Nigel T. Goode

Molecular aspects of the metastatic cascade.

III, Regulation of cell growth 68 A, Signal transduction pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6B B. Protein kinase C activity and turnout maligna,lcy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 C. Effect of organ mierocnvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Isoform specificity of activators and inhibitors of protein kinase C γ and δ

Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol. Biol. Cell 5 (1994) 907–920). We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening. This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCδ than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCγ. We have also shown that the anti‐leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform‐specific manner.

A phorbol ester-responsive PKC-ζ generated by fusion with the regulatory domain of PKC-δ

A hybrid molecule generated by fusing the regulatory domain of PKC‐δ with the catalytic domain of PKC‐ζ is, like PKC‐δ but unlike PKC‐ζ, a phorbol ester‐dependent enzyme. However, the substrate specificity of this hybrid resembles that of PKC‐ζ. Expression of mammalian PKC‐δ, but not PKC‐ζ, m the fission yeast Schizosaccharomyces pombe causes growth retardation and phorbol esters amplify the PKC‐δ phenotype without affecting that of PKC‐ζ (Goode et al., submitted). The chimaeric molecule also inhibited growth and this effect was phorbol‐ester dependent. Both the hybrid and PKC‐δ holoenzyme, in contrast to PKC‐ζ, down‐regulate upon prolonged exposure to phorbol esters in vivo. Thus, this hybrid retains the regulatory properties conferred by PKC‐δ but the catalytic properties of PKC‐ζ. This regulatable chimaeric molecule will be useful in assessing the function of PKC‐ζ.

The role of protein kinase C in regulating equine eosinophil adherence and superoxide production.

Abstract.Objective: To determine if protein kinase C (PKC) regulates equine eosinophil function.Material or subjects: Blood eosinophils were obtained from healthy ponies.Methods: IL-5- and histamine-induced adherence to serum-coated plastic was measured as the eosinophil peroxidase content of adherent cells and serum treated zymosan (STZ)-and IL-5-induced superoxide production by the reduction of cytochrome C. Eosinophil PKC activity was quantitated as the rate of transfer of 32P from ATP to substrate. The effects of Ro31-8220 (isotype non-selective PKC inhibitor), Gö6976 (conventional PKC inhibitor), and rottlerin (PKCδ inhibitor) were determined by ANOVA and Bonferroni’s or Dunnett’s test.Results: Ro31-8220 and Gö6976 reduced superoxide production whereas only Gö6976 inhibited adherence. Rottlerin inhibited histamine-induced adherence and increased STZ-induced superoxide production. Ro31-8220 and Gö6976, but not rottlerin, inhibited PKC activity.Conclusions: PKC is involved in regulating equine eosinophil adherence and superoxide production. The role of PKCδ appears to depend upon the stimulus used and response measured.

Cholesterol esterification in human monocyte-derived macrophages is inhibited by protein kinase C with dual roles for mitogen activated protein kinases.

The possible role of protein kinase C (PKC) and mitogen activated protein (MAP) kinases in the stimulation of cholesterol esterification by acetylated low density lipoprotein (acLDL) in human monocyte‐derived macrophages (HMDM) was studied. Cholesterol esterification, as assessed by the rate of incorporation of [3H]‐oleate into cholesteryl ester, was markedly higher in HMDM incubated with acLDL as compared to native LDL (nLDL). In the presence of the phorbol ester, phorbol 12‐myristate 13‐acetate (PMA, 100 nM), however, the rate of incorporation was reduced by about 50% and 85% in incubations with nLDL and acLDL, respectively. Thus, the difference in the rate of cholesteryl esterification induced by the two types of lipoprotein was abolished by PMA, indicating that PKC activation inhibits the process, and this was confirmed by the finding that the PKC inhibitor calphostin C reversed the PMA‐induced inhibition of cholesterol esterification. Incubation of HMDM with PMA was found to cause a considerable increase in the activation of p42/44 extracellular signal‐regulated MAP kinases (ERK) and p38 MAP kinases, reaching a maximum at 30 min. In the presence of acLDL, the ERK inhibitor PD98059 decreased cholesterol esterification in HMDM by about 35%. In contrast, the p38 inhibitor SB203580 had no effect. However, when PMA was present in addition to SB203580, esterification was reduced to a level lower than that observed with PMA alone. These findings suggest that activation of ERK, but not p38, MAP kinases is involved in the induction of cholesterol esterification by acLDL in HMDM, while p38 MAP kinases may modulate the inhibitory effect of PKC, and thus provide evidence that MAP kinases play a role in the regulation of foam cell formation in human macrophages.

Cloning, expression and biological activity of equine interleukin (IL)-5

The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.

Differential localization of protein kinase C isotypes in equine eosinophils and neutrophils.

Phorbol esters, which activate protein kinase C (PKC), stimulate equine eosinophil superoxide production and adherence. After showing that superoxide production could be inhibited by the nonselective PKC inhibitors, staurosporine and bisindolymaleimide I, the PKC isotypes in equine eosinophils were characterized, because evidence suggests that individual isotypes may play distinct roles in regulating eosinophil function. Western blots demonstrated that equine eosinophils expressed PKC α, β, δ, ɛ, ι, and ζ. However, unlike the equine neutrophil, the majority of the PKC was detected in the particulate fraction of the cell. Despite this unusual location, the PKC in equine eosinophils was activatable, suggesting that it is functionally competent. The regulatory role of PKC in equine eosinophils may reflect the association of activity with the particulate fraction and the profile of isotype expression.

