Decrease in the phosphorylation of the proteins associated with heterogeneous nuclear RNA after the application of 3'-methyl-4-dimethylaminoazobenzene

The effect of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) on the phosphorylation of the proteins of the nuclear ribonucleoprotein (RNP) particles was studied in liver of rats. Forty eight hours after the application of 4 mg of the hepatocarcinogen per 100 g of body wt. by stomach intubation the particle proteins contained only 7% as much phosphate per mg of protein as the proteins of the same particles isolated from liver of control animals. Determination of the protein kinase and protein phosphatase activities in the total fraction of the non-histone nuclear proteins 48 h after the application of the carcinogen have shown an increase (200% and 159%, respectively) in both enzymatic activities. These results suggest that the hepatocarcinogen could induce the observed high turnover of the phosphates on the proteins of the liver nuclear ribonucleoprotein particles and the resulting dephosphorylation of these particles by stimulation of nuclear protein kinases and phosphatases. Qualitatively the same, but quantitatively much smaller changes were also observed 48 h after the application of the non-carcinogenic p-aminoazobenzene (AB) by stomach intubation and in regenerating liver. After the application of AB phosphorylation of the proteins of rat liver nuclear ribonucleoprotein particles decreased to 70% and in regenerating liver to 61% of the phosphorylation of particle proteins in control liver. Since it is assumed that nuclear RNP particles are involved in the processing and transport of newly synthesized premessenger RNA it is possible that the drastic dephosphorylation of the particle proteins induced by the carcinogen could be connected with the distortion of RNA processing which is observed in liver of animals treated with hepatocarcinogens.     

Changes in liver nuclear protein metabolism after a single dose of urethane to suckling mice

Treatment of 8-9-day-old C57BL/A mice with a single carcinogenic dose of urethane, at 1.2 mg/g body wt., resulted in an immediate decrease in liver DNA synthesis reaching a maximum at about 16-18 h after injection, the rate of synthesis returning to normal after 48 h. When the nuclear proteins were radiolabelled, the non-histone protein (NHP) fraction showed a significant decrease in specific activity 8-18 h after injection of urethane and slight increase in specific activity after 24 h. Histone and residual proteins did not show any significant change. The liver NHP were analysed by isoelectric focusing (IEF) and sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gels. The latter technique failed to show any distinctive differences but IEF results indicated some quantitative and qualitative changes in protein content and synthesis were induced by the urethane treatment. The most noticeable change in the stained gels was an increase in a protein component having a pI of 7.35 and the appearance of new bands at pI's of 7.85 and 5.55 in the 18 h treated livers. However, the [3H]tryptophan labelling pattern indicated that this was not due to an increased synthesis of these components. 24 h after urethane there appeared to be an increased rate of synthesis of some of the major components of the mixture, particularly at the pI 5.65 region. Histone and residual protein fractions were also analysed by electrophoresis and showed no difference between treated and control livers.     

Changes in protein synthesis during maturation of sheep oocytes in vivo and in vitro.

The qualitative profiles of the proteins synthesized by sheep oocytes at various stages of maturation were determined by electrophoretic separation in one dimension on polyacrylamide SDS gels. No change in protein synthetic pattern was observed in ooce changes had taken place in at least 12 separate protein bands. Marked alterations in the synthesis of some proteins were apparent 15 h after LH; formation of proteins in 5 of the original bands was either reduced or not detectable, while new synthesis was evident from the appearance of 7 additional bands. The pattern of proteins produced by oocytes cultured within the follicle corresponded closely with that observed in vivo: changes in synthesis were initiated about 9 h after addition of gonadotrophin and were completed by 15 h. Oocytes cultured outside the follicle in a gonadotrophin-containing medium did not exhibit a change in protein synthesis and at 15 h only those proteins produced during the early stages of maturation were being synthesized.     

Circadian rhythms in the phosphorylation of rat liver histones and similar basic proteins

In the rat liver, the phosphorylation of histones is subject to a circadian rhythm. Most classes of histones, which had been obtained by polyacrylamide gel electrophoresis of nuclear HCl-extracts, exhibited maximum phosphorylation at 21.00 h and at 08.00 h. This is in correlation to maxima of RNA synthesis (23.00 h) and protein synthesis (24.00 h and 12.00 h), reported in the literature. A series of basic proteins, not as yet described, could be separated from the histones. These proteins exhibit a high extent of phosphorylation and a rhythmicity analogous to that of the histones. It is suggested that these proteins are of importance in the expression of genetic information.     

