Acute mesenteric ischemia following lancehead snakebite: an unusual case report in the Northernmost Brazilian Amazon.

Snakebites have a great impact in the Brazilian Amazon, being the lancehead Bothrops atrox the species responsible for most accidents, disabilities, and deaths. This study shows a case report of an indigenous patient from the Yanomami ethnicity, male, 33 years-old, envenomed by a B. atrox snake. Envenoming caused by B. atrox are characterized by local manifestations (e.g., pain and edema) and systemic manifestations, mainly coagulation disorders. The indigenous victim was admitted in the main hospital of Roraima and evolved with an unusual complication, an ischemia and necrosis of the proximal ileum, requiring segmental enterectomy with posterior side-to-side anastomosis. The victim was discharge after 27 days of hospitalization with no complaints. Snakebite envenomations may evolve with life-threatening complications, which can be treated by the antivenom following access to a healthcare unit, often late in indigenous population. This clinical case shows the need of strategies that aim improvement in the access to the healthcare by indigenous people, as well as demonstrates an unusual complication that may result from lancehead snakebites. The article also discusses the decentralization of snakebites clinical management to indigenous community healthcare centers to mitigate complications.

DOC export is exceeded by C fixation in May Creek: A late-successional watershed of the Copper River Basin, Alaska.

Understanding the entirety of basin-scale C cycling (DOC fluxes and CO2 exchanges) are central to a holistic perspective of boreal forest biogeochemistry today. Shifts in the timing and magnitude of dissolved organic carbon (DOC) delivery in streams and eventually into oceans can be expected, while simultaneously CO2 emission may exceed CO2 fixation, leading to forests becoming stronger CO2 sources than sinks amplifying rising trace gases in the atmosphere. At May Creek, a representative late-successional boreal forest watershed at the headwaters of the Copper River Basin, Alaska, we quantified the seasonality of DOC flux and landscape-scale CO2 exchange (eddy covariance) over two seasonal cycles. We deployed in situ fDOM and conductivity sensors, performed campaign sampling for water quality (DOC and water isotopes), and used fluorescence spectroscopy to ascertain DOC character. Simultaneously, we quantified net CO2 exchange using a 100 ft eddy covariance tower. Results indicate DOC exports were pulse-driven and mediated by precipitation events. Both frequency and magnitude of pulse-driven DOC events diminished as the seasonal thaw depth deepened, with inputs from terrestrial sources becoming major contributors to the DOC pool with decreasing snowmelt contribution to the hydrograph. A three-component parallel factorial analysis (PARAFAC) model indicated DOC liberated in late-season may be bioavailable (tyrosine-like). Combining Net Ecosystem Exchange (NEE) measurements indicate that the May Creek watershed fixes 142-220 g C m-2 yr-1 and only 0.40-0.57 g C m-2 yr-1 is leached out as DOC. Thus, the May Creek watershed and similar mature spruce forest dominated watersheds in the Copper River Basin are currently large ecosystem C sinks and exceeding C conservative. An understanding of DOC fluxes from Gulf of Alaska watersheds is important for characterizing future climate change-induced seasonal shifts.

Effects of microbial phytase on mucin synthesis, gastric protein hydrolysis, and degradation of phytate along the gastrointestinal tract of growing pigs.

