Species specificity of iron delivery in hybridomas

Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that HxH hybridomas do not respond to bovine transferrin a⁺ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.     

Bud dormancy and vegetative growth in Salix polaris as affected by temperature and photoperiod

Summary Vegetative growth of two ecotypes (lat. 78° 15N and 69°37N) of Salix polaris L. was studied in phytotron experiments. Dormancy of the winter buds was broken by chilling at 0.5°C for 14 to 42 days. Chilling requirement increased with decreasing growth temperature. The optimum temperature for bud break and shoot growth was about 15°C for both ecotypes. Cessation of apical shoot growth and abscission of shoot tip was not prevented by long photoperiods. However, at high temperature, 15°C or more, and in 18 to 24 h photoperiod, two or three growth flushes occurred frequently in both ecotypes. Leaf abscission in the arctic ecotype from lat. 78°N was not affected by photoperiod when grown at 6°C, but was stimulated by short photoperiod when grown at 15°C. In the ecotype from lat. 69°N leaf abscission was enhanced by short photoperiod even at 6°C.     

Interactions stabilizing DNA tertiary structure in the Escherichia coli chromosome investigated with ionizing radiation

The structure of the bacterial chromosome was investigated after introducing breaks in the DNA with gamma irradiation. It is demonstrated that irradiation of the chromosome in the cell prior to isolation results in partial unfolding of the isolated condensed DNA, while irradiation of the chromosome after it is released from the cell has no demonstrable effect on DNA folding. The results indicate that RNA/DNA interactions which stabilize DNA folds are unstable when breaks are introduced in the DNA prior to isolation of the chromosome. It is suggested that the supercoiled state of the DNA is required for the initial stabilization of some of the critical RNA/DNA interaction in the isolated nucleoid. However, some of these interactions are not affected by irradiation of the cells. Remnant supercoiling in partially relaxed chromosomes containing a limited number of DNA breaks has the same superhelical density as the unirradiated chromosome. This suggests that restraints on rotation of the packaged DNA are formed prior to the physical unwinding which occurs at the sites of the radiation induced DNA breaks. — Analysis of the in vitro irradiated chromosomes shows that there are 100+-30 domains of supercoiling per genome equivalent of DNA. The introduction of up to 50 double-strand breaks per nucleoid does not influence rotor speed effects of the sedimentation coefficient of the chromosome.     

Hsp90 inhibition and co‐incubation with pertuzumab induce internalization and degradation of trastuzumab: Implications for use of T‐DM1

The receptor tyrosine kinase HER2 is associated with a number of human malignancies and is an important therapeutic target. The antibody‐drug conjugate trastuzumab emtansine (T‐DM1; Kadcyla^®) is recommended as a first‐line treatment for patients with HER2‐positive metastatic breast cancer. T‐DM1 combines the antibody‐induced effects of the anti‐HER2 antibody trastuzumab (Herceptin^®) with the cytotoxic effect of the tubulin inhibitor mertansine (DM1). For DM1 to have effect, the T‐DM1‐HER2 complex has to be internalized and the trastuzumab part of T‐DM1 has to be degraded. HER2 is, however, considered endocytosis‐resistant. As a result of this, trastuzumab is only internalized to a highly limited extent, and if internalized, it is rapidly recycled. The exact reasons for the endocytosis resistance of HER2 are not clear, but it is stabilized by heat‐shock protein 90 (Hsp90) and Hsp90 inhibitors induce internalization and degradation of HER2. HER2 can also be internalized upon activation of protein kinase C, and contrary to trastuzumab alone, the combination of two or more anti‐HER2 antibodies can induce efficient internalization and degradation of HER2. With intention to find ways to improve the action of T‐DM1, we investigated how different ways of inducing HER2 internalization leads to degradation of trastuzumab. The results show that although both Hsp90 inhibition and activation of protein kinase C induce internalization of trastuzumab, only Hsp90 inhibition induces degradation. Furthermore, we find that antibody internalization and degradation are increased when trastuzumab is combined with the clinically approved anti‐HER2 antibody pertuzumab (Perjeta^®).     

Metabolic fingerprinting reveals differences between northern and southern strains of the cryptic diatom Chaetoceros socialis

Morphology and molecular phylogeny constitute the structural elements of diatom taxonomy. These approaches do not, however, give information on the functioning of taxa. Additional methods to serve a more integrated and wide-ranging taxonomy have therefore been called for. Metabolic fingerprinting is one approach used within the field of metabolomics, often applied in classification of samples. Here we apply metabolic fingerprinting in a taxonomic study of a cryptic diatom species. Strains of the cosmopolitan diatom Chaetoceros socialis from two geographical areas; the north-east Atlantic and Arctic and the Gulf of Naples, were cultivated at three different temperatures; 2.5, 8 and 13°C. The strains from the two different geographical areas exhibited different growth rates as well as different photosynthetic efficiencies. Algal extracts, collected at the end of the growth experiments, were analysed by Ultra-Performance Liquid Chromatography High Resolution Mass Spectrometry. The two groups of strains were separated by principal component analysis of their metabolic fingerprints. Analysis of the data revealed both qualitative and quantitative differences in metabolite markers. These phenotypic differences reinforce differences also found for morphology, phylogenetic markers and growth rates, and point at different adaptive characteristics in organisms living under different temperature regimes.     

