A Drosophila CREB/CREM homolog encodes multiple isoforms, including a cyclic AMP-dependent protein kinase-responsive transcriptional activator and antagonist.

We have characterized a Drosophila gene that is a highly conserved homolog of the mammalian cyclic AMP (cAMP)-responsive transcription factors CREB and CREM. Uniquely among Drosophila genes characterized to date, it codes for a cAMP-responsive transcriptional activator. An alternatively spliced product of the same gene is a specific antagonist of cAMP-inducible transcription. Analysis of the splicing pattern of the gene suggests that the gene may be the predecessor of the mammalian CREB and CREM genes.

Lumen loss in transplant coronary artery disease is a biphasic process involving early intimal thickening and late constrictive remodeling: results from a 5-year serial intravascular ultrasound study.

BACKGROUND: Coronary artery disease is the major cause of late cardiac allograft failure. However, few data exist regarding the natural history of changes in intimal and external elastic membrane (EEM) areas after heart transplantation. METHODS AND RESULTS: In 38 transplant recipients, serial intravascular ultrasound examinations were performed 3.7+/-2.2 weeks after transplantation and annually thereafter for 5 years. In 59 coronary arteries, we compared 135 matched segments among serial studies. In each segment, intravascular ultrasound images were digitized at 1-mm intervals, and mean values of EEM and lumen and intimal areas were analyzed. In the first year after transplantation, the intimal area increased significantly from 1.8+/-1.6 to 3.0+/-2.1 mm(2) (P<0.001). Subsequently, the annual increase in intimal area decreased. EEM area did not change during the first year; however, between years 1 and 3, significant expansion of EEM area occurred (15.4+/-4.6 to 17.2+/-5.4 mm(2), P<0.001). Thereafter, EEM area decreased significantly from 17.2+/-5.4 mm(2) (year 3) to 15.1+/-4.9 mm(2) (year 5, P=0.01). Different mechanisms of lumen loss were observed during 2 phases after transplantation: early lumen loss primarily caused by intimal thickening and late lumen loss caused by EEM area constriction. CONCLUSIONS: This serial ultrasound study revealed that most of the intimal thickening occurred during the first year after heart transplantation. Changes in the EEM area showed a biphasic response, consisting of early expansion and late constriction. Thus, different mechanisms of lumen loss were observed during the early and late phases after transplantation.

Differential requirements for segment polarity genes in wingless signaling.

The segment polarity genes wingless and engrailed are required throughout development of Drosophila. During early embryogenesis, these two genes are expressed in adjacent domains, in an inter-dependent way. Later, their expression is regulated by different mechanisms and becomes maintained by auto-regulation. To dissect the genetic requirements for the initial signaling between wingless and engrailed expressing cells, we have previously used a transgenic Drosophila strain that expresses wingless under the control of the heat shock promoter (HS-wg). Focusing on the later phases of wingless and engrailed regulation, we have now extended these studies, using embryos carrying various combinations of segment polarity mutations and the HS-wg transgene. We confirm some of the existing models of regulation of the expression of wingless and engrailed. In addition, we find that HS-wg embryos require engrailed for induction of ectopic endogenous wingless expression. Signaling from engrailed cells to this novel wingless expression domain is dependent on hedgehog but also on porcupine. We further demonstrate a novel requirement for hedgehog in maintenance of expression of engrailed itself.

Conservation of dishevelled structure and function between flies and mice: isolation and characterization of Dvl2.

The segment polarity gene dishevelled (dsh) of Drosophila is required for pattern formation of the embryonic segments and the adult imaginal discs. dsh encodes the earliest-acting and most specific known component of the signal transduction pathway of Wingless, an extracellular signal homologous to Wnt1 in mice. We have previously described the isolation and characterization of the Dvl1 mouse dsh homolog. We report here the isolation of a second mouse dsh homolog, Dvl2, which maps to chromosome 11. The Dvl2 amino acid sequence is equally related to the dsh sequence as is that of Dvl1, but Dvl2 is most similar to the Xenopus homolog Xdsh. However, unlike the other vertebrate dsh homologs. Like the other genes, Dvl2 is ubiquitously expressed throughout most of embryogenesis and is expressed in many adult organs. We have developed an assay for dsh function in fly embryos, and show that Dvl2 can partially rescue the segmentation defects of embryos devoid of dsh. Thus, Dvl2 encodes a mammalian homolog of dsh which can transduce the Wingless signal.

