Planning Benchmark Study for Stereotactic Body Radiation Therapy of Pancreas Carcinomas with Simultaneously Integrated Boost and Protection: Results of the DEGRO/DGMP Working Group on Stereotactic Radiation Therapy and Radiosurgery.

PURPOSE/OBJECTIVE: The proximity or overlap of PTV and OAR poses a major challenge in SBRT of pancreatic cancer (PACA). This international treatment planning benchmark study investigates whether Simultaneously Integrated Boost (SIB) and Protection (SIP) concepts in PACA SBRT can lead to improved and harmonized plan quality. MATERIALS/METHODS: A multiparametric specification of desired target doses (GTVD50%, GTVD99%, PTVD95%, PTV0.5cc) with two prescription doses of GTVD50%=5×9.2Gy (46Gy) and GTVD50%=8×8.25Gy (66Gy) and OAR limits were distributed with planning CT and contours from 3 PACA patients. In phase 1, plans were ranked using a scoring system for comparison of trade-offs between GTV/PTV and OAR. In phase 2, re-planning was performed for the most challenging case and prescription with dedicated SIB and SIP contours provided for optimization after group discussion. RESULTS: For all 3 cases and both phases combined, 292 plans were generated from 42 institutions in 5 countries using commonly available treatment planning systems. The GTVD50% prescription was performed by only 76% and 74% of planners within 2% for 5 and 8 fractions, respectively. The GTVD99% goal was mostly reached, while the balance between OAR and target dose showed initial SIB/SIP-like optimization strategies in about 50% of plans. For plan ranking, 149 and 217 score penalties were given for 5 and 8 fractions, pointing to improvement possibilities. For phase 2, the GTVD50% prescription was performed by 95% of planners within 2% and GTVD99% as well as OAR doses were better harmonized with notable less score penalties. Fourteen of 19 planners improved their plan rank, 9 of them by at least 2 ranks. CONCLUSION: Dedicated SIB/SIP concepts in combination with multiparametric prescriptions and constraints can lead to overall harmonized and high treatment plan quality for PACA SBRT. Standardized SIB/SIP treatment planning in multicenter clinical trials appears feasible after group consensus and training.

A machine learning tool for re-planning and adaptive RT: A multicenter cohort investigation.

PURPOSE: To predict patients who would benefit from adaptive radiotherapy (ART) and re-planning intervention based on machine learning from anatomical and dosimetric variations in a retrospective dataset. MATERIALS AND METHODS: 90 patients (pts) treated for head-neck cancer (H&N) formed a multicenter data-set. 41 H&N pts (45.6%) were considered for learning; 49 pts (54.4%) were used to test the tool. A homemade machine-learning classifier was developed to analyze volume and dose variations of parotid glands (PG). Using deformable image registration (DIR) and GPU, patients' conditions were analyzed automatically. Support Vector Machines (SVM) was used for time-series evaluation. "Inadequate" class identified patients that might benefit from replanning. Double-blind evaluation by two radiation oncologists (ROs) was carried out to validate day/week selected for re-planning by the classifier. RESULTS: The cohort was affected by PG mean reduction of 23.7±8.8%. During the first 3weeks, 86.7% cases show PG deformation aligned with predefined tolerance, thus not requiring re-planning. From 4th week, an increased number of pts would potentially benefit from re-planning: a mean of 58% of cases, with an inter-center variability of 8.3%, showed "inadequate" conditions. 11% of cases showed "bias" due to DIR and script failure; 6% showed "warning" output due to potential positioning issues. Comparing re-planning suggested by tool with recommended by ROs, the 4th week seems the most favorable time in 70% cases. CONCLUSIONS: SVM and decision-making tool was applied to overcome ART challenges. Pts would benefit from ART and ideal time for re-planning intervention was identified in this retrospective analysis.

Comparative study of permanent interstitial prostate brachytherapy post-implant evaluation among seven Italian institutes.

