Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Insights into the conformational changes of several human lysozyme variants associated with hereditary systemic amyloidosis

In this study, various ethanol- and temperature-induced molecular dynamics simulations were conducted to investigate the conformational changes of several human lysozyme variants (I56T, D67H, and T70N) associated with hereditary systemic amyloidosis. The results show that these variants are all more sensitive to conditions affecting the structural integrity of this protein. The structural analyses of these variants reveal a high population of more unstable beta-domain and distorted hydrophobic core compared to the wild-type human lysozyme, particularly for the two natural amyloidogenic variants D67H and I56T. For the D67H variant, the distance between the mass centers of residues 54 and 67 was found to elongate as a result of the destruction of the hydrogen-bonding network stabilizing the two long loops in the beta-domain. It further accelerates the unfolding of this variant, starting from the hydrophobic core between the alpha- and beta-domains. For the I56T variant, the introduction of a hydrophilic residue in the hydrophobic core directly destroys the native contacts in the alpha-beta interface, leading to fast unfolding. The present results are consistent with the previous hypothesis suggesting that the distortion of the hydrophobic core at the alpha-beta interface putatively results in the formation of the initial "seed" for amyloid fibrils.     

Src phosphorylates Grb2-associated binder 1 upon hepatocyte growth factor stimulation

Grb2-associated binder 1 (Gab1) is known to play an important role in hepatocyte growth factor (HGF) signaling, which rapidly becomes tyrosine-phosphorylated upon HGF stimulation. In this study, we found that the tyrosine phosphorylation of Gab1 in the cells derived from Src/Yes/Fyn null mouse embryos was approximately 40% lower than that in their wild type counterparts upon HGF stimulation. Increased expression of wild-type Src enhanced HGF-induced phosphorylation of Gab1, and, in contrast, expression of the Src kinase-deficient mutant or treatment of the specific Src inhibitor PP1 suppressed it. Expression of a constitutively active Src mutant (Y527F) or oncogenic v-Src led to a prominent increase in Gab1 phosphorylation independent of HGF stimulation. Moreover, Src interacted with Gab1 via both its Src homology 2 and 3 domains and was capable of phosphorylating purified Gab1 in vitro. Finally, the increased phosphorylation of Gab1 by Src selectively potentiated HGF-induced activation of ERK and AKT. Taken together, our results establish a new role for Src in HGF-induced Gab1 phosphorylation.     

Reciprocal raft-receptor interactions and the assembly of adhesion complexes

Cell adhesion complexes are critical for the physical coordination of cell-cell interactions and the morphogenesis of tissues and organs. Many adhesion receptors are anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) moiety and are thereby partitioned into membrane rafts. In this review, we focus on reciprocal interactions between rafts and adhesion molecules, leading to receptor clustering and raft expansion and stability. A model for a three-stage adhesion complex assembly process is also proposed. First, GPI-anchored adhesion molecules are recruited into rafts, which in turn promote receptor cis-oligomerization and thereby produce precursory complexes primed for avid trans-interactions. Second, trans-interactions of the receptors cross-link and stabilize large amalgams of rafts at sites of adhesion complex assembly. Finally, the enlarged and stabilized rafts acquire enhanced abilities to recruit the cytoskeleton and induce signaling. This process exemplifies how the domain structure of the plasma membrane can impact the function of its receptors.     

Purification and characterization of a novel 7-kDa non-specific lipid transfer protein-2 from rice (Oryza sativa)

A novel 7-kDa non-specific lipid transfer protein-2 (nsLTP2) has been isolated from rice (Oryza sativa) seeds. In contrast to nsLTP1s, few nsLTP2s have been purified and characterized. Complete amino acid sequence of rice nsLTP2 was determined by N-terminal Edman degradation of the intact protein as well as the peptide fragments resulted from trypsin digestions. Rice nsLTP2 consists of 69 amino acid residues with eight conserved cysteines forming four disulfide bonds. The secondary structure of rice nsLTP2 is predominantly alpha-helical as determined by circular dichroism spectroscopy. Cysteine pairings of nsLTP2 have one miss match at Cys(35)-X-Cys(37) motif compared to nsLTP1. Primary structure analysis of various plant nsLTP2s revealed an interesting conservation of sequence features among nsLTP2 family.     

Antibiotic amoxicillin induces DNA lesions in mammalian cells possibly via the reactive oxygen species

Amoxicillin is a commonly prescribed drug for anti- bacterial infection. In this study, we are interested in the effect of the drug on the cellular DNA integrity. Amoxicillin was added to the human or hamster cells in culture, and the DNA lesions induced by the drug were assessed by a comet assay with nuclear extract incubation (Wang et al., 2005 Anal Biochem 337: 70-75). Amoxicillin at 5mM rapidly induced DNA lesions in human AGS cells. The level of DNA lesions attained a maximum at about 1h, and then declined steadily and reached almost the basal level at 6h following the drug treatment. Similar induction pattern of DNA lesions was found with amoxicillin-related antibiotics such as ampicillin but not with the unrelated antibiotics such as kanamycin. For studying the repair kinetics, the cells were treated with amoxicillin for only 1h and continued culture in the absence of the drug for a certain period of time before subsequent analysis. Repair of the amoxicillin-induced DNA lesions was essentially completed within 4h. Such repair may not involve nucleotide excision repair (NER) pathway because the repair was completed with similar kinetics in both NER proficient Chinese hamster CHO-K1 cells and its isogenic NER deficient UV24 cells. Instead, the repair may involve base excision repair (BER) pathway because immunodepletion of OGG1/2, glycosylases involved in BER rendered the nuclear extract unable to excise DNA lesions induced by amoxicillin in the modified comet assay. Furthermore, amoxicillin induced intracellular reactive oxygen species (ROS) at the tempo similar to that of DNA lesions induction. Thus, we hypothesize that amoxicillin causes oxidative DNA damage in mammalian cells via ROS.     

A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase

Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ^−/− cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.     

High yield expression in a recombinant <Emphasis Type="Italic">E. coli</Emphasis> of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

Background

Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

Results

Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.

Conclusions

Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

     

Molecular mechanisms of survival and apoptosis in RAW 264.7 macrophages under oxidative stress

Organisms living in an aerobic environment are continuously exposed to reactive oxygen species (ROS). Apoptosis of cells can be induced by ROS and cells also develop negative feedback mechanisms to limit ROS induced cell death. In this study, RAW264.7 murine macrophage cells were treated with H₂O₂ and cDNA microarray technique was used to produce gene expression profiles. We found that H₂O₂ treatment caused up-regulation of stress, survival and apoptosis related genes, and down-regulation of growth and cell cycle promoting genes. Numerous genes of metabolism pathways showed special expression patterns under oxidative stress: glycolysis and lipid synthesis related genes were down-regulated whereas the genes of lipid catabolism and protein synthesis were up-regulated. We also identified several signaling molecules as ROS-responsive, including p53, Akt, NF- B, ERK, JNK, p38, PKC and INF- . They played important roles in the process of apoptosis or cell survival. Finally, an interactive pathway involved in cellular response to oxidative stress was proposed to provide some insight into the molecular events of apoptosis induced by ROS and the feedback mechanisms involved in cell survival.Y. Zhang and C.C. Fong contributed equally to this work.