A regional Australasian experience of extended endoscopic transsphenoidal surgery for craniopharyngioma: Progression of the mentoring model.

Endoscopic endonasal transsphenoidal approaches to craniopharyngioma has become increasingly popular over the last 15 years. We present the results of our retrospective series of craniopharyngiomata resected by an endoscopic, endonasal approach at a low-volume service in Australasia. Between the years of 2009 and 2017, 11 patients underwent pure endoscopic endonasal transsphenoidal resection for a craniopharyngioma at our institutions. The medical records, histopathology, intraoperative findings and patient imaging were retrospectively assessed. 11 patients were included with 5 male and 6 female patients. The mean age was 32.0 years (range 14-68 years). Of this patient series a gross total resection of the tumour was achieved in 8 of 11 patients (73%). In the immediate postoperative phase, 10 of the 11 patients developed diabetes insipidus (91%). The pituitary stalk was formally not seen in 4 patients and all were treated with vasopressin. Of the 7 patients where the pituitary stalk was identified it was formally divided in 6 and preserved in 1 patient where the tumour was separate to the stalk. The endoscopic endonasal transsphenoidal approach for excision of craniopharyngioma, utilising and progressing the surgical mentoring model, can achieve adequate decompression of critical structures. Furthermore, our aggressive approach to divide and remove the involved pituitary stalk results in high rates of gross macroscopic resection with excellent long-term disease control with a greater risk of postoperative diabetes insipidus and panhypopituitarism.

Protein kinase Cε and protein kinase Cθ double-deficient mice have a bleeding diathesis.

BACKGROUND: In comparison to the classical isoforms of protein kinase C (PKC), the novel isoforms are thought to play minor or inhibitory roles in the regulation of platelet activation and thrombosis. OBJECTIVES: To measure the levels of PKCθ and PKCε and to investigate the phenotype of mice deficient in both novel PKC isoforms. METHODS: Tail bleeding and platelet activation assays were monitored in mice and platelets from mice deficient in both PKCθ and PKCε. RESULTS: PKCε plays a minor role in supporting aggregation and secretion following stimulation of the collagen receptor GPVI in mouse platelets but has no apparent role in spreading on fibrinogen. PKCθ, in contrast, plays a minor role in supporting adhesion and filopodial generation on fibrinogen but has no apparent role in aggregation and secretion induced by GPVI despite being expressed at over 10 times the level of PKCε. Platelets deficient in both novel isoforms have a similar pattern of aggregation downstream of GPVI and spreading on fibrinogen as the single null mutants. Strikingly, a marked reduction in aggregation on collagen under arteriolar shear conditions is observed in blood from the double but not single-deficient mice along with a significant increase in tail bleeding. CONCLUSIONS: These results reveal a greater than additive role for PKCθ and PKCε in supporting platelet activation under shear conditions and demonstrate that, in combination, the two novel PKCs support platelet activation.

A Dictyostelium nuclear phosphatidylinositol phosphate kinase required for developmental gene expression.

The generation of diacylglycerol (DAG) in response to receptor stimulation is a well-documented signalling mechanism that leads to activation of protein kinase C (PKC). Putative alternative effectors contain sequences that interact with DAGs, but the mechanisms of signal transduction are unknown. We have identified a Dictyostelium gene encoding a novel protein which contains a domain with high identity to the DAG-binding domain of PKC. It does not encode a PKC homologue as the conservation does not extend outside this region. We confirm that the proposed DAG-binding domain is sufficient to mediate interaction of a fusion protein with vesicles containing DAG. The protein also shows significant homology to mammalian phosphatidylinositol phosphate (PIP) kinases and we show that this domain has PIP kinase activity. The protein, PIPkinA, is enriched in the nucleus and abrogation of gene function by homologous recombination inhibits early developmental gene expression, blocking development at an early stage. Thus, we have identified a PIP kinase from Dictyostelium which is required for development, is a candidate effector for DAG and has the potential to synthesize nuclear PIP(2).

Signalling events underlying platelet aggregation induced by the glycoprotein VI agonist convulxin.