Destruction of hepatic mixed-function oxygenase parameters by CCl4 in rats following acute treatment with chlordecone, Mirex, and phenobarbital.

Previous work has established the marked potentiation of CCl4 hepatoxicity by prior exposure to chlordecone (CD). This study was conducted to determine if prior exposure to CD results in enhancement of CCl4-induced destruction of the hepatic microsomal mixed-function oxygenase (MFO) system. Male Sprague-Dawley rats received a single oral dose of CD (10 mg/kg) or corn oil vehicle alone (1 ml/kg) 24 hr prior to a single ip injection of CCl4 (0-100 microliter/kg). Mirex (M; 10 mg/kg) and phenobarbital (PB; 80 mg/kg/day for two days) were used as negative and positive controls respectively for the potentiation of CCl4 hepatotoxicity. Hepatotoxicity was evaluated 24 hrs after CCl4 administration by elevations of three serum enzymes (GPT, GOT, and ICD). The key hepatic microsomal MFO parameters measured were microsomal protein, cytochrome P-450 content, glucose-6-phosphatase (G-6-Pase), and aminopyrine demethylase (APD). As previously demonstrated using a subchronic dietary pretreatment protocol, CD potentiated CCl4 hepatotoxicity over a range of CCl4 doses to a greater extent than PB or M, as judged by elevations in serum enzymes. PB caused the greatest increase in total P-450 content and the greatest increase in CCl4-mediated destruction of microsomal protein and APD activity. M caused the least destruction of total hepatic cytochrome P-450, despite the same level of cytochrome P-450 as in the PB group. CD treatment caused the greatest decrease in G-6-Pase activity in comparison to PB or M pretreatments and a similar degree of P-450 destruction as observed with the PB group. These findings suggest that in general, CCl4-induced destruction of hepatic MFO parameters measured in this study is disproportional to the known degree of potentiated hepatotoxicity by the pretreatments and does not accurately reflect the potentiation of CCl4 hepatotoxicity by CD.

Evaluation of three-dimensional segmentation algorithms for the identification of luminal and medial-adventitial borders in intravascular ultrasound images.

Intravascular ultrasound (IVUS) provides direct depiction of coronary artery anatomy, including plaque and vessel area, which is important in quantitative studies on the progression or regression of coronary artery disease. Traditionally, these studies have relied on manual evaluation, which is laborious, time consuming, and subject to large interobserver and intraobserver variability. A new technique, called active surface segmentation, alleviates these limitations and makes strides toward routine analyses. However, for three-dimensional (3-D) plaque assessment or 3-D reconstruction to become a clinical reality, methods must be developed which can analyze many images quickly. Presented is a comparison between two active surface techniques for three-dimensional segmentation of luminal and medial-adventitial borders. The force-acceleration technique and the neighborhood-search technique accurately detected both borders in vivo (r2 = 0.95 and 0.99, Williams' index = 0.67 and 0.65, and r2 = 0.95 and 0.99, WI = 0.67 and 0.70, respectively). However, the neighborhood-search technique was significantly faster and required less computation. Volume calculations for both techniques (r2 = 0.99 and r2 = 0.99) also agreed with a known-volume phantom. Active surface segmentation allows 3-D assessment of coronary morphology and further developments with this technology will provide clinical analysis tools.

Potentiation of CCl4 lethality by chlordecone.

Previous studies have dealt with the propensity of chlordecone (CD) to potentiate the hepatotoxicity of CCl4. The effect of this interaction upon CCl4 lethality has never been reported. The objective of the present study was to evaluate the effect of CD on CCl4 lethality and several parameters associated with cytochrome P-450 activity. Male Sprague-Dawley rats (150-200 g) were fed powdered diets containing 0 to 10 ppm CD or 225 ppm phenobarbital (PB) for 15 days. On day 15, rats from each group received an i.p. injection of CCl4 in corn oil and the 48-h LD50 determined. Additional animals were used to measure hepatic microsomal levels cytochrome P-450 and aminopyrine demethylase. CD decrease the LD50 of CCl4 from 2.8 ml/kg to 0.042 ml/kg, representing a 67-fold increase in toxicity, as assessed by lethality. PB in this protocol decreased the LD50 to 1.7 ml/kg, representing a 1.6-fold increase in lethality. However, the induction of cytochrome P-450 and associated parameters of mixed-function oxidase (MFO) activity did not correlate with the lethality data. Hepatic microsomal P-450 was increased over controls by 40% and 76% in CD and PB rats respectively. PB also stimulated aminopyrine demethylase activity to a much greater extent (190%) than CD (49%) relative to controls. These findings provide evidence that the CD-CCl4 interaction results in a marked increase in CCl4 lethality, in addition to the previously reported marked increase in hepatotoxicity. These results remain consistent with the proposal that bioactivation is the mechanisms underlying enhanced CCl4 toxicity, but suggest that specific effects of CD upon CCl4 metabolism may be the pivotal mechanism underlying potentiation.

