Mass spectrometry based method to increase throughput for kinome analyses using ATP probes.

Protein kinases play critical roles in many biological and pathological processes, making them important targets for therapeutic drugs. Here, we desired to increase the throughput for kinome-wide profiling. A new workflow coupling ActivX ATP probe (AAP) affinity reagents with isotopic labeling to quantify the relative levels and modification states of kinases in cell lysates is described. We compared the new workflow to a classical proteomics approach in which fractionation was used to identify low-abundance kinases. We find that AAPs enriched approximately 90 kinases in a single analysis involving six cell lines or states in a single run, an 8-fold improvement in throughput relative to the classical approach. In general, AAPs cross-linked to both the active and inactive states of kinases but performing phosphopeptide enrichment made it possible to measure the phospho sites of regulatory residues lying in the kinase activation loops, providing information on activation state. When we compared the kinome across the six cell lines, representative of different breast cancer clinical subtypes, we observed that many kinases, particularly receptor tyrosine kinases, varied widely in abundance, perhaps explaining the differential sensitivities to kinase inhibitor drugs. The improved kinome profiling methods described here represent an effective means to perform systematic analysis of kinases involved in cell signaling and oncogenic transformation and for analyzing the effect of different inhibitory drugs.

Dissecting variability in responses to cancer chemotherapy through systems pharmacology.

Variability in patient responses to even the most potent and targeted therapeutics is now the primary challenge facing drug discovery and patient care, particularly in oncology and immune therapy. Variability with respect to mechanisms of induced resistance is observed both in drug-naive patients and among those who are initially responsive. Genomics has developed powerful tools for systematic interrogation of disease genotype and transcriptional states (particularly in cancer) and for correlation of these measures with parameters of disease such as histological diagnosis and outcome. In contrast, mechanistic preclinical studies remain relatively narrowly focused, leading to many apparent contradictions and poor understanding of the determinants of response. We describe the emergence of a systems pharmacology approach that is mechanistic, quantitative, probabilistic, and postgenomic and promises to do for mechanistic pharmacology what genomics is doing for correlative studies. We focus on studies in cell lines (which currently dominate mechanism-oriented analysis), but our arguments are equally valid for real tumors studied in short-term culture as xenografts and, perhaps some time in the future, in humans.

Independent manipulation of stimulus change and unexpectedness dissociates indices of the orienting response.

Results obtained with the standard repetition-change paradigm of orienting research cannot be attributed unambiguously to either stimulus change or to unexpectedness. By adding announcement conditions, in which participants were told about an impending stimulus change, these two factors were disentangled. In Experiment 1, reaction times (RTs) were longer and ratings of surprise were higher with unannounced than with announced stimulus change. In contrast, larger skin conductance response (SCR) magnitudes occurred following change, irrespective of its congruence with participants' expectations. Experiment 2 replicated the results for SCR magnitude and, furthermore, revealed the same pattern of results for the evoked cardiac response. Surprise ratings again reflected the unexpectedness of stimulus presentations. The dissociation between RT and autonomic measures provides difficulties for resource allocation accounts of the orienting response.

The role of 5'-leader length, secondary structure and PABP concentration on cap and poly(A) tail function during translation in Xenopus oocytes.

The 5'-cap structure and poly(A) tail of eukaryotic mRNAs function synergistically to promote translation initiation through a physical interaction between the proteins that bind to these regulatory elements. In this study, we have examined the effect of leader length and the presence of secondary structure on the translational competence and the function of the cap and poly(A) tail for mRNAs microinjected into Xenopus oocytes. Increasing the length of the 5'-leader from 17 to 144 nt resulted in a 2- to 4-fold increase in expression from an mRNA containing an unstructured leader but increased expression up to 20-fold for an mRNA containing 5'-proximal structure. Consequently, the presence of secondary structure was less inhibitory for those mRNAs with a longer 5'-leader. Co-injection of poly(A)-binding protein (PABP) mRNA increased the function of the cap and poly(A) tail in promoting translation from poly(A)(+) but not poly(A)(-) mRNAs, particularly for mRNAs containing secondary structure. In the absence of an internal ribosome entry site, expression from the distal cistron of a dicistronic mRNA increased as a function of the length of the intercistronic region and the concentration of PABP. The inhibitory effect of intercistronic located secondary structure on translation was position-dependent. Indeed, the effect of secondary structure was abolished if positioned 134 nt upstream of the distal cistron. These data suggest that the length of a leader, the presence of secondary structure and the concentration of PABP determine the extent to which the cap and poly(A) tail regulate translation.

Secondary structure in the 5'-leader or 3'-untranslated region reduces protein yield but does not affect the functional interaction between the 5'-cap and the poly(A) tail.

