Plasma and pulmonary disposition of ceftiofur and its metabolites after intramuscular administration of ceftiofur crystalline free acid in weanling foals.

The objectives of this study were to determine the plasma and pulmonary disposition of ceftiofur crystalline free acid (CCFA) in weanling foals and to compare the plasma pharmacokinetic profile of weanling foals to that of adult horses. A single dose of CCFA was administered intramuscularly to six weanling foals and six adult horses at a dose of 6.6 mg/kg of body weight. Concentrations of desfuroylceftiofur acetamide (DCA) were determined in the plasma of all animals, and in pulmonary epithelial lining fluid (PELF) and bronchoalveolar lavage (BAL) cells of foals. After intramuscular (IM) administration to foals, median time to maximum plasma and PELF concentrations was 24 h (12-48 h). Mean (± SD) peak DCA concentration in plasma (1.44 ± 0.46 µg/mL) was significantly higher than that in PELF (0.46 ± 0.03 µg/mL) and BAL cells (0.024 ± 0.011 µg/mL). Time above the therapeutic target of 0.2 µg/mL was significantly longer in plasma (185 ± 20 h) than in PELF (107 ± 31 h). The concentration of DCA in BAL cells did not reach the therapeutic level. Adult horses had significantly lower peak plasma concentrations and area under the curve compared to foals. Based on the results of this study, CCFA administered IM at 6.6 mg/kg in weanling foals provided plasma and PELF concentrations above the therapeutic target of 0.2 µg/mL for at least 4 days and would be expected to be an effective treatment for pneumonia caused by Streptococcus equi subsp. zooepidemicus at doses similar to the adult label.

Plasma pharmacokinetics, pulmonary distribution, and in vitro activity of gamithromycin in foals.

The objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) and Rhodococcus equi. A single dose of gamithromycin (6 mg/kg of body weight) was administered intramuscularly. Concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils were determined using HPLC with tandem mass spectrometry detection. The minimum inhibitory concentration of gamithromycin required for growth inhibition of 90% of R. equi and S. zooepidemicus isolates (MIC(90)) was determined. Additionally, the activity of gamithromycin against intracellular R. equi was measured. Mean peak gamithromycin concentrations were significantly higher in blood neutrophils (8.35±1.77 µg/mL) and BAL cells (8.91±1.65 µg/mL) compared with PELF (2.15±2.78 µg/mL) and plasma (0.33±0.12 µg/mL). Mean terminal half-lives in neutrophils (78.6 h), BAL cells (70.3 h), and PELF (63.6 h) were significantly longer than those in plasma (39.1 h). The MIC(90) for S. zooepidemicus isolates was 0.125 µg/mL. The MIC of gamithromycin for macrolide-resistant R. equi isolates (MIC(90)=128 µg/mL) was significantly higher than that for macrolide-susceptible isolates (1.0 µg/mL). The activity of gamithromycin against intracellular R. equi was similar to that of azithromycin and erythromycin. Intramuscular administration of gamithromycin at a dosage of 6 mg/kg would maintain PELF concentrations above the MIC(90) for S. zooepidemicus and phagocytic cell concentrations above the MIC(90) for R. equi for approximately 7 days.

Effect of Propionibacterium acnes-containing immunostimulant on interferon-gamma (IFNγ) production in the neonatal foal.

Production of the Th1 cytokine interferon gamma (IFNγ) is associated with resistance to intracellular pathogens, including Rhodococcus equi. While neonatal foals are initially deficient in IFNγ production, expression of this cytokine increases throughout their first year of life. This is presumably the result of stimulation by environmental antigens including pathogen associated molecular patterns (PAMPS) signaling through toll-like receptors (TLR). This increased expression of IFNγ is likewise associated with an age-related resistance to R. equi infection. While immunostimulants containing PAMPS have been administered to adult horses in an attempt to modify their immune response, the effect of these materials on IFNγ expression in foals is unknown. The main objective of this study was to determine the effect of administering a commercial immunomostimulant EqStim® (Propionibacterium acnes) on IFNγ production measured using intracellular staining (IFNγ) and RT-PCR.

Influenza A viruses with truncated NS1 as modified live virus vaccines: pilot studies of safety and efficacy in horses.

