Disturbance in function and expression of condensin affects chromosome compaction in HeLa cells.

Condensin, a major non-histone protein complex on chromosomes, is responsible for the formation of rod-shaped chromosome in mitosis. A heterodimer composed of SMC2 (structural maintenance of chromosomes) and SMC4 subunits constitutes the core part of condensin. Although extensive studies have been done in yeast, fruit fly and Xenopus to uncover the mechanisms and molecular nature of SMC proteins, little is known about the complex in mammalian cells. We have conducted a series of experiments to unveil the nature of condensin complex in human chromosome formation. The results show that overexpression of the C-terminal domain of SMC subunits disturbs chromosome condensation, leading to formation of swollen chromosomes, while knockdown of SMC subunits severely disturbs mitotic chromosome formation, resulting in chromatin bridges between daughter cells and multiple nuclei in single cells. The salt extraction assay indicates that a fraction of the condensin complex is bound to chromatin in interphase, but most of the condensin bind to chromatin at the onset of mitosis. Thus, disturbance in condensin function or expression affects chromosome condensation and influences mitotic progression.

Platelets and fibrinogen facilitate each other in protecting tumor cells from natural killer cytotoxicity.

The functions of platelets and fibrinogen in protecting tumor cells from natural killer cytotoxicity have been discussed for more than 20 years. However, their exact roles and relationships in the process are still not clear. In this study, we show that tumor cells prefer to adhere to fibrinogen than to platelets, and fibrinogen can enhance the adhesion of tumor cells to platelets. Beta3 integrin plays an important role in the adhesion of B16F10 to platelets enhanced by fibrinogen. In the presence of thrombin, fibrinogen forms dense fibrin(ogen) layers around tumor cells. Tumor cells can induce platelets to aggregate and form thrombin. Platelets, as well as thrombin, can help fibrinogen protect tumor cells from lethal contact with natural killer cells and natural killer cytotoxicity. Hirudin, a specific inhibitor of thrombin, can reverse the effect of platelets on fibrinogen in blocking natural killer cytotoxicity. Our results suggest that fibrinogen helps platelets to adhere to tumor cells, and platelets in turn promote more fibrinogen to aggregate around tumor cells by forming thrombin. They facilitate each other in protecting tumor cells from natural killer cytotoxicity.

Dynamic changes of nucleolar DNA configuration and distribution during the cell cycle in Allium sativum cells.

To investigate the correlation between subnucleolar structure and function, the precise distribution and configuration of nucleolar DNA during the cell cycle of Allium sativum were determined using the NAMA-Ur DNA-specific staining technique. We showed that nucleolar DNA is present in two forms: compacted chromatin clumps and a decondensed DNA cloud. The form of the DNA within the nucleolus varied greatly as the cell cycle progressed. During telophase, chromosomes extended into the prenucleolar body. In early G1 phase, DNA was only located in the fibrillar centers in the form of the condensed chromatin clump, while in mid-G1, S and G2 phases, the two forms of DNA were distributed in the fibrillar centers (FC) and dense fibrillar component (DFC). In prophase of mitosis, nucleolar DNA, along with FC and DFC, was linked into a network structure and condensed into a large chromatin clump. The area of the DNA cloud in the dense fibrillar component changed during different phases of the cell cycle. Our results demonstrated that the configuration of nucleolar DNA undergoes a series of decondensations and condensations during the cell cycle to fulfill the function of the nucleoli during the different phases.

In situ comparative studies on subnucleolar distribution and configuration of plant rDNA.

The distribution and configurations of nucleolar DNA in Pisum sativum L., Allium sativum L., Triticum aestivum L. were analyzed by specific cytochemical staining using NAMA-Ur. It has been observed that in the nucleoli of different plant species, the DNA occupied different positions in different areas, which may imply a different status and strategy of rDNA transcription. Our results showed irregular clumps of rDNA surrounding FCs in semi-circular formations in P. sativum and T. aestivum, indicating a regular pattern of rDNA distribution and supporting the helix model of rDNA configuration. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, which suggests a possible origin for nucleolar DNA.

