Proteome analysis of nuclear matrix proteins during apoptotic chromatin condensation.

The nuclear matrix (NM) is considered a proteinaceous scaffold spatially organizing the interphase nucleus, the integrity of which is affected during apoptosis. Caspase-mediated degradation of NM proteins, such as nuclear lamins, precedes apoptotic chromatin condensation (ACC). Nevertheless, other NM proteins remain unaffected, which most likely maintain a remaining nuclear structure devoid of chromatin. We, therefore, screened various types of apoptotic cells for changes of the nuclear matrix proteome during the process of apoptotic ACC. Expectedly, we observed fundamental alterations of known chromatin-associated proteins, comprising both degradation and translocation to the cytosol. Importantly, a consistent set of abundant NM proteins, some (e.g. hNMP 200) of which displaying structural features, remained unaffected during apoptosis and might therefore represent constituents of an elementary scaffold. In addition, proteins involved in DNA replication and DNA repair were found accumulated in the NM fraction before cells became irreversibly committed to ACC, a time point characterized in detail by inhibitor studies with orthovanadate. In general, protein alterations of a consistent set of NM proteins (67 of which were identified), were reproducibly detectable in Fas-induced Jurkat cells, in UV-light treated U937 cells and also in staurosporine-treated HeLa cells. Our data indicate that substantial alterations of proteins linking chromatin to an elementary nuclear protein scaffold might play an intriguing role for the process of ACC.

Continuum saltation model for sand dunes.

We derive a phenomenological continuum saltation model for aeolian sand transport that can serve as an efficient tool for geomorphological applications. The coupled differential equations for the average density and velocity of sand in the saltation layer reproduce both the known equilibrium relations for the sand flux and the time evolution of the sand flux as predicted by microscopic saltation models. The three phenomenological parameters of the model are a reference height for the grain-air interaction, an effective restitution coefficient for the grain-bed interaction, and a multiplication factor characterizing the chain reaction caused by the impacts leading to a typical time or length scale of the saturation transients. We determine the values of these parameters by comparing our model with wind tunnel measurements. Our main interest are out of equilibrium situations where saturation transients are important, for instance at phase boundaries (ground/sand) or under unsteady wind conditions. We point out that saturation transients are indispensable for a proper description of sand flux over structured terrain, by applying the model to the windward side of an isolated dune, thereby resolving recently reported discrepancies between field measurements and theoretical predictions.

Elevated plasma levels of crosslinked fibrinogen gamma-chain dimer indicate cancer-related fibrin deposition and fibrinolysis.

Cancer-related fibrin deposition and fibrinolysis were investigated by two-dimensional gel electrophoresis of human solid tumor and effusion specimen in addition to plasma samples. Fibrinogen gamma-chain dimer indicating fibrin deposition and plasmin-generated fibrinogen beta-chain fragments were identified in various solid tumor types by amino acid sequencing, mass spectrometry analysis and Western blotting. In tumor-associated effusions, these techniques allowed to observe plasmin-generated fragments of fibrinogen alpha, beta and gamma-chains in addition to elevated levels of acute-phase proteins. Similar observations were made in case of inflammation-associated effusions. No fibrin degradation product was observed in plasma samples, however, high amounts of fibrinogen gamma-chain dimer crosslinked by transglutaminase were detected in plasma from tumor patients, but not in plasma from controls and patients suffering acute infections and/or inflammations. This finding demonstrated that high transglutaminase activity may be associated with cancer. The presented data indicate that the amount of crosslinked fibrinogen gamma-chain dimer in plasma may correlate with tumor-associated fibrin deposition. The tumor-biological relevance of this potential marker protein is discussed.

hNMP 200: a novel human common nuclear matrix protein combining structural and regulatory functions.

