Reversibility of phorbol diester-induced macrophage spreading

Phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate induced spreading of mouse macrophages with 50% effective concentrations of 3 nM and 35 nM, respectively. Macrophages treated with 100 or 1000 nM phorbol 12, 13- dibutyrate showed a time related decrease in spreading after washout. Spreading induced by 1, 10, or 100 nM phorbol 12-myristate 13-acetate was irreversible; however, washed phorbol 12,13-dibutyrate-treated cells respread after a second exposure to this compound. Washout of ³[H]phorbol diesters corroborated these observations in that 5% of ³H-phorbol 12-myristate 13-acetate and only 0.1% ³[H]phorbol, 12,13-dibutyrate remained associated with washed cells. Since phorbol 12-myristate 13 acetate is much more lipophilic than phorbol 12,13-dibutyrate, the reversibility of phorbol diester-induced macrophage spreading may depend upon the lipophilicity of the derivative utilized.Abbreviations DMEM Dulbecco's Minimal Essential Medium - PDA phorbol 12,13-diacetate - PDBu phorbol 12, 13-dibutyrate - PMA phorbol 12 myristate, 13 acetate - 4PDD phorbol 12, 13 didecanoate     

Fast tandem mass spectra-based protein identification regardless of the number of spectra or potential modifications examined

MOTIVATION: Comparing tandem mass spectra (MSMS) against a known dataset of protein sequences is a common method for identifying unknown proteins; however, the processing of MSMS by current software often limits certain applications, including comprehensive coverage of post-translational modifications, non-specific searches and real-time searches to allow result-dependent instrument control. This problem deserves attention as new mass spectrometers provide the ability for higher throughput and as known protein datasets rapidly grow in size. New software algorithms need to be devised in order to address the performance issues of conventional MSMS protein dataset-based protein identification. METHODS: This paper describes a novel algorithm based on converting a collection of monoisotopic, centroided spectra to a new data structure, named 'peptide finite state machine' (PFSM), which may be used to rapidly search a known dataset of protein sequences, regardless of the number of spectra searched or the number of potential modifications examined. The algorithm is verified using a set of commercially available tryptic digest protein standards analyzed using an ABI 4700 MALDI TOFTOF mass spectrometer, and a free, open source PFSM implementation. It is illustrated that a PFSM can accurately search large collections of spectra against large datasets of protein sequences (e.g. NCBI nr) using a regular desktop PC; however, this paper only details the method for identifying peptide and subsequently protein candidates from a dataset of known protein sequences. The concept of using a PFSM as a peptide pre-screening technique for MSMS-based search engines is validated by using PFSM with Mascot and XTandem. AVAILABILITY: Complete source code, documentation and examples for the reference PFSM implementation are freely available at the Proteome Commons, http://www.proteomecommons.org and source code may be used both commercially and non-commercially as long as the original authors are credited for their work.     

Myosin VI regulates endocytosis of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl(-) channel expressed in the apical plasma membrane in fluid-transporting epithelia. Although CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated in clathrin-mediated endocytosis in epithelial cells. The goal of this study was to determine whether myosin VI regulates CFTR endocytosis. Endogenous, apical membrane CFTR in polarized human airway epithelial cells (Calu-3) formed a complex with myosin VI, the myosin VI adaptor protein Disabled 2 (Dab2), and clathrin. The tail domain of myosin VI, a dominant-negative recombinant fragment, displaced endogenous myosin VI from interacting with Dab2 and CFTR and increased the expression of CFTR in the plasma membrane by reducing CFTR endocytosis. However, the myosin VI tail fragment had no effect on the recycling of endocytosed CFTR or on fluid-phase endocytosis. CFTR endocytosis was decreased by cytochalasin D, an actin-filament depolymerizing agent. Taken together, these data indicate that myosin VI and Dab2 facilitate CFTR endocytosis by a mechanism that requires actin filaments.     

The mandatory CAM testing of cells and scaffolds for tissue engineering: benefits for the three Rs of cooperation with the vaccine industry

In cooperation with BAXTER Vaccine AG, which supplies incubated special pathogen-free chicken eggs (including a full veterinary record), a permanent hen's egg chorio-allantoic membrane test (HET-CAM) unit has been established, where angiogenesis testing, cell culture, and digital and histological analyses are performed. At the Core Unit for Biomedical Research, the location of the animal testing facility of the Medical University Vienna, cell-scaffold constructs must be evaluated in vitro and in ovo prior to eventual in vivo tissue engineering experiments. The animal testing advisory committee requires that new test proposals are first evaluated by using cell culture and HET-CAM models. Approvals for in vivo experiments are postponed and not issued prior to in vitro/in ovo evaluation. Examples are presented of protocols planned for in vivo studies on cell seeded scaffolds, which were refined after in vitro/in ovo evaluations.     