[Localization of nucleolar DNA and transcription sites of rRNA genes in situ in wheat cells].

By using the conventional electron microscopic technique and DNA specific cytochemical staining method (NAMA-Ur), we directly observed the arrangement and location of intranucleolar DNA in situ in wheat (Triticum aestivum L.) cells. The results showed that nucleolar DNA was found in Fibrillar Centers (FC), Dense Fibrillar Component (DFC) and the transitional region between FC and DFC. Moreover, the nucleolar DNA was distributed along the periphery of FC and by surrounding FC. We employed RNP preference staining (Bernhard staining) method to visualize the distribution and position of RNP in situ in nucleoli of wheat cells. The results directly showed that RNP mainly located in the transitional region between FC and DFC, in DFC and in Granular Component (GC). Moreover, RNP was irregularly distributed around FC. By employing anti-RNA/DNA hybrid antibodies, we directly and selectively labeled transcription sites of rRNA genes and testified that localization of transcription sites was not only in the transitional region between DFC and FC but also in DFC of nucleoli in wheat cells.

c-Abl is required for the signaling transduction induced by L-selectin ligation.

Lymphocyte recruitment onto inflamed tissues requires cells tethering to and rolling on vascular surfaces under flow. L-selectin is constitutively expressed on leukocytes to mediate the leukocytes' initial capture and subsequent rolling along the vessel. Apart from its adhesive function, engagement of L-selectin also results in cell activation, which is related to the completed signaling transduction. Here we show that ligation of L-selectin with its mAb increases c-Abl kinase activity, and that the activated c-Abl kinase can be recruited to and phosphorylate the cytoplasmic domain of L-selectin. In addition, the activated c-Abl kinase can regulate Zap70 kinase by increasing the phosphorylation of the Y319 site of Zap70 kinase and connect with Zap70 kinase through its SH2 domain. These results indicate that c-Abl kinase plays an important role in accepting and transferring the upstream activation events induced by L-selectin ligation.

c-Abl tyrosine kinase activates p21 transcription via interaction with p53.

c-Abl non-receptor tyrosine kinase has been implicated in many cellular processes including cell differentiation, stress response and regulating gene transcription. The mechanism by which c-Abl is involved in the regulation of gene transcription remains to be elucidated. In this study, we investigated the functions of c-Abl in the activation of p21 promoter. Our results showed that overexpression of c-Abl tyrosine kinase activated p21 promoter and endogenous p21 transcription in U2OS cells. We found that p53 is involved in the activation of p21 promoter by c-Abl, and integrative structure of p53 is required for regulating p21 transcription. In addition, the chromatin immunoprecipitation study demonstrated that c-Abl and p53 can be recruited to the region containing p53 binding site of p21 promoter, and c-Abl increases the DNA binding activity of p53 to the p21 promoter. Furthermore, not only the activation of p21 promoter but also the recruitment to p21 promoter by c-Abl is dependent on the interaction between c-Abl and p53 protein.

Nuclear actin is involved in the regulation of CSF1 gene transcription in a chromatin required, BRG1 independent manner.

Actin is an important protein in nucleus and has been implicated in transcription, however, the mechanism of its function in transcription is still not clear. In this article, we studied the role of actin in the regulation of human CSF1 gene transcription. Our results showed that nuclear actin stimulates the activity of CSF1 promoter, and the role in augmenting CSF1 gene transcription requires the formation of chromatin and Z-DNA structure. The ATP binding motifs of nuclear actin are essential for its function in regulating CSF1 gene transcription, and upon actin overexpression, there is an increase in the ATPase activity of nuclear proteins. Further investigation revealed that nuclear actin regulates CSF1 gene transcription in a BRG1 independent manner. Together, these original results have provided evidence for further understanding the mechanism of nuclear actin in regulating gene transcription.

