Effects of RANKL on the proliferation and apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis through regulating the NF-κB signaling pathway.

OBJECTIVE: The aim of this study is to investigate the regulatory effects of receptor activator of nuclear factor-kappa B ligand (RANKL) on the proliferation and apoptosis of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA), and to explore its regulatory mechanism. MATERIALS AND METHODS: Synoviocytes were primarily cultured in rats of recognized collagen-induced arthritis (CIA) model. Meanwhile, they were induced into FLS models by lipopolysaccharides (LPS). All cells were divided into three groups, including blank group, model group and RANKL inhibitor group. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1ß (IL-1ß) in the cells were detected by enzyme-linked immunosorbent assay (ELISA). The proliferation and apoptosis of FLS were detected via 3-(4,5)-dimethylthiazol (-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was conducted to measure the messenger ribonucleic acid (mRNA) expression levels of nuclear factor-kappa B ligand (NF-κB) and Caspase-3 in FLS. Furthermore, Western blotting was adopted to detect the protein expression levels of NF-κB and Caspase-3 in FLS. RESULTS: Compared with the blank group, the expression levels of TNF-α and IL-1ß in the cells of the model group increased significantly. Cell proliferation rate increased significantly, whereas the cell apoptosis rate decreased remarkably in the model group. Meanwhile, the mRNA and protein levels of NF-κB and Caspase-3 in FLS were significantly up-regulated. Compared with the model group, the levels of TNF-α and IL-1ß in cells of RANKL inhibitor group notably declined. Similarly, cell proliferation rate was significantly reduced, whereas the cell apoptosis rate increased significantly. Furthermore, the mRNA and protein levels of NF-κB and Caspase-3 in FLS were evidently down-regulated. CONCLUSIONS: RANKL inhibitors can inhibit the proliferation and promote the apoptosis of FLS in RA. In addition, its mechanism may be related to the inhibition of NF-κB signaling pathway.

Fabrication and measurement of nanostructures on the micro ball surface using a modified atomic force microscope.

In order to machine and measure nanostructures on the micro ball surface, a modified atomic force microscope (AFM) combining a commercial AFM system with a home built precision air bearing spindle is established. Based on this system, motions of both the AFM scanner and the air bearing spindle are controlled to machine nanostructures on the micro ball based on the AFM tip-based nano mechanical machining approach. The eccentric error between the axis of the micro ball and the axis of the spindle is reduced to 3-4 µm by the provided fine adjusting method. A 1000 nano lines array, 36 square pits structure, 10 square pits structure, and a zig-zag structure on the circumference of the micro ball with the diameter of 1.5 mm are machined successfully. The measurement results achieved by the same system reveal that the profiles and mode-power spectra curves of the micro ball are influenced by the artificially machined nanostructures significantly according to their distributions. This work is an useful attempt for modifying the micro ball profile and manufacture of the spherical modulation targets to study the experimental performance of the micro ball in implosion.

Magnetic resonance imaging of cerebellar liponeurocytoma. A case report and review of the literature.

Cerebellar liponeurocytoma is a rare benign neuroepithelial tumour. We describe the case of a 50-year-old man presenting with signs of increased intracranial pressure and cerebellar dysfunction. Magnetic resonance imaging showed a heterogeneous, well-circumscribed cerebellar mass with a predominant adipose content. Diffusion-weighted imaging showed an isointense mass with a hyperintense rim. Craniotomy demonstrated a soft grey mass with intratumoral bright patchy yellow areas. Histological and immunohistochemical findings indicated an advanced neuronal, glial and focal lipomatous differentiation with a low level of mitotic activity.

Inhibitors from natural products to HIV-1 reverse transcriptase, protease and integrase.

Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus type 1 (HIV-1) infection, is still one of the most challenging diseases of the early 21st century. Reverse transcriptase (RT), protease (PR) and integrase (IN) are three key enzymes of HIV-1. Despite the shortcomings of chemical drugs such as toxicity, lack of curative and multiple effects, the search for more and better anti-HIV agents has been focused on natural products. Many natural products have been shown to possess promising activities that could assist in the prevention and amelioration of the disease. Most of these natural anti-HIV agents have other medicinal values as well, which afford them further prospective as novel lead compounds for the development of new drugs. These natural products can deal with both the virus and the various disorders that are caused by HIV. In this review, natural inhibitors of RT, PR and IN have been found to be classified and the relationship between structure and inhibitory activity is discussed.

