Preventing and reversing the cellular consequences of Z alpha-1 antitrypsin accumulation by targeting s4A.

BACKGROUND & AIMS: The Z variant (Glu342Lys) of α(1)-antitrypsin (AT) polymerizes and accumulates in the hepatocyte endoplasmic reticulum (ER) predisposing to neonatal hepatitis and liver cirrhosis. The resultant secretory defect leaves the lungs vulnerable to elastolysis and early-onset emphysema. Our aim in this study was to evaluate the effect of targeting strand 4a (s4A) as a strategy to inhibit polymerization and restore plasma secretion. METHODS: HEK293 cells and HepG2 cells were transfected with Z-AT (Z-AT cells) or control M-AT (M-AT cells). The effect of Ac-TTAI-NH(2) (4M), Ac-FLEAIG-NH(2) (6M), and Ac-SEAAASTAVVIA-NH(2) (12M) on preventing and reversing intracellular Z-AT polymers and secretion of AT was evaluated by pulse-chase/immunoprecipitation, ELISA, and immunoblot with a polymer-specific antibody (ATZII). The ER overload response was assessed by RT-PCR for PERK, calnexin, and RGS16, and ELISA for NF-κB, IL-6, and IL-8. RESULTS: All peptides prevented the intracellular accumulation of Z-AT (4M>6M>12M) in comparison with control peptides, with detection of the AT-Inhibitor complex in inclusion bodies. In so doing, 4M also significantly increased the concentration of secreted Z-AT and the elastase inhibitory activity. Furthermore, the 4M peptide was able to reverse the intracellular aggregation of Z-AT. The ER accumulation of Z-AT was shown to induce PERK-dependent NF-κB, IL-6, IL-8, and RGS16 and calnexin; all of which could be abrogated effectively by 4M. 4M had no effect on apoptosis or cell viability. CONCLUSIONS: These findings are the first evidence that targeting s4A can prevent the cellular accumulation and deleterious effects of Z-AT and restore its plasma concentrations. As such, this is a major step towards treatment of patients with Z-AT-related disease.

Enhanced removal of trivalent chromium from leather wastewater using engineered bacteria immobilized on magnetic pellets.

Leather wastewater contains various toxic contaminants, with trivalent chromium (Cr(III)) having high concentration and adversely affecting wastewater treatment. In this study, a Cr(III) adsorption protein (MerP) was displayed on the cell surface of Escherichia coli and then coupled with a magnetic pellet system to facilitate Cr(III) adsorption. The results showed the engineered strain M-BL21 achieved an in vitro Cr(III) adsorption capacity of 2.38 mmol/g. Next, the magnetic pellets were prepared as component ratios of sodium alginate (2.5%), polyvinyl alcohol (8%), Fe3O4 nanoparticles (3.5%), and M-BL21 at 3 g/L. The optimized system was capable of Cr(III) adsorption at an efficiency of 91.29%, which was substantially higher than that of the magnetic carrier alone (67%). Results of scanning electron microscopy with energy-dispersive X-ray analysis proved that Cr(III) was absorbed on the magnetic pellet. The recyclable performance of magnetic property (13.34185 emu/g) and high Cr(III) adsorption efficiency (68.75%) remained after five cycles of Cr(III) absorption. In the medium-scale experiment, 25 L of leather wastewater were treated with magnetic pellet and the Cr(III) removal efficiency reached 88.2%. Thus, our results present an advanced, fully operational, and eco-friendly method for in situ removal of Cr(III) from contaminated wastewater.