Expression of nuclear matrix proteins in rat liver tissue

We have studied the synthesis of nuclear matrix proteins as it occurs in the rat liver. To investigate their kinetics in tissue, nuclear matrix proteins were prepared from liver of rats injected with radioactive methionine. Synthesis of lamins was not observed in quiescent hepatocytes although they were the principal proteins of this subcellular fraction, suggesting that lamins are very stable in the liver. When hepatocytes were stimulated to divide by partial hepatectomy, only synthesis of lamin B was initiated. Many proteins not visible on Coomassie blue-stained gels were detectable by autoradiography. In the nuclear matrix extracts of quiescent hepatocytes, one of the most prominently labeled ones was a protein of 70 kDa. After hepatectomy, an additional protein of 62 kDa was detectable. These proteins were visible 1 h after the injection of radioactivity, but were no longer observed in nuclear matrices prepared 24 h after injection. These experiments indicate that in addition to lamins, two nuclear matrix proteins are present in the rat liver that were not detected previously, perhaps because of their rapid turnover.     

Diurnal variation of cytosolic fatty acid-binding protein content and of palmitate oxidation in rat liver and heart

Delipidated proteins from albumin-free liver and heart cytosol obtained from rats sacrificed at the mid-dark or the mid-light phase of the light cycle were assayed for their palmitate-binding capacity. In both tissues a marked variation of this binding capacity was observed from about 3-4 nmol/mg of protein in the mid-light phase of the cycle to about 7-8 nmol/mg of protein in the mid-dark phase. Sephadex G-75 chromatography of the cytosolic proteins revealed that the palmitate binding could in all cases almost entirely be attributed to proteins of Mr = 12,000-14,000, suggesting that the observed diurnal variations are related to differences in the content of fatty acid-binding protein (FABP). In both rat liver and heart FABP represents about 4 (mid-light) to 8% (mid-dark) of the total soluble proteins. Cholestyramine feeding increased the FABP content of liver cytosol from rats sacrificed at the mid-light phase, but not in those sacrificed at the mid-dark phase, in such a way that the diurnal variation of the FABP content virtually disappeared. The palmitate oxidation capacity and citrate synthase activity also exhibited a concomitant diurnal periodicity in rat liver and, to a lesser extent, in rat heart. The results provide additional evidence for an important role of FABP in cellular fatty acid metabolism in both liver and heart and for the similarity of FABP with sterol carrier protein.     

Diurnal variations in endogenous RNA polymerase activity and amounts of nuclear non-histone protein, DNA and cytoplasmic protein in rat liver.

From rats fed ad libitum and kept under a 12 + 12 h light/dark regimen, the DNA dependent RNA polymerase activity of liver cell nuclei was determined avery four hours. From identical rats, nuclear non-histone protein and DNA, and cytoplasmic protein was determined by Feulgen-Naphthol Yellow S cytophotometry of isolated liver cells. The minimum: maximum ratio of the RNA polymerase activity is 0.77; the min:max ratio of nuclear non-histone protein is 0.84. These two parameters have identical time courses with a gradual decline during the light period and a sharp rise after the onset of the dark period. The variations in nuclear DNA content, estimated as the amount of Feulgen stain bound, closely parallel those of the RNA polymerase activity and nuclear non-histone protein content (min:max = 0.96). The amount of cytoplasmic protein per cell also varies throughout the day, but its time curve lags behind those of nuclear non-histone content and RNA polymerase activity. These results are consistent with the concept of nuclear non-histone proteins as de-repressors of the DNA template in differentiated, non-proliferating cells, and support the validity of using Feulgen-Naphthol Yellow S cytophotometry of nuclear non-histone proteins as an estimate of gene expression in such cells.     

