Ligand- and kinase activity-independent cell survival mediated by the epidermal growth factor receptor expressed in 32D cells

To investigate the intrinsic activities of the epidermal growth factor receptor and the role of its kinase domain in these functions within a cellular environment lacking endogenous ErbB protein expression, wild-type EGF receptor (WT-EGFR) and two kinase-impaired mutants, D813A and K721R, were expressed in 32D murine hematopoietic cells, a line which is normally dependent on interleukin 3 (IL3) for growth and survival. Addition of EGF in the absence of IL3 stimulates receptor autophosphorylation and, in the presence of serum, mitosis in cells expressing WT-EGFR, but not in cells expressing D813A or K721R. Unexpectedly, cells expressing WT-EGFR or K721R exhibited IL3-independent survival in the presence of fetal bovine serum; parental 32D cells and cells expressing D813A did not survive, apparently undergoing apoptosis in the absence of IL3, whether or not serum was present. Addition of EGF did not prevent the apoptosis of WT-EGFR or K721R cells in serum-free medium. Activation of Akt was not necessary to mediate the prosurvival activity of EGF receptor expression. These results suggest that the EGF receptor can mediate the prevention of apoptosis independently of both receptor-ligand binding and receptor kinase activity, and this activity is disrupted by the D813A mutation.     

The anatomy of the stigma and style from Cyclamen persicum (Mill.) cv. "pure white" and its relation to pollination success

Old flowers of Cyclamen generate few or no seeds. To understand the pollination problems of Cyclamen we investigate the general anatomy of the stigma and the style of Cyclamen persicum by scanning electron microscopy at different stages of floral maturity. Our investigations confirm that there is a hollow style. Against data commonly found in the literature, we present evidence of pollen germination and tube growth that show the stigma is not outside the style but inside it. Furthermore the maturation process of the style during the flowering time indicates a mechanism by which the stigma becomes shut off through closure at the terminal aperture of the style. At 3 to 5 days after anthesis there was the beginning closure of the style which was nearly completed at 21 days. The substance which leads to the closure is still unknown. The closure of the hollow style is a probable cause for failure of seed set in flowers not pollinated early in anthesis.     

Transfer of amino acids and nitrate from the roots into the xylem of Ricinus communis seedlings

The loading of amino acids and nitrate into the xylem was investigated by collection and analysis of root-pressure exudate from the cut hypocotyl stumps of seedlings of Ricinus communis L. Glutamine was found to be the dominant amino acid in the exudate and also to be the amino acid which is transferred to the xylem most rapidly and accumulated to the greatest extent. The comparison between uptake and xylem loading showed significant differences in specificity between these two transport reactions, indicating a different set of transport systems. Nitrate is transferred to the xylem at a higher relative rate than any amino acid despite the great nitrate-storage capacity of the root system. Thus the supply of nitrate to Ricinus plants leads to enhanced nitrogen allocation to the shoots.     

<Emphasis Type="Italic">Paenibacillus</Emphasis>—a predominant endophytic bacterium colonising tissue cultures of woody plants

High densities of endophytic bacteria were found in plant material from poplar, larch and spruce that had been micropropagated for at least 5 years. The majority of these bacteria were assigned to the genus Paenibacillus based on the sequencing of the 16S rRNA genes. Other endophytic bacteria such as Methylobacterium, Stenotrophomonas or Bacillus could also be found but only in some tissue cultures. Certain species or strains of Paenibacillus, especially those with a close relationship to P. humicus, seemed to accumulate under in vitro conditions without visible negative influences on the plant’s development. Poplar microcuttings inoculated with the endophytic Paenibacillus isolate 22 showed significantly more roots per cutting and higher root length in comparison to the control plants after 3 weeks.     

Intracellular recording from a spider vibration receptor

The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ’s cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.     

Metabolism and toxicological detection of the designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) in rat urine using gas chromatography-mass spectrometry

The amphetamine-derived designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) is an upcoming substance on the illicit drug market. In the current study, the identification of its metabolites in rat urine and their toxicological detection in the authors' systematic toxicological analysis (STA) procedure were examined. DOI is extensively metabolized by O-demethylation and beside small amounts of parent compound it was found to be excreted mainly in form of metabolites. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug users' dose of DOI by detection of the two O-demethyl metabolite isomers in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of DOI in human urine.     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies