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Normal adult rat chromaffin cells show a robust proliferative response in vitro to nerve growth factor (NGF) and other mitogens. In contrast, PC12 rat pheochromocytoma cells proliferate in the absence of exogenous mitogens and undergo neuronal differentiation in response to NGF. We demonstrate in this work that the antiproliferative drug rapamycin suppresses normal chromaffin cell proliferation. This effect is blocked by FK 506, indicating that it occurs via interaction of rapamycin with its intracellular binding protein, FKBP. Rapamycin must be added within 2 days of mitogen stimulation in order to be fully effective. PC12 cells are refractory to the antiproliferative effect of rapamycin, although rapamycin does exert its expected inhibitory effect in PC12 cells on both basal and NGF-stimulated activation of one of its biochemical targets, the 70-kDa S6 protein kinase (p70S6K). The discordant findings suggest that a proliferative signal normally requiring activation of p70S6K either is unnecessary in PC12 cells or is provided by a downstream or cross-communicating pathway. They also suggest that p70S6K does not participate in the morphological responses of PC12 cells to NGF. Determining the basis for rapamycin resistance in PC12 cells might help to identify signaling abnormalities involved in the pathogenesis of pheochromocytoma.

Adult rat chromaffin cells may proliferate or extend neurites when stimulated by nerve growth factor (NGF) but their response is predominantly proliferative, making them a unique model for studying how mitogenic specificity is achieved. We examined contributions of the NGF receptors trk and p75 and of the major NGF signaling pathways to proliferation versus neurite outgrowth. The type of initial NGF response does not correlate with intensity of immunoreactivity for trk or p75. However, proliferation is initiated at lower NGF concentrations than neurite outgrowth, suggesting that it requires a less intense signal. Mitogenic cooperativity between receptors at low NGF concentrations is suggested by inhibitory effects of p75-blocking antibodies, but responses to trk-agonist antibody indicate that trk activation alone can induce proliferation. NGF-induced phosphorylation of ras-mediated mitogen-activated protein kinases (MAPK) Erk1 and Erk2 is as prolonged in normal chromaffin cells as in PC12 cells, where NGF is neuritogenic. Trk-agonist antibody, which is as mitogenic as NGF but less neuritogenic, causes equally prolonged but less intense ERK phosphorylation. The MAPK kinase(MEK-1) inhibitor PD98059 partially inhibits Erk phosphorylation and does not inhibit chromaffin cell proliferation, while depolarization selectively inhibits proliferation without blocking Erk phosphorylation. Proliferation is markedly reduced by the phosphoinositol-3 (PI-3) kinase inhibitor LY294002 while downregulation of protein kinase C (PKC) causes no change. These findings suggest that low-level, rather than short-duration, stimulation of NGF signaling pathways causes NGF to be mitogenic. Ras-mediated MAPK activation may be more critical in neurite outgrowth than in proliferation and PI-3 kinase may be the major mitogenic determinant.

Chronic consumption by rats of diets rich in sugars or sugar alcohols leads to an increased incidence of pheochromocytomas. This relationship is hypothesized to be based on altered Ca 2+ homeostasis due to increased intestinal Ca 2+ absorption. Other agents associated with pheochromocytomas in rats in long-term toxicity studies have been shown to increase chromaffin cell proliferation, leading to the suggestion that the tumors occur secondarily to increased chromaffin cell turnover. We have demonstrated marked stimulation of chromaffin cell proliferation by vitamin D 3 , a potent stimulus to Ca 2+ absorption not previously associated with adrenal medullary toxicity. This effect is detectable during the first week of dietary supplementation and persists throughout a 4-week time course. Lactose and xylitol, representative of sugars and sugar alcohols associated with pheochromocytomas, are also mitogenic but to a lesser extent, with their effects first detectable during Week 4 of dietary supplementation. Vitamin D 3 , its active metabolite calcitriol, lactose, and xylitol all fail to stimulate proliferation of rat chromaffin cells in vitro. The mitogenic effects of these agents may be mediated presynaptically in vivo. The data suggest that altered Ca 2+ homeostasis may increase chromaffin cell proliferation and support the hypothesis that diets containing high concentrations of sugars and sugar alcohols cause pheochromocytomas in rats secondarily by this mechanism.

This report describes the localization of the REST (RE1-silencing transcription factor) gene to human chromosome 14q12 using fluorescence in situ hybridization. This gene plays a role in the regulation of certain genes exclusively in neurons. 12 refs., 1 fig.

