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Les fusions du gène de la neureguline 1(NRG1) en activant les voies de signalisation d’ErbB2/ErbB3 peuvent être responsables d’une addiction oncogénique. Les fusions NRG1 sont devenues une cible thérapeutique potentielle pour de multiples types de tumeurs, y compris les adénocarcinomes (ADC) pulmonaires. L’afatinib, un inhibiteur pan-ErbB, pourrait être une option thérapeutique pour les patients atteints d’un ADC NRG1+comme le suggèrent des données précliniques et la publication de 7 cas cliniques (Tableau 1).

Kinetic energy release distributions (KERDs) upon dissociation of proton-bound dimers are utilized along with finite heat bath theory analysis to obtain relative proton affinities of monomeric species composing the dimer. The proposed approach allows accurate measurement of relative proton affinities based on KERD measurements for the compound with unknown thermochemical properties vs a single reference base. It also allows distinguishing the cases when dissociation of proton-bound dimers is associated with a reverse activation barrier, for which both our approach and the kinetic method become inapplicable. Results are reported for the n-butanol−n-propanol dimer, for which there is no significant difference in entropy effects for two reactions, and for the pyrrolidine−1,2-ethylenediamine dimer, which is characterized by a significant difference in entropy effects for the two competing reactions. Relative protonation affinities of −1.0 ± 0.3 kcal/mol for the n-butanol−n-propanol pair and 0.27 ± 0.10 kcal/mol for the pyrrolidine−1,2-ethylenediamine pair are in good agreement with literature values. Relative reaction entropies were extracted from the branching ratio and KERD measurements. Good correspondence was found between the relative reaction entropies for the n-butanol−n-propanol dimer (Δ(ΔS^‡) = −0.3 ± 1.5 cal/mol K) and the relative protonation entropy for the two monomers (Δ(ΔSp) = 0). However, the relative reaction entropy for the pyrrolidine−1,2-ethylenediamine dimer is higher than the difference in protonation entropies (Δ(ΔS^‡) = 8.2 ± 0.5 cal/mol K vs Δ(ΔSp) = 5 cal/mol K).

Normal-incidence surface-induced dissociation of singly protonated des-Arg¹-bradykinin (PPGFSPFR) and des-Arg⁹-bradykinin (RPPGFSPF) has been studied using a specially designed Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). We found that, with a reaction time of 1 s, the collision-energy-resolved fragmentation efficiency curve (FEC) for des-Arg⁹-bradykinin is shifted to lower energies by about 4 eV relative to the FEC for des-Arg¹-bradykinin. Because the Arrhenius activation energies found by Williams and co-workers in BIRD experiments are 0.82 and 1.2 eV for des-Arg¹- and des-Arg⁹-bradykinin, respectively, we expected to find the reverse order in our long-reaction-time FTICR-SID experiments. We rationalize the difference between our data and the thermal kinetics using Tolman's theorem to calculate threshold energies from the Arrhenius activation parameters. We found that, when the preexponential factor for reaction is very high (>10¹⁷ s^-1) or very low (<10¹⁰ s^-1), Tolman's correction becomes fairly large. In this case, the Arrhenius parameters are strongly correlated, and the activation energy depends on the reaction dynamics and temperature. This can result in the reversal of the order of the Arrhenius activation energies for different systems relative to the order of the threshold energies, meaning that conclusions about relative stabilities of large molecules should be based not on the thermal activation energies but rather on actual threshold energies, which can be calculated using Tolman's correction. The threshold energies extracted from the Arrhenius activation parameters are 1.15 and 1.24 eV for des-Arg¹- and des-Arg⁹-bradykinin, respectively, assuming a temperature of 450 K. However, des-Arg¹-bradykinin dissociates via a very tight transition state. Consequently, microcanonical rate−energy dependencies for these two peptides cross at very low internal energies and des-Arg⁹-bradykinin has a larger rate constant at internal energies sampled experimentally.

Energy transfer in collisions of benzene molecular ions with a fluorinated self-assembled monolayer surface at normal incidence was studied over the range of collision energies from 0 to 75 eV in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Rice−Ramsperger−Kassel−Marcus (RRKM) theory was used to calculate breakdown graphs for a simplified decomposition scheme of benzene on the time scale of ICR MS detection. Statistical partitioning of excess internal energy between the neutral and the ionic products was included in the theoretical model. Internal energy distributions of the predissociating parent ions were iteratively calculated using the recursive internal energy distribution search (RIEDS) method for each collision energy. On the basis of the above measurements, the collision-energy-dependent ESID → Eint energy conversion efficiency was found to maximize at about 19.5% under the conditions of 31 eV SID collision energy.