Previously we have reported about human nuclear matrix proteins (hNMPs) with increased reassembling and potential filament-forming capability [C. Gerner et al., 1999, J. Cell. Biochem. 74, 145-151]. Here, we cloned the cDNA of one of these proteins, hNMP 200, following partial amino acid sequencing of the novel 56-kDa nuclear protein. Sequence alignments show hNMP 200-related proteins in metazoans, plants, and yeast, the homologous Saccharomyces cerevisiae protein prp19 being an accessory, but essential, factor for pre-mRNA processing. Evidence for any enzymatic activity was not detected. However, the hNMP 200 primary sequence contained five consensus WD-repeat sequences, indicative of participation and regulatory function in larger protein assemblies. Northern blot analysis and 2D protein electrophoresis showed ubiquitous expression of hNMP 200 in a variety of cell types. (35)S labeling studies indicated a high metabolic stability of the protein. The hNMP 200 gene was assigned to chromosomal region 11q12.2. Confocal laser scanning microscopy revealed that the intracellular localization conformed with that reported for other structural nuclear proteins. In interphase cells, green fluorescent protein-tagged hNMP 200 was predominantly nucleoplasmic. Structures with speckled appearance extended through several sections of in situ-isolated nuclear matrices. During cell division hNMP 200 became irregularly distributed in prophase, sparing regions of condensing chromatin. In anaphase it was concentrated in the spindle midzone. The putative dual function of the novel NMP is discussed. Being a component of the nuclear framework, it may provide structural support for components of the RNA-processing machinery, thereby also modulating splicing activities.

Differential nuclear localization and nuclear matrix association of the splicing factors PSF and PTB.

A monoclonal antibody raised against nuclear matrix proteins detected a protein of basic pI in human nuclear matrix protein samples of various cellular origin. The ubiquitously occurring (common) nuclear matrix protein was identified as splicing factor PSF (PTB associated splicing factor). The interaction between the splicing factors PSF and PTB/hnRNP I was confirmed by co-immunoprecipitation from nuclear salt extracts. However, the nuclear localization of PSF and PTB and their distribution in subnuclear fractions differed markedly. Isolated nuclear matrices contained the bulk of PSF, but only minor amounts of PTB. In confocal microscopy both proteins appeared in speckles, the majority of which did not co-localize. Removing a large fraction of the soluble PTB structures by salt extraction revealed some colocalization of the more stable PTB fraction with PSF. These PTB/PSF complexes as well as the observed PSF-PTB interaction may reflect the previously reported presence of PTB and PSF in spliceosomal complexes during RNA processing. The present data, however, point to different cellular distribution and nuclear matrix association of the majority of PSF and PTB.

A human common nuclear matrix protein homologous to eukaryotic translation initiation factor 4A.

Amino acid sequencing and mass spectrometry revealed identity of a human nuclear matrix protein, termed hNMP 265, with a predicted protein of gene KIAA0111. Two-dimensional electrophoresis and Northern hybridization showed the protein to ubiquitously occur in various human cell types. Exhibiting DEAD-box motifs characteristic for RNA helicases, hNMP 265 is highly similar to the human initiation factors eIF4A-I and -II. On the other hand, hNMP 265 greatly differs from the initiation factors by a N-terminal sequence rich in charged amino acids. Sequence searches and alignments indicate proteins related to hNMP 265 in other eukaryotes. Chimeras between hNMP 265 and green fluorescence protein or hapten appeared as speckles in extranucleolar regions in the nucleus, but not in the cytoplasm. Experiments with tagged deletion mutants indicated that the N-terminal amino acid sequence is necessary for nuclear localization. A putative role of hNMP 265 in pre-mRNA processing is discussed.

Reassembling proteins and chaperones in human nuclear matrix protein fractions.

To detect putative filament forming components, nuclear matrix proteins were searched for proteins extensively reassembling from urea solution. Eight proteins, ubiquitously occurring in various human cell types, but not apparent in the cytosol, were registered by means of two-dimensional gel electrophoresis. They consisted of a protein exhibiting a novel amino acid sequence; of nuclear lamin B2, RbAp46, and RbAp48; and of four as yet unknown proteins. Furthermore, partial sequencing, mass spectrometry, and immunodetection of proteins demonstrated the presence of molecular chaperones and protein folding catalysts in the nuclear matrix fractions. In addition to a TCP-1-related protein, certain members of the heat shock, PDI, and calreticulin family of proteins were detected. On the basis of the absence of several other heat shock proteins in the nuclear matrix fraction, a general contamination by cytoplasmic chaperones appears unlikely.