We have investigated the role of secretion and intracellular signalling events in aggregation induced by the glycoprotein (GP)VI-selective snake venom toxin convulxin and by collagen. We demonstrate that aggregation induced by threshold concentrations of convulxin undergoes synergy with ADP acting via the P2Y12 receptor whereas there is no synergy via the P2Y1 receptor or with thromboxanes. On the other hand, apyrase, the P2Y12 receptor antagonist, AR-C67085, and indomethacin only marginally inhibit aggregation induced by convulxin. In comparison, these inhibitors severely attenuate the response to collagen. In order to investigate whether the weak inhibitory action against convulxin is due to release of agonists other than ADP from dense granules, experiments were performed on murine platelets deficient in this organelle (pearl mice platelets). A slightly greater reduction in aggregation induced by convulxin was observed in pearl platelets than in the presence of inhibitors of ADP, but a maximal response was still attained. Importantly, inhibition of protein kinase C further reduced the response to convulxin in pearl platelets demonstrating a direct role for the kinase in aggregation. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N',N'-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM) abolished aggregation induced by convulxin under all conditions. Activation of phospholipase C by convulxin was potentiated by ADP acting through the P2Y12 receptor. In conclusion, we show that Ca2+ and protein kinase C, but not release of the secondary agonists ADP and thromboxane A2, are required for full aggregation induced by convulxin, whereas the response induced by collagen shows a much greater dependence on secretion of secondary agonists.

Signalling components underlying platelet aggregation to a Ca2+ ionophore and a phorbol ester.

Although it is well established that G(q)- and G(i)-coupled receptors can combine to mediate platelet aggregation, the signalling events underlying the synergy are not fully characterised. This study has used the calcium ionophore, A23187, and phorbol ester, PMA, to investigate this question. We show that aggregation to submaximal but not maximal concentrations of ionophore is partially inhibited by antagonism of the P2Y(12) ADP receptor or PKC blockade. However, a full aggregation response can be restored under these conditions by addition of PMA or ADP. Studies using PI 3-kinase inhibitors demonstrate that this is the second messenger pathway that restores aggregation by the G(i)-coupled receptor in the presence of PKC blockade. However, under normal circumstances, PI 3-kinase activity is not a major requirement for aggregation to the ionophore. PMA stimulates a weak aggregation which takes several minutes to reach a maximum. Threshold concentrations of PMA and a G(i)-coupled receptor agonist when added alone show no effect on aggregation, but when combined induce aggregation responses. This study demonstrates that calcium and PKC interact synergistically with a G(i)-coupled receptor agonist to mediate aggregation, and also with each other. Activation of G(i) supports aggregation in part through the PI 3-kinase pathway. High concentration of ionophore on their own can induce aggregation independent of PKC and activation of G(i). Multiple signalling pathways mediate platelet aggregation and their relative importance depends on experimental conditions.

Cell cycle-dependent regulation of early developmental genes.

Cell cycle phase at the onset of development in Dictyostelium influences cell fate. Cells in the G2 phase, which tend to become spores, show a more rapid induction of expression of the cell surface receptor involved in the chemotaxis. We show that differential induction of developmental expression is restricted to some transcripts, including those encoding proteins required for chemotaxis, and thus is not due to general transcriptional repression during mitosis. We also show that cells showing rapid induction of one such gene are preferentially located at the centre of early aggregates. These results are consistent with cells derived from G2 phase being at the centre of early aggregates because selective differences in gene regulation render them more efficient at aggregation.

Protein kinase C-alpha is essential for Ramos-BL B cell survival.

The Ramos-Burkitt lymphoma (BL) B cell line is driven into growth arrest and apoptosis by cross-linking surface immunoglobulin. We demonstrate that protein kinase C (PKC) activity is required for Ramos B cell proliferation and survival. A variety of PKC inhibitors trigger a significant decrease in [(3)H]thymidine incorporation with a concomitant increase in cell death. Antisense depletion of expression of the PKC-alpha isoform is sufficient to trigger cell death in the absence of any other signal, demonstrating a requirement for this isoform for survival of Ramos-BL B cells. Cross-linking surface immunoglobulin also leads to depletion of PKC-alpha levels, suggesting that this may be one mechanism by which this signals for cell death in Ramos-BL B cells.

Negative influence of RasG on chemoattractant-induced ERK2 phosphorylation in Dictyostelium.

