TSH overcomes Braf(V600E)-induced senescence to promote tumor progression via downregulation of p53 expression in papillary thyroid cancer.

The BRAF(V600E) mutation is found in approximately 40% of papillary thyroid cancers (PTC). Mice with thyroid-specific expression of Braf(V600E) (TPO-Braf(V600E)) develop PTC rapidly with high levels of serum thyroid-stimulating hormone (TSH). It is unclear to what extent the elevated TSH contributes to tumor progression. To investigate the progression of Braf(V600E)-induced PTC (BVE-PTC) under normal TSH, we transplanted BVE-PTC tumors subcutaneously into nude and TPO-Braf(WT) mice. Regression of the transplanted tumors was observed in both nude and TPO-Braf(WT) mice. They were surrounded by heavy lymphocyte infiltration and oncogene-induced senescence (OIS) was demonstrated by strong ß-gal staining and absence of Ki-67 expression. In contrast, BVE-PTC transplants continued to grow when transplanted into TPO-Braf(V600E) mice. The expression of Trp53 was increased in tumor transplants undergoing OIS. Trp53 inactivation reversed OIS and enabled tumor transplants to grow in nude mice with characteristic cell morphology of anaplastic thyroid cancer (ATC). PTC-to-ATC transformation was also observed in primary BVE-PTC tumors. ATC cells derived from Trp53 knockout tumors had increased PI3K/AKT signaling and became resistant to Braf(V600E) inhibitor PLX4720, which could be overcome by combined treatment of PI3K inhibitor LY294002 and PLX4720. In conclusion, BVE-PTC progression could be contained via p53-dependent OIS and TSH is a major disruptor of this balance. Simultaneous targeting of both MAPK and PI3K/AKT pathways offer a better therapeutic outcome against ATC. The current study reinforces the importance of rigorous control of serum TSH in PTC patients.

Pleiotropic effects of YC-1 selectively inhibit pathological retinal neovascularization and promote physiological revascularization in a mouse model of oxygen-induced retinopathy.

Vascular endothelial growth factor (VEGF) and inducible nitric-oxide synthase (iNOS) have been implicated in ischemia-induced retinal neovascularization. Retinal ischemia has been shown to induce VEGF and iNOS expression. It has been postulated that one of the crucial consequences of iNOS expression in the ischemic retina is the inhibition of angiogenesis. Furthermore, iNOS was shown to be overexpressed in Müller cells from patients with diabetic retinopathy. YC-1, a small molecule inhibitor of hypoxia-inducible factor (HIF)-1 alpha, has been shown to inhibit iNOS expression in various tissue models. Our aim was to assess the pleiotropic effects of YC-1 in an oxygen-induced retinopathy (OIR) mouse model and evaluate its therapeutic potential in HIF-1- and iNOS-mediated retinal pathologies. Dual-injections of YC-1 into the neovascular retinas decreased the total retinopathy score, inhibited vaso-obliteration and pathologic tuft formation, and concomitantly promoted physiological retinal revascularization, compared with dimethyl sulfoxide (DMSO)-treated group. Furthermore, YC-1-treated retinas exhibited a marked increase in immunoreactivities for CD31 and von Willebrand factor and displayed significant inhibition in HIF-1alpha protein expression. Furthermore, YC-1 down-regulated VEGF, erythropoietin, endothelin-1, matrix metalloproteinase-9, and iNOS message and protein levels. When hypoxic Müller and neuoroglial cells were treated with YC-1, iNOS mRNA and protein levels were reduced in a dose-dependent fashion. We demonstrate that YC-1 inhibits pathological retinal neovascularization by exhibiting antineovascular activities, which impaired ischemia-induced expression of HIF-1 and its downstream angiogenic molecules. Furthermore, YC-1 enhanced physiological revascularization of the retinal vascular plexuses via the inhibition of iNOS mRNA and protein expressions. The pleiotropic effects of YC-1 allude to its possible use as a promising therapeutic iNOS inhibitor candidate for the treatment of retinal neovascularization.

RNase L downmodulation of the RNA-binding protein, HuR, and cellular growth.

