Application and feasibility of systemic lupus erythematosus reproductive health care quality indicators at a public urban rheumatology clinic.

OBJECTIVES: Quality indicators (QIs) are evidence-based processes of care designed to represent the current standard of care. Reproductive health QIs for the care of patients with systemic lupus erythematosus (SLE) have recently been developed, and examine areas such as pregnancy screening for autoantibodies, treatment of pregnancy-associated antiphospholipid syndrome, and contraceptive counseling. This study was designed to investigate our performance on these QIs and to explore potential gaps in care and demographic predictors of adherence to the QIs in a safety-net hospital. METHODS: We performed a record review of patients with a diagnosis of SLE at Denver Health Medical Center (DH) through an electronic query of existing medical records and via chart review. Data were limited to female patients between the ages of 18 and 50 who were seen between July 2006 and August 2011. RESULTS: A total of 137 female patients between the ages of 18 and 50 were identified by ICD-9 code and confirmed by chart review to have SLE. Of these, 122 patients met the updated 1997 American College of Rheumatology SLE criteria and had intact reproductive systems. Only 15 pregnancies were documented during this five-year period, and adherence to autoantibody screening was 100 percent. We did not have any patients who were pregnant and met criteria for pregnancy-associated antiphospholipid syndrome. Sixty-five patients (53%) received potentially teratogenic medications, and 30 (46%) had documented discussions about these medications' potential risk upon their initiation. Predictors of whether patients received appropriate counseling included younger age (OR 0.92, CI 0.87-0.98) and those who did not describe English as their primary language (OR 0.24, CI 0.07-0.87) in the multivariate analysis. CONCLUSIONS: We were able to detect an important gap in care regarding teratogenic medication education to SLE patients of childbearing potential in our public health academic clinic, as only one in two eligible patients had documented appropriate counseling at the initiation of a teratogenic medication.

Requirement of circadian genes for cocaine sensitization in Drosophila.

The circadian clock consists of a feedback loop in which clock genes are rhythmically expressed, giving rise to cycling levels of RNA and proteins. Four of the five circadian genes identified to date influence responsiveness to freebase cocaine in the fruit fly, Drosophila melanogaster. Sensitization to repeated cocaine exposures, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for period, clock, cycle, and doubletime, but not in flies lacking the gene timeless. Flies that do not sensitize owing to lack of these genes do not show the induction of tyrosine decarboxylase normally seen after cocaine exposure. These findings indicate unexpected roles for these genes in regulating cocaine sensitization and indicate that they function as regulators of tyrosine decarboxylase.

Aspirin: its effect on platelet glycolysis and release of adenosine diphosphate.

Incubation of human platelets with aspirin inhibited glycolysis and produced a fall in the concentration of adenosine triphosphate. When platelets were exposed to collagen there was an increase in glycolysis and release of adenosine diphosphate. Prior incubation of the platelets with aspirin for 5 minutes did not totally suppress the increase in glycolysis after exposure to collagen but completely inhibited the collagen-induced reaction of the release of adenosine diphosphate. It is suggested that aspirin acts on human platelets by inhibiting both release of adenosine diphosphate and the transport of glucose across the platelet membrane.

CNS and hypoderm regulatory elements of the Drosophila melanogaster dopa decarboxylase gene.

Expression of the dopa decarboxylase gene (Ddc) is regulated in a tissue- and developmental stage-specific manner throughout the life cycle of the fruit fly, Drosophila melanogaster. Essential Ddc regulatory elements lie within 208 base pairs upstream from the RNA start point. Functional elements within this 5' flanking region were mapped by deletion analysis, which assayed expression in vivo after germline integration via P element vectors. One of the elements is essential for expression in both the larval and adult central nervous system, and at least two other elements are necessary for quantitatively normal expression in the hypoderm. Within each of the intervals that have regulatory effects are found sequence elements conserved between the Ddc genes of two distantly related species of flies. On the basis of this correlation, regulatory functions for these sequence elements can be postulated.