Oxidized {alpha}1-antitrypsin stimulates the release of monocyte chemotactic protein-1 from lung epithelial cells: potential role in emphysema.

alpha(1)-Antitrypsin (AT) is a major elastase inhibitor within the lung. Oxidation of critical methionine residues in AT generates oxidized AT (Ox-AT), which has a greatly diminished ability to inhibit neutrophil elastase. This process may contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD) by creating a functional deficiency of AT permitting lung destruction. We show here that Ox-AT promotes release of human monocyte chemoattractant protein-1 (MCP-1) and IL-8 from human lung type epithelial cells (A549) and normal human bronchial epithelial (NHBE) cells. Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion. These findings were supported by the fact that instillation of Ox-AT into murine lungs resulted in an increase in JE (mouse MCP-1) and increased macrophage numbers in the bronchoalveolar lavage fluid. The effect of Ox-AT was dependent on NF-kappaB and activator protein-1 (AP-1)/JNK. These findings have important implications. They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the antielastase lung protection, but also converts AT into a proinflammatory stimulus. Ox-AT generated in the airway interacts directly with epithelial cells to release chemokines IL-8 and MCP-1, which in turn attracts macrophages and neutrophils into the airways. The release of oxidants by these inflammatory cells could oxidize AT, perpetuating the cycle and potentially contributing to the pathogenesis of COPD. Furthermore, these data demonstrate that molecules such as oxidants, antiproteinases, and chemokines, rather than act independently, are likely to interact to cause emphysema.

A Review on Microbial Electrocatalysis Systems Coupled with Membrane Bioreactor to Improve Wastewater Treatment.

Microbial electrocatalysis is an electro reaction that uses microorganisms as a biocatalyst, mainly including microbial electrolytic cells (MEC) and microbial fuel cells (MFC), which has been used for wastewater treatment. However, the low processing efficiency is the main drawback for its practical application and the additional energy input of MEC system results in high costs. Recently, MFC/MEC coupled with other treatment processes, especially membrane bioreactors (MBR), has been used for high efficiency and low-cost wastewater treatment. In these systems, the wastewater treatment efficiency can be improved after two units are operated and the membrane fouling of MBR can also be alleviated by the electric energy that was generated in the MFC. In addition, the power output of MFC can also reduce the energy consumption of microbial electrocatalysis systems. This review summarizes the recent studies about microbial electrocatalysis systems coupled with MBR, describing the combination types and microorganism distribution, the advantages and limitations of the systems, and also addresses several suggestions for the future development and practical applications.

Effects of antisense-myc-conjugated single-walled carbon nanotubes on HL-60 cells.

Antisense therapy is limited in application in clinical therapy because of two existing problems: rapid degradation of antisense nucleic acids and poor diffusion across the cell membrane. Here we report the use of single-walled carbon nanotubes as delivery system for transporting antisense myc into HL-60 cells. Antisense-myc-conjugated single-walled carbon nanotubes were synthesized, characterized by atomic force microscopy (AFM), fluorescent microscopy, and Raman spectroscopy. Incubated with HL-60 cells at 37 degrees C, the single-walled carbon nanotubes (SWNTs) inside HL-60 cells were measured quantitatively by HPLC. Expression of c-myc gene and protein were analyzed by reverse transcriptase PCR and Western blot, respectively. Results showed that as-myc-conjugated SWNTs dropped character peaks at quadruple wavenumber (cm(-1)) compared with SWNTs, and could enter into HL-60 cells within 15 min after incubation, whose uptake amounts by HL-60 cells increased as the incubation time increased. After 48 hours, the amount of SWNTs in HL-60 cells began to decrease. Compared with as-myc and SWNTs, as-myc-conjuagted SWNTs exhibited the strongest inhibition on the proliferation of HL-60 cells, induced cell apoptosis, and down-regulated lowest expression of c-myc gene and C-MYC protein. The SWNTs can directly deliver as-myc into HL-60 cells, enhance the inhibition of as-myc on HL-60 cells, and is likely a better delivery system for antisense therapy. Antisense modified SWNTs can be used for intracellular gene regulation with potential applications in tumor therapy and drug delivery.

PH domain of G protein-coupled receptor kinase-2 binds to protein kinase C (PKC) and negatively regulates activity of PKC kinase.

G protein-coupled receptor kinase-2 (GRK),also known as beta1-adrenergic receptor kinase(beta-ARK1), plays an important role in agonist-induced desensitization of the beta-adrenergic receptors. Activation of protein kinase C (PKC) is able to stimulate phosphorylation and activation of GRKs and induce desensitization of G protein-coupled receptor. However, detail mechanism of interaction between PKC and GRK2 and the effect of GRK2 on activity of PKC remain unknown. Pleckstrin homology (PH) domain is a kind of functionally domain containing about 120 amino acids, which exists on many protein molecules that involve in cellular signal transduction. A PH domain located in GRK2 residue 548 to 660 may play a significant role in mediating interaction between PKC and GRK2. In present study, we revealed that PKC could associate with PH domain of GRK2 in pull-down assay in vitro. Co-immunoprecipitation displayed binding of PKC to GRK2 in intact Jurkat cells after prolonged stimulation of epinephrine. Assay of PKC beta1 kinase activity indicated that the binding of the PH domain of GRK2 to PKC beta 1 could down-regulate activity of PKC beta 1 kinase. Thus, GRK2 may play a negative feedback regulatory role on PKCbeta1 activity in interaction between GRK2 and PKCbeta 1.

Rheumatoid arthritis is auto-immunoreaction to collagen II in cartilage happened in synovial tissue.

Rheumatoid arthritis is complex and not clear on the mechanism of pathogenesis. On the basis of analysis of the symptom and pathology of rheumatoid arthritis patients, we raised a new hypothesis. The content of the hypothesis is as follows: (A) Collagen II or collagen II-Iike substance in human cartilage is the cross-autoantigen of some infecting virus or bacteria because of the structure's similarity. (B) The inflammation in synovial tissue is auto-immunoreaction to collagen II in cartilage. (C) The proliferation and attachment of synovial tissue to the surface of cartilage is due to the chemotaxis of collagen II in cartilage for the immunocytes in synovial tissue. (D) The collagenase secreted from synovial cells and immunocytes are the direct elements in the destruction of cartilage. The fallen collagen II from cartilage is one of the most important inducer on the synovial cells and immunocytes for the production of collagenase.

Binding of Insulin Receptor Substrate 1 PH Domain to Protein Kinase C.

To screen for novel ligands of insulin receptor substrate 1(IRS-1)PH domains, to investigate the role of the PH domains in signal transduction processes and to examine association of the PH domain with protein kinase C(PKC), rat liver was employed to clone the gene encoding for the PH domains. Total RNAs were isolated and purified from fresh liver and mRNAs were reversely transcribed into cDNAs. After PCR the fragments of the DNA were cloned into vector pUC19. The sequence of the fusion gene was confirmed by sequencing, and the gene was correctly expressed in E.coli as fusion protein with glutathione S- transferase(GST). The fusion protein was purified by glutathione agarose beads, then was incubated with the lysate of Jurkat cells. After SDS-PAGE, the proteins were transferred to PVDF membrane, and an anti-PKC antibody was used to detect binding between the PH domain with PKC. The sequence of the gene encoding for the PH domains was confirmed to be correct, and the PH domain was successfully expressed in E.coli JM 109 in soluble form. Western blots confirmed the binding of the PH domain with PKC in vitro. In conclusion, purified IRS-1 PH domain GST fusion protein was obtained and its biological activity was confirmed.

[Inhibitive effect on airway mucus overproduction with DNA vaccine based on xenogeneic homologous calcium-activated chloride channel in asthmatic mice].

OBJECTIVE: To observe the inhibitive effect on airway mucus overproduction with DNA vaccine based on human calcium-activated chloride channel 1 (hCLCA1) in asthmatic mice, who own mCLCA3 being xenogeneic homology of hCLCA1 in airway goblet cell. METHODS: The DNA vaccine was made with hCLCA1 gene inserted into pSecTag2B, and then BALB/c mice were vaccinated by i.m. once every two weeks. When serum antibody showed binding activity to mCLCA3 with ELISA analysis, asthma will be induced with ovalbumin in the vaccinated mice. To detect mucus production, lung sections were PAS stained and their MUC5AC mRNA levels were investigated by reverse transcription polymerase chain reaction (RT-PCR). Mice in control groups were injected with pSecTag2B/mCLCA3, pSecTag2B and saline, respectively. RESULTS: Antiserum of vaccine group after three times vaccination showed good binding activity to three mCLCA3 extracellular domains (ED), and the activity to N-terminal C-terminal ED was stronger than middle-ED. After induced to asthma, the number of goblet cell and MUC5AC mRNA level in vaccine group were lower than these in control group. CONCLUSION: hCLCA1 DNA vaccine can induce mouse to produce serum antibody binding itself mCLCA3, and thus airway mucus overproduction of asthmatic mouse is effectively inhibited.

[Cloning tumor-related genes and tumor suppressor genes in glioma with polymerase chain reaction-based subtractive hybridization].

BACKGROUND & OBJECTIVE: Glioma is a common tumor in central nervous system with no specific clinical therapy. Its pathogenesis is unclear. This study was to clone tumor-related genes and tumor suppressor genes in glioma with polymerase chain reaction (PCR)-based subtractive hybridization, and to explore the molecular biological mechanism of tumorigenesis of glioma. METHODS: mRNA was isolated from a sample of human glioma, and reversely transcribed into cDNA. PCR-based subtractive hybridization was used to clone tumor-related genes and tumor suppressor genes from it. RESULTS: In tumor-related candidate gene group, phospho-protein enriched in astrocytes of 15 (PEA15) and homology of acid fibroblast growth factor (aFGF) were picked up. Whereas, in tumor suppressor gene group, interferon-induced protein 17 and ndr2 were picked up. ndr2 was widely expressed in normal brain tissue, but absent in glioma tissue. CONCLUSION: ndr2 gene is a candidate tumor suppressor gene, and may play a role in tumorigenesis of glioma.

High expression of bcl-x(L) in K562 cells and its role in the low sensitivity of K562 to realgar-induced apoptosis.

Arsenic compounds (As(2)O(3 )or()As(4)S(4)) have been used successfully for the treatment of acute promyelocytic leukemia (APL) for quite a long time. It has been noticed that the sensitivity to apoptosis induced by As(2)O(3 )varies among various leukemia cells. It was reported by several groups that As(2)O(3) could induce apoptosis in APL-derived NB4 cells at concentrations of 0.5-1 mumol/l, whereas in other leukemia cells like K562, As(2)O(3) has no effects at the same concentration. K562 cells undergo apoptosis only when the concentration of As(2)O(3 )is greater than 2 mumol/l. Another arsenic compound, realgar (As(4)S(4)), a traditional Chinese mineral medicine, has been used to treat APL effectively and demonstrated to have lower toxicity than As(2)O(3). It would be interesting to know whether NB4 and K562 cells will show different sensitivity to realgar as well and if there is a difference, what is the cellular mechanism of it. In our present study, K562 cells were much less sensitive than NB4 cells to apoptosis induced by realgar. We confirm that the expression of bcl-x(L) is significantly higher in K562 cells than that in NB4 cells and is not downregulated upon realgar treatment. K562 cells become sensitive to realgar at clinically acceptable concentrations when bcl-x(L) expression level is downregulated by transfecting bcl-x(L) antisense RNA vector into the cells. Our results suggest that the increased bcl-x(L) expression in K562 cells contributes to its insensitivity to realgar-induced apoptosis.

[Cloning and expression of the extracellular domain of calcium-activated chloride channel in mouse airway goblet cells].