The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis

The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin-dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin.     

Studying properties of neurotransmitter receptors by non-stationary noise analysis of spontaneous postsynaptic currents and agonist-evoked responses in outside-out patches

Chemical synaptic transmission depends on neurotransmitter-gated ion channels concentrated in the postsynaptic membrane of specialized synaptic contacts. The functional characteristics of these neurotransmitter receptor channels are important for determining the properties of synaptic transmission. Whole-cell recording of postsynaptic currents (PSCs) and outside-out patch recording of transmitter-evoked currents are important tools for estimating the single-channel conductance and the number of receptors contributing to the PSC activated by a single transmitter quantum. When single-channel activity cannot be directly resolved, non-stationary noise analysis is a valuable tool for determining these parameters. Peak-scaled non-stationary noise analysis can be used to compensate for quantal variability in synaptic currents. Here, we present detailed protocols for conventional and peak-scaled non-stationary noise analysis of spontaneous PSCs and responses in outside-out patches. In addition, we include examples of computer code for individual functions used in the different stages of non-stationary noise analysis. These analysis procedures require 3-8 h.     

Protection of atlantic salmon Salmo salar against infectious pancreatic necrosis after DNA vaccination

Although vaccines against infectious pancreatic necrosis (IPN) based on inactivated virus or recombinant structural viral proteins are commercially available, the protection is not complete and the disease is still a problem for the Atlantic salmon Salmo salar farming industry. In the present study, 5 different plasmids that expressed whole or parts of the large open reading frames (ORF) of Segment A of the IPN virus (IPNV) were constructed. The plasmids were shown to express proteins in cell cultures and in zebrafish Danio rerio in vivo. The specificities of the expressed proteins were confirmed by staining with IPNV-specific monoclonal antibodies (MAb) The plasmids were then used alone or in different combinations to vaccinate groups of Atlantic salmon, which subsequently were challenged in an experimental assay for IPN. A high level of protection was induced only by the plasmid combination that contained a plasmid expressing all the large ORF polyprotein.     

Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK), which is regulated by protein stability. However, its function is unknown and no physiological substrates for ERK3 have yet been identified. Here we demonstrate a specific interaction between ERK3 and MAPK-activated protein kinase-5 (MK5). Binding results in nuclear exclusion of both ERK3 and MK5 and is accompanied by ERK3-dependent phosphorylation and activation of MK5 in vitro and in vivo. Endogenous MK5 activity is significantly reduced by siRNA-mediated knockdown of ERK3 and also in fibroblasts derived from ERK3-/- mice. Furthermore, increased levels of ERK3 protein detected during nerve growth factor-induced differentiation of PC12 cells are accompanied by an increase in MK5 activity. Conversely, MK5 depletion causes a dramatic reduction in endogenous ERK3 levels. Our data identify the first physiological protein substrate for ERK3 and suggest a functional link between these kinases in which MK5 is a downstream target of ERK3, while MK5 acts as a chaperone for ERK3. Our findings provide valuable tools to further dissect the regulation and biological roles of both ERK3 and MK5.     

Mutants of the protein serine kinase PSKH1 disassemble the Golgi apparatus

We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring nonreceptor tyrosine kinases. Biochemical studies demonstrated that PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual amino-terminal acylation targets PSKH1 to Golgi as shown by colocalization with beta-COP and GM130, while nonpalmitoylated (myristoylated only) PSKH1 targets intracellular membranes colocalizing with protein disulphide isomerase (PDI, a marker for ER). Immunoelectron microscopy revealed that the dually acylated amino-terminal domain (in fusion with EGFP) was targeted to Golgi membranes as well as to the plasma membrane (PM), suggesting that the amino-terminal domain provides PSKH1 with membrane specificity dependent on its fatty acylation status. Subcellular fractionation by sucrose gradient analysis confirmed the impact of dual fatty acylation on endomembrane targeting, while cytosol and membrane fractioning revealed that myristoylation but not palmitoylation was required for general membrane association. A minimal region required for proper Golgi targeting of PSKH1 was identified within the first 29 amino acids. Expression of a PSKH1 mutant where the COOH-terminal kinase domain was swapped with green fluorescent protein and cysteine 3 was exchanged with serine resulted in disassembly of the Golgi apparatus as visualized by redistribution of beta-COP and GM130 to a diffuse cytoplasmic pattern, while leaving the tubulin skeleton intact. Our results suggest a structural and regulatory role of PSKH1 in maintenance of the Golgi apparatus, a key organelle within the secretory pathway.