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P Loidl

Publications and source records attributed to P Loidl.

At least 55 records · Page 3Linked to original sources
PubMed1992

Enzymes involved in the dynamic equilibrium of core histone acetylation of Physarum polycephalum.

DEAE-Sepharose chromatography of extracts from plasmodia of the myxomycete Physarum polycephalum revealed the presence of multiple histone acetyltransferases and histone deacetylases. A cytoplasmic histone acetyltransferase B, specific for histone H4, and two nuclear acetyltransferases A1 and A2 were identified; A1 acetylates all core histones with a preference for H3 and H2A, whereas A2 is specific for H3 and also slightly for H2B. Two histone deacetylases, HD1 and HD2, could be discriminated. They differ with respect to substrate specificity and pH dependence. For the first time the substrate specificity of histone deacetylases was determined using HPLC-purified individual core histone species. The order of acetylated substrate preference is H2A much greater than H3 greater than or equal to H4 greater than H2B for HD1 and H3 greater than H2A greater than H4 for HD2, respectively; HD2 is inactive with H2B as substrate. Moreover histone deacetylases are very sensitive to butyrate, since 2 mM butyrate leads to more than 50% inhibition of enzyme activity.

Acetylation↗
PubMed1992

Investigation of nuclear c-MYC oncoprotein expression in human hematopoiesis: suitability of a rapid and reliable semiquantitative evaluation system.

The biologic functions mediated by the nuclear protooncogene, c-MYC are correlated to gene dosage. Since automated quantification programs are expensive, time-consuming and not easily available, and since analysis by flow cytometry is difficult in the case of nuclear antigens, we examined the suitability and reproducibility of a semiquantitative in situ evaluation system. This system was based on the percentage of nuclear area staining positively, and comprised the following categories: 0: negative, 1+: single scattered grains of the immunocytochemical staining product, 2+: confluence of grains to patches but less than 50% nuclear area positive, and 3+: greater than 50% positive nuclear area. In addition, sensitivity and specificity of two anti-c-MYC antibodies were investigated. Although both antibodies differed slightly in staining pattern and sensitivity, the four quantification categories were applicable for immunostainings of both antisera and highly reproducible when re-evaluated by the same observer (r = 0.98; p = 0.0001) or a second investigator (rAb155 = 0.98, rAb DCPm = 0.96; p = 0.0001), both reading blindly and independently. Comparing our semiquantitative evaluation categories and results of computer-assisted image analysis, the percentage of positive nuclear area (p less than 0.0001), the median staining intensity (p less than 0.0001), and the product of both (p less than 0.0001) differed significantly in the four evaluation categories. This result still held true after correction for nuclear size, which differed appreciably in various cell types (p less than 0.0001). The product of positive nuclear area, staining intensity and nuclear size (microns 2), which best approximates the absolute amount of c-MYC within a certain cell, was clearly different within the four staining categories (p less than 0.0001) and did not depend on cellular morphology within the staining categories 0 to 2. Also, the immunocytochemical technique proved highly reproducible (median day/day variance 0.65% (0-13); r = 0.995). The practicability of this system for semiquantification was demonstrated by (a) correlation of H score values of immunocytochemical stainings with densitometric scans of Western blots and (b) by the fact that peripheral blood lymphocytes, Phytohemagglutinin stimulated blasts, 13 cases of multiple myeloma and HL-60 cells differed concerning their estimated c-MYC amounts (p = 0.0125). This confirms on the effector molecule level results previously reported from mRNA in situ and Northern blotting analyses. We conclude that a simple and highly reproducible evaluation system can be used for in situ comparison of nuclear oncogene dosage.

Gene Expression↗
PubMed1991

Histone acetylation in Zea mays.I. Activities of histone acetyltransferases and histone deacetylases.

