[Mismatch repair (MMR) efficiency and MSH2 gene mutation in human colorectal carcinoma cell line COLO320HSR].

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLHL. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT 116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.

[Dystrophin gene expression in patients with Duchenne muscular dystrophy after myoblast transplantation]. / Ekspressiia gena distrofina u bol'nykh miodistrofiei Diushenna posle transplantatsii mioblastov.

Based on originally designed technique of myoblast cultivation and in accordance with the approved by the Russian Ministry of Health "one muscle treatment" protocol of myoblast transplantation to the Duchenne muscular dystrophy patients, the first in Russia clinical trial of this gene correction method was carried out. Immonologically related myoblast cultures (30 to 90 million cells per patient) were injected after all preliminary procedures into tibialis anterior muscles of four boys selected from a group of volunteer recipients (Duchenne muscular dystrophy patients) based on the analysis of a number of surface antigens in donor-recipient pairs. The condition of the patients remained satisfactory during the whole period of post-transplantation follow-up (from 6 months to 1.5 years). Six months after myoblast transplantation the presence of donor DNA or dystrophin synthesis was demonstrated in muscle biopsies of three out of four patients. This result confirms efficacy and safety of the procedure used.

[Human myoblast culture as muscle stem cells in medical and biological studies]. / Kul'tiviruemye mioblasty cheloveka kak stvolovye kletki myshechnoi tkani v medikobiologicheskikh issledovaniiakh

The method for obtaining human myoblast culture has been modified to consider the specific histological localization of the satellite cells as well as their growth properties; the cultivation conditions have been selected to grow up to 150000 cells/cm2. At high densities, the cells remain mononuclear and preserve their typical myoblast morphology as well as the capacity for fusion and the formation of myotubes. By contrast to fibroblasts, up to 80% of the cells in the myoblast culture were positive in the acid phosphatase test, which indicates their stem nature. The obtained myoblast cultures were used in the clinical tests of cell-mediated gene therapy of Duchenne's muscular dystrophy as well as in the bioassay for the effects of biologically active compounds.

The band areas of proteins determined by fluorescent scanning in the commercial automated gel electrophoresis apparatus.

An automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)-proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self-quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near-constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.

Interpretation of electrophoretic band shapes by a partition chromatographic model.

Measurements of the shape of electrophoretic bands of phycoerythrin and conalbumin have been made at regular intervals during migration in agarose gels. Analysis of the peak shapes suggests the existence of a significant degree of asymmetry. This is to be contrasted with the symmetry around the peak associated with the generally assumed Gaussian band. The degree of asymmetry of the bands decreased as a function of time and increased with agarose concentration. A similar experiment on DNA indicated constancy of the degree of asymmetry as a function of time. These results can be interpreted as, but do not prove the validity of, a nonlocal diffusion equation which generalizes a theory originally put forth by Giddings and Eyring (J. Am. Chem. Soc. 1955, 59, 416-420). The results may be significant in framing a measure of the resolvability of electrophoretic peaks.

Separation and isolation of subcellular-sized particles by electrophoresis in polymer solution using the commercial scanning apparatus.

Electrophoresis of fluorescently labeled rat liver microsomes and polystyrene carboxylates of 10 and 30 nm radius was conducted in buffered 10-15% polyvinylpyrrolidone (M(r) = 10(6) solutions, using a horizontal gel electrophoresis apparatus with intermittent scanning of fluorescence (HPGE-1000, LabIntelligence). Banding, constant migration rates and Ferguson plots were obtained in these polymer solutions. The major microsome band detected by the automated scan was located visually on the gel by means of its fluorescein label and was isolated by volumetric withdrawal, recovery was monitored by scanning and ascertained to be near quantitative after three consecutive steps, in each of which 30 microL were withdrawn. This preparative method promises to be generally applicable to particles that are too large to enter into gels.

Reproducibility of mobility in gel electrophoresis.

