Mapping inhibitor response to the in-frame deletions,insertions and duplications of epidermal growth factor receptor (EGFR) in non-small cell lung cancer

Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of non-small cell lung cancer (NSCLC). However, a large number of in-frame deletion, insertion and duplication mutations in the EGFR tyrosine kinase (TK) domain have been observed to alter drug response to such a kinase target. Thus, a systematic investigation of the intermolecular interactions between the clinical small-molecule agents and various EGFR in-frame mutants would help to establish a complete picture of drug response to kinase mutations in lung cancer, and to design new EGFR inhibitors with high potency and selectivity to target drug-resistant mutants. Here, we describe a combined pipeline to explore the drug response of five representative EGFR inhibitors, including three FDA-approved agents (gefitinib, erlotinib and lapatinib) and two compounds under clinical development (AEE788 and TAK-285) to a number of clinically relevant EGFR in-frame mutations, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying drug resistance and sensitivity to EGFR in-frame mutations. It was found that the insertion and duplication mutations in exon 20 can generally cause drug resistance to EGFR due to the reduced size of kinase’s active pocket, while deletion mutations in exon 19 associate closely with increased inhibitor sensitivity to EGFR by establishing additional non-bonded interactions across complex interface, including hydrogen bonds, cation–π interactions and hydrophobic contacts.     

Vibrational sensitivity of the wing of the pigeon (Columba livia) — a study using heart rate conditioning

Summary The vibrational sensitivity of awake pigeons was tested with the heart rate conditioning method. This method proved to be a very sensitive instrument for the behavioural measurement of the pigeon's sensitivity to mechanical stimuli.Sine wave vibrational stimuli between 50 and 2000 Hz were applied to the 1st, 7th and 16th primary feathers of the wing. The resulting threshold curves were U- or V-shaped with 3 characteristics: (i) The frequency of best response was either 800 or 900 Hz. (ii) Within the broad frequency range of vibrational sensitivity (50 to 2000 Hz) the sensitivity was extremely high at the best frequency: threshold amplitudes lay between 0.5 and 0.09 m. (iii) The threshold curves showed very sharp tuning at best frequency with bandwidths between 0.3 and 0.7 octaves (measured at best frequency 10fold above threshold). The functional meaning of the characteristic vibrational sensitivity of the wing for flight control is discussed; it is interpreted as an adaptation to special situations occurring during flight.Abbreviations CR conditioned response - CS conditioned stimulus - ECG electrocardiogram - HC(s) Herbst corpuscle(s) - UCS unconditioned stimulus All experiments were carried out at the Ruhr-Universität Bochum, Lehrstuhl für Allgemeine Zoologie     

Spatial orientation of rainbow trout to plane-polarized light: The ontogeny of E-vector discrimination and spectral sensitivity characteristics

Summary The results of this study demonstrate that trout (Salmo gairdneri) are capable of orienting to polarized light fields. The spectral composition of the polarized light fields can significantly influence the orientation of trout. Rainbow trout exhibit ontogenetic losses in orientation to polarized light fields which appears coincident with the ontogenetic loss of the UV-sensitive cones. Trout were trained to swim to a refuge located at one end of the training tank under a polarized light field. The E-vector of the polarized light field was oriented parallel or perpendicular to the long axis of the training tank. Trained fish were released in a circular test tank and their angular response scored. Under a white plus ultraviolet polarized light field, trout oriented in the trained E-vector orientation. For instance, fish trained under a parallel E-vector orientation exhibited angular responses close to parallel in the test tank. However, when the spectral composition of the polarized light field was manipulated, the accuracy of spatial orientation of the trout varied. Trout weighing about 30 g exhibited accurate orientation to the white plus UV polarized light field. The trout were incapable of orientation at a body weight of 50 to 60 g.     

Different modes of action of substance P in the motor control of the feline stomach and pylorus

In an experimental in vivo model to study gastropyloric motility in the cat a contraction of the stomach and the pyloric sphincter was regularly obtained in animals subjected to electrical vagal nerve stimulation or local intraarterial (i.a.) injection of substance P (SP). Much more infrequently contractile motor responses were recorded at splanchnic nerve stimulation. The contractile effects of SP were sensitive to atropine or local infusion of a SP analogue, (d-Pro²,d-Trp^7,9)-SP, indicating that SP activated a final common cholinergic neuron in both stomach and pylorus. However, there seemed to be separate transmission mechanisms in these two regions based on the results of the physiological studies. The vagally induced pyloric contraction was noncholinergic, nonadrenergic, but sensitive to ganglionic blockade (hexamethonium) or the SP analogue, indicating involvement of SP in a peptidergic pathway to the sphincter. The infrequent splanchnically induced pyloric contraction was sensitive to atropine, the SP analogue or ganglionic blockade (hexamethonium) in favour of SP acting on a final cholinergic neuron in this system. On the other hand the gastric contraction, obtained at either extrinsic nerve stimulations or local i.a. injection of SP, was sensitive to atropine or the SP analogue but hexamethonium resistant. These findings suggest antidromic activation of SP-containing axon collaterals of the extrinsic nerves terminating on cholinergic neurons of the gastric wall. When afferent C-fibres of the vagal nerve were selectively activated by local heating, pyloric contraction and gastric relaxation were obtained via vago-vagal reflexes. After cervical vagotomy heating of the distal end of the vagal nerve elicited a gastric contraction, previously demonstrated to be atropine sensitive and hexamethonium resistant, but no pyloric motor response. This suggests that the antidromic activation mechanism was present only in the stomach, not in the pylorus.     