DEAE-Sepharose chromatography of extracts from Zea mays meristematic cells revealed multiple histone acetyltransferase and histone deacetylase enzyme forms. An improved method for nuclear isolation allowed us to discriminate nuclear and cytoplasmic enzymes. Two nuclear histone acetyltransferases, A1 and A2, a cytoplasmic B-enzyme and two nuclear histone deacetylases, HD1 and HD2, have been identified. The histone specificity of the different enzyme forms has been studied in an in vitro system, using chicken erythrocyte histones as substrate. The cytoplasmic histone acetyltransferase B is the predominant enzyme, which acetylates mainly histone H4 and to a lesser extent H2A. The nuclear histone acetyltransferase A1 preferentially acetylates H3 and also H4, whereas enzyme A2 is specific for H3. This substrate specificity was confirmed with homologous Z. mays histones. The two histone deacetylases differ from each other with respect to ionic strength dependence, inhibition by acetate and butyrate, and substrate specificity. The strong inhibitory effect of acetate on histone deacetylases was exploited to distinguish different histone acetyltransferase forms.

Acetylation↗
PubMed1991

Histone acetylation in Zea mays. II. Biological significance of post-translational histone acetylation during embryo germination.

Multiple forms of histone acetyltransferases and histone deacetylases, which have been separated and characterized in the accompanying manuscript (López-Rodas, G., Georgieva, E. I., Sendra, R., and Loidl, P. (1991) J. Biol. Chem. 266, 18745-18750), together with in vivo acetate incorporation, were studied during the germination of Zea mays embryos. Total histone acetyltransferase activity increases during germination with two maxima at 40 and 72 h after start of germination. This fluctuation is mainly due to the cytoplasmic B-enzyme which predominantly acetylates histone H4 up to the diacetylated form. The nuclear histone acetyltransferase A2, specific for H3, is low throughout germination, except at 24 h, when it transiently becomes the main activity. Both enzymes are also present in the dry embryo, whereas the second nuclear enzyme A1, specific for H3 and H4, is absent in the initial stage of differentiation. The two histone deacetylases, HD1 and HD2, exhibit entirely different patterns. Whereas HD1 activity is low in the dry embryo and increases during germination, HD2 is the predominant enzyme at the start of differentiation, but almost disappears at later stages. Analysis of the in vivo acetate incorporation reveals that H4 is present in up to tetraacetylated subspecies. The pattern of acetate incorporation into core histones closely resembles the fluctuations of histone acetyltransferase B. Based on the analysis of thymidine kinase activity a close correlation was established between histone acetyltransferase B and DNA replication, whereas the A2 enzyme is associated with transcriptional activity. Histone deacetylase HD1 obviously serves a specific function in the dry embryo and could be a prerequisite for DNA repair processes. The study confirms the idea of DNA repair processes. The study confirms the idea of multiple functions of histone acetylation and assigns distinct enzymes, involved in this modification, to certain nuclear processes.

Acetylation↗
PubMed1991

Expression of the c-myc proto-oncogene in multiple myeloma and chronic lymphocytic leukemia: an in situ analysis.

The c-myc gene plays a pivotal role in mediating the competence state for cell cycle transversion. This biologic role is in contradiction to reports of elevated expression of the gene in multiple myeloma, a tumor with restricted self-renewal capacity. To more clearly define the role of this gene in plasma cells of myeloma patients, c-myc messenger RNA (mRNA) and/or oncoprotein expression were semiquantitatively analyzed on the single cell level in 19 cases of multiple myeloma, among them 1 biclonal case and 1 case with coexistent chronic lymphocytic leukemia (CLL). Performing anti-sense/mRNA in situ hybridization, mature c-myc gene transcripts were detected in 92% (12 of 13) of cases and could definitely be attributed to the plasma cells by our study. The number of Ki 67-positive plasma cells actively passing the cell cycle was less than 1% and independent of c-myc gene expression. However, because the presence of the 152-c-MYC epitope was correlated to extent of marrow plasmacytosis (r = .64; P = .043) and content of plasmablasts (P = .09), the c-myc gene might serve a function different from proliferative activity, but also associated with tumor cell mass. In CLL cells (21 of 22 cases) and their benign counterparts, ie, bone marrow and peripheral blood lymphocytes, the anti-sense/c-myc mRNA hybridization signals remained below the threshold considered as cutpoint between negative and positive. The low amounts of c-myc transcripts were correlated to neither stage of disease (P = .52) nor lymphocyte counts (P = .24). Because the numbers of peripheral blood lymphoma cells were independent of tumor mass and of c-myc gene transcripts expressed, peripheral blood lymphocytosis might more likely reflect homing processes than proliferative activity in CLL.