The trace amine tyramine is essential for sensitization to cocaine in Drosophila.

BACKGROUND: Sensitization to psychostimulant drugs of abuse is thought to be an important aspect of human addiction, yet how it develops is still unclear. The development of sensitization to cocaine in the fruit fly Drosophila melanogaster is strikingly similar to that observed in vertebrates. By taking advantage of the powerful genetic approaches that are possible in Drosophila, we are able to identify and characterize mutants that fail to develop sensitization. RESULTS: We found that the Drosophila mutant inactive (iav) failed to become sensitized to cocaine. Mutant flies had reduced amounts of the trace amine tyramine in the brain because of reduced activity of the enzyme tyrosine decarboxylase (TDC), which converts tyrosine to tyramine. Furthermore, cocaine exposure induced TDC enzyme activity in a time-dependent manner that paralleled the development of behavioral sensitization. The sensitization failure of iav flies could be rescued by feeding the flies with tyramine; other biogenic amines or amine precursors did not have the same effect. CONCLUSIONS: These results indicate an essential role for tyramine in cocaine sensitization in Drosophila.

Stereotypic behavioral responses to free-base cocaine and the development of behavioral sensitization in Drosophila.

Cocaine abuse is a large social and economic problem that has received much public and scientific attention in recent years. Rodent and primate models have been used to study the behavioral and neurological effects of cocaine. Repeated intermittent doses of cocaine lead to progressive increases in both locomotor activity and stereotyped behaviors known as 'reverse tolerance' or behavioral sensitization, which may model the behavioral and neurochemical processes occurring in cocaine-addicted humans [1]. The biological basis of sensitization is poorly understood. We report that free-base cocaine administered in volatile form to the fruit fly Drosophila melanogaster induces multiple reflexive motor responses that resemble cocaine-induced behaviors in rodents. These behaviors are both dose dependent and sexually dimorphic. Furthermore, Drosophila develops a behavioral sensitization to intermittent doses of cocaine. These results suggest that the pathways leading to cocaine-induced responses and sensitization are evolutionarily conserved between Drosophila and higher vertebrates, and that this genetically tractable animal can be used as a new model system to help determine the biological mechanisms underlying these processes.

Ectopic G-protein expression in dopamine and serotonin neurons blocks cocaine sensitization in Drosophila melanogaster.

Sensitization to repeated doses of psychostimulants is thought to be an important component underlying the addictive process in humans [1] [2] [3] [4]. In all vertebrate animal models, including humans [5], and even in fruit flies, sensitization is observed after repeated exposure to volatilized crack cocaine [6]. In vertebrates, sensitization is thought to be initiated by processes occurring in brain regions that contain dopamine cell bodies [2] [7]. Here, we show that modulated cell signaling in the Drosophila dopamine and serotonin neurons plays an essential role in cocaine sensitization. Targeted expression of either a stimulatory (Galpha(s)) or inhibitory (Galpha(i)) Galpha subunit, or tetanus toxin light chain (TNT) in dopamine and serotonin neurons of living flies blocked behavioral sensitization to repeated cocaine exposures. These flies showed alterations in their initial cocaine responsiveness that correlated with compensatory adaptations of postsynaptic receptor sensitivity. Finally, repeated drug stimulation of a nerve cord preparation that is postsynaptic to the brain amine cells failed to induce sensitization, further showing the importance of presynaptic modulation in sensitization.

Alpha 2-antiplasmin supplementation inhibits tissue plasminogen activator-induced fibrinogenolysis and bleeding with little effect on thrombolysis.

