The glycosylation characteristic of hepatoma-associated antigen HAb18G/CD147 in human hepatoma cells

HAb18G/CD147 is a highly glycosylated transmembrane protein belonging to the immunoglobulin superfamily. Our previous studies have demonstrated that overexpressing HAb18G/CD147 enhances the metastatic potentials of human hepatoma cells. In the present study, to investigate the glycosylation characteristic of HAb18G/CD147 in human hepatoma cells, HAb18G/CD147 was first purified from human FHCC-98 hepatoma cells by immunoaffinity chromatography, and then introduced into human fibroblasts culture system for matrix metalloproteinases induction. As a result, the elevated levels of matrix metalloproteinases secreted by fibroblasts were detected by gelatin zymography. The lysates of human hepatoma FHCC-98 cell revealed two major forms of HAb18G/CD147 (43-66 and 35 kDa) by western blot assay. To elucidate whether the variation of molecule size were caused by different glycosylation, two different approaches were employed to accomplish this goal: deglycosylation with N-glycosylation inhibitor tunicamycin or endoglycosidases. A single deglycosylated core protein with molecular weight approximately 27 kDa was obtained from both methods. Furthermore, the results of endoglycosidases treatment also showed that two forms of HAb18G/CD147 contain different types of oligosaccharide chains, thus sensitive to different endoglycosidase. In conclusion, the present study demonstrated that purified native HAb18G/CD147 has the bioactivity of stimulating human fibroblasts to produce elevated levels of matrix metalloproteinases, and that the two different forms of HAb18G/CD147 are derived from the single core protein but differ in their degree and types of glycosylation.     

Erk is involved in the differentiation induced by diallyl disulfide in the human gastric cancer cell line MGC803

Diallyl disulfide (DADS) is a major constituent of garlic. Previously, we found that DADS both inhibited proliferation in human gastric cancer cells in vitro and in vivo, and induced G2/M arrest. In this study, we investigated whether this differentiation effect was induced by DADS in human gastric cancer MGC803 cells, and whether it was related to an alteration in ERK activity. The results showed that the growth of MGC803 cells was inhibited by DADS. Cells treated with DADS displayed a lower nucleocytoplasmic ratio and tended to form gland and intercellular conjunction structures. The ConA-mediated cell agglutination ratio and cells’ ALP specific activity decreased. In MGC803 cells, dye transfer was limited to a few cells neighbouring the dye-injected cell and to a depth of 1–2 layers beneath the scrape site. However, after treatment with DADS, the LY (Lucifer Yellow) was transferred to several cells immediately neighbouring the microinjected cell and to a depth of 2–4 cell layers from the scrape site. This indicated that DADS induced differentiation in MGC803 cells. Western blot analysis revealed that although DADS did not influence the quantity of ERK1/2 protein expressed, it did decrease its phosphorylation in a concentration-dependent manner, compared with the controls. At 30 mg·L⁻¹, DADS inhibited the activation of ERK1/2 in 15–30 min. These results suggested that the DADS-induced differentiation of MGC803 cells involved an alteration of the ERK1/2 signaling pathway.     

Application of "oxygen uptake rate-amino acids" associated mode in controlled-fed perfusion culture

Controlled-fed perfusion, a new operation mode, which combines the advantages of fed-batch and perfusion, has been reported to enhance monoclonal antibody productivity. The aim of the present study was to further enrich this mode by an "oxygen uptake rate-amino acids (OUR-AA)" strategy in which the feeding of amino acids was controlled according to the variation of OUR during perfusion. And the effects of this strategy on bioreactor productivity and product quality were evaluated. Experimental results indicated that by using this "OUR-AA" approach in controlled-fed perfusion mode a high viable cell density of more than 1.9 x 10(7)cells/ml was achieved and the productivity of mAb reached 325 mg/l/d, which was significantly increased by nearly twofold over those of the perfusion and fed-batch process. The residual concentrations of selected amino acids were controlled at a relative steady level by OUR during the culture. The immunoreactivity and the purity of the antibody were well preserved as the culture process was evolving from flask to the controlled-fed perfusion mode. The primary application of "OUR-AA" approach in controlled-fed perfusion mode may present a novel control strategy to enhance the culture performance and to display the potential of this approach in automatic control field.     

