Facilitators and barriers to a dietitian-implemented blended care weight-loss intervention (SMARTsize): a qualitative study.

BACKGROUND: Dietitians play an important role in the intervention and prevention of being overweight and obesity. More and more blended care interventions are being implemented. The present study aimed to evaluate the delivery by Dutch dietitians of a multicomponent, evidence-based weight-loss programme (SMARTsize), including counselling for relapse prevention. The aim of this qualitative study was to identify facilitators and barriers to the delivery of SMARTsize. METHODS: Nine semi-structured interviews were conducted with 10 dietitians who participated in a larger implementation study. Each interview was recorded and transcribed verbatim. Determinants of theory of implementation, including characteristics of the user, the innovation, organisational context and setting, and innovation strategy guided interviews and analysis. Data were coded and analysed using the framework approach. RESULTS: According to dietitians, the SMARTsize intervention had a positive influence on patients. The main implementation facilitators were the availability of implementation materials, such as a manual, training in relapse prevention and social support from other dietitians. The main barriers to implementation were organisation and financial reimbursement of cooking classes, the belief that patients need more individual counselling in the starting phase, and the unsuitability for people with low levels of health literacy. CONCLUSIONS: Most dietitians considered that implementation of the SMARTsize intervention consisting of e-health, written information and cooking classes and face-to-face counselling is challenging but feasible. Further development of the SMARTsize intervention and implementation tools is needed to lower experienced barriers. It is also recommended that a version of the intervention to be developed that is suitable for patients with lower levels of health literacy.

Using citrate-functionalized TiO2 nanoparticles to study the effect of particle size on zebrafish embryo toxicity.

TiO2 nanoparticles (NPs) are photoactive, potentially producing toxicity in vivo in the presence of sunlight. We have previously demonstrated photodependent toxicity in zebrafish embryos exposed to TiO2 NPs. Here we investigate the effect of particle size on developing zebrafish exposed to 6, 12 and 15 nm citrate-functionalized anatase TiO2 NPs under either simulated sunlight illumination or in the dark. All three sizes of TiO2 NPs caused photo-dependent toxicity. Under simulated sunlight illumination, the acute toxicity of the 6 nm citrate-TiO2 NPs (120 h LC50 of 23.4 mg L(-1)) exceeded that of the 12 and 15 nm citrate-TiO2 NPs. Exposure to 6 nm particles under illumination also caused a higher incidence of developmental defects than the larger particles. These abnormalities included pericardial edema, yolk-sac edema, craniofacial malformation, and opaque yolk. To gain insight into the mechanisms of toxicity, we measured hydroxyl radicals (˙OH) generated by NPs in vitro and reactive oxygen species (ROS) produced in vivo. We found that on a mass basis, smaller particles generated higher levels of ROS both in vitro and in vivo, and the 6 nm citrate-TiO2 NPs induced more oxidative stress than larger particles in the zebrafish embryo. We examined oxidative DNA damage by measuring 8-hydroxydeoxyguanosine in zebrafish exposed to different-sized citrate-TiO2 NPs and found that 6 nm particles caused more DNA damage than did larger particles (12 and 15 nm) under illumination. Our results indicate a photo-dependent toxicity of citrate-TiO2 NPs to zebrafish embryos, with an inverse relationship between particle size and toxicity. Production of more ROS, resulting in more oxidative stress and more DNA damage, represents one possible mechanism of the higher toxicity of smaller citrate-TiO2 NPs. These results highlight the relationship between citrate-TiO2 NP size and toxicity/oxidative stress in developing zebrafish embryos.

The frequency, magnitude and timing of post-diagnosis body weight gain in Dutch breast cancer survivors.

To evaluate the association between systemic treatments and post-diagnosis weight gain in breast cancer patients during longer follow-up periods, we conducted a retrospective cohort study (n=271). Information on adjuvant systemic treatments and repeated body weight measurements was obtained from medical records, and analysed using multi-level regressions. During the first year, a mean weight change of +2.0kg (SD 4.9) was observed. Overall, 29% of all breast cancer patients had gained 5kg or more in body weight during total follow-up (median: 3 years). In multi-level analyses, women who received combined systemic treatment gained significantly more weight as compared with women who received no systemic treatment (4.5kg versus 2.0kg at 5 years post-diagnosis, p<0.05). Significant weight gain occurs in breast cancer patients in the Netherlands during the first year post-diagnosis. After the first year, further weight gain mainly occurs in women who receive chemotherapy in combination with endocrine therapy.

