Mapping of atypical protein kinase C within the nerve growth factor signaling cascade: relationship to differentiation and survival of PC12 cells.

The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-iota was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-iota with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-iota by NGF. At the level of Raf-1, neither PKC-iota nor PI3 kinase was required for activation; however, PKC-iota could weakly activate MEK. Inhibitors of PKC-iota activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-iota were likewise required for NGF-induced NF-kappaB activation and cell survival, whereas Ras was not required for either survival or NF-kappaB activation but was required for differentiation. IKK existed as a complex with PKC-iota, Src and IkappaB. Consistent with a role for Src in regulating NF-kappaB activation, an absence of Src activity impaired recruitment of PKC-iota into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-kappaB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-kappaB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-iota and the NF-kappaB pathway.

Nerve growth factor stimulates multisite tyrosine phosphorylation and activation of the atypical protein kinase C's via a src kinase pathway.

Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-iota becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-iota were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-iota in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-iota were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-iota. Recruitment of PKC-iota into the complex was dependent on the tyrosine phosphorylation state of PKC-iota. The association of src and PKC-iota was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-iota (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-iota. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-iota, whereas the Y325F mutation significantly reduced src-induced activation of PKC-iota. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-kappaB, with significant impairment by the Y325F PKC-iota mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.

Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses.

REASONS FOR PERFORMING STUDY: Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. HYPOTHESIS: Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. METHODS: Twelve healthy horses received an i.v. infusion either of saline or a phospholipid emulsion (100 mg/kg), followed immediately by E. coli 055:B5 LPS (30 ng/kg). Clinical parameters, haematological profiles, serum tumour necrosis factor (TNF) activity, serum lipid profiles, urine analyses and severity of haemolysis were monitored before and at selected times after LPS. Monocytes were also incubated in vitro with LPS in the presence or absence of emulsion, after which TNF and tissue factor activities were determined. RESULTS: Clinical signs of endotoxaemia were reduced in horses receiving the emulsion, including clinical score, heart rate, rectal temperature, serum TNF activity, and the characteristic leucopenic response to LPS, when compared to horses not receiving the emulsion. Three horses receiving the emulsion had none, 2 had mild and one had moderate haemolysis. There were no differences in urinalysis results and creatinine concentrations, either within the groups over time or between the groups. Serum concentrations of phosphatidylcholine, bile acids and triglycerides peaked immediately after the infusion; there were no significant changes in concentrations of nonesterified fatty acids or cholesterol. Incubation of equine monocytes with emulsion prevented LPS-induced TNF and tissue factor activities. CONCLUSIONS: Rapid administration of emulsion significantly reduced inflammatory effects of LPS in vivo and caused a clinically insignificant degree of haemolysis. The results of the in vitro studies indicate that emulsion prevents not only LPS-induced synthesis of cytokines, but also expression of membrane-associated mediators (i.e. tissue factor). POTENTIAL RELEVANCE: Rapid i.v. administration of emulsions containing phospholipids that bind endotoxin may provide a clinically useful method of treating endotoxaemia in horses.

Overexpression of atypical PKC in PC12 cells enhances NGF-responsiveness and survival through an NF-kappaB dependent pathway.

Removal of atypical PKC blocks NGF-induced differentiation of PC12 cells.1 We now examine the consequences that overexpression of atypical PKCs had upon NGF responses. PC12 cells were stably transfected with either PKC-iota or PKC-zeta. Overexpression of atypical PKCs markedly enhanced NGF- induced neurite outgrowth as well as enhanced NGF-stimulated JNK kinase. Cotransfection of HA-JNK1 along with increasing concentrations of PKC-iota, resulted in dose-dependent phosphorylation of GST c-Jun (1 - 79). NGF treatment of PC12 cells resulted in activation of NF-kappaB. In comparison, overexpression of atypical PKC-iota was by itself sufficient to activate NF-kappaB and shift the kinetics of NGF-induced kappaB activity. Furthermore, transfection of full-length antisense PKC-iota blocked basal and NGF-stimulated NF-kappaB. Differentiated and undifferentiated PC12 cells overexpressing atypical PKC-iota were protected from serum deprivation-induced cell death. Collectively, these findings demonstrate that atypical PKC-iota lies in a pathway that regulates NF-kappaB and contributes to both neurotrophin-mediated differentiation and survival signaling.