The mechanism of alkylation of DNA by 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC), a metabolite of DIC (NSC-45388). Non-involvement of diazomethane

Comparison of the nuclear magnetic resonance spectra of chemically synthesized methyl-d₁-methanol with the methanol produced in the solvolytic decompostion of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) in D₂O under acidic, basic or neutral conditions indicated that no deuterium was exchanged for the hydrogens on the methyl group. Diazomethane can therefore be ruled out as an intermediate in this reaction.The methyl-d₃-guanine isolated after incubation of methyl-d₃-MIC with calfthymus DNA in vitro displayed, on chemical ionization mass spectrometry, a quasimolecular ion (MH⁺) at m/e 169, which was 3 mass units higher than the quasimolecular ion for an undeuterated 7-methylguanine standard. The major fragment ions for 7-methyl-d₃-guanine on electron impact mass spectrometry likewise were situated at positions 3 mass units higher than the fragment ions for 7-methylguanine itself.These data indicate that the methylation of biological macromolecules by MIC must involve the transfer of an intact methyl group.     

Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast

Summary The outer mitochondrial membranes of all organisms so far examined contain a protein which forms voltage-dependent anion selective channels (VDAC) when incorporated into planar phospholipid membranes. Previous reports have suggested that the yeast (Saccharomyces cerevisiae) outer mitochondrial membrane component responsible for channel formation is a protein of 29,000 daltons which is also the major component of this membrane. In this report, we describe the purification of this 29,000-dalton protein to virtual homogeneity from yeast outer mitochondrial membranes. The purified protein readily incorporates into planar phospholipid membranes to produce ionic channels. Electrophysiological characterization of these channels has demonstrated they have a size, selectivity and voltage dependence similar to VDAC from other organisms. Biochemically, the purified protein has been characterized by determining its amino acid composition and isoelectric point (pI). In addition, we have shown that the purified protein, when reconstituted into liposomes, can bind hexokinase in a glucose-6-phosphate dependent manner, as has been shown for VDAC purified from other sources. Since physiological characterization suggests that the functional parameters of this protein have been conserved, antibodies specific to yeast VDAC have been used to assess antigenic conservation among mitochondrial proteins from a wide number of species. These experiments have shown that yeast VDAC antibodies will recognize single mitochondrial proteins fromDrosophila, Dictyostelium andNeurospora of the appropriate molecular weight to be VDAC from these organisms. No reaction was seen to any mitochondrial protein from rat liver, rainbow trout,Paramecium, or mung bean. In addition, yeast VDAC antibodies will recognize a 50-kDa mol wt protein present in tobacco chloroplasts. These results suggest that there is some antigenic as well as functional conservation among different VDACs.     

Specifically deuterated and tritiated auxins

Regiospecific syntheses of monodeuterated and monotritiated natural auxin (indole- 3-acetic acid), a synthetic auxin (naphthalene-1-acetic acid) and a photoaffinity labeling auxin (5-azidoindole-3-acetic acid) are described. These syntheses provide benzene-ring tritiated auxins for use in reversible and covalent binding studies.     

Reactivation of a glycerinated model of Amoeba

Summary Motile models ofAmoeba proteus prepared by extraction at –20 °C with a 50% buffered glycerol solution showed remarkable contraction on addition of Mg-ATP with retainment of Ca⁺⁺ sensitivity. The initial contraction around the nucleus occurred on addition of Mg-ATP independently of the Ca⁺⁺ concentration, and was followed by contractions of three different patterns. In 10^–8M Ca⁺⁺, the granuloplasm contracted as a whole and separated from the membrane leaving no detectable particles in the anterior region. In 10^–6M Ca⁺⁺, the granuloplasm contracted leaving some granules which showed active and vigorous two-directional streaming similar to that in the living cell. Sometimes a part of the plasma membrane twitched. The streaming lasted up to 20 minutes. In 10^–4M Ca⁺⁺, after the initial contraction around the nucleus, no significant movement was observed. All these movements in the models were inhibited by cytochalasin B or N-ethylmaleimide. The relationship between the Mg-ATP and the Ca⁺⁺ concentrations was examined.     

Voltage dependence of current through the Na,K-exchange pump ofRana oocytes

Summary We have studied current (I _Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I _Str was measured as the strophanthidin-sensitive current in the presence of Ba^2–, Cd²⁺ and gluconate (in place of external Cl^–). In addition,I _Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK _0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na⁺-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI _Str depended directly onV _m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na⁺] markedly decreased the voltage sensitivity ofI _p , while producing only a small change in the maximal outwardI _Str. In contrast, decreasing the external [K⁺] from 25 to 2.5mm reducedI _Str at 0 mV without substantially affecting its voltage dependence. At K⁺ concentrations of 1mm, both the absolute value ofI _Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI _Str by [K⁺] _o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K⁺ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K⁺] _o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na⁺ and K⁺ are consistent with results obtained in other tissues and may reflect an ion-well effect.     

Fine Needle Aspiration Cytology of the Breast: Factors Affecting Sensitivity

At Northampton General Hospital a pathologist takes, stains and immediately reports aspirates at a fine needle aspiration clinic which is run in conjunction with a busy surgical breast clinic. the effect of various factors on the sensitivity of the technique have been quantified. Small tumour size, certain types of tumour and lesions difficult to palpate are causes of reduced sensitivity. Acellular samples had little effect on sensitivity. In this clinic trainee aspirators achieved good results early in their experience. After one year each had improved to the level of an experienced aspirator.