An experiment was conducted to test the hypothesis that pigs fed diets supplemented with exogenous phytase reduce mucin synthesis in the small intestine, increase protein hydrolysis in the stomach, increase breakdown of phytate along the gastrointestinal tract, and increase mineral and AA digestibility. A diet based on corn, soybean meal, and canola meal was formulated to meet requirements for growing pigs except for Ca and P, which were lower than requirements. Three additional diets were formulated by adding 750, 1,500, or 3,000 units of phytase (FTU) per kilogram to the basal diet. Eight growing barrows (38.45 ± 3.06 kg) were prepared with a T-cannula in the duodenum and another T-cannula in the distal ileum. Pigs were housed individually and allotted to a replicated 4 × 4 Latin square design with four pigs and four periods in each square. Each period lasted 14 d with the initial 7 d being the adaptation period to the diets. Pigs were fed twice daily in combined amounts equal to 3.2 times the estimated requirement for maintenance energy. Results indicated that the apparent ileal digestibility (AID) and the apparent total tract digestibility (ATTD) of Ca and P increased (linear and quadratic, P ≤ 0.05) as phytase inclusion increased. However, values for AID of Ca and P were not different from values for ATTD of Ca and P, indicating that there is no net absorption of Ca and P in the hindgut. The apparent duodenal digestibility (ADD) of Ca and P was ~30% and 10% to 20%, respectively, indicating some digestion in the stomach of both Ca and P. A quadratic increase (P < 0.05) of the AID of GE was observed with the breakpoint around 1,500 FTU, but there was a negative linear (P ≤ 0.001) effect of dietary phytase on the ATTD of GE. Phytase did not affect mucin synthesis in the small intestine, protein hydrolysis in the stomach, or ileal digestibility of dispensable and indispensable AA. However, degradation of higher phytate esters (IP6 and IP5) into lower phytate esters (IP4 and IP3) and inositol increased as dietary phytase increased, indicating that it is possible to completely degrade dietary phytate if microbial phytase is included by at least 3,000 FTU in the diet. In conclusion, supplementing diets with phytase resulted in increased degradation of phytate and phytate esters and improved digestibility of Ca and P, but phytase did not change intestinal mucin synthesis, gastric protein hydrolysis, or the AID of AA.

Sex-based differential regulation of oxidative stress in the vasculature by nitric oxide.

BACKGROUND: Nitric oxide ((•)NO) is more effective at inhibiting neointimal hyperplasia following arterial injury in male versus female rodents, though the etiology is unclear. Given that superoxide (O2(•-)) regulates cellular proliferation, and (•)NO regulates superoxide dismutase-1 (SOD-1) in the vasculature, we hypothesized that (•)NO differentially regulates SOD-1 based on sex. MATERIALS AND METHODS: Male and female vascular smooth muscle cells (VSMC) were harvested from the aortae of Sprague-Dawley rats. O2(•-) levels were quantified by electron paramagnetic resonance (EPR) and HPLC. sod-1 gene expression was assayed by qPCR. SOD-1, SOD-2, and catalase protein levels were detected by Western blot. SOD-1 activity was measured via colorimetric assay. The rat carotid artery injury model was performed on Sprague-Dawley rats ±(•)NO treatment and SOD-1 protein levels were examined by Western blot. RESULTS: In vitro, male VSMC have higher O2(•-) levels and lower SOD - 1 activity at baseline compared to female VSMC (P < 0.05). (•)NO decreased O2(•-) levels and increased SOD - 1 activity in male (P<0.05) but not female VSMC. (•)NO also increased sod- 1 gene expression and SOD - 1 protein levels in male (P<0.05) but not female VSMC. In vivo, SOD-1 levels were 3.7-fold higher in female versus male carotid arteries at baseline. After injury, SOD-1 levels decreased in both sexes, but (•)NO increased SOD-1 levels 3-fold above controls in males, but returned to baseline in females. CONCLUSIONS: Our results provide evidence that regulation of the redox environment at baseline and following exposure to (•)NO is sex-dependent in the vasculature. These data suggest that sex-based differential redox regulation may be one mechanism by which (•)NO is more effective at inhibiting neointimal hyperplasia in male versus female rodents.

Efficacy of the investigational mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute lymphoblastic leukemia.

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anticancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here, we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib (DA) in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of DA. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

Fetal exposure to propoxur and abnormal child neurodevelopment at 2 years of age.