Study of serum amyloid A concentrations as a means of achieving early diagnosis of Rhodococcus equi pneumonia.

REASONS FOR PERFORMING STUDY: Prognosis of Rhodococcus equi pneumonia can be challenging because the course of the disease is often insidious and overt clinical signs are subtle. Early diagnosis is considered desirable because it may offer the chance of more successful implementation of treatment and, thereby, improved outcome. Serological tests have previously failed to be accurate for early detection or diagnosis. Measurement of serum amyloid A (SAA) prior to and at the time of clinical signs was therefore chosen in order to assess its potential clinical use. OBJECTIVE: To determine whether SAA concentrations differentiate foals affected with R. equi pneumonia from unaffected foals, either prior to the onset of disease or at the time of onset of clinical signs. HYPOTHESIS: SAA concentrations are significantly higher among foals that develop R. equi pneumonia than in foals from the same environment that remain clinically unaffected. METHODS: Serum samples were obtained from 212 foals 7-14 days and 196 foals 21-28 days post partum, and from affected foals and age-matched controls at the time of onset of signs of pneumonia. SAA concentration was determined for each sample. RESULTS: There were no significant differences between SAA concentrations of foals with R. equi and clinically unaffected foals during the 2 periods of examination or at the time of onset of clinical signs of R. equi pneumonia. CONCLUSIONS: Concentrations of SAA are variable among foals with R. equi pneumonia and cannot be used reliably either as an ancillary diagnostic tool or to screen for early detection of disease during the first month post partum. POTENTIAL RELEVANCE: Bimonthly monitoring concentration of SAA is not useful as a screening test for early detection of R. equi pneumonia and does not facilitate diagnosis of this disease when used according to the protocol of this study.

Characterization of the messenger RNA population coding for components of viperid snake venom.

Total polyadenylated messenger RNA was prepared from the milked venom glands of the South African puff adder (Bitis arietans) and translated in an in vitro translation system. The products of cell free synthesis were immunoprecipitable with puff adder venom antiserum. Treatment of these cell free products with a dog pancreas microsomal membrane preparation demonstrated the presence of signal peptides. Northern blot hybridization of total puff adder venom gland mRNA to its complementary single stranded copy DNA revealed two discrete mRNA populations coding for the major components of puff adder venom. The relative amounts of these polyadenylated mRNA sequences changed during the onset of venom synthesis, suggesting mRNA deadenylation, general endonucleolytic RNA degradation and selective degradation of high molecular weight message components.

Genomic characterization of equine interleukin-4 receptor alpha-chain (IL4R).

Three overlapping fragments of the equine interleukin-4 receptor alpha chain gene (IL4R) were cloned and sequenced. The resulting 3553 bp cDNA sequence exhibited homology to human, murine and bovine IL4R. The equine IL4R exhibits many conserved features when compared to other species, including intron-exon boundary positions and amino acid sequence motifs characteristic of type I cytokine receptors. The IL4R gene was localized to horse chromosome ECA13 by synteny mapping on a somatic cell hybrid panel. Evidence for an alternative splice variant of IL4R was found in the genomic sequence and subsequently verified using RT-PCR on equine monocyte RNA. A polymorphism screen of the largest exon, homologous to exon 12 of the human IL4R gene, was performed using DNA from 60 horses of various breeds which yielded 11 coding-region single nucleotide polymorphisms (SNPs), 7 synonymous and 4 non-synonymous. Three of the four non-synonymous SNPs occur at high frequencies and are found very near a conserved tyrosine residue.

