Changes of proteins induced by anticoagulants can be more sensitively detected in urine than in plasma

The most fundamental property of biomarkers is change. But changes are hard to maintain in plasma since it is strictly controlled by homeostatic mechanisms of the body. There is no homeostatic mechanism for urine. Besides, urine is partly a filtration of blood, and systematic information can be reflected in urine. We hypothesize that change of blood can be reflected in urine more sensitively. Here we introduce the interference into the blood by two anticoagulants heparin or argatroban. Plasma and urine proteins were profiled by LC-MS/MS and then validated by Western blot in totally six SD female rats before and after the drug treatments. In argatroban treated group, with exactly the same experimental procedure and the same cutoff value for both plasma and urine proteins, 62 proteins changed in urine, only one of which changed in plasma. In heparin treated group, 27 proteins changed in urine but only three other proteins changed in plasma. Both LC-MS/MS and Western blot analyses demonstrated drug-induced increases in transferrin and hemopexin levels in urine but not in plasma. Our data indicates that urine may serve as a source for more sensitive detection of protein biomarkers than plasma.     

Structure, subnuclear distribution, and nuclear matrix association of the mammalian telomeric complex

Mammalian telomeres are composed of long arrays of TTAGGG repeats complexed with the TTAGGG repeat binding factor, TRF. Biochemical and ultrastructural data presented here show that the telomeric DNA and TRF colocalize in individual, condensed structures in the nuclear matrix. Telomeric TTAGGG repeats were found to carry an array of nuclear matrix attachment sites occurring at a frequency of at least one per kb. The nuclear matrix association of the telomeric arrays extended over large domains of up to 20-30 kb, encompassing the entire length of most mammalian telomeres. TRF protein and telomeric DNA cofractionated in nuclear matrix preparations and colocalized in discrete, condensed sites throughout the nuclear volume. FISH analysis indicated that TRF is an integral component of the telomeric complex and that the presence of TRF on telomeric DNA correlates with the compact configuration of telomeres and their association with the nuclear matrix. Biochemical fractionation of TRF and telomeric DNA did not reveal an interaction with the nuclear lamina. Furthermore, ultrastructural analysis indicated that the mammalian telomeric complex occupied sites throughout the nuclear volume, arguing against a role for the nuclear envelope in telomere function during interphase. These results are consistent with the view that mammalian telomeres form nuclear matrix- associated, TRF-containing higher order complexes at dispersed sites throughout the nuclear volume.     

Changes in distribution of nuclear matrix antigens during the mitotic cell cycle

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.     

Modification of nuclear matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix

Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed.     

Cancer cachexia modifies the zonal distribution of lipid metabolism-related proteins in rat liver

Cancer cachexia is a syndrome that causes profound metabolic disruption. Lipid metabolism in the liver is markedly affected. We investigated the effect of cachexia upon liver-acinus lipid-metabolism zonation in Walker 245 carcinosarcoma-bearing rats (TB). The expression of protein (by Western blotting) and mRNA (by semi-quantitative polymerase chain reaction) of the enzymes of the carnitine palmitoyltransferase system (CPT I and CPT II) and of liver fatty-acid-binding protein (L-FABP) was studied. Although no changes were found for these parameters, the maximal activities (by radioassay) of CPT I and II were reduced (P<0.05) in TB compared with controls. CPT II activity in the perivenous (PV) region was higher in TB compared with controls. The distribution of CPT II and L-FABP (by immunohistochemistry) within the acinus was modified by cachexia: whereas CPT II positivity was restricted to the PV zone, L-FABP labelling shifted from periportal (control) to perivenous (TB) zone. These changes in metabolic zonation, together with decreased CPT II activity, may contribute to the aggravation of cachexia. The authors thank FAPESP for financial support (00/03761-2) and for the scholarship awarded to M. Kazantzis.     

Changes of nuclear matrix proteins following the differentiation of human osteosarcoma MG-63 cells

Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide （HMBA）. Their nuclear matrix proteins （NMPs） were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.     

