Information flow during gene activation by signaling molecules: ethylene transduction in Arabidopsis cells as a study system

Background  

We study root cells from the model plant Arabidopsis thaliana and the communication channel conformed by the ethylene signal transduction pathway. A basic equation taken from our previous work relates the probability of expression of the gene ERF1 to the concentration of ethylene.     

Helicobacter pylori induces urease subunit B-specific CD8+ T cell responses in infected individuals via cytosolic pathway of cross-presentation

Background

Urease subunit B (UreB), a conserved and key virulence factor of Helicobacter pylori (H. pylori), can induce the host CD4⁺ T cell immune responses to provide protection, but less is known regarding CD8⁺ T cell responses. The characteristics of H. pylori-specific CD8⁺ T cell responses and the mechanism underlying antigen processing and presentation pathways remain unclear. This study was focus on protective antigen recombinant UreB (rUreb) to detect specific CD8⁺ T cell responses in vitro and elucidate the mechanism of UreB antigen processing and presentation.

Methods

The peripheral blood mononuclear cells (PBMCs) collected from H. pylori-infected individuals were stimulated with rUreB in vitro to detect specific CD8⁺ T cell responses after co-culture with rUreB-pulsed autologous hMDCs. Through blocking assay, we investigated the potential pathway of UreB antigen processing and presentation via the cytosolic pathway or vacuolar pathway. The cytokines production of UreB specific CD8⁺ T cell were evaluated as well.

Results

We demonstrated UreB can induce specific CD8⁺ T cell immune responses in H. pylori infected individuals. Importantly, we characterized that UreB were mainly processed by proteasome instead of lysosomal proteases and presented through cytosolic pathway of cross-presentation, which requires endoplasmic reticulum–Golgi transport and newly synthesized MHC-I molecules, to induce functional-specific CD8⁺ T cell (IFN-γ + TNF-α + Grz A+ Grz B+) responses.

Conclusions

These results suggest that H. pylori UreB induces specific CD8⁺ T cell responses through cytosolic pathway of cross-presentation in infected individuals.     

Identification and biochemical analysis of a homolog of a sulfate transporter from a vanadium-rich ascidian Ascidia sydneiensis samea

Background

Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians.

Methods

We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed.

Results

The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively.

General significance

This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study.     

Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development

Background  

Vertebrate neural development requires precise coordination of cell proliferation and cell specification to guide orderly transition of mitotically active precursor cells into different types of post-mitotic neurons and glia. Lateral inhibition, mediated by the Delta-Notch signaling pathway, may provide a mechanism to regulate proliferation and specification in the vertebrate nervous system. We examined delta and notch gene expression in zebrafish embryos and tested the role of lateral inhibition in spinal cord patterning by ablating cells and genetically disrupting Delta-Notch signaling.     

Straw blood cell count,growth, inhibition and comparison to apoptotic bodies

Background  

Mammalian cells transform into individual tubular straw cells naturally in tissues and in response to desiccation related stress in vitro. The transformation event is characterized by a dramatic cellular deformation process which includes: condensation of certain cellular materials into a much smaller tubular structure, synthesis of a tubular wall and growth of filamentous extensions. This study continues the characterization of straw cells in blood, as well as the mechanisms of tubular transformation in response to stress; with specific emphasis placed on investigating whether tubular transformation shares the same signaling pathway as apoptosis.     

Molecular understanding of sterically controlled compound release through an engineered channel protein (FhuA)

Background  

Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Δ1-160; Onaca et al., 2008). Compound fluxes within the FhuA Δ1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester). Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems.     

Protein expression of G-protein inwardly rectifying potassium channels (GIRK) in breast cancer cells

Background  

Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK) channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established risk factor for breast cancer and has been found to open GIRK. In order to further investigate GIRK channels in breast cancer and possible alteration by ethanol, we identified GIRK channel protein expression in breast cancer cells.     

The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction

Background  

In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level.     