AIM: To clone and express the extracellular domain of murine calcium-activated chloride channel (mCLCA3) in airway goblet cell of mouse. METHODS: According to the gene sequence of mCLCA3 the PCR primers for N-terminal, middle and C-terminal extracellular domains were designed. Using recombinant plasmid pcDNA3.1(-)/mCLCA3 as template, the DNAs coding for the three extracellular domains were amplified. And then the DNAs encoding N-terminal and C-terminal extracellular domains were inserted into expression vector pRSET-A, while the middle extracellular domain DNA was inserted into pGEX-T1. E.coli. BL21(DE3) were transformed with the three recombinant plasmids, respectively, and were induced with IPTG for expression. RESULTS: DNA sequencing showed that the cloned DNAs encoding extracellular domains were identical with those in GenBank (GenBank accession No. NM-017474 ). The 3 domains were expressed in E.coli and most of the expressed products existed in the form of inclusion body. CONCLUSION: The expression of three extracellular domains of mCLCA3 lays the foundation for further preparing anti-mCLCA3 antibody and exploring the mechanism of modulation of mCLCA3.

Functional analysis of discoidin domain receptor 2 in synovial fibroblasts in rheumatoid arthritis.

In order to know whether any protein tyrosine kinase (PTK) is involved in the over-proliferation and erosiveness of synovial fibroblasts (SF) of rheumatoid arthritis (RA) patients, RT-PCR and RNA dot blotting were done to analyse PTKs profile in RA SF. Platelet-derived growth factor receptor A (PDGFRA), insulin-like growth factor 1 receptor (IGF-1R), Janus kinase 1 (JAK1), TYK2, discoidin domain receptor 2 (DDR2), and Lyn were expressed in SF, and the expression of PDGFRA, IGF-1R, and DDR2 in SF of RA were higher than that of osteoarthritis (OA, as control). Immunoblotting and gelatinase zymography showed that DDR2 in RA SF, which still secreted active matrix metalloproteinase 1 (MMP-1) in vitro, were in active form. Stimulation of collagen II could make NIH-3T3 cells (as control) produce MMP-1, which could be inhibited by soluble extracellular part of DDR2. These results indicated that the over-expression of MMP-1 in RA SF might be related to the activation of DDR2, and collagen II, act as DDR2 ligand, might be one of the stimulators of the over-expression of MMP-1 of RA SF.

[Inducible expression of reconstructed human caspase-6 gene in Hela cells].

AIM: To observe the pro-apoptotic effect of RCasp-6 gene in Hela cells. METHODS: RCasp-6 gene was amplified by PCR and cloned into the pIND vector. Hela cells were transfected with pIND-RCasp-6 and then inducced with ecdyson analogue. Expression of target gene was detected by immunocytochemical staining. Changes of the morphology and growth of Hela cells subjected to transfection of target gene were observed by HE staining and viable cell counting. RESULTS: RCasp-6 gene was cloned by PCR and its eukaryotic expression vector was successfully reconstructed. Expression of RCasp-6 gene in the transfected Hela cells leads to the morphological changes of the cells. Many of the transfected Hela cells shrunk and some cells were died. CONCLUSION: The expression of reconstructed human caspase-6 gene can efficiently accelerate death of Hela cells.

N-Myc downstream-regulated gene 2 (NDRG2) inhibits glioblastoma cell proliferation.

The most severe form of brain glioma, glioblastoma (GBM), is highly malignant and usually resistant to chemotherapy. Therefore, discovery of new targets for gene therapy is important. Using subtraction cloning, we identified the human N-Myc downstream-regulated gene 2 (hNDRG2), located at chromosome 14q11.2, as a gene that is significantly suppressed in GBM tissues. Semiquantitative RT-PCR showed that the hNDRG2 gene transcript is expressed in normal brain tissue and low-grade gliomas but is present at low levels in 15 of 27 (56%) human GBM tissues and all of the 6 human glioblastoma cell lines examined. Furthermore, transfection of human glioblastoma U373 and U138 cells with a cDNA encoding hNDRG2 markedly reduced the cell proliferation. Our findings provide the first evidence to suggest that hNDRG2 may play a role in glioblastoma carcinogenesis.