Inhibition of anaplastic lymphoma kinase (ALK) activity provides a therapeutic approach for CLTC-ALK-positive human diffuse large B cell lymphomas

ALK positive diffuse large B-cell lymphomas (DLBCL) are a distinct lymphoma subtype associated with a poor outcome. Most of them feature a t(2;17) encoding a clathrin (CLTC)-ALK fusion protein. The contribution of deregulated ALK-activity in the pathogenesis and maintenance of these DLBCLs is not yet known. We established and characterized the first CLTC-ALK positive DLBCL cell line (LM1). LM1 formed tumors in NOD-SCID mice. The selective ALK inhibitor NVP-TAE684 inhibited growth of LM1 cells in vitro at nanomolar concentrations. NVP-TAE684 repressed ALK-activated signalling pathways and induced apoptosis of LM1 DLBCL cells. Inhibition of ALK-activity resulted in sustained tumor regression in the xenotransplant tumor model. These data indicate a role of CLTC-ALK in the maintenance of the malignant phenotype thereby providing a rationale therapeutic target for these otherwise refractory tumors.     

Transfection of a phosphatidyl-4-phosphate 5-kinase gene into rat atrial myocytes removes inhibition of GIRK current by endothelin and alpha-adrenergic agonists

GIRK (G protein-activated inward-rectifying K(+) channel) channels, important regulators of membrane excitability in the heart and in the central nervous, are activated by interaction with betagamma subunits from heterotrimeric G proteins upon receptor stimulation. For activation interaction of the channel with phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P(2)) is conditional. Previous studies have provided evidence that in myocytes PtIns(4,5)P(2) levels relevant to GIRK channel regulation are under regulatory control of receptors activating phospholipase C. In the present study a phosphatidyl-4-phosphate 5-kinase was expressed in atrial myocytes by transient transfection. This did not affect basal properties of GIRK current activated by acetylcholine via M(2) receptors but completely abolished inhibition of guanosine triphosphate-gamma-S activated current by endothelin-1 or alpha-adrenergic agonists. We conclude that though PtIns(4,5)P(2) is conditional for channel gating, its normal level in the membrane is not limiting basal function of GIRK channels. Moreover, our data provide further evidence for a regulation of GIRK channels by alpha(1A) receptors and endothelin-A receptors, endogenously expressed in atrial myocytes, via depletion of PtIns(4,5)P(2).     

Intestinal Epithelial Cell Autophagy Is Required to Protect against TNF-Induced Apoptosis during Chronic Colitis in Mice

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Functional Expression of Odorant Receptors of the Zebrafish Danio rerio and of the Nematode C. elegans in HEK293 Cells

Odorant receptors of zebrafish and C. elegans were functionallyexpressed in vertebrate kidney cells (HEK293) using the eucaryoticexpression vector pSMyc. Receptor-encoding cDNA cloned intothis vector was expressed as a fusion protein with the N-terminalmembrane import sequence of the guinea-pig serotonin receptorfollowed by a myc tag. Immunocytochemical evidence indicatesthat this strategy directs a protein with the predicted immunoreactivityand approximate molecular weight to the plasma membrane. Fishfood extract (TetraMin) evoked a transient increase in intracellular[Ca²⁺] in HEK293 cells transiently transfected with plasmidscontaining cDNA for three fish odorant receptors and convertedto stable cell lines. The effect of the extract was concentrationdependent and limited to the fraction of the extract <5 kDa.Pretreating the transfected cells with the PLC inhibitor U73122reduced the odor-evoked signal. Fish food extract also evokeda transient increase in intracellular [Ca²⁺] in HEK293 cellstransiently transfected with plasmids containing cDNA for singlefish odorant receptors. Diacetyl evoked a transient increasein intracellular [Ca²⁺] in HEK293 cells transiently transfectedwith plasmids encoding the cDNA of ODR10, an odorant receptorof C. elegans suggested in other work to be specific for diacetyl.These results strongly imply that odorant receptors can be functionallyexpressed in HEK293 cells using this novel expression protocol.Chem. Senses 22: 467–476, 1997.     

Muscle metabolism from near infrared spectroscopy during rhythmic handgrip in humans

The rate of metabolism in forearm flexor muscles (MO2) was derived from near-infrared spectroscopy (NIRS-O2) during ischaemia at rest rhythmic handgrip at 15% and 30% of maximal voluntary contraction (MVC), post-exercise muscle ischaemia (PEMI), and recovery in seven subjects. The MO2 was compared with forearm oxygen uptake (VO2) [flow x (oxygen saturation in arnterial blood-oxygen saturation in venous blood, SaO2 - SvO2)], and with the 31P-magnetic resonance spectroscopy-determined ratio of inorganic phosphate to phosphocreatine (P(I):PCr). During ischaemia at rest, the fall in NIRS-O2 was more pronounced [76 (SEM 3) to 3 (SEM 1)%] than in SvO2 [71 (SEM 3) to 59 (SEM 2)%]. During the handgrip, NIRS-O2 was lower at 30% compared to 15% MVC [58 (SEM 3) v.s. 67 (SEM 3)%] while the SvO2 was similar [29 (SEM 3) v.s. 31 (SEM 4)%]. Accordingly, MO2 as well as P(I):PCr increased twofold, while VO2 increased only 30%. During PEMI after 15% and 30% MVC, NIRS-O2 fell to 9 (SEM 1)% and "0", but the use of oxygen by forearm muscles was not reflected in SvO2. During reperfusion after PEMI, the peak NIRS-O2 was lowest after intense exercise, while for SvO2 the reverse was seen. The discrepancies between NIRS-O2 and SvO2, and therefore between the estimates of the metabolic rate, would suggest significant limitations in sampling venous blood which is representative of the flexor muscle capillaries. In support of this contention, SvO2 and venous pH decreased during the first seconds of reperfusion after PEMI. To conclude, NIRS-O2 of forearm flexor muscles closely reflected the exercise intensity and the metabolic rate determined by magnetic resonance spectroscopy but not that rate derived from flow and the arterio-venous oxygen difference.     

A comparative study of the action of melittin on sphingomyelin and phosphatidylcholine bilayers

To investigate whether lipid solubilization is of relevance in describing the interaction between melittin and biological membranes, we studied melittin-induced polymorphism using model membranes composed of the biological lipid sphingomyelin (bovine brain). The behavior of the system was monitored by solid state ³¹P-NMR and turbidity measurements and compared to the peptides well-characterized action on the synthetic lipid dipalmitoylphosphatidylcholine. It was found that melittin-induced macroscopic changes of sphingomyelin membranes are qualitatively the same as in the case of dipalmitoylphosphatidylcholine bilayers. The sphingomyelin/melittin system is thus proposed to show a reversible vesicle-to-disc transition (fluid-to-gel phase) through an intermediate fusion or aggregation event centered at the main transition temperature, T_m, as reported in the case of saturated phosphatidylcholine. In the case of spontaneous disc formation at 37 °C, the lipid-to-peptide molar ratio in the discoidal objects was determined to be approximately 20 for dipalmitoylphosphatidylcholine and about 12 in the case of natural sphingomyelin. Melittin partition coefficients between membranes and the aqueous medium at 37 °C were found to be 6.1±0.8 mm ^–1 and 3.7±0.4 mm ^–1 for sphingomyelin and dipalmitoylphosphatidylcholine, respectively. For very high peptide quantities (lipid-to-peptide molar ratio, R_i≤5) mixed micelles are formed over the entire temperature range (20° to 60 °C) for both kinds of lipids.     

An analysis of Bat Hawk Macheiramphus alcinus diet in the Melaky Region of lowland western Madagascar

We present information on the prey taken by the Bat Hawk Macheiramphus alcinus in two different areas of lowland western central Madagascar. These are the first dietary data from Madagascar for this widespread Old World species. The recovered remains were almost exclusively of bats and birds, with a few examples of reptiles and insects. In total, 178 pellets were analysed. On the basis of minimum number of individuals and biomass, bats accounted for 58.3% and 30.3%, respectively, and birds 36.1% and 69.7%, respectively. Amongst the nine species of bats recovered from the pellets, four were represented by multiple individuals, particularly taxa belonging to the families Molossidae and Vespertilionidae that fly in open areas, and for the 11 species of identified birds, all were represented by a single individual. These patterns are interpreted as a specialisation of feeding on bats during a narrow window of time at dusk, as they leave day roost sites, and then using birds in a more general manner to fill in nutritional needs.     

Bending elasticities of model membranes: influences of temperature and sterol content

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.