Protein kinase C (PKC) isotype profile in eosinophils from ponies with sweet itch and role in histamine-induced eosinophil activation

Eosinophils have been implicated in the pathogenesis of the seasonal equine allergic skin disease, sweet itch. Protein kinase C (PKC) is involved in regulating eosinophil function and antigen challenge has been reported to alter PKC isotype expression in blood eosinophils from allergic human subjects. Here we have compared the pattern of PKC isotype expression in eosinophils from sweet itch ponies with that in cells from normal ponies both during the active and inactive phases of the disease. A role for PKC in histamine-induced eosinophil activation was also investigated. Conventional PKCs alpha and beta, novel PKCs delta and epsilon and atypical PKCs iota and zeta were identified in eosinophils pooled from four allergic ponies during the inactive phase, when no clinical signs were evident. The PKC isotypes, like those in eosinophils from normal ponies, were located primarily in the particulate fraction of the cell. Isotype expression in cells from normal and allergic animals did not appear to be different. In contrast, during the active phase of the disease, when the sweet itch ponies had clinical signs, the expression of PKCs beta, epsilon and iota in eosinophils from these animals appeared to be increased relative to that in cells from normal ponies. When PKC expression in eosinophils from five individual normal and sweet itch ponies was compared, small, but statistically significant, increases in PKC epsilon and PKCdelta expression were evident in eosinophils from the sweet itch ponies during the active and inactive phases, respectively. The non-selective PKC inhibitors, staurosporine and Ro31-8220, significantly reduced histamine-induced superoxide production. Use of Gö6976, an inhibitor of conventional PKCs, suggested that PKCalpha and/or beta were involved and that there was significantly greater inhibition of the response in eosinophils obtained from sweet itch ponies during the active phase. There was no significant difference in histamine-induced superoxide production by eosinophils from allergic and normal ponies and the functional significance of the increased PKC isotype expression in eosinophils from sweet itch ponies relative to that in cells from healthy animals remains to be established.

Regulation of bone mineral density in the grey squirrel, Sciurus carolinensis: Bioavailability of calcium oxalate, and implications for bark stripping

Summary The damage caused when grey squirrels strip the outer bark off trees and ingest the underlying phloem can result in reduced timber quality or tree death. This is extremely costly to the UK forestry industry and can alter woodland composition, hampering conservation efforts. The calcium hypothesis proposes that grey squirrels ingest phloem to ameliorate a seasonal calcium deficiency. Calcium in the phloem predominantly takes the form of calcium oxalate (CaOx), however not all mammals can utilise CaOx as a source of calcium. Here, we present the results of a small‐scale study to determine the extent to which grey squirrels can utilise CaOx. One of three custom‐made diets containing calcium in varying forms and quantities (CaOx diet, Low‐calcium carbonate (CaCO 3) diet and Control diet) were fed to three treatment groups of six squirrels for 8 weeks. Bone densitometric properties were measured at the end of this time using peripheral quantitative computed tomography and micro‐computed tomography. Pyridinoline—a serum marker of bone resorption—was measured regularly throughout the study. Bone mineral density and cortical mineralisation were lower in squirrels fed the CaOx diet compared to the Control group but similar to that of those on the Low‐calcium diet, suggesting that calcium from calcium oxalate was not effectively utilised to maintain bone mineralisation. Whilst no differences were observed in serum pyridinoline levels between individuals on different diets, females had on average higher levels than males throughout the study. Future work should seek to determine if this apparent lack of ability to utilise CaOx is common to a large sample of grey squirrels and if so, whether it is consistent across all areas and seasons.

Structural Studies on the Protein Kinase C Gene Family

Protein kinase C (PKC) consists of a family of at least nine members which can be subdivided into two main groups on the basis of sequence homology. Modification is required for the enzyme to be functional as the primary translation product (~77kD) appears to express no kinase activity. PKC isolated from tissue sources is a phosphoprotein of a higher molecular weight (~80kD). Use of a kinase deficient mutant of PKC indicates that the primary translation product undergoes an initial phosphorylation by a separate kinase with subsequent autophosphorylation to generate the “mature” protein. The primary translation products expressed in E. coli bind phorbol esters with apparently normal affinity, consistent with this family forming a major class of phorbol ester receptors. Isolation of the individual isotypes of PKC from tissue sources or after expression of cDNA sequences in mammalian cells has revealed differences in substrate specificity and effector dependence of kinase activity. The fusion of regulatory and catalytic domains from different isotypes has revealed that the regions of the protein responsible for the limited substrate range of PKC-e in vitro lie within the regulatory domain of the enzyme, suggesting a role for this region in limiting access to the active site. The substrate selectivity may be extended towards a physiologically relevant substrate. Activated versions of the various isotypes of PKC are being constructed by mutagenesis in order to study the functions of the individual isotypes following expression in mammalian cells.