Two-generation reproductive study in mink fed diisopropyl methylphosphonate (DIMP).

Two generations of "Ranch Wild" mink (Mustela vison) were fed the organophosphate diisopropyl methylphosphonate (DIMP) at 0, 150, 450, or 1250ppm, to determine potential toxicity to the dams. Chemical, hematologic, necropsy, and microscopic examinations were performed on all parental animals and representative kits. The F0 and F1 dams had 3.4 and 4.6% mortality, respectively, distributed among all groups and not attributed to DIMP exposure. Adverse effects were mild and limited to the highest dose group. Plasma cholinesterase was reduced 40% (F0) and 31% (F1), as was whole blood cholinesterase (16 and 8.5%). Heinz bodies were present in 2.8% (F0) and 1.3% (F1) of erythrocytes. The erythrocyte count was reduced 6.3% in the F0. Reproductive efficiency was not affected. The mink were not uniquely susceptible to DIMP, relative to the literature on other species. The no observed adverse effect level (NOAEL), based on the 450ppm group of F1 females, was 56.5mg DIMP/kgBW per day; the lowest observed adverse effect level (LOAEL) was 329.5mg DIMP/kgBW per day.

Distribution of methylene and nonmethylene-interrupted dienoic fatty acids in polar lipids and triacylglycerols of selected tissues of the hardshell clam (Mercenaria mercenaria).

Fatty acid profiles of polar lipids and triacylglycerols were determined for 6 tissues of the hardshell clam (Mercenaria mercenaria), namely, mantle, gill, mouth, foot, digestive tract/gonadal tissue and adductor muscle. The largest concentrations of nonmethylene-interrupted dienoic (NMID) fatty acids were found in the gill, mantle, and foot. Structural analyses were undertaken to determine the double bond configurations of the various NMID isomers. The major 22C NMID species were Δ7,13- and Δ7,15-docosadienoic acid. The major 20C NMID species were Δ7,11- and Δ7,13-eicosadienoic acid and Δ5,11-eicosadienoic acid.

Alternative tertiary care pathways for a rural Department of Veterans Affairs Medical Center.

This study addresses the cost of rural health care delivery where veterans do not have ready access to tertiary Department of Veterans Affairs Medical Centers (VAMCs) but where local community health care is available. The study sample was 209 patients referred for tertiary care to a VAMC 50 miles distant from the referring rural VAMC. The cost of tertiary referral VAMC care was retrospectively compared with the cost had the patients received the tertiary care in the local community hospital located in the immediate vicinity. In addition, the cost of travel resulting from the remote access was also computed. Findings indicate that a savings of +309,293 could have been obtained had a local community hospital provided the tertiary care utilizing the Health Care Financing Administration Medicare rate. Data generated by the methodology of this study are expected to provide a baseline for policy decisions relating to alternative pathways for tertiary care in the Department of Veterans Affairs.

Sonic hedgehog is required for cardiac outflow tract and neural crest cell development.

The Hedgehog signaling pathway is critical for a significant number of developmental patterning events. In this study, we focus on the defects in pharyngeal arch and cardiovascular patterning present in Sonic hedgehog (Shh) null mouse embryos. Our data indicate that, in the absence of Shh, there is general failure of the pharyngeal arch development leading to cardiac and craniofacial defects. The cardiac phenotype results from arch artery and outflow tract patterning defects, as well as abnormal development of migratory neural crest cells (NCCs). The constellation of cardiovascular defects resembles a severe form of the human birth defect syndrome tetralogy of Fallot with complete pulmonary artery atresia. Previous studies have demonstrated a role for Shh in NCC survival and proliferation at later stages of development. Our data suggest that SHH signaling does not act directly on NCCs as a survival factor, but rather acts to restrict the domains that NCCs can populate during early stages (e8.5-10.5) of cardiovascular and craniofacial development.