Kinetic energy releases (KERs) in unimolecular fragmentations of singly and multiply charged ions provide information concerning ion structures, reaction energetics and dynamics. This topic is reviewed covering both early and more recent developments. The subtopics discussed are as follows: (1) introduction and historical background; (2) ion dissociation and kinetic energy release: kinematics; potential energy surfaces; (3) the kinetic energy release distribution (KERD); (4) metastable peak observations: measurements on magnetic sector and time-of-flight instruments; energy selected results by photoelectron photoion coincidence (PEPICO); (5) extracting KERDs from metastable peak shapes; (6) ion structure determination and reaction mechanisms: singly and multiply charged ions; biomolecules and fullerenes; (7) theoretical approaches: phase space theory (PST), orbiting transition state (OTS)/PST, finite heat bath theory (FHBT) and the maximum entropy method; (8) exit channel interactions; (9) general trends: time and energy dependences; (10) thermochemistry: organometallic reactions, proton-bound clusters, fullerenes; and (11) the efficiency of phase space sampling. Copyright © 2001 John Wiley & Sons, Ltd.

Multiple-collision activation (MCA-CID) and surface-induced dissociation (SID) of protonated trialanine, (AAA)H⁺; tetraalanine, (AAAA)H⁺; pentaalanine, (AAAAA)H⁺; and prolyltetraalanine, (PAAAA)H⁺ were studied using a 7 T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Fragmentation efficiency curves obtained using both techniques were compared by converting the SID collision energy into an “effective” center-of-mass frame using an arbitrary neutral partner of mass, MN. The best overlap between the SID and MCA-CID fragmentation efficiency curves was obtained using very similar values of MN for all the peptides, indicating that all the protonated precursor ions were undergoing similar interaction with terminal chemical groups on the fluorinated self-assembled monolayer (SAM) surface. Collision energy resolved fragmentation efficiency curves obtained using both methods are very similar, and both methods of collisional activation result in a quasi-thermal population of ion internal energies. We suggest that normal-incidence ion−surface collisions with self-assembled monolayer surface involve interactions of the projectile ion with chemical groups on the surface with efficient transfer of impact energy into the surface and into the internal energy of the ion. This mechanism, except for time frame for the activation process, is analogous to the sequential gas phase collisional activation of these model peptides.

We present a rigorous theoretical basis of the kinetic method, commonly used for thermochemical determinations in mass spectrometry, based on finite heat bath theory (FHBT) developed by Klots. A simple analytical expression for the branching ratio is derived from FHBT formalism. This expression simplifies to the expression given by the absolute reaction rate theory (1) for very large clusters or (2) for reactions having a negligible kinetic shift. The reacting population is described by two different temperatures rather than by the “effective” temperature as suggested previously. Simulations performed using both RRKM and FHBT revealed that the kinetic plots are slightly nonlinear. The observed curvature is related to the changes in the transition state temperature as a function of the critical energy for fragmentation. The curvature of the plots decreases for larger clusters. We show that the “effective” temperature closely resembles the average value of the transition state temperature. This allows us to assign a new definition of the effective temperature and predict its properties. The results of simulations confirm that the extended version of the kinetic method introduced by Fenselau and co-workers provides accurate relative energetics for competitive reactions for both small and large ions. However, accurate thermochemical information can be obtained from the kinetic method only if reactions under investigation have negligible reverse activation energies. A new approach for extracting relative fragmentation energetics and entropy differences for two competing reactions is proposed. This approach requires a measurement of kinetic energy release distributions (KERDs) for the two fragmentation channels; the relative energetics and dynamics can be extracted from a single measurement.

