Biomedical subjects
L Böhm
Publications and source records attributed to L Böhm.
Irradiation damage in chromatin isolated from V-79 Chinese hamster lung fibroblasts.
The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl2. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure.
Functional effects of CRP binding to nuclei.
Binding of human CRP and rat CRP to their respective autologous liver nuclei has been demonstrated. The binding was shown to consist of both a calcium- and non-calcium-dependent component. The co-precipitation of human CRP with histones in physiological calcium concentrations suggests that the calcium-independent binding to chromatin is mediated by the CRP-polycation site. Complement-dependent chromatin solubilization by human CRP bound to nuclei was confirmed and show that the solubilized material is intact chromatin with a large proportion of small monosome structures of 180 base pair repeat. The binding of human and rat CRP to nuclei enhanced micrococcal nuclease digestion, suggesting that CRP binding in both these species alters chromatin structure by increasing linker DNA exposure. The suppressed transcription of DNA, after saturating CRP binding to nuclei, could limit aberrant transcription of damaged chromatin.
Structural properties of the methionine-containing subfraction of rat liver histone H1.
Cyanogen bromide (CNBr) cleavage of total rat liver histone H1 generates a C-terminal peptide which originates from a methionine-containing subfraction. This subfraction comprises approx. 20% of the whole rat liver H1 population, resembles calf thymus CTL-1 in size but contains methionine and histidine, higher proportions of serine and less alanine and proline. Edman degradation established the N-terminal sequence of the CNBr peptide as Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val-Glu. By alignment with calf thymus CTL-1, methionine was identified as residue 30 replacing alanine in a non-conservative replacement. Residue 40 is deleted but sequence homology near the double proline sequence in the G-domain is retained. The CNBr peptide is estimated at 177-181 residues and comprises the complete G- and C-domain and two arginines from the basic cluster in the N-domain. Removal from H1 of all but two residues of the N-domain does not abolish secondary and tertiary folding. This GC-peptide opens new approaches to the study of the function of H1 in chromatin.
Sequence conservation in the N-terminal domain of histone H1.
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Structural studies of chromatin by using proteases.
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Proteases as structural probes for chromatin: the domain structure of histones.
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Effect of irradiation and endogenous nucleases on rat liver chromatin.
These assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease, the levels of monosomes and oligosomes are identical to suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease. Histones isolated from monosomes of irradiated and unirradiated nuclei were intact, showing no fragmentation or loss of residues, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis.
Precise elimination of the N-terminal domain of histone H1.
The proteinase from mouse submaxillary gland was used to cleave total calf thymus histone H1 between residues 32 and 33. The C-terminal peptide, comprising residues 33 to the C-terminus, was purified and identified by amino acids analysis and Edman degradation. Spectroscopic characterization by n.m.r. for tertiary structure and by c.d. for secondary structure shows the globular domain of the parent histone H1 to be preserved intact in the peptide. It has therefore lost only the N-terminal domain and is a fragment of histone H1 comprising the globular plus C-terminal domains only. Precise elimination of only the N-terminal domain makes the fragment suitable for testing domain function in histone H1.
Proteolytic digestion studies of chromatin core-histone structure. Identification of limit peptides from histone H2B.
Trypsin digestion of chicken erythrocyte chromatin results in a well-defined set of limit peptides derived from the core histones (P1-P5). Two peptides running in sodium dodecyl sulphate gel electrophoresis at the positions of P2 and P3 have been identified as histone H2B residues 21-125 and 24-125. No C-terminal residues are therefore lost from histone H2B. The N-terminal sequence removed correlates well with that having low sequence conservation and not with that showing the greatest basicity. The same correlation was reported previously by us for histone H2A.
Studies on the ultrastructure of blood cells and the microaggregate fraction in stored human blood.
Electron microscopy of citrate-phosphate-dextrose (CPD) buffered bank blood performed over 21 days shows that the normal architecture of erythrocytes, platelets and leucocytes disappears and that deformed organelles, leucocyte "ghosts", cell fragments and microaggregates accumulate. Leucocytes and platelets emerge as the most sensitive indicators of blood deterioration showing profound morphological changes from the first day. Microaggregates which passed through a 20-micrometer high capacity transfusion filter were identified as platelet conglomerates, leucocyte "ghost" and platelet-leucocyte "ghost" coaggregates with diameters of 6--20, 20--30 and 30--40 micrometers respectively. Of these aggregates present in the filtrate, 62% fell into the size range of 20--40 micrometers. The composition of microaggregates varies with storage time, the platelet conglomerates appear first during or after Day 1, leucocyte "ghosts" after Day 5 followed by platelet-leucocyte "ghost" coaggregate formation. At this stage the number of intact leucocytes and platelets is reduced and the filtrate shows an abundance of leucocyte debris. Microfiltration would thus appear to reduce but not eliminate the danger of microembolism and damage to capillary endothelium.