Applying antitrust concepts to the acute care hospital industry: defining the relevant market for hospital services.

In considering the possible antitrust implications of a merger of two or more competing hospitals, the courts have generally found that hospitals provide a cluster of services which have significant peculiar characteristics that allow them to be considered a single product market. Spurred by changes in their environments, hospitals during the last decade have become markedly less homogeneous in their range of products and geographic markets. As a result, the impact of hospital mergers in the future may need to be assessed in multiple, more narrowly defined relevant markets, for which several possible definitional bases are suggested in this paper. The increased precision associated with such multidimensional antitrust analysis should permit a more effective consideration of the trade-offs between increases in hospitals' market power and advances in their relative operating efficiency and/or quality of services.

Signaling by wingless in Drosophila.

Wingless, a member of the Wnt gene family, is an essential gene for segmentation in Drosophila, and is also involved in many other patterning events. The gene encodes a secreted protein that can regulate gene expression in adjacent cells. Recently, significant progress has been made in elucidating the signal transduction pathway of wingless, mainly by genetic experiments but increasingly also at the biochemical level. While many components of wingless signaling, in particular a receptor, remain to be identified, our current understanding of wingless pathway is more advanced than that of other Wnt genes. We will give an overview of the various roles of wingless in development, and we will then summarize the wingless signaling pathway as it has emerged from genetic and biochemical studies. Where appropriate, wingless signaling will be compared to the activity of vertebrate Wnt proteins.

In vivo metabolism of CCl4 by rats pretreated with chlordecone, mirex, or phenobarbital.

The propensity of chlordecone (CD) to potentiate hepatotoxic and lethal effects of CCl4 is well established. Mirex (M), a close structural analogue of CD, or phenobarbital (PB), powerful inducers of hepatic microsomal drug metabolizing enzymes, are much weaker potentiators of CCl4 toxicity. The purpose of this study was to test the possibility that CD potentiates the toxicity of CCl4 by increasing the metabolism of CCl4 to a greater degree than either PB or M. We compared the in vivo metabolism of CCl4 in rats pretreated with CD, M, or PB, by measuring the hepatic content of 14CCl4, the expiration of 14CCl4, expiration of 14CCl4-derived 14CO2, and lipid peroxidation. Male Sprague-Dawley rats (250-270 g) were pretreated with a single oral dose of CD (10 mg/kg), M (10 mg/kg), or corn oil vehicle (1 ml/kg). PB pretreatment consisted of an ip injection of sodium PB (80 mg/kg) in saline (0.9%) for 2 successive days. Twenty-four hours later, 14CCl4 (0.1 ml/kg; sp act: 0.04 mCi/mmol) was administered ip in corn oil and the radioactivity present in the expired air was collected for 6 hr. Excretion of the parent compound as represented by the 14C label in the toluene trap was unchanged by any of the pretreatments. Expiration of 14CO2 measured during the 6 hr after CCl4 administration was increased in animals pretreated with PB or CD. In vivo lipid peroxidation measured as diene conjugation in lipids extracted from the livers was increased to a similar extent in animals pretreated with PB and CD, whereas the serum transaminases (ALT, AST) were significantly elevated only in animals pretreated with CD.M did not affect 14CO2 production and was without a significant effect on the lipid peroxidation. The radiolabel present in the liver at 6 hr showed no difference in hepatic content of free 14CCl4 among the groups, but the covalently bound label present in the lipid fractions of the livers pretreated with PB was elevated in comparison to CD and M treatments. These data indicate that a single oral administration of CD (10 mg/kg) 24 hr prior to CCl4 administration (100 microliter/kg) enhances the oxidative metabolism of CCl4 but to a lesser extent than PB (80 mg/kg, ip, twice), which is in inverse relationship to the potentiation of the hepatotoxic and lethal effects of CCl4 associated with these pretreatments.

The segment polarity phenotype of Drosophila involves differential tendencies toward transformation and cell death.