BACKGROUND AND PURPOSE: The purposes of this multicentric study are (a) the evaluation of four different commercially available treatment planning systems (TPSs) and (b) to verify whether the dosimetric results are comparable, also when considering the inter-observer variabilities and the different scanning protocols used. This work is to be considered a first step to test the value of multicentric studies based on dosimetric evaluation of the quality of the implants. PATIENTS AND METHODS: Four different TPSs were used and the following tests were performed:Comparison of the parameters and mathematical algorithms used; comparison of the dose distributions generated by three different geometries of sources based on 32 dose-points on each source geometry. An octagonal geometric phantom was used to compare volume algorithms and dose-volume histogram (DVH) calculations (V150(Gy), V100(Gy), V50(Gy) and V25(Gy)). Comparison of the post-plan source distribution performed on a prostate-phantom implanted with (125)I seeds. A CT scan of the phantom was obtained at each participating center. Both the geometrical coordinates (with respect to the most caudal one), and the spread of the geometrical distribution, were calculated. The volumes included within different isodoses were also collected. Comparison of the post-plan source distribution performed on an actual patient. Post-plan V100% and D90(Gy) derived from seed distributions obtained by different operators were calculated, using the same target delineation. RESULTS: All the considered TPSs satisfied the AAPM dosimetric parameter recommendations. Point-dose examinations revealed differences smaller than 5%, except for one of the systems. Although the volume algorithm was not the same for all systems, no statistically significant difference was found in the volume measurements. The DVHs also presented differences smaller than 5%, except for one TPS. The distances between the seeds, based on the same CT images, showed a mean SD of 0.13 mm. The mean maximum difference of the position of each seed was 0.36 mm. The most significant errors were made in the cranio-caudal direction (mean maximal difference: 0.44 mm); here the size of the step between slices played an important role. The algorithm of source positioning of the different TPSs may also help explain this difference. The compiled DVHs showed differences smaller than 5%. Post-plans derived from different seed distributions showed a mild dependence upon operators. We obtained a mean value of 97.8 and 152.7 with a percentage of SD of 0.43 and 1.7, respectively, for V100% and D90(Gy). CONCLUSIONS: Three-dimensional (3D) geometric reconstructions of seed distributions are slightly dependent upon the operators and the scanning protocols have little effect on the dosimetric evaluation. Some relevant discrepancies were found between one of the TPSs and the other three if few sources were used; increasing the number of seeds those differences became less pronounced. Multicentric studies on the quality of prostate implants based on post-implant dosimetry are feasible, provided an accurate step-wise evaluation of the procedure be performed.

Differential development of cholinergic nerve terminal markers in rat brain regions: implications for nerve terminal density, impulse activity and specific gene expression.

During critical developmental periods, cholinergic activity plays a key role in programming the development of target cells. In the current study, ontogeny of cholinergic terminals and their activity were contrasted in 4 brain regions of the fetal and neonatal rat using choline acetyltransferase activity, which is unresponsive to changes in impulse flow, and [3H]hemicholinium-3 binding, which labels the high-affinity choline transporter that upregulates in response to increased neuronal stimulation. In all 4 regions (cerebral cortex, midbrain + brainstem, striatum, hippocampus) choline acetyltransferase activity increased markedly from late gestation through young adulthood, but generally did so in parallel with the expansion of total membrane protein, reflective of axonal outgrowth and synaptic proliferation. In contrast, [3H]hemicholinium-3 binding was extremely high in late gestation and immediately after birth, declined in the first postnatal week and then rose again into young adulthood. The ontogenetic changes reflected alterations primarily in the number of binding sites (Bmax) and not in binding affinity. Only the latter phase of development of [3H]hemicholinium-3 binding corresponded to the ontogenetic changes in choline acetyltransferase activity; in the hippocampus, there were disparities even in young adulthood, where [3H]hemicholinium-3 binding showed a spike of activity centered around the 5th to 6th postnatal week, whereas choline acetyltransferase did not. Correction of binding for membrane protein development did not eliminate any of the major differences in developmental patterns between the two markers. These results suggest that development of the choline transporter binding site is regulated independently of the outgrowth of the bulk of cholinergic nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual control of DNA synthesis by alpha- and beta-adrenergic mechanisms in normoxic and hypoxic neonatal rat brain.

To examine how catecholamines influence cell replication in the developing brain, we examined regional [3H]thymidine incorporation into DNA after acute challenge with an alpha-adrenergic blocking agent (phenoxybenzamine) or a beta-blocker (propranolol). Phenoxybenzamine inhibited DNA synthesis in 1-day-old rat pups but the effect was less pronounced at 8 days; regional differences corresponded to transient expression of alpha-receptors and their subsequent maturational decline. Propranolol given at 1 day of age exerted a regionally selective, promotional effect on DNA synthesis; in contrast, at 8 days, propranolol inhibited DNA synthesis in all brain regions. Propranolol, but not phenoxybenzamine, also exacerbated the reduction in DNA synthesis caused by neonatal hypoxia, and again the effect was limited to the 1-day-old group. These results indicate that catecholamines exert a dual action on DNA synthesis; the effects are dependent upon maturational profiles of specific receptor populations which are either transiently expressed or which couple to cell replication only during a critical period.

Fetal nicotine exposure ablates the ability of postnatal nicotine challenge to release norepinephrine from rat brain regions.

Exposure of the fetus to nicotine is known to affect the function of noradrenergic pathways in the central nervous system. In the current study, synaptic mechanisms underlying the functional defects were evaluated in the offspring of pregnant rats given nicotine infusions of 2 mg/kg/day throughout gestation, administered by osmotic minipumps. At 30 days postpartum, norepinephrine levels in brain regions of the offspring were significantly reduced. More importantly, acute challenge with either 0.1 mg/kg or 0.3 mg/kg of nicotine evoked significant norepinephrine release from brain regions of control animals, but failed to do so in the fetal nicotine cohort. These results suggest that prenatal exposure to nicotine produces a deficit in subsequent noradrenergic responsiveness, deficits which may participate in behavioral and neuroendocrine abnormalities.

Altered development of basal and forskolin-stimulated adenylate cyclase activity in brain regions of rats exposed to nicotine prenatally.

Exposure of the fetus to nicotine is known to affect cellular development, synaptogenesis and synaptic activity of a wide variety of neurotransmitter pathways in the central nervous system. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipumps. After birth, offspring of the nicotine infused dams displayed marked alterations in membrane-associated adenylate cyclase activity; the regional selectivity correlated both with nicotinic cholinergic receptor concentration and the maturational timetable of each region. In the midbrain and brainstem, which display relatively high receptor concentrations and earliest cell development, basal adenylate cyclase activity in the nicotine group was elevated in the immediate period postpartum, returned to normal by the end of the first month, but then became subnormal in young adulthood. The initial promotion of basal activity was mirrored by forskolin-stimulated activity, suggesting that in this phase, the alterations were occurring at the level of the adenylate cyclase catalytic unit itself. The lack of effect on forskolin stimulation in the later phase, where basal activity was subnormal in the nicotine group, suggests that some alterations in regulatory subunits are responsible for the maturational switch in nicotine's effects on adenylate cyclase. In the cerebellum, where cell replication occurs primarily after birth and receptor concentrations are low, basal adenylate cyclase showed only a deficit in the nicotine group; again, although forskolin stimulation was significantly affected, the actions on basal activity were much more prominent, suggesting defects at the level of G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

In search of a mechanism for receptor-mediated neurobehavioral teratogenesis by nicotine: catecholamine release by nicotine in immature rat brain regions.

Nicotine disrupts central nervous system development through interactions with nicotinic cholinergic receptors found in immature brain, leading to discoordination of target cell replication and differentiation. However, it is unclear whether the net result is achieved by nicotine's actions on its specific target cells, or indirectly through receptor-mediated release of other neurotransmitters, such as catecholamines, that possess neurotrophic properties. In the current study, developing rats (1, 7, 14 and 21 days old) were challenged acutely with nicotine (0.3 mg/kg) and the release of catecholamines was evaluated in vivo (AMPT method) in three brain regions that differ in nicotinic receptor concentrations. Nicotine did not stimulate catecholamine release at birth, but developed the capacity to do so in parallel with the ontogeny of nicotinic cholinergic receptors in the midbrain+brainstem and in the forebrain. In the cerebellum, which remains poor in nicotinic receptors, no response was obtained at any age. Superimposed on this general pattern, changes in sensitivity to nicotine were also seen that corresponded to ontogenetic changes in endogenous cholinergic tone, suggesting that receptor desensitization occurs normally during developmental stages in which neuronal activity is high. The absence of a catecholamine response to nicotine at birth in the rat indicates that neurobehavioral teratology associated with fetal nicotine exposure does not reflect secondary actions mediated through catecholamines. However, because brain development in the neonatal rat corresponds to fetal stages in man, the onset of these mechanisms may be relevant to human fetal exposure.

Impact of fetal nicotine exposure on development of rat brain regions: critical sensitive periods or effects of withdrawal?

Fetal nicotine exposure evokes alterations in central nervous system structural, neurochemical, and behavioral development. In the current study, the relative importance of critical developmental exposure periods and withdrawal were examined by infusing nicotine to pregnant rats using osmotic minipumps beginning on the fourth day of gestation. Infusions were confined to either the first 8 days (withdrawal on gestational day 13), to nearly all of gestation (withdrawal on gestational day 21), or throughout gestation and continued into the first 2 postnatal weeks. Maternal weight gain was retarded by nicotine, with a hierarchy corresponding to the duration of nicotine exposure. Similarly, fetal and neonatal body weights were unaffected in the group receiving the shortest duration of nicotine exposure, and were less affected by the intermediate infusion regimen than by the longest regimen; brain region weights were reduced significantly only with the longest regimen. Using ODC activity, a sensitive marker for altered brain cell development, we found little change in animals exposed to nicotine in early gestation and undergoing withdrawal on day 13. However, in the groups receiving nicotine through the end of gestation or through gestation and into the postnatal period, ODC activity was significantly elevated. These results indicate that withdrawal from nicotine contributes little, if any, effect either to the growth deficits or to abnormalities of brain cell development. Instead, the most important factor appears to be exposure within the developmental period corresponding to the proliferation of nicotinic receptors and the timing of receptor control of cell replication and differentiation.

Loss of neonatal hypoxia tolerance after prenatal nicotine exposure: implications for sudden infant death syndrome.

Maternal cigarette smoking has a high correlation with sudden Infant Death Syndrome, a condition in which cardiorespiratory failure occurs during an hypoxic episode, as in sleep apnea. Pregnant rats were given nicotine infusions of 2 or 6 mg/kg/day throughout gestation, regimens that produce plasma nicotine levels spanning the range in smokers. The day after birth, animals in the high dose group displayed excessive mortality during hypoxic challenge. These animals were found to be deficient in an essential response component, namely adrenomedullary catecholamine release that is required to maintain neonatal cardiac rhythm during hypoxia; the defect was in adrenal cell function rather than in altered innervation or nicotinic receptor desensitization. We also examined brainstem and forebrain noradrenergic mechanisms that are involved in neonatal respiratory control. The nicotine group showed suppressed spontaneous neuronal activity, but were hyperresponsive to hypoxia. As these projections are inhibitory for respiration, the nicotine-induced sensitization would be expected to contribute to respiratory arrest during hypoxia. Prenatal nicotine exposure may thus provide a useful animal model with which to study the physiological mechanisms that underlie Sudden Infant Death Syndrome, while at the same time providing a biological explanation for the association of the syndrome with smoking.

Prenatal terbutaline exposure in the rat: selective effects on development of noradrenergic projections to cerebellum.

