Photoluminescence and magnetism integrated multifunctional black phosphorus probes through controllable PO bond orbital hybridization.

Biological probes with integrated photoluminescence and magnetism characteristics play a critical role in modern clinical diagnosis and surgical protocols combining fluorescence optical imaging (FOI) with magnetic resonance imaging (MRI) technology. However, traditional magnetic semiconductors can easily generate a spin splitting at the Fermi level and half-metallic electronic occupation, which will sharply reduce the radiation recombination efficiency of photogenerated carriers. To overcome this intrinsic contradiction, we propose a controllable oxidation strategy to introduce some particular PO bonds into black phosphorus nanosheets, in which the p orbital hybridization between P and O atoms not only provides some carrier recombination centers but also leads to a room-temperature spin polarization. As a result, the coexistence of photoluminescence and magnetism is realized in multifunctional black phosphorus probes with excellent biocompatibility. This work provides a new insight into integrating photoluminescence and magnetism together by intriguing atomic orbital hybridization.

Function of GATA transcription factors in hydroxyurea-induced HEL cells.

HEL cells, a human erythroleukemia cell line, mainly express the fetal (gamma) globin gene and trace amount of the embryonic (epsilon) globin gene, but not adult (beta) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (beta) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of human beta-globin gene, including the proximal regulatory element (the beta-promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU-induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human beta-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of human beta-globin gene in HU-induced HEL cells.

[The apoptosis of K562 cells induced by IL-3 and hydroxyurea cooperatively].

The effect of IL-3 and hydroxyurea on human erythroleukemia cell line (K562 cells) was demonstrated by using the electro-microscopy and flow cytometry. Our data showed that neither IL-3 nor hydroxyurea could induce the apoptosis of K562 cells alone. However, the IL-3 and hydroxyurea could induce the apoptosis of K562 cells cooperatively. Analysis with flow cytometry showed that the percentage of apoptotic cells was about 31.90% after K562 cells were induced by IL-3 and hydroxyurea cooperatively for 5 days, and the sub-G1 peak (apoptotic peak) was detected in the induced K562 cells. Meanwhile, the percentage of S-phase in the IL-3 and hydroxyurea induced K562 cells was increased, and the proliferation of the induced K562 cells was inhibited significantly. Furthermore, the IL-3 and hydroxyurea induced K562 cells showed chromatin condensation with regular crescents at the nuclear edges and apoptotic bodies. It suggested that IL-3 could enhance the sensitivity of K562 cells to hydroxyurea and the apoptosis of K562 cells could be induced by IL-3 and hydroxyurea cooperatively.

The structure of the nucleosome core particle of chromatin in chicken erythrocytes visualized by using atomic force microscopy.

The structure of the nucleosome core particle of chromatin in chicken erythrocytes has been examined by using AFM. The 146 bp of DNA wrapped twice around the core histone octamer are clearly visualized. Both the ends of entry/exit of linker DNA are also demonstrated. The dimension of the nucleosome core particles is approximately 1-4 nm in height and approximately 13-22 nm in width. In addition, superbeads (width of approximately 48-57 nm, height of approximately 2-3 nm) are occasionally revealed, two turns of DNA around the core particles are also detected.

[Analysis of the binding of GATA factors to the regulatory elements of human beta-globin gene in hydroxyurea-induced and uninduced HEL cells].