Tissue plasminogen activator (t-PA) causes fibrinogen proteolysis when alpha 2-antiplasmin levels fall, and this may contribute to t-PA-induced hemorrhage. Because clot-bound plasmin is protected from alpha 2-antiplasmin inhibition, we tested the possibility that alpha 2-antiplasmin supplementation would block t-PA-induced fibrinogenolysis and bleeding without affecting thrombolysis. When added to human or rabbit plasma, alpha 2-antiplasmin inhibits t-PA-induced fibrinogenolysis, but hat little effect on the lysis of 125I-fibrin clots. To examine its effect in vivo, rabbits with preformed 125I-labeled-jugular vein thrombi were randomized to receive t-PA, t-PA and alpha 2-antiplasmin, or saline. alpha 2-Antiplasmin infusion produced a modest decrease in t-PA-induced thrombolysis (from 40.2% to 30.1%, P = 0.12), but reduced fibrinogen consumption from 87% to 27% (P = 0.0001), and decreased blood loss from standardized ear incisions from 5,594 to 656 microliter (P < 0.0001). We hypothesize that alpha 2-antiplasmin limits t-PA-induced hemorrhage by inhibiting fibrinogenolysis and subsequent fragment X formation because (a) SDS-PAGE and immunoblot analysis indicate less fragment X formation in alpha 2-antiplasmin treated animals, and (b) when added to a solution of fibrinogen and plasminogen clotted with thrombin in the presence of t-PA, fragment X shortens the lysis time in a concentration-dependent fashion. These findings suggest that fragment X incorporation into hemostatic plugs contributes to t-PA-induced bleeding. By blocking t-PA-mediated fibrinogenolysis, alpha 2-antiplasmin supplementation may improve the safety of fibrin-specific plasminogen activators.

The effect of platelet age on platelet adherence to collagen.

The adherence to collagen of rabbit platelets labeled in vivo with (35)SO(4) (=) has been studied both in vitro and in vivo. The young platelets are labeled with (35)SO(4) (=) 2-3 days after administration of the isotope to the animals. We exposed platelet-rich plasma (ethylenediamine-tetraacetate, EDTA, as anticoagulant), prepared from blood taken from rabbits 54 hr after giving the (35)SO(4) (=), to collagen in vitro. There was a fall in the specific radioactivity of the nonadherent platelets which indicated a selective adhesion of young platelets to the collagen. In experiments designed to have most of the (35)S label in the oldest platelets it was found that exposure of plasma containing these platelets to collagen resulted in an increase in the specific radioactivity of the nonadherent platelets. Similar observations were obtained when glycine-(14)C was used as a platelet label. However, when DF(32)P (di-isopropyl phosphorofluoridate-(32)P), which is thought to label platelets of all ages equally, was used, the adherence of platelets to collagen did not result in any changes in the specific activity of the nonadherent platelets. In in vivo studies in which we infused a collagen suspension into rabbits 54 hr after giving (35)SO(4) (=) we found that the specific radioactivity of the platelets remaining in the circulation fell. This did not occur when we infused the collagen 96 hr after giving the (35)SO(4) (=). The results from these studies indicate that young platelets adhere to collagen more readily than older platelets.

Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors.

Propagation of venous thrombi or rethrombosis after coronary thrombolytic therapy can occur despite heparin administration. To explore potential mechanisms, we set out to determine whether clot-bound thrombin is relatively protected from inhibition by heparin-antithrombin III but susceptible to inactivation by antithrombin III-independent inhibitors. Using plasma fibrinopeptide A (FPA) levels as an index of thrombin activity, we compared the ability of thrombin inhibitors to block FPA release mediated by fluid-phase thrombin with their activity against the clot-bound enzyme. Incubation of thrombin with citrated plasma results in concentration-dependent FPA generation, which reaches a plateau within minutes. In contrast, there is progressive FPA generation when fibrin clots are incubated with citrated plasma. Heparin, hirudin, hirudin dodecapeptide (hirugen), and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK) produce concentration-dependent inhibition of FPA release mediated by fluid-phase thrombin. However, heparin is much less effective at inhibiting thrombin bound to fibrin because a 20-fold higher concentration is necessary to block 70% of the activity of the clot-bound enzyme than is required for equivalent inhibition of fluid-phase thrombin (2.0 and 0.1 U/ml, respectively). In contrast, hirugen and PPACK are equally effective inhibitors of fluid- and solid-phase thrombin, while hirudin is only 50% as effective against the clot-bound enzyme. None of the inhibitors displace bound 125I-labeled thrombin from the clot. These studies indicate that (a) clot-bound thrombin is relatively protected from inhibition by heparin, possibly because the heparin binding site on thrombin is inaccessible when the enzyme is bound to fibrin, and (b) clot-bound thrombin is susceptible to inactivation by antithrombin III-independent inhibitors because the sites of their interaction are not masked by thrombin binding to fibrin. For these reasons, antithrombin III-independent inhibitors may be more effective than heparin in certain clinical settings.