Changes in distribution of extracellular matrix proteins during wound repair in corneal endothelium

The distribution of fibronectin (FN) and laminin (LM) in non-injured and injured rat corneal endothelium in vivo was investigated by light microscopy using immunoperoxidase cytochemistry. In non-injured tissues, both FN and LM have distinct pericellular staining patterns and exhibit some diffuse cytoplasmic staining. After a circular freeze injury, cells migrating into the wound area at 24 hr lack the characteristic pericellular staining observed in non-injured cells but show cytoplasmic staining for both extracellular matrix glycoproteins. Endothelial cells on the periphery of such preparations do not partake in wound repair and retain their pericellular staining patterns. Forty-eight hours after injury, cells have filled in the wound area but are disorganized. They display intracellular FN and LM staining but do not demonstrate any pericellular staining. When observed 10 days after injury, a uniform monolayer has formed but neither FN nor LM is detected pericellularly. By 14 days post injury, endothelial cells in the wound area display pericellular FN patterns but not LM patterns. This may reflect differences in the function of each glycoprotein in maintaining the attachment of the endothelium to Descemet's membrane.     

Dynamic changes in the length distribution of actin filaments during polymerization can be modulated by barbed end capping proteins

During actin polymerization, it has been theorized that the actin filament length distribution initially grows in the form of a Gaussian before converting to produce that of an exponential. However, it has been difficult to demonstrate this experimentally. In this study, we use modern fluorescence microscopy techniques to observe the changing actin filament length distribution during and subsequent to the polymerization process. Nucleated actin filament growth using barbed end capping proteins (gelsolin and erythrocyte capping protein) leads to Gaussian length distributions that are relatively stable. As predicted, nucleated actin filament growth using actin/spectrin complexes follows a similar process until polymerization reaches equilibrium whereafter the Gaussian length distribution rapidly converts to that of an exponential. This study provides direct confirmation of the original theories for the mode of actin polymerization but raises doubts regarding the mechanism of the length distribution conversion from Gaussian to exponential.     

Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii

Dinolflagellate is one of the primitive eukaryotes,whose nucleus may represent one of the transition stages from prokaryotic nucleoid to typical eukaryotic nucleus,Using selective extraction together with embeddment-free section and whole mount electron microscopy,a delicate nuclear matrix filament network was shown,for the first time,in dinoflagellate Crypthecodinium cohnii nucleus,Chromosome residues are connected with nuclear matrix filaments to form a complete network spreading over the nucleus,Moreover,we demonstrated that the dinoflagellate chromosome retains a protein scafflod after the depletion of DNA and soluble proteins.This scaffold preserves the characterstic morphology of the chromosome.Two dimensional electrophoreses indicated that the nuclear matrix and chromosome scaffold are mainly composed of acidic proteins.Our results demonstrated that a framework similar th the nuclear matrix and chromosome scaffold in mammalian cells appears in this primitive eukaryote,suggesting that these structures may have been originated from the early stages of eukaryote evolution.     

Plant distribution can be reflected by the different growth and morphological responses to water level and shade in two emergent macrophyte seedlings in the Sanjiang Plain

In the Sanjiang Plain (North East China), narrowleaf small reed (Deyeuxia angustifolia) usually distributes widely in typical meadow or marsh, while reed (Phragmites australis), the concomitant species, is distributed sparsely in the D. angustifolia communities or relative open sites. To date, the mechanisms responsible for their different distribution patterns are far from clear. Both water level and light are important factors determining plant distribution in wetland ecosystems and therefore, the aim of this paper was to identify the role of these two factors and their potential interaction on plant distribution in this plain. Growth responses and biomass allocation of the two macrophytes were investigated by growing them in three irradiances (300, 100, 20 μmol m⁻² s⁻¹) and two water levels (0 and 5 cm) under greenhouse conditions. Biomass accumulation, mean relative growth rate (RGR), height and mean relative elongation rate (RER) of both species significantly decreased with the reduction of light availability. Biomass accumulation, RGR, height and RER of P. australis were significantly inhibited by higher water level. However, water level had no effect on the growth of D. angustifolia. Stem mass fraction was higher at 0-cm water level in D. angustifolia, and was not affected by water level in P. australis. These data suggest that D. angustifolia has a higher adaptive ability to acclimate to flooding and shade stresses than does P. australis, which might be an important reason for their different distribution patterns.     

Crosslinking of nuclear proteins to DNA by cis-diamminedichloroplatinum in intact cells. Involvement of nuclear matrix proteins.

In order to detect the nuclear matrix proteins involved in DNA binding, avoiding possible artifacts derived from the disruption of nuclei, proteins were crosslinked to DNA by the action of cis-diamminedichloroplatinum on intact chicken liver cells and analyzed by two-dimensional gel electrophoresis. At least eleven species of crosslinked proteins were found to derive from the nuclear matrix prepared from the same cell type, and five of these were found also among the proteins crosslinked to DNA in intact liver cells from ox and pig. This subset of common proteins, conserved in different animal species, is likely to have a fundamental role for the anchorage of DNA to the nuclear matrix.     

Variations in ADP-ribosylation of nuclear scaffold proteins during the HeLa cell cycle

Cell cycle variations in ADP-ribosylation of nuclear scaffold proteins were determined. Nuclei of synchronized cells were isolated and labeled with [32P]NAD before nuclear scaffolds were obtained by digestion of DNA with DNase I and extraction of proteins with 2M NaCl. Autoradiograms revealed the three groups of "lamins" and a species identified as poly (ADP-ribose) polymerase to be the primary ADP-ribosylated proteins. The patterns of modification of nuclear scaffold proteins displayed similar features through the cell cycle. Radioactivity in the lamins increased from 20% in early-S phase to 40% in G1 phase of the next cell cycle.     

No discrete complexes containing DNA polymerase α activity can be solubilized from the heat-stabilized nuclear matrix prepared from HeLa S3 cells

Most of the DNA polymerase α activity, bound to the heat-stabilized nuclear matrix prepared from HeLa S3 cells, was released as a matrix extract by sonication. When the extract was centrifuged in a 5–20 per cent linear sucrose gradient no definite peaks of activity could be identified. Most of the activity sedimented to the bottom of the tube under all the conditions tested, whilst the remaining activity was associated with matrix fragments of various and irregular size. No 10 S complexes, containing polymerase activity, were seen after incubation of the extract for 16 h before centrifugation. Other solubilization procedures (i.e. treatment of the matrix with chelating agents, high pH associated with reducing agents, ionic and nonionic detergents) failed to produce release of matrix-bound DNA polymerase α activity. In contrast, we released 10 S complexes, containing polymerase activity, from the matrix prepared from nuclei not exposed to heat. We conclude that a 37°C incubation of isolated nuclei before extraction with 2 M NaCl and DNase I digestion causes DNA polymerase α to bind to the nuclear matrix in a form that cannot subsequently be released as discrete components, at variance with previous results obtained with the matrix prepared from regenerating rat liver.     

Changes in the biosynthesis of nuclear matrix proteins in the thymocytes of mice exposed to radiation and hydrocortisone

Using the method of two-dimensional electrophoresis two newly synthesized proteins with pI 6.6 and 4.8 were identified whose synthesis had been induced by gamma-rays and hydrocortisone. The elucidation of the role of the newly synthesized polypeptides may be important in understanding the mechanisms of cell death.     

Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum.The nuclear matrix and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl.SD-PAGE analyses revealed that the nuclear matrix and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight.Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence,suggesting the presence of the antigen in them.Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold.Immunoelectron microscopic observations further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.     

Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii~1

Dinoflagellate is one of the primitive eukaryotes,whosenucleus may represent one of the transition stages fromprokaryotic nucleoid to typical eukaryotic nucleus.Usingselective extraction together with embeddment-free sectionand whole mount electron microscopy,a delicate nuclearmatrix filament network was shown,for the first time,indinoflagellate Crypthecodinium cohnii nucleus.Chromosomeresidues are connected with nuclear matrix filaments to forma complete network spreading over the nucleus.Moreover,we demonstrated that the dinoflagellate chromosome retainsa protein scaffold after the depletion of DNA and solubleproteins.This scaffold preserves the characteristic mor-phology of the chromosome.Two dimensional elec-trophoreses indicated that the nuclear matrix and chromo- some scaffold are mainly composed of acidic proteins.Ourresults demonstrated that a framework similar to the nuclearmatrix and chromosome scaffold in mammalian cells appearsin this primitive eukaryote,suggesting that these structuresmay have been originated from the early stages of eukaryoteevolution.     

Nuclear matrix and chromosome scaffold preparations of in vitro cultured bovine liver cells have two proteins in common

Nuclear matrices and chromosome scaffolds of in vitro cultured bovine liver cells were prepared under conditions that preserve the specific binding of the DNA. Protein compositions were analysed by electrophoresis and peptide mapping. Two slightly acidic polypeptides of apparent molecular masses 47 and 53 kDa were present in nuclear matrix as well as chromosome scaffold preparations. The corresponding matrix and scaffold proteins had identical peptide maps. Their putative function in the spatial organization of the DNA during the cell cycle is considered.