The collision energy dependence of the fragmentation of the 1-bromonaphthalene radical cation was studied using sustained off-resonance excitation (SORI) in a 7 T Fourier transform ion cyclotron resonance mass spectrometer (FTMS). Fragmentation efficiency curves were obtained as a function of collision energy at four different pressures of Ar bath gas corresponding to collision numbers of 3, 5, 15, and 20. The results were modeled using RRKM/QET formalism. A refined analytical form for the collisional energy deposition function is proposed. The ability to obtain accurate fragmentation energetics of a complex system using the present approach is demonstrated. The “effective temperatures” deduced from the average internal energies for C6H5Br^+• and C10H7Br^+• were found to be the same for both ions provided the bath gas pressure and the maximum value of center-of-mass collision energy were the same. The range of effective temperatures from 1000 to 3700 K sampled in the present study significantly exceeds the temperature range accessible by blackbody infrared radiative dissociation (BIRD). We anticipate that the present approach can be used to study fragmentation energetics of biomolecules.

Using a high performance electron gun, electron-induced ionization and excitation of fullerene ion beams is achieved in the second field-free region of a reversed-geometry double-focusing mass spectrometer. Excitation of mass-selected C60z+ion beams leads to consecutive C2eliminations, which are observed in mass-analyzed ion kinetic energy (MIKE) spectra. Electron-induced decay of the ions takes place on a short time scale of 0–3 μs. Average kinetic energy release (KER) values were determined for the electron-induced C2eliminations from C60z+ions with z= 2–4 and were invariably found to be higher than for the corresponding spontaneous eliminations. The results are interpreted as being due to a time-dependent KER, since the non-fixed energy in the transition state is increased for higher internal energies and shorter lifetimes of the reactant ions. These results are compared with time-resolved KER values obtained previously, using an ion trap/reflectron instrument, for C2elimination from C60+. The two sets of data are modeled by finite heat bath theory (FHBT) using calculated rate energy dependencies. Calculations are performed using currently available information on the activation parameters. The dissociation is modeled with an Arrhenius pre-exponential factor, A= 5 × 1019s−1(at a temperature KBT= 0.2 eV) and a vaporization (activation) energy ΔEvap= 10.3 eV. Modeling the data necessitates inclusion of radiative decay in parallel with the dissociation. Radiative decay is dominant at times longer than a few microseconds.

Psoralens in combination with ultraviolet light (UVA; 320-400 nm) are used in the photochemical treatment of a variety of skin diseases including vitiligo, a skin depigmentational disorder, and psoriasis, a disease of accelerated epidermal cell proliferation. Although it is generally assumed that the major site of action of the psoralens is DNA, we have obtained evidence that another site may be the primary target for these compounds. We have identified specific, saturable, high-affinity binding sites for 8-methoxypsoralen on HeLa cells and have detected specific binding of 8-methoxypsoralen to four other human cell lines and five mouse cell lines. In HeLa cells, specific binding is reversible and independent of the ability of the compound to intercalate into DNA. In addition, binding sites become covalently modified by the psoralen after UVA exposure. Specific binding of 8-[methoxy-3H]methoxypsoralen constitutes 79% of the label bound to the cells. Scatchard analysis indicated two classes of psoralen binding sites: high-affinity sites with a Kd of 19 X 10(-9) M (1.8 X 10(5) sites per cell) and low-affinity sites with a Kd of 4 X 10(-6) M (7.1 X 10(6) sites per cell). Four structurally related psoralen analogs block 8-methoxypsoralen binding in a manner that parallels their biological activity. Based on these findings, we hypothesize that specific binding sites for psoralens on mammalian cells mediate, at least in part, psoralen-induced phototoxicity.

We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed that there are differences between the binding characteristics in intact cells and in membrane fractions prepared from these cells. Scatchard analysis of the binding data from membrane fractions gave a linear plot (Kd = 9.1 X 10(-8) M). With intact cells, a curvilinear Scatchard plot was obtained. This was resolved into two components defining binding sites with affinity constants of 1.7 X 10(-9) M and 4.6 X 10(-7) M. Thus, it appears that [3H]diazepam binding in intact cells is more complex than in isolated membranes. Several related benzodiazepines, including flunitrazepam, Ro-5-4864, nitrazepam, oxazepam, lorazepam, Ro-5-3072, chlordiazepoxide, and clonazepam also induced melanogenesis. When these compounds were tested for their ability to inhibit [3H]diazepam binding, flunitrazepam, diazepam, and Ro-5-4864 were found to be the most effective inhibitors. These three compounds were also the most potent in inducing melanogenesis. Our results suggest that the benzodiazepines modulate cell differentiation. The presence of high-affinity binding sites in this homogeneous, easily grown cell line may provide a useful model for studies on the mechanism of action of these compounds.