Terbutaline, used in the treatment of premature labor and asthma, crosses the placenta and can stimulate beta 2-adrenergic receptors in the fetus. This study examines the effects of prenatal exposure to terbutaline (10 mg/kg SC on gestational days 17, 18 and 19) on the development of noradrenergic projections in brain regions of the fetal and neonatal rat, using synaptosomal uptake of [3H]norepinephrine as a marker for synaptogenesis. Although terbutaline exposure did not compromise body or brain region growth, uptake was adversely affected selectively in the cerebellum, a region which also displays close coupling of fetal beta 2-receptors to control of cell development near term. These results thus provide biochemical evidence that terbutaline may be a neurobehavioral teratogen.

Prenatal nicotine exposure impairs beta-adrenergic function: persistent chronotropic subsensitivity despite recovery from deficits in receptor binding.

Gestational exposure to nicotine has been shown to interfere with biochemical markers of development of central and peripheral noradrenergic activity. The current study examines the development and function of cardiac beta-adrenergic receptors in the offspring of pregnant rats given nicotine infusions of 6 mg/kg/day from gestational days 4 through 20, administered by subcutaneously implanted osmotic minipumps. Prenatal nicotine exposure delayed the development of beta-adrenergic receptor binding capabilities, as assessed with [125I]pindolol in membrane preparations from heart and kidney. The deficits in receptor binding were associated with marked subsensitivity of chronotropic responses to administration of a beta-adrenergic agonist, isoproterenol. Although the effects on receptor binding resolved after weaning, functional deficiencies in responsiveness to isoproterenol or to preganglionic electrical stimulation of sympathetic nerves to the heart persisted into adulthood. These results indicate that prenatal exposure to nicotine produces long-term alterations in adrenergic responsiveness of sympathetic target tissues.

Fetal terbutaline exposure causes selective postnatal increases in cerebellar alpha-adrenergic receptor binding.

beta-Adrenergic agonists used in therapy of premature labor and asthma cross the placenta and can affect development of the fetal nervous system. In the current study, pregnant rats were given 10 mg/kg of terbutaline on gestational days 17, 18 and 19 and adrenergic receptor binding capabilities examined in brain regions of the offspring. Despite the absence of body or brain growth impairment, selective increases were seen postnatally in cerebellar alpha 1- and alpha 2-receptor subtypes, whereas the same receptor populations were decreased by small amounts in cerebral cortex and midbrain + brainstem. beta-Adrenergic receptors showed little or no change in any region. The regional and subtype selectivity are compatible with primary deficits in the development of noradrenergic projections to the cerebellum identified in previous studies and provide further evidence that therapeutic use of beta-adrenergic agonists may produce neurobehavioral teratology.

Chronic prenatal nicotine exposure sensitizes rat brain to acute postnatal nicotine challenge as assessed with ornithine decarboxylase.

Prenatal exposure to nicotine has been shown to produce postnatal up-regulation of central nervous system nicotinic receptors and to alter subsequent differentiation of neural tissues. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants; the postnatal development of cholinergic receptor reactivity was examined through measurements of the ability of acute nicotine administration to stimulate midbrain + brainstem ornithine decarboxylase (ODC) activity, a key regulatory enzyme in neural cell differentiation and growth. In control rats, the ODC response to nicotine was absent at birth and developed during the second postnatal week in parallel with the known ontogenetic rise of nicotinic receptors. Offspring of the nicotine-infused dams exhibited hyper-reactivity of ODC to postnatal acute nicotine challenge: the response developed earlier than in controls and subsequently the magnitude of the effect was 2-3 times greater. Since the development of cholinergic transmission influences differentiation of target cells, alterations in cholinergic nicotinic receptor mediated responses likely explain the delayed appearance of abnormal cell differentiation associated with prenatal nicotine.

Deficits in development of central cholinergic pathways caused by fetal nicotine exposure: differential effects on choline acetyltransferase activity and [3H]hemicholinium-3 binding.

Nicotine has been hypothesized to induce neurobehavioral teratology by mimicking prematurely the natural developmental signals ordinarily communicated by the ontogeny of cholinergic synaptic transmission. In the current study, the effects of fetal nicotine exposure (2 mg/kg/day or 6 mg/kg/day) on development of central cholinergic pathways were examined in striatum and hippocampus of animals exposed from gestational days 4 through 20, using maternal infusions with osmotic minipumps. Brain region weights and choline acetyltransferase activity, an enzymatic marker for development of cholinergic nerve terminals, were within normal limits in the nicotine-exposed animals. However, development of [3H]hemicholinium-3 binding which labels the presynaptic high affinity cholinergic transporter, was deficient in both striatum and hippocampus. Abnormalities occurred during two distinct phases; in the early neonatal period, when [3H]hemicholinium-3 binding sites are transiently overexpressed, and during or after the period of rapid synaptogenesis, when binding in controls is rising consequent to the increase in nerve impulse activity. These data thus indicate that fetal nicotine exposure, even at doses that do not cause overt signs of maternal/fetal/neonatal toxicity or growth impairment, influences both specific gene expression of cholinergic nerve terminal markers, as well as indices of neuronal function. Comparison of regional selectivity at the two dose levels indicated greater sensitivity of the striatum, a region with a prenatal peak of neuronal mitosis, as compared to hippocampus, where mitosis peaks postnatally; the regional differences are consistent with vulnerability to nicotine during a critical phase of cell development.

Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding.

A bis-quaternary fluorescence probe, propidium diiodide, has been found to exhibit a tenfold enhancement of fluorescence when bound to acetylcholinesterase from Torpedo california. The complex is characterized by a high affinity, KD = 3.0 times 10-7 M, and 1:1 stoichiometry with the 82,000 molecular weight subunit of acetylcholinesterase. A wide variety of other quaternary ammonium ligands such as decamethonium, gallamine, d-tubocurarine, tetraethylammonium, and tetramethylammonium will completely dissociate propidium from the enzyme as will monovalent and divalent inorganic cations. The competitive dissociation does not show cooperative behavior or a distinct, requirement for occupation of multiple sites of different affinity to produce displacement. While a directly competitive relationship can be illustrated macroscopically, the various quaternary ligands show a different susceptibility toward inorganic cation displacement. The affinity of propidium relative to gallamine increases with ionic strength. This finding indicates that there is not complete equivalence in the negative subsites to which quaternary groups bind. Although edrophoniumwill also displace propidium from the enzyme, the dissociation constant obtained from this competitive relationship is 3.5 orders of magnitude greater than the constants obtained for inhibition of catalysis. By competitive displacement titrations it is shown that the primary binding site of edrophonium is distinct from that of propidium and a ternary complex with the two ligands can form on each subunit. In contrast to edrophonium, the binding of propidium is unaffected by methanesulfonylation of the active center serine and is uncompetitive with the carbamylating substrate, N-methyl-7-dimethylcarbamoxyquinolinium. Thus, it appears that propidium associates with a peripheral anionic center on the enzyme. Although propidium and edrophonium associate at separate sites on acetylcholinesterase, bis-quaternary ligands where the quaternary nitrogens are separated by 14 A displace both ligands from the enzyme with equal effectiveness.

Role of thyroid hormone in the development of beta adrenergic control of ornithine decarboxylase in rat heart and kidney.

The role of thyroid status in the ontogeny of beta adrenergic receptor control of ornithine decarboxylase (ODC) activity was assessed in hearts and kidneys of neonatal rats. Hyperthyroidism induced by administration of tri-iodothyronine on postnatal days 1 to 5 caused a reduction in the ability of isoproterenol to stimulate cardiac ODC but subsequently accelerated the onset of the postweaning peak of the response; the latter effect was even more prominent when tri-iodothyronine administration was given on postnatal days 14 to 18. Hypothyroidism induced by propylthiouracil administration led to persistent subsensitivity of the cardiac ODC response to beta receptor stimulation. Kidney ODC, which does not become subject to beta receptor regulation until after weaning, was resistant to hyperthyroid-induced changes in reactivity, but hypothyroidism still resulted in long-term response deficits. These results suggest that thyroid hormone is permissive for normal development of the beta receptor-ODC link, and that the euthyroid state provides the optimal conditions for maturation of this signal transduction mechanism. The relative resistance of kidney ODC responses to alterations by hyperthyroidism further indicates that the effects of excess hormone can only be expressed when the receptor-enzyme link is already competent. Finally, thyroid status had equivalent effects on the abilities of vasopressin or angiotensin to stimulate ODC, suggesting that the site of thyroid hormone action is at a transduction locus common to several different receptor types.

Beta-adrenergic control of c-fos expression in fetal and neonatal rat tissues: relationship to cell differentiation and teratogenesis.

beta-Adrenergic receptors appear in noradrenergic target tissues well before the arrival of nerve terminals, and are thought to play a role in the control of cell differentiation. We examined the ability of beta-agonists to stimulate expression of the nuclear transcription factor, c-fos, in developing rat liver and heart. This factor has been shown to associated with trophic activation of genes involved in both cell differentiation and cell growth. In response to terbutaline, a beta 2-selective agonist, marked stimulation of c-fos was demonstrated in the liver, which contains beta 2-receptors, on Gestational Day 20, as well as on Postnatal Days 1 and 8. In the heart, which contains predominantly beta 1-receptors, isoproterenol (a non-subtype-selective beta-agonist) was more effective that terbutaline, indicating that either receptor subtype can elicit stimulation of c-fos. In both tissues, the response magnitude increased with age, rather than following changes in receptor number, which increase in the heart but decrease in the liver; the same pattern has been seen for the ability of beta-agonists to promote cell differentiation at the expense of replication; the implication is that ontogenetic changes in post-receptor coupling are much more important than is the number of receptors in determining neurotrophic influences on gene expression and cell development. In keeping with the view that fetal/neonatal beta-adrenergic stimulation of c-fos is related to cell differentiation rather than simply to growth, repeated administration of isoproterenol to neonatal rats did not elicit cardiac hypertrophy, whereas the same treatment did produce hypertrophy in adult rats. The intracellular signaling cascade from beta-receptor to c-fos expression may thus provide one of the basic cellular mechanisms for trophic control of differentiation by biogenic amines, and for the teratologies associated with beta-adrenergic agonist therapy.

Molecular forms of acetylcholinesterase from Torpedo californica: their relationship to synaptic membranes.

The 16S and 8S forms of acetylcholinesterase (AchE), which are composed of an elongated tail structure in addition to the more globular catalytic subunits, were extracted and purified from membranes from Torpedo californica electric organs. Their subunit compositions and quaternary structures were compared with 11S lytic enzyme which is derived from collagenase or trypsin treatment of the membranes and devoid of the tail unit. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of reducing agent, appreciable populations of monomeric through tetrameric species are observed for the 11S form. Under the same conditions, the 16S form yields only monomer and dimer in addition to a higher molecular weight species. If complete reduction is effected, only the 80,000 molecular weight monomer is dominant for both the 11S and 16S forms. Cross-linking of the 11S form by dimethyl suberimidate followed by reduction yields monomer through tetramer in descending frequency, while the 16S form again shows a high molecular weight species. A comparison of the composition of the 11S and 16S forms reveals that the latter has an increased glycine content, and 1.1 and 0.3 mol % hydroxyproline and hydroxylysine, respectively. Collagenases that have been purified to homogencity and are devoid of amidase and caseinolytic activity, but active against native collagen, will convert 16S acetylcholinesterase to the 11S form. Thus, composition and substrate behavior of the 16S enzyme are indicative of the tail unit containing a collagen-like sequence. A membrane fraction enriched in acetylcholinesterase and components of basement membrane can be separated from the major portion of the membrane protein. The 16S but not the 11S form reassociates selectively with this membrane fraction. These findings reveal distinct similarities between the tail unit of acetylcholinesterase and basement membrane components and suggest a primary association of AchE with the basement membrane.