Insulin treatment enhances casein kinase II (CKII) activity in 3T3-L1 mouse adipocytes and H4-IIE rat bepatoma cells, the magnitude of the activation varying from 30% to 150%. Activation of CKH was apparent after 5 min of exposure of 3T3-L1 cells to insulin, was maximal by 10 min, and persisted through 90 min. The insulin-stimulated activity was inhibited by low concentrations of heparin and was stimulated by spermine. Activation of CKII was effected by physiological concentrations of insulin'(EC50 = 0.15 nM), suggesting that the effect is a true insulin response and not one mediated through insulin-like growth factor receptors. Epidermal growth factor (100 ng/ml for 10 min) also activated CKII in A431 human carcinoma cells, which is consistent with other observations that insulin and epidermal growth factor may have some common effects. Insulin stimulation of CKII activity was due to an increase in the maximal velocity of the kinase; the apparent Km for peptide substrate was not altered. Enhanced activity did not appear to result from increased synthesis of CKII protein, because cycloheximide did not block the effect and because an immunoblot developed with antiserum to CKII showed no effect of insulin on the cytosolic'concentration of CKII. Because insulin-stimulated CKII activity was maintained after chromatography ofcell extracts on Sephadex G-25, it is unlikely that the effect is mediated by a low-molecularweight activator ofthe kinase. Rather, the results are consistent with the possibility that insulin activates CKII by promoting a covalent modification of the kinase.There is considerable evidence that reversible protein phosphorylation contributes to the mechanism of insulin action (reviewed in ref. 1). The f3 subunit of the insulin receptor is a protein-tyrosine kinase that is activated by insulin, and the hormone promotes phosphorylation of several proteins on tyrosine residues (1). Insulin also enhances phosphorylation of sefine and threonine residues in proteins, including the insulin receptor (1), ribosomal protein S6 (2-4), ATP-citrate lyase (5, 6), membrane-bound cAMP phosphodiesterase (7) Although CKII has been implicated in the regulation of a wide variety of cellular processes, including the synthesis of glycogen, fatty acids, RNA, and protein (11, 12), there have been few studies of its regulation. Such studies have been facilitated, however, by the development of a specific peptide substrate for CKII (13) that was useful for estimating changes in kinase activity during differentiation of 3T3-L1 cells (14). Because CKII appears to phosphorylate an insulin-stimulated phosphorylation site and because it has been implicated in the regulation of a variety of fundamental cellular processes, we undertook studies of the short-term regulation of CKII activity and found that the kinase is rapidly activated by insulin (12). In this paper, we report in detail the characteristics of the response of CKII to insulin and that the kinase is also activated by epidermal growth factor. EXPERIMENTAL PROCE...

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Migraine is a debilitating disease that affects almost 15% of the population worldwide and is the first cause of disability in people under 50 years of age, yet its etiology and pathophysiology remain incompletely understood. Recently, small molecules and therapeutic antibodies that block the calcitonin gene-related peptide (CGRP) signaling pathway have reduced migraine occurrence and aborted acute attacks of migraine in clinical trials and provided prevention in patients with episodic and chronic migraine. Heterogeneity is present within each diagnosis and patient's response to treatment, suggesting migraine as a final common pathway potentially activated by multiple mechanisms, e.g., not all migraine attacks respond to or are prevented by anti-CGRP pharmacological interventions. Consequently, other unique mechanisms central to migraine pathogenesis may present new targets for drug development. Pituitary adenylate cyclase-activating peptide (PACAP) is an attractive novel target for treatment of migraines. We generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1910) with reactivity to both PACAP38 and PACAP27. In vitro, ALD1910 effectively antagonizes PACAP38 signaling through the pituitary adenylate cyclase-activating peptide type I receptor, vasoactive intestinal peptide receptor 1, and vasoactive intestinal peptide receptor 2. ALD1910 recognizes a nonlinear epitope within PACAP and blocks its binding to the cell surface. To test ALD1910 antagonistic properties directed against endogenous PACAP, we developed an umbellulone-induced rat model of neurogenic vasodilation and parasympathetic lacrimation. In vivo, this model demonstrates that the antagonistic activity of ALD1910 is dosedependent, retaining efficacy at doses as low as 0.3 mg/kg. These results indicate that ALD1910 represents a potential therapeutic antibody to address PACAP-mediated migraine.

ABSTRACrCell wall composition of a stable suspension of proso millet (Panicum miliaceum L. cv Abarr) cells is similar to those of tissues and cell suspensions of other graminaceous species. Extraction of hemicelluloses with step-wise increasing concentrations of alkali yields materials that, like those of embryonal cells of maize coleoptiles, comprise mostly glucuronoarabinoxyla, xyloglucan, and small amounts of (1-3),(14)-f- MATERIALS AND METHODS Development of Cell Suspension Cultures. Embryogenic calli of proso millet (Panicum miliaceum L. cv Abarr) were obtained from mature seed (11) and were transferred monthly to LS medium (12) containing 2.5 mg/L 2,4-D, 4% (w/v) sucrose, and 0.8% (w/v) agar. About 1 g ofthe light yellow callus was dispersed into liquid medium in which LS medium was replaced by MS liquid medium (20; commercially packaged by GibCo) and 3% (w/v) sucrose. The suspension was initiated in 20 ml of medium in a 250-ml Erlenmeyer flask on a gyratory shaker at 100 cycles/ min with a 2.5 cm displacement. Ten ml of fresh medium was added three times at 4 to 5 d intervals, and after 2 weeks, 10 ml of the suspension was transferred with a sterile open-end pipet to 10 ml of fresh medium. The process was repeated for three culture cycles until the culture had stabilized. About 0.5 g of the cells was then transferred to 40 ml of fresh medium, and cells were subcultured biweekly. To yield suspensions of smaller cell colonies, a serological pipet was used to transfer cultures. Alternatively, small colonies that accumulated as a 'film' on the flask at or near the surface of the liquid medium were also collected and transferred to fresh medium. Both methods yielded stable cell suspensions that were maintained for 9 months before beginning these experiments. From observations by light microscopy,