Antibodies, Monoclonal↗
PubMed1991

Cell cycle dependent association of c-myc protein with the nuclear matrix.

We demonstrate the presence of a c-myc protein with an apparent molecular weight of 67 kd in the lower eukaryote Physarum polycephalum with polyclonal and monoclonal anti-c-myc-antibodies. It is shown that the amount of myc protein present in the nucleus does not fluctuate during the naturally synchronous cell cycle of Physarum. The myc protein remains firmly associated with the nuclear matrix after a variety of matrix preparation procedures. Although the level of c-myc protein is invariant during the cell cycle, the matrix-bound portion of c-myc protein is higher during S-phase as compared to G2-period. In immunoelectron microscopy the immunosignal was considerably stronger in the nuclear matrix compared with unfractionated nuclei, indicating increased accessibility of c-myc protein in nuclear scaffold structures. We show that the nuclear localization of c-myc protein changes during the cell cycle, being transiently but specifically bound to the periphery of the nuclear matrix structure during S-phase.

Blotting, Western↗
PubMed1990

Morphology of the differentiation and maturation of LLC-PK1 epithelia.

In the present study, a stereologic approach was utilized to quantitatively assess morphological changes during the differentiation of LLC-PK1 cells into an epithelial membrane. This renal epithelial cell line has been described to undergo morphological changes during differentiation and maturation from subconfluent culture to a confluent epithelial layer. An increase in the number of apical microvilli, interpreted as an areal increase in this membrane domain was reported. This morphological differentiation was found to be accompanied by an increase in the expression of apical Na(+)-dependent hexose transport and the activities of certain brush border enzymes. Since no data are available that quantify the morphologic changes during LLC-PK1 differentiation, a quantitative morphologic-stereologic-investigation was performed for an early (6 days) and a late (12 days) state of confluence of LLC-PK1 monolayer cultures. The following morphological parameters were determined by light and electron microscopic morphometry: volume fractions (Vv) of nuclei, mitochondria, and lysosomes, and surface densities (Sv) of the apical and basolateral cell membrane domains. For the apical membrane surface, the microvillous fraction has been measured separately. Since the stereologic approach used in the present study allows the determination of absolute cell volumes, the absolute measures of organelle volumes (V) and membrane surfaces (S) per average cell can be calculated from volume and surface densities. Although no changes in cell density were found for 6 and 12 day old LLC-PK1 monolayers, indicating ceased cell proliferation due to contact inhibition, remarkable changes were found concerning the absolute cell volume and apical membrane surface. The observed increase in the apical cell surface was exclusively due to the enlarged microvillous surface fraction. This finding is in good agreement with the increased number of Na(+)-dependent hexose transporters as well as with the increased expression of apical membrane marker enzymes observed during the differentiation of LLC-PK1 monolayers.

Animals↗
PubMed1988

ADP-ribosylation in isolated nuclei of Physarum polycephalum.

ADP-ribosylation of histones and non-histone nuclear proteins was studied in isolated nuclei during the naturally synchronous cell cycle of Physarum polycephalum. Aside from ADP-ribosyltransferase (ADPRT) itself, histones and high mobility group-like proteins are the main acceptors for ADP-ribose. The majority of these ADP-ribose residues is NH2OH-labile. ADP-ribosylation of the nuclear proteins is periodic during the cell cycle with maximum incorporation in early to mid G2-phase. In activity gels two enzyme forms with Mr of 115,000 and 75,000 can be identified. Both enzyme forms are present at a constant ratio of 3:1 during the cell cycle. The higher molecular mass form cannot be converted in vitro to the low molecular mass form, excluding an artificial degradation during isolation of nuclei. The ADPRT forms were purified and separated by h.p.l.c. The low molecular mass form is inhibited by different ADPRT inhibitors to a stronger extent and is the main acceptor for auto-ADP-ribosylation. The high molecular mass form is only moderately auto-ADP-ribosylated.