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits regression of the common cardinal vein in developing zebrafish.

A role for the aryl hydrocarbon receptor (AHR) pathway in vascular maturation has been implicated by studies in Ahr-null mice. In this study the hypothesis that activation of AHR signaling by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters common cardinal vein (CCV) development in the zebrafish embryo was investigated. The CCV is a paired vessel that grows across the yolk, connecting to the heart. It is extensively remodeled and regresses as the heart migrates dorsally within the pericardium. TCDD significantly reduced CCV growth as early as 44 h post fertilization (hpf), and CCV area was reduced to 63% of control at 62 hpf. This vascular response to TCDD was at least as sensitive as previously defined endpoints of TCDD developmental toxicity in zebrafish. TCDD also blocked regression of the CCV (by 80 hpf), possibly contributing to the "string-like" heart phenotype seen in TCDD-exposed zebrafish larvae. Dependence of the block in CCV regression on zebrafish (zf) AHR2 was investigated using a zfahr2 specific morpholino to knock down expression of AHR2. The zfahr2 morpholino had no effect on CCV regression in the absence of TCDD, but did protect against the TCDD-induced block of CCV regression. This demonstrates that the TCDD-induced block in CCV regression is AHR2 dependent. It is significant that decreased CCV growth occurs before and inhibition of CCV regression occurs concurrent with overt signs of TCDD developmental toxicity. This suggests that alterations of vascular growth and remodeling may play a role in TCDD developmental toxicity in zebrafish.

Disruption of erythropoiesis by dioxin in the zebrafish.

2,3,7,8-Tetrachlorodibenzo-p- dioxin (TCDD, or dioxin) causes early life stage mortality in a variety of fish species. We have used the zebrafish (Danio rerio) to study the cardiovascular effects of TCDD treatment over the time course of zebrafish development. Early TCDD exposure (6 ng/ml) starting at 4 hr postfertilization (hpf) produced reductions in blood flow and in the number of circulating erythrocytes. These defects were consistently observable by 72 hpf. However, these responses were not observed when TCDD exposure was delayed until 96 hpf or later. These results suggest a model in which TCDD interferes with cardiovascular and erythropoietic developmental processes that are normally completed by 96 hpf. This model is strengthened by the finding that TCDD exposure blocks the step in hematopoiesis in which developing zebrafish switch from the primitive phase to the definitive phase of erythropoiesis. We observed no effect of TCDD on the levels of circulating primitive erythrocytes before 72 hpf and the expression of markers for early hematopoiesis, GATA-1 and GATA-2. However, early TCDD exposure prevented the appearance of definitive phase erythrocytes. TCDD produced a small delay in the migration of blood cells expressing SCL from the intermediate cell mass to the dorsal mesentery and dorsal aorta. Despite the decrease in blood flow produced by TCDD, confocal microscopy of the trunk vasculature by using a Tie2/green fluorescence protein endothelial marker at 48, 60, 72, and 96 hpf of TCDD-exposed (4 hpf) revealed no apparent defects in blood vessel structure.

Relative potencies of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners to induce cytochrome P4501A mRNA in a zebrafish liver cell line.

Induction of cytochrome P4501A (CYP1A) mRNA by polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF), and polychlorinated biphenyl (PCB) congeners was measured in a zebrafish liver (ZF-L) cell line. The ZF-L cells were far less sensitive to PCDD, PCDF, and PCB congeners than were other fish cell lines. The 2,3,7,8-PCDDs, 2,3,7,8-PCDFs, and PCB 126 caused dose-related induction. All other PCBs tested, including other coplanar as well as ortho-substituted congeners, were ineffective at inducing CYP1A. The potency of each congener that gave a response, relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin, was determined. The ZF-L cell-derived relative potency values (REPs) are similar to other in vitro REPs in that the ZF-L cell-derived REPs are generally higher than those derived from in vivo models. Furthermore, the ZF-L cell-derived REPs are generally within fivefold of REPs determined in a variety of rainbow trout systems when the same endpoint in the same tissue are compared. Analysis of these data indicates that REPs based on molecular and biochemical responses in sensitive and insensitive species are similar, but overestimate relative in vivo toxicity in the rainbow trout. The ZF-L cell-derived REPs expand the database of REPs, providing additional information that will be useful in quantifying the uncertainty associated with applying consensus fish-specific toxic equivalency factors in ecological risk assessment.

Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions.

In order to further establish zebrafish as a vertebrate model for studying the mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity it is necessary to characterize the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) signaling pathways in this species. In this study, three zfARNT2 cDNAs were isolated, expressed, and characterized and named zfARNT2b, zfARNT2c, and zfARNT2a. zfARNT2b, zfARNT2c, and zfARNT2a encode proteins with theoretical molecular weights of 81, 79, and 45 kDa, respectively. zfARNT2b and zfARNT2a proteins are identical over the first 403 amino acids but differ in their C-terminal domains as a result of alternative mRNA splicing. zfARNT2c is nearly identical to zfARNT2b, with the exception of an in frame 15 amino acid deletion adjacent to the basic region of zfARNT2c. Using quantitative RT-PCR methods the tissue distribution of each zfARNT2 isoform was determined. In COS-7 cells expressing zfARNT2b and zfAhR2, 10 nM TCDD causes a nine-fold induction of a dioxin responsive reporter gene. In COS-7 cells expressing zfARNT2a or zfARNT2c, TCDD does not induce reporter gene expression. In contrast, all three zfARNT2 proteins induce reporter gene activity under control of hypoxia responsive elements when cotransfected with the zebrafish endothelial specific PAS protein 1. DNA gel shift analysis suggests that the decreased function of zfARNT2a is due to inefficient binding of zfARNT2a/zfAhR2 complexes to dioxin responsive elements. These results also indicate that alternative mRNA splicing results in formation of ARNT proteins with distinct functional properties.

Regulation of gene expression by glucose in Saccharomyces cerevisiae: a role for ADA2 and ADA3/NGG1.

When Saccharomyces cerevisiae cells are transferred from poor medium to fresh medium containing glucose, they rapidly increase the transcription of a large group of genes as they resume rapid growth and accelerate progress through the cell cycle. Among those genes induced by glucose is CLN3, encoding a G(1) cyclin that is thought to play a pivotal role in progression through Start. Deletion of CLN3 delays the increase in proliferation normally observed in response to glucose medium. ADA2 and ADA3/NGG1 are necessary for the rapid induction of CLN3 message levels in response to glucose. Loss of either ADA2 or ADA3/NGG1 also affects a large number of genes and inhibits the rapid global increase in transcription that occurs in response to glucose. Surprisingly, these effects are transitory, and expression of CLN3 and total poly(A)(+) RNA appear normal when ADA2 or ADA3/NGG1 deletion mutants are examined in log-phase growth. These results indicate a role for ADA2 and ADA3/NGG1 in allowing rapid transcriptional responses to environmental signals. Consistent with the role of the Ada proteins in positive regulation of CLN3, deletion of RPD3, encoding a histone deacetylase, prevented the down regulation of CLN3 mRNA in the absence of glucose.

Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners.

Transactivation assays were used to compare the potency and efficacy of polychlorinated dibenzo-p-dioxin (PCDD), dibenzofuran (PCDF), and biphenyl (PCB) congeners in activating aryl hydrocarbon receptors (AhRs) from rainbow trout (rtAhR2alpha and rtAhR2beta), zebrafish (zfAhR2), and human (huAhR), respectively. All AhRs were expressed with their species-specific AhR nuclear translocator (ARNT) in COS-7 cells. Transactivation activity was determined for two PCDD, two PCDF, and seven PCB congeners with each of the four AhR/ARNT pairs using prt1Aluc, a luciferase reporter driven by two dioxin-responsive enhancer elements (DREs) from the rainbow trout cyp1A gene. Maximal-fold induction, EC50, and relative potency values were calculated for congeners that exhibited dose-related activity in the assay. Of the four AhR/ARNT pairs tested with PCDD, PCDF, and non-ortho PCB congeners, three exhibited high activity (rainbow trout AhR2alpha, zebrafish AhR2, and human AhR), while rainbow trout AhR2beta had very weak or no activity. Comparisons between these AhRs showed that while mono-ortho PCBs were able to activate the human AhR, they were generally ineffective in activating rainbow trout and zebrafish AhR2s. This supports the hypothesis that structural differences between mammalian and fish AhRs may account for differences in relative potencies of the mono-ortho PCBs between mammals and fish. Another important finding was a significant difference in transactivation activity between the two rainbow trout AhR2 isoforms despite the fact that they are 95% identical at the amino acid level. For all PCDD, PCDF, and PCB agonists tested, rainbow trout AhR2alpha was significantly more active than AhR2beta. However, rainbow trout AhR2beta is active as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-activated transcription factor, with enhancer elements from the mouse cyp1A gene. This suggests that AhR2beta may have evolved to serve a different physiological function than AhR2alpha in salmonid fish species.

Two forms of aryl hydrocarbon receptor type 2 in rainbow trout (Oncorhynchus mykiss). Evidence for differential expression and enhancer specificity.

Two aryl hydrocarbon receptors (AhRs), rtAhR2alpha and rtAhR2beta, were cloned from rainbow trout (rt) cDNA libraries. The distribution of sequence differences, genomic Southern blot analysis, and the presence of both transcripts in all individual rainbow trout examined suggest that the two forms of rtAhR2 are derived from separate genes. The two rtAhR2s have significant sequence similarity with AhRs cloned from mammalian species, especially in the basic helix-loop-helix and PAS functional domains located in the amino-terminal 400 amino acids of the protein. In contrast, the Gln-rich transactivation domain found in the carboxyl-terminal half of mammalian AhRs is absent from both rtAhR2s. Both clones were expressed by in vitro transcription/translation and proteins of approximately 125 kDa were produced. These proteins bind 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and are able to bind dioxin response elements in gel shift assays. rtAhR2alpha and rtAhR2beta are expressed in a tissue-specific manner with the highest expression of rtAhR2beta in the heart. Expression of rtAhR2alpha and rtAhR2beta mRNAs is positively regulated by TCDD. Both rtAhR2alpha and rtAhR2beta produced TCDD-dependent activation of a reporter gene driven by dioxin response elements. Surprisingly, the two receptors showed distinct preferences for different enhancer sequences. These results suggest that the two receptor forms may regulate different sets of genes, and may play different roles in the toxic responses produced by AhR agonists such as TCDD.

The DPL1 gene is involved in mediating the response to nutrient deprivation in Saccharomyces cerevisiae.

Sphingosine-1-phosphate is a sphingolipid metabolite involved in the regulation of cell proliferation in mammalian cells. The major route of sphingosine-1-phosphate degradation is through cleavage at the C, bond by sphingosine phosphate lyase. The recent identification of the first dihydrosphingosine/sphingosine phosphate lyase gene in Saccharomyces cerevisiae establishes that phosphorylated sphingoid base metabolism is conserved throughout evolution. The dpl1delta deletion mutant, which accumulates endogenous phosphorylated sphingoid bases, exhibits unregulated proliferation upon approach to stationary phase. The increased proliferation rate during respiratory growth was associated with failure to appropriately recruit cells into the G1 phase of the cell cycle. Several genes were found to be overexpressed or prematurely expressed during nutrient deprivation in the dpl1delta strain, including glucose-repressible genes and G1 cyclins. These studies implicate a role for DPL1 and phosphorylated sphingoid bases in the regulation of global responses to nutrient deprivation in yeast.

Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) mediates the toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in vertebrates. To further establish zebrafish as a vertebrate model to study the molecular mechanism of TCDD toxicity, we have isolated and characterized the cDNA encoding the zebrafish aryl hydrocarbon receptor (zfAhR2). Analysis of the deduced protein sequence revealed the 1027 amino acid protein is approximately 200 amino acids longer than previously isolated receptors. zfAhR2 is homologous to previously cloned PAS proteins within the basic helix-loop-helix and PAS domains. The C-terminal domain of zfAhR2 diverges from the mammalian AhR at position 420, and does not contain a Q-rich domain. zfAhR2 mRNA is first detected by Northern blot analysis at 24 h post fertilization, and expression increases throughout early development. Treatment of zebrafish embryos and zebrafish liver cells with graded doses of TCDD results in a dose-dependent increase in zfAhR2 mRNA. The time course for zfAhR2 and cytochrome P4501A mRNA induction by TCDD are similar. In vitro produced zfAhR2 protein dimerizes with the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNTb) and binds dioxin response elements derived from the rainbow trout CYP1A gene. Finally, transient coexpression of zfAhR2 and rtARNTb in COS-7 cells results in a TCDD dose-related increase in transcription driven by the rainbow trout CYP1A promoter and enhancer.