Thrombogenic effect of high-dose aspirin in rabbits. Relationship to inhibition of vessel wall synthesis of prostaglandin I2-like activity.

Aspirin is a promising antithrombogenic agent. It inhibits the generation of thromboxane A(2) by acetylating platelet cyclo-oxygenase. Aspirin also inhibits vessel wall production of PGI(2) which is an inhibitor of platelet aggregation, and therefore is potentially thrombotic. To investigate these two opposing effects we studied the effects of aspirin upon fibrin accretion onto experimentally induced venous thrombi in rabbits and on the PGI(2)-like activity of vessel wall using the thrombin-induced [(14)C]serotonin release assay. A 200-mg/kg dose of aspirin significantly augmented thrombus size when compared to (a) sodium salicylate administered in equal doses, (b) aspirin in a 10-mg/kg dose or (c) controls (P < 0.001). A 200-mg/kg dose of aspirin totally inhibited vessel wall PGI(2)-like activity whereas aspirin in a 10-mg/kg dose produced less inhibition, and 200 mg/kg sodium salicylate had no effect. Local instillation of tranylcypromine, an inhibitor of PGI(2) formation, also significantly augmented thrombus size compared to saline-treated controls and totally inhibited the production of PGI(2)-like activity. The thrombogenic effect of high dose aspirin was lost if an interval of 2.5 h or longer elapsed between vessel damage and drug administration, indicating that in contrast to the platelet, the effect of aspirin on vessel wall prostaglandin synthesis is relatively short-lived. It is concluded that aspirin, in doses higher than those used clinically, can augment experimental thrombosis, presumably by inhibiting the synthesis of vessel wall PGI(2).

Hemostatic function, survival, and membrane glycoprotein changes in young versus old rabbit platelets.

Although in vitro studies have demonstrated functional differences between young and old platelets, in vivo differences have not been precisely established. Therefore the in vivo hemostatic function of young and old platelets and the survival time have been examined in rabbits. The hemostatic function was measured by performing serial ear bleeding times in irradiation-induced thrombocytopenic rabbits. After irradiation with 930 rad the platelet count gradually diminished reaching a nadir ( approximately 20 x 10(3)/mul) at 10 d. The platelets present in the circulation, 7-10 d after irradiation, were considered old platelets, and the platelets present after recovery, 11-14 d postirradiation, young platelets. The measurement of platelet size was consistent with the hypothesis that platelets become smaller with age: the mean size was 3.84 mum(3) for old platelets and 5.86 mum(3) for young platelets. Regression analysis of the relationship between the bleeding time and the platelet count in 18 rabbits showed a significantly different slope for rabbits with predominantly old platelets compared with rabbits with predominantly young platelets (P < 0.001). Young platelets were more effective giving much shorter bleeding times than old platelets at comparable platelet counts. Survival times of young and old platelets were measured using platelets harvested on day 8 postirradiation (old platelets) and day 12 postirradiation (young platelets) that were labeled and then reinjected into normal recipient animals. The mean platelet survival time, calculated by gamma function, of old platelets was 28.8 h; of young platelets, 87.4 h; and of normally circulating heterogeneous platelets, (normal platelets) 53.0 h. Notably, the survival of old platelets was found to be exponential, and of young platelets, linear. Analysis of the membrane glycoproteins in young, old and normal platelets indicated that there was no qualitative difference amongst the young, normal, and old platelets. The relative relationship among all the glycoprotein peaks was equal and the only changes observed were quantitative, with young platelets having significantly more membrane glycoprotein per cell than old platelets and normal platelets. Normal platelets had intermediate concentrations of each glycoprotein. These results demonstrate that young platelets are hemostatically more effective in vivo than old platelets. The data are compatible with the hypothesis that platelets age in the circulation by losing membrane fragments and then after becoming senescent, are removed from the circulation by a random process.

Resolution of experimental pulmonary emboli with heparin and streptokinase in different dosage regimens.

Thrombolytic agents may be useful in acute pulmonary embolism, but their optimal dosage remains uncertain. We have examined the relative efficacy of heparin and different doses of streptokinase, either alone or in combination, in acute experimental pulmonary embolism. A standardized massive embolus of autologous blood clot incorporating canine [(125)I]-fibrinogen was given to 40 dogs; the degree of resolution after 24 h was quantitated by measuring the radioactivity in the lungs and was compared with detailed postmortem observations. The amount of residual embolus was 49% in control animals, 28% after heparin (200 U/kg loading dose and 800 U/kg/24 h maintenance dose), and 6% after high dose streptokinase (250,000 U loading dose and 100,000 U/h maintenance dose); it was 31% after low dose streptokinase (25,000 U loading dose and 10,000 U/h maintenance dose), 7% after low dose streptokinase with heparin, 14% after very low dose streptokinase (5,000 U/h without a loading dose) with heparin, and 9% after short course streptokinase (250,000 U loading dose and no maintenance dose) with heparin. The combination of heparin and low doses or brief courses of streptokinase appeared to be synergistic and produced as much resolution as did standard high dose streptokinase alone. The enhanced resolution of pulmonary emboli in heparin-treated animals may have been due to the prevention by heparin of further deposition of fibrin on the embolus. It appears that dosage regimens of thrombolytic therapy other than those in current use may be worthy of clinical examination.

Shortening of the bleeding time in rabbits by hydrocortisone caused by inhibition of prostacyclin generation by the vessel wall.

The effect of hydrocortisone on thrombocytopenic bleeding has been studied in rabbits using a jugular vein bleeding-time technique and a microvascular bleeding-time technique. An inverse relationship was found between the bleeding time and platelet count with both techniques in rabbits made thrombocytopenic by either X-irradiation or injection of heterologous platelet antiserum. Hydrocortisone shortened both bleeding times in thrombocytopenic animals when given in single large doses intravenously (25-100 mg/kg), in daily doses (6 mg/kg) intramuscularly, and shortened the jugular bleeding time when applied to the outside of the jugular vein or instilled intraluminally into the vein. This effect was also noted in normal animals. The effect on thrombocytopenic bleeding was dose related. When given daily, the effect was greater when hydrocortisone was given for 10 d than for 5 d. Both indomethacin and tranylcypromine also reduced the jugular vein bleeding time when instilled intraluminally into the jugular vein, whereas exogenously provided arachidonic acid reversed the effect of hydrocortisone but did not reverse the effect of indomethacin or tranylcypromine. Exogenously provided linoleic acid did not have any effect. Perfusion of the vessel segment with prostacyclin (PGI(2)) reversed the effect of intraluminally administered hydrocortisone, indomethacin, and tranylcypromine. Similarly, hydrocortisone, indomethacin, and tranylcypromine all reduced the rate of loss of fluid from a standard wound in isolated vessels emptied of blood and perfused with saline under constant pressure. PGI(2) reversed the action of these three agents, however, arachidonic acid reversed only the effect of hydrocortisone and did not reverse the effect of indomethacin and tranylcypromine. The generation of PGI(2)-like material and 6-keto-prostaglandinF(1) alpha from jugular vein strips was prevented by prior exposure of the animals or vessel wall to hydrocortisone. These results are compatible with the hypothesis that the vessel wall releases smooth muscle-relaxing prostaglandins when injured and that inhibition of prostaglandin formation by hydrocortisone enhances hemostasis by allowing vasoconstriction to be maintained.