ATM-Chk2-p53 activation prevents tumorigenesis at an expense of organ homeostasis upon Brca1 deficiency

BRCA1 is a checkpoint and DNA damage repair gene that secures genome integrity. We have previously shown that mice lacking full-length Brca1 (Brca1(delta11/delta11)) die during embryonic development. Haploid loss of p53 completely rescues embryonic lethality, and adult Brca1(delta11/delta11)p53+/- mice display cancer susceptibility and premature aging. Here, we show that reduced expression and/or the absence of Chk2 allow Brca1(delta11/delta11) mice to escape from embryonic lethality. Compared to Brca1(delta11/delta11)p53+/- mice, lifespan of Brca1(delta11/delta11)Chk2-/- mice was remarkably extended. Analysis of Brca1(delta11/delta11)Chk2-/- mice revealed that p53-dependent apoptosis and growth defect caused by Brca1 deficiency are significantly attenuated in rapidly proliferating organs. However, in later life, Brca1(delta11/delta11)Chk2-/- female mice developed multiple tumors. Furthermore, haploid loss of ATM also rescued Brca1 deficiency-associated embryonic lethality and premature aging. Thus, in response to Brca1 deficiency, the activation of the ATM-Chk2-p53 signaling pathway contributes to the suppression of neoplastic transformation, while leading to compromised organismal homeostasis. Our data highlight how accurate maintenance of genomic integrity is critical for the suppression of both aging and malignancy, and provide a further link between aging and cancer.     

Use of recombinant calpain-2 siRNA adenovirus to assess calpain-2 modulation of lung endothelial cell migration and proliferation

In the Streptozotocin-induced diabetic rat heart, a decrease in the conductivity and suppression of electrical cell-to-cell coupling were observed. To clarify this mechanism, the present study was performed to investigate alterations of the gap junction connexin 43 (C×43) using immunoblotting, immunohistochemistry, electron-microscopic analyses. An enhanced activation of PKCε, an augmentation of PKCε-mediated phosphorylation of C×43, a decrease in the total amount of C×43, a reduction in the area of immunoreactive particles for C×43 at the intercalated disk, distribution of C×43 to cell periphery or cytoplasm and the internalization∼annular profiles of the gap junction were all characteristically recognized in the diabetic heart. Such abnormalities in the expression of C×43 were alleviated by treatment with either lysosomal (NH₄Cl, Leupeptin) or proteasomal inhibitor (ALLN). These results suggest that the PKCε-mediated hyperphosphorylation of C×43 makes C×43 vulnerable to proteolytic degradation and that a decrease in the conductivity in the diabetic heart is also caused by a decrease in the number of gap junction channels due to an acceleration of the proteolytic degradation of C×43. The remodeling of C×43 induced by the activation of PKCε may therefore contribute to the formation of the arrhythmogenic substrate in the diabetic heart. The cardioprotective effect of the remodeling of C×43 by PKCε is discussed.     

Effect of C-terminal truncation on the molecular chaperone function and dimerization of Escherichia coli trigger factor

To examine the role of the C-terminal domain in the chaperone function of trigger factor (TF), a number of truncation mutants were constructed, namely: TF419, TF389, TF380, TF360, TF344, and TF251, in which the C-terminal 13, 43, 52, 72, 88 residues or the entire C-domain were deleted, respectively. Co-expression of mutant chicken adenylate kinase (AK) with TF and the C-terminal truncation mutants was achieved using a plasmid pBVAT that allows expression of TF and AK from a single plasmid. The results show that truncation of the C-terminus of TF has only minor effect on its ability to assist AK refolding in vivo. Further, ribosome-binding experiments indicate that C-terminal truncation mutants can still bind to the ribosome and the presence of the C-terminus may in fact lower the affinity of TF for the ribosome in vivo. This indicates that the C-domain of trigger factor may not be essential for the ribosome-associated molecular chaperone function of TF. However, the purified TF C-terminal truncation mutants had a dramatically reduced ability to assist rabbit muscle GAPDH refolding in vitro and a reduced tendency to dimerize. This shows that the structural integrity of the C-terminus contributes to both the chaperone function of TF and the stability of the dimeric form.     

Effects of selective estrogen receptor modulator raloxifene plus 17-beta estradiol in aorta and mammary gland of female experimental atherosclerosis rabbits and possible involvement of ERK signal transduction pathway

The present study investigated the effects of raloxifene, a second generation selective estrogen receptor modulator (SERM), plus 17-betaE2 on aortic atherosclerosis and mammary gland hyperplasia in ovariectomized, cholesterol-fed rabbits. Following 10 weeks of raloxifene, 17-betaE2, or raloxifene plus 17-betaE2 administration, serum total cholesterol, triglyceride, low density lipoprotein were significantly decreased in the drug groups compared to the placebo group. Consistent with serum lipid results, the total lesion area for each aorta of the drug groups decreased significantly as compared to the placebo group. HE staining of aorta paraffin section showed that in the drug groups the endothelial monolayer was almost continuous. While in mammary gland, HE staining of paraffin sections indicated the hyperplasia of epithelial cells (in 17-betaE2 group) was obviously inhibited in raloxifene plus 17-betaE2 group. In cultured vascular smooth muscle cell (VSMC), the results of MTT and [3H]TdR incorporation showed that the drug groups could inhibit AngII-induced proliferation of VSMC. Western blotting proved that raloxifene plus 17-betaE2 inhibited the expression of phosphorylated ERK protein, similar to 17-betaE2 but different from raloxifene. This effect was inhibited by PD98059 (inhibitor of MAPK) or ICI182780 (ER antagonist). In conclusion, this study suggests that SERM raloxifene plus 17-betaE2 improves the lipid metabolism and relieves the aorta changes of female experimental atherosclerosis rabbits, which are partly implemented by the inhibition of VSMC growth through ERK cascade. The hyperplasia of mammary gland epithelial cells could be significantly inhibited by raloxifene plus 17-betaE2.     

Oligonucleotide Microarray with RD-PCR Labeling Technique for Detection and Typing of Human Papillomavirus

Currently, screening for high-risk human papillomavirus (HPV) infection remains an important health concern throughout the world, because of the close association between certain types of HPV and cervical cancer. In this study, we explore the possibility of using ∼70mer oligonucleotide microarray for detection and genotyping of HPV. The ∼70mer type-specific oligonucleotide probes of four different types HPV were designed by using biological software Arraydesigner 2.0, which analyzed the whole genome sequences of HPV and selected optimal probes. These probes were synthesized and printed onto the surface of glass slides in order to prepare a low-density microarray. HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction display polymerase chain reaction (RD-PCR). HPV plasmid DNA was restricted with Sau3A I to produce multiple fragments that were ligated to adaptors subsequently and used as PCR template. PCR labeling was performed with the fluorescently labeled universal primer (Cy3-UP) whose sequence is designed according to the adaptor of the RD-PCR approaches. The labeled samples were hybridized with the oligonucleotide microarray. The scanning results showed that HPV DNA hybridized specifically with multiple spots correspondingly to show positive signals, whereas no signals were detected of all the negative and blank controls. These results demonstrated that ∼70mer oligonucleotide microarray can be applied to HPV detection and genotyping. The application of RD-PCR in the sample labeling can increase significantly the sensitivity of the assay and will be especially useful for the discriminate diagnosis of multiple pathogens.