Tumor necrosis factor (TNF‐alpha) stimulates a number of signal transduction pathways in which phospholipases produce lipid second messengers. However, the immediate molecular targets of these messengers, in particular those of ceramide and arachidonic acid (AA) and their role in TNF signaling are not well defined. In this study we investigated the relationship of ceramide and AA in regulating an atypical PKC isozyme, PKC zeta. U937 cells responding to TNF‐alpha treatment with NF kappa B activation displayed enhanced phosphorylation of PKC zeta, which is already detectable 30 s after stimulation. [14C]ceramide specifically binds to and regulates kinase activity of PKC zeta in a biphasic manner. Binding studies indicate high and low affinity binding with bmax values of 60 and 600 nM and Kd values of 7.5 and 320 nM respectively. At ceramide concentrations as low as 0.5 nM an up to 4‐fold increase in autophosphorylation is obtained, which, at concentrations > 60 nM, again declines to basal levels. Interestingly, AA competes for ceramide binding and inhibits basal and ceramide‐stimulated PKC zeta kinase activity at < 100 nM. Metabolism of [14C]ceramide in cells is slow and is inhibited in the presence of equimolar concentrations of lyso‐phosphatidylcholine. Based on the bifunctional modulation of PKC zeta by the lipid messengers ceramide and AA, a model of TNF signal pathways is suggested in which PKC zeta takes a central position, acting as a molecular switch between mitogenic and growth inhibitory signals of TNF‐alpha.

BackgroundUrea cycle disorders (UCDs) are rare inherited metabolic defects of ammonia detoxification. In about half of patients presenting with a UCD, the first symptoms appear within a few days after birth. These neonatal onset patients generally have a severe defect of urea cycle function and their survival and outcome prognoses are often limited.To understand better the current situation of neonatal onset in UCDs, we have performed a multicentre, retrospective, non-interventional case series study focussing on the most severe UCDs, namely defects of carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), and argininosuccinate synthetase (ASS).Methods and resultsData of 63 patients were collected (27 patients with ASS deficiency, 23 patients with OTC deficiency, and 12 patients with CPS1 deficiency, one patient definite diagnosis not documented). The majority of patients (43/63, 68 %) had an initial ammonia concentration exceeding 500 μmol/L (normal < 100), of which most (26/43, 60.5 %) were also encephalopathic and were treated with hemodialysis. In patients surviving the initial crisis, recurrence of hyperammonemic events within the first 1.5 years of life occurred frequently (mean 3.6 events, range 0–20). Of all patients, 16 (25.4 %) died during or immediately after the neonatal period.ConclusionWe observed in this cohort of neonatal onset UCD patients a high rate of initial life-threatening hyperammonemia and a high risk of recurrence of severe hyperammonemic crises. These corresponded to a high mortality rate during the entire study period (30.2 %) despite the fact that patients were treated in leading European metabolic centers. This underlines the need to critically re-evaluate the current treatment strategies in these patients.

) -EJB 96 1293/4 13 murine tissues and 12 cell lines were tested for the expression of the novel protein kinase C (PKC) isoenzyme p . Using two different PKCp antibodies (sc-639 and P26720), PKCp was detected in all tissues and cells and thus proved to be an ubiquitous PKC isotype. However, in some tissues, PKCp was recognized only by the antibody P26720 and not by sc-639. Thymus, lung and peripheral blood mononuclear cells expressed the greatest amount of PKCp.Recognition of PKCp by the antibody sc-639 was drastically impaired when treating keratinocytes or mouse skin in vivo with the phorbol ester 12-0-tetradecanoy1phorbo1-13-acetate (TPA), thus mimicking down-regulation of PKCp. The lack of a decrease in the PKCp amount and, thus, the lack of downregulation could be proved using the antibody P26720. This antibody was able to recognize PKCp in extracts of untreated as well as TPA-treated tissues or cells. Phosphorylation of proteins in a cell-free system (cell or tissue extracts) in the presence and absence of TPA or other PKC activators and various protein kinase inhibitors indicated that phosphorylation of activated PKCp caused its reduced interaction with the antibody sc-639. Therefore, this antibody might present a well suited tool for the detection of activated PKCp in vivo. Moreover, our results clearly show that some antibodies, such as sc-639, might be able to selectively detect non-phosphorylated or phosphorylated forms of a protein, and that such properties of an antibody have to be studied carefully before the latter can be used for reliable quantitative determination of this protein. We consider this information important to avoid misinterpretation of data concerning the immunological quantification of proteins such as PKCp.

In order to gain insight into the biological function of a PKC iso‐enzyme, the protein kinase Cμ, we analyzed the expression pattern of this protein in mouse epidermis and keratinocytes in culture. Daily analysis of neonatal mouse epidermis immediately after birth showed a time‐dependent reduction in the PKCμ content. Expression of the proliferating‐cell nuclear antigen (PCNA), indicative of the proliferative state of cells, was reduced synchronously with PKCμ as the hyperplastic state of the neonatal tissue declined. In epidermal mouse keratinocytes, fractionated according to their maturation state, PKCμ expression was restricted to PCNA‐positive basal‐cell fractions. In primary cultures of those cells, growth arrest and induction of terminal differentiation by Ca2+ resulted in strongly reduced PKCμ expression, concomitantly with the loss of PCNA expression. Treatment of PMK‐R1 keratinocytes with 100 nM of the mitogen 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) resulted in activation of PKCμ, reflected by translocation from the cytosolic to the particulate fraction and by shifts in electrophoretic mobility. DNA synthesis was significantly inhibited by the PKCμ inhibitor Goedecke 6976, while Goedecke 6983 did not inhibit PKCμ. Carcinomas generated according to the 2‐stage carcinogenesis protocol in mouse skin consistently exhibited high levels of PKCμ. These data correlate PKCμ expression with the proliferative state of murine keratinocytes and point to a role of PKCμ in growth stimulation. A correlation between PKCμ expression and enhanced cell proliferation was also observed for NIH3T3 fibroblasts transfected with and over‐expressing human PKCμ. Int. J. Cancer 80:98–103, 1999. © 1999 Wiley‐Liss, Inc.