A Residue Quartet in the Extracellular Domain of the Prolactin Receptor Selectively Controls Mitogen-activated Protein Kinase Signaling

Cytokine receptors elicit several signaling pathways, but it is poorly understood how they select and discriminate between them. We have scrutinized the prolactin receptor as an archetype model of homodimeric cytokine receptors to address the role of the extracellular membrane proximal domain in signal transfer and pathway selection. Structure-guided manipulation of residues involved in the receptor dimerization interface identified one residue (position 170) that in cell-based assays profoundly altered pathway selectivity and species-specific bio-characteristics. Subsequent in vitro spectroscopic and nuclear magnetic resonance analyses revealed that this residue was part of a residue quartet responsible for specific local structural changes underlying these effects. This included alteration of a novel aromatic T-stack within the membrane proximal domain, which promoted selective signaling affecting primarily the MAPK (ERK1/2) pathway. Importantly, activation of the MAPK pathway correlated with in vitro stabilities of ternary ligand·receptor complexes, suggesting a threshold mean lifetime of the complex necessary to achieve maximal activation. No such dependence was observed for STAT5 signaling. Thus, this study establishes a residue quartet in the extracellular membrane proximal domain of homodimeric cytokine receptors as a key regulator of intracellular signaling discrimination.     

Design of growth‐dependent biosensors based on destabilized GFP for the detection of physiological behavior of Escherichia coli in heterogeneous bioreactors

In this work, we present the design and characterization of Green Fluorescent Protein (GFP)‐based reporter systems designed to describe cellular activity in “complex,” heterogeneous bioreactors. The reporter systems consist of Escherichia coli strains carrying growth dependent promoters fused to genes expressing stable and unstable variants of GFP, respectively. The response of Escherichia coli cells to transient exposure to glucose was studied in a two‐compartment scale down bioreactor (SDR) consisting of a well‐stirred tank reactor (STR) connected to a plug‐flow reactor (PFR). Such a SDR system is employed to mimic the situation of high glucose concentration and oxygen limitation that often encountered in large‐scale, fed‐batch bioreactors and the response of E. coli was simulated by continuously pumping microbial cells from STR to the PFR. We found that repeated addition of concentrated glucose pulses with varied frequency at the entrance of the PFR had consequences on strain physiological behavior. The GFP expressions were significantly marked after 10 h of cultivation in STR (control reactor) and SDR, whereas, growth rates were rather similar. Additional experiments in chemostat with programmed glucose perturbation suggested that the activities of the promoters were linked with the substrate limitation signal. Taken together with immunoblot analysis, we suppose protein leakage is responsible for the overexpression of fis and the related promoters, such as rrnB in this case study, but additional works are required in order to confirm this relationship. This investigation is useful for a better understanding of the fast dynamic phenomena occurring in heterogeneous large‐scale bioreactors. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 553–563, 2013     

Type of chemotherapy has substantial effects on the immune system in ovarian cancer

Chemotherapy induces a variety of immunological changes. Studying these effects can reveal opportunities for successful combining chemotherapy and immunotherapy. Immuno-chemotherapeutic combinations in ovarian cancer are currently not generating the anticipated positive effects. To date, only scattered and inconsistent information is available about the immune-induced changes by chemotherapy in ovarian cancer. In this study, we compared six common chemotherapeutics used in ovarian cancer patients (carboplatin, paclitaxel, pegylated liposomal doxorubicin, gemcitabine, carboplatin-paclitaxel and carboplatin-gemcitabine) and studied their effects on the immune system in an ovarian cancer mouse model. Mice received a single chemotherapy or vehicle injection 21 days after tumor inoculation with ID8-fluc cells. One week after therapy administration, we collected peritoneal washings for flow cytometry, serum for cytokine analysis with cytometric bead array and tumor biopsies for immunohistochemistry. Carboplatin-paclitaxel showed the most favorable profile with a decrease in immunosuppressive cells in the peritoneal cavity and an increase of interferon-gamma in serum. In contrast, carboplatin-gemcitabine seemed to promote a hostile immune environment with an increase in regulatory T-cells in tumor tissue and an increase of macrophage-inflammatory-protein-1-beta in the serum.     

Studies on the ecology and host specificity of Balbiania investiens (Rhodophyceae)

Finds of the rare epiphytic rhodophyte Balbiania investiens (Acrochaetiales) from River Österdalälven in Dalecarlia, Sweden, are described. Its distribution, history and taxonomy are presented and discussed. Information on its host, represented by certain species of the genus Batrachospermum (Nemalionales), is given with particular reference to ecology and distribution in Sweden. The algal composition and water chemistry of one new Swedish location are presented and categorized according to Israelson's classification of Scandinavian running waters.     

DOES DAPI DETECT CYTOPLASMIC DNA?

Ultrastructural observations have revealed that plastids are present in orchid pollen tubes, but the DNA-binding fluorochrome 4',6-diamidino-2-phenylindole (DAPI) does not localize any DNA in the pollen tube plastids at optimum binding and flourescence conditions. However, the plastids do contain DNA since the gene coding for the large subunit of rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, rbcL) has been amplified by the polymerase chain reaction from orchid pollen tubes. It is therefore concluded that DAPI is an unreliable fluorochrome for detecting plastid DNA.