Ribonuclease L (RNase L) is an intracellular enzyme that is vital in innate immunity, but also is a tumor suppressor candidate. Here, we show that overexpression of RNase L decreases cellular growth and downmodulates the RNA-binding protein, HuR, a regulator of cell-cycle progression and tumorigenesis. The effect is temporal, occurring in specific cell-cycle phases and correlated with the cytoplasmic localization of RNase L. Both cellular growth and HuR were increased in RNASEL-null mouse fibroblast lines when compared to wild-type cells. Moreover, the stability of HuR mRNA was enhanced in RNASEL-null cells. The HuR 3' untranslated region (UTR), which harbors U-rich and adenylate-uridylate-rich elements, was potently responsive to RNase L when compared to control 3' UTR. Our results may offer a new explanation to the tumor suppressor function of RNase L.

Cannabinoid 2 receptor induction by IL-12 and its potential as a therapeutic target for the treatment of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is the most aggressive type of thyroid malignancies. Previously, we demonstrated that tumorigenicity of anaplastic thyroid carcinoma cell line ARO was significantly reduced following interleukin (IL)-12 gene transfer. We suspected that tumor target structure in ARO/IL-12 cells might be changed and such a change may make them more susceptible to be killed through mechanisms apart from natural killer-dependent pathway. To identify genes involved, we examined gene expression profile of ARO and ARO/IL-12 by microarray analysis of 3757 genes. The most highly expressed gene was cannabinoid receptor 2 (CB2), which was expressed eightfold higher in ARO/IL-12 cells than ARO cells. CB2 agonist JWH133 and mixed CB1/CB2 agonist WIN-55,212-2 could induce significantly higher rate of apoptosis in ARO/IL-12 than ARO cells. Similar results were obtained when ARO cells were transfected with CB2 transgene (ARO/CB2). A considerable regression of thyroid tumors generated by inoculation of ARO/CB2 cells was observed in nude mice following local administration of JWH133. We also demonstrated significant increase in the induction of apoptosis in ARO/IL12 and ARO/CB2 cells following incubation with 15 nM paclitaxel, indicating that tumor cells were sensitized to chemotherapy. These data suggest that CB2 overexpression may contribute to the regression of human anaplastic thyroid tumor in nude mice following IL-12 gene transfer. Given that cannabinoids have shown antitumor effects in many types of cancer models, CB2 may be a viable therapeutic target for the treatment of anaplastic thyroid carcinoma.

Correlation of antigenic expression with progress in antibiotic therapy of acute human brucellosis.

Human brucellosis is a zoonotic disease which is endemic in Saudi Arabia. The aim of this study was to investigate the humoral immune responses and identify the target antigens that persist at different stages in human brucellosis during antibiotic therapy. To do this, an acute case of accidental nosocomial infection was studied experimentally. Blood was collected from the patient at the time of diagnosis, and at weekly intervals during therapy until remission. IgG and IgM immunoblotting was used to characterize specific antigenic determinants, and ELISA antibody titration was performed to quantify the circulating antibodies. Results indicated that protein bands of 12-13.5 kDa bound IgG in the patient's sera but did not bind IgM on immunoblots and are probably not specific for, or important in, early stage infections. However, an 18 kDa band persisted during infection through remission. The pivotal and most important findings were that the number of protein bands seen on immunoblots, the magnitude of ELISA antibody titres and the concomitant changes in the intensity of the polypeptide bands of 42-43 kDa were positively correlated with the stage of infection. High numbers of anti-IgG and -IgM immunoblot bands coupled with high ELISA antibody titres and a concomitant increase in intensity of the 42-43 kDa bands were positively correlated with acute and severe infection. Conversely, a reduction in the number of polypeptide bands as well as a decrease in the intensity, until the complete disappearance of the 42-43 kDa bands, coupled with low (baseline) ELISA antibody titration values indicated successful treatment and remission. The routine use of the methods described here to ascertain the stage of the disease, assess the progress of antimicrobial therapy and monitor cases of relapse in human brucellosis is suggested.

Cross-reactivities between date palm (Phoenix dactylifera L.) polypeptides and foods implicated in the oral allergy syndrome.

BACKGROUND: Date fruit and pollen antigens share a number of cross-reactive epitopes. Date pollen has been shown to cross-react with antigens from Artemisia, cultivated rye (Secale cereale), Timothy grass (Phleum pratense), Sydney golden wattle (Acacia longifolia) and Bermuda grass (Cynodon dactylon) pollen. The present study was carried out to examine any cross-reactivities between date palm polypeptides and antigens of some common foods and vegetables that have been implicated in the oral allergy syndrome (OAS). Because most of such cross-reactivities in other allergens are attributable to the presence of carbohydrate chains and profilin, their role was also investigated. METHODS: Fresh extracts of 20 common fruits and vegetables were prepared. Putative date profilins were isolated by affinity chromatography using a poly L-proline column. Date fruit extracts were digested by various endoglycosidases and the immunoglobulin (Ig)E binding of the postdigest products was assessed in immunoblots. Rabbit antisera to whole date fruit extracts, Timothy grass profilin and putative date profilins, as well as human sera from date sensitive individuals were used in immunoblotting, ELISA and in inhibition experiments. RESULTS: IgG, ELISA and immunoblot results with the different rabbit antisera and date-sensitive atopic sera showed several antigenic cross-reactivities and similar cross-reactivities were seen with birch, date and timothy grass profilins. IgE, ELISA and immunoblot experiments with pooled date sensitive human sera showed a range of cross-reactivities with some food extracts. A number of the IgE cross-reactivities could be inhibited after preabsorption of pooled sera with date extracts. Sixty-six percent of individual date hypersensitive human sera bound IgE in putative date fruit profilin and their pooled sera bound IgE in birch pollen profilin. IgE-binding of the endoglycosidase digested date fruit extracts to atopic serum pool was restricted to only a very low molecular weight band of 6.5-8 kDa. CONCLUSION: These results indicate that date palm polypeptides share cross-reactive IgG and IgE epitopes with a number of foods implicated in the oral allergy syndrome, bind to birch and Timothy grass profilins and bind IgE through glycosyl residues. The clinical relevance of these cross-reactivities needs to be further elucidated.

Cultivar-specific IgE-epitopes in date (Phoenix dactylifera L.) fruit allergy. Correlation of skin test reactivity and ige-binding properties in selecting date cultivars for allergen standardization.

BACKGROUND: Date fruits are allergenic and standardized extracts are required for diagnosis and therapy of this allergy. Since there are several cultivars of dates, this study was carried out to assess the allergenicity of different cultivars in order to select suitable source material for standardization. METHODS: The protein profiles of 18 of the most commonly sold varieties were compared by SDS-PAGE and their relative allergenicity assessed by SPT and IgE-based ELISA and immunoblotting. Thirty-two date fruit-sensitive patients were skin tested with a pooled extract from all the cultivars. Six of the patients with high SPT results (> or =3+) who volunteered were further tested with the 18 cultivars and their sera used in ELISA and immunoblotting. RESULTS: Six of the cultivars gave high SPT-positive reactions in > or =4 of patients. Five of these high SPT-reactive cultivars gave high IgE ELISA scores (> or =0.58) but individual cultivars varied in their number of IgE immunoblot bands. Cultivar-specific IgE-binding patterns indicated that only certain cultivars bound IgE at molecular weights of < or =14.3 and 27-33 kDa whilst all cultivars bound to a 54-58 kDa doublet. Cultivars that bind to the < or =14.3 and 27-33 kDa bands appeared to form the majority of the high SPT-reactive cultivars. When individual sera of 24 of the 32 SPT-positive patients were used in IgE immunoblots with the pooled cultivar extract, all sera bound IgE at < or =14.3 and 27-33 kDa and about 60% of sera bound to a 54-58 kDa doublet bands. CONCLUSIONS: These results indicate that allergenicity of date fruits is a cultivar-specific phenomenon. Sixty to 100% of sera from date fruit-allergic patients bind IgE to three major allergens of < or =14.3, 27-33 and 54-58 kDa. Five of the cultivars that evoke high SPT reactions, high IgE ELISA scores and bind IgE to the major allergens, can be selected for the preparation of 'in-house' allergen extracts and for allergen standardization.

Allergy to date fruits: characterization of antigens and allergens of fruits of the date palm (Phoenix dactylifera L.).

BACKGROUND: Date-palm (Phoenix dactylifera L.) fruits are eaten daily by most inhabitants of the Middle East and the neighboring countries. Recent reports have indicated that dates are allergenic. This study aimed to investigate the antigenic and allergenic potential of date fruits. METHODS: Date-fruit extracts from eight cultivars were evaluated in skin prick tests (SPT) in an atopic population, used to produce antisera, analyzed by SDS-PAGE, and fractionated by gel-filtration chromatography. Sera from SPT-positive individuals were evaluated by ELISA and RAST, and in anti-igE immunoblot experiments. RESULTS: About 13% of patients were SPT-positive for at least two extracts. SDS-PAGE of whole extracts revealed 15-18 protein bands of 6.5->100 kDa, and Sephacryl S-200 fractions gave distinct peptide bands. RAST and anti-IgE ELISA gave a range of positive results, which could be abrogated when sera were preabsorbed with fruit extracts. IgE immunoblots of different extracts with pooled positive sera revealed different anti-IgE-binding immunoprints. All the positive sera from fruit-allergic and pollen-allergic individuals bound strongly to two anti-IgE reactive bands of 6.5 to 12-14 kDa and 28-33 kDa, respectively, and about 50% of sera bound to a 54-58-kDa band. CONCLUSIONS: These results strongly indicate that 1) date-palm fruit is a potent allergen 2) sera from fruit-allergic as well as pollen-allergic patients recognize common fruit-specific epitopes 3) there is heterogeneity in patient responses to the different extracts.

Aeroallergens and viable microbes in sandstorm dust. Potential triggers of allergic and nonallergic respiratory ailments.

Aeroallergens and antigens in sandstorm dust, extracts of which were skin prick test (SPT) positive in allergic patients, were detected by rocket immunoelectrophoresis and ELISA. Fungi and bacteria isolated by agar settle plates and soil dilution and soil washing methods were enumerated and identified. Cat dander, Acacia, Alternaria, Aspergillus, Chenopodium, Cladosporium, Bermuda grass, Pithecellobium, Prosopis, Rumex, cultivated rye, and Washingtonia palm allergens were detected by both methods. Viable microbes including 1892 +/- 325 colony-forming units (cfu) of bacteria, and 869 +/- 75 cfu of fungi were isolated per gram of dust by the soil dilution method. Randomly selected microbial colonies on streaking and subculture were found to consist of between two and seven mixed colonies. Fungi including Alternaria, Aspergillus, Botrytis, Cladosporium, Mortierella, Mucor, Mycelia sterilia, Penicillium, Pythium, Ulocladium, Verticillium, and some yeasts were isolated. Actinomyces, Bacillus, Pseudomonas, and mostly coagulase-negative Staphylococcus species were identified, but the bulk of unidentified bacterial isolates were mainly mixed colonies of rods, cocci, coccobacilli, and some filamentous types. Six-hour agar settle-plate counts during sandstorms were 100 and 40% higher for bacteria and fungi, respectively, than without sandstorms. The most abundant aeroallergens were those of Acacia, Alternaria, Aspergillus, Bermuda grass, Cladosporium, cultivated rye, Prosopis, and cat dander. Pithecellobium dulce, Rumex crispus, and Washingtonia palm allergens were detectable for the first time in Riyadh. IgE reactivities of the dust in man were demonstrated by ELISA using sera from atopic, exposed, and normal subjects. These results indicate that sandstorm dust is a prolific source of potential triggers of allergic and nonallergic respiratory ailments, and the methods mentioned here should be routinely used for quick sampling of the environment.

Does actin polymerization status modulate Ca2+ storage in human neutrophils? Release and coalescence of Ca2+ stores by cytochalasins.

The aim of this paper was to establish whether actin polymerization modulated cytosolic Ca2+ storage in human neutrophils. Over the concentration ranges which inhibit actin polymerization, cytochalasins A, B, and D liberated Ca2+ from membrane-bound stores within neutrophils. Two Ca2+ storage sites were identified in neutrophils by the accumulation of the Ca2+ binding probe, chlortetracycline: one at the center of the cell and the other at the cell periphery. Confocal imaging demonstrated that cytochalasins released Ca2+ from the neutrophil periphery, but not from the central Ca2+ store. Ca2+ store release was coupled to Ca2+ influx, suggesting that the peripheral site may be a physiological store containing a Ca2+ influx factor. 3,3'-Dihexyloxacarbocyanine iodide staining organelles, which correlate with Ca2+ release sites, coalesced in neutrophils after treatment with cytochalasins. We propose that peripheral Ca2+ storage sites are restricted from coalescence by cortical polymerized actin and that Ca2+ store coalescence and Ca2+ release are coupled events.