REASONS FOR PERFORMING STUDY: Three previously described NS1 mutant equine influenza viruses encoding carboxy-terminally truncated NS1 proteins are impaired in their ability to inhibit type I IFN production in vitro and are replication attenuated, and thus are candidates for use as a modified live influenza virus vaccine in the horse. HYPOTHESIS: One or more of these mutant viruses is safe when administered to horses, and recipient horses when challenged with wild-type influenza have reduced physiological and virological correlates of disease. METHODS: Vaccination and challenge studies were done in horses, with measurement of pyrexia, clinical signs, virus shedding and systemic proinflammatory cytokines. RESULTS: Aerosol or intranasal inoculation of horses with the viruses produced no adverse effects. Seronegative horses inoculated with the NS1-73 and NS1-126 viruses, but not the NS1-99 virus, shed detectable virus and generated significant levels of antibodies. Following challenge with wild-type influenza, horses vaccinated with NS1-126 virus did not develop fever (>38.5 degrees C), had significantly fewer clinical signs of illness and significantly reduced quantities of virus excreted for a shorter duration post challenge compared to unvaccinated controls. Mean levels of proinflammatory cytokines IL-1beta and IL-6 were significantly higher in control animals, and were positively correlated with peak viral shedding and pyrexia on Day +2 post challenge. CONCLUSION AND CLINICAL RELEVANCE: These data suggest that the recombinant NS1 viruses are safe and effective as modified live virus vaccines against equine influenza. This type of reverse genetics-based vaccine can be easily updated by exchanging viral surface antigens to combat the problem of antigenic drift in influenza viruses.

In vitro and in vivo modulation of the equine immune response by parapoxvirus ovis.

REASON FOR PERFORMING STUDY: While immune modulators are used routinely in equine medicine, their mechanism of action is not always known. OBJECTIVES: To determine the effect of a commercial preparation of inactivated parapoxvirus ovis (Orf virus; PPVO) on cytokine gene expression by equine peripheral blood mononuclear cells (PBMC) both in vitro and in vivo. METHODS: PBMC were prepared from 6 mixed-breed yearlings and cultured in vitro with PPVO with or without Concanavalin A (Con A) for 24 h. Effects on the expression of IFNalpha, IFNbeta IFNgamma, TNFalpha and IL-18 were analysed by real time quantitative PCR (RT-PCR). In addition, 12 yearling horses were treated with PPVO and whole blood RNA samples were prepared at regular intervals to assess effects on in vivo cytokine gene expression. Six of those yearlings were later treated with saline and served as treatment controls. Nine additional yearlings were injected intradermally with a single dose and their injection sites biopsied at 24 and 48 h for cytokine expression. RESULTS: In vitro culture of PBMC with PPVO led to a significant increase in IFNalpha and IFNbeta gene expression compared to mock-stimulated cultures. In addition, expression of IFNgamma and TNFalpha was significantly higher in PBMC stimulated with PPVO and Con A, than those stimulated with Con A alone. No changes were observed in IL-18 gene expression in vitro. Treatment of horses with a 3-dose regimen of PPVO resulted in elevation of IFNgamma gene expression, which was detected 24 h after the first dose and declined thereafter. Intradermal inoculation led to increased expression of IFNgamma along with IFNbeta, IL-15 and IL-18. CONCLUSIONS: Together these results indicate that PPVO stimulated IFNgamma production both in vitro and in vivo. Increased cytokine expression could account for its immunomodulatory activity. POTENTIAL RELEVANCE: The absence of adverse reactions and clear indications of increased expression of cytokine gene expression supports previous clinical uses for this immune modulator in those situations when increased expression of IFNgamma is warranted.

Anatomy of a malpractice lawsuit.

Given the prevalence of medical malpractice lawsuits, physicians are often thrust into the legal world without the education of a juris doctor. The risk of facing suit varies among specialties, but there is no guarantee any physician will proceed through his or her career without being a defendant in a lawsuit. Every physician stands a significant chance of being sued. Although a lawsuit is an exhausting and intimidating situation in and of itself, the ramifications of a plaintiff's verdict could have chilling effects on the physician's life, both professionally and personally. Therefore, it is imperative that every physician have an understanding of the legal process of which he or she may become involved. This article provides a practical guide for the physician, including the fundamental procedures of a medical malpractice lawsuit, the behavior that will be expected or required of the defendant physician, and the effect of disobeying the required procedures.

