one-step-negative-chromatographic-purification-helicobacter-pylori

One-step Negative Chromatographic Purification of <em>Helicobacter pylori</em> Neutrophil-activating Protein Overexpressed in <em>Escherichia coli</em> in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

National Tsing Hua University

CASK acts as a coactivator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK coactivation effect is unclear. Here, we report that CASK binds to another nuclear protein, CINAP, which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b and reelin. A knockdown of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1/CASK/CINAP protein complex and thus modifies expression of the NR2b gene.

Transcriptional modification by a CASK-interacting nucleosome assembly protein.

CASK interacting nucleosome assembly protein (CINAP) modulates gene expression and its abundance in cultured neurons is regulated by synaptic activity. To further study the function of CINAP in vivo, we examined the temporal and spatial expression profiles of CINAP. CINAP was widely expressed in different regions of adult mouse brain, including the cerebral cortex, hippocampus, striatum, hypothalamus, cerebellum, and two adult brain regions known to generate progenitor neurons. During early development, CINAP was also expressed in regions where neuronal progenitor cells were actively dividing, the ventricular and subventricular zones, suggesting that in addition to regulating gene expression in mature neurons, CINAP may also play a role in dividing cells. Since the hypothalamus responds to several physiological responses, we examined whether CINAP protein levels in the paraventricular nucleus (PVN) of the hypothalamus are regulated by changes in osmolality achieved through oral administration of hypertonic saline. Compared with control mice, mice treated with hypertonic saline expressed higher CINAP protein levels in the PVN, supporting a role of CINAP in neural response in vivo. Using confocal microscopic analysis, a significant amount of CINAP was found in the cytoplasm of neurons. Biochemical fractionation further confirmed that CINAP was associated with synapses, suggesting a translocation of CINAP protein from synapse to the nucleus. Consistent with this speculation, nuclear CINAP levels in the PVN were higher in hypertonic saline-treated mice than those who drank water. CINAP may be regulated through changes in protein stability and nuclear translocation in neurons.

Neural activity- and development-dependent expression and distribution of CASK interacting nucleosome assembly protein in mouse brain.

Bcl11A/Evi9/CTIP1, a Kruppel-like zinc finger gene, plays an important role in B-cell development. In addition to expression in B lymphocytes, Bcl11A/Evi9/CTIP1 is also highly expressed in the brain, although its function there is still unclear. In the present study, regional and subcellular distributions of Bcl11A/Evi9/CTIP1 in rat brain were investigated by immunostaining and biochemical fractionation. Using antibodies recognizing the first 18 amino acid residues of Bcl11A/Evi9/CTIP1, the distribution of 2 isoforms of Bcl11A/Evi9/CTIP1 gene products, Bcl11A-L/Evi9a and Bcl11A-S/Evi9c, was examined. In rat brain, both Bcl11A-L/Evi9a and Bcl11A-S/Evi9c were expressed, although the amount of Bcl11A-S/Evi9c protein was higher. Bcl11A-S/Evi9c was widely expressed in different regions of the rat brain. In contrast, Bcl11A-L/Evi9a was more restricted, being expressed in the cerebral cortex, hippocampus, and olfactory bulb. At the subcellular level, biochemical fractionation and confocal analysis of adult rat brain revealed that, in addition to being in the nuclei of neurons, fractions of Bcl11A-L/Evi9a and Bcl11A-S/Evi9c could be found in extranuclear locations. Double staining with the synaptic marker synaptophysin indicated a synaptic distribution of Bcl11A/Evi9/CTIP1. Postsynaptic density was also biochemically purified and subjected to immunoblotting using Bcl11A/Evi9/CTIP1 antibodies. The results showed that Bcl11A-L/Evi9a was enriched in the PSD I and PSD II fractions. In contrast, only a trace amount of Bcl11A-S was detected in PSD fractions. Our study also indicated that a fraction of Bcl11A/Evi9/CTIP1 was present in the cytoplasm, even at synapses. To regulate gene expression in the nuclei, nuclear translocation of Bcl11A/Evi9/CTIP1 may be one of the mechanisms controlling neuronal Bcl11A/Evi9/CTIP1 function.

Expression of zinc finger transcription factor Bcl11A/Evi9/CTIP1 in rat brain.

