The use of isobaric tag peptide labeling (iTRAQ) and mass spectrometry to examine rare,primitive hematopoietic cells from patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease, associated with a t(9, 22) chromosomal translocation leading to formation of the BCR/ABL chimeric protein, which has an intrinsic tyrosine kinase activity. Recently, the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (imatinib) has been successfully used clinically, although, disease relapse can still occur. The precise detail of the mechanism by which CML cells respond to imatinib is still unclear. We therefore systematically examined the effects of imatinib on the primitive CML cell proteome, having first established that the drug inhibits proliferation and induces increased apoptosis and differentiation. To define imatinib-induced effects on the CML proteome, we employed isobaric tag peptide labeling (iTRAQ) coupled to two-dimensional liquid chromatography/tandem mass spectrometry. Given the limited clinical material available, the isobaric tag approach identified a large population of proteins and provided relative quantification on four samples at once. Novel consequences of the action of imatinib were identified using this mass spectrometric approach. DEAD-box protein 3, heat shock protein 105 kDa, and peroxiredoxin-3 were identified as potential protein markers for response to imatinib. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Stephen D. Griffiths and John Burthem contributed equally to this publication. This work is supported by The Leukaemia Research Fund (UK).     

Lymphokine regulation of human lymphocyte proliferation: Formation of resting G0 cells by removal of interteukin 2 in cultures of proliferating T lymphocytes

The question of whether lymphocytes which have once been activated and have completed one or several cell cycle(s) can return to the G₀ phase and stay ready for a new activation (G₀-G₁ transition), rather than simply die, was investigated. To do so interleukin 2 (IL-2) was removed from cultures of continuously proliferating human T lymphocytes and the formation of resting (G₀) cells was measured. Kinetic analyses in freshly prepared peripheral blood lymphocytes (PBL) revealed that the onset of detectable RNA synthesis and the appearance of structures binding the anti-Tac antibody occurred simultaneously. This allowed the expansion of the definition of G₀ T lymphocytes as cells having a low RNA (and DNA) content, and no Tac antigen. When cultured human T cells proliferating continuously by means of IL-2 were characterized in terms of their distribution in the cell cycle, 7 days after the initial PHA stimulation, it could be demonstrated that very few cells were in the G₀ phase, supporting the concept of direct S/G₂/M-G₁ transition. However, when IL-2 was removed from the cultures, the [³H]thymidine incorporation per 10⁴ cells and correspondingly the number of cells in the S/G₂/M and G₁ phases were reduced drastically and during the following 72-hr period, the number of G₀ cells increased markedly. Restimulation of such in vitro formed G₀ cells, under conditions permitting observation of their shift from the G₀ to G₀ phase, demonstrated that most cells could respond normally. Based on these observations, it was concluded that IL-2 not only ensures T-lymphocyte survival and proliferation, but IL-2 starvation induces many continuously proliferating T lymphocytes to stop cycling and to return to the G₀ phase of the cell cycle where they remain functional.     

Ethanol-induced hypothermia and ethanol consumption in the rat are affected by low-level microwave irradiation

Microwave irradiation of rats by circularly polarized, 2,450-MHz, pulsed waves (2-μs pulses; 500 pps) was performed in waveguides to determine effects on ethanol-induced hypothermia and on ethanol consumption. Rats injected intraperitoneally with ethanol (3 g/kg in a 25% v/v water solution) immediately after 45 min of microwave irradiation exhibited attenuation of the initial rate of fall in body temperature, which was elicited by the ethanol, but exhibited no significant difference in maximal hypothermia as compared with that of sham-irradiated rats. Microwave irradiation did not affect the consumption of a 10% sucrose (w/v) solution by water-deprived rats. However, it enhanced the consumption of a solution of 10% sucrose (w/v) + 15% ethanol (v/v) by water-deprived animals. These results were obtained at a specific absorption rate (SAR) of 0.6 W/kg, which rate of energy dosing would require a power density of 3–6 mW/cm² if exposure of the animals had occurred to a 12-cm plane wave.     

Preliminary results from a controlled evaluation of thermal biofeedback as a treatment for essential hypertension

In a controlled trial, thermal biofeedback (n=20) and abbreviated progressive relaxation (n=22) were compared in the treatment of mild to moderate hypertensive patients whose blood pressures (BP) were initially controlled on two medications. For the clinical end point of maintaining control of BP on a single drug after treatment, biofeedback was superior to relaxation training (at 3 months, 47% success for biofeedback versus 23% for relaxation). This same result tended to be true for patient-measured home BPs. BPs from laboratory psychophysiological testing showed no consistent advantage for one treatment over the other.This research was supported by a grant from NHLB1, HL-27622.     

On-line tools for sequence retrieval and multivariate statistics in molecular biology

We have developed a World-Wide Web server for browsing sequencecollections structured under the ACNUC format and for performingmultivariate analyses on sequences. General collections (likeGenBank or EMBL), as well as specialized data banks (like Hovergenand NRSub) can be accessed. This system allows complex queriesto be constructed, and the result of each query, representedby a list of sequences, is stored on the server. It is thenpossible to reuse this list to compute multivariate analyseson the sequences. Two examples of applications are shown. Thefirst one consists in a study of codon usage with correspondenceanalysis on all the protein genes of Haemophilus influenzaeRd. This study allows the highly expressed genes and the integralmembrane proteins of this organism to be identified. The secondone consists in an ordering of 70 aligned protein sequencesof growth hormone with principal coordinate analysis. With thismethod, we are able to re-establish the patterns of relationshipsbetween the sequences previously determined with tree buildingprograms.     

The P2X7 receptor antagonist JNJ-47965567 administered thrice weekly from disease onset does not alter progression of amyotrophic lateral sclerosis in SOD1G93A mice

Purinergic Signalling - The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate...     

Integration of biological networks and gene expression data using Cytoscape

Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.     

Plasmid-mediated resistance to fosfomycin in Staphylococcus epidermidis

Staphylococcus epidermidis strain BM2641, isolated from a patient, was resistant to penicillin G, methicillin, aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin B-type (MLS) antibiotics, and to high levels of fosmycin. Resistance to forsfomycin and/or to MLS was lost at low frequencies either spontaneously or after curing with novobiocin. The plasmid DNA from BM2641 and its cured derivatives was purified, analyzed by agarose gel electrophoresis and transferred to a nitrocellulose sheet. Comparative analysis of the resistance phenotypes with the plasmid content of the strains indicated that fosfomycin and MLS resistance were encoded by plasmids pIP1842 (2.5 kb) and pIP1843 (2.6 kb), respectively. Southern hybridization with a probe specific for gene fosA of Serratia marcescens showed that the fosfomycin resistance determinant in Staphylococcus is not homologous to that of Gram-negative bacteria.     

M-CSF stimulated differentiation requires persistent MEK activity and MAPK phosphorylation independent of Grb2-Sos association and phosphatidylinositol 3-kinase activity

Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.     

Characterization of phosphoinositide kinases in normal and sickle anaemia red cells

PtdIns and PtdInsP kinases from normal erythrocyte (AA) membranes and sickle cell anaemia erythrocyte (SS) membranes have been characterized. PtdIns kinase was studied in native membranes under conditions in which PtdInsP kinase and PtdInsP phosphatase do not express any activity. Kinetic analysis of the AA and SS PtdIns kinases indicate similar K_m values for PtdIns and ATP but higher V_max values for SS PtdIns kinase. PtdInsP kinase was partially purified from erythrocyte ghosts by NaCl extraction. The kinetic parameters of PtdInsP kinase determined under these conditions were similar in AA and SS NaCl extracts. These data suggest the presence of some effector of PtdIns kinase in SS cell membranes, resulting in a greater activity of the enzyme. This leads consequently, to increase the PtdInsP pool and to activate PtdInsP kinase, in agreement with our previous observations of a greater [³²P]Pi incorporation in both polyphosphoinositides in SS cells relatively to AA cells.