Masitinib mesylate does not enhance sensitivity to radiation in three feline injection-site sarcoma cell lines under normal growth conditions.

Masitinib, a selective tyrosine kinase inhibitor, was investigated as a radiosensitizer in three primary feline injection-site sarcoma (ISS) cell lines. Sensitivity to masitinib was previously assessed via cell growth inhibition assays with mean IC50 values of 5.5-8.6µM. Clonogenic assays were performed to determine the effect of masitinib and radiation on cell survival. Single dose radiation (0-12Gy) experiments were carried out under normal growth conditions in control ISS cells and in cells incubated with 1 or 6µM masitinib for 72h prior to irradiation. Radiation administered either alone or in combination with masitinib induced a dose-dependent reduction in clonogenic survival. Survival from the combined masitinib and radiation treatment was not significantly different from that of radiation alone. Results suggest that masitinib does not directly enhance ISS cell radiosensitivity under normal in vitro conditions, although this does not preclude the utility of further investigations to assess sensitization properties under altered conditions.

Disparate effects of LPS infusion and carbohydrate overload on inflammatory gene expression in equine laminae.

Although clinical evidence of endotoxemia has been associated with the development of acute laminitis in hospitalized horses with gastrointestinal diseases and endotoxins have been detected in the circulation of horses with experimentally-induced laminitis, it is unclear what role, if any, endotoxins have play the pathogenesis of the disease. Therefore, in the present study we compared the effects of endotoxin infusion to that of intra-gastric administration of mixed carbohydrate (CHO) on clinical signs of laminitis, plasma concentrations of TNF-α and IL-10, and laminar tissue expression of 20 genes associated with inflammation. Horses were divided into 4 groups: Control (water placebo, n=7), endotoxin infusion (LPS, n=6), CHO/Developmental (30% decrease in central venous pressure, n=6) and CHO/Lame (Obel grade I laminitis, n=7). Horses in the LPS group developed clinical signs consistent with systemic inflammation, had rapid increases in plasma concentrations of both TNF-α and IL-10, and leukopenia, but did not have any changes in laminar tissue expression of the genes associated with inflammation. In contrast, horses administered CHO developed clinical signs consistent with systemic inflammation, had more delayed increases in TNF-α, IL-10 and total leukocyte counts, and had marked increases in laminar tissue expression of the genes associated with inflammation. Only the horses administered CHO developed clinical signs of laminitis, providing additional credence to the concept that factors other than endotoxin are responsible for the changes in laminar tissue gene expression that occur during the development of acute equine laminitis.

Masitinib demonstrates anti-proliferative and pro-apoptotic activity in primary and metastatic feline injection-site sarcoma cells.

Dysregulation of platelet-derived growth factor receptor (PDGFR) may play a role in feline injection-site sarcoma (ISS) cell growth and viability. Masitinib, a tyrosine kinase inhibitor approved for treatment of canine mast cell tumours, is highly selective for the PDGFR signalling pathway and may offer a new therapeutic approach for this disease. The in vitro effects of masitinib on growth, apoptosis and PDGFR signalling in two novel ISS cell lines were investigated. PDGFR expression was confirmed by Western blot in cell lines derived from a primary ISS tumour (JB) and a corresponding, histologically confirmed ISS lung metastasis (JBLM). Masitinib inhibited cell growth and PDGFR phosphorylation in both cell lines. Higher drug concentrations were required to inhibit growth than to modulate ligand-induced autophosphorylation of PDGFR. These in vitro data suggest that masitinib displays activity against both primary and metastatic ISS cell line and may aid in the clinical management of ISS.

Validation of a reliable set of primer pairs for measuring gene expression by real-time quantitative RT-PCR in equine leukocytes.