OBJECTIVE: Our aim was to determine the effects of fetal exposure to propoxur and pyrethroids, on child neurodevelopment at 2 years of age. PATIENTS AND METHODS: Mothers were prospectively recruited during mid-pregnancy in Bulacan, Philippines where multiple pesticides including propoxur, cyfluthrin, chlorpyrifos, cypermethrin, pretilachlor, bioallethrin, malathion, diazinon and transfluthrin are used. To detect prenatal exposure to these pesticides, maternal hair and blood, infant's hair, cord blood, and meconium were analyzed for the pesticides by gas chromatography/mass spectrometry. Infants were examined at 2 years of age with 95.1% follow up rate and their neurodevelopment outcome was assessed by the Griffiths mental developmental scale (N=754). RESULTS: Meconium analysis was the most sensitive method to detect fetal exposure to pesticides and exposure was highest for propoxur (21.3%) and the grouped pyrethroids (2.5% - bioallethrin, transfluthrin, cyfluthrin and cypermethrin). Path analysis modeling was performed to determine the effects of fetal exposure to propoxur and pyrethroids on the child's neurodevelopment at 24 months of age while controlling for confounders. Only singletons and those with complete data for the path analysis were included (N=696). Using a path analysis model, there was a significant negative (ß=-0.14, p<0.001) relationship between prenatal pesticide exposure to propoxur and motor development at 2 years of age after controlling for confounders, e.g., infant gender, socioeconomic status, maternal intelligence, home stimulation (HOME), postnatal exposure to propoxur and blood lead level at 2 years of age. CONCLUSION: At 2 years of age, prenatal exposure to propoxur was associated with poorer motor development in children.

Combined analysis of prenatal (maternal hair and blood) and neonatal (infant hair, cord blood and meconium) matrices to detect fetal exposure to environmental pesticides.

OBJECTIVE: The aim of this study was to determine optimum biomarkers to detect fetal exposure to environmental pesticides by the simultaneous analysis of maternal (hair and blood) and infant (cord blood, infant hair or meconium) matrices and to determine if a combination of these biomarkers will further increase the detection rate. PATIENTS AND METHODS: Pregnant women were prospectively recruited from an agricultural site in the Philippines with substantial use at home and in the farm of the following pesticides: propoxur, cyfluthrin, chlorpyrifos, cypermethrin, pretilachlor, bioallethrin, malathion, diazinon and transfluthrin. Maternal hair and blood were obtained at midgestation and at delivery and infant hair, cord blood and meconium were obtained after birth. All samples were analyzed by gas chromatography/mass spectrometry (GC/MS) for the above pesticides and some of their metabolites. RESULTS: A total of 598 mother/infant dyads were included in this report. The highest rates of pesticide exposure were detected in meconium (23.2% to propoxur, 2.0% to pretilachlor, 1.7% to cypermethrin, 0.8% to cyfluthrin, 0.7% to 1,1,1-trichloro-2,2-bis, p-chlorophenylethane (DDT) and 0.3% to malathion and bioallethrin) and in maternal hair (21.6% to propoxur, 14.5% to bioallethrin, 1.3% to malathion, 0.8% to DDT, 0.3% to chlorpyrifos and 0.2% to pretilachlor). Combined analysis of maternal hair and meconium increased detection rate further to 38.5% for propoxur and to 16.7% for pyrethroids. Pesticide metabolites were rarely found in any of the analyzed matrices. CONCLUSIONS: There is significant exposure of the pregnant woman and her fetus to pesticides, particularly to the home pesticides, propoxur and pyrethroids. Analysis of meconium for pesticides was the single most sensitive measure of exposure. However, combined analysis of maternal hair and meconium significantly increased the detection rate. A major advantage of analyzing maternal hair is that prenatal pesticide exposure in the mother can be detected and intervention measures can be initiated to minimize further exposure of the fetus to pesticides.

Decision-making of Chinese caregivers for adult out-of-home placement.

BACKGROUND: In the light of the existence of lengthy waiting lists for out-of-home placements, this study examines the factors affecting the decision to apply or not to apply for this facility in a city traditionally characterized by a cultural and policy emphasis on family responsibility and by relatively low levels of welfare commitment to residential services. METHOD: A sample of 321 people responsible for providing care for adults with moderate to severe intellectual disability (ID) from 22 service units of seven non-governmental organizations in Hong Kong was surveyed by means of a structured questionnaire. RESULTS: The non-application group reported better emotional ties with the people with ID and greater confidence in their caregiving skills, but also tended to be caring for more challenging people with poorer health and higher or more frequent levels of self-harm behaviour than the application group who exhibited higher levels of worry and fear. Discriminant analysis successfully predicted 80% of non-application cases, while logistic regression revealed that decline in perceived competence to care, absence of other health problems and at least one parent of the client having long-term illness were better predictors of the decision to apply than handicap-specific characteristics of the people with ID themselves. CONCLUSION: The implications of this finding are discussed, and consideration given to the possibility of developing policies designed to strengthen and treasure family values while not detracting from the importance of providing proper community support.

A selective inhibitor of the p110delta isoform of PI 3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic effects of VP16.

Current therapy for acute myeloid leukaemia (AML) is suboptimal with a high incidence of relapse. There is strong evidence that constitutive phosphoinositide 3-kinase (PI3K) activity plays a significant role in the pathophysiology of AML. PI3K products are derived from the activity of a number of PI3K catalytic isoforms (class I, II and III) but the relative contribution of these enzymes in AML remains unknown. As non-isoform-selective inhibitors of PI3K such as LY294002 may produce unwanted toxicity to normal tissues, we have investigated the role of the leukocyte-restricted p110delta PI3K isoform in 14 cases of AML. p110delta was detected in all cases whereas the expression levels of the other class I PI3Ks varied more widely, and were often undetectable. The p110delta-selective compound IC87114 inhibited constitutive phosphorylation of the PI3K target Akt/PKB and reduced cell number to a mean of 66+/-5% (range 14-88%). In eight cases, the combination of IC87114 and VP16 (a topoisomerase II inhibitor) was synergistic in reducing viable cell number, and was associated with a reduction in constitutive NF-kappaB activity. IC87114 did not have direct adverse effects or enhance the activity of VP16 on the proliferation and survival of normal haemopoietic progenitors. Overall, our results identify the p110delta isoform as a potential therapeutic target in AML and support a clinical approach to use isoform-selective over broad-spectrum PI3K inhibitors.

Chemokine inhibition--why, when, where, which and how?

Chemokines are small chemoattractant cytokines that control a wide variety of biological and pathological processes, ranging from immunosurveillance to inflammation, and from viral infection to cancer. Genetic and pharmacological studies have shown that chemokines are responsible for the excessive recruitment of leucocytes to inflammatory sites and damaged tissue. In the present paper, we discuss the rationale behind interfering with the chemokine system and introduce various points for therapeutic intervention using either protein-based or small-molecule inhibitors. Unlike other cytokines, chemokines signal via seven-transmembrane GPCRs (G-protein-coupled receptors), which are favoured targets by the pharmaceutical industry, and, as such, they are the first cytokines for which small-molecule-receptor antagonists have been developed. In addition to the high-affinity receptor interaction, chemokines have an in vivo requirement to bind to GAGs (glycosaminoglycans) in order to mediate directional cell migration. Prevention of the GAG interaction has been shown to be a viable therapeutic strategy. Targeting chemokine intracellular signalling pathways offers an alternative small-molecule approach. One of the key signalling targets downstream of a variety of chemokine receptors identified to date is PI3Kgamma (phosphoinositide 3-kinase gamma), a member of the class I PI3K family. Thus the chemokine system offers many potential entry points for innovative anti-inflammatory therapies for autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis and allergic contact dermatitis.

Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways.

Skeletal muscle is composed of multinucleated fibres, formed after the differentiation and fusion of myoblast precursors. Skeletal muscle atrophy and hypertrophy refer to changes in the diameter of these pre-existing muscle fibres. The prevention of atrophy would provide an obvious clinical benefit; insulin-like growth factor 1 (IGF-1) is a promising anti-atrophy agent because of its ability to promote hypertrophy. However, the signalling pathways by which IGF-1 promotes hypertrophy remain unclear, with roles suggested for both the calcineurin/NFAT (nuclear factor of activated T cells) pathway and the PtdIns-3-OH kinase (PI(3)K)/Akt pathway. Here we employ a battery of approaches to examine these pathways during the hypertrophic response of cultured myotubes to IGF-1. We report that Akt promotes hypertrophy by activating downstream signalling pathways previously implicated in activating protein synthesis: the pathways downstream of mammalian target of rapamycin (mTOR) and the pathway activated by phosphorylating and thereby inhibiting glycogen synthase kinase 3 (GSK3). In contrast, in addition to demonstrating that calcineurin does not mediate IGF-1-induced hypertrophy, we show that IGF-1 unexpectedly acts via Akt to antagonize calcineurin signalling during myotube hypertrophy.

Ras controls growth, survival and differentiation in the Drosophila eye by different thresholds of MAP kinase activity.

Ras mediates a plethora of cellular functions during development. In the developing eye of Drosophila, Ras performs three temporally separate functions. In dividing cells, it is required for growth but is not essential for cell cycle progression. In postmitotic cells, it promotes survival and subsequent differentiation of ommatidial cells. In the present paper, we have analyzed the different roles of Ras during eye development by using molecularly defined complete and partial loss-of-function mutations of Ras. We show that the three different functions of Ras are mediated by distinct thresholds of MAPK activity. Low MAPK activity prolongs cell survival and permits differentiation of R8 photoreceptor cells while high or persistent MAPK activity is sufficient to precociously induce R1-R7 photoreceptor differentiation in dividing cells.

Retroviral gene transfer of dominant negative raf-1 mutants suppresses ha-ras-induced transformation and delays tumor formation.

Activating mutants of ras are among the most frequently found genetic alterations in human cancers. Therefore, Ras appears to be an attractive target for therapeutic intervention using gene transfer. The protein kinase Raf-1 acts as a direct downstream effector of Ras and is involved in Ras-induced cellular transformation. Using the NIH3T3 fibroblast-derived tumor cell line PEJ, which expresses oncogenic Ha-rasG12V, we analyzed whether dominant negative mutants of Raf-1 can inhibit Ras-mediated transformation. Retroviral gene transfer was used to stably transduce PEJ cells with three different dominant negative mutants of Raf-1. This resulted in reversion of the transformed phenotype in vitro as evidenced by an increase in contact inhibition and reduced anchorage-independent growth. However, tumor formation in nude mice was significantly delayed only by one of these mutants. Therefore, dominant negative mutants of the oncoprotein Myc, which is known to synergize with Raf-1 in tumor formation, were transduced into PEJ cells expressing a dominant negative Raf mutant. This leads to killing of the cells. These results indicate that although interference with Ras-induced transformation using dominant negative mutants of Raf is feasible and effective in vitro using retroviral vectors, an additional block (e.g., that of Myc) is necessary to kill PEJ cells. These results also indicate that interference with Ras-dependent signaling is not sufficient for inhibition of tumor formation of PEJ cells in vivo.

Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt.

Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is phosphatidylinositol 3-kinase). However, these two signaling pathways were shown to exert opposing effects on muscle cell hypertrophy. Furthermore, the PI3K-Akt pathway was shown to inhibit the Raf-MEK-ERK pathway; this cross-regulation depended on the differentiation state of the cell: Akt activation inhibited the Raf-MEK-ERK pathway in differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlated with its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex.

Psychophysical measures of sensitization to tonic heat discriminate chronic pain patients.

Sensitization to continued nociceptive stimulation is supposed to be involved in the development of chronic pain at several levels of the CNS, but experimental studies investigating the perceptual dynamics of sensitization in humans are rare, and the diagnostic validity of experimental pain models is not known. The present study used a tonic heat paradigm to assess early sensitization (15-100 s) to experimental pain in 30 chronic pain patients (15 musculoskeletal/back pain, 15 headache) and 23 healthy controls. Change in pain sensation during prolonged stimulation was measured by a dual sensitization method which combines subjective ratings and behavioural responses in an indirect psychophysical protocol protected against response bias. Phasic and tonic pain thresholds were measured for control purposes. The degree of sensitization was linearly related to stimulus temperature, and groups differed significantly in this 'sensitization gradient': chronic pain patients sensitized earlier and stronger than healthy subjects, musculoskeletal pain patients showed the strongest effect. Pain thresholds were lowered in headache patients only. Discriminant analysis demonstrated good sensitivity and specificity of individual sensitization measures for distinguishing pain syndromes, particularly in combination with pain thresholds. The results are in accordance with current models of spinal plasticity contributing to pathological pain states. They argue for the diagnostic value of psychophysical measures of sensitization.