Diurnal variations in endogenous RNA polymerase activity and amounts of nuclear non-histone protein,DNA and cytoplasmic protein in rat liver

Summary From rats fed ad libitum and kept under a 12+12 h light/dark regimen, the DNA dependent RNA polymerase activity of liver cell nuclei was determined every four hours. From identical rats, nuclear non-histone protein and DNA, and cytoplasmic protein was determined by Feulgen-Naphtol Yellow S cytophotometry of isolated liver cells. The minimum: maximum ratio of the RNA polymerase activity is 0.77; the min:max ratio of nuclear non-histone protein is 0.84. These two parameters have identical time courses with a gradual decline during the light period and a sharp rise after the onset of the dark period. The variations in nuclear DNA content, estimated as the amount of Feulgen stain bound, closely parallel those of the RNA polymerase activity and nuclear non-histone protein content (min:max=0.96). The amount of cytoplasmic protein per cell also varies throughout the day, but its time curve lags behind those of nuclear nonhistone content and RNA polymerase activity. These results are consistent with the concept of nuclear non-histone proteins as de-repressors of the DNA template in differentiated, non-proliferating cells, and support the validity of using Feulgen-Naphthol Yellow S cytophotometry of nuclear non-histone proteins as an estimate of gene expression in such cells.     

The relationship between DNA synthesis and the synthesis of nuclear proteins in rat brain during development

—The incorporation of [³H]thymidine into nuclear DNA of rat brain progressively increased from birth until the 8th postnatal day and it was lowest in the adult brain. When isolated nuclei from brain cells were separated into a neuronal- and a glial-rich fraction (composed of glial and neuroblast nuclei in young animals), the specific radioactivity of the DNA was higher in the glial-rich fraction at all ages investigated. The incorporation of [³H]leucine into proteins of rat brain was considerably higher in the 8-than in the 1-day-old rat. The greatest difference in the rate of protein synthesis between 8- and 1-day-old brain occurred in the nuclear proteins, especially those associated with DNA. There was an accumulation of protein and RNA in nuclei from brain cells from birth to the 8th postnatal day. The increased content of proteins occurred primarily in the fraction soluble in buffered saline (nuclear sap).     

Primary biliary cirrhosis sera recognize not only gp210 but also proteins of the p62 complex bearing N-acetylglucosamine residues from rat liver nuclear envelope

We have recently observed reactivity of primary biliary cirrhosis (PBC) sera with several proteins bearing N-acetylglucosamine residues from rat liver nuclear envelopes. The aim of this study was to characterize the reactive antigens. Sera from 31 patients with PBC, 30 with rheumatoid arthritis (RA) and 30 with Sjögren's syndrome (SS) were examined. Rim-like immunofluorescence staining was observed in 15 of 31 (48%) sera from patients with PBC, in 1 of 30 with RA and in 1 of 30 with SS. Upon immunoblotting using preparations of whole rat liver nuclear envelopes and their Triton X 100-KCl extract as antigen souces, a 200 kDa protein band was observed in 9 of sera with PBC. Furthermore, upon immunoblotting using the wheat germ aggulutinin-bound fraction of rat liver envelope as antigen, 62, 60 and 54 kDa protein bands corresponding to components of the p62 complex in the nuclear pore complex (Kita et al. Biochem. 113, 377–382) were observed in 7, 5 and 6 samples respectively, of the 31 PBC sera. Our data suggest that PBC sera recognize not only the 210 kDa protein but also the p62 complex proteins.Abbreviations ANA antinuclear antibody - AMA anti-mitochondrial antibodies - IF immunofluorescence - LAP2 lamina-associated polypeptide 2 - LBR lamin B receptor - anti-NBP 60 anti-nuclear localization signal binding protein 60 - NE nuclear envelope - NPC nuclear pore complex - PBC primary biliary cirrhosis - RA rheumatoid arthritis - SLE systemic lupus erythematosus - SS Sjögren's syndrome - WGA wheat germ agglutinin     

Cytophotometric study of nuclear proteins during gametogenesis in Ascaris lumbricoides.