Phosphate-uptake behaviour of a mutant of Synechococcus sp. PCC 7942 lacking one protein of the high-affinity phosphate-uptake system

The phosphate-uptake behaviour of a sphX mutant of the cyanobacterium Synechococcus leopoliensis (Raciborski) Komarek, strain PCC 7942 has been studied. This mutant lacks the high-affinity phosphate-binding protein that is abundantly expressed under phosphate-deficient growth conditions. The following observations have been made: (i) The mutant is still capable of utilizing phosphate at nanomolar external concentrations. (ii) Elimination of the sphX gene leads to an increase in the Michaelis constant and the maximum velocity of the initial influx of ³²P-phosphate. (iii) The capacity of the wild type to adapt within a few minutes to a transitory increase in the external phosphate concentration in an energetically efficient way (G. Falkner et al. 1994, C R Acad Sci Paris, Life sciences 317: 535–541) is lost in the mutant. As a result, the mutant can no longer attain pulse-adapted states that reflect in a characteristic way preceding exposures to higher phosphate concentrations. Received: 6 February 1998 / Accepted: 8 May 1998     

A Modified-Whittaker nested vegetation sampling method

A standardized sampling technique for measuring plant diversity is needed to assist in resource inventories and for monitoring long-term trends in vascular plant species richness. The widely used Whittaker plot (Shmida 1984) collects species richness data at multiple spatial scales, using 1 m², 10 m², and 100 m² subplots within a 20 m × 50 m (1000 m²) plot, but it has three distinct design flaws involving the shape and placement of subplots. We modified and tested a comparable sampling design (Modified-Whittaker plot) that minimizes the problems encountered in the original Whittaker design, while maintaining many of its attractive attributes. We overlaid the two sampling methods in forest and prairie vegetation types in Larimer County, Colorado, USA (n=13 sites) and Wind Cave National Park, South Dakota, USA (n=19 sites) and showed that the modified design often returned significantly higher (p<0.05) species richness values in the 1 m², 10 m², and 100 m² subplots. For all plots, except seven ecotone plots, there was a significant difference (p<0.001) between the Whittaker plot and the Modified-Whittaker plot when estimating the total number of species in the 1000 m² plots based on linear regressions of the subplot data: the Whittaker plot method, on average, underestimated plant species richness by 34%. Species-area relationships, using the Modified-Whittaker design, conformed better to published semilog relationships, explaining, on average, 92% of the variation. Using the original Whittaker design, the semilog species-area relationships were not as strong, explaining only 83% of the variation, on average. The Modified-Whittaker plot design may allow for better estimates of mean species cover, analysis of plant diversity patterns at multiple spatial scales, and trend analysis from monitoring a series of strategically-placed, long-term plots.     

Lack of interaction between ErbB2 and insulin receptor substrate signaling in breast cancer

Background

ErbB2 Receptor Tyrosine Kinase 2 (ErbB2, HER2/Neu) is amplified in breast cancer and associated with poor prognosis. Growing evidence suggests interplay between ErbB2 and insulin-like growth factor (IGF) signaling. For example, ErbB2 inhibitors can block IGF-induced signaling while, conversely, IGF1R inhibitors can inhibit ErbB2 action. ErbB receptors can bind and phosphorylate insulin receptor substrates (IRS) and this may be critical for ErbB-mediated anti-estrogen resistance in breast cancer. Herein, we examined crosstalk between ErbB2 and IRSs using cancer cell lines and transgenic mouse models.

Methods

MMTV-ErbB2 and MMTV-IRS2 transgenic mice were crossed to create hemizygous MMTV-ErbB2/MMTV-IRS2 bigenic mice. Signaling crosstalk between ErbB2 and IRSs was examined in vitro by knockdown or overexpression followed by western blot analysis for downstream signaling intermediates and growth assays.

Results

A cross between MMTV-ErbB2 and MMTV-IRS2 mice demonstrated no enhancement of ErbB2 mediated mammary tumorigenesis or metastasis by elevated IRS2. Substantiating this, overexpression or knockdown of IRS1 or IRS2 in MMTV-ErbB2 mammary cancer cell lines had little effect upon ErbB2 signaling. Similar results were obtained in human mammary epithelial cells (MCF10A) and breast cancer cell lines.