HEL cells, a human erythroleukemia cell line, expressing mainly the gamma-globin genes, small amount of epsilon-globin gene, but not beta-globin gene. Our previous studies demonstrated that beta-globin gene could be expressed in HEL cells induced by hydroxyurea. However, the molecular mechenism is still unknown. Here the binding patterns of GATA factors (GATA-1 and GATA-2) to the regulatory elements of human beta-globin gene were examined with the nuclear extracts from hydroxyurea-induced and uninduced HEL cells. Our results showed in EMSA assay that GATA factors could bind to the core sequence of HS2(-10681 to -10971 bp), the 3' flanking sequence of HS2 core(-10323 to -10680 bp) and the promoter of human beta-globin gene(+20 to -112 bp). However, the binding patterns between hydroxyurea-induced and uninduced HEL cells were different. Furthermore, by using Western-blot analysis, our data showed that the amount of GATA-2 was decreased in hydroxyurea-induced HEL cells. In contrast to GATA-2, the amount of GATA-1 was increased in hydroxyurea-induced HEL cells. These results showed that the different members of GATA family might play different roles during the differentiation of erythrocytes. GATA-1 may stimulate the differentiation of HEL cells, while GATA-2 can probably inhibit the differentiation of HEL cells.

The 5'-flanking cis-acting elements of the human epsilon-globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins.

The nuclear matrix attachment regions(MARs) and the binding nuclear matrix proteins in the 5'-flanking cis-acting elements of the human epsilon-globin gene have been examined. Using in vitro DNA-matrix binding assay, it has been shown that the positive stage-specific regulatory element (epsilon-PREII, -446 bp(-)-419 bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating that epsilon-PREII may be an erythroid-specific facultative MAR. In gel mobility shift assay and Southwestern blotting assay, an erythroid-specific nuclear matrix protein (epsilon-NMP kappa) in K562 cells has been revealed to bind to this positive regulatory element (epsilon-PREII). Furthermore, we demonstrated that the silencer (-392 bp(-)-177 bp) upstream of the human epsilon-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nuclear matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element might be a constitutive nuclear matrix attachment region (constitutive MAR). Our results demonstrated that the nuclear matrix and nuclear matrix proteins might play an important role in the regulation of the human epsilon-globin gene expression.

[Studies on DNA-protein interactions in the HS3 of the human beta-LCR].

Upstream of the human epsilon-globin gene is the Locus Control Region (LCR) of the human beta-globin cluster, which consists of four DNase-I hypersensitive sites(HS1-HS4). It has been reported in transgenic experiments that HS3 preferentially regulates epsilon-globin gene expression. In order to elucidate the regulatory function of HS3 in the expression of globin gene, nuclear extracts from mouse hematopoietic tissues at several developmental stages were prepared and the binding of the nuclear factors to HS3 was analysed by using electrophoresis mobility shift assay(EMSA). Our results showed that the binding patterns of HS3 with nuclear extracts of mouse hematopoietic tissues at day 13 and day 18 of gestation were completely different; furthermore, by Southwestern Blot, the distinction between both stages was also demonstrated. It has been known that GATA and CACCC binding motifs are contained within HS3 core region. Using competitive gel-retardation assay, we found that no shift bands could be competed by using CACCC motif as a competitor. However one shift band at day 13 and day 18 of gestation could be competed respectively by using GATA motif as a competitor. We suggested that the shift bands, which could not be competed by both motifs, might be novel and stage-specific factors. In addition, by using Western Blot, we demonstrated that the two shift bands at day 13 and day 18 of gestation, competed by GATA motif, were GATA-2 and GATA-1 respectively: GATA-1 was expressed in mouse hematopoietic tissues at day 18 of gestation and not expressed at day 13 of gestation; however, GATA-2 was only expressed in mouse hematopoietic tissues at day 13 of gestation. According to these results, we speculated that HS3 might play an important role in regulation of stage-specific expression of globin genes through interaction between stage-specific nuclear factors and HS3.

Characterization of antigen-antibody complexes by size-exclusion chromatography coupled with low-angle light-scattering photometry and viscometry.

In this paper, the molecular masses (M(r)s) of the complexes of monoclonal anti-BSA (antibody to bovine serum albumin) (clone: 33) and monomer BSA were determined on-line by using size-exclusion chromatography (SEC) coupled with a low-angle laser light-scattering (LALLS) detector and two concentration detectors, ultraviolet (UV) and refractive index (RI) (SEC-LALLS/UV/RI system). Also, the size and M(r)s of the complexes were evaluated by the SEC-LALLS/UV/viscometer (VISC) system. This study demonstrated that, for small size macromolecules, the combination of light scattering and viscosity detection was a suitable choice for determining their M(r)s and sizes.