Regulation of the Cln3-Cdc28 kinase by cAMP in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae grows at widely varying rates in different growth media. In order to maintain a relatively constant cell size, yeast cells must regulate the rate of progress through the cell cycle to match changes in growth rate, moving quickly through G1 in rich medium, and slowly in poor medium. We have examined connections between nutrients, and the expression and activity of Cln3-Cdc28 kinase that regulates the G1-S boundary of the cell cycle in yeast, a point referred to as Start. We find that Cln3 protein levels are highest in glucose and lower in poorer carbon sources. This regulation involves both transcriptional and post-transcriptional control. Although the Ras-cAMP pathway does not appear to affect CLN3 transcription, cAMP increases Cln3 protein levels and Cln3-Cdc28 kinase activity. This regulation requires untranslated regions of the CLN3 message, and can be explained by changes in protein synthesis rates caused by cAMP. A model for CLN3 regulation and function is presented in which CLN3 regulates G1 length in response to nutrients.

Transcriptional regulation of CLN3 expression by glucose in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the transition from the G1 phase of the mitotic cycle into S phase is controlled by a set of G1 cyclins that regulate the activity of the protein kinase encoded by CDC28. Yeast cells regulate progress through the G1/S boundary in response to nutrients, moving quickly through G1 in glucose medium and more slowly in poorer medium. We have examined connections between glucose and the level of the message encoding Cln3, a G1 cyclin. We found that glucose positively regulates CLN3 mRNA levels through a set of repeated AAGAAAAA (A2GA5) elements within the CLN3 promoter. Mutations in these sequences reduce both transcriptional activation and specific interaction between CLN3 promoter elements and proteins in yeast extracts. Creation of five point mutations, replacing the G's within these repeats with T's, in the CLN3 promoter substantially reduces CLN3 expression in glucose medium and inhibits the ability of the cells to maintain a constant size when shifted into glucose.

Growth-independent regulation of CLN3 mRNA levels by nutrients in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells regulate progress through the G1 phase of the cell cycle in response to nutrients, moving quickly through G1 in rich medium and slowly in poor medium. Recent work has shown that the levels of Cln3 protein, a G1 cyclin, are low in cells growing in poor medium and high in cells growing rapidly in rich medium, consistent with the previously recognized role of Cln3 in promoting passage through Start. Cln3 protein levels appear to be regulated both transcriptionally and posttranscriptionally. We have worked to define the nutrient signals that regulate CLN3 mRNA levels. We find that CLN3 mRNA levels are high during log-phase growth in glucose medium, low in postdiauxic cells growing on ethanol, and slightly lower still in cells in stationary phase. CLN3 mRNA levels are induced by glucose in a process that involves transcriptional control, requires metabolism of the glucose, and is independent of the Ras-cyclic AMP pathway. CLN3 mRNA levels are also positively regulated by nitrogen sources, but phosphorus and sulfur limitation do not affect CLN3 message levels.

Indomethacin and epinephrine effects on outflow facility and cyclic adenosine monophosphate formation in monkeys.

PURPOSE: To investigate the effect of indomethacin inhibition of prostanoid production on the epinephrine-stimulated increase in outflow facility and cyclic adenosine monophosphate (cAMP) production in the anterior segment of the monkey eye. METHODS: Topical indomethacin was given 1 hour before the intracameral administration of epinephrine to living cynomolgus monkeys. Outflow facility was measured for 45 to 60 minutes, beginning 3 hours after epinephrine administration, by two-level constant pressure perfusion of the anterior chamber. Cyclic adenosine monophosphate formation was measured in cell membranes isolated from rhesus monkey ciliary muscle, ciliary processes, trabecular meshwork, and iris in the presence of forskolin, indomethacin, epinephrine, or indomethacin and epinephrine combined. RESULTS: Three hours after the intracameral administration of 5.5 micrograms epinephrine, facility increased by approximately 40%, a putatively maximal response, at which time the intracameral epinephrine concentration was approximately 15 microM. Pretreatment with topical indomethacin produced a dose-dependent inhibition of epinephrine's facility-increasing effect; the maximum inhibition of 50% to 70% occurred at an indomethacin dose of 50 to 125 micrograms. Doubling the indomethacin dose (250 micrograms) produced no further inhibition, whereas a fivefold larger epinephrine dose (27.5 micrograms) did not overcome the inhibition. Forskolin and epinephrine both stimulated cAMP production in vitro, whereas [indomethacin] > or = 10(-4) M partially inhibited both basal and epinephrine-stimulated cAMP production in all four tissues. CONCLUSIONS: Approximately half of the epinephrine-induced facility increase is inhibited by indomethacin, but it is unclear whether the indomethacin-inhibitable fraction is mediated by epinephrine-stimulated prostanoid production or release.

Activation of the Ras/cyclic AMP pathway in the yeast Saccharomyces cerevisiae does not prevent G1 arrest in response to nitrogen starvation.

Cells carrying mutations that activate the Ras/cyclic AMP (Ras/cAMP) pathway fail to accumulate in G1 as unbudded cells and lose viability in response to nitrogen starvation. This observation has led to the idea that cells carrying this type of mutation are sensitive to nitrogen starvation because they are unable to appropriately arrest in G1. In this study, we tested predictions made by this model. We found that cells with activating Ras/cAMP pathway mutations do not continue to divide after nitrogen starvation, show a normal decrease in steady state levels of START-specific transcripts, and are not rescued by removal of cAMP during nitrogen starvation. These findings are inconsistent with the idea that activation of the Ras/cAMP pathway prevents growth arrest in cells starved for nitrogen. Our finding that cells with an active Ras/cAMP pathway have dramatically reduced amino acid stores suggests an alternative model. We propose that cells at high cAMP levels are unable to store sufficient nutrients to allow return to the G1 phase of the cell cycle when they are suddenly deprived of nitrogen. It is this inability to return to G1, rather than a failure to arrest, which leaves cells at different points in the cell cycle following nitrogen starvation.

The effect of age on muscarinic receptor transcripts in rat brain.

The expression of five muscarinic receptor mRNAs (m1, m2, m3, m4, m5) was examined in neostriatum, hippocampus and frontal cortex of 3, 18, and 33 month old rats. Transcripts for ml (3.0 kb), m2 (6.2 kb), m3 (4.5 kb) and m4 (3.3 kb) were detected in all brain regions studied. A 6.0 kb transcript for the m5 muscarinic receptor was observed in hippocampus and neostriatum, but was not detected in frontal cortex. Age-related changes in muscarinic receptor transcript expression were restricted to the neostriatum, where m3 and m4 mRNAs were decreased at both 18 and 33 months of age. The reduction in transcripts of m3 and m4 receptors may contribute to changes in cholinergic system function with age.

Connections between the Ras-cyclic AMP pathway and G1 cyclin expression in the budding yeast Saccharomyces cerevisiae.

We have identified two processes in the G1 phase of the Saccharomyces cerevisiae cell cycle that are required before nutritionally arrested cells are able to return to proliferative growth. The first process requires protein synthesis and is associated with increased expression of the G1 cyclin gene CLN3. This process requires nutrients but is independent of Ras and cyclic AMP (cAMP). The second process requires cAMP. This second process is rapid, is independent of protein synthesis, and produces a rapid induction of START-specific transcripts, including CLN1 and CLN2. The ability of a nutritionally arrested cell to respond to cAMP is dependent on completion of the first process, and this is delayed in cells carrying a CLN3 deletion. Mating pheromone blocks the cAMP response but does not alter the process upstream of Ras-cAMP. These results suggest a model linking the Ras-cAMP pathway with regulation of G1 cyclin expression.

Changes in gene expression in the Ras/adenylate cyclase system of Saccharomyces cerevisiae: correlation with cAMP levels and growth arrest.

Levels of cyclic 3',5'-cyclic monophosphate (cAMP) play an important role in the decision to enter the mitotic cycle in the yeast, Saccharomyces cerevisiae. In addition to growth arrest at stationary phase, S. cerevisiae transiently arrest growth as they shift from fermentative to oxidative metabolism (the diauxic shift). Experiments examining the role of cAMP in growth arrest at the diauxic shift show the following: 1) yeast lower cAMP levels as they exhaust their glucose supply and shift to oxidative metabolism of ethanol, 2) a reduction in cAMP is essential for traversing the diauxic shift, 3) the decrease in adenylate cyclase activity is associated with a decrease in the expression of CYR1 and CDC25, two positive regulators of cAMP levels and an increase in the expression of IRA1 and IRA2, two negative regulators of intracellular cAMP, 4) mutants carrying disruptions in IRA1 and IRA2 were unable to arrest cell division at the diauxic shift and were unable to progress into the oxidative phase of growth. These results indicate that changes cAMP levels are important in regulation of growth arrest at the diauxic shift and that changes in gene expression plays a role in the regulation of the Ras/adenylate cyclase system.