Phorbol 12-myristate 13-acetate and serum synergize to promote rapamycin-insensitive cell proliferation via protein kinase C-eta.

Previously, we have shown that PKC-eta (protein kinase C-eta) positively regulates glioblastoma proliferation and confers resistance to irradiation-induced apoptosis. In this study, we investigated the efficacy of rapamycin in inhibiting cell proliferation in two glioblastoma cell lines U-251MG (PKC-eta expressing) and U-1242MG (PKC-eta deficient) following PKC-eta activation. In U-251MG cells, rapamycin (10 nM) treatment was less effective as an antiproliferative agent when cells were concurrently stimulated with 10% serum and phorbol 12-myristate 13-acetate (PMA, 100 nM), a potent activator of PKC isozymes. Rapamycin-insensitive growth was owing to PKC-eta, as U-1242MG and U-251MG cells infected with a kinase-dead form of PKC-eta (U-251kr) were susceptible to rapamycin-induced inhibition of cell proliferation. Furthermore, U-251MG cells transfected with PKC-eta antisense oligonucleotides were sensitive to rapamycin. PKC-eta-expressing cells stimulated with PMA maintained p70S6K phosphorylation on Thr389 and phosphorylation of rpS6 (ser235/36), suggesting p70S6K kinase activity was still intact. Inhibition of p70S6K expression with small interfering RNA oligonucleotides inhibited cell proliferation greater than 50% in the presence of a combination of PMA and serum. Additionally, p70S6K co-precipitated with PKC-eta, suggesting a physical interaction between PKC-eta and p70S6K regulates the observed phosphorylation. Taken together, these data demonstrate that rapamycin-insensitive glioblastoma proliferation involves PKC-eta signaling.

Analyses of the effects of Rck2p mutants on Pbs2pDD-induced toxicity in Saccharomyces cerevisiae identify a MAP kinase docking motif, and unexpected functional inactivation due to acidic substitution of T379.

Rck2p is a Ser/Thr kinase that binds to, and is activated by, Hog1p. Expression of the MAP kinase kinase Pbs2pDD from a GAL1-driven plasmid hyperactivates the HOG MAP kinase pathway, and leads to cessation of growth. This toxic effect is reduced by deletion of RCK2. We studied the structural and functional basis for the role of Rck2p in mediating the growth arrest phenotype associated with overexpression of Pbs2pDD. Rck2p kinase activity is required for the effect, because Rck2p(Delta487-610), as well as full-length Rck2p, is toxic with Pbs2pDD, but kinase-defective versions of either protein with a K201R mutation are not. Thus, the C-terminal portion of Rck2p is not required provided the protein is activated by removal of the autoinhibitory domain. Relief of inhibition in Rck2p normally requires phosphorylation by Hog1p, and Rck2p contains a putative MAP kinase docking site (TILQR589R590KKVQ) in its C-terminal segment. The Rck2p double mutant R589A/R590A expressed from a centromeric plasmid did not detectably bind Hog1p-GFP and was functionally inactive in mediating the toxic effect of Pbs2pDD, equivalent to an RCK2 deletion. However, overexpression of Rck2p R589A/R590A from a multicopy plasmid restored function. In contrast, RCK2-K201R acted as a multicopy suppressor of PBS2DD, markedly reducing its toxicity. This suppressor activity required the K201R mutation, and the effect was largely lost when the docking site was mutated, suggesting suppression by inhibition of Hog1p functions. We also studied the effect of replacing the predicted T379 and established S520 phosphorylation sites in Rck2p by glutamic acid. Surprisingly, the T379E mutant markedly reduced Pbs2pDD toxicity, and toxicity was only partially rescued by S520E. Rck2 T379E was sufficiently inactive in an rck2Delta strain to allow some cells to survive PBS2DD toxicity even when overexpressed. The significance of these findings for our understanding of Rck2p function is discussed.

ERK2- and p90(Rsk2)-dependent pathways regulate the CCAAT/enhancer-binding protein-beta interaction with serum response factor.