Spatial organization of calcium dynamics in growth cones of sensory neurones.

The concentration of calcium ions in the cytosol ([Ca2+]i) has a dominant influence on neuronal development. A [Ca2+]i rise can, depending on the amplitude and location, promote outgrowth or dramatically inhibit it. We have used the fluorescent calcium indicators Fura-2 and Fura-2 dextran to measure [Ca2+]i dynamics in sensory neurones from the adult rat. [Ca2+]i was low and uniform in advancing growth cones, even during specific behaviours such as protrusion, filling and consolidation. A brief train of action potentials caused [Ca2+]i to rise at the extreme leading edge of the growth cone. [Ca2+]i changes in more proximal regions of the growth cone were much smaller. This spatially organized [Ca2+]i change, which may result from a concentration of calcium channels at the growth cone leading edge, is likely to function in spontaneously active regenerating axons in vivo to specifically activate calcium-dependent processes at the growth cone tip.

Monomeric human IgE evokes a transient calcium rise in individual human neutrophils.

Digital fluorescence calcium imaging was used to investigate and identify the primary biological responses of human neutrophils to monomeric immunoglobulin E (IgE). Treatment of neutrophils with IgE caused a transient rise in the level of intracellular calcium that was inhibited by pertussis toxin. The calcium rise was due mainly to release from an intracellular membrane-enclosed store that is also sensitive to the chemotactic peptide formyl-Met-Leu-Phe. The IgE-induced calcium transient was independent of Fc gamma receptors and of Fc epsilon receptor ligation. Our data suggest that the mere binding of IgE to neutrophils is sufficient to evoke a biological response without the need for IgE/receptor cross-linking.

The nucleus is insulated from large cytosolic calcium ion changes.

Extracellular events regulate functions in the cell nucleus by means of calcium ions acting through effector enzymes. Recently, the traditional view of the nuclear pore as freely permeable to small ions has been questioned as a result of reports that nuclear calcium can be regulated independently of cytosolic calcium. We have used confocal microscopy of fluorescent Ca2+ indicators to investigate the Ca2+ dynamics between cytosol and nucleus in neurons. We find that a previously reported amplification of Ca2+ changes in the nucleus is a measurement artefact. Small changes of cytosolic Ca2+ cause equally rapid changes in nuclear Ca2+, consistent with the free diffusion of Ca2+ through nuclear pores. In contrast, large cytosolic Ca2+ increases (above 300 nM) are attenuated in the nucleus. Our results show the nuclear envelope shapes but does not block the passage of Ca2+ signals from cytosol to nucleus.

A narrow window of intracellular calcium concentration is optimal for neurite outgrowth in rat sensory neurones.

We have examined neurite outgrowth in rat sensory neurones when cytosolic free calcium concentration ([Ca2+]i) was varied in the range 0-60 nM. Neurite outgrowth was maximal at 35 nM [Ca2+]i and was reduced at higher and lower values of [Ca2+]i. These results provide direct evidence for Mattson and Kater's suggestion of an optimal calcium range for growth cone function.

The effect of haemodialysis on the pharmacokinetics of tenoxicam in patients with end-stage renal disease.

We have studied the pharmacokinetics of tenoxicam after single and multiple oral doses of 20 mg in five patients (2 men and 3 women) with end-stage renal disease undergoing haemodialysis. After a single dose, tenoxicam had a half-life (t1/2) of 33 h, an apparent clearance (CL.f-1) of 4.3 ml.min-1, and an apparent volume of distribution (Vz.f-1) of 11.81. The maximum tenoxicam concentration (Cmax) was 4.3 mg.l-1 at a median tmax of 1.7 h. There were no significant differences between the values calculated from the pre- or post-dialyser port plasma samples. Tenoxicam plasma concentrations measured during once daily dosing before and after haemodialysis showed that tenoxicam does not accumulate. Our findings suggest that dosage adjustment may not be required in patients with end-stage renal disease on haemodialysis taking tenoxicam.

"Clamping" actin in polymerized form in electro-permeabilized neutrophils inhibits oxidase activation.

