Fibronectin induction abrogates the BRAF inhibitor response of BRAF V600E/PTEN-null melanoma cells.

The mechanisms by which some melanoma cells adapt to Serine/threonine-protein kinase B-Raf (BRAF) inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF mutant and PTEN null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma tissue microarray showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5ß1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of FN expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

Does assimilation into schemas involve systems or cellular consolidation? It's not just time.

A comment by Rudy and Sutherland [Rudy, J. R., & Sutherland, R. J. (2008). Is it systems or cellular consolidation? Time will tell. An alternative interpretation of the Morris Group's recent Science Paper. Neurobiology of Learning and Memory] has suggested an alternative account of recent findings concerning very rapid systems consolidation as described in a recent paper by Tse et al [Tse, D., Langston, R. F., Kakeyama, M., Bethus, I., Spooner, P. A., & Wood, E. R., et al. (2007). Schemas and memory consolidation. Science, 316, 76-82]. This is to suppose that excitotoxic lesions of the hippocampus cause transient disruptive neural activity outside the target structure that interferes with cellular consolidation in the cortex. We disagree with this alternative interpretation of our findings and cite relevant data in our original paper indicating why this proposal is unlikely. Various predictions of the two accounts are nonetheless outlined, together with the types of experiments needed to resolve the issue of whether systems consolidation can occur very rapidly when guided by activated neural schemas.

The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: potential therapy for cancer.

The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC(50)s <80 nM. Growth of ErbB-2- and EGFR-expressing tumor cell lines is inhibited at concentrations <0.5 microM. Selectivity for tumor cell growth inhibition versus normal human fibroblast growth inhibition ranges from 10- to >75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.

Habituation of the proleg withdrawal reflex in Manduca sexta does not involve changes in motoneuron properties or depression at the sensorimotor synapse.

Larvae of the hawkmoth, Manduca sexta, exhibit a defensive proleg withdrawal reflex in which deflection of mechanosensory hairs on the proleg tip (the planta) evokes retraction of the proleg. A previous behavioral study showed that this reflex habituates in response to repeated planta hair deflection and exhibits several other defining features of habituation. In a semi-intact preparation consisting of a proleg and its associated segmental ganglion, repeated deflection of a planta hair or electrical stimulation of its sensory neuron causes a neural correlate of habituation, manifested as a decrease in the number of action potentials evoked in the proleg motor nerve. Monosynaptic connections from planta hair sensory neurons to the principal planta retractor motoneuron exhibit several forms of activity-dependent plasticity. In the present study we recorded intracellularly from this motoneuron during repetitive electrical stimulation of a planta hair sensory neuron. The number of action potentials evoked in the motoneuron decreased significantly, representing a neural correlate of habituation. The motoneuron's resting membrane potential, input resistance. and spike threshold measured before and after repetitive stimulation did not differ between the stimulated group and a control group. Furthermore, the amplitude of the monosynaptic excitatory postsynaptic potential, as well as the magnitude of paired-pulse facilitation, evoked in the motoneuron by the sensory neuron did not change after repetitive stimulation. These results suggest that depression at the sensorimotor synapse does not contribute to reflex habituation. Rather, other mechanisms in the ganglion of the stimulated segment, such as changes in polysynaptic reflex pathways, appear to be responsible.

The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo.

The epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinases are currently being targeted by various mechanisms in the treatment of cancer. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains with IC50 values against purified EGFR and ErbB-2 of 10.2 and 9.8 nM, respectively. This report describes the efficacy in cell growth assays of GW2016 on human tumor cell lines overexpressing either EGFR or ErbB-2: HN5 (head and neck), A-431 (vulva), BT474 (breast), CaLu-3 (lung), and N87 (gastric). Normal human foreskin fibroblasts, nontumorigenic epithelial cells (HB4a), and nonoverexpressing tumor cells (MCF-7 and T47D) were tested as negative controls. After 3 days of compound exposure, average IC50 values for growth inhibition in the EGFR- and ErbB-2-overexpressing tumor cell lines were < 0.16 microM. The average selectivity for the tumor cells versus the human foreskin fibroblast cell line was 100-fold. Inhibition of EGFR and ErbB-2 receptor autophosphorylation and phosphorylation of the downstream modulator, AKT, was verified by Western blot analysis in the BT474 and HN5 cell lines. As a measure of cytotoxicity versus growth arrest, the HN5 and BT474 cells were assessed in an outgrowth assay after a transient exposure to GW2016. The cells were treated for 3 days in five concentrations of GW2016, and cell growth was monitored for an additional 12 days after removal of the compound. In each of these tumor cell lines, concentrations of GW2016 were reached where outgrowth did not occur. Furthermore, growth arrest and cell death were observed in parallel experiments, as determined by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 treatment inhibited tumor xenograft growth of the HN5 and BT474 cells in a dose-responsive manner at 30 and 100 mg/kg orally, twice daily, with complete inhibition of tumor growth at the higher dose. Together, these results indicate that GW2016 achieves excellent potency on tumor cells with selectivity for tumor versus normal cells and suggest that GW2016 has value as a therapy for patients with tumors overexpressing either EGFR or ErbB-2.

Hippocampal neurons encode information about different types of memory episodes occurring in the same location.

Firing patterns of hippocampal complex-spike neurons were examined for the capacity to encode information important to the memory demands of a task even when the overt behavior and location of the animal are held constant. Neuronal activity was recorded as rats continuously alternated left and right turns from the central stem of a modified T maze. Two-thirds of the cells fired differentially as the rat traversed the common stem on left-turn and right-turn trials, even when potentially confounding variations in running speed, heading, and position on the stem were taken into account. Other cells fired differentially on the two trial types in combination with behavioral and spatial factors or appeared to fire similarly on both trial types. This pattern of results suggests that hippocampal representations encode some of the information necessary for representing specific memory episodes.

The discovery of potent cRaf1 kinase inhibitors.

A series of benzylidene-1H-indol-2-one (oxindole) derivatives was synthesized and evaluated as cRaf-1 kinase inhibitors. The key features of the molecules were the donor/acceptor motif common to kinase inhibitors and a critical acidic phenol flanked by two substitutions. Diverse 5-position substitutions provided compounds with low nanomolar kinase enzyme inhibition and inhibited the intracellular MAPK pathway.

Neurotoxic hippocampal lesions have no effect on odor span and little effect on odor recognition memory but produce significant impairments on spatial span, recognition, and alternation.

Recent work has shown that lesions of the hippocampus in monkeys cause deficits in the capacity to remember increasing numbers of objects, colors, and spatial locations (). However, others have observed that hippocampectomized monkeys can show intact memory for a list of objects or locations (). We wished to explore the effects of hippocampal damage on the capacity of memory in the rodent and, to do so, developed novel "span" tasks in which a variable number of odors or locations had to be remembered. In the odor span task (experiment 1), rats were trained on a nonmatching to sample task in which increasing numbers of odors had to be remembered. Half of the trained rats received ibotenic acid lesions of the hippocampus. Postoperatively, hippocampectomized animals did not differ from control animals even when required to remember up to 24 odors. However, when tested on delayed retention of a list of 12 odors, rats with hippocampal lesions were impaired at a long delay. Also, these rats were impaired on a subsequent test of delayed spatial alternation. In a spatial span task (experiment 2), naive rats were trained on a nonmatching to sample task in which a variable number of locations had to be remembered. After this, half of the animals received ibotenic acid lesions. Postoperatively, hippocampectomized animals performed above chance levels when required to remember a single cup location, but were unable to remember more. Subsequent testing on another spatial delayed alternation task suggested that hippocampectomized rats could recognize, but could not inhibit their approach to previously visited locations.

The global record of memory in hippocampal neuronal activity.

In humans the hippocampal region of the brain is crucial for declarative or episodic memory for a broad range of materials. In contrast, there has been controversy over whether the hippocampus mediates a similarly general memory function in other species, or whether it is dedicated to spatial memory processing. Evidence for the spatial view is derived principally from the observations of 'place cells'-hippocampal neurons that fire whenever the animal is in a particular location in its environment, or when it perceives a specific stimulus or performs a specific behaviour in a particular place. We trained rats to perform the same recognition memory task in several distinct locations in a rich spatial environment and found that the activity of many hippocampal neurons was related consistently to perceptual, behavioural or cognitive events, regardless of the location where these events occurred. These results indicate that nonspatial events are fundamental elements of hippocampal representation, and support the view that, across species, the hippocampus has a broad role in information processing associated with memory.

A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors.

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.

Genetic responses of the thermophilic archaeon Sulfolobus acidocaldarius to short-wavelength UV light.

The archaea which populate geothermal environments are adapted to conditions that should greatly destabilize the primary structure of DNA, yet the basic biological aspects of DNA damage and repair remain unexplored for this group of prokaryotes. We used auxotrophic mutants of the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius to assess genetic and physiological effects of a well-characterized DNA-damaging agent, short-wavelength UV light. Simple genetic assays enabled quantitative dose-response relationships to be determined and correlated for survival, phenotypic reversion, and the formation of genetic recombinants. Dose-response relationships were also determined for survival and phenotypic reversion of the corresponding Escherichia coli auxotrophs with the same equipment and procedures. The results showed S. acidocaldarius to be about twice as UV sensitive as E. coli and to be equally UV mutable on a surviving-cell basis. Furthermore, UV irradiation significantly increased the frequency of recombinants recovered from genetic-exchange assays of S. acidocaldarius. The observed UV effects were due to the short-wavelength (i.e., UV-C) portion of the spectrum and were effectively reversed by subsequent illumination of S. acidocaldarius cells with visible light (photoreactivation). Thus, the observed responses are probably initiated by the formation of pyrimidine dimers in the S. acidocaldarius chromosome. To our knowledge, these results provide the first evidence of error-prone DNA repair and genetic recombination induced by DNA damage in an archaeon from geothermal habitats.

Neural correlates of habituation of the proleg withdrawal reflex in larvae of the hawk moth, Manduca sexta.

The larval proleg withdrawal reflex of the hawk moth, Manduca sexta, exhibits robust habituation. This reflex is evoked by deflecting one or more mechanosensory planta hairs on a proleg tip. We examined neural correlates of habituation in an isolated proleg preparation consisting of one proleg and its segmental ganglion. Repeated deflection of a single planta hair caused a significant decrease in the number of action potentials evoked in the proleg motor nerve (which carries the axons of proleg retractor motor neurons). Significant response decrement was seen for interstimulus intervals of 10 s, 60 s and 5 min. Response decrement failed to occur in the absence of repetitive stimulation, the decremented response recovered spontaneously following a rest, and electrical stimulation of a body wall nerve facilitated the decremented response (a neural correlate of dishabituation). Adaptation of sensory neuron responses occurred during repeated hair deflections. However, when adaptation was eliminated by direct electrical stimulation of sensory neurons, the response in the proleg motor nerve still decreased significantly. Muscle recordings indicated that the response of an identified proleg retractor motor neuron decreased significantly during habituation training. Thus, habituation of the proleg withdrawal reflex includes a central component that is apparent at the level of a single motor neuron.

Neural mechanisms of behavioral plasticity: metamorphosis and learning in Manduca sexta.

This review summarizes our current understanding of the neural circuit underlying the larval proleg withdrawal reflex (PWR) of Manduca sexta and describes how PWR function changes in two contexts: metamorphosis and learning. The first form of PWR plasticity occurs during the larval-pupal transformation, when the reflex is lost. One mechanism that contributes to this loss is the weakening of monosynaptic excitatory connection from proleg sensory neurons to proleg retractor motor neurons. This change is associated with the hormonally-mediated regression of proleg motor neuron dendrites, which may break synaptic contacts between the sensory and motor neurons. After pupation, some of the proleg motor neurons die in a segment-specific pattern that persists even after individual motor neurons are isolated from the nervous system and exposed to hormones in vitro. The second form of PWR plasticity involves short-term, activity-dependent changes in neural function during the larval stage. The nicotinic cholinergic connections from proleg sensory neurons to motor neurons exhibit several forms of plasticity including facilitation, depression, post-tetanic potentiation and two types of muscarinic modulation. Larval PWR behavior exhibits two simple forms of learning-habituation and dishabituation-which involve alterations in the central PWR circuit. These studies of a simple circuit illustrate neural mechanisms by which behaviors undergo both short- and long-term modifications.

Acute care costs of the oldest old: they cost less, their care intensity is less, and they go to nonteaching hospitals.

BACKGROUND: The cost of acute hospital care is often believed to increase with age among older persons. Our clinical experience in the acute hospital setting suggested that people aged 90 years and older may be a distinct cohort who have different health care needs and who use health resources differently from younger aged groups. METHODS: To determine the cost of care for the oldest old and younger old patients in Massachusetts acute care hospitals, 1992 and 1993 discharge data from all nonfederal Massachusetts hospitals were examined (678 954 discharges) according to five age groups: 60 to 69 years (n=210 270), 70 to 79 years (n=256 781), 80 to 89 years (n=171 725), 90 to 99 years (n=39 170), and 100 or more years (n=1008). Average estimated total and ancillary costs per discharge and per diagnosis related group were calculated. Differences by gender and survivorship were also examined. RESULTS: Hospitalization costs peaked in the 70- to 79-year age group and declined with age thereafter. Case mix was an important determinant of this trend. Despite lower cost per stay, average length of stay was longer for the oldest age groups. Ancillary costs accounted for 53% of the total costs per stay among the 60- to 69-year-olds and only 32% among the 100 or more-year- olds. For hospitalizations during which the patient died, the average cost per discharge decreased 61%, from $16886 for 60- to 69-year-olds to $6523 for centenarians. Costs were greater for decedents than for survivors, although these differences decreased dramatically with increasing age. Those aged 80 years and older tended to be hospitalized in nonteaching hospitals. CONCLUSIONS: In the acute hospital setting, the oldest old cost less per admission than younger elderly patients. This finding must be considered when future health care costs are predicted for this fastest growing segment of our population.

