Improved low pH bicelle system for orienting macromolecules over a wide temperature range

We have prepared and characterized a novel bicelle system composed of 1,2-di-O-dodecyl-sn-glycero-3-phos- phocholine (DIODPC) and 3-(chloramidopropyl)dimethylammonio-2-hydroxyl-1-propane sulfonate (CHAPSO). At the optimal DIODPC/CHAPSO molar ratio of 4.3:1, this medium becomes magnetically oriented from pH 6.5 down to pH 1.0. Unlike previously reported bicelle preparations, these bicelles are chemically stable at low pH and are capable of inducing protein alignment, as illustrated by the large residual dipolar couplings measured for rusticyanin from Thiobacillus ferrooxidans at pH 2.1. The DIODPC/CHAPSO system is particularly useful for measuring residual dipolar couplings of macromolecules that require very acidic conditions.     

Quantifying information transfer by protein domains: Analysis of the Fyn SH2 domain structure

Background  

Efficient communication between distant sites within a protein is essential for cooperative biological response. Although often associated with large allosteric movements, more subtle changes in protein dynamics can also induce long-range correlations. However, an appropriate formalism that directly relates protein structural dynamics to information exchange between functional sites is still lacking.     

Cdh1 Regulates Cell Cycle through Modulating the Claspin/Chk1 and the Rb/E2F1 Pathways

APC/Cdh1 is a major cell cycle regulator and its function has been implicated in DNA damage repair; however, its exact role remains unclear. Using affinity purification coupled with mass spectrometry, we identified Claspin as a novel Cdh1-interacting protein and further demonstrated that Claspin is a novel Cdh1 ubiquitin substrate. As a result, inactivation of Cdh1 leads to activation of the Claspin/Chk1 pathway. Previously, we demonstrated that Rb interacts with Cdh1 to influence its ability to degrade Skp2. Here, we report that Cdh1 reciprocally regulates the Rb pathway through competing with E2F1 to bind the hypophosphorylated form of Rb. Although inactivation of Cdh1 in HeLa cells, with defective p53/Rb pathways, led to premature S phase entry, acute depletion of Cdh1 in primary human fibroblasts resulted in premature senescence. Acute loss of many other major tumor suppressors, including PTEN and VHL, also induces premature senescence in a p53- or Rb-dependent manner. Similarly, we showed that inactivation of the p53/Rb pathways by overexpression of SV40 LT-antigen partially reversed Cdh1 depletion–induced growth arrest. Therefore, loss of Cdh1 is only beneficial to cells with abnormal p53 and Rb pathways, which helps explain why Cdh1 loss is not frequently found in many tumors.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

ETISEQ – an algorithm for automated elution time ion sequencing of concurrently fragmented peptides for mass spectrometry-based proteomics

Background  

Concurrent peptide fragmentation (i.e. shotgun CID, parallel CID or MS^E) has emerged as an alternative to data-dependent acquisition in generating peptide fragmentation data in LC-MS/MS proteomics experiments. Concurrent peptide fragmentation data acquisition has been shown to be advantageous over data-dependent acquisition by providing greater detection dynamic range and providing more accurate quantitative information. Nevertheless, concurrent peptide fragmentation data acquisition remains to be widely adopted due to the lack of published algorithms designed specifically to process or interpret such data acquired on any mass spectrometer.     

Reactivity of an antimetastatic organometallic ruthenium compound with metallothionein-2: relevance to the mechanism of action

The reaction of metallothionein-2 (MT-2) with the organometallic antitumour compound [Ru(η(6)-p-cymene)Cl(2)(pta)], RAPTA-C, was investigated using ESI MS and ICP AES. The studies were performed in comparison to cisplatin and significant differences in the binding of the two complexes were observed. RAPTA-C forms monoadducts with MT-2, at variance with cisplatin, that has been observed to form up to four adducts. These data, combined with ICP AES analysis, show that binding of both RAPTA-C and cisplatin to MT-2 requires the displacement of an equivalent amount of zinc, suggesting that Cys residues are the target binding sites for the two metallodrugs. The competitive binding of RAPTA-C and cisplatin towards a mixture of ubiquitin (Ub) and MT-2 was also studied, showing that MT-2 can abstract RAPTA-C from Ub more efficiently than it can abstract cisplatin. The mechanistic implications of these results are discussed.     

Efficacy of several insecticides alone and with horticultural mineral oils on light brown apple moth (Lepidoptera: Tortricidae) eggs

The aim of the research was to identify efficacious and less environmentally harmful treatments than the standard chlorpyrifos sprays used for the control light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), eggs on nursery stock. A series of dip experiments showed a range of responses when comparing the efficacy of insecticides on egg hatch of E. postvittana. The insecticides that compared most favorably with chlorpyrifos were lamda-cyhalothrin and gamma-cyhalothrin, and thiacloprid. Indoxacarb, novaluron, and spinosad caused significant mortality only when combined with All Seasons mineral oil. All Seasons, showed ovicidal properties when evaluated alone and demonstrated adjuvant properties when combined with the above-mentioned insecticides, except gamma-cyhalothrin and thiacloprid. Several other horticultural mineral oils performed similarly, except the efficacy of spinosad varied with the oil product used, suggesting that the oil type selected is important for some insecticide and oil combinations. Several insecticides evaluated in this study are likely candidates for further work to develop treatments against E. postvittana eggs on nursery plants. Mineral oils are ovicidal and combinations with insecticides are likely to be advantageous.     

Identification of a proline-rich inositol polyphosphate 5-phosphatase (PIPP)•collapsin response mediator protein 2 (CRMP2) complex that regulates neurite elongation

Neuron polarization is essential for the formation of one axon and multiple dendrites, establishing the neuronal circuitry. Phosphoinositide 3-kinase (PI3K) signaling promotes axon selection and elongation. Here we report in hippocampal neurons siRNA knockdown of the proline-rich inositol polyphosphate 5-phosphatase (PIPP), which degrades PI3K-generated PtdIns(3,4,5)P(3), results in multiple hyperelongated axons consistent with a polarization defect. We identify collapsin response mediator protein 2 (CRMP2), which regulates axon selection by promoting WAVE1 delivery via Kinesin-1 motors to the axon growth cone, as a PIPP-interacting protein by Y2H screening, direct binding studies, and coimmunoprecipitation of an endogenous PIPP, CRMP2, and Kinesin-1 complex from brain lysates. The C-terminal growth cone-targeting domain of PIPP facilitates its interaction with CRMP2. PIPP growth cone localization is CRMP2-dependent. PIPP knockdown in PC12 cells promotes neurite elongation, WAVE1 and Kinesin-1 growth cone localization, whereas knockdown of CRMP2 exhibits the opposite phenotype, with shorter neurites and decreased WAVE1/Kinesin-1 at the growth cone. In contrast, CRMP2 overexpression promotes neurite elongation, a phenotype rescued by full-length PIPP, or expression of the CRMP2-binding PIPP domain. Therefore this study identifies PIPP and CRMP2 exert opposing roles in promoting axon selection and neurite elongation and the complex between these proteins serves to regulate the localization of effectors that promote neurite extension.