Adenosine Diphosphate↗
PubMed1988

Periodic fluctuations of nuclear high mobility group like proteins during the cell cycle of Physarum polycephalum.

High mobility group like (HMG-like) nuclear proteins were isolated from plasmodia of the lower eucaryote Physarum polycephalum and characterized by different types of polyacrylamide gel electrophoresis. The synthesis of these proteins was measured during the naturally synchronous cell cycle of Physarum. The four HMG-like proteins (AS1-4) exhibit a pronounced cell cycle dependent pattern of synthesis: AS1 and AS4 have a clear maximum of synthesis in mid S phase with a basal synthesis during the entire G2 period. In contrast, AS2 and AS3 have little synthesis in S phase but a broad maximum in mid G2 period. The four HMG-like proteins have a very low synthesis in early S phase and late G2 period. In addition, other non-histone proteins, which are coextracted with the HMG proteins, exhibit distinct periodic synthesis patterns. A novel non-histone protein, which is the most abundant protein species in 0.35 M NaCl extracts, was detected. It exhibits a high rate of synthesis around the time of mitosis. In general, the results indicate that, in contrast to the main cytoplasmic proteins, most nuclear proteins are phase-specific with respect to their synthesis in the cell cycle.

Amino Acids↗
PubMed1988

Towards an understanding of the biological function of histone acetylation.

A model is presented which explains the biological function of posttranslational acetylation of core histones in chromatin. Along the lines of this model histone acetylation serves as a general mechanism to destabilize nucleosome core particles during various processes occurring in chromatin. Acetylation acts as a signal that modulates histone-protein and histone-DNA interactions and finally leads to the displacement of particular histones from nucleosome cores. The high specificity of the acetylation signal for different processes (DNA replication, transcription, differentiation-specific histone replacement) is achieved by site specificity and asymmetry of acetylation in nucleosomes. The essential features of this model are in accord with the more recent results on histone acetylation.

Acetylation↗
PubMed1988

In situ preparation of the nuclear matrix of Physarum polycephalum: ultrastructural and biochemical analysis of different matrix isolation procedures.

A novel method for in situ preparation of nuclear matrix from whole plasmodia of Physarum polycephalum without isolation of nuclei is presented. Plasmodia are encapsulated in agarose beads and after solubilization of the cytoplasm the nuclear matrix is prepared. With this quick and easy technique nuclear matrix can be reproducibly prepared with perfect recovery. We compared the ultrastructural and biochemical properties of the matrix after three different matrix isolation procedures: preparation with high salt, ammonium sulphate and lithium diiodosalicylic acid. The results show that the ultrastructure and protein composition of the three types of matrix are very similar or even identical. We conclude that many of the conflicting results on nuclear matrix in the literature are due to perturbations of nuclear integrity during the isolation of nuclei. For this reason the new in situ method is an important approach in the standardization of nuclear matrix isolation.

Cell Nucleus↗
PubMed1987

Postsynthetic acetylation of histones during the cell cycle: a general function for the displacement of histones during chromatin rearrangements.

Postsynthetic acetylation of core histones exhibits a peak during S-phase of the Physarum cell cycle. The maximum 3H-acetate incorporation precedes the maximum of histone synthesis. Acetate is incorporated into all core histones during S-phase, but only into H2A and H2B during G2-period. Resolution of acetylated H4-subspecies reveals acetate incorporation into preexisting H4, but not into newly synthesized molecules during mitosis and early S-phase. In a protamine competition assay histones from S-phase chromatin are released at lower protamine concentrations as compared to the lower acetylated G2-chromatin. We demonstrate a preferential release of highly acetylated H4-subspecies at low protamine concentrations. Our results fit into a general model of the relationship between histone acetylation and chromatin assembly. According to this model acetylation of core histones would serve as a signal for displacement of histones from nucleosomes by modulating histone-protein or histone-DNA interactions. We propose that this mechanism operates during DNA-replication and transcription, as well as during other chromatin rearrangements.