Combined antiplatelet and anticoagulant therapy: clinical benefits and risks.

The combination of anticoagulant and antiplatelet therapy is more effective than antiplatelet therapy alone for the initial and long-term management of acute coronary syndromes but increases the risk of bleeding. Antiplatelet therapy is often combined with oral anticoagulants in patients with an indication for warfarin therapy (e.g. atrial fibrillation) who also have an indication for antiplatelet therapy (e.g. coronary artery disease) but the appropriateness of such an approach is unresolved. Anticoagulation appears to be as effective as antiplatelet therapy for long-term management of acute coronary syndrome and stroke, and possibly peripheral artery disease, but causes more bleeding. Therefore, in such patients who develop atrial fibrillation, switching from antiplatelet therapy to anticoagulants might be all that is required. The combination of anticoagulant and antiplatelet therapy has only been proven to provide additional benefit over anticoagulants alone in patients with prosthetic heart valves. The combination of aspirin and clopidogrel is not as effective as oral anticoagulants in patients with atrial fibrillation, whereas the combination of aspirin and clopidogrel is more effective than oral anticoagulants in patients with coronary stents. Whether the benefits of triple therapy outweigh the risks in patients with atrial fibrillation and coronary stents requires evaluation in randomized trials.

Association between asymptomatic deep vein thrombosis detected by venography and symptomatic venous thromboembolism in patients undergoing elective hip or knee surgery.

BACKGROUND: Venography is commonly used to compare the efficacy of different thromboprophylaxis strategies for preventing deep vein thrombosis (DVT) in patients undergoing total hip replacement (THR) or total knee replacement (TKR). METHODS: We explored the relation between asymptomatic DVT and symptomatic venous thromboembolism (VTE) in patients undergoing THR or TKR treated with standard doses of enoxaparin (30 mg b.i.d. or 40 mg o.d.) by comparing the incidence of asymptomatic DVT in venographic studies with the incidence of symptomatic VTE in studies where venography was not performed. RESULTS: In 10 venographic studies involving 5796 patients, the incidence of asymptomatic DVT after THR was 13.2% [95% CI, 12.2-14.2%] and after TKR was 38.1% (95% CI, 35.5-40.8%). In two studies involving 3500 patients who did not undergo venography, the 90-day incidence of symptomatic VTE after THR was 2.7% (95% CI, 2.1-3.4%) and after TKR was 1.8% (95% CI, 0.9-2.7%). For every symptomatic VTE in THR studies where venography was not performed there were five asymptomatic DVTs in the venographic studies; for TKR, the ratio was 1:21. The incidence of asymptomatic DVT and the symptomatic VTE/asymptomatic DVT ratio was influenced by the venogram reading committee (Gothenburg vs. Hamilton: total DVT after THR, 19.5% vs. 8.7%, P < 0.0001; for TKR, 42.7% vs. 27.2%, P < 0.0001). CONCLUSIONS: Comparisons across trials show a consistent relation between asymptomatic venographic DVT in patients undergoing elective THR or TKR surgery and symptomatic VTE in patients not undergoing venography. Differences exist in the strength of the relation depending on the type of surgery and the venogram reading committee.

cis-regulatory sequences responsible for alternative splicing of the Drosophila dopa decarboxylase gene.