The 5'-cap structure and poly(A) tail of eukaryotic mRNAs cooperate to promote translation initiation but whether this functional interaction benefits certain classes of mRNAs has not been investigated. In this study, we investigate whether a structured 5'-leader or 3'-untranslated region (UTR) affects the cap/poly(A) tail interaction. A structured leader reduced the degree to which the 5'-cap promoted translation in plant cells and inhibited translation from capped and uncapped mRNAs equally in yeast. Secondary structure within the 3'-UTR reduced translational efficiency when adjacent to the stop codon but had little effect on the cap/poly(A) tail synergy. The functional interaction between the cap and poly(A) tail was as important for an mRNA with a structured leader or 3'-UTR as it was for an unstructured mRNA in either species, suggesting that these structures can reduce translation without affecting the functional interaction between the cap and poly(A) tail. However, the loss of Xrn1p, the major 5'-->3' exoribonuclease in yeast, abolished cap-dependent translation and the functional interaction between the cap and poly(A) tail, suggesting that the cap/poly(A) tail synergy is of particular importance under conditions of active RNA turnover.

Identification and characterization of the functional elements within the tobacco etch virus 5' leader required for cap-independent translation.

Translation in plants is highly cap dependent, and the only plant mRNAs known to naturally lack a cap structure (m(7)GpppN) are viral in origin. The genomic RNA of tobacco etch virus (TEV), a potyvirus that belongs to the picornavirus superfamily, is a polyadenylated mRNA that is naturally uncapped and yet is a highly competitive mRNA during translation. The 143-nucleotide 5' leader is responsible for conferring cap-independent translation even on reporter mRNAs. We have carried out a deletion analysis of the TEV 5' leader to identify the elements responsible for its regulatory function and have identified two centrally located cap-independent regulatory elements (CIREs) that promote cap-independent translation. The introduction of a stable stem-loop structure upstream of each element demonstrated that CIRE-1 is less 5' end dependent in function than CIRE-2. In a dicistronic mRNA, the presence of the TEV 5' leader sequence in the intercistronic region increased expression of the second cistron, suggesting that the viral sequence can function in a 5'-distal position. Interestingly, the introduction of a stable stem-loop upstream of the TEV leader sequence or upstream of either CIRE in dicistronic constructs markedly increased their regulatory function. These data suggest that the TEV 5' leader contains two elements that together promote internal initiation but that the function of one element, in particular, is facilitated by proximity to the 5' end.

A dissociation between reaction time to sinusoidal gratings and temporal-order judgment.

The effect of the spatial frequency (SF) of visual gratings on reaction time (RT) and temporal-order judgment (TOJ) was examined in three experiments. In experiment 1 the visual stimuli were vertical sinusoidal gratings with SFs between 2 and 8 cycles deg-1 and the comparison stimulus in the TOJ task was a 2300 Hz tone. Whereas SF had a highly significant effect on RT, it left TOJ completely unaffected. To test whether this dissociation was due to the sharp (high SF) horizontal edges of the gratings, a second experiment was carried out with circular stimuli with no sharp edges. These stimuli did produce an effect of SF on TOJ, but it was significantly smaller than was the effect on RT. In experiment 3 we confirmed that this difference was not due to differences in grating orientation between the first two experiments. These findings (a) solve discrepancies between findings reported in the literature and (b) strongly suggest that RT and TOJ cannot be regarded as converging operations for determining 'visual latency'. This dissociation can best be accounted for by assuming that the output of early stimulus analysis can feed directly into the motor system (direct parameter specification), whereas the conscious representation that is used for TOJ is based on later integrative processes.

[Reaction time and temporal serial judgment: corroboration or dissociation?]. / Reaktionszeit und zeitliches Reihenfolgeurteil: Ubereinstimmung oder Dissoziation?

Dissociations between a motor response and the subject's verbal report have been reported from various experiments that investigated special experimental effects (e.g., metacontrast or induced motion). To examine whether similar dissociations can also be observed under standard experimental conditions, we compared reaction times (RT) and temporal order judgments (TOJ) to visual and auditory stimuli of three intensity levels. Data were collected from six subjects, each of which served for nine sessions. The results showed a strong, highly significant modality dissociation: While RTs to auditory stimuli were shorter than RTs to visual stimuli, the TOJ data indicated longer processing times for auditory than for visual stimuli. This pattern was found over the whole range of intensities investigated. Light intensity had similar effects on RT and TOJ, while there was a marginally significant tendency of tone intensity to affect RT more strongly than TOJ. It is concluded that modality dissociation is an example of "direct parameter specification", where the pathway from stimulus to response in the simple RT experiment is (at least partially) separate from the pathway that leads to a conscious, reportable representation. Two variants of this notion and alternatives to it are discussed.