Transformation of selenate and selenite to elemental selenium byDesulfovibrio desulfuricans

Summary Desulfovibrio desulfuricans (DSM 1924) can be adapted to grow in the presence of 10 mM selenate or 0.1 mM selenite. This growth occurred in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor. As determined by electron microscopy with energy-dispersive X-ray analysis, selenate and selenite were reduced to elemental selenium which accumulated inside the cells. Selenium granules resulting from selenite metabolism were cytoplasmic while granules of selenium resulting from selenate reduction appeared to be in the periplasmic region. The accumulation of red elemental selenium in the media following stationary phase resulted from cell lysis with the liberation of selenium granules. Growth did not occur with either selenate or selenite as the electron acceptor and¹³C nuclear magnetic resonance indicated that neither selenium oxyanion interfered with fumarate respiration. At 1 M selenate and 100 M selenite, reduction byD. desulfuricans was 95% and 97%, respectively. The high level of total selenate and selenite reduced indicated the suitability ofD. desulfuricans for selenium detoxification.     

Attenuation of a virulent Aeromonas hydrophila with novobiocin and pathogenic characterization of the novobiocin‐resistant strain

Aim

To determine whether novobiocin resistance strategy could be used to attenuate a virulent Aeromonas hydrophila AH11P strain and to characterize the growth and pathogenic differences between the novobiocin‐resistant strain and its virulent parent strain AH11P.

Methods and Results

A novobiocin‐resistant strain AH11NOVO was obtained from a virulent Aer. hydrophila strain AH11P through selection of resistance to novobiocin. AH11NOVO was found to be avirulent to channel catfish (Ictalurus punctatus), whereas AH11P was virulent. When AH11NOVO vaccinated channel catfish were challenged with AH11P at 14 days postvaccination, relative per cent of survival of vaccinated fish was 100%. The cell proliferation rate of AH11NOVO was found to be significantly (P < 0·05) less than that of AH11P. In vitro motility assay revealed that AH11NOVO was nonmotile, whereas AH11P was motile. AH11NOVO had significantly (P < 0·05) lower in vitro chemotactic response to catfish mucus than that of AH11P. Although the ability of AH11NOVO to attach catfish gill cells was similar to that of AH11P, the ability of AH11NOVO to invade catfish gill cells was significantly (P < 0·05) lower than that of AH11P.

Conclusions

The novobiocin‐resistant AH11NOVO is attenuated and different from its parent AH11P in pathogenicity.

Significance and Impact of the Study

The significantly lower chemotactic response and invasion ability of AH11NOVO compared with that of its virulent parent strain AH11P might shed light on the pathogenesis of Aer. hydrophila.     

Potential involvement of serine/threonine protein phosphatases in apoptosis of HepG2 cells during selenite treatment

Selenium, an essential biological trace element present in both prokaryotic and eukaryotic cells, exerts its regulatory effect in a variety of cellular events, including cell growth, survival, and death. Selenium compunds have been shown in different cell lines to inhibit apoptosis by several mechanisms. Serine/threonine phosphatases (STPs) are potentially important in selenite-induced apoptosis because of their role in regulation of diverse set of cellular processes. In this study, the regulatory role of STPs in selenite-induced apoptosis has been implied by the use of two specific inhibitors: ocadaic acid and calyculin A. Our results show a decrease in cell density in HepG2 cells under selenite treatment. Resulting specific enzyme activities showed a concentration-dependent increase in all three phosphatase activities after 24 h in cells treated with 5 μM selenite and these activities decreased at 48 and 72 h. However, in cells treated with 10μM selenite, PP2A and PP2B decreased at 48 h, whereas PP2C activity did not change at this dose. In cells treated with 25μM, there was not a significant change in PP2C activity. These data suggest that the most specific response to selenite treatment was in PP2A and PP2B activities in a dose-dependent manner. Our results with OA and Cal-A further support the view that PP1 and PP2A might act as negative regulators of growth. With these data, we have first demonstrated the role of serine/threonine protein phosphatases in the signaling pathway of selenite-induced apoptosis and resulting cytotoxicity     

Sub-cellular internalization and organ specific oral delivery of PABA nanoparticles by side chain variation

Background  

Organic nanomaterials having specific biological properties play important roles in in vivo delivery and clearance from the live cells. To develop orally deliverable nanomaterials for different biological applications, we have synthesized several fluorescently labelled, self-assembled PABA nanoparticles using possible acid side chain combinations and tested against insect and human cell lines and in vivo animal model. Flurophores attached to nanostructures help in rapid in vivo screening and tracking through complex tissues. The sub-cellular internalization mechanism of the conjugates was determined. A set of physio-chemical parameters of engineered nanoskeletons were also defined that is critical for preferred uptake in multiple organs of live Drosophila.     