The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in specific sets of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. The Ddc CNS mRNA contains all four exons (A through D), whereas the hypodermal mRNA contains only three exons (A, C, and D). To localize cis-regulatory sequences responsible for Ddc alternative splicing, a Ddc minigene and several fusion genes containing various amounts of Ddc sequences fused to fushi tarazu (ftz) exon 1 were constructed and introduced into flies by P-element-mediated germ line transformation. We find that Ddc intron ab and exon B are sufficient to regulate Ddc alternative splicing, since transcripts of a minimal fusion gene containing most of Ddc intron ab and exon B are spliced to exon B in the CNS but not in the hypoderm. These results indicate that Ddc alternative splicing is regulated by either a negative mechanism preventing splicing to exon B in the hypoderm or a positive mechanism activating splicing to exon B in the CNS. Our previous data suggest that Ddc hypodermal splicing is the actively regulated splicing pathway (J. Shen, C. J. Beall, and J. Hirsh, Mol. Cell. Biol. 13:4549-4555, 1993). Here we show that deletion of Ddc intron ab sequences selectively disrupts hypodermal splicing specificity. These results support a model in which Ddc alternative splicing is negatively regulated by a blockage mechanism preventing splicing to exon B in the hypoderm.

Isolation and characterization of the dopa decarboxylase gene of Drosophila melanogaster.

We have isolated chromosomal deoxyribonucleic acid clones containing the Drosophila dopa decarboxylase gene. We describe an isolation procedure which can be applied to other nonabundantly expressed Drosophila genes. The dopa decarboxylase gene lies within or very near polytene chromosome band 37C1-2. The gene is interrupted by at least one intron, and the primary mode of regulation is pretranslational. At least two additional sequences hybridized by in vivo ribonucleic acid-derived probes are found within a 35-kilobase region surrounding the gene. The developmental profile of ribonucleic acid transcribed from one of these regions differs from that of the dopa decarboxylase transcript.

Tissue-specific alternative splicing of the Drosophila dopa decarboxylase gene is affected by heat shock.

The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in a small number of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. We investigated whether Ddc splicing in the CNS is a general property of the CNS or a unique property of the cells that normally express Ddc by expressing the Ddc primary transcript ubiquitously under the control of an Hsp70 heat shock promoter. Under basal expression conditions, Ddc splicing shows normal tissue specificity, indicating that the regulation of Ddc splicing in the CNS is tissue specific rather than cell specific. Previous studies have shown that severe heat shock blocks mRNA splicing in cultured Drosophila melanogaster cells. Our results show that splicing of the heat shock-inducible Hsp83 transcript is very resistant to heat shock. In contrast, under either mild or severe heat shock, the splicing specificity of the heat shock-induced Ddc primary transcript is affected, leading to the accumulation of inappropriately high levels of the CNS splice form in non-CNS tissues. The chromosomal Ddc transcript is similarly affected. These results show unexpected heterogeneity in the splicing of individual mRNAs as a response to heat shock and suggest that the Ddc CNS-specific splicing pathway is the default.

High levels of intron-containing RNAs are associated with expression of the Drosophila DOPA decarboxylase gene.

We have examined the structure and expression during embryonic development of the Drosophila DOPA decarboxylase gene, Ddc. The Ddc gene is transcribed to make at least five different size classes of RNA. These RNA species first appear late in embryogenesis, coincident with induction of Ddc enzyme activity. The most abundant and smallest RNA appears to be Ddc mRNA. The sequences encoding this RNA are split by two intervening sequences. Each of the larger RNA species contains some or all of the intervening sequences. We have noted two unusual features of Ddc expression during embryogenesis. First, the intervening-sequence-containing RNAs are present as 20% or more of the polyadenylated Ddc RNA molecules, an exceptionally high proportion. Second, these RNAs do not disappear as rapidly as Ddc mRNA after Ddc enzyme activity reaches fully induced levels. These observations indicate slow rates of RNA processing relative to mRNA half-life and suggest that post-transcriptional steps participate in regulating Ddc expression. Although four of the five RNA species were detected at multiple developmental stages during which Ddc is expressed, one was found uniquely during embryogenesis. This RNA differs from Ddc mRNA in length and in time of expression during embryogenesis but is transcribed in the same orientation and from the same genomic sequences as the Ddc primary transcript.