The extension of axon branches is important for target innervation but how axon branching is regulated is currently not well understood. Here, we report that Bcl11A/CTIP1/Evi9, a zinc finger transcription factor, downregulates axon branching. Knockdown of Bcl11A induced axon branching and multi-axon formation, as well as dendrite outgrowth. Due to alternative splicing, a single Bcl11A gene encodes two protein products, Bcl11A-L and -S. Bcl11A-L was found to be the main Bcl11A player in regulation of neurite arborization; Bcl11A-S is an antagonist of Bcl11A-L. Time-lapse study further suggests that Bcl11A-L knockdown enhances axon dynamics and increases the duration of axon outgrowth. Finally, the expression of DCC and MAP1b, two molecules involved in direction and branching of axon outgrowth, is controlled by Bcl11A-L. DCC overexpression rescues the phenotype induced by Bcl11A-L knockdown. In conclusion, this report provides the first evidence that Bcl11A is important for neurite arborization.

Bcl11A/CTIP1 regulates expression of DCC and MAP1b in control of axon branching and dendrite outgrowth.

While neuronal activity regulates neurite outgrowth and branching, the details of the underlying mechanisms are still largely unclear. This study investigated the effect of the glutamate receptors on neuritogenesis using cultured hippocampal neurons. At 3-4 days in vitro, NMDA treatment promoted axon branching but not primary axon extension. In contrast, blockade of the NMDA receptor (NMDAR) by AP5 treatment enhanced primary axon extension. NMDAR activation also increased dendrite number and total dendrite length. These results suggest that NMDAR controls axon and dendrite morphogenesis. A previous study demonstrated that knockdown of the zinc finger transcription factor B cell lymphoma 11A-long (Bcl11A-L) reduces deleted in colorectal cancer (DCC) and microtubule-associated protein (MAP) 1b expression, thereby promoting axon branching. Here, glutamate stimulation down-regulated the levels of the Bcl11A-L, DCC, MAP1b, and MAP2c proteins. Over-expression of either Bcl11A-L or DCC countered the effect of NMDA or glutamate on axon branching and dendrite outgrowth, indicating that the Bcl11A-L/DCC pathway is an important downstream effector of glutamate receptors in neurite arborization. Because knockdown of Bcl11A-L did not down-regulate MAP2c, our results suggest that glutamate receptors also use a Bcl11A-L-independent pathway to control dendrite outgrowth. To summarize, this study reveals novel pathways downstream of glutamate receptors that regulate axon and dendrite arborization.

Bcl11A/CTIP1 mediates the effect of the glutamate receptor on axon branching and dendrite outgrowth.

Calcium/calmodulin-dependent serine kinase (CASK), a causative gene in X-linked mental retardation, carries out multiple functions in neurons, including vesicle trafficking of ion channels, synapse formation, and gene transcription. From a yeast two-hybrid screen, Krüppel-like zinc finger protein B cell lymphoma/COUP-TF-interacting protein 1 (Bcl11A/CTIP1) was identified as a CASK binding protein. Through alternative splicing, a single Bcl11A gene encodes two major protein products in neurons, Bcl11A-S and Bcl11A-L. CASK interacted with both Bcl11A-S and Bcl11A-L in transfected COS cells and brain. Immunofluorescence staining further indicated the colocalization of CASK and Bcl11A in the nuclei of neurons. These studies supported an interaction between CASK and Bcl11A in vivo. Bcl11A-L has previously been shown to play a role in gene transcription as well as axon outgrowth and branching. Here, we further show that Bcl11A-L rearranges the distribution of nuclear actin, which may be related to the function of Bcl11A-L in gene expression. More importantly, using cultured hippocampal neurons as a model system, we show that CASK enhances the ability of Bcl11A-L to restrict axon outgrowth and branching. Interruption of the interaction between CASK and Bcl11A increased the outgrowth and branching of axons, suggesting that the interaction between CASK and Bcl11A controls axon arborization. In conclusion, our results suggest that, through the interaction with Bcl11A, CASK plays a role in axonogenesis, which may be related to brain anatomical characteristics in humans.

