Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.     

Reactivity of histidine and lysine side-chains with diethylpyrocarbonate -- a method to identify surface exposed residues in proteins

The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.     

Primary Succession of Soil Rotifers in Clays of Brown Coal Post‐Mining Dumps

Changes in rotifer soil communities along a primary succession chronosequence was studied on brown coal post mining areas near Sokolov, NW part of the Czech Republic. The chronosequence of unreclaimed plots was 2, 11, 14, 20, 43 years old. The rotifers were extracted from soil samples using a modification of the Baermann funnel method with combined light and temperature gradients. In total, 34 taxa of soil rotifers were identified throughout the study. The most common species were Encentrum arvicola, Adineta vaga, A. steineri, Habrotrocha rosa, H. elegans, H. filum, Macrotrachela quadricornifera and M. nana. Rotifer abundance varied from 4 ± 2 · 10³ to 516 ± 488 · 10³ individuals m^–2. Species number per sample increased with age of the plot (r = 0.45, P = 0.003). The most important environmental variables which significantly affected rotifer community were wood cover, sodium concentration and age of the plot. Pioneer plant species occupied 2 and 11 year old plots, 14–20 year old plots were covered by Salix caprea shrubs and a forest formed by Betula pendula and Populus tremuloides developed on the 43 year old plot. Some species were ubiquitous and present throughout the chronosequence (Macrotrachela quadricornifera). Among the pioneer species were Encentrum incisum, Habrotrocha rosa and Macrotrachela papillosa, 14–20 years old plots were preferred by Adineta vaga, E. arvicola, H. filum and M. nana, while the oldest plot was dominated by Adineta steineri and Encentrum mucronatum. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Reversible coupling of individual phycobiliprotein isoforms during state transitions in the cyanobacterium Trichodesmium analysed by single-cell fluorescence kinetic measurements

In the non-heterocyst, marine cyanobacterium Trichodesmium nitrogen fixation is confined to the photoperiod and occurs coevally with oxygenic photosynthesis although nitrogenase is irreversibly inactivated by oxygen. In previous studies it was found that regulation of photosynthesis for nitrogen fixation involves Mehler reaction and various activity states with reversible coupling of photosynthetic components. We now investigated these activity states in more detail. Spectrally resolved fluorescence kinetic measurements of single cells revealed that they were related to alternate uncoupling and coupling of phycobilisomes from and to the photosystems, changing the effective cross-section of PSII. Therefore, we isolated and purified the phycobiliproteins of Trichodesmium via ion exchange chromatography and recorded their UV/VIS absorption, fluorescence excitation and fluorescence emission spectra. After describing these spectra by mathematical equations via the Gauss-Peak-Spectra method, we used them to deconvolute the in vivo fluorescence spectra of Trichodesmium cells. This revealed that the contribution of different parts of the phycobilisome antenna to fluorescence quenching changed during the daily activity cycle, and that individual phycobiliproteins can be reversibly coupled to the photosystems, while the expression levels of these proteins did not change much during the daily activity cycle. Thus we propose that variable phycobilisome coupling plays a key role in the regulation of photosynthesis for nitrogen fixation in Trichodesmium.     

Destabilization of the secondary structure of RNA by ribosomal protein S1 from escherichia coli

S1 is an acidic protein associated with the 3′ end of 16S RNA; it is indispensable for ribosomal binding of natural mRNA. We find that S1 unfolds single stranded stacked or helical polynucleotides (poly rA, poly rC, poly rU). It prevents the formation of poly (rA + rU) and poly (rI + rC) duplexes at 10–25 mM NaCl but not at 50–100 mM NaCl. Partial, salt reversible denaturation is also seen with coliphage MS2 RNA, $\text{E. coli}$ rRNA and tRNA. Generally, only duplex structures with a Tm greater than about 55° are formed in the presence of S1. The protein unfolds single stranded DNA but not poly d(A·T).     

Spatial distribution and seasonal variation in fluoride enrichment in groundwater and its associated human health risk assessment in Telangana State,South India

Groundwater is a vital source of drinking water in Siddipet rural and urban regions of Central Telangana, South India and it is a major cause of fluoride toxicity in humans. The intake of elevated fluoride has a significant impact on human health, especially immediate problems that are seen in children's teeth. The primary aim of the study was to identify the seasonal variation in fluoride concentration and associated health risks in the residents of the study region. To assess the fluoride contamination in groundwater, a total of 158 samples were analyzed in two seasons. The mean concentrations of fluoride 1.26 mg/L and 2.21 mg/L were 1.46 and 2.8 times higher than the acceptable limit of 1.5 mg/L, before and after monsoon respectively. To estimate the human health risks due to the ingestion of elevated fluoride through drinking water, hazard quotient fluoride (HQ_Fluoride) was calculated using the United States Environmental Protection Agency method. HQ_Fluoride values were 0.44–2.44 and 0.89–4.67 for children, 0.36–2.00 and 0.73–3.82 for females, and 0.41–2.26 and 0.82–4.31 for males in pre- and post-monsoon seasons respectively, suggesting emphatically greater risk than the acceptable limits (HQ_Fluoride > 1), which generates health risks.     

Coordinated effects of electromagnetic field exposure on erythropoietin-induced activities of phosphatidylinositol-phospholipase C and phosphatidylinositol 3-kinase

Initial studies with the erythropoietin-sensitive human hematopoietic cell line, TF1, demonstrated both multifarious effects of pulsed electromagnetic field (EMF) exposure on lipid signal transduction and antiproliferative effects of EMF. Stimulation of TF1 cells with erythropoietin resulted in increased phosphatidylinositol 3-kinase activity within 2 min. Addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, produced a decrease in cell proliferation as measured by accumulation of cells in the G₀/G₁ phase of the cell cycle and suppression of erythropoietin-induced DNA synthesis. Similar effects on cell proliferation were seen under EMF treatment. Phosphatidylinositol 3-kinase activity in erythropoietin-stimulated TF1 cells, measured in whole-cell extracts, increased 34% within 2 min and remained above basal levels for at least 20 min. EMF decreased erythropoietin-stimulated phosphatidylinositol 3-kinase activity to lower than basal levels. Additionally, translocation of the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase to the membrane was prevented by EMF. Phosphatidylinositol-specific phospholipase C was activated, as reflected by increases in diacylglycerol and inositol trisphosphate at 15–60 s after EMF treatment. These results provide the first evidence of subtle coordinated changes by EMF associated with loss of phosphatidylinositol 3-kinase activity, inhibition of the translocation of p85 to the membrane, and activation of phosphatidylinositol-phospholipase C.