Dynamic change and derivation process of FC and DFC through G1, S and G2 phases in HeLa cells.

In order to get a deeper understanding of the relationship between nucleolus structure and its function, the dynamic change and derivation of FC (fibrillar center) and DFC (dense fibrillar component) through interphase were investigated in HeLa cells synchronized at the ultrastructural level. The results showed that there was a process of FC and DFC derivation in the nucleolus of HeLa cells during interphase. In G1 phase there were a few big FCs in the nucleolus of the HeLa cell. In S phase DFC around the FC got thickened and the configuration of the DFC changed. A lot of tiny FCs were derived from parts of the thickened DFC. We called the FC and DFC formed in G1 phase as primary FC (pri-FC) and primary DFC (pri-DFC) and the FC and DFC derived from the thickened pri-DFC as secondary FC (sec-FC) and secondary DFC (sec-DFC). In G2 phase sec-FC and sec-DFC were gradually separated from pri-DFC and scattered evenly in the nucleolus. Few large pri-FCs coexisted with numerous tiny sec-FCs in the nucleolus of HeLa cells in G2 phase. Based on the results of our observation, we suggest here a model of the dynamic change and the process of derivation of FC and DFC through interphase.

c-Abl is involved in the F-actin assembly triggered by L-selectin crosslinking.

L-selectin is a cell adhesion molecule mediating the initial capture and subsequent rolling of leukocytes along the endothelial cells expressing L-selectin ligands. In addition to its action in adhesion, an intracellular signaling role for L-selectin has been recognized. Its cytoplasmic domain is involved in signal transduction following antibody crosslinking and in the regulation of receptor binding activity in response to intracellular signals. In this work, we demonstrated that L-selectin crosslinking led to F-actin polymerization and redistribution in human neutrophils. Using immuno-fluorescence microscopy, we observed that F-actin redistribution spatiotemporally related to the polarization of L-selectin. STI571, a specific inhibitor for cytoplasmic tyrosine kinase c-Abl, can inhibit F-actin polymerization and c-Abl redistribution in the activated neutrophils. Furthermore, we determined that c-Abl redistributed to the region where L-selectin polarized and associated with L-selectin in the activated neutrophils. The association between L-selectin and c-Abl was reduced by cytochalasin B. These results suggested that c-Abl was involved in the F-actin alteration triggered by L-selectin crosslinking in human neutrophils.

Chemically modified heparin inhibits the in vitro adhesion of nonsmall cell lung cancer cells to P-selectin.

PURPOSE: Several independent studies have indicated that tumor metastasis can be inhibited by chemically modified heparin with low anticoagulant activity in the different tumor models. The mechanism of inhibition by the heparin derivatives in part accounts for the interference of tumor cell-platelet interaction mediated by P-selectin. METHODS: In the present study, we demonstrated that both heparin and chemically modified heparins inhibited the adhesion of nonsmall cell lung cancer (NSCLC) cells to P-selectin under static or flow conditions in vitro. RESULTS: Flow cytometric analysis with the heparan sulfate-specific monoclonal antibody revealed that both NSCLC cells express heparan sulfate-like proteoglycans. Furthermore, heparinase treatment impaired P-selectin binding, indicating that heparan sulfate-like proteoglycans on the tumor cell surface are implicated in the adhesion of NSCLC cells to P-selectin. CONCLUSIONS: These findings suggest that some chemically modified heparins with low anticoagulant activity may deserve further testing in the experimental NSCLC treatment protocols.

[The relationship between abnormity of synaptonemal complex and male fertility impairment in human].

It was said that 11% of all men showed their infertility. The genetic origins of male infertility may be classified into three main groups: chromosome abnormalities, microdeletions and gene mutations. Growing literature has shown that spermatogenesis failure or reproductive failure (pregnancy wastages) occurred in male carriers of chromosomal distortion/aberration. But the mechanisms remain largely unsolved. Synaptonemal complex (SC) of human spermatocytes from such carriers offers new information. This review aims to summarize recent development of SC analysis for male infertile diagnosis and sum up our results obtained recently. The relationship between male infertility/sterility and SC abnormity was discussed and reviewed as following five aspects. (1) The association of XY-bivalent and the rearranged autosomes interfere with or affect, by their contact, X chromosome normal functions. (2) Male infertility is related to the incomplete pairing in break regions of rearranged autosomes. (3) SC fragmentation, lateral elements (LEs) swelling and pairing disorder result in spermatogenic failure. (4) This heterosynapsis at early stage of meiosis in rearranged autosomes, results in unbalanced germs and pregnancy wastages. (5) Gene mutations of SC proteins result in male infertility.

Selectively desulfated heparin inhibits P-selectin-mediated adhesion of human melanoma cells.

Accumulating evidence has suggested that one of the mechanisms by which heparin inhibits metastasis is by blocking the P-selectin-based interaction of platelets with tumor cells. Here we demonstrate that the sulfate groups at C6/N and especially C6, but not C2 and C3, of heparin play a critical role in P-selectin recognition and that 2-O,3-O-desulfated heparin can block P-selectin-mediated A375 human melanoma cell adhesion. Our findings show that chemical modification of heparin, especially 2-O,3-O-desulfation, may result in a therapeutic agent that is anti-metastatic because it blocks unwanted P-selectin-dependent adhesion but that lacks dose-limiting anticoagulant effects.

c-Abl tyrosine kinase regulates c-fos gene expression via phosphorylating RNA polymerase II.

c-Abl tyrosine kinase, predominantly distributed in nucleus, has been implicated in many important cellular processes including the regulation of gene transcription. In this study, we showed that c-Abl promoted the transcription of c-fos gene, both exogenously and endogenously. The nuclear localization and tyrosine kinase activity of c-Abl were required for the activation of c-fos gene. c-Abl was associated with RNA polymerase II (RNAP II) in vivo and augmented the tyrosine phosphorylation of the largest subunit of RNAP II. In addition, c-Abl and RNAP II could be recruited to the region of c-fos promoter. The combined results suggest that c-Abl plays an important role in the transcriptional regulation of c-fos gene and the tyrosine phosphorylation of the largest subunit of RNAP II by c-Abl is involved in the regulating process.

Identification and characterization of coiled body-like structures in pea (Pisum sativum L.).

Coiled bodies (CBs) are nuclear organelles which were considered as "universal" nuclear structures in eukaryotic cells, but the formation and function of CBs, especially in plant cells, remained unclear. In this article we reported that CBs in meristematic cells of pea are oval to round obstacles in nucleus and in adjacent to nucleolus, often have the same electron density with nucleolus. We found that CBs could be stained by the rRNP preference staining method, but no rDNA was detected in the structure. Furthermore, our results of immunoelectron microscopy showed that several processing factors, include fibrillarin, U3 snoRNA and ITS1, were present in CB. It seems probable that CBs is derived structurally from nucleolus and act as transport, storage and processing subnucleolar organelles.

Subnucleolar distribution and organization of Vicia faba L. rDNA in situ.

The distribution and organization of nucleolar DNA in Vicia faba L. was analyzed by specific cytochemical staining using NAMA-Ur. The results showed that nucleolar DNA was distributed in the FCs and at the FC/DFC junctions. Statistical analysis showed that the rRNA genes occupied about one-third of the total dense fibrillar component region. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, suggesting a possible origin for nucleolar DNA.

Actin is closely associated with RNA polymerase II and involved in activation of gene transcription.

Biochemical and morphological studies have demonstrated the presence of actin in the nucleus of different eukaryotic cells, whereas its role remains unclear. In this work, we studied the interaction and the functional relationship between nuclear actin and RNA polymerase II (RNAP II). The immunofluorescence study demonstrated a clear co-localization of nuclear actin with RNAP II in HeLa cells. Meanwhile, actin can be immunoprecipitated by anti-RNAP II antibody, indicating that they could interact with each other. Treatment of cells with alpha-amanitin induced the formation of actin bundle network in the nucleoplasm. Blocking of the formation of filamentous actin (F-actin) by cytochalasin B modified the distribution of actin. Although the actin content remained unchanged in resting and concanavalinA stimulated mouse lymphocytes, the actin content in the nuclei showed a progressive increase after stimulation. Furthermore, the antibody against actin blocked RNA synthesis in a eukaryotic in vitro transcription system. These observations implicate that nuclear actin interacts with RNAP II and may have function on the RNAP II-mediated transcription.

Extensive alterations in DNA methylation and transcription in rice caused by introgression from Zizania latifolia.

It has been demonstrated that insertion of foreign DNA into mammalian genome can profoundly alter the patterns of DNA methylation and transcription of the host genome. Introgression of alien DNA into plant genomes through sexual crossing and genetic engineering are commonly used in breeding, but it is not known if plant genomes have similar responses to alien DNA introgression as those of animals. Two stable rice lines with introgression from wild rice, Zizania latifolia, were analyzed for patterns of cytosine DNA methylation and transcription of a set of selected sequences, including cellular genes and transposable element (TE)-related DNA segments. In 21 of the 30 studied sequences, marked changes in DNA methylation and/or transcription were observed compared with those of the rice parent. In all analyzed sequences, the absence of Zizania homologues in the introgression lines was confirmed. No change in DNA methylation and expression patterns was detected in randomly selected individuals of the rice parent nor in two sibling lines without introgressed Zizania DNA. The changed methylation patterns in both introgression lines were stably maintained in all five randomly sampled individuals of a given line, as well as in selfed progenies of the lines. Changed patterns in methylation and expression were also found in an independently produced asymmetric somatic nuclear hybrid (SH6) of rice and Z. latifolia that involves a different rice genotype but also contains a small amount of Z. latifolia DNA integrated into the rice genome. Thus, we have demonstrated that alien DNA introgression into a plant genome can induce extensive alterations in DNA methylation and transcription of both cellular genes and TE-related DNA segments in a genotype-independent manner.

Unpolymerized nuclear actin is involved in the activation of CSF-1 gene transcription.

Nuclear actin is a nuclear component in many kinds of eukaryotic cells but its function is still not clear. In this study, we overexpressed actin in nuclei and found that it promoted transcription of the CSF-1 gene, both exogenous and endogenous, approximately two-fold. Cytochalasin B did not affect this function of nuclear actin. Our results suggest that nuclear actin plays a role in regulating CSF-1 gene transcription, and this role does not depend on actin polymerization.

Modified heparin inhibits P-selectin-mediated cell adhesion of human colon carcinoma cells to immobilized platelets under dynamic flow conditions.

Accumulating evidence indicates that the formation of tumor cell platelet emboli complexes in the blood stream is a very important step during metastases and that the anti-metastasis effects of heparin are partially due to a blockade of P-selectin on platelets. In this study, heparin and chemically modified heparins were tested as inhibitors of three human colon carcinoma cell lines (COLO320, LS174T, and CW-2) binding to P-selectin, adhering to CHO cells expressing a transfected human P-selectin cDNA, and adhering to surface-anchored platelets expressing P-selectin under static and flow conditions. The aim was to screen for heparin derivatives with high anti-adhesion activity but negligible anticoagulant activity. In this study, four modified heparins with high anti-adhesion activity were identified including RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin. NMR analysis proved the reliability of structure of the four modified heparins. Our findings suggested that the 6-O-sulfate group of glucosamine units in heparin is critical for the inhibition of P-selectin-mediated tumor cell adhesion. Heparan sulfate-like proteoglycans on these tumor cell surfaces are implicated in adhesion of the tumor cells to P-selectin. Some chemically modified heparins with low anticoagulant activities, such as 2/3ODS-heparin, may have potential value as therapeutic agents that block P-selectin-mediated cell adhesion and prevent tumor metastasis.