Electro-permeabilized neutrophils take-up small membrane-impermeant molecules into their cytoplasm, yet retain the ability to activate their oxidase and to transiently polymerize actin in response to f-met-leu-phe (fmlp). Using this system phalloidin was introduced into the cytosol in order to determine whether polymerization of actin affects oxidase activation. Cytosolic phalloidin prevented the depolymerization of actin following stimulation with fmlp, which was consequently "clamped" in the polymerized form during oxidase activation. Under these conditions oxidase activation was inhibited, the extent of inhibition being related to the level of polymerization at which the actin was "clamped". It was concluded that the actin polymerization which accompanies stimulation with fmlp interacts with other intracellular signals to limit oxidase activation.

Actin polymerization in neutrophils is triggered without a requirement for a rise in cytoplasmic Ca2+.

Stimulation of rat neutrophils with the peptide fMetLeuPhe caused (i) the appearance of a 40 kDa protein in the Triton-X-100-insoluble cytoskeleton, (ii) the disappearance of DNAase inhibition from the cytosol and (iii) the appearance of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD-phallacidin) binding sites. All three observations were consistent with a rapid and transient assembly of polymerized actin, peaking at approximately 5 s and returning to near resting levels within 40 s. By experimentally depleting the cells of Ca2+ and increasing the cytoplasmic Ca2+ buffering capacity, the peptide-induced Ca2+ transient was reduced from a peak of 900 nM to 250 nM, without inhibiting actin polymerization, and this peak was sustained for at least 2 min. A further dissociation between the triggering of actin polymerization and peptide-induced Ca2+ elevation and oxidase activation was demonstrated at low concentrations of peptide (1-100 pM), actin polymerization being triggered without an elevation in Ca2+ or activation of the oxidase. Two other agents which induced actin polymerization, phorbol 12-myristate 13-acetate and latex beads, failed to elevate cytoplasmic Ca2+. It was therefore concluded that neither Ca2+ nor those intracellular messengers which act with Ca2+ to trigger the neutrophil oxidase are responsible for triggering actin polymerization in neutrophils.

The use of fura-2 to determine the relationship between cytoplasmic free Ca2+ and oxidase activation in rat neutrophils.

Incubation of rat neutrophils with fura-2-acetoxy-methyl ester (fura-2/AM) resulted in the loading of fura-2 almost exclusively into the cytoplasm. Despite the additional presence of fura-2/AM esterase activity in the granules, only 1.5% of cell-associated fura-2 was located within these organelles. Fura-2 leaked from neutrophils at an acceptably low rate 0.16 +/- 0.05% min-1 at 37 degrees C. At intracellular concentrations of fura-2 up to 500 microM, there was no effect on oxidase activation; although the cellular ATP content was reduced to approximately 50%. The peptide, f-met-leu-phe (fmlp), 1 microM, produced intensity changes of fluorescence excited at 340nm and 380nm which were consistent with a cytoplasmic Ca2+ rise from the resting level of 94 +/- 13nM to 768 +/- 173nM (n = 6). Intracellular concentrations of fura-2 greater than 1mM were required to buffer effectively this rise, and it was estimated that an intracellular fura-2 concentration required for a high signal:autofluorescence ratio (100 microM) the cytoplasmic Ca2+ buffering capacity of the cells was increased by only 10%. The rise in cytoplasmic free Ca2+ induced by the peptide preceded activation of the oxidase by several seconds, and the magnitude of the response was dependent on the extent of the Ca2+ rise, half-maximal activation being achieved at approx. 600nM. These data were therefore consistent with a secondary messenger role for cytoplasmic Ca2+ in triggering neutrophil oxidase activation.

Diacylglycerol kinase inhibitor, R59022, potentiates neutrophil oxidase activation by Ca2+-dependent stimuli. Evidence for two separate but convergent pathways.

An inhibitor of diacylglycerol kinase, R59022, enhanced activation of the neutrophil oxidase stimulated by the Ca2+-ionophore, A23187 (1 microM), and by N-formyl-methionyl leucyl-phenylalanine (1 microM). The enhancement was reversed by two inhibitors of c-kinase, retinal (10 microM), and gossypol (20 microM). Activation by phorbol-myristyl-acetate and unopsonised latex beads were not enhanced. It was concluded that the chemotactic peptide generated diacylglycerol, but that maximum activation of c-kinase by this route was not achievable. The role of diacylglycerol in activation by beads remained unclear.