Acetates↗
PubMed1987

Histone acetyltransferase activity during the cell cycle.

Histone acetyltransferase activity was measured in isolated nuclei during the synchronous cell cycle of the myxomycete Physarum polycephalum. Nuclei were incubated with [14C]acetyl-coenzyme A and an excess of exogenous calf thymus histones. The activity is periodic during the cell cycle; it rises during the S-phase to reach a maximum in the early G2-period with a decline in mid and late G2. Comparison of the pattern of enzyme activity with the in vivo acetylation of histones during the cell cycle reveals that the enzyme activity does not wholly determine the acetylation state, indicating that other factors, including possibly the structural state of chromatin, are responsible for the observed cell cycle pattern of in vivo histone acetylation.

Acetates↗
PubMed1987

Histone synthesis during the cell cycle of Physarum polycephalum. Synthesis of different histone species is not under a common regulatory control.

The synthesis of histones and nonhistone nuclear proteins was studied during the naturally synchronous cell cycle of Physarum polycephalum. Contrary to the commonly accepted idea of a tight coupling of histone biosynthesis and DNA replication during the somatic cell cycle we found that 40% of total histone synthesis takes place in the G2 period in the complete absence of DNA synthesis. The core histones exhibit a maximum of synthesis during S-phase. The synthesis of histones H2A and H2B continues during the G2 period, but synthesis of H4 and H3 is restricted to the S-phase of the cell cycle. Experiments with hydroxyurea demonstrated that the synthesis of H4 and H3 is completely dependent on unperturbed DNA synthesis, whereas synthesis of H2A and H2B is independent from DNA synthesis during the entire cell cycle. This implicates significant differences between the arginine-rich histones H4 and H3 and the moderately lysine-rich histones H2A and H2B with respect to the control mechanisms of their synthesis, the metabolic stability, and the function for chromatin structure. The nonhistone nuclear proteins are synthesized throughout the cell cycle with a broad maximum in the early G2 period. The cell cycle pattern of synthesis of H1 rather resembles the pattern of the nonhistone proteins than of core histones.

Cell Cycle↗
PubMed1986

Biosynthesis and posttranslational acetylation of histones during spherulation of Physarum polycephalum.

Plasmodia of Physarum polycephalum can be induced to differentiate into dormant spherules: DNA-, RNA- and protein-synthesis cease during this process. Analysis of the histone H4 acetylation during spherulation revealed no significant changes of the relative acetate content and percentage of acetylated H4 subspecies. This result does not support a close correlation of histone acetylation and transcriptional activity. Posttranslational incorporation of 3H-acetate into core histones decreased rapidly after start of spherulation. However, acetate incorporation increased significantly at a late stage of spherulation (30 h). To elucidate the role of this elevated acetate incorporation we followed histone synthesis during spherulation. Histone synthesis decreased upon induction of differentiation and stopped after 12 h. After 38 h of spherulation histone synthesis again occurred in the absence of DNA synthesis. The peak of acetate incorporation into core histones clearly preceded this late histone synthesis, indicating acetylation of preexisting histones. We suggest, that this acetate incorporation is part of the mechanism, by which preexisting histones are replaced by newly synthesized histones. Pulse treatment with actinomycin D or cycloheximide during spherulation suggested, that the observed histone synthesis is essential for the germination of spherules. Obviously, new histones have to be synthesized for the coordinate course of the differentiation program.

Acetylation↗
PubMed1985

ADP-ribosyltransferase in isolated nuclei during the cell cycle of Physarum polycephalum.

ADP-ribosyltransferase was measured in isolated nuclei of Physarum polycephalum. Activity was determined with and without exogenous DNA and histones. During the synchronous cell cycle the activity measured with exogenous substrates exhibited a typical peak enzyme pattern with a maximum of activity in S-phase, whereas activity measured without exogenous substrates displayed a step enzyme pattern. Both activities doubled in each cell cycle.

Cell Cycle↗