Proteolytic digestion studies of chromatin core-histone structure. Identification of the limit peptides of histones H3 and H4.
Trypsin digestion of chromatin results in a well-defined set of limit peptides (P1--P5) derived from the four core histones. Those from histones H3 and H4 have been identified. P1 is H3 residues 27--129; P4 is H4 residues 18--102 and P5 is H4 residues 20--102. The N-terminal sequences removed correlate well with the regions that undergo post-synthetic acetylation and which show the greatest degree of sequence conservation. Autolytic digestion of chromatin releases a peptide (P1') from H3 representing residues 21--135. The implications of protease digestion for the higher order structure of chromatin are discussed.
Proteolytic digestion studies of chromatin core-histone structure. Identification of a limit peptide of histone H2A.
Tryptic digestion of chicken erythrocyte nuclei, to a level at which no intact histone remained, resulted in a set of resistant peptides. These were partially separated by exclusion chromatography. One of the peptides was shown to represent the central sequence 12--118 of histone H2A. This was established by amino acid analysis and by Edman degradations. Comparison of the sequence of histone H2A from a wide range of cell types shows that the tryptic cleavage points correspond closely to the limits of the highly conserved central sequence and not to the limits of the strongly basic regions. It is proposed that the 11 N-terminal and 10 C-terminal residues cleaved by trypsin are exposed in chromatin and play a structural and functional role different from the central 107 residues. The exposed position of the 118--119 bond accords with the known linkage point of ubiquitin to residue 119 of histone H2A in the semi-histone A24.
The composition of stigmatic exudate and the ultrastructure of the stigma papillae in Aptenia cordifolia.
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Sites of histone/histone interaction in the H3 - H4 complex.
Sites of interaction between histones H3 and h4 have been probed by investigating complex formation, firstly between histone H4 and three peptides cleaved by chemical means from histone H3 (residues 1-90 and 1-120 using cyanogen bromide and residues 42-135 using N-bromosuccinimide), secondly between histone H3 and two peptides cleaved from histone H4 (residues 1 - 84 using cyanogen bromide and residues 38-102 using chymotrypsin) and thirdly between the H4 peptide (residues 38-102) and the three H3 peptides (residues 1-90, 1-120 and 42-135). The criterion for complex formation is the appearance of characteristic perturbed resonances in the aromatic region of the 270 - MHZ proton resonance spectrum of the peptide mixture. It is concluded that loss of 37 N-terminal residues from histone H4 and 41 N-terminal residues from histone H3 does not prevent complex formation, whilst the loss of 18 C-terminal residues from H4 and 45 C-terminal residues from H3 does prevent it; that last 15 C-terminal residues of H3 are, however, not required for forming a complex. The regions important for complex formation are therefore defined as residues 42-120 in histone H3 and residues 38-102 in histone H4.
Proteolytic degradation of histones and site of cleavage in histone F2al and F3.
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Flow cytometric evaluation of apoptosis and cell viability as a criterion of anti-tumour drug toxicity.
BACKGROUND: Determination of the drug concentration required to kill 50% of the tumour cells (EC50) does not take into account the propensity of cells to undergo apoptosis and necrosis. These 2 parameters and the viable cells are here assessed by a flow cytometric (FC) approach using propidium iodide (PI) and FITC-Annexin V staining. MATERIALS AND METHODS: A number of carcinoma cell lines of defined p53 status were exposed to cis-PtII for 24 hours, stained with PI and FITC-Annexin V and analyzed by FC. Unstained viable cells, early apoptotic cells and necrotic cells were scored separately in dual parameter plots of green fluorescence (FITC) against red fluorescence (PI) to generate dose response curves. RESULTS: EC50 values for cell viability were found to be 1-4 times higher than survival data from colony assays resembling data obtained by MTT or Crystal Violet vital dye staining. Percentage apoptosis measured by Annexin V binding was in agreement with microscopic scoring of apoptotic cells after Acridine Orange staining. CONCLUSION: The FC assay described gives a good estimate of cell viability resembling data from vital dye staining assays and provides additional information on apoptosis and necrosis. FC data from Annexin V binding and microscopic scoring after Acridine Orange staining were in excellent agreement.