Biochemical assay-based selectivity profiling of clinically relevant kinase inhibitors on mutant forms of EGF receptor

Gefitinib and erlotinib are potent EGFR tyrosine kinase inhibitors (potentially) useful for the treatment of non-small-cell lung cancer (NSCLC). Clinical responses, however, in NSCLC patients have been linked to the presence of certain activating mutations of EGFR. We used an ELISA-based biochemical assay to confirm the selective inhibitory efficacy of gefitinib and erlotinib on the activated mutant receptor. Our results are in line with the clinical observations providing evidence for the predictive power of the kinase assay. Four additional compounds were also investigated: CI-1033 and EKB-569 had dramatic inhibitory effects on all EGFR forms, whereas PD153035 and AG1478 were active on wild-type and activating mutant protein. In docking simulations with wild-type EGFR, our inhibitory data are in good agreement with the binding scores. These data confirm that anilinoquinazolines are good starting structures for the next generation of selective drugs against mutant EGFR, whereas CI-1033 and EKB-569 may represent advances for patients with both wild-type and anilinoquinazoline-resistant mutant tumors.     

Antibacterial peptides and proteins with multiple cellular targets.

Native antimicrobial peptides and proteins represent bridges between innate and adaptive immunity in mammals. On the one hand they possess direct bacterial killing properties, partly by disintegrating bacterial membranes, and some also by inhibiting functions of intracellular biopolymers. On the other, native antimicrobial peptides and proteins upregulate the host defense as chemoattractants or by various additional immunostimulatory effects. Structure-activity relationship studies indicate that residues responsible for the activities on bacterial membranes or for the secondary functions do not perfectly overlap. In reality, in spite of the relatively short size (18-20 amino acid residues) of some of these molecules, the functional domains can frequently be separated, with the cell-penetrating fragments located at the C-termini and the protein binding domains found upstream. As a cumulative effect, multifunctional and target-specific (agonist or antagonist) antimicrobial peptides and proteins interfere with more than one bacterial function at low concentrations, eliminating toxicity concerns of the earlier generations of antibacterial peptides observed in the clinical setting.     

Exploring Leptin Antagonism in Ophthalmic Cell Models

Background

Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions.

Methods and Results

The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca.

Conclusions

Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models.     

Nutrition of winter wheat during the life cycle. I. Yield and accumulation of dry matter and minerals

Mineral uptake by winter wheat (Trilicum aestivum L. cv. Martonvasari 8) was studied throughout the life cycle. Accumulation of macronutrients (i.e. total nitrogen, phosphorus, potassium, sodium, magnesium and calcium) and the water content of roots and shoots of plants grown in complete nutrient solution were higher than those of plants grown in two types of soils. The supply of macronutrients was in some cases limiting for soil-grown plants as revealed by a comparison of available and accumulated amounts of these nutrients. Their supply was abundant, however, for solution-grown plants. This led to a doubling of grain yield for the latter plants with a three fold increase in accumulation of dry matter and a five-fold increase in fresh weight. The efficiency ratios of solution-grown plants to soil-grown plants were approximately 1 for N and Na, 0.5 for Mg and Ca, and 0.3 for P and K.     

Different mechanisms influencing permeation of PDGF-AA and PDGF-BB across the blood-brain barrier

Platelet-derived growth factor (PDGF) exerts neurotrophic and neuromodulatory effects on the CNS. To determine the permeability of the blood-brain barrier (BBB) to PDGF, we examined the blood-to-brain influx of radioactively labeled PDGF isoforms (PDGF-AA and PDGF-BB) by multiple-time regression analysis after intravenous (i.v.) injection and by in-situ perfusion, and also determined the physicochemical characteristics which affect their permeation across the BBB, including lipophilicity (measured by octanol:buffer partition coefficient), hydrogen bonding (measured by differences in octanol : buffer and isooctane : buffer partition coefficients), serum protein binding (measured by capillary electrophoresis), and stability of PDGF in blood 10 min after i.v. injection (measured by HPLC). After i.v. bolus injection, neither 125I-PDGF-AA nor 125I-PDGF-BB crossed the BBB, their influx rates being similar to that of the vascular marker 99mTc-albumin. 125I-PDGF-AA degraded significantly faster in blood than 125I-PDGF-BB. PDGF-BB, however, was completely bound to a large protein in serum whereas PDGF-AA showed no binding. Thus, degradation might explain the poor blood-to-brain influx of PDGF-AA, whereas protein binding could explain the poor influx of circulating PDGF-BB. Despite their lack of permeation in the intact mouse, both 125I-PDGF-AA and 125I-PDGF-BB entered the brain by perfusion in blood-free buffer, and the significantly faster rate of 125I-PDGF-AA than 125I-PDGF-BB may be explained by the lower hydrogen bonding potential of 125I-PDGF-AA. Thus, the lack of significant distribution of PDGF from blood to brain is not because of the intrinsic barrier function of the BBB but probably because of degradation and protein binding. Information from these studies could be useful in the design of analogues for delivery of PDGF as a therapeutic agent.     

Histochimie des colorations dans le bloc à base de métaux,application aux coupes semi-fines

Résumé Les auteurs décrivent une nouvelle méthode de coloration dans le bloc de tissu avec différents sels métalliques (alun de fer, tétroxyde d'osmium, acétate d'uranyl et chromeosmium) et l'hématoxyline. Cette technique est utilisée sur des tissus qui sont ensuite inclus en Araldit et permet d'obtenir des coupes semi-fines déjà colorées. Les auteurs ont cherché à préciser la spécificité de fixation du fer dans le tissu au moyen de méthodes d'extraction et de blocage. D'après leurs observations, le fer se fixerait aux groupes tissulaires chargés négativement. La nature des liaisons semble être de type électrostatique et complexe. Les liaisons complexes semblent prédominer au niveau des structures contenant des acides nucléiques.

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Histochemistry of block staining with metals for semifine sections

Summary The authors describe a new method of block staining for semifine sections with various metal salts (ferriammonium-sulphate, osmium tetroxyde, uranyl acetate, chromeosmium) and hematoxylin. They investigated the specificity of iron-alaun-hematoxylin with several extractions and blocking methods. According to their observations the dissociated iron is bounded to the negatively charged tissular groups. The nature of binding is thought to be electrostatic and coordinativ (complex). The last one is probably predominant in nucleic acids containing structures. This method seems to be encouraging for electron-histochemical investigations.