The quantitative exploitation of gel electrophoresis to yield molecular and gel fiber properties rests on the assumption that mobility is characteristic of the macromolecule migrating as a band and is a physical constant for any system defined by pH, ionic strength and temperature. This assumption has not been tested intra-experimentally in previous literature. With the commercial introduction of automated gel electrophoresis apparatus, the collection of multiple mobility data during a single run without additional expense of labor has made it possible to test the assumption. As a start, we undertook that test for three proteins and their sodium dodecyl sulfate (SDS) derivatives, in agarose and polyacrylamide gel electrophoresis, various field strengths, continuous and discontinuous buffers, as well as intra- and interexperimentally. It was found that in agarose gel electrophoresis conducted in a single buffer, the standard deviation of mobility over a wide concentration range ranges intra-experimentally from 0.2 to 1.3% for two globular proteins and 1.4 to 5.3% for the same proteins derivatized with SDS. Interexperimentally, it was 3% in the single case tested to date. The standard deviation in polyacrylamide appears to be higher, varies in inverse relation to the mobility value, i.e. increases with gel concentration in the range of 11 to 19%T, and varies substantially between the two SDS-proteins investigated. Mobility in a discontinuous buffer system decreases continuously due to the decreasing leading phase/trailing phase ratio along the migration path. The decrease is sharpest in the "nonrestrictive" stacking gel.

Determination of optimally resolving gel concentration and migration time (path) in gel electrophoresis.

The notion of a mathematically defined optimally resolving gel concentration for components of a pair of molecular species of any given size was developed by Rodbard et al. (Electrophoresis and Isoelectric Focusing on Polyacrylamide Gel, pp. 28-62, de Gruyter, Berlin, 1974) 21 years ago. The mathematical treatment was incorporated into a computer program (T-OPT) for mainframe computers which upon input of the slope and intercept on the mobility axis of the Ferguson plots of the two components, electrophoresis time and temperature, yielded plots of resolution vs gel concentration. The same algorithms were later incorporated into the program ELPHOFIT for personal computers. Ideality of diffusion spreading and zero initial zone width were assumed along with a Gaussian peak distribution and an equal area for both components. Moreover, these programs failed to respond to the practical question of the migration time (or path) required for the resolution at the optimal gel concentration, although an independent program predicting the course of resolution under the assumption of free diffusion band spreading in gels (DAR-001) had been devised by Rodbard for application in preparative elution-PAGE. The present work advances the technology for predicting resolving conditions by presenting a computer program which allows the user (i) to predict the gel concentration which is optimal for obtaining the desired degree of resolution at any migration time, (ii) to prescribe the minimal degree of resolution between two band distributions one wishes to achieve, and (iii) to predict the migration time (or path) required at the optimal gel concentration for the resolution of the two components. The program is written in MATLAB and can be used by any computer that supports MATLAB language.

Commercial automated gel electrophoresis apparatus: application to DNA, band dispersion, nonlinear Ferguson curves, and isolation.

Recently available commercial automated gel electrophoresis apparatus with intermittent scanning of fluorescently labeled gel patterns (the HPGE-1000 apparatus of LabIntelligence, Menlo Park CA) was tested with regard to (i) its applicability to DNA in its native conformation, (ii) its ability to recognize the correct number of components, (iii) its capability to evaluate the width and shape of bands detected during electrophoresis, (iv) its ability to yield nonlinear Ferguson plots in a labor-saving fashion, and (v) its preparative potential. Ethidium homodimer (EtD) DNA (bp) ratios were systematically varied and the mobility of DNA fragments labeled at each ratio was measured in order to find a ratio which provided an unaltered mobility and presumably therefore an unaltered conformation of the fragment. That ratio was found to be 1/40 EtD/DNA (bp) or less. With such weak labeling of DNA, a representative fragment of 527 bp length requires a minimum load of 200 ng and a 2 micrograms load for a full-scale peak height. Using the baseline automatically selected by the software of the apparatus, the band areas of the 17 components of a DNA digest were consistently evaluated by the software, as evidenced by the proportionality between DNA length and area. The areas of the separated bands of DNA fragments of 1857 and 121 bp length were found to be constant with time of electrophoresis. The dispersion coefficient was found to decrease with agarose concentration in electrophoresis at 1 V/cm; however, at higher field strength, the band width of the 1857 bp fragment was surprisingly found to increase with gel concentration, presumably due to stretching.(ABSTRACT TRUNCATED AT 250 WORDS)

Recovery of SDS-protein and DNA using commercial automated gel electrophoresis apparatus.

The HPGE-1000 apparatus (LabIntelligence, Menlo Park, CA) is a gel electrophoresis instrument with intermittent fluorescence scanning of the migration path and with preparative capability. An electroelution cup sealed with gel is placed onto the band of interest, identified and located under computer control, and the band is electroeluted into the cup at a right angle to the orientation of the resolving gel. The correct location of the eluted band and the degree of its recovery into the elution cup are then verified on the gel pattern, visualized on the computer screen. Using that procedure, SDS-conalbumin-FLUOS was electrophoresed at 5 V/cm in a discontinuous tricinate-chloride-Tris system at loads of 0.25 to 20 micrograms, using 5% agarose (MetaPhor, FMC), 0.03% SDS gel at 5 degrees C. The horizontal gel was partitioned at the sample loading slit between a gel in Tris-tricinate (prepared at the concentrations of an operative phase ZETA) and in Tris-chloride (prepared as phase BETA). The elution cup was sealed with the latter gel and overlayered with buffer of the composition of the former. This arrangement should provide for electroelution of the band as a highly concentrated stack. At electroelution times of 2, 3.5, 4-5, 12, 15 and 15 min at 15 V/cm yields were 58, 60, 54-76, 99, 99 and 84% for loads of 0.25, 0.5, 1, 4, 10 and 20 micrograms, respectively. At the most sensitive scale of detection (13), a full-scale peak was obtained at a load of 1.7 micrograms when the fluorophore (FLUOS, Boehringer-Mannheim) to protein ratio was 10:1. Similarly, homogeneous nucleosomal DNA (146 bp), electrophoresed in 0.2 x TBE buffer at a load of 5 micrograms, was near-quantitatively recovered into the same buffer by electroelution at 15 V/cm for 2.5 or 6 min.

A computer program for predicting recovery of SDS-protein in the automated HPGE-1000 apparatus.

The commercial automated gel electrophoresis apparatus (HPGE-1000 of LabIntelligence, Menlo Park, CA) allows one to recover the material migrating and visualized as a fluorescent-labeled band by electrophoresis into a collection cup located above the band at a right angle to the orientation of the separation path. The degree of recovery is a function of sample load (peak area), electrophoresis time at constant field strength, the mobility of the material and band width. Neglecting the latter, recovery of several SDS-proteins was measured as a function of the first three parameters. These measurements were used as a data base for a computer program capable of predicting, by interpolation of the experimental values, the time of electrophoresis needed to obtain a specified degree of recovery, or the degree of recovery obtained after a desired time of electrophoresis into the collection cup.

Fluorescent labeling of DNA with ethidium homodimer without measurable decrease in DNA mobility: application to automated gel electrophoresis apparatus.

Mobilities of DNA, fluorescently labeled with ethidium homodimer (EtD), and normalized to the mobility of the 50-bp fragment [Rf(50)], are constant with time of electrophoresis, permitting one to conduct studies on DNA mobility in an automated electrophoresis apparatus with fluorescence detection. DNA mobility decreases with an increasing binding density of ethidium homodimer, as previously reported by others and expected since the dye decreases both the net charge and the conformational flexibility of the DNA. However, it was found that this effect of ethidium binding on electrophoretic mobility becomes undetectable at binding densities less than 1 EtD/40 bp. This implies that for the purposes of electrophoretic analysis in an apparatus with fluorescence detector, DNA with ethidium homodimer intercalated at dye-DNA ratios less than 1/40 bp behaves like unlabeled DNA and may be assumed to be in a conformation similar to that of the unliganded DNA. Necessarily, the low labeling ratio lowers the sensitivity of detection and, in particular, increases the load requirement for a full-scale band height required for the analysis of bandwidth and band shape during electrophoresis.

[Study of protein products of gene expression in cells with altered chromosome sets for genetic mapping]. / Izuchenie belkovykh produktov gennoi ékspressii v kletkakh s izmenennym naborom khromosom dlia geneticheskogo kartirovaniia.

Two-dimensional electrophoresis was used for analyzing proteins in hybrid cells that contained single human chromosomes (chromosome 5, chromosome 21, or chromosomes 5 and 21) against the background of the mouse genome. By comparing the protein patterns of hybrid and parent cells (about 1000 protein fractions for each kind of cell), five fractions among proteins of hybrid cells were supposedly identified as human proteins. The genes of two of them are probably located on chromosome 5, and those of other three, on chromosome 21. Moreover, analysis of proteins in fibroblasts of patients with the cri-du-chat syndrome (5p-) revealed a decrease in the content of two proteins, as compared with those in preparations of diploid fibroblasts. This fact was regarded as evidence that two corresponding genes are located on the short arm of chromosome 5. Methodological problems associated with the use of protein pattern analysis in cells with altered chromosome sets for the purposes of genetic mapping are discussed.

Detection of a single base mismatch in double-stranded DNA by electrophoresis on uncrosslinked polyacrylamide gel.

Uncrosslinked polyacrylamide forms gels in the concentration range of 15-40% acrylamide. Electrophoresis in these gels of a commercially available 350 bp heteroduplex DNA preparation separates it from the homoduplex DNA of the same size. The separation is qualitatively equivalent to that previously achieved in a commercial proprietary gel ("Mutation Detection Gel" of AT-Biochem), or in an equivalent 14% T, 0.15% C (N,N'-methylenebisacrylamide) gel, but the mechanical stability of mutation detection electrophoresis (MDE) gels or 0.15% C gels is better than that of uncrosslinked polyacrylamide gels. The separation in any of these three gel media can be carried out in short gel tubes within a few hours of electrophoresis time. In both uncrosslinked polyacrylamide and MDE gel media, the Ferguson plots [log(mobility) vs. gel concentration] and the plots of effective molecular radius vs. gel concentration ("T-plots") of both the heteroduplex and homoduplex DNA indicate an augmented size but similar flexibility upon passage through the gel than exhibited by the components of a DNA standard ladder. Homoduplex and heteroduplex DNA correspondingly exhibit a parallelism of their Ferguson curves in transverse MDE pore gradient gel electrophoresis, suggesting a surface net charge difference, possibly due to a conformational reorientation too subtle to be detected by a shift in the slope of the Ferguson plot, as has been observed once previously with a "kinked" DNA species. The gel fiber radius or length per unit volume of uncrosslinked polyacrylamide and MDE gels do not differ significantly within confidence limits, which are wide compared to unconventionally crosslinked gels, presumably because of their greater swelling.

The relative separation efficiencies of highly concentrated, uncrosslinked or low-crosslinked polyacrylamide gels compared to conventional gels of moderate concentration and crosslinking.

The joint report [1] has shown that the separation of heteroduplex DNA from homoduplex DNA can be achieved by uncrosslinked polyacrylamide gels or gels of a very low degree of crosslinking (0.15%) with N,N'-methylenebisacrylamide (Bis), while conventional polyacrylamide gels of 2-5% crosslinking with Bis are incapable of such a separation in the absence of added denaturing agents. This result raised the question whether in application to other separation problems the same superiority of uncrosslinked or low-crosslinked polyacrylamide existed. To test that question, Ferguson plots were determined for the members of a DNA ladder (50 to 1000 bp) in polyacrylamide with 0, 0.1, 0.2, 0.3, 0.5% C (Bis), and the separation efficiency function, S, was evaluated in comparison with that in conventional 2-5% C (Bis) gels. S was found to be lower, not higher, in gels of low crosslinking at the respective maximally effective gel concentrations. However, the range of gel concentrations in which gels of low or no crosslinking were effective extended over a range of at least 10% T, while conventionally crosslinked gels were most effective over a range of 3 to 1 units of %T.

[Erythrocyte membrane proteins in patients with hereditary spherocytosis]. / Belki membran éritrotsitov u bol'nykh nasledstvennym sferotsitozom.

Red blood cell membrane proteins were studied in a group of patients with hereditary spherocytosis, in comparison with normal donors, to reveal anomalous proteins associated with this disease. For this purpose red blood cells of the patients and normal donors were fractionated, by the age, in Ficoll-400 gradient, as a result red blood cell membranes were obtained with proteins that were investigated by the method of two-dimensional electrophoresis. In comparison of two-dimensional electrophoregrams of red blood cell membrane proteins of normal donors and those of microspherocytosis patients it was found that the latters had additional peptides in the area of glyceraldehyde-3-phosphate hydrogenase and pyruvate kinase. The changes detected in the red blood cell membrane protein composition might be caused by age shifts in the red blood cell population or by the disease type.

[Comparative study of human erythrocyte membranes in normal people and in Huntington's chorea patients]. / Sravnitel'noe izuchenie membran éritrotsitov krovi chekoveka v norme ibol'nykh khoreei gentingtona.

Erythrocytes of healthy volunteers and of patients with hereditary chorea were studied. Evaluation of the state of cellular membrane was carried out by measuring osmotic resistance, activity of Na+, K(+)-ATPase and protein composition. In the patients osmotic resistance of erythrocytes was distinctly decreased down to 66.3 +/- 3.3, while the Na+, K(+)-ATPase activity was increased 4-fold as compared with controls. Protein composition of erythrocyte membranes, studied by means of two-dimensional electrophoresis, was similar both in healthy persons and in patients with hereditary chorea when the electrophoretograms were analyzed visually. An additional protein with Mr = 30,000 and r-1-0.25 was detected in one of the patients. Slowly sedimenting fraction of erythrocytes was found in almost all the patients with hereditary chorea when erythrocytes aging was studied by means of fractionation in Ficoll density gradient. The fraction was not observed in healthy persons. These data suggest that the cell membranes in Huntington's chorea are altered as compared with normal state.

Endogenous proteolysis of the human erythrocyte membrane as studied by two-dimensional gel electrophoresis and electron spin resonance.

1. Endogenous proteolysis in human erythrocyte membranes was studied in human erythrocyte membranes incubated at 37 degrees C by monitoring changes in 2-D electrophoretic pattern of membrane polypeptides and in the spectra of maleimide-spin labeled membranes. 2. A strong effect of exogenous proteases derived from contaminating other blood elements was found, resulting in formation of specific spots on 2-D electropherograms, requiring very careful leukocyte removal in investigations of red cell membrane protein composition and proteolysis. 3. Studies of the effects of protease inhibitors and Ca2+ confirmed a complex pattern of endogenous red cell membrane proteolysis ("self-digestion") involving many substrates and enzymes. 4. A promoting effect of high concentrations (150 mM) of Ca2+ on endogenous red cell membrane proteolysis was found.

[Characteristics of endogenous proteolysis in preparations of human erythrocyte membranes]. / Kharakteristika éndogennogo proteoliza v preparatakh membran éritrotsitov cheloveka.

Endoproteolytic activity in human erythrocyte membrane preparations has been examined at 37 degrees C by one- and two-dimensional electrophoresis. Two-dimensional mapping has shown that the presence of leukocyte enzymes in erythrocytes prepared in a regular manner (centrifugation) cannot be excluded. Sedimentation in the 1.5% dextran 500,000 with the following erythrocyte purification on HBS-cellulose has made it possible to prepare erythrocyte membranes characterized by low level endoproteolytic activity without leukocyte enzymes. The marker peptide has been found. It is likely to be a specific product of the enzyme activity of membrane localization.

[Two-dimensional map of membrane proteins from human erythrocytes]. / Dvumernaia karta membrannykh belkov éritrotsitov cheloveka.

Human erythrocyte membrane proteins were analyzed by a modified two-dimensional electrophoresis performed according to O'Farrell. This method was used to construct a two-dimensional map of human erythrocyte membrane proteins. The map plotted in the coordinates "relative molecular mass versus relative electrophoretic mobility during IEF" was used for the characterization of 189 proteins. The position of major membrane proteins in the map was determined on the basis of their Mr, pI as well as literature data. Carboanhydrase was positioned by coelectrophoresis. A comparative analysis of erythrocyte membrane and cytosol preparations by two-dimensional protein mapping revealed that some of erythrocyte proteins have dual localization.