X-linked mental retardation gene CASK interacts with Bcl11A/CTIP1 and regulates axon branching and outgrowth.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-induced gastric inflammation. Due to its immunogenic and immunomodulatory properties, HP-NAP has been used for developing vaccines against H. pylori infection and new drugs for cancer therapy.

High yield purification of Helicobacter pylori neutrophil-activating protein overexpressed in Escherichia coli.

Helicobacter pylori neutrophil-activating protein (HP-NAP) activates several innate leukocytes including neutrophils, monocytes, and mast cells. It has been reported that HP-NAP induces degranulation and interleukin-6 (IL-6) secretion of rat peritoneal mast cells. However, the molecular mechanism is not very clear. Here, we show that HP-NAP activates human mast cell line-1 (HMC-1) cells to secrete histamine and IL-6. The secretion depends on pertussis toxin (PTX)-sensitive heterotrimeric G proteins but not on Toll-like receptor 2. Moreover, HP-NAP induces PTX-sensitive G protein-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38-mitogen-activated protein kinase (p38 MAPK), and Akt in HMC-1 cells. Inhibition of ERK1/2, p38 MAPK, or phosphatidylinositol 3-kinase (PI3K) suppresses HP-NAP-induced release of histamine and IL-6 from HMC-1 cells. Thus, the activation of HMC-1 cells by HP-NAP is through Gi-linked G protein-coupled receptor-mediated MAPKs and PI3K/Akt pathways.

Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells.

Patients with risk factors including younger age, mastoiditis, external ear disease, treatment by older surgeons, and concomitant mastoidectomy should receive detailed management to minimize the probability of re-operation for chronic otitis media (COM).

Chronic otitis media surgery and re-operation risk factor analysis: A nationwide retrospective cohort study of 18 895 patients.

We introduce an idea of synthesizing a class of genetic registers based on the existing sequential biological circuits, which are composed of fundamental biological gates. In the renowned literature, biological gates and genetic oscillator have been unveiled and experimentally realized in recent years. These biological circuits have formed a basis for realizing a primitive biocomputer. In the traditional computer architecture, there is an intermediate load-store section, i.e. a register, which serves as a part of the digital processor. With which, the processor can load data from a larger memory into it and proceed to conduct necessary arithmetic or logic operations. Then, manipulated data are stored back to the memory by instruction via the register. We propose here a class of bio-registers for the biocomputer. Four types of register structures are presented. In silicon experiments illustrate results of the proposed design.

Implementation of a genetic logic circuit: bio-register.

Hedgehog (HH) pathway plays an important role in embryonic development, but is largely inactive in adult except for tissue repair. Aberrant activation of HH pathway has been found in a variety of cancer types. In non-small cell lung cancer, however, the role and importance of HH pathway remain controversial. In the current study, we found that HH pathway was maintained in low activity in lung adenocarcinoma (LAC) cells under normal culture condition, but was highly induced in response to stress conditions. Activation of HH pathway promoted cell survival, growth, and invasion partially through HGF and MET signaling. Hedgehog-Interacting Protein (HHIP), a cell-surface negative regulator of HH pathway, was epigenetically silenced in LAC. Overexpression of HHIP blocked the activation of HH and HGF/MET pathways, and made cells significantly more susceptible to stress conditions. In LAC cells with acquired resistance to Epidermal Growth Factor Receptor Tyrosin Kinase Inhibitor (EGFR-TKI), we found that a part of tumor cells were much more sensitive to HH or HGF/MET inhibitors, suggesting an oncogenic addiction shift from EGFR to HH and HGF/MET pathways. In conclusion, this study showed that HH pathway is a survival signaling that drives LAC cell growth under stress conditions, and HHIP is a key regulator to block the induction of HH pathway. Targeting the HH pathway through inhibitors or HHIP thus holds promise to address EGFR-TKI resistance in LAC in clinic.

Hedgehog pathway maintains cell survival under stress conditions, and drives drug resistance in lung adenocarcinoma.

Palatoplasty can significantly decrease their middle ear re-intubation rate with a relatively lower hazard ratio compared to children who underwent VTI only.

Ting-Yu Kuo

Institute of Molecular and Cellular Biology

National Tsing Hua University

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Ting-Yu Kuo

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Molecular and Cellular Biology

National Tsing Hua University

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Institute of Molecular and Cellular Biology