Protein Phosphatase 2C of Toxoplasma Gondii Interacts with Human SSRP1 and Negatively Regulates Cell Apoptosis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability.</p><p><b>METHODS</b>The yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry.</p><p><b>RESULTS</b>We identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1.</p><p><b>CONCLUSION</b>TgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth.</p>

Inhibitory effects of high mobility group chromosomal protein N2 on human tongue carcinoma transplanted in nude mice / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the inhibitory effect of high mobility group chromosomal protein N2 (HMGN2) on human tongue carcinoma tumor in nude mice.</p><p><b>METHODS</b>A transplantation tumor model in nude mice was constructed by injecting Tca8113 cells. After a week, negative control groups, masculine control groups, and HMGN2 groups were established. Cell culture of the three groups were separately injected with washing buffer II, cis-dichlorodiamineplatinum (DDP), and HMGN2 protein. The tumors were moved after four treatments, and then analyzed by hematoxylin-eosin (HE) staining.</p><p><b>RESULTS</b>A transplanted tumor model was established successfully. The volumes of HMGN2 groups and masculine control groups were smaller than those of the negative groups. Mouse weight did not differ among the three groups. Average tumor weight of the negative group was (0.38 +/- 0.19)g, that of the HMGN2 group was (0.21 +/- 0.15)g, and that of the DDP group was (0.23 +/- 0.16)g. These factors indicated no statistically significant difference among the three groups. The tumor inhibitory rate of HMGN2 group was 45.71%, and that of the positive group was 39.44%. Based on evaluation by naked eye, the tumor in the negative group was larger than that in other groups. In addition, cell necrosis was observed during HE staining.</p><p><b>CONCLUSION</b>HMGN2 could significantly inhibit growth of the transplanted tumor in nude mice.</p>

Study on prohibition of high mobility group chromosomal protein N2 against human oral squamous cell carcinoma in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Take human oral squamous cell carcinoma Tca8113 as experimental model, and study the anti oral squamous cell carcinoma activity of high mobility group chromosomal protein N2 (HMGN2) molecule.</p><p><b>METHODS</b>Train a large number of recombinant human HMGN2 expression vector Escherichia coli BL21. HMGN2 was expressed under isopropyl-1-thio-beta-galactopyranoside (IPTG) induction and purified by B-PER GST Fusion Protein Purification Kit. A variety of concentrations HMGN2 were added to cell culture medium, cells were tested by MTT, Hoechst 33342 fluorescence staining, flow cytometry assay and Western-blot.</p><p><b>RESULTS</b>MTT results proved that HMGN2 could significantly inhibit human oral squamous cell carcinoma Tca8113 growth. Hoechst 33342 fluorescence staining, flow cytometry assay test and Western-blot proved HMGN2 could make Tca8113 cells morphological change, make Tca8113 cells block in S period of cell cycle and strongly promote Tca8113 cells to apoptosis.</p><p><b>CONCLUSION</b>HMGN2 can promote apoptosis of oral squamous cell carcinoma cells.</p>

Genes related with male gonadal morphogenesis in mammals / 中华男科学杂志

Gene expressions are sex-specific in the sex development of mammals. Different genes express in different phases and tend to change with the time. The functions of some genes, such as SRY, SOX9, SOX8, DAX1, and FGF9, have already been defined in male gonadal morphogenesis. This paper presents a review of the genes involved in the formation of the male gonad in mammals.

Differentiation of mesenchymal stem cells into nucleus pulposus cells in vitro / 华中科技大学学报（医学）（英德文版）

To find a new source of seed cells for constructing tissue-engineered intervertebral disc, nucleus pulposus (NP) cells and mesenchymal stem cells (MSCs) were isolated from New Zealand white rabbits. The nucleus pulposus cells population was fluorescence-laelled and co-cultured with MSCs with or without direct contact. Morphological changes were observed every 12 h. Semi-quantitative reverse transcriptase-polymerase chain reaction was performed to assess the expression levels of Sox-9, aggreacan and type II collagen every 24 h after the co-culture. MSCs treated with direct contact rounded up and presented a ring-like appearance. The expression of marker genes was significantly increased when cells were co-cultured with direct contact for 24 h. No significant change was found after coculture without direct contact. Co-culture of NP cells and MSCs with direct contact is a reliable method for generating large amount of NP cells used for cell-based tissue engineering therapy.

Cloning and study of adult-tissue-specific expression of Sox9 in Cyprinus carpio.

The Sox9 gene is one of the important transcription factors in the development of many tissues and organs, particularly in sex determination and chondrogenesis. We amplified the genomic DNA of Cyprinus carpio using degenerate primers, and found that there were two versions of Sox9 in this species: Sox9a and Sox9b, that differ in having an intron of different length (704 bp and 616 bp, respectively) in the conserved HMG box region that codes for identical amino acid sequences. We used a two-phase rapid amplification of cDNA ends (RACE) for the isolation of full-length cDNA of Sox9b. Sequence analyses revealed a 2447-bp cDNA containing 233-bp 5' untranslated region, a 927-bp 3' untranslated region, including poly(A), and a 1287 bp open reading frame (ORF) encoding a protein of 428 amino acids. The HMG box of 79 amino acid motif was confirmed from positions 96-174. Sequence alignment showed that the identity of amino acids of Sox9 among ten animal species, including C. carpio, is 75%, indicating that the Sox9 gene is evolutionarily quite conserved. The expression level of Sox9b gene varied among several organs of adult C. carpio, with the level of expression being highest in the brain and testis.

Construction of recombinant baculovirus Ac-CMV-hSox9 for gene therapy of intervertebral disc degeneration / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration.</p><p><b>METHODS</b>Bac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDuljgreen fluorescene protein (GFP)-CMV (pFGC)-hSox9. The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani (LB) agarose culture medium containing isopropyl-beta-D-thiogalactoside (IPTG), X-gal, gentamicin, kanamycin and tetracycline. The white colonies were selected and cultured for amplification, and the hSox9Bacmid DNA was extracted. After verification, recombinant baculovirus Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells. The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining.</p><p><b>RESULTS</b>Polymerase chain reaction (PCR) confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein. Western blotting and immunohistochemical staining analysis indicated that exogenous hSox9 gene was expressed in the disc cells.</p><p><b>CONCLUSIONS</b>The successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.</p>

Recent integrations of mammalian Hmg retropseudogenes.

We propose that select retropseudogenes of the high mobility group nonhistone chromosomal protein genes have recently integrated into mammalian genomes on the basis of the high sequence identity of the copies to the cDNA sequences derived from the original genes. These include the Hmg1 gene family in mice and the Hmgn2 family in humans. We investigated orthologous loci of several strains and species of Mus for presence or absence of apparently young Hmg1 retropseudogenes. Three of four analysed elements were specific to Mus musculus, two of which were not fixed, indicative of recent evolutionary origins. Additionally, we datamined a presumptive subfamily (Hmgz) of mouse Hmg1, but only identified one true element in the GenBank database, which is not consistent with a separate subfamily status. Two of four analysed Hmgn2 retropseudogenes were specific for the human genome, whereas a third was identified in human, chimpanzee and gorilla genomes, and a fourth additionally found in orangutan but absent in African green monkey. Flanking target-site duplications were consistent with LINE integration sites supporting LINE machinery for their mechanism of amplification. The human Hmgn2 retropseudogenes were full length, whereas the mouse Hmg1 elements were either full length or 3'-truncated at specific positions, most plausibly the result of use of alternative polyadenylation sites. The nature of their recent amplification success in relation to other retropseudogenes is unclear, although availability of a large number of transcripts during gametogenesis may be a reason. It is apparent that retropseudogenes continue to shape mammalian genomes, and may provide insight into the process of retrotransposition, as well as offer potential use as phylogenetic markers.

Isolation and sequencing of seven Sox genes from the lacertid lizard Eremias breuchleyi

The Sox family of genes shares a high sequence similarity with the HMG box region of the human Y chromosomal gene, SRY. We used highly degenerate primers to clone and sequence seven Eremias breuchleyi Sox genes (EbSox2, EbSox3, EbSox4, EbSox11, EbSox12, EbSox14 and EbSox21). A database search for the cloned sequences revealed the following percentage identity with the homologous human SOX genes: EbSox2 = 96 percent, EbSox3 = 88 percent, EbSox4 = 94 percent, EbSox11 = 99 percent, EbSox12 = 96 percent, EbSox14 = 98 percent, EbSox21 = 97 percent. Cluster analysis indicates that they seem to belong to group B and group C of Sox gene family, respectively.

The regulation effect of interleukin-1 on expression of cartilage specificity gene Sox9 mRNA in the human intervertebral discs / 中华外科杂志

<p><b>OBJECTIVE</b>To assess the regulating effects of interleukin-1 (IL-1) on gene expression of cartilage specificity gene Sox9 and type II collagen mRNA in the human intervertebral discs.</p><p><b>METHODS</b>RT-PCR were used to investigate the effects of IL-1 on gene expression of Sox9 and type II collagen mRNA in intervertebral discs cells cultures of embryo.</p><p><b>RESULTS</b>The Sox9 and type II collagen mRNA in intervertebral discs were decreased progressively along with the addition concentrations of IL-1 than the controls. And the mRNA of Sox9 and type II collagen also markedly decreased with the time of culture.</p><p><b>CONCLUSIONS</b>IL-1 could cause dose-dependent and time-dependent inhibition effects on Sox9 and type II collagen gene expression in human intervertebral discs.</p>

Overexpression of SOX9 in mouse embryonic stem cells directs the immediate chondrogenic commitment

Mouse embryonic stem (mES) cells are capable of undergoing chondrogenesis in vitro. To enhance this process, the human SOX9 (hSOX9) cDNA was delivered into mES cells and the clones overexpressing hSOX9 (denoted as mES-hSOX9 cells) were verified by Western blot analysis. The transcripts of collagen IIA (a juvenile form), aggrecan and Pax1 were expressed in mES-hSOX9 cells grown on feeder layers, suggesting the immediate effect of exogenous SOX9 on chondrogenesis. However, SOX9 overexpression did not affect the cell cycle distribution in undifferentiated mES cells. Upon differentiation, collagen IIB (an adult form) was detected in day 3 immature embryoid bodies. In addition, the overexpression of exogenous SOX9 significantly induced transcriptional activity driven by SOX9 binding site. Taken together, we for the first time demonstrated that constitutive overexpression of exogenous SOX9 in undifferentiated mES cells might have dual potentials to induce both chondrogenic commitment and growth capacity in the undifferentiated status.

Advances in gonadal differentiation regulated by SRY / 中华男科学杂志

Gondadal differentiation is genetically determined in humans. Sex is determined when the bipotential embryologic tissues differentiate into testes or ovary. SRY, a gene located on the Y chromosome, triggers a complex genetic cascade leading to testicular differentiation. However, only a minority of 46, XY sex reversal patients can be explained by SRY mutations, suggesting that other genes influencing sex determination are to be discovered. Recent studies show that testis differentiation requires insulin receptor family function in mice. SRY normally requires two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for gonadal differentiation.

Involvement of Sox-4 in the cytochrome c-dependent AIF-independent apoptotic pathway in HeLa cells induced by delta12-prostaglandin J2

delta12-Prostaglandin (PG) J2 is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed delta12-PGJ2-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of delta12-PGJ2- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated delta12-PGJ2-induced caspase activation, loss of mitochondrial transmembrane potential (delta psi m), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the delta psi m was dissipated. One of the earliest events observed in delta12-PGJ2-induced apoptotic events was dissipation of delta psi m, the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of delta psi m depolarization in delta12-PGJ2- induced apoptosis. Up-regulation of Sox-4 protein by delta12-PGJ2 was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that delta12-PGJ2 induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that delta12-PGJ2-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by delta12-PGJ2.

Sox-4 is a positive regulator of Hep3B and HepG2 cells' apoptosis induced by prostaglandin (PG)A2 and 12-PGJ2

We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A2 and delta(12)-PGJ(12) induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca(2+) ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA(2) and delta(12)-PGJ(12) in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.

Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells

Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG) are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 µM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994) 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980) 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30 per cent H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

The expression of the high mobility group I(Y) mRNA in thyroid cancers: useful tool of differential diagnosis of thyroid nodules

OBJECTIVE: Thyroid nodule is frequent and occurs in about 5+ACU- of the general population. In contrast, thyroid cancer is much less frequent and occurs in about 5-10+ACU- of thyroid nodules. Distinguishing between benign and malignant lesions is an important task that is best accomplished by fine needle aspiration. Recently, Chiappetta et al. reported that the expression of the high mobility group (HMG) I(Y) proteins correlates with the malignant phenotype of human thyroid neoplasia, and suggested that the detection of the HMG I(Y) proteins might be a valid tool for an easy and sensitive discrimination assay between benign and malignant neoplastic thyroid disease. METHODS: We evaluated the expression of the HMG I(Y) mRNA in 39 frozen thyroid tissues from patients with thyroid nodule by semiquantitative RT-PCR. RESULTS: The expression of the HMG I(Y) mRNA was low in all of 10 normal thyroid tissues. In all of 3 adenomatous goiters, 6 follicular adenomas and 2 Hurthle cell adenomas, the HMG I(Y) mRNA expression level was low. In 11 of 13 papillary carcinomas and all of 5 follicular carcinomas, the HMG I(Y) mRNA expression level was high. CONCLUSION: These results indicate that there is a correlation between the expression of HMG I(Y) and the malignant phenotype of thyroid cancer, suggesting that these proteins may be useful as a marker in thyroid cancer.

Separation of nonhistone high mobility group [HMG] from human lymphocytes by high performance liquid chromatography

The high mobility group [HMG] of nonhistone proteins have been investigated using two high performance liquid chromatographic techniques [HPLC]. Reversed-phase HPLC under conditions of 50 mM triethylamine adjusted to pH 2.2 with phosphoric acid [solvent A] and 95% acetonitrile in water [solvent B] was used to separate proteins primarily on the basis of differences in the overall hydrophobicity. Size exclusion HPLC under conditions of two different solvents [A, 0.1% trifluoroacetic acid TFA; B, 1.0% sodium dodecyl sulphate, SDS] was used to separate proteins. HMG proteins from human lymphocytes were separated into the HMG 1, HMG 2, HMG 14 and HMG 17 components. RP-HPLC is a proper method to resolve all the human lymphocyte HMG-proteins. Size exclusion HPLC was employed to resolve the HMG-protein subunits and determine their molecular weights. Ideal SE-HPLC is not capable of resolving HMG 1 from HMG 2 or HMG 14 from HMG 17 due to their molecular weight similarities. The purity of protein fractions were examined by acetic acid-urea-triton X-100 gel electrophoresis