One of the more prominent clinical treatments for skin diseases such as psoriasis and vitiligo involves the use of a combination of psoralens and UV light, a procedure referred to as PUVA chemotherapy. This drug regimen markedly alters epidermal cell growth and differentiation. In many cell types, an early cellular event following treatment of cells with PUVA is inhibition of binding of epidermal growth factor (EGF) to its receptor. To examine the mechanism underlying this effect, we used A431 cells, a human epidermal cell line known to express large numbers of EGF receptors. We found that exposure of A431 cells to PUVA caused a dramatic inhibition of EGF-stimulated EGF receptor tyrosine kinase activity. Inhibition required intact cells and did not appear to be mediated by protein kinase C, because this inhibition was apparent in cells in which the enzyme was down-regulated by phorbol ester pretreatment and in cells treated with inhibitors of protein kinase C. Inhibition of tyrosine kinase activity by PUVA was distinct from other inhibitors of EGF receptor function in that it was associated with a rapid increase in the amount of phosphate incorporated into serine residues of the EGF receptor. This suggested that PUVA-induced serine phosphorylation may mediate EGF receptor kinase activity. These results demonstrate that alterations in EGF receptor function may contribute to the therapeutic efficacy of PUVA in photo-chemotherapy.

Functional electrical stimulation (FES) assisted resistance training has been effective in increasing muscular strength and endurance in spinal cord injured men and women in preparation for FES-assisted cycle programs and for FES-assisted standing and walking. Increases in blood pressure and a concomitant bradycardia suggestive of autonomic dysreflexia have been reported during FES-assisted resistance training. Self-induced autonomic dysreflexia in athletes who use wheelchairs suppressed the normal exercise induced serum testosterone increase. We, therefore, examined the changes in hematocrit and circulating levels of testosterone, sex hormone binding globulin (SHBG), Cortisol, prolactin, norepinephrine and epinephrine during FES assisted resistance exercise in five high spinal cord injured men (SCI) and comparable maximal exercise in five able bodied controls (AB). Mean serum testosterone levels significantly increased with FES-assisted resistance training in SCI and maximal resistance exercise in AB with no significant change in hematocrit or SHBG. Prolactin, Cortisol and epinephrine levels were unchanged while norepinephrine levels were significantly increased in SCI and AB. These findings suggest that there is no concern over inadequate physiological androgen response to an exercise stimulus in SCI. The data do not support the previous findings that elevated levels of norepinephrine in autonomic dysreflexia suppress testosterone response to exercise.

Shoulder dysfunction due to regular wheelchair use is a common problem among people with spinal cord injuries. As a remedial measure, strengthening of the scapular retractor muscles has been suggested. Electromyographical analysis was utilized to examine scapular retraction muscle use during rowing, backward wheeling and a standardized scapular retraction exercise in seven people with spinal cord injuries and seven able bodied subjects. In addition, a pilot study using indwelling electrodes was completed to validate the use and placement of surface electrodes. Both rowing and the standardized scapular retraction exercise recruited higher levels of retractor involvement than backward wheeling. We suggest that rowing, because of its value as a cardiovascular exercise and high level of retractor recruitment, is an appropriate and effective means of remediating scapular retractor weakness.

The psoralens, when activated by ultraviolet light of 320-400 nm (UVA light), are potent modulators of epidermal cell growth and differentiation. Previously, we reported that, in mammalian cells, these compounds bind to specific saturable high-affinity cellular receptor sites. In the present studies, we demonstrate that binding of psoralens to their receptors followed by UVA light activation is associated with inhibition of epidermal growth factor (EGF) receptor binding. Inhibition of EGF binding, which required UVA light, was rapid and dependent on the dose of UVA light (0.5-2.0 J/cm2), as well as the concentration of psoralens (10 nM to 1 microM). Higher doses of UVA light (2.0-6.0 J/cm2) by themselves were also inhibitory, indicating that psoralens potentiate the UVA-induced inhibition of EGF binding. A number of biologically active analogs of psoralen, including 8-methoxypsoralen, 5-methoxypsoralen, and 4,5',8-trimethylpsoralen, when activated by UVA light, were found to be inhibitors of binding. Inhibition of EGF binding by psoralens was observed in a variety of human and mouse cell culture lines known to possess psoralen receptors. In the epidermal-derived line PAM 212, at least two populations of receptors with different affinities for EGF were found. Psoralens and UVA light selectively inhibited binding to the higher-affinity EGF receptors, an effect analogous to that of the phorbol ester tumor promoters. As observed with phorbol esters, photoactivated psoralens appeared to inhibit EGF binding by an indirect mechanism. These data demonstrate that the psoralens and UVA light have direct biological effects on cell-surface membranes. Since EGF is a growth-regulatory peptide, the ability of psoralens and UVA light to inhibit EGF binding may underlie the biologic effects of these agents in the skin.

