as a tool for the choice of the restriction enzyme that yields the lowest probability of hazardously obtaining coincidences of chromosomic patterns. The applicability of this model has been exemplified by determining the optimal number of fragments for some well-characterized bacteria and by comparing these values with those that have been experimentally used.

Bacteria↗

PubMed1996

Modeling for intergroup comparisons of imaging data.

Intergroup comparisons pose unique challenges in the analysis of functional imaging data. Imperfections in intersubject stereotaxis can give rise to artifactual results and make it particularly important to allow for intersubject differences in task-related changes when formulating statistical models. Because intergroup comparisons generally involve inferences about the populations from which the subjects were drawn rather than inferences about the particular subjects themselves, subjects must be treated as random rather than fixed effects in the statistical model. These requirements, when combined with the need to adjust for multiple spatial comparisons, result in low statistical power when the number of subjects in each group is small. Functional imaging studies to identify differences between groups generally require many more subjects than other types of functional imaging studies and require careful advance planning to maximize the likelihood of reaching meaningful conclusions.

Brain Mapping↗

PubMed2000

Selection of an adaptive test statistic for use with multiple comparison analyses of neuroimaging data.

Statistical analysis of neuroimages is commonly approached with intergroup comparisons made by repeated application of univariate or multivariate tests performed on the set of the regions of interest sampled in the acquired images. The use of such large numbers of tests requires application of techniques for correction for multiple comparisons. Standard multiple comparison adjustments (such as the Bonferroni) may be overly conservative when data are correlated and/or not normally distributed. Resampling-based step-down procedures that successfully account for unknown correlation structures in the data have recently been introduced. We combined resampling step-down procedures with the Minimum Variance Adaptive method, which allows selection of an optimal test statistic from a predefined class of statistics for the data under analysis. As shown in simulation studies and analysis of autoradiographic data, the combined technique exhibits a significant increase in statistical power, even for small sample sizes (n = 8, 9, 10).

Algorithms↗

PubMed2001

A tool for comparison of PET and fMRI methods: calculation of the uncertainty in the location of an activation site in a PET image.

A technique for calculating the uncertainty in the location of an activation site in a PET image, without performing repeated measures, is presented. With the development of new fMRI methods for measuring cerebral hemodynamics, demonstration of the efficacy of these techniques will be critical to establish clinical utility. Comparisons with PET are a powerful tool for validating these new fMRI techniques. In addition to the fact that PET techniques are well-established methods for making physiological measurements in vivo, PET methods are also free of the geometric distortions and nonuniform signal-to-noise artifacts (due to signal dropout) common in fMRI techniques. Comparisons reported previously have been limited by the large number of trials acquired in single-subject fMRI studies and the small number of trials in a PET study (due to the radiation dose to the patient or the interscan delays for tracer decay). Our method calculates both the center of mass (CM) of a predefined region of interest and the uncertainty in the location of the CM using the preimage PET data (sinograms). Results of phantom studies demonstrate that our method is an unbiased measurement equivalent to that of repeated measures with a large number of images. Extension of this technique to estimate the uncertainty in the location of an activation site in a PET statistical parametric map will permit precise rigorous comparisons of PET and fMRI methods in single subjects without the constraints imposed by the relatively small number of PET measurements.

Brain Mapping↗

PubMed1998

Use of the mouse intranasal test (MINT) to determine the allergenic potency of detergent enzymes: comparison to the guinea pig intratracheal (GPIT) test.

A mouse intranasal test (MINT) was developed to determine the relative allergenicity of detergent enzymes. In this simple method, various doses of the enzymes are administered in a detergent matrix, via intranasal instillation, on days 1, 3, and 10, with serum samples collected on day 15. The sera are then analyzed for enzyme specific IgG1 antibody by an antigen specific enzyme immunoassay. The protease enzyme Alcalase (protease Subtilisin Carlsberg) has been used as a benchmark enzyme for development and characterization of the model. The objective of the current studies was to obtain potency comparisons with other protease and nonprotease enzymes and to begin to assess the validity of the model by comparison with potency determinations obtained with the guinea pig intratracheal (GPIT) test. The range of potencies detected among several enzymes of different classes was approximately 60-fold (compared with Alcalase). Modification of the dosing regimen to permit slightly more extended dosing did not change the relative potency determination. Comparison of data from the MINT and GPIT methods indicate that both the mouse and the guinea pig recognize the bacterial amylase Termamyl and a fungal exocellulase as more potent than Alcalase, a serine protease (Subtilisin B) and a fungal alpha-amylase (Fungamyl) as less potent than Alcalase, and the serine protease, Savinase, as equivalent to Alcalase. Also, these data are in alignment with clinical data on the prevalence of occupational enzyme sensitization. Given the simplicity and low cost of the MINT method compared with the GPIT test, these results support continued development of the method as an alternative approach for assessing the allergenicity of enzymes.