Ras--a versatile cellular switch.

With the number of known roles played by Ras proteins increasing rapidly, finding answers to how the diverse cellular responses are triggered is becoming increasingly pertinent. Although our understanding of the control of specificity of signal transduction is still small, the combination of biochemical, structural and genetic analyses is starting to reveal how the cell-specific responses to Ras activation are controlled.

14-3-3 is phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in vivo regulates Raf/14-3-3 interaction.

14-3-3 proteins mediate interactions between proteins involved in signal transduction and cell cycle regulation. Phosphorylation of target proteins as well as 14-3-3 are important for protein-protein interactions. Here, we describe the purification of a protein kinase from porcine brain that phosphorylates 14-3-3 zeta on Thr-233. This protein kinase has been identified as casein kinase Ialpha (CKIalpha) by peptide mapping analysis and sequencing. Among mammalian 14-3-3, only 14-3-3 tau possesses a phosphorylatable residue at the same position (Ser-233), and we show that this residue is also phosphorylated by CKI. In addition, we show that 14-3-3 zeta is exclusively phosphorylated on Thr-233 in human embryonic kidney 293 cells. The residue 233 is located within a region shown to be important for the association of 14-3-3 to target proteins. We showed previously that, in 293 cells, only the unphosphorylated form of 14-3-3 zeta associates with the regulatory domain of c-Raf. We have now shown that in vivo phosphorylation of 14-3-3 zeta at the CKIalpha site (Thr-233) negatively regulates its binding to c-Raf, and may be important in Raf-mediated signal transduction.

MEK1 mediates a positive feedback on Raf-1 activity independently of Ras and Src.

Growth factor stimulated receptor tyrosine kinases activate a protein kinase cascade via the serine/threonine protein kinase Raf-1. Direct upstream activators of Raf-1 are Ras and Src. This study shows that MEK1, the direct downstream effector of Raf-1, can also stimulate Raf-1 kinase activity by a positive feedback loop. Activated MEK1 mediates hyperphosphorylation of the amino terminal regulatory as well as of the carboxy terminal catalytic domain of Raf-1. The hyperphosphorylation of Raf-1 correlates with a change in the tryptic phosphopeptide pattern only at the carboxy terminus of Raf-1 and an increase in Raf-1 kinase activity. MEK1-mediated Raf-1 activation is inhibited by co-expression of the MAPK specific phosphatase MKP-1 indicating that the MEK1 effect is exerted through a MAPK dependent pathway. Stimulation of Raf-1 activity by MEK1 is independent of Ras, Src and tyrosine phosphorylation of Raf-1. MEK1 can however synergize with Ras and leads to further increase of the Raf-1 kinase activity. Thus, MEK1 can mediate activation of Raf-1 by a novel positive feedback mechanism which allows fast signal amplification and could prolong activation of Raf-1.

Negative regulation of Raf activity by binding of 14-3-3 to the amino terminus of Raf in vivo.

In the developing eye of Drosophila the protein kinase D-Raf controls the specification of the R7 photoreceptor cells. We show that overexpression of wild-type D-Raf inhibits the formation of R7 cells in a dose-dependent manner. Conversely, overexpression of mutant D-Raf proteins in which the conserved S388 is replaced by A or by D promotes the formation of supernumerary R7 cells, indicating increased D-Raf activity in vivo. S388 in D-Raf corresponds to S259 in c-Raf; shown to be involved in binding of 14-3-3. We show that analogous substitutions of S259 in c-Raf prevent binding of 14-3-3 zeta to the amino terminus of c-Raf and cause a Ras-independent constitutively increased c-Raf kinase activity. Binding of 14-3-3 zeta to the second binding site at the carboxy terminal catalytic domain was unaffected by these mutations. These results suggest that the increased kinase activity of mutant D-Raf is caused by the selective loss of 14-3-3 binding to its amino terminus. Therefore, binding of 14-3-3 to the amino terminus of Raf appears to negatively regulate Raf kinase activity in vivo.