The initial event and site of cocaine-induced hepatic injury have not been elucidated. In an attempt to identify the minimal effective dose and the site of injury, we have examined the livers of mice exposed to small daily doses of cocaine, using morphological and biochemical methods. All doses of cocaine greater than 5 mg/kg were able to cause significant elevation of serum glutamic pyruvic transaminase. Light microscopy revealed a progression of centrilobular necrosis as the dose increased from 10-30 mg/kg. The initial morphologic changes observed prior to necrosis included aggregation of intermediate filaments and dilation of rough endoplasmic reticulum with loss of ribosomes. Immunohistochemistry, using antibodies to cytokeratins, showed staining of individual hepatocytes in livers from cocaine-treated animals but not in controls. In contrast to earlier reports, we found little, if any, disruption of mitochondria. In vitro, the direct application of cocaine, norcocaine, and N-hydroxynorcocaine on isolated mitochondria had no effect on the ADP:O or respiratory control ratios, at concentrations up to 2.0 mM. Our studies demonstrate that any early cellular alterations in cocaine-induced hepatic injury are manifested in intermediate filaments and endoplasmic reticulum with no evidence of mitochondrial involvement.

The medieval kingdoms of Portugal and Leon faced a common Muslim enemy on their southern frontiers. They also viewed each other as potential threats, along a boundary which grew in length as the Muslims were pushed back. Military preparedness was in these circumstances a major preoccupation of the monarchs in the two kingdoms. Offensive forces were needed for continued territorial expansion, and defensive forces were needed to protect lands that had already been gained, whether from Muslim counterattack or from inroads by the Christian neighbor. The towns and their militias figured prominently in royal military policy, in Portugal and Leon as elsewhere in the Iberian Peninsula. This is most evident in the charters awarded to the towns, which frequently contain provisions governing obligations for offensive and defensive military service. These provisions are pervasive enough and significant enough to provide information not only about the changing military role of the towns but also more generally about municipal legal history and royal strategies for settling and holding newly acquired territory. Municipal charters the foral in Portugal (plural forais) and the fuero in Leon, terms derived from the Latinforum survive in substantial numbers in the period between 1055 and 1279, the years which mark the limits of this study. The documentation is more complete for Portugal than for Leon, because the Portuguese royal archives have been less beset by natural and man-made disasters. The discussion accordingly gives more space to the Portuguese materials, but my purpose is comparative. In order to investigate the legal interaction between Portugal and Leon, I have attempted to summarize developments on both sides of the border to the extent that the surviving documents allow. My approach is to identify families of charters containing identical or nearly identical military law, a method well established for Leon and Castile but less

In modern society, enmeshed with confrontations involving the individual, military service and the state, historians are often inclined to make comparisons with the distant past which offer relief from the pressures of contemporary history. Regarding military service, the Middle Ages are occasionally suggested as an age when combat was sporadic, when only the small feudal aristocracy encountered a martial obligation, and when the remainder of society could concentrate on the other burdens of life, free of the paraphernalia of war, hot or cold. As with many romantic generalizations concerning the period, the comparative bliss of the medieval non-combatant is open to question. Many would note, however, that the feudal classes did possess a monopoly on warfare for several centuries in parts of Continental Europe, and would tend to place all discussion of military institutions within a feudal context.

THE TWELFTH AND THIRTEENTH CENTURIES were pivotal in the history of the Iberian Peninsula. The Reconquest created several large Christian principalities in the territory recovered from Islam, imposing a large number of northern Spaniards upon the south and fundamentally shaping the outlook of peninsular natives from that day to this.' As the Christian states drove to the south in an uneven series of thrusts, the Reconquest drained off the northern population, a drain which was never entirely replaced by extrapeninsular colonists and crusaders. During the struggle for the Guadiana and lower Ebro valleys in the mid-twelfth century, the north's population shortage made it difficult to find sufficient colonists for the conquered regions. The rulers of Leon, Castile, Aragon, and Catalonia were compelled to attract migrants by granting liberal municipal charters-fueros-that enlarged personal freedoms and immunities. The conquered towns of the south drew their Christian populations from an adventurous, mobile segment of the northern population willing to relocate on the edge of European civilization. In the southern municipalities aggressive Christian conquerors came into daily contact with an older, more urbane, and recently defeated native populace. The farther south the northern Christians penetrated, the older and deeper were the Arab and Berber civilizations they encountered. The Iberian municipalities that were the scene of this Muslim-Christian confrontation have received some attention from scholars over the last several decades, although a fresh examination in depth is still badly needed.2 The exploration of munici