Psoralens in combination with ultraviolet light are potent modulators of epidermal cell growth and differentiation. Responsive cell types contain specific, saturable, high-affinity binding sites for the psoralens. These binding sites become covalently modified by the psoralen molecule following ultraviolet light exposure. In the present studies the psoralen receptor, labeled with [3H]8-methoxypsoralen, was visualized in the cytoplasmic and plasma membrane fractions of HeLa cells following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor had an apparent molecular mass of approximately 22,000 daltons and was shown to be sensitive to protease, but not nuclease treatment. The radiolabeled receptor could not be visualized in nuclear extracts of cells. Covalent binding of the radioligand to the receptor protein was inhibited by excess unlabeled 8-methoxypsoralen, indicating that covalent psoralen-receptor binding was saturable. In addition, the covalently modified receptor was found to persist in cells for over 5 h. The presence of a cellular protein that exhibits specific affinity for the psoralens and becomes photoalkylated by these compounds, together with previous data showing that the psoralens have direct effects on the cell surface membranes, supports our model that some of the biological effects of photoactivated psoralens are receptor-mediated.

Previous investigations have demonstrated close genetic linkage between loci governing expression of strain-specific idiotypes and immunoglobulin heavy (H) chain allotype (i.e., the CHlocus). This linkage is presumed to reflect polymorphism of VHgenes (or of their expression) linked to the polymorphic CHlocus. That there was no apparent involvement of light (L) chain loci (thought to be unlinked to H chain) in inheritance of the idiotype-positive (Id+) phenotype was surprising, because the L chain is required for formation of each of the idiotypes studied at the chemical level. However, previous studies involving backcrosses of F1(Id+× Id-) mice to the Id-parental strain have never employed as the Id-parent one of several inbred strains shown by G. M. Edelman and P. D. Gottlieb [(1970) Proc. Natl. Acad. Sci. USA67, 1191-1199] to express a VL-region polymorphism. Among backcrosses performed in the present study, one involved the A/J strain as the Id+parent and the PL/J strain, one of the several strains with and L chain polymorphism, as the Id-parent. Whereas in three other backcrosses performed, idiotype expression segregated with H chain allotype, in the backcross to PL/J, all mice producing the characteristic A/J Id+phenotype were A/J allotype+, but not all A/J allotype+mice were Id+. Typing of backcross progeny for the Ly-3 thymocyte alloantigens, controlled by a locus closely linked to that governing the L chain polymorphism (called the VK-1locus), indicated that only the Ly-3 heterozygotes expressed the characteristic A/J Id+phenotype. Thus, all Id+backcross mice inherited both the H chain allotype and the Ly-3locus (and closely linked L chain-related locus) from the Id+A/J strain. This suggests that, when strains with L chain polymorphisms are included in genetic studies of idiotype expression, segregation of loci governing L chain expression may be found to contribute to inheritance of the Id+character. In addition, these studies may be taken as further evidence that the VL-region repertoire of the PL/J strain may differ considerably from that of most other inbred strains of mice.

The purpose of the investigation was to examine the safety and efficacy of functional electrical stimulation (FES)-assisted hydraulic resistance training in improving cardiovascular fitness in persons with spinal cord injuries. The cardiopulmonary responses of 10 high spinal cord injured (SCI) and five able bodied (AB) subjects were assessed during three bouts of FES-assisted leg extension exercise. The protocol involved three 30-minute tests: (1) unloaded leg extension, (2) hydraulically-resisted leg extension (loaded), and (3) a reproduction of the unloaded and loaded protocols to measure cardiac output (Q). Pre-measurements were made of body mass, mean limb weight, maximal force output and maximal oxygen uptake (incremental arm ergometry). Oxygen uptake (VO2), minute ventilation (Ve), respiratory exchange ratio (RER), heart rate (HR), blood pressure (BP) were recorded before, during and after tests. There was a significant difference in VO2 max between SCI and AB subjects. Cardiac output significantly increased between the loaded and unloaded tests. The significant increases from rest to unloaded and loaded exercise pointed to the potential value of adding resistance to a leg extension training regime. Heart rate and BP of the participants with SCI consistently demonstrated a response suggestive of autonomic dysreflexia. Upon stimulation an immediate increase in (predominantly systolic) BP was observed, followed by a fall in HR. On cessation of stimulation HR exhibited a substantial rebound effect and BP returned to normal levels. This response was highly reproducible and suggests caution be exercised in the use of FES for people with SCI with lesion levels above the major splanchnic outflow (T6).