The apoptosis of HEL cells induced by hydroxyurea.

Hydroxyurea has been used to synchronize cultured cells to S-phase and used to treat patients with sickle-cell anemia. Recently, we found that hydroxyurea can induce the apoptosis of HEL (human erythroleukemia) cells. The induced HEL cells showed ultrastructurally chromatin condensation with regular crescents at the nuclear edges and apoptotic bodies. However, the cells of K562, another human erythroleukemia cell line, did not show such morphological changes. Under fluoroscope, the HEL cells after induction often displayed a clear reduction in nuclear diameter and nuclear chromatin cleavage and condensation and the presence of nuclear ring and apoptotic bodies. Analysis with flow cytometry showed that the percentage of apoptotic cells is about 30-40% after HEL cells were induced by hydroxyurea for 3 days. DNA ladder can be observed by electrophoretic analysis.

[Transacting factors binding to the positive control region (PCR) in the 5' flanking sequence of human beta-globin gene].

Using gel mobility shift assay, three protein factors (Pa, Pb, Pc) binding to the positive control region (PCR) in the 5' flanking sequence of human beta-globin gene were detected in the nuclear extracts from mouse fetal liver at d 18 or d 19 of gestation. Competitive experiment showed that Pb and Pc could bind to GATA-1 motif, therefore could be the members of GATA family. While Pa was a new and developmental stage specific trans-acting factor, we suggested that the factor Pa was related to the activation of beta-globin gene.

[The effects of hydroxyurea on cell-cycle distribution and the expression of human beta-globin gene in K 562 cells].

Although the hydroxyurea (HU) has been extensively studied, little is known of its molecular mechanism in controlling the expression of human globin gene and in modulating the progression of cell-cycle in K 562 cell. In the present study, the effect of hydroxyurea on proliferative kinetics of K 562 cells was examined by monitoring the number of cells during a period of 8 day's cell culture. Our results showed that there was a dose related decrease in cell growth when K562 cells were incubated with HU. Moreover, cell-cycle analysis demonstrated that HU had profound effect on cell-cycle distribution. In the case of the induced K 562 cells, there was an increased accumulation of cells in S phase and a decreased fraction of cells in G 1 and G 2 + M phase. Furthermore, HU could induce the expression of human beta-globin gene in the induced K 562 cells. Our results indicate that HU has a potential to inhibit the proliferation of K 562 cells and to stimulate the terminal differentiation of this cell.

[The role of hydroxyurea in globin gene expression and cell differentiation of human erythroleukemia cell line].

The HEL cells, a human erythroleukemia cell line, express mainly the fetal globin gene and small amount of the embryonic (epsilon) globin gene, but not the adult (beta) globin gene. Hydroxyurea, a small organic compound, has been successfully used to treat sickle cell anemia and beta-thalessaemia. Our data demonstrated that the growth rate of HEL cell proliferation was inhibited by different doses of hydroxyurea (from 50 mumol/L to 200 mumol/L). Using both routine RT-PCR and quantitative PCR analyses, we revealed that the expression of beta-globin gene was sharply activated and alpha-globin gene was almost completely silenced when HEL cells were induced for 3 or 5 days. Meanwhile, gamma-globin gene was expressed with no much difference between induced and uninduced HEL cells. We also demonstrated that the expression of GATA-1 and NF-E 2, which were two of the most important transcription factors in erythrocyte development, was activated about 3 folds in the induced cells. We suggested that the induction of GATA-1 and NF-E 2 expression by hydroxyurea might lead to activation of the adult beta-globin gene through some pathways of signal transduction, therefore, hydroxyurea might play a role to induce HEL cells to terminal differentiation.

Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy (AFM).