ISG15 expression in response to double-stranded RNA or LPS in cultured Fetal bovine lung (FBL) cells.

Fetal bovine lung (FBL) cells are used in the culture of viruses which infect cattle and ISG15 plays a role in innate immunity against viral infections. However, whether the expression of ISG15 gene can be induced in FBL cells is still unknown. In this work, the expression of ISG15 in cultured FBL cells was detected after stimulated with poly I:C or LPS. Real-time PCR analyses revealed that the transcript of ISG15 can be induced by poly I:C or LPS. The increased expression of free ISG15 was confirmed via Western blotting. Furthermore, immunofluorescence assays demonstrated that IRF-3 was translocated from the cytoplasm to the nucleus in the FBL cells treated with poly I:C. Chromatin immunoprecipitation assays showed that IRF-3 can bind to the promoter of the bISG15 gene. To demonstrate IRF-3 can promote the expression of bISG15, we establish a luciferase-reporter system of bovine ISG15 gene in 293 T cells. The luciferase assay showed that the over-expression of bovine IRF-3 could activate the promoter of bISG15 gene. Taken together, these results suggest that the expression of bISG15 can be induced in FBL cells stimulated with poly I:C or LPS, and IRF-3 may play a role in inducing the expression of ISG15 in FBL cells.

Behavioral study of calorie-restricted rats from early old age.

PURPOSE: To investigate the effect of Calorie Restriction (CR) on learning and memory ability of early aged rats. METHODS: 18-month rats were subjected to restricted intake by 60% comparing with that of rats fed ad libitum (AL) for 6 months. We compared the overall health status, including survival rate and locomotor activity by open-field test. We examined the spatial cognition ability of the rats by Morris Water Maze. RESULTS: Our results showed that CR rats had higher survival rate and spontaneous locomotor activity compared with AL rats. CR rats slowed the inability of spatial learning and reference memory. CONCLUSION: These findings demonstrated that CR in early old rats delayed the declination of spatial cognition.

The requirements and mechanism for capsid assembly and budding of bovine foamy virus.

Little is known about assembly of non-primate foamy virus (FV) such as bovine foamy virus (BFV). To help determine the requirements for assembly of BFV, we constructed BFV-Gag expression plasmids containing all or part of the gag gene, with or without modification by addition of myristate (Myr). Each construct was transfected alone, and with pFenv, into Sf-9 insect cells. The results showed that only the entire Gag could transit through nucleus, which is required for BFV viral assembly in the cytoplasm. Unlike other retroviruses (but like primate foamy viruses), BFV requires the coexpression of the Env protein for viral particle budding. In the case of BFV, this occurs at the plasma membrane rather than the endoplasmic reticulum (ER), due to lack of a functional ER retrieval signal (ERRS). The results also showed that addition of a Myr-membrane targeting signal to the C-terminus of Gag could restore the budding from plasma membrane, implying that Myr-membrane targeting signal could substitute for Env protein in budding.

[Construction of B. thuringiensis shuttle vector and expression of the cry1C gene].

We have constructed the E. coli-Bt shuttle vector pHV-1 by cloning the replicon (approximately 1.6 kb) of Bt ken-Ag and the aphI gene of pUC4K into pUC19. The rate of plasmid maintenance is more than 80% after 100 generations in E. coli, whereas 80% after 40 generations in Bti 4Q8. We have also constructed pHV-cry1C through cloning the alpha-amylase promoter from B. licheniformis and the cry1C gene from Bt 9510 into pHV-1 and introduced it into Bti 4Q8 by means of electroporation. Under the microscope, we can see that there is no crystal in Bti 4Q8, however, there are many rhomboid crystals in Bti 4Q8 (pHV-cry1C), which are smaller than those of Bt 9510. The bioassay result of Bti 4Q8 (pHV-cry1C) demonstrates that the expressed crystal protein is insecticidally active against Spodoptera exigue.

Expression of membrane-associated macrophage colony-stimulating factor (M-CSF) in Hodgkin's disease and other hematologic malignancies.

We have identified a membrane-bound form of M-CSF (m-M-CSF) from an established human leukemic J6-1 cell line. To further understand its biological significance, we studied the expression of this membrane-associated growth factor in the lymph nodes of lymphoma patients and bone marrow smears from patients with hematologic diseases by immunohistochemical staining using anti-M-CSF MAb. We detected a high incidence of m-M-CSF expression in 75% (9/12) of the lymph node sections from patients with Hodgkin's Disease (HD). The antigens were detected primarily in large clusters of mononuclear Hodgkin's cells and the extracellular matrix (EM) surrounding them. In one HD patient with abundant multinucleated Reed-Sternberg (R-S) cells, all of them were intensely stained with anti-M-CSF MAb. In non-Hodgkin's lymphomas (NHL), the incidence (17.6 %) of m-M-CSF expression was lower (3/17). Yet, no m-M-CSF antigens were detected in the lymph nodes from six cases of non-hematologic malignancies and other diseases. A high response also was detected in bone marrow smears obtained from patients with hematologic malignancies, which include myeloid leukemias (32.5%), lymphomas with bone marrow metastasis (50%) and myelodysplastic syndromes (MDS) (37.5 %). By comparison, only 6.8 % of bone marrow smears from non-malignant hematologic diseases and 2.7% of lymphoid leukemias showed positive staining with anti-M-CSF MAb. Our results showed that high expression of m-M-CSF antigens is linked to some types of lymphomas, especially HD. and myeloid leukemias, and may play a role in the development of these hematologic malignancies.

[Effect of changes of amino acids of N-terminal region of the mature protein on secretion of alpha-amylase in B. subtilis].

The mutant plasmid pAmy413C, in which G takes the place of A at the 271 position of alpha-amylase gene on the pAmy413 from B. licheniformis, was constructed by site-direct mutagenesis. At the N-terminus of the mature alpha-amylase, amino acid +2Asn was substituted by +3Asp in the wild type protein. Then, the alpha-amylase output of the mutant plasmid pAmy413C in B. subtilis was 2.02-2.57 times higher than that of the wild type pAmy413C in the same strain. The amino acid sequencing at the N-terminus of the matural alpha-amylase revealsed that the recognition site of signal peptidase I moved one amino acid upstream, from Ala-(+2)Asn to AlaAla-(+3) Asp. That is, the +2Asn of the wild type was changed to the +3Asp of the mutant. The secondary structural analysis showed that a 14-cycle structure formed in the alpha-amylase mRNA when the free energy was -51.7 kcal. In this case, the mutant is identical with the wild type. The difference between them is that G at 271 position is no longer paired with U at 211 position, hence, a G-overhang is formed. The secondary structural analysis of protein showed that one amino acid diminished in the turn structure of amino acid at 33-37 position, and this very amino acid is involed in an alpha-helix structure. In short, all the changes mentioned above in conformation and charged amino acids contribute to the increase in the protein secretion in B. subtilis.

Enhancement of J6-1 human leukemic cell proliferation by cell-cell contact: role of an M-CSF-like membrane-associated growth factor MAF-J6-1.

Density-dependent cell proliferation and cluster formation are growth phenotypes frequently associated with leukemia cells. The secretion of autocrine growth factor, such as granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 1 (IL-1), has been implicated as one possible mechanism in leukemogenesis. In many cases, however, leukemia cells do not appear to produce autocrine growth stimulators. J6-1 is an established human myeloid leukemia cell line that exhibits both density-dependent and cluster-forming growth characteristics. The effect of direct cell-cell contact on J6-1 cell proliferation was investigated. We have isolated from J6-1 cells a membrane-bound factor (designated as MAF-J6-1) that promoted the colony formation by both J6-1 cells and mouse bone marrow CFU-GM. The growth-promoting activity of MAF-J6-1 can be neutralized by either anti-macrophage-CSF (M-CSF or CSF-1) or anti-MAF-J6-1 monoclonal antibodies (MAb), suggesting that MAF-J6-1 is related to M-CSF. Using an immunoblot analysis with anti-MAF-J6-1 MAb, the MW of this membrane-associated factor was estimated to be 80 kDa. Both antibodies also induced a modest growth inhibition on J6-1 cells in vitro. Similarly, addition of exogenous recombinant human M-CSF augmented the colony formation by J6-1 cells, an effect also neutralized by both antibodies. Using an in situ hybridization technique, J6-1 cells were found to express a high level of c-fms proto-oncogene, which encodes the receptor for the M-CSF. Taken together, our results suggest that the membrane-bound MAF-J6-1 promote J6-1 cell proliferation and cluster formation through a 'juxtacrine' mechanism.

Identification and characterization of a human herpesvirus 6 gene segment that trans activates the human immunodeficiency virus type 1 promoter.

Human herpesvirus 6 (HHV-6) is a lymphotropic herpesvirus, and in vitro, HHV-6 can productively infect many of the same cell types as can human immunodeficiency virus (HIV). Coinfection by both viruses in vitro can lead to both activation of the HIV promoter and acceleration of cytopathic effects. We have previously demonstrated that a large, 22.25-kb cloned HHV-6 fragment, pZVB70, can trans activate HIV promoter expression in vitro. In this study, we show that the pZVB70 fragment can trans activate the HIV promoter in human T-cell lines as well as in the monkey kidney cell line CV-1. The pZVB70 insert was digested with various restriction enzymes, and individual fragments were transfected into cells to test for their ability to trans activate the HIV promoter. By this method, we have identified a 1.8-kb subfragment, B701, that is involved in trans activation. Sequence analyses show that B701 potentially encodes a 143-amino-acid protein. This protein shares no homology with other herpesvirus proteins, such as ICP0 and ICP4, that have been shown to trans activate the HIV promoter. However, it shows weak sequence homology with the gene products encoded by the cytomegalovirus early US22 gene family, suggesting that the putative B701 protein may be an HHV-6 early regulatory protein. The 143-amino-acid coding sequence of B701 was cloned by polymerase chain reaction, and transfection of this construct into cells activated HIV promoter expression. The target site on the HIV promoter for the putative B701 protein is mapped to the NF-kappa B binding site. Our results suggest that the putative B701 protein may function by directly binding to the NF-kappa B site or may involve cellular factors, such as NF-kappa B, either directly or indirectly.

[Stability and gene expression of foreign plasmid in Bacillus subtilis BF7658].

The plasmid pAmy411 carrying thermostable alpha-amylase gene has been transduced into B. subtilis BF7658 by bacteriophage PBS1. The transduction frequency was 10(-9) transductants per PFU. The stability of plasmid pAmy411 in B. subtilis BF7658 was lower than that in B. subtilis AS1.1176, whereas the copy numbers were double than that in B. subtilis AS1.1176. The expressive level of thermostable alpha-amylase gene was about 6 times higher than that in B. subtilis AS1.1176.

[Study on Bacillus pumilus as a recipient strain for genetic engineering of Bacillus].

Bacillus pumilus 289 can be transformed easily by plasmid pUB110 through protoplast transformation with the frequency of 10(-5)--10(-3), similar to B. subtilis AS 1.1176, a derivative strain of B. subtilis 168. The regeneration frequency of its protoplast is only slightly lower than B. subtilis AS1.1176 (0.3-12.0% compare to 1.53--24.16%). Plasmid pUB110 can be maintained in both bacterial strains stably. The frequency of loss of the plasmid in both strains is lower than 3% after 45 generations in LB medium. But it is quite different that the hybrid plasmid (pUB110 with 3.9 kb foreign DNA fragment) can be maintained much more stably in B. pumilus 289 than in B. subtilis AS1.1176. The frequency of loss of the plasmid is lower than 5% in B. pumilus 289 and 24% in B. subtilis AS1.1176 after 25 generations when they grown in SH medium. The expression level of foreign gene in B. pumilus 289 is also much higher than that in B. subtilis AS1.1176. Therefore B. pumilus 289 is valuable to be exploited as recipient strain for genetic engineering of Bacillus in the future.