Coenzyme Q10, a cutaneous antioxidant and energizer.

The processes of aging and photoaging are associated with an increase in cellular oxidation. This may be in part due to a decline in the levels of the endogenous cellular antioxidant coenzyme Q10 (ubiquinone, CoQ10). Therefore, we have investigated whether topical application of CoQ10 has the beneficial effect of preventing photoaging. We were able to demonstrate that CoQ10 penetrated into the viable layers of the epidermis and reduce the level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was also shown. CoQ10 was determined to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, activation of specific phosphotyrosine kinases and prevention of oxidative DNA damage. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging.

Nuclear matrix proteins specific for subtypes of human hematopoietic cells.

Nuclear matrices were prepared from isolated subtypes of human hematopoietic cells and from cultured leukemia cells. The nuclear matrix proteins were analyzed by high-resolution two-dimensional gel electrophoresis and computer-assisted image analysis. While more than 200 protein spots were shared among the cells, about 50 distinct spots were found characteristic for individual cells or groups of related cells. This allowed to differentiate between hematopoietic cells and nonhematopoietic cells, lymphocytes and myeloid cells, monocytes, neutrophils, and promyelocytic leukemia cells. B and T lymphocytes could not be differentiated. Myeloid cells with their polymorph nuclei were characterized by the presence of 13 and by the absence of seven distinct spots, as well as by low concentrations of nuclear lamins and of heterogeneous nuclear ribonucleoproteins. Neutrophils with multilobular nuclei displayed six additional spots, while lacking 18 nuclear matrix protein spots. The nuclear matrix of proliferating cells showed three distinct spots in addition to proliferating cell nuclear antigen, increased concentrations of numatrin (B23), and heterogeneous nuclear ribonucleoproteins. The described cell-specific nuclear matrix proteins may represent new markers for hematopoietic cells.

Identification and characterization of the ubiquitously occurring nuclear matrix protein NMP 238.

By systematic comparison of two-dimensional electrophoretic patterns of nuclear matrix proteins an ubiquitously occurring (common) nuclear matrix protein, termed NMP 238, was detected. Localization of the protein in isolated nuclear matrices and in nuclear and cytoplasmic regions of cells was determined by confocal immunofluorescence microscopy. N-terminal protein sequencing, mass spectrometry, and sequencing of a human EST cDNA clone showed identity of the protein with a nuclear protein, termed TIP49, of as yet uncertain function. Expression of the corresponding gene in diverse human and rat cells was confirmed by Northern blotting. The protein displays two nuclear localization signals. Sequence homologies indicate evolutionary related proteins in nematodes, yeast, and archaebacteria. Similarities to the AAA family of proteins and to a subgroup of chaperones suggest that the nuclear matrix protein may play a role in the assembly and ATP-dependent anchorage of proteins.

Similarity between nuclear matrix proteins of various cells revealed by an improved isolation method.

Comparative analysis of nuclear matrix proteins by two-dimensional electrophoresis may be greatly impaired by copurifying cytoskeletal proteins. The present data show that the bulk of adhering cytofilaments may mechanically be removed by shearing of nuclei pretreated with vanadyl ribonucleoside complexes. Potential mechanisms of action not based on ribonuclease inhibition are discussed. To individually preserve the integrity of nuclear structures, we developed protocols for the preparation of nuclear matrices from three categories of cells, namely leukocytes, cultured cells, and tissue cells. As exemplified with material from human lymphocytes, cultured amniotic cells, and liver tissue cells, the resulting patterns of nuclear matrix proteins appeared quite similar. Approximately 300 spots were shared among the cell types. Forty-nine of these were identified, 21 comprising heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoproteins L and nuclear lamin B2 isoforms were identified by amino acid sequencing and mass spectrometry. However, individually expressed proteins, such as the proliferating cell nuclear antigen, also pertained following application of the protocols. Thus, enhanced resolution and comparability of proteins improve systematic analyses of nuclear matrix proteins from various cellular sources.

A novel spliced transcript of human CLAPS2 encoding a protein alternative to clathrin adaptor protein AP17.

Transcripts of genes encoding proteins of clathrin complexes have been reported to undergo tissue-specific alternative splicing. AP17, encoded by human CLAPS2 cDNA, is the small chain of the major clathrin adaptor complex AP-2 associated with mammalian plasma membranes. In this study, two cDNAs were isolated from a cDNA library of human blood cells. Whereas one cDNA encoded AP17, the other cDNA encoded a putative novel protein variant, termed AP17Delta. Both coding regions were completely sequenced. Consisting of 142aa residues, the predicted protein AP17Delta of 12kDa lacks 38aa residues of AP17. Using specific primers for RT-PCR, mRNAs for AP17Delta and AP17 were found in leukocytes and cultured leukemia cells. The finding of a putative intron in a human EST cDNA clone suggests that mRNAs for AP17 and AP17Delta are formed by alternative splicing. In addition, the identity of human and rat AP17 amino acid sequences is demonstrated.

Alteration of nuclear matrix protein composition during apoptosis in rat embryo cells.

Alteration of the nuclear matrix protein composition during active cell death was investigated by high resolution 2-dimensional gel electrophoresis and computer-assisted image analysis. Nuclear matrices were isolated from purified nuclei of a rat embryo cell line showing an immediate apoptotic response to serum reduction. While cell shrinkage and cytoplasmic compaction, characteristic features of apoptosis, were induced, the nuclear matrix protein pattern was not altered 1 h after induction of apoptosis. However, two sets of novel nuclear matrix protein spots appeared with differing kinetics within the following 5 h of apoptosis. They consisted of five and six protein spots, respectively. In addition, the intensity of five nuclear matrix protein spots that had already been present in the uninduced cells increased continuously within an observation period of 12 h. These coincidences point to a potential involvement of the described nuclear matrix proteins in the apoptotic process.

Identification of human common nuclear-matrix proteins as heterogeneous nuclear ribonucleoproteins H and H' by sequencing and mass spectrometry.

Nuclear-matrix proteins were prepared from different rat and human cells and separated by two-dimensional gel electrophoresis. By computer-assisted analysis of the images, two of the proteins were identified as ubiquitously occurring (common) nuclear-matrix proteins, which appeared in tissue-dependent concentrations. The two proteins that originated from human blood mononuclear cells were analyzed further. Tryptic digests of the blotted proteins were analyzed by partial peptide sequencing and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The two human common nuclear-matrix proteins were identified as heterogeneous nuclear ribonucleoproteins (hnRNP) H and H' or their variants. Furthermore, mass analysis revealed details on the N terminus of hnRNP H.

Common nuclear matrix proteins in rat tissues.

Nuclear matrix proteins have been defined as insoluble residual proteins resulting from treatment of isolated nuclei with nucleases, detergents and high ionic strength buffers. They are considered as in part representing the proteins constituting the three-dimensional framework of the interphase nucleus. Though cell-specific nuclear matrix proteins have been differentiated from ubiquitously occurring (common) nuclear matrix proteins, the number and types of common nuclear matrix proteins have not yet been unequivocally established. In the present study nuclear matrix proteins were prepared from isolated nuclei of rat kidney, liver, lung, spleen and testes. The matrix proteins were separated by two-dimensional (2-D) electrophoresis and silver stained. Then the spot patterns were compared by computer-assisted image analysis. Composite images were derived for nuclear matrix proteins of individual tissues. Finding between 396-483 spots per tissue, a total of 964 individual spots were registered. Of these, 102 were common nuclear matrix proteins, as appearing in each of the tissue-characteristic images. The apparent molecular mass and pI data may serve for further identification of these nuclear proteins.

