The adaptor Grb7 is a novel calmodulin-binding protein: functional implications of the interaction of calmodulin with Grb7

We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243-256) predicted to form an alpha-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Delta and Grb7VDelta) have less capacity to be associated to membranes. Grb7Delta maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin beta1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Delta instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Delta and Grb7VDelta lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.     

Minimal residual disease as the target for immunotherapy and gene therapy of cancer (review)

Local recurrences at the site of tumour resection as well as distant micrometastases manifested after surgery represent major problems in oncology. Adjuvant immunotherapy and gene therapy may help to cope, at least partially, with these problems. Adjuvant modalities may be more effective in treating residual tumour disease compared to bulky tumours, owing to a favourable effector/target cell ratio. The purpose of this review was to summarize, evaluate and discuss the results obtained with adjuvant immunotherapy and immunomodulatory gene therapy of surgical minimal residual tumour disease in experimental and clinical tumour systems. The prospects and limitations of adjuvant therapeutic modalities will be considered.     

Investigation of histopathological and cytogenetic effects on Lepomis gibbosus (Pisces: Perciformes) in the Cine stream (Aydin/Turkey) with determination of water pollution

Water quality and the distribution of some heavy metals in three different organs of Lepomis gibbosus from the Cine Stream were studied. Also, histopathological changes in gill, liver, and muscle tissue were examined at light microscopical level. Micronucleus (MN) formation in fish erytrocytes, as an indicator of chromosomal damage, has been increasingly used to detect the genotoxic potential of environmental contaminants. The frequency of MN was examined from samples of fish from the Cine Stream and a control group. MN frequency was higher in fish samples caught from the Cine Stream than that in the control group. The chemicals ammonia, nitrite, nitrate, orthophosphate, and sulphate were determined as parameters that possibly affect the gill, liver, and muscle morphology. Zn was the most accumulated metal in tissues as well as in water. Maximum metal accumulation occurred in both liver and gills. For histopathological examinations, samples of gills, liver, and muscle tissues of L. gibbosus were studied by using light microscopy. In this study, a significant decrease in mean length of primary and secondary lamellae were observed. Moreover, cellular proliferation developed with secondary lamellae fusion, ballooning degenerations or club deformation of secondary lamellae, as well as distribution of necrotic, hyperplastic and clavate secondary lamellae. In the liver, altered staining, swollen and ruptured parenchymal cells, loss of cord structure, reduce of glycogen in hepatocytes, and vacuolar structure filled with cellular debris and many dark particles were seen. In muscle tissue, focal necrosis, cellular dissolution, and a decline or loss of striation in muscle fibres were found.     

Effects of sub-chronic antipsychotic drug treatment on body weight and reproductive function in juvenile female rats

Rationale Weight gain caused by some antipsychotics is not only confined to adults but can also adversely affect both children and adolescents. Indeed, olanzapine and risperidone have been associated with extreme weight gain in adolescents even greater than that reported in adults. We have recently shown substantial weight gain in adult female rats following treatment with olanzapine and risperidone but not ziprasidone. Objectives The aim of the present study was to compare the effects of several antipsychotics on weight gain and reproductive function in juvenile (aged 7 weeks) female hooded Lister rats. Methods Olanzapine (4 mg/kg), risperidone (0.5 mg/kg), ziprasidone (2.5 mg/kg), sulpiride (10 mg/kg), haloperidol (0.5 mg/kg) or vehicle was administered i.p. once per day for 21 days. Body weight, food and water intake were measured daily, in addition to the determination of stage of the oestrous cycle. Results Sub-chronic administration of olanzapine, risperidone, sulpiride and haloperidol, but not ziprasidone, significantly increased body weight compared to vehicle-treated animals during weeks 1–3. Sulpiride significantly increased food and water intake. Significantly increased percentage intra-abdominal fat weight was observed in olanzapine, risperidone, sulpiride and haloperidol, but not ziprasidone-treated animals. Marked disruption of the oestrous cycle was observed in all but the ziprasidone-treated group, which continued to have regular 4-day oestrous cycles. Conclusions Weight gain observed in these juvenile animals was 1.5–2 times greater than that previously observed in adult rats. These findings have important implications for the use of antipsychotics in children and adolescent patients.