Differentiation of Tumorigenic C6 Glioma Cells Induced by Enhanced IL-6 Signaling.

Background and objectives: Cancer stem cells (CSCs) are obstacles to cancer therapy due to their therapeutic resistance, ability to initiate neoplasia, and roles in tumor relapse and metastasis. Efforts have been made to cure CSCs, such as the use of differentiation therapy, which induces cancer stem-like cells to undergo differentiation and decrease their tumorigenicity. Interleukin 6 (IL-6) upregulates the expression of glial fibrillary acidic protein (GFAP) in C6 glioma cells, indicating that it is able to induce the differentiation of these cells. The C6 glioma cell line forms a high percentage of cancer stem-like cells, leading us to speculate whether IL-6 signaling could modulate the differentiation of tumorigenic C6 glioma cells. However, we observed that IL-6 alone could not efficiently induce the differentiation of these cells. Therefore, different IL-6 signaling elicitors, including IL-6 alone, a combination of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), and tumor necrosis factor-α (TNF-α) plus IL-6/sIL-6R (TNF-α/IL-6/sIL-6R), were evaluated for their potential use in differentiation therapy. Materials and Methods: The potential of IL-6 signaling elicitors in differentiation therapy were examined by assessing changes in biomarker levels, the rate of cell proliferation, and tumorigenicity, respectively. Results: Enhanced IL-6 signaling could effectively induce C6 glioma cell differentiation, as determined by observed variations in the expression of differentiation, cell cycle, and stem cell biomarkers. Additionally, the total cell population and the tumorigenicity of glioma cells were all considerably reduced after TNF-α/IL-6/sIL-6R treatment. Conclusions: Our findings provide evidence that enhanced IL-6 signaling can efficiently promote tumorigenic C6 glioma cells to undergo differentiation.

Overexpression of the peanut CLAVATA1-like leucine-rich repeat receptor-like kinase AhRLK1 confers increased resistance to bacterial wilt in tobacco.

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease affecting hundreds of plant species, yet the host factors remain poorly characterized. The leucine-rich repeat receptor-like kinase gene AhRLK1, characterized as CLAVATA1, was found to be up-regulated in peanut upon inoculation with R. solanacearum. The AhRLK1 protein was localized in the plasma membrane and cell wall. qPCR results showed AhRLK1 was induced in a susceptible variety but little changed in a resistant cultivar after inoculated with R. solanacearum. Hormones such as salicylic acid, abscisic acid, methyl jasmonate, and ethephon induced AhRLK1 expression. In contrast, AhRLK1 expression was down-regulated under cold and drought treatments. Transient overexpression of AhRLK1 led to a hypersensitive response (HR) in Nicotiana benthamiana. Furthermore, AhRLK1 overexpression in tobacco significantly increased the resistance to R. solanacearum. Besides, the transcripts of most representative defense responsive genes in HR and hormone signal pathways were significantly increased in the transgenic lines. EDS1 and PAD4 in the R gene signaling pathway were also up-regulated, but NDR1 was down-regulated. Accordingly, AhRLK1 may increase the defense response to R. solanacearum via HR and hormone defense signaling, in particular through the EDS1 pathway of R gene signaling. These results provide a new understanding of the CLAVATA1 function and will contribute to genetic enhancement of peanut.

Substrate-induced changes in domain interaction of vacuolar H⁺-pyrophosphatase.

Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H(+)-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H(+)-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H(+)-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H(+)-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H(+)-pyrophosphatase in the presence and absence of substrate and its analog.

Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway.

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif ((161)DDDDDGDD(168)). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp(132), Thr(133), Asp(164), Asp(165), and Tyr(178). The goals of this study were to determine possible roles of the Ca(2+)-binding cluster for the interaction of LipL32 and Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca(2+)-binding residues of LipL32 to Ala, and their effects subsequently were observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells.

CɛmX peptide-carrying HBcAg virus-like particles induced antibodies that down-regulate mIgE-B lymphocytes.

Type-I hypersensitivity reactions play a critical role in the pathogenesis of various allergic diseases. The successful development of the anti-IgE antibody, omalizumab, has validated IgE as an effective therapeutic target for the treatment of various IgE-mediated allergic diseases. Two research groups have reported that mAbs specific for certain parts of CɛmX, a domain of 52 aa residues in human membrane-bound IgE (mIgE), can cause the lysis of mIgE-B cells by apoptosis and antibody-dependent cellular cytotoxicity (ADCC). Herein, we explore virus-like particles formed by hepatitis B virus core antigen (HBcAg) that harbors the entire CɛmX peptide or its fragments as immunogens for inducing anti-CɛmX antibodies. The results showed that mice immunized subcutaneously with these immunogens produced antibodies that bind to recombinant CɛmX-containing human IgE.Fc in ELISA and Western blot analyses, and to genetically engineered human mIgE-expressing Ramos B cell line in fluorescence flow cytometric assays. The IgG antibodies purified from the sera of immunized mice were able to cause the apoptosis of mIgE-expressing Ramos cells through a BCR-dependent caspase pathway. Furthermore, the IgG could mediate ADCC in human mIgE-expressing A20 murine B-cell lymphoma. These studies suggest that HBcAg-CɛmX peptide immunogens warrant further investigation as a therapeutic modality for modulating IgE in patients with IgE-mediated allergic diseases.

Abi plays an opposing role to Abl in Drosophila axonogenesis and synaptogenesis.

Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. Here we show that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, we show that mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled (ena) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization.

Identification and characterization of a novel fibril forming peptide in fungal starch binding domain.

Scanty information is available regarding the chemical basis for structural alterations of the carbohydrate-binding modules (CBMs). The N-terminal starch binding domain (SBD) of Rhizopus oryzae glucoamylase (GA) forms fibrils under thermal stress, presenting an unusual conformational change from immunoglobulin-like to beta-sheet-rich structure. Site-directed mutagenesis revealed that the C-terminal Lys of SBD played a crucial role in the fibril formation. The synthetic peptide (DNNNSANYQVSTSK) representing the C-terminal 14 amino acid residues of SBD was further demonstrated to act as a fibril-forming segment, in which terminal charges and an internal NNNxxNYQ motif were key fibril-forming determinants. The formation of fibril structure in a fungal SBD, caused by its chemical and biophysical requirements, was demonstrated for the first time.

Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.

Radiation inactivation analysis of progesterone receptor in human uterine cytosol.

It is believed that human progesterone receptor (PR) contains a ligand binding subunit A (83 kDa) or subunit B (120 kDa) and 2 copies of heat shock proteins (hsp90) of molecular weight 90 kDa. To elucidate the mechanism of hormone binding, we employed radiation inactivation to determine its functional size. The functional masses determined in the presence of glycerol, molybdate and potassium chloride were 120+/-14, 124+/-13 and 130+/-20 kDa, respectively. From scatchard plot analysis, the radiation decreased the binding sites and increased the binding affinity of PR with ligand. The functional masses of PR dissolved in the three variant buffers were similar to the molecular weight of PR subunit B. The results implied that PR subunit B could bind with ligand despite hsp90 and hsp90 was not involved in the PR binding to progesterone.