The segment polarity genes of Drosophila are required for intrasegmental organization, as revealed by their abnormal cuticular morphology in mutant embryos. Lesions in most of these loci result in a similar cuticular phenotype, in which the normally naked, posterior region of the segment is covered to varying degrees by ectopic denticles. A temperature-sensitive allele of armadillo, which allows us to vary the level of arm+ activity, generates this entire range of phenotypes, suggesting that these genes affect a common pathway. Previous work with a strong allele of arm revealed the locus to be cell-autonomous, in that small homozygous epidermal clones secreted denticles. We have conducted a similar clonal analysis at all levels of arm+ activity. This shows a differential tendency toward cell transformation and cell death within the segment. Antibodies to segmentation gene-fusion products show that the cell death is primarily in the most posterior region of the segment. We suggest that differential cell respecification, resulting in transformation or death, is involved in generating the segment polarity phenotype.

The Drosophila segment polarity gene dishevelled encodes a novel protein required for response to the wingless signal.

The Drosophila Wnt-1 homolog, wingless (wg), is involved in the signaling of patterning information in several contexts. In the embryonic epidermis, Wg protein is secreted and taken up by neighboring cells, in which it is required for maintenance of engrailed transcription and accumulation of Armadillo protein. The dishevelled (dsh) gene mediates these signaling events as well as wg-dependent induction across tissue layers in the embryonic midgut. dsh is also required for the development processes in which wg functions in adult development. Overall, cells lacking dsh are unable to adopt fates specified by Wg. dsh functions cell autonomously, indicating that it is involved in the response of target cells to the Wg signal. dsh is expressed uniformly in the embryo and encodes a novel protein with no known catalytic motifs, although it shares a domain of homology with several junction-associated proteins. Our results demonstrate that dsh encodes a specific component of Wg signaling and illustrate that Wnt proteins may utilize a novel mechanism of extracellular signal transduction.

Chlordecone-induced fat depletion in the male rat.

The principal objective of this study was to quantitate the anatomical and biochemical parameters associated with the depletion of body fat by the pesticide, chlordecone (CD, or Kepone). Groups of 5 male Sprague-Dawley rats (150-175 g) were fed a control diet or a diet containing 100 ppm CD for 5, 15, or 20 d. After the treatment period, animals were killed by exsanguination. Weight of the period epididymal fat pads was taken as the anatomical marker for the depletion of body fat. Blood levels of acetoacetate, 3-hydroxybutyrate, and nonesterified fatty acids (NEFA) were measured as biochemical as biochemical markers for lipolysis of body fat depots. Serum enzymes (SGPT, ICD) and serum triglycerides were measured to assess liver damage. A consistent and significant difference was observed in the weight of epididymal fat pads between the control and each of the treatment groups. The reduction in epididymal fat reached a maximum of 60% in the CD-fed animals after 20 d. Circulating ketone bodies were not different in any of the treated-animal groups, indicating that CD treatment does not result in metabolic ketosis. Serum triglycerides and NEFA levels were not significantly different in treatment groups versus in controls. Serum transaminases and isocitrate dehydrogenase were not elevated by exposure to CD. These findings indicate that metabolic ketosis is not induced by dietary exposure to CD. Utilization of lipids as energy substrates appears to be the primary underlying mechanism responsible for the loss of body fat observed in CD-treated rats. It appears that CD induces a depletion of body fat stores as a consequence of altered energy balance of the animal.

Lipid composition of selected tissues of the hardshell clam, Mercenaria mercenaria.

1. The total lipids and lipid classes from six tissues of Mercenaria mercenaria were determined. 2. Polar lipids accounted for the largest fraction of lipids. The highest concentration of polar lipids was found in the gill. 3. Free sterols were found only in trace amounts in the gill but were found in much higher quantities (up to 22.6%) in other tissues. 4. The largest stores of triacylglycerols were in the digestive tract-gonads and adductor muscle.

dishevelled and armadillo act in the wingless signalling pathway in Drosophila.

The Wnt genes encode conserved secreted proteins that play a role in normal development and tumorigenesis. Little is known about the signal transduction pathways of Wnt gene products. One of the best characterized Wnt family members is the Drosophila segment polarity gene wingless. We have investigated whether segment polarity genes with a wingless-like phenotype mediate the wingless signal. We used a wingless transgene controlled by a heat-shock promoter for genetic epistasis experiments. We show that wingless acts through dishevelled and armadillo to affect the expression of the homeobox gene engrailed and cuticle differentiation.