The organization of the higher order structure of chromatin in chicken erythrocytes has been examined with tapping-mode scanning force microscopy under conditions close to their native environment. Reproducible high-resolution AFM images of chromatin compaction at several levels can be demonstrated. An extended beads-on-astring (width of approximately 15-20 nm, height of approximately 2-3 nm for each individual nucleosome) can be consistently observed. Furthermore, superbeads (width of approximately 40 nm, height of approximately 7 nm) are demonstrated. Visualization of the solenoid conformation at the level of 30 nm chromatin fiber is attained either by using AFM or by using electron microscopy. In addition, tightly coiled chromatin fibers (approximately 50-60 nm and approximately 90-110 nm) can be revealed. Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning the folding pattern of interphase chromatin fibers.

Trans-acting factors from the human fetal liver binding to the human epsilon-globin gene silencer.

The developmental stage-specific silencing of the human epsilon-globin gene during embryonic life is controlled, in part, by the silencer (-392 bp approximately -177 bp) upstream of this gene. In order to elucidate its role, the nuclear extract from the human fetal liver has been prepared and the interactions between trans-acting factors and this silencer element have been examined. By using DNaseI footprinting assay, a major protected region from -278 bp to -235 bp within this silencer element was identified. Furthermore, we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MW approximately 32, 28, 26 and 22 kD) in the nuclear extract isolated from the human fetal liver, which could specifically bind to this region. Our results suggested that these trans-acting factors might play an important role in silencing the human embryonic epsilon-globin gene expression at the fetal stage through the interactions with this silencer.

[Interaction of cis-regulatory element in the 5' flanking sequence of human beta-globin gene with trans-acting factors at different developmental stages].

Human beta-globin gene mainly expressed in the adult bone marrow, while not expressed in fetal liver, adult liver and K562 cells. Using gel mobility shift assay, different protein factors binding to the regulatory elements (from -372 to -194 bp) in the 5' flanking sequence of human beta-globin gene were detected in the nuclear extracts from human fetal liver, adult liver and K562 cells respectively. Competitive experiments showed that both the protein factors from adult and K562 cells not only bind to negative control region 2 (NCR 2, from -372 to -224 bp), but also bind to positive control region (PCR, from -223 to -194 bp), suggesting that there may be similar mechanism to silence the expression of human beta-globin gene in these two kind of cells. The protein factor in human fetal liver only binds to NCR 2, indicating that there is a particular silencing mechanism for beta-globin gene expression in human fetal stage.

Interaction between HMG proteins (1 + 2) and the negative regulatory region 1 (NCR1) in the 5'-flanking sequence of the human beta-globin gene.

The pattern of high mobility group proteins 1 and 2 (HMG1,2) interaction with the 5'-flanking sequence of the human beta-globin gene has been analyzed by scanning tunnelling microscopy (STM). A 200 bp negative regulatory region in the 5'-flanking sequence of the human beta-globin gene can be folded by HMG proteins 1 and 2 into a circular structure (diameter 70 +/- 6 A) with a linear tail which seems to be a left-handed double helix structure.

Binding of HMG proteins to the 5'-flanking sequence of human beta-globin gene.

Our previous studies have identified that there are at least three regulatory regions (two negative regions and one positive region) in the 5'-flanking sequence of human beta-globin gene (-610 to +1 bp). The binding of HMG proteins to both negative regulatory regions was examined by the gel mobility shift and DNase I protection assays. In gel mobility shift assay, we observed that HMG proteins 1 and 2 could bind to both negative regulatory regions (NCR1 and NCR2). Using the gel shift competition assay, we identified that the binding proteins between the two regions are different from each other. DNase I protection analysis shows that HMG proteins 1 and 2 only bind to one site (between -560 and -533 bp) in NCR1. However, two protected regions can be detected in NCR2, one between -272 and -252 bp relative to the cap site, the other between -306 and -329 bp. We also observed that HMG proteins 14 and 17 could not bind to both negative regions, so it seems that HMG proteins 1 and 2 may play an important role in the regulation of beta-globin expression through DNA-protein interaction or through protein-protein interaction.