Abstract. Four mutant PC 12 pheochromocytoma cell lines that are nerve growth factor (NGF)-nonresponsive (PC12 n"r) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC 12 ""~ cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC 12 ~nr cells closely resembled non-NGFtreated PC 12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC 12 nnr lines differed from PC 12 cells in three ways in addition to failure of NGF response: (a) PC12 n"r cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC I2 cells. (b) The PC 12 nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC I2 cells possess both high-and low-affinity NGF binding sites. (c) The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC 12 n"r cells compared to PC I2 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC I2 TM cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity.

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Oxygen consumption is used to study the dynamics of dissolved organic carbon (DOC) utilization by bacteria. Preliminary incubation experiments examining oxygen consumption and bacterial growth demonstrated that a small labile fraction of the total DOC pool typically supports bacterial metabolism. Bacterial growth and respiration rates were frequently fastest within the first few hours of incubation experiments suggesting that the pool of organic matter used for growth was rapidly consumed. Comparisons of bacterial production and respiration with total DOC concentrations suggested that approxinlately 1 to 3 % of the pool supports bacterial growth. The presence of a small labile component of the DOC pool suggests a close coupl~ng between bacteria and sources of substrate. Bacterial coupling to phytoplankton was examined in mesocosm experiments in which phytoplankton were enriched with nutrients and bacterial production and oxygen consumption were followed over diel cycles. Bacterial production, oxygen consumption and the availability of organic matter were highest during daylight, when phytoplankton production was assumed to be greatest. The effect of a varying carbon supply over diel cycles on bacterial growth efficiency was examined. During the mesocosm experiment, growth efficiencies were greatest during daylight when substrate availability was greatest. At several estuarine sites, efficiencies varied markedly over daily and seasonal temporal scales. These results suggest that growth efficiency is an important consideration when estimating the bacterial role in aquatic carbon cycles.

The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the PC 12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 d of exposure to NGF, these cells acquire many of the properties of normal sympathetic neurons . However, by phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, it seemed likely that some of the initial responses to the factor may be surface related .PC 12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface . Ruffles appear over the dorsal surface of the cells within 1 min, become prominent by 3 min, and almost disappear by 7 min. Microvilli, conversely, disappear as the dorsal ruffles become prominent. Ruffles are seen at the periphery of the cell at 3 min, are prominent on most of the cells by 7 min and are gone by 15 min . The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli reappear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h.These changes were not observed with other basic proteins, with a-bungarotoxin (which binds specifically to PC 12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earliest NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor's action. 870J . CELL BIOLOGY

Tests have been made of the action of the methyltransferase inhibitors 5'-S-methyl adenosine, 5'-S-(2-methyl-propyl)-adenosine, and 3-deaza-adenosine ± L-homocysteine thiolactone, on nerve growth factor (NGF)-dependent events in the rat pheochromocytoma line PC12 . Each of these agents inhibited NGF-dependent neurite outgrowth at concentrations of the order of millimolar. Slow initiation of neurite outgrowth over several days and more rapid regeneration of neurites (=1 d) were blocked, as was the priming mechanism necessary for genesis of neurites . The inhibitions were reversible in that PC12 cells maintained for several days in the presence of inhibitors grew neurites normally after washout of these agents. Other NGF-dependent responses of the PC12 line (i.e., induction of ornithine decarboxylase activity [over 4 h], enhancement of tyrosine hydroxylase phosphorylation [over 1 h], and rapid changes in cell surface morphology [30 s onward]) were inhibited by each of the agents . In contrast, corresponding epidermal growth factor-dependent responses in ornithine decarboxylase activity, phosphorylation, and cell surface morphology were not blocked, but instead either unaffected or enhanced, by the methylation inhibitors. These inhibitors did not act by blockade of binding of NGF to high-,or low-affinity cell surface receptors, though they partially inhibited internalization of [ 125 1]NGF . The inhibition of rapidly-induced NGF-dependent events and the differential inhibition of responses to NGF and epidermal growth factor imply that the methyltransferase inhibitors specifically block one of the first steps in the mechanistic pathway for NGF .