Quantification of gene expression using real-time reverse transcription quantitative PCR (RT-qPCR) is a reliable method to monitor cellular responses to pro-inflammatory stimuli. The main objective of this study was to validate a set of equine primer pairs that can be routinely used to monitor expression of genes that are central to inflammatory and immune responses. This paper describes the steps used to optimize and validate 29 equine primer pairs for RT-qPCR assays using SYBR Green detection. To validate these assays, monocytes were isolated from three horses and stimulated with Escherichia coli LPS. Because four of the 29 genes demonstrated poor amplification efficiency due to weak induction of gene expression by LPS, monocytes were stimulated with alternative agents (PMA and Poly I:C) known to induce gene expression in monocytes. These agents, acting through different pathways than LPS, improved the level of gene expression and yielded good amplification efficiency for these genes. PCR efficiency was based on a standard curve for each gene and the calculated efficiency was approximately 100% for all 29 genes. The PCR efficiencies for the majority of the target genes were equivalent to that of the housekeeping gene (18S rRNA) used in all experiments. Dissociation curve analysis and gel electrophoresis revealed a single product for each gene analyzed. To exemplify utilization of the validated primer pairs in studies of inflammatory cell activation, temporal changes in mRNA expression of a subset of genes were monitored in equine monocytes incubated with LPS. The availability of the 29 validated primer pairs reported herein will allow investigators to elucidate the response of horses to a variety of inflammatory stimuli and will further our understanding of disease pathogenesis in horses.

Atypical PKC zeta is activated by ceramide, resulting in coactivation of NF-kappaB/JNK kinase and cell survival.

Both protein kinase C (PKC) and ceramide play a critical role in cell signaling, but the relationship between PKC and ceramide is unclear. Low concentrations of ceramide were observed to transiently stimulate PKC zeta activity in vitro and in vivo, whereas high doses of ceramide lead to inhibition of PKC zeta. Inhibition of activity was accompanied by enhanced binding of the negative regulator, Par4 to PKC zeta. Treatment of PC12 cells with low doses of ceramide promoted survival in serum-free media and activation of nuclear factor-KB, whereas higher doses (>2.5 microM) resulted in cell death. Overexpression of either aPKC isoform, PKC zeta or iota, resulted in enhanced survival of PC12 cells at high doses of ceramide and in ceramide-stimulated Jun N-terminal kinase (JNK), without any apparent effect on mitogen-activated kinase. These findings support a role for ceramide-induced PKC zeta activity in the control of cell survival signaling via a pathway that also activates JNK kinase.

Increased intracellular Ca2+ is necessary for maximal expression of the proto-oncogene c-jun in the Jurkat T-cell line.

The time course and signal-transduction requirements for proto-oncogene c-jun expression in T-cells were investigated. Expression of c-jun mRNA was evident at 30 min after stimulation. Both the activation of Ca2+/phospholipid-dependent kinase as well as an increased intracellular free Ca2+ concentration were necessary for the maximal induction of c-jun mRNA and synthesis of Jun protein 1 h after stimulation.

Identification of Src as a novel atypical protein kinase C-interacting protein.

Atypical protein kinase C-zeta (PKC-zeta) participates in nerve growth factor (NGF) signaling and is required for NGF-induced differentiation of PC12 cells. The biological activity of PKC-zeta is likely mediated by interaction of PKC-zeta with specific proteins. Affinity column chromatography employing the PKC-zeta regulatory domain coupled to glutathione-agarose was used to search for proteins that bound PKC-zeta. Two proteins (59/60 kDa) were recovered from NGF-stimulated PC12 cell lysates that bound the matrix. Western blot analysis of pooled column fractions identified these proteins as tubulin and src, respectively. Using purified preparations of src and tubulin, PKC-zeta was shown to interact with both proteins using blot overlay. To demonstrate a functional interaction in vivo, PC12 cells expressing a temperature-sensitive v-src were shifted to the permissive temperature (37 degrees C), followed by immunoprecipitation. At the permissive temperature where src was active, PKC-zeta was tyrosine phosphorylated and coassociated with src in vivo; by comparison, at the nonpermissive temperature (40 degrees C) PKC-zeta was not tyrosine phosphorylated. Taken together, these findings support a novel role for the interaction of src and atypical PKC in vivo, which is dependent upon the activity of src and the tyrosine phosphorylation state of PKC-zeta.