Reduction in the number of nucleoli/nucleus and increase in their size were usually observed in rat liver after partial hepatectomy. These changes of nucleoli were greatest 16–18 h after the operation, when RNA biosynthesis in the nucleoli is reported to be highest. Approx. 50% of the nuclei had one enlarged nucleolus at this time but after the increase in nuclear DNA synthesis less than 15% of the nuclei had one nucleolus, as in normal liver. Before the next peak of nuclear DNA synthesis, nucleolar changes appeared again, though less conspicuously.The enlarged nucleoli of regenerating liver were separated from smaller ones by discontinuous sucrose gradient centrifugation and the contents of nucleic acid and ribosomal cistrons in different-sized nucleoli were measured. The large nucleoli in regenerating liver were found to have increased DNA content, whereas smaller ones had the normal content. The total number of ribosomal cistrons in the enlarged nucleoli from regenerating liver was also increased roughly in proportion to the DNA content. No significant difference was found between the percentages of ribosomal cistrons in whole nuclear DNAs from regenerating and normal liver. Small but reproducible [³H]TdR incorporation into nucleolar DNA was observed and this was similar in normal liver and regenerating liver 12 h after partial hepatectomy. Therefore, the nucleolar changes in regenerating liver were not accompanied by any particular DNA synthesis in the nucleolus itself. These results suggest that in the nuclei of regenerating liver nucleolar chromatins may be redistributed and assembled into large nucleoli, rather than that any amplification of ribosomal cistrons occurs.     

Differences in the binding of 3-methylcholanthrene and phenobarbital to rat liver cytosolic and nuclear protein fractions

The inducers of cytochrome P-450c and P-450b, 3-methylcholanthrene and phenobarbital, respectively, have been studied in their interaction with subcellular fractions from rat liver. 3-Methylcholanthrene bound to both nuclear and cytoplasmic components as demonstrated by DNA-cellulose chromatography. The binding of 3-methylcholanthrene to cytosolic proteins, on DNA-cellulose, was approximately 27 fmol/mg of applied protein, whereas the binding to nuclear proteins was 250–570 fmol/mg applied protein. Phenobarbital did not bind to proteins of rat serum, rat liver cytosol, or rat liver nuclei which could bind to DNA-cellulose. Further examination of the potential interaction of phenobarbital to rat liver cytosolic proteins was carried out using either DEAE A-50 Sephadex chromatography, charcoal dextran analysis, or sucrose density gradients. No binding of phenobarbital to rat liver cytosolic proteins was observed under these experimental conditions. In contrast, the binding of 3-methylcholanthrene to cytosolic proteins showed four peaks of radioactivity after DEAE A-50 Sephadex chromatography, two peaks by sucrose density gradient analysis, and specific binding (0.13 pmol/mg protein) was observed using the charcoal dextran technique. One of the peaks on sucrose gradients was labile in the presence of salt. The uptake and intranuclear distribution of 3-methylcholanthrene and phenobarbital were markedly different after incubation with whole nuclei: 64% of the available 3-methylcholanthrene but only 3% of the available phenobarbital radioactivity became associated with nuclei. Of this radioactivity, the highest specific activity of the 3-methylcholanthrene radioactivity was associated with the 2 m KCl-resistant nuclear pellet and the highest specific activity of the phenobarbital radioactivity was associated with the nuclear fraction soluble in the absence of salt. These results are interpreted in regard to the induction of cytochrome P-450c.     

Preparation in undenatured form of the main protein bound to heterogeneous nuclear RNA in liver and hepatoma cells

Particles carrying heterogeneous nuclear RNA (30 S-particles) were prepared from rat liver and Zajdela hepatoma ascites cell nuclei after ultrasonic disruption. The ribonucleoprotein structures were disintegrated in the presence of 100 mM spermidine. Using chromatography on Sepharose-polyadenylate a protein component has been obtained which possessed high affinity for heterogeneous nuclear RNA, polyuridylate and polyadenylate, and double-stranded DNA. This protein was the main species of the ribonucleoprotein studied; it showed bands with molcular weights of 37000 and 40000 respectively in SDS gel electrophoresis. The RNA-binding proteins isolated from liver and hepatoma had identical molecular weights and the same affinity for Sepharose-polyadenylate used in the isolation.Abbreviations hnRNA heterogeneous nuclear RNA - hnRNP ribonucleoprotein which contains hnRNA     

Enhancement of nuclear Ca2+-ATPase activity in regenerating rat liver: involvement of nuclear DNA increase

The alteration of calcium content, Ca²⁺-ATPase activity, DNA content and DNA fragmentation in the nuclei of regenerating rat liver was investigated. Liver was surgically removed about 70% of that of sham-operated rats. the reduced liver weight by partial hepatectomy was completely restored at 3 days after the surgery. Regenerating liver significantly increased Ca²⁺-ATPase activity and DNA content in the nuclei between 1 and 5 days after hepatectomy. The nuclear calcium content was clearly increased from 2 days after hepatectomy. The increase of Ca²⁺-ATPase activity in regenerating liver was clearly inhibited by the presence of trifluoperazine (10 M), staurosporine (2.5 M) and dibucaine (10 M), which are inhibitors of calmodulin and protein kinase, in the enzyme reaction mixture. However, the nuclear enzyme activity in normal rat liver was not significantly altered by these inhibitors. Meanwhile, the increase of nuclear DNA content in regenerating liver was completely blocked by the administration of trifluoperazine (2.5 mg/100 g body weight), suggesting an involvement of calmodulin. Now, the nuclear DNA fragmentation was significantly decreased in regenerating liver, suggesting that this decrease is partly contributed to the increase in nuclear DNA content. The present study clearly demonstrates that regenerating liver enhances nuclear Ca²⁺-ATPase activity and induces a corresponding elevation of nuclear calcium content. This Ca²⁺-signaling system may be involved in the regulation of nuclear DNA functions in regenerating rat liver.     

Induction of unscheduled DNA synthesis in liver and micronucleus in bone marrow of rats exposed in vivo to the benzidine-derived azo dye, Direct Black 38

The genotoxic activity of the benzidine-derived azo dye, Direct Black 38 (DB38), was studied in vivo, using two different genetic end-points: unscheduled DNA synthesis in liver (UDS) and bone marrow micronucleus (MN). Exposure times were 12, 24 or 36 h. Both assays were performed in the same rat, except for the 24-h exposure when only MN was investigated. For the liver UDS assay, the rat hepatocarcinogen, 6-dimethylaminophenylazobenzthiazole (6BT), was used as positive control and for the MN assay, cyclophosphamide (CP). In agreement with earlier results, 6BT gave rise to a dose-related increase in liver UDS after 12-h exposure to 25 or 50 mg/kg bw. After 36-h exposure, there was still an indication of a weak dose-response effect between 0 and 5 net nuclear grains (NG). DB38 induced liver UDS at the higher dose levels used (500 and 1000 mg/kg), and after both 12- and 36-h exposure. With the longer exposure time, a weak induction of UDS was also observed at 100 mg/kg. The strongest UDS induction (12.2 NG), was obtained in one rat after 36-h exposure to 500 mg/kg. DB38 also had a weak effect on the MN induction, which was statistically significant at the higher concentrations used. A dose-related response was observed at all exposure times used.     

Tissue-specific binding of nuclear factors to the 5′-flanking region of rat gene for calcium-binding protein regucalcin

The existence of nuclear factors which bind to the 5-flanking region of calcium-binding protein regucalcin gene in rats was investigated. We previously reported that rat regucalcin mRNA is expressed in a highly tissue-specific manner; the mRNA was mainly present in the liver but only slightly in the kidney. When the nuclear proteins extracted from the liver and kidney of rats were used in the gel mobility shift assays, a protein-DNA complex was uniquely formed with the DNA fragment containing the upstream region from the first exon of rat regucalcin gene. On the other hand, this complex was not found by using the nuclear extracts from rat brain, spleen, and heart. The nuclear proteins of these extracts, however, could specifically bind to the DNA fragment containing the first exon region of rat regucalcin gene, although Northern blot analysis did not show detectable amount of regucalcin mRNA levels in rat brain, spleen, and heart. The present study demonstrates that the existence of nuclear protein components which bind to the regucalcin gene. These identified components may be involved in the tissue-specific regulation of regucalcin gene expression.     

Interaction of dexamethasone-receptor complexes with rat liver nuclei and DNAs]

The role of DNAs in the nuclear binding of dexamethasone-receptor complexes (DRC) was studied. The cytosolic receptors from rat liver have a sedimentation coefficient of about 7S, the Stock's radius--of about 50 A and possess a high affinity to dexamethasone (Kas = 2,6 X 10(8) M-1). Their capacity is 3 X 10(-13) and 5.5--7.0 X 10(-12) mole of dexamethasone per mg cytosolic protein and mg DNA, respectively. DRC has the ability to bind to the nuclei of rat liver. DRC binding to nuclei is increased approximately 3-fold by temperature activation of cytosol. The nuclear acceptor sites are saturated at the level of 16.2 pmoles of bound DRC per mg nuclear DNA. Free DNA has the ability to compete with nuclei for binding with DRC. Temperature-activated DRC can bind both with homo- and heterologous DNAs. Secondary DRC-DNA complexes were isolated by means of gel filtration on Sepharose 4B. Thermal denaturation of DNA decreases its ability to bind DRC approximately 2-fold. DNAs of a similar nucleotide composition, i.e. DNA from rat liver (GC = 43 mole%) and DNA from Photobacterium belozerskii (GC = 44 mole%), have a close DRC-binding ability. At the same time, these DNAs bind about 1.5-fold less DRC, as compared to DNA from Pseudomonas aeruginosa (GC = 67 mole%) and about 1.5-fold more, than does DNA from T2 phage (GC = 35 mole%). Thus the positive correlation between the GC composition of DNA and its DRC-binding ability was established. Unique sequences (Cot greater than 600) bind several times less DRC than the reiterated sequences (also denaturated) (Cot = O--600) of rate liver DNA. Thus, DNA can be considered as a nuclear acceptor of DRC. It is assumed, that DRC is able to recognise in DNA certain short GC-rich sequences, distributed in the rate genome in a non-random fashion.     

Thymidine kinase and deoxyribonucleic acid metabolism in growing and regenerating livers from rats on controlled feeding schedules

Rats accustomed to eating during the first 8h of a daily 12h dark period re-established about 80% of intact liver weight, protein and DNA within 4 days following partial hepatectomy; further increases were not observed. Liver thymidine kinase activity and thymidine incorporation into liver DNA exhibited marked daily oscillations during liver regeneration. Maximum values were observed near the end of the dark period both in intact growing rats and in rats partially hepatectomized 2h before the end of the dark period. The time of day of surgery affected thymidine kinase activity and thymidine incorporation into DNA at specific times following partial hepatectomy. This seriously affects the interpretation of reports of experiments where the time of day of killing has been held constant and time of surgery varied. Highly significant correlation coefficients were observed for thymidine incorporation before killing versus thymidine kinase activity at time of killing and for thymidine versus orotic acid incorporation into DNA of livers from rats partially hepatectomized 2h before the end of the dark period and killed at 12h intervals. Thymidylate phosphatase activity returned to the normal amount at a rate similar to that for liver protein. Thymidylate phosphatase did not affect the validity of the thymidine kinase assay. The relationship of [(14)C]orotic acid to [(3)H]thymidine incorporation into liver DNA varied with the time of day, with the age of the rat and during the regeneration of the liver.     

Quantitative solubilization of nonhistone chromosomal proteins without denaturation using zwitterionic detergents

Of three kinds of commercial zwitterionic detergents [SB 12, SB 14, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)], SB 12 and Chaps were more useful than SB 14 because of high solubility and less interference with protein assay. Efficiency for protein solubilization at pH 6-9 was higher for SB 12 than for Chaps with either calf thymus chromatin or rat liver nuclei. At pH 9 and ionic strength (I) = 0.35, 1% SB 12 and 1% Chaps were capable of solubilizing about 70% and about 47% of total proteins in rat liver nuclei, respectively. Core histones in rat liver nuclei were extracted to a lesser extent with Chaps than with SB 12. DNA-dependent RNA polymerase and isopeptidase activities were barely inactivated by 1% Chaps at pH 8-9, but isopeptidase activity was inhibited by 0.3% SB 12. These facts indicate that whereas SB 12 is effective for solubilization of whole nuclear proteins, Chaps is suitable for the selective extraction of nonhistone chromosomal proteins without denaturation.     

Formation of DNA adducts in liver of rats treated with 2-aminofluorene. Differences between total and matrix attached DNA fractions

The level of adducts in total DNA and the nuclear matrix-attached DNA was compared at 12, 24, 48 and 72 h after a single injection of a hepatocarcinogen, 2-aminofluorene. In rat liver, the amount of adducts in total DNA increased gradually up to 72 h, reaching the value of about 54 per 10(7) nucleotides, whereas the maximal level of adducts in matrix DNA was observed 48 h after injection. Matrix and total DNA differed, also in the level of adducts in particular spots in four dimensional chromatography on PEI cellulose plates.