The Dictyostelium ERK2 protein is transiently activated when cells are treated with the chemotactic agents cAMP or folic acid. Activating phosphorylation is markedly inhibited in strains overexpressing the constitutively activated RasG protein. This is in marked contrast to mammalian cells where the highly related mitogen-activated protein kinases (MAPKs) are stimulated by Ras activation.

Isoform specificity of activators and inhibitors of protein kinase C gamma and delta.

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 PKCS than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCgamma. We have also shown that the anti-leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform-specific manner.

Chemoattractants induce tyrosine phosphorylation of ERK2 in Dictyostelium discoideum by diverse signalling pathways.

Two homologues of mitogen-activated protein kinases have been identified in Dictyostelium discoideum (ERK1 and EKR2). We here demonstrate transient tyrosine phosphorylation of ERK2 in response to the chemoattractants cAMP and folic acid that correlates with activity. To investigate the signalling pathways, we studied the response in strains with altered cAMP-dependent protein kinase (PKA) status. The degree of cAMP-induced ERK2 tyrosine phosphorylation was increased in cells overexpressing PKA activity but no such increase was observed in the response to folic acid. Our observations suggest that cAMP-induced ERK2 tyrosine phosphorylation is positively modulated by a PKA-regulated step which is not involved in the response to folic acid, suggesting the presence of diverse signalling pathways leading to ERK2 activation.

A protein kinase C-like activity involved in the chemotactic response of Dictyostelium discoideum.

During the developmental life cycle of the cellular slime mould Dictyostelium discoideum cells aggregate in response to pulses of extracellular cAMP. This chemotactic agent stimulates a number of signalling pathways in the cell including the activation of a phospholipase C activity leading to the transient generation of inositol 3,4,5-trisphosphate and diacylglycerol. The role of diacylglycerol in chemotactic response and development of Dictyostelium is not known. We have evidence to suggest that two protein kinase C-like enzymes exist in Dictyostelium due to the different cellular responses to two inhibitors specific for protein kinase C. One enzyme is preferentially sensitive to D-erythro-sphingosine, a diacylglycerol analogue, and is required for growth. A second is preferentially inhibited by bisindolylmaleimide GF109203X and is required for chemotaxis. We have identified protein kinase C-like kinase activity in Dictyostelium cell extracts which appears as the cells aggregate. This activity is stimulated by diacylglycerol, especially biologically relevant diacylglycerol species, and phosphorylates a peptide substrate which is an efficient substrate for mammalian protein kinase Cs. This activity is a candidate for the effector of diacylglycerol generated during the aggregative phase of Dictyostelium development and defines a role for diacylglycerol in the chemotactic response.

Cells at the center of Dictyostelium aggregates become spores.

The cellular slime mold Dictyostelium discoideum undergoes a developmental life cycle on starvation to generate a fruiting body consisting of a mass of spores supported on a stalk of dead, vacuolated cells. The choice between alternative cell fates is influenced by a variety of factors including cell cycle position at the onset of starvation. We present evidence to suggest that the cell cycle position influences cell fate by determining the position of cells in the early aggregate. The existence of a strain which cannot initiate development on its own but which can respond to signals generated by nonmutant cells has allowed us to investigate the eventual cell fate of the initiating cells which are, by definition, at the center of the early aggregate. Cells which have a propensity to become prespore cells show an increased efficiency in initiating development of this strain. Labeling the initiating cells by the expression of green fluorescent protein reveals that these cells become spores. The higher levels of expression of genes characteristic of early development in cells with a prespore tendency are consistent with the earlier expression of the components of relay in prespore cells.

Studies on the phosphorylation of protein kinase C-alpha.

A kinase-defective protein kinase C-alpha mutant is shown to be a phosphoprotein when expressed in COS-1 cells, indicating that intramolecular phosphorylation does not fully account for the phosphate content of protein kinase C-alpha. Furthermore, evidence is presented that the intermolecular phosphorylation of protein kinase C-alpha is due to an activity other than protein kinase C-alpha itself, and this phosphorylation appears to be necessary for protein kinase C-alpha activity. By contrast, the characteristic shift in apparent molecular mass consequent on phosphorylation in vivo can be accounted for by autophosphorylation, as demonstrated in vitro. The relationship between these phosphorylated protein kinase C-alpha species is discussed.