Recently a FES (functional electrical stimulation)-assisted rowing machine was developed to enhance cardiovascular training in people with spinal cord injuries. The machine was assessed in terms of its efficacy as a training tool. Six patients who were quadriplegic (C6–T1) and 2 who were paraplegic (T3–6) completed a series of three tests in succession: (1) leg stimulation only (quadriceps and hamstring groups)—‘Stim’, (2) arm row only—‘Row’ and (3) simultaneous row and stimulation—‘R & S’. Measurements recorded included oxygen uptake (VO2), minute ventilation (Ve), respiratory exchange ratio (RER), heart rate (HR) and blood pressure (BP). In addition, 6 out of the 8 subjects took part in a qualitative assessment comprising a guided interview exploring the subject's perception of the machine and test.Significant increases in VO2 were demonstrated between the three tests with R & S producing mean steady-state values of 16.34 nm (± 0.74) ml/kg/min (83% of maximum). These values represented a 12% increase over Row alone. Of interest was the qualitative assessment which revealed that subjects perceived R & S to be easier than Row despite the higher levels of VO2 observed. The results suggest that the rowing machine represents a potentially valuable hybrid training device that may significantly reduce risk factors for cardiovascular disease and improve the quality of life of people with SCI.

The B16/C3 murine melanoma is a pigmented tumor that is rich in the copper-containing enzyme, tyrosinase. This enzyme, which converts tyrosine to melanin precursors, is largely associated with membrane fractions of cells and exists in a number of discrete isozymic forms ranging in molecular mass from 58,000 to 150,000 daltons and pI from 3.4 to 5.2. One of these isozymes (Mr = 58,000, pI 3.4) has been purified to homogeneity. The purified enzyme catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA to dopaquinone. Ascorbic acid, tetrahydrofolate, and dopamine can serve as cofactors in the hydroxylase reaction. The Michaelis constants for the purified enzyme were 7 X 10(-4) M for L-tyrosine and 6 X 10(-4) M for L-DOPA. The Vmax for L-DOPA was much greater than the Vmax for L-tyrosine indicating that tyrosine hydroxylation is rate-limiting in melanin precursor biosynthesis. Two putative copper chelators, phenylthiourea and diethyldithiocarbamide inhibited both the tyrosine hydroxylase and L-DOPA oxidase activities of the enzyme. Phenylthiourea was a noncompetitive inhibitor while diethyldithiocarbamide was a competitive inhibitor indicating that these agents act by different mechanisms. When digested with proteases and glycosidases, higher molecular weight forms of tyrosinase co-migrated with the purified enzyme in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the isozyme was derived from larger precursors. Thus, post-translational processing of tyrosinase may underlie isozyme diversity and this may be important in the control of melanogenesis in this tumor model.

Topical application of beta-naphthoflavone to CD-1 mice induced an 87-fold increase in epidermal 7-ethoxyresorufin O-dealkylation activity per cell and a many-fold increase in epidermal cytochrome P-4501A1 (CYP1A1) concentration. Flow cytometric analysis of individual epidermal cells from acetone-treated and beta-naphthoflavone-treated mice using a monoclonal antibody for CYP1A1 indicated that 50% to 60% of the isolated epidermal cells were refractory to beta-naphthoflavone induction of CYP1A1. Examination of the differences between responsive and nonresponsive epidermal cells from beta-naphthoflavone-treated mice revealed that 70% of the low CYP1A1-containing cells (noninduced) separated by flow cytometry were basal cells and only 12% were suprabasal differentiated cells. In contrast, about 50% of the high CYP1A1-containing induced cells separated by flow cytometry from the epidermis of mice treated with beta-naphthoflavone were suprabasal cells and 35% were basal cells. These results indicate that topical application of beta-naphthoflavone increased the level of CYP1A1 in about 80% of the separated suprabasal cells and in about 35% of the separated basal cells.