Administration, Intranasal↗

PubMed2001

Mammalian reovirus L2 gene and lambda2 core spike protein sequences and whole-genome comparisons of reoviruses type 1 Lang, type 2 Jones, and type 3 Dearing.

The reovirus L2 genome segment encodes the core spike protein lambda2, which mediates enzymatic reactions in 5' capping of the viral plus-strand transcripts. Complete nucleotide-sequence determinations were made for the L2 genome segments of eight mammalian reoviruses, including the prototype isolates of serotypes 1 and 2: Lang (T1L) and Jones (T2J), respectively. Each L2 segment was found to be 3912 or 3915 bases in length. Partial nucleotide-sequence determinations were also made for the 3916-base L2 segment of reovirus type 3 Dearing (T3D), the prototype isolate of serotype 3. The whole-genome sequence of reovirus T3D was reported previously. The T1L L2 analysis represents completion of the whole-genome sequence of that isolate as well. The T2J L2 analysis leaves only the sequence of the M1 segment yet to be reported from the genome of that isolate. The T2J M1 sequence made available from analysis in another lab was used for initiating whole-genome comparisons of reoviruses T1L, T2J, and T3D in this report. The nine L2 gene sequences and deduced lambda2 protein sequences were used to gain further insights into the biological variability, structure, and functions of lambda2 through comparisons of the sequences and reference to the crystal structure of core-bound lambda2. Phylogenetic comparisons suggest the presence of three evolutionary lines of divergent L2 alleles among the nine isolates. Localized regions of conserved amino acids in the lambda2 crystal structure include active-site clefts of the RNA capping enzyme domains, sites of interactions between lambda2 domains within the pentameric spike structure, and sites of interaction between lambda2 subunits and other proteins in viral particles.

Amino Acid Sequence↗

PubMed1998

Comparison of the di- and trinucleotide frequencies from the genomes of nine different coronaviruses.

As an alternative to protein alignments for the comparison of sequences, the reiterations of mono- di- and trinucleotide frequencies were used for the comparison of coronavirus sequences. The relative abundance of the di- and trinucleotide frequencies within the 3' part from nine coronavirus genomes were determined. The patterns of dinucleotide frequencies and the trinucleotide frequencies showed some common features for all coronaviruses but also differences between the groups formerly defined on the base of antigenic relatedness. The normalised dinucleotide frequencies were further used to calculate the distances between coronavirus sequences. Based on the dinucleotide frequency distances, coronaviruses can be divided into two groups which roughly reflect the taxonomic groups. In this kind of evaluation, however, IBV occupies a position different to the one that it would take based on most protein sequence comparisons. Based on similarities within coding sequences and antigenic properties IBV occupies a place outside of both groups. Based on the dinucleotide frequencies IBV gained a position in between of the TGEV-related and the MHV-clustered coronaviruses.

Animals↗

PubMed1996

Hydroxyl and water molecule orientations in trypsin: comparison to molecular dynamic structures.