Conclusion

Despite previous evidence suggesting that ErbB receptors can bind and activate IRSs, our findings indicate that ErbB2 does not cooperate with the IRS pathway in these models to promote mammary tumorigenesis.

     

Monomethylarsonous Acid (MMAIII) Has an Adverse Effect on the Innate Immune Response of Human Bronchial Epithelial Cells to Pseudomonas aeruginosa

Arsenic is the number one contaminant of concern with regard to human health according to the World Health Organization. Epidemiological studies on Asian and South American populations have linked arsenic exposure with an increased incidence of lung disease, including pneumonia, and chronic obstructive pulmonary disease, both of which are associated with bacterial infection. However, little is known about the effects of low dose arsenic exposure, or the contributions of organic arsenic to the innate immune response to bacterial infection. This study examined the effects on Pseudomonas aeruginosa (P. aeruginosa) induced cytokine secretion by human bronchial epithelial cells (HBEC) by inorganic sodium arsenite (iAs^III) and two major metabolites, monomethylarsonous acid (MMA^III) and dimethylarsenic acid (DMA^V), at concentrations relevant to the U.S. population. Neither iAs^III nor DMA^V altered P. aeruginosa induced cytokine secretion. By contrast, MMA^III increased P. aeruginosa induced secretion of IL-8, IL-6 and CXCL2. A combination of iAs^III, MMA^III and DMA^V (10 pbb total) reduced IL-8 and CXCL1 secretion. These data demonstrate for the first time that exposure to MMA^III alone, and a combination of iAs^III, MMA^III and DMA^V at levels relevant to the U.S. may have negative effects on the innate immune response of human bronchial epithelial cells to P. aeruginosa.     

Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection

The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system.Avian H5N1 influenza viruses, currently circulating mainly in southeast Asia, are likely to cause the next influenza pandemic (18, 26, 37). The supply of embryonated eggs for traditional influenza vaccine production may be endangered in this case. Efforts to produce inactivated H5N1 vaccines in permanent cells have resulted in large-scale manufacturing, for instance, in Vero cells (21). This approach, based either on fermentation of H5N1 wild-type (wt) viruses (21) or on viruses attenuated by reverse genetics (9, 31), is the most straightforward strategy for egg-independent, rapid vaccine production.A further approach that may result in more widely available, egg-independent H5 vaccines is the use of recombinant viral vectors expressing protective antigens. Promising protection results were obtained so far with adenovirus-based vectors in mouse models (13, 14). Adenovirus vectors are usually produced in permanent complementing cell lines (11) and have been widely used in clinical trials. Cancellation of a recent trial involving human immunodeficiency virus adenovirus vectors due to suspected enhancement of disease, however, may complicate the future use of these vectors (41). Poxvirus vectors, including recombinant modified vaccinia virus Ankara (MVA) (1, 43), constitute a further class of vectors that have been used to express H5N1 influenza virus antigens (5, 22, 44, 46). Usually, however, the large-scale production of MVA is carried out in primary chicken cells, since these are the most efficient production substrates and are also accepted by regulators. In a pandemic, this production platform may not be available because permanent nontumorigenic avian cell lines are currently not available for production.In this study, we used a permanent cell line, modified Vero cells, to produce nonreplicating vaccinia virus vectors expressing the H5 hemagglutinin (HA), the major influenza virus protective antigen. The defective vaccinia virus (dVV) vectors are safe due to their lack of replication capacity in normal hosts, while they share the superior immunizing properties of poxviral live vaccines (15, 33). Previously, a permanent cell line based on rabbit kidney cells was engineered to express the essential vaccinia virus D4R gene encoding the enzyme uracil-DNA-glycosylase. This cell line allowed the construction of replication-deficient vaccinia virus vectors (15). In this work, a complementing system based on Vero cells was established and used to produce the defective vaccinia virus vector dVV-HA5. The vector was used to immunize mice and was compared to an inactivated whole-virus (whv) vaccine and to replicating control viruses. The dVV-HA5 candidate vaccine induced neutralizing antibodies and full protection, similar to results with an inactivated whv vaccine. Further, it is important to ensure that the immune responses generated by a pandemic influenza vaccine give long-lived, broad, cross-clade protection. While antibody responses to influenza virus provide protective immunity, T-cell responses are also thought to play an important role in clearance of and recovery from infections. Thus, a vaccine which can produce both effective humoral and T-cell responses would be advantageous. A vaccinia virus vector-based pandemic influenza vaccine has the potential to provide this advantage.