Two-dimensional electrophoresis reveals a nuclear matrix-associated nucleolin complex of basic isoelectric point.

A monoclonal antibody was raised against a salt-extractable fraction of nuclear matrix / intermediate filament scaffolds of polarized MDCK cells. The antibody recognized an approximately 100 kDa protein in total cell lysates and nuclear matrices of various human cells and tissues and stained nucleolar structures in immunofluorescence microscopy. By partial sequencing of five peptides derived from immunoprecipitated protein, the targeted antigen was found to be homologous to human nucleolin. After two-dimensional electrophoresis of total HeLa cell lysates, immunoreactive bands were detected at isoelectric point (pI) 5.5--6.1, characteristic for nucleolin, and at pI 8.5--9. Whereas the protein focusing at acidic pI was found in Triton X-100-soluble cellular fractions, the antigen focusing at basic pI was exclusively contained in the residual nuclear fraction and was solubilized upon treatment of nuclear matrices with RNAse. The component solubilized by RNAse treatment was still detected at basic pI in two-dimensional electrophoresis. However, upon immunoprecipitation of the antigen from the RNAse-released fraction in the presence of sodium dodecyl sulfate (SDS), the nuclear matrix-derived antigen was positioned at pI 5--6. The present data indicate that the nuclear matrix-bound nucleolin is associated with ribonucleoproteins and a basic component resisting dissociation under conditions of isoelectric focusing.

Consequences for sun protection factors when solar simulator spectra deviate from the spectrum of the sun.

Synopsis The sun protection factor (SPF) of two products, one with an expected SPF of 4 and another with an expected SPF of 15 were determined, using two solar simulators: Multiport Solar UV Simulator (xenon, Solar Light, Philadelphia, PA, USA), and Supersun 5000 (metal halide, Mutzhas, Munich, Germany). The mean SPFs using the Multiport were: 4.8 for the low SPF product and 19.4 for the high SPF one. The results using the Supersun were lower: 2.6 for the low SPF product and 7.2 for the high SPF one. Relative emission spectra of the two sources were recorded using a fluorescence spectrophotometer in bioluminescence mode. Efficacy spectra were calculated and compared with the corresponding spectrum of natural sunlight. It was evident that the spectral power of the xenon source is too high in the UVB, leading to overestimation of SPFs, whereas the Supersun irradiator emits too much in the UVA, resulting in too low SPFs. Heat effects and photodegradation of UV filters are discussed as further possible reasons for the discrepancies between the experimentally determined SPFs. Our results confirm a recent publication about theoretical SPFs, calculated with emission spectra of a xenon source and spectra of the sun at different elevation angles, where the authors provide evidence that in natural sunlight the contribution of UVA to total UV radiation is twice as high as in a xenon source. This may contribute to an understanding of why sunscreens tested according to the FDA method (xenon sources) often yield higher SPFs than those obtained from European testing procedures.

The use of biotinylated poly(ADP-ribose) for studies on poly(ADP-ribose)-protein interaction.

Poly(ADP-ribose) is routinely detected by the use of radioactive polymers formed from labeled substrates. In this report a simple and time-saving method for the biotinylation and the detection of poly(ADP-ribose) on blots is described. The polymer modified by light-induced reaction with photobiotin was colorimetrically detected and quantified, using streptavidine-alkaline phosphatase conjugates. The separation of poly(ADP-ribose) chains on polyacrylamide gels was not affected by the biotinylation of the polymers. When biotinylated poly(ADP-ribose) was used to detect the poly(ADP-ribose) binding capability of proteins in ligand blots, the results were comparable to those obtained with poly([32P]ADP-ribose). Experiments with histones and rat liver nuclear proteins demonstrate that in studies on poly(ADP-ribose)-protein interaction, this method is applicable to the detection of poly(ADP-ribose) binding proteins.