A comparison is presented of experimentally observed hydroxyl and water hydrogens in trypsin determined from neutron density maps with the results of a 140ps molecular dynamics (MD) simulation. Experimental determination of hydrogen and deuterium atom positions in molecules as large as proteins is a unique capability of neutron diffraction. The comparison addresses the degree to which a standard force-field approach can adequately describe the local electrostatic and van der Waals forces that determine the orientations of these hydrogens. The molecular dynamics simulation, based on the all-atom AMBER force-field, allowed free rotation of all hydroxyl groups and movement of water molecules making up a bath surrounding the protein. The neutron densities, derived from 2.1A D2O-H2O difference Fourier maps, provide a database of 27 well-ordered hydroxyl hydrogens. Virtually all of the simulated hydroxyl orientations are within a standard deviation of the experimentally-observed positions, including several examples in which both the simulation and the neutron density indicate that a hydroxyl group is shifted from a 'standard' rotamer. For the most highly ordered water molecules, the hydrogen distributions calculated from the trajectory were in good agreement with neutron density; simulated water molecules that displayed multiple hydrogen bonding networks had correspondingly broadened neutron density profiles. This comparison was facilitated by development of a method to construct a pseudo 2A density map based on the hydrogen atom distributions from the simulation. The degree of internal water molecules is shown to result primarily from the electrostatic environment surrounding that water molecule as opposed to the cavity size available to the molecule. A method is presented for comparing the discrete observations sampled in a dynamics trajectory with the time-averaged data obtained from X-ray or neutron diffraction studies. This method is particularly useful for statically-disordered water molecules, in which the average location assigned from a trajectory may represent a site of relatively low occupancy.

Crystallography↗

PubMed1991

[Early stomach cancer in comparison with advanced stomach cancer. Results of a prospective study of diagnosis and 5-year survival of 131 patients with early stomach cancer and 795 patients with advanced stomach cancer].

A multicentre prospective observational study with 22 surgical and 14 pathological units in West-Germany gathered data from 1420 patients with gastric cancer between April 1982 and October 1989, 131 patients with early gastric cancer (EGC) and 795 patients with a resectable advanced gastric cancer (AGC) were selected for comparison. Patients with EGC were younger than those with AGC (49% vs 37% younger than 60 years) and symptoms of an ulcer were found twice in comparison to AGC (40.7% vs 23.7%). EGC in comparison to AGC were frequently multifocal (9.2% vs 3.0%) and located in the middle and lower part of the stomach (83.9% vs 56.8%). Five-year-survival rates of mucosa carcinoma was 84% and of submucosal carcinoma 69% (p = 0.0741). WHO-typing of EGC and AGC were identical. But according to Laurén's classification there were more intestinal types with EGC than with AGC (60.3% vs 51.5%) and less diffuse or mixed types with EGC than with AGC (33.6% vs 44.0%). Five-year-survival rates of diffuse and intestinal types of EGC showed no significant difference (p = 0.19). Extended lymph node dissection was done in only one third of EGC and AGC. Five-year-survival rates of 36 EGC patients with and 95 EGC patients without extended lymph node dissection were 85% versus 72% (p = 0.0916). These results are a hint that systematic lymphadenectomy may have a beneficial effect on survival.

Adult↗

PubMed1990

Comparisons of genetic variability detected among mouse blood proteins using one- and two-dimensional electrophoreses.

Comparisons of the sensitivities of one-dimensional (1D) and two-dimensional (2D) electrophoreses to detect genetic variability have generally shown that the 2D approach appears to be two- to five-fold less sensitive than conventional 1D approaches. Concerns about the validity of this conclusion have arisen because such comparisons have involved mainly enzymic proteins in 1D approaches versus a complex mixture of soluble proteins in most 2D analyses. Comparisons involving the absolute number of variants detected, using 1D and 2D sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), denatured mouse blood proteins isolated from C3HeB/FeJ and C57B1/6J inbred strains of mice, and highly sensitive silver staining, indicate that the latter uncovers at least as much variability as the former. Although the relative percentage of variable bands (1D SDS-PAGE) was greater than the relative percentage of variable spots (2D SDS-PAGE) when proteins of intact erythrocytes were surveyed, both techniques uncovered approximately equal percentages of variable proteins when the mouse erythrocyte proteins were partitioned into membrane and lysate components. Therefore, the simpler 1D SDS-PAGE was found to be as effective as 2D SDS-PAGE in detecting protein variability. Since 1D SDS-PAGE separates proteins primarily on the basis of molecular weight and to a lesser degree on other primary protein sequence alterations, much of the variability observed by 2D SDS-PAGE may be due to these same features and unit charge differences may not play a significant role in detecting variability in the proteins studied. This differs from enzymic proteins, where such charge differences appear to be responsible for much of the variability. This study also indicated that decreasing the number of proteins in samples (membranes and lysates vs whole erythrocytes) increased the ability of both of these techniques to resolve differences. Mating studies indicated that most of the differences detected with both techniques were inherited and were not artifacts.

Animals↗