Ischemia-induced object-recognition deficits in rats are attenuated by hippocampal ablation before or soon after ischemia.

The literature on the role of the hippocampus in object-recognition contains a paradox: Transient forebrain ischemia (ISC) produces hippocampal damage and severe deficits on the delayed nonmatching-to-sample (DNMS) task, yet hippocampal ablation (ABL) produces milder deficits. Experiment 1 confirmed that pretrained rats display severe DNMS deficits following ISC, but not ABL. Ischemia produced loss of CA1 neurons, but no obvious extrahippocampal damage. In Experiments 2 and 3, ISC rats from Experiment 1 received ABL, and ABL rats received ISC; neither treatment affected DNMS performance. In Experiment 4, rats that received ISC followed 1 hr later by ABL displayed only mild deficits. It is hypothesized that ISC-induced DNMS deficits are due to extrahippocampal damage produced by pathogenic processes that involve the hippocampus.

Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.

Correlation of striatal dopamine release and peripheral hypertension after transient ischemia in gerbils.

PURPOSE: Intracranial norepinephrine release has been associated with post-carotid endarterectomy hypertension in human beings. To study this phenomenon under more controlled conditions, we studied the relationship of cerebral catecholamines and blood pressure in gerbils, whose cerebral circulation is similar to that in human beings. METHODS: Twelve anesthetized gerbils underwent iliac artery blood pressure monitoring and in vivo electrochemistry catecholamine monitoring with use of catecholamine-specific electrodes placed stereotactically into the cerebral striatum. Six gerbils underwent 10 minutes of bilateral carotid artery occlusion (ischemic), whereas six underwent carotid artery dissection without occlusion (control). RESULTS: The control group demonstrated a continuous gradual decline in blood pressure and striatal catecholamine during the 150-minute observation period. In contrast the ischemic gerbils demonstrated a sharp catecholamine rise during ischemia, a marked catecholamine drop shortly after carotid artery unclamping, and then a secondary larger catecholamine release that peaks in 60 minutes and gradually returns to baseline in 120 minutes. The blood pressure closely followed the catecholamine levels, with a sharp 20 mm Hg rise in blood pressure above baseline during carotid artery occlusion, followed by a dramatic 10 mm Hg drop below baseline after carotid artery unclamping and then a gradual rise of the blood pressure 25 mm Hg above baseline, which peaks in 80 minutes, with a gradual decline to the same blood pressure as in the control subjects 120 minutes after ischemia. CONCLUSION: We conclude that striatal catecholamine release correlates with peripheral blood pressure during transient cerebral ischemia and reperfusion. This phenomenon may explain the mechanism of post-carotid endarterectomy hypertension in human beings, and this gerbil model can be used to study its prevention and treatment.

Targets of nitric oxide in a mouse model of liver inflammation by Corynebacterium parvum.

Treatment of mice with Corynebacterium parvum induces chronic inflammation. This treatment followed by an injection of lipopolysaccharide (LPS) produces hepatic necrosis and death. We examined liver tissue by using electron paramagnetic resonance (EPR) spectroscopy and found that, in addition to the previously reported nonheme nitrosyl complexes, heme nitrosyl complexes were also formed. Hemoglobin nitrosyl complexes measured in the whole blood of mice treated with C. parvum were not increased after additional LPS treatment. However, this treatment significantly increased the heme nitrosyl complexes in the liver, whereas the nonheme nitrosyl complex concentration was unaffected. EPR signals from whole blood and liver tissues from mice treated with C. parvum and C. parvum + LPS were inhibited by prolonged treatment with NG-monomethyl-L-arginine (L-NMA). Nitric oxide (.NO) is known to bind to cytochrome P450 heme, and we consistently found a suppression of EPR signals attributable to ferric low-spin cytochrome P450/P420 peaks in the livers of mice treated with C. parvum and C. parvum + LPS. By performing analyses of EPR spectra obtained from hepatocytes exposed to .NO, we were able to unambiguously identify EPR signals attributable to cytochrome P420 and nonheme nitrosyl complexes in the livers of both treatments. Deconvolution of the composite in vivo EPR spectra indicated that hemoglobin nitrosyl complexes contributed weakly in the C. parvum livers, but threefold more in the C. parvum + LPS livers, suggesting that hemorrhage may have occurred. Experiments with L-NMA treatment revealed that this additional .NO production did not correlate with hepatic necrosis and onset of death. Immunoprecipitation of liver cytosols from C. parvum- and (C. parvum + LPS)-treated mice using an antibody against mouse inducible nitric oxide synthase showed that this enzyme was indeed present in the cytosolic fractions and was absent in those from control livers. Our novel detection of cytochrome P420 nitrosyl complex in vivo may be linked to any role of hepatic P450's functions during liver inflammation.