The serum response element (SRE) of the c-fos promoter is a convergence point for mitogenic signaling pathways. Several transcription factors regulate SRE, including serum response factor (SRF), ternary complex factors, and CCAAT/enhancer-binding protein-beta (C/EBPbeta). C/EBPbeta can interact with both SRF and the ternary complex factor family member Elk-1, but only in response to activated Ras. Transactivation of the SRE by C/EBPbeta is also greatly stimulated by Ras. The Ras effectors that signal to C/EBPbeta are unknown. In this report, we demonstrate that a consensus MAPK site in C/EBPbeta is necessary for Ras stimulation of both C/EBPbeta-SRF interaction and transactivation of the SRE by C/EBPbeta. To dissect signaling pathways activated downstream of Ras, different Ras effector constructs were analyzed. We show that activated forms of Raf and phosphatidylinositol 3-kinase stimulate C/EBPbeta-SRF interaction. We also show a novel selectivity for the MAPK family member ERK2, where dominant-negative ERK2, but not dominant-negative ERK1, blocks Ras stimulation of C/EBPbeta-SRF interaction. In addition, recombinant C/EBPbeta protein is phosphorylated by ERK2, but not by ERK1, in vitro. Finally, we demonstrate a requirement for p90(Rsk2) in regulation of C/EBPbeta-SRF interaction. These data show that multiple Ras effectors are required to regulate C/EBPbeta and SRF association.

Identification of the S6 kinase activity stimulated in quiescent brine shrimp embryos upon entry to preemergence development as p70 ribosomal protein S6 kinase: isolation of Artemia franciscana p70S6k cDNA.

We previously demonstrated that a protein kinase responsible for phosphorylating 40S ribosomal subunits is activated in quiescent Artemia franciscana embryos within 15 min of restoration of normal tonicity and incubation at 30 degrees C. Here, we identify the activated S6 kinase as A. franciscana p70 ribosomal S6 kinase (p70S6k) subsequent to the isolation of an Artemia p70S6k cDNA. The protein conceptually translated from cDNA has 70% similarity and 64% identity to both Drosophila melanogaster and human p70S6k. Southern blot analysis is consistent with presence of a single p70S6k gene. Two transcripts of 5.4 and 2.7 kb were found. Abundance of both mRNAs increased dramatically around 4 h of preemergence development, and exhibited different steady-state level variation thereafter. Stimulated S6 kinase activity, partially purified by Superose 6 chromatography, correlated best with the slowest migrating, approximately 65 kDa, form detected by Western analysis using a specific polyclonal antibody made to a peptide from the predicted p70S6k NH2-terminus. Furthermore, the A. franciscana p70S6k was immunoprecipitated with the same antibody, showing in parallel an S6 kinase activity similar to peak profiles. We conclude that the stimulated S6 kinase activity is that of an ortholog of human p70S6k that may be involved in the regulation of protein synthesis during preemergence development in A. franciscana species.

A different function for a critical tryptophan in c-Raf and Hck.

The similarity of the catalytic domains of Raf and Src family members suggests that functions of homologous residues may be similar in both kinase families. A tryptophan residue, W260, in the WEI region of the Src family kinase Hck has an important role in regulating ATP binding. We tested the hypothesis that the tryptophan, W342, in the WEI region of c-Raf may have a similar role to the W260 of Hck. Mutation of W260 to A in Hck activates kinase activity, but we found that mutation of W342 to A in c-Raf inactivates the kinase activity. Mutating W342 to aspartate (D), lysine (K) or histidine (H) also inactivated c-Raf whether assayed as a purified immunoprecipitate or when recruited to the plasma membrane. A constitutively active c-Raf can be generated by mutating two regulatory tyrosines to aspartate. When placed into this active c-Raf mutant, mutation of W342 to D, K or H enabled phosphorylation and activation of the c-Raf substrate MEK at the plasma membrane but not in an immunoprecipitation assay. We conclude that (1) Tryptophan has a different role in the WEI regions of c-Raf and Hck, (2) W342 is not directly involved in MEK binding as both positive and negative residues at 342 are permissive for MEK activation at the membrane in a constitutively active c-Raf mutant, (3) Factors at the membrane are capable of potentiating activation of c-Raf containing mutated W342 in a hyperactivated c-Raf, but not in a wild type c-Raf and (4) There is a stringent structural requirement for W at residue 342 in c-Raf.

Creation of a stress-activated p90 ribosomal S6 kinase. The carboxyl-terminal tail of the MAPK-activated protein kinases dictates the signal transduction pathway in which they function.

Mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) lie immediately downstream of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38 MAPK. Although the family of MAPKAPKs shares sequence similarity, it demonstrates selectivity for the upstream activator. Here we demonstrate that each of the ERK- and p38 MAPK-regulated MAPKAPKs contains a MAPK docking site positioned distally to the residue(s) phosphorylated by MAPKs. The isolated MAPK docking sites show specificity for the upstream activator similar to that reported for the full-length proteins. Moreover, replacement of the ERK docking site of p90 ribosomal S6 kinase with the p38 MAPK docking site of MAPKAPK2 converts p90 ribosomal S6 kinase into a stress-activated kinase in vivo. It is apparent that mechanisms controlling events downstream of the proline-directed MAPKs involve specific MAPK docking sites within the carboxyl termini of the MAPKAPKs that determine the cascade in which the MAPKAPK functions.

Interaction among mitochondria, mitogen-activated protein kinases, and nuclear factor-kappaB in cellular models of Parkinson's disease.

Oxidative stress induced by acute complex I inhibition with 1-methyl-4-phenylpyridinium ion activated biphasically the stress-activated c-Jun N-terminal kinase (JNK) and the early transcription factor nuclear factor-kappaB (NF-kappaB) in SH-SY5Y neuroblastoma cells. Early JNK activation was dependent on mitochondrial adenine nucleotide translocator (ANT) activity, whereas late-phase JNK activation and the cleavage of signaling proteins Raf-1 and mitogen-activated protein kinase (MAPK) kinase (MEK) kinase (MEKK)-1 appeared to be ANT-independent. Early NF-kappaB activation depended on MEK, later activation required an intact electron transport chain (ETC), and Parkinson's disease (PD) cybrid (mitochondrial transgenic cytoplasmic hybrid) cells had increased basal NF-kappaB activation. Mitochondria appear capable of signaling ETC impairment through MAPK modules and inducing protective NF-kappaB responses, which are increased by PD mitochondrial genes amplified in cybrid cells. Irreversible commitment to apoptosis in this cell model may derive from loss of Raf-1 and cleavage/activation of MEKK-1, processes reported in other models to be caspase-mediated. Therapeutic strategies that reduce mitochondrial activation of proapoptotic MAPK modules, i.e., JNK, and enhance survival pathways, i.e., NF-kappaB, may offer neuroprotection in this debilitating disease.

Generation of constitutively active p90 ribosomal S6 kinase in vivo. Implications for the mitogen-activated protein kinase-activated protein kinase family.

p90 ribosomal S6 kinases (RSKs), containing two distinct kinase catalytic domains, are phosphorylated and activated by extracellular signal-regulated kinase (ERK). The amino-terminal kinase domain (NTD) of RSK phosphorylates exogenous substrates, whereas the carboxyl-terminal kinase domain (CTD) autophosphorylates Ser-386. A conserved putative autoinhibitory alpha helix is present in the carboxyl-terminal tail of the RSK isozymes ((697)HLVKGAMAATYSALNR(712) of RSK2). Here, we demonstrate that truncation (Delta alpha) or mutation (Y707A) of this helix in RSK2 resulted in constitutive activation of the CTD. In vivo, both mutants enhanced basal Ser-386 autophosphorylation by the CTD above that of wild type (WT). The enhanced Ser-386 autophosphorylation was attributed to disinhibition of the CTD because a CTD dead mutation (K451A) eliminated Ser-386 autophosphorylation even in conjunction with Delta alpha and Y707A. Constitutive activity of the CTD appears to enhance NTD activity even in the absence of ERK phosphorylation because basal phosphorylation of S6 peptide by Delta alpha and Y707A was approximately 4-fold above that of WT. A RSK phosphorylation motif antibody detected a 140-kDa protein (pp140) that was phosphorylated upon epidermal growth factor or insulin treatment. Ectopic expression of Delta alpha or Y707A resulted in increased basal phosphorylation of pp140 compared with that of WT, presenting the possibility that pp140 is a novel RSK substrate. Thus, it is clear that the CTD regulates NTD activity in vivo as well as in vitro.

Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo.

Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-terminal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3) interacted with extracellular signal-regulated kinase (ERK) but not c-Jun-NH2-kinase (JNK) or p38 mitogen-activated protein kinase (MAPK). Within the carboxyl-terminal residues of the RSK isozymes is a region of high conservation corresponding to residues 722LAQRRVRKLPSTTL735 in RSK1. Truncation of the carboxyl-terminal 9 residues, 727VRKLPSTTL735, completely eliminated the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues 731PSTTL735 had no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunoprecipitated with hemagglutinin-tagged wild type RSK2 (HA-RSK2) in BHK cell cytosol. However, ERK did not co-immunoprecipitate with HA-RSK2((1-729)), a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminated in vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase activity of HA-RSK2 increased 6-fold in response to insulin. HA-RSK2((1-729)) had a similar basal kinase activity to that of HA-RSK2 but was not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2((1-729)) could be activated to the same extent in vitro by active ERK2, demonstrating that HA-RSK2((1-729)) was properly folded. These data suggest that the conserved region of the RSK isozymes (722LAQRRVRKL730 of RSK1) provides for a specific ERK docking site approximately 150 amino acids carboxyl-terminal to the nearest identified ERK phosphorylation site (Thr573). Complex formation between RSK and ERK is essential for the activation of RSK by ERK in vivo. Comparison of the docking site of RSK with the carboxyl-terminal tails of other MAPK-activated kinases reveals putative docking sites within each of these MAPK-targeted kinases. The number and placement of lysine and arginine residues within the conserved region correlate with specificity for activation by ERK and p38 MAPKs in vivo.

pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167.

The estrogen receptor alpha (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1 in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

Ribosomal S6 kinase is activated as an early event in preemergence development of encysted embryos of Artemia salina.

Dormant Artemia salina cysts contain desiccated gastrulae that are metabolically inactive, and physiologically arrested. Following rehydration, embryos resume development via alterations in protein expression, in the complete absence of cell division. In mammals, activation of p70 ribosomal S6 kinase (p70S6k) has been implicated in translational control, in particular the selective up-regulation of translation of mRNAs with polypyrimidine tracts at their 5' start sites. We therefore investigated ribosomal S6 kinase activity in preemergence development. We demonstrate that an S6 kinase activity is rapidly stimulated (within < 15 min) following rehydration and coincides with the onset of ribosomal S6 subunit phosphorylation. This S6 kinase activity displays chromatographic and biochemical characteristics that are similar to those of mammalian p70S6k. Partially purified Artemia S6 kinase was inactivated by treatment with protein phosphatase 2A. Activation of S6 kinase activity was shown to be due to an enzymatic step(s), and not simply rehydration of stored, active enzyme. The temporal profile of activation of S6 kinase activity is compatible with a regulatory function for p70S6k in early preemergence development of encysted Artemia. These studies identify activated Artemia cysts as a system for biochemical studies of p70S6k regulation.

Activation in vitro of somatostatin receptor subtypes 2, 3, or 4 stimulates protein tyrosine phosphatase activity in membranes from transfected Ras-transformed NIH 3T3 cells: coexpression with catalytically inactive SHP-2 blocks responsiveness.

Somatostatin receptors (sstr) subtypes 1-5 were transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras(G12V) to assess the ability of each receptor to stimulate protein tyrosine phosphatase (PTPase) activity in vitro. Treatment of membranes from sstr2-, sstr3-, or sstr4-expressing cells with somatostatin-14 plus guanyl-5'-yl imidodiphosphate (GMPPNP) increased PTPase activity, and this stimulation was pertussis toxin-sensitive. Somatostatin alone, GMPPNP alone, or somatostatin plus GDP were ineffective under these conditions. sstr1 and sstr5 failed to increase PTPase activity although both receptors were expressed, as assessed by appearance of high-affinity binding sites for [125I-Tyr11]somatostatin-14. Somatostatin plus GMPPNP stimulated PTPase activity in vitro when sstr2 was coexpressed with wild type PTP1B or a Cys to Ser (C/S), catalytically inactive PTP1B or with wild type SH2-domain containing PTPase SHP-2. However, coexpression with catalytically inactive C/S SHP-2 abrogated this response. Thus, three of the five cloned sstr's can couple to activate PTPase in this cellular background. Abrogation of the response by C/S SHP-2 strongly suggests, but does not prove, a role for SHP-2 in the mechanism.

Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway.

Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways.