Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells.

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.     

Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors.

The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of these sites by truncation of the carboxyl-terminal 123 amino acid residues, resulted in reduced receptor phosphorylation of an in vivo specific substrate phospholipase C-gamma 1 to less than 50% compared to the wild-type receptor. The internalization rate constant Ke was also significantly lower in these mutants (0.15/min) compared to cells transfected with wild-type receptor (0.27/min). Additional mutation of tyrosine 992 to phenylalanine in the truncated receptor mutant (Dc-123F) further decreased the receptor internalization rate to a minimal level (ke = 0.07-0.10/min), equivalent to the ke measured for cells expressing kinase-negative receptor (A721). Moreover, tyrosine kinase activity of the Dc-123F receptor toward phospholipase C-gamma 1, compared to wild-type receptor, was reduced by 90%. Taken together, these results show that EGF receptor lacking five autophosphorylation sites functions similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites.     

Evaluation of matrix-assisted laser desorption/ionization time of flight mass spectrometry for characterization of Culicoides nubeculosus biting midges

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has shown promise in species identification of insect species. We evaluated its potential to consistently characterize laboratory-reared biting midges of the species Culicoides nubeculosus (Meigen) (Diptera: Ceratopogonidae). Twenty-one reproducible potential biomarker masses for C. nubeculosus were identified under different experimental treatments. These treatments included the homogenization of insects in either water or known concentrations of formic acid. The biomarker masses were present independent of age, gender and different periods of storage of individuals in 70% ethanol (a standard preservation method). It was found that the presence of blood in females reduced the intensity of the MALDI-TOF pattern, necessitating the removal of the abdomen before analysis. The protein profiles of a related non-biting midge, Forcipomyia sp. (Diptera: Ceratopogonidae), and of Aedes japonicus japonicus (Theobald) (Diptera: Culicidae) mosquitoes were also examined and were distinctly different. These findings provide preliminary data to optimize future studies in differentiation of species within the Culicoides genus using MALDI-TOF MS which is a rapid, simple, reliable and cost-effective technique.     

Epidermal growth factor (EGF) stimulates inositol trisphosphate formation in cells which overexpress the EGF receptor

EGF is a low molecular weight polypeptide hormone which acts as a regulator of cell growth and differentiation. The A-431 cell line has been used frequently to examine receptor-mediated biochemical effects of EGF, since this cell line has an increased (20-50 fold) level of EGF receptors. We have utilized A-431 cells to examine the influence of EGF on formation of an intracellular second messenger, inositol, 1,4,5-trisphosphate (Ins-1,4,5-P3), and other inositol phosphates. The results show that EGF induces rapid formation of Ins-1,4,5-P3 as well as Ins-1,3,4-P3 and Ins-1,3,4,5-P4. There is a concurrent decrease in the level of the lipid precursor for Ins-1,4,5-P3, phosphatidylinositol 4,5-biphosphate (PIP2). Furthermore, we have examined five other cell lines that overexpress the EGF receptor and find that EGF treatment induces formation of inositol polyphosphates in those cell lines also.     

Stabilization of phosphofructokinase during air-drying with sugars and sugar/transition metal mixtures

Phosphofructokinase (PFK) purified from rabbit skeletal muscle is fully inactivated after air-drying and rehydration. The addition of trehalose, maltose, or sucrose to the enzyme solution prior to rapid drying results in a recovery of almost 70% of the original activity, whereas about 30% is recovered during slow drying. Similar stabilization is seen with up to 200 mM lactose, but at higher concentrations the sugar comes out of solution during drying, and there is a dramatic drop in the activity recovered. Glucose at concentrations up to 500 mM is ineffective at protecting air-dried PFK. Addition of ionic zinc to enzyme-sugar mixtures prior to drying greatly enhances the stabilization imparted by the above sugars, but zinc alone affords no protection. Several other organic solutes (proline, glycine, trimethylamine N-oxide, glycerol, and myo-inositol) that afford cryoprotection to PFK, an effect enhanced by the addition of zinc, do not stabilize the enzyme during air-drying, even if zinc is present. The addition of ionic copper, cobalt, or nickel to trehalose-PFK solution prior to rapid drying results in a large increase in the activity recovered, and the presence of cadmium or manganese leads to a minor increase. Magnesium and calcium are ineffective in this respect. During slow drying, the presence of cadmium or calcium leads to increased preservation, magnesium and manganese have no influence on stabilization, and copper and nickel inactive the enzyme.     

Parasite manipulation of brain monoamines in California killifish (Fundulus parvipinnis) by the trematode Euhaplorchis californiensis

California killifish (Fundulus parvipinnis) infected with the brain-encysting trematode Euhaplorchis californiensis display conspicuous swimming behaviours rendering them more susceptible to predation by avian final hosts. Heavily infected killifish grow and reproduce normally, despite having thousands of cysts inside their braincases. This suggests that E. californiensis affects only specific locomotory behaviours. We hypothesised that changes in the serotonin and dopamine metabolism, essential for controlling locomotion and arousal may underlie this behaviour modification. We employed micropunch dissection and HPLC to analyse monoamine and monoamine metabolite concentrations in the brain regions of uninfected and experimentally infected fish. The parasites exerted density-dependent changes in monoaminergic activity distinct from those exhibited by fish subjected to stress. Specifically, E. californiensis inhibited a normally occurring, stress-induced elevation of serotonergic metabolism in the raphae nuclei. This effect was particularly evident in the experimentally infected fish, whose low-density infections were concentrated on the brainstem. Furthermore, high E. californiensis density was associated with increased dopaminergic activity in the hypothalamus and decreased serotonergic activity in the hippocampus. In conclusion, the altered monoaminergic metabolism may explain behavioural differences leading to increased predation of the infected killifish by their final host predators.