Phosphorylation of human serum amyloid A protein by protein kinase C.

Monokine-induced hepatic secretion of serum amyloid A protein (apo-SAA), an acute-phase reactant, is followed by rapid association with high-density lipoprotein (HDL) in plasma. Plasma clearance of apo-SAA is more rapid than any of the other HDL apolipoproteins. It has been shown that, of the acute-phase HDL3 apolipoproteins, apo-SAA preferentially associates with neutrophil membranes. HDL apolipoproteins have been shown to activate protein kinase C in endothelial cells. We therefore investigated potential phosphorylation of HDL3 apolipoproteins by protein kinase C. Apo-SAA was the only apolipoprotein phosphorylated (Km = 12 mM). Phosphorylation of the apo-SAA-containing HDL3 particle was selective for the more basic isoforms of apo-SAA (pI 7.0, 7.4, 7.5 and 8.0), with more acidic isoforms being phosphorylated when delipidated acute-phase apolipoproteins were used as substrate. However, phosphorylation was not in itself responsible for the establishment of the apo-SAA isoforms.

The early bactericidal activity of isoniazid related to its dose size in pulmonary tuberculosis.

Collections of sputum from 105 patients with newly diagnosed pulmonary tuberculosis were made before and at 1 and 2 d after the start of chemotherapy with isoniazid (INH) alone given to groups of patients in doses of 600 mg, 300 mg, 150 mg, 75 mg, 37.5 mg, 18.75 mg, and 9 mg daily, as well as from an untreated group. Counts of colony forming units (cfu) of Mycobacterium tuberculosis in the collections were set up on plates of selective 7H10 medium. The early bactericidal activity (EBA) of INH was defined as the decrease in log10 cfu/ml sputum/day during the first 2 d of treatment. A smooth curve relating EBA to log dose was obtained, with 600 mg INH yielding the highest mean EBA of 0.539, and 18.75 mg INH yielding the lowest EBA (0.111) that could be distinguished from the bactericidal activity of the untreated group. The ratio of the usual dose of 300 mg INH to the lowest dose, of 18.75 mg, that produced a detectable EBA, termed the therapeutic margin, was therefore 16, in contrast to the lower therapeutic margin of 4 for rifampin. The EBA was related to the INH acetylator genotype of patients treated with 600 mg or 9 mg INH.

Trimodality of isoniazid elimination: phenotype and genotype in patients with tuberculosis.

The study was undertaken to show that polymorphic isoniazid elimination in humans is trimodal; that the acetylator genotype and eliminator phenotype of the individual patient are concordant; and that the differences in the pharmacokinetic parameters of fast, intermediate, and slow eliminator subgroups are statistically significant. Sixty adult patients of both sexes and of mixed race with tuberculosis participated in the trial. The apparent elimination rate constant (k, h(-1)) and the area under the isoniazid concentration-time curve (AUC, mg/L/h), over the interval 2 to 6 h after oral isoniazid were determined in all patients; NAT2 allele composition was determined in 47 patients. Serum INH concentrations were determined by HPLC and genotypes by PCR/restriction enzyme analysis. Three eliminator phenotypes could be distinguished, and concordance between the phenotype and the genotype of the individual could be demonstrated. The isoniazid concentration-time profiles of the three eliminator subgroups were significantly different (p < 0.05). The NAT2*12A allele, which codes for fast acetylation, has a high frequency in the population studied, the intermediate acetylator genotype is constituted of codominant fast and slow alleles, and the distribution of phenotypes/genotypes in the population is consistent with Hardy-Weinberg predictions. The therapeutic implications of polymorphic isoniazid metabolism are discussed.