Treatment of A431 human epidermoid cells with epidermal growth factor (EGF; 20 nM) results in decreased proliferation. This is associated with blockage of the cells in the S and/or G2 phases of the cell cycle. We found that tyrphostin, a putative tyrosine kinase inhibitor, in the range of 50 to 100 microM, partially reversed the growth-inhibitory and cell cycle changes induced by EGF. By using high-pressure liquid chromatography with electrochemical detection, we found that tyrphostin was readily incorporated into A431 cells, reaching maximal levels within 1 h. Although tyrphostin (50 to 100 microM) had no effect on high-affinity binding of EGF to its receptor in A431 cells for up to 24 h, the compound partially inhibited EGF-stimulated EGF receptor tyrosine kinase activity. However, this effect was evident only after prolonged treatment of the cells (4 to 24 h) with the drug. When the peak intracellular concentration of tyrphostin occurred (1 h), no inhibition of tyrosine kinase activity was observed. After both 1 and 24 h, tyrphostin was a less effective inhibitor of tyrosine kinase activity than the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate, which almost completely blocked EGF receptor autophosphorylation. On the basis of our data, we hypothesize that tyrphostin is not a competitive inhibitor of the EGF receptor tyrosine kinase in intact cells and that it functions by an indirect mechanism.

MELITTIN, a 26-amino acid polypeptide of known sequence, is the major constituent of bee venom1. Its primary amino acid sequence reveals that the molecule has both basic and hydro-phobic termini, which give the molecule an amphipathic character. Consequently melittin can insert into phospholipid bilayers and exhibits surfactant activity. There is evidence that the association of melittin with cellular membranes results in: (1) disturbance of the acyl groups of phospholipids such that they assume a broader range of angles with respect to each other2,3; (2) increased phospholipid susceptibility to hydrolysis by phospholipase4,5; and (3) increased synthesis of prostaglandins from the arachidonic acid released from the phospholipids6. Another amphipathic but structurally unrelated group of compounds consists of esters of the diterpene alcohol phorbol. These compounds are well known tumour promoters and have also recently been shown to have the ability to enhance phospholipid deacylation7,19. It is therefore of interest to determine whether melittin can produce some of the other phenotypic effects that can be induced in cells in culture by phorbol ester tumour promoters8,9. We report here that both melittin and the phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), inhibit differentiation of mouse melanoma cells, enhance anchorage-independent growth of adenovirus-transformed rat embryo cells, and induce arachidonic acid release and prostaglandin synthesis from C3H/10T½ mouse embryo fibroblasts.

We describe the case of an 11-month-old girl with a rare cerebellar glioblastoma driven by a NACC2-NTRK2(Nucleus Accumbens Associated Protein 2-Neurotrophic Receptor Tyrosine Kinase 2) fusion. Initial workup of our case demonstrated homozygous CDKN2Adeletion, but immunohistochemistry for other driver mutations, including IDH1 R132H, BRAF V600E, and H3F3A K27M were negative, and ATRX was retained. Tissue was subsequently submitted for personalized oncogenomic analysis, including whole genome and whole transcriptome sequencing, which demonstrated an activating NTRK2fusion, as well as high PD-L1 expression, which was subsequently confirmed by immunohistochemistry. Furthermore, H3and IDHdemonstrated wildtype status. These findings suggested the possibility of treatment with either NTRK- or immune checkpoint- inhibitors through active clinical trials. Ultimately, the family pursued standard treatment that involved Head Start III chemotherapy and proton radiotherapy. Notably, at most recent follow upapproximately two years from initial diagnosis, the patient is in disease remission and thriving, suggesting favorable biology despite histologic malignancy. This case illustrates the value of personalized oncogenomics, as the molecular profiling revealed two actionable changes that would not have been apparent through routine diagnostics. NTRKfusions are known oncogenic drivers in a range of cancer types, but this is the first report of a NACC2-NTRK2fusion in a glioblastoma.LEARNING OBJECTIVESThis presentation will enable the learner to:1.Explore the current molecular landscape of pediatric high grade gliomas2.Recognize the value of personalized oncogenomic analysis, particularly in rare and/or aggressive tumors3.Discuss the current status of NTRK inhibitor clinical trials