Murine mesenchymal progenitor cells from different tissues differentiated via mesenchymal microspheres into the mesodermal direction

Background  

Because specific marker molecules for phenotypical identification of mesenchymal stem and progenitor cells are missing, the assessment of the in vitro-differentiation capacity is a prerequisite to characterize these cells. However, classical differentiation protocols are often cell-consuming and time intensive. Therefore, the establishment of novel strategies for differentiation is one topic of current efforts in stem cell biology. The goal of this study was to demonstrate the practicability of a new differentiation test using plastic adherent cell isolates from different tissues.     

Brief report: Lactobacillus bulgaricus GLB44 (Proviotic™) plus esomeprazole for Helicobacter pylori eradication: A pilot study

Background

Recent studies of Lactobacillus delbrueckii subsp. bulgaricus GLB44 plus a proton‐pump inhibitor (PPI) reported cures of more than 90% of patients with active Helicobacter pylori infections.

Aim

To confirm the high H. pylori cure rates reported previously.

Method

A pilot study was done in healthy H. pylori‐infected volunteers using 3‐gram sachet (3 billion cells) of L. delbrueckii GLB44 plus 22.3 mg of esomeprazole b.i.d., for 14 days. The result was determined by urea breath testing 4 weeks after therapy. Stopping rules required for ending enrollment if less than 3 of the first 10 subjects were cured.

Results

Nine subjects were entered and because all failed to achieve negative urea breath test, the stopping rule required the study to end.

Conclusion

We were unable to confirm reports of achieving a high H. pylori cure rate with L. delbrueckii GLB44 plus a PPI.     

Optimization of conditions for cadmium selenide quantum dot biosynthesis in Saccharomyces cerevisiae

The biosynthesis of quantum dots has been explored as an alternative to traditional physicochemical methods; however, relatively few studies have determined optimal synthesis parameters. Saccharomyces cerevisiae sequentially treated with sodium selenite and cadmium chloride synthesized CdSe quantum dots in the cytoplasm. These nanoparticles displayed a prominent yellow fluorescence, with an emission maximum of approximately 540 nm. The requirement for glutathione in the biosynthetic mechanism was explored by depleting its intracellular content through cellular treatments with 1-chloro-2,4-dinitrobenzene and buthionine sulfoximine. Synthesis was significantly inhibited by both of these reagents when they were applied after selenite treatment prior to the addition of cadmium, thereby indicating that glutathione contributes to the biosynthetic process. Determining the optimum conditions for biosynthesis revealed that quantum dots were produced most efficiently at entry into stationary phase followed by direct addition of 1 mM selenite for only 6 h and then immediately incubating these cells in fresh growth medium containing 3 mM Cd (II). Synthesis of quantum dots reached a maximum at 84 h of reaction time. Biosynthesis of 800-μg g⁻¹ fresh weight cells was achieved. For the first time, significant efforts have been undertaken to optimize each aspect of the CdSe biosynthetic procedure in S. cerevisiae, resulting in a 70% increased production.

     

Exostosin-like 2 regulates FGF2 signaling by controlling the endocytosis of FGF2

Background

Heparan sulfate proteoglycans are ubiquitously expressed on cell surfaces and in extracellular matrices, and are engaged in heparin-binding growth factor-related signal transduction. Thus, changes in the amounts, structures, and chain lengths of heparan sulfate have profound effects on aspects of cell growth controlled by heparin-binding growth factors such as FGF2. Exostosin glycosyltransferases (EXT1, EXT2, EXTL1, EXTL2, and EXTL3) control heparan sulfate biosynthesis, and the expression levels of their genes regulate the amounts, chain lengths, and sulfation patterns of heparan sulfate. Unlike EXT1, EXT2, and EXTL3, EXTL2 functions chain termination of heparan sulfate. Here, we examined the importance of EXTL2 in FGF2-dependent signaling.

Analysis of gene expression in cotton fiber initials

Background  

Cotton (Gossypium hirsutum L.) fibers are trichomes that initiate from the ovule epidermis. Little is known about the developmental pathway causing fiber to differentiate from ovular epidermal cells even though limits on the number of cells that differentiate into fiber will limit yield.     

A quantitative real-time RT-PCR assay for mature C. albicans biofilms

Background  

Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells.