Muscle-specific, liver-detargeted adeno-associated virus gene therapy rescues Pompe phenotype in adult and neonate Gaa-/- mice.

Pompe disease (PD) is a neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency. Reduced GAA activity leads to pathological glycogen accumulation in cardiac and skeletal muscles responsible for severe heart impairment, respiratory defects, and muscle weakness. Enzyme replacement therapy with recombinant human GAA (rhGAA) is the standard-of-care treatment for PD, however, its efficacy is limited due to poor uptake in muscle and the development of an immune response. Multiple clinical trials are ongoing in PD with adeno-associated virus (AAV) vectors based on liver- and muscle-targeting. Current gene therapy approaches are limited by liver proliferation, poor muscle targeting, and the potential immune response to the hGAA transgene. To generate a treatment tailored to infantile-onset PD, we took advantage of a novel AAV capsid able to increase skeletal muscle targeting compared to AAV9 while reducing liver overload. When combined with a liver-muscle tandem promoter (LiMP), and despite the extensive liver-detargeting, this vector had a limited immune response to the hGAA transgene. This combination of capsid and promoter with improved muscle expression and specificity allowed for glycogen clearance in cardiac and skeletal muscles of Gaa-/- adult mice. In neonate Gaa-/- , complete rescue of glycogen content and muscle strength was observed 6 months after AAV vector injection. Our work highlights the importance of residual liver expression to control the immune response toward a potentially immunogenic transgene expressed in muscle. In conclusion, the demonstration of the efficacy of a muscle-specific AAV capsid-promoter combination for the full rescue of PD manifestation in both neonate and adult Gaa-/- provides a potential therapeutic avenue for the infantile-onset form of this devastating disease.

Malaria in Refugee Children Resettled to a Holoendemic Area of Sub-Saharan Africa.

BACKGROUND: Malaria is a leading cause of morbidity and mortality in refugee children in high-transmission parts of Africa. Characterizing the clinical features of malaria in refugees can inform approaches to reduce its burden. METHODS: The study was conducted in a high-transmission region of northern Zambia hosting Congolese refugees. We analyzed surveillance data and hospital records of children with severe malaria from refugee and local sites using multivariable regression models and geospatial visualization. RESULTS: Malaria prevalence in the refugee settlement was similar to the highest burden areas in the district, consistent with the local ecology and leading to frequent rapid diagnostic test stockouts. We identified 2197 children hospitalized for severe malaria during the refugee crisis in 2017 and 2018. Refugee children referred from a refugee transit center (n = 63) experienced similar in-hospital mortality to local children and presented with less advanced infection. However, refugee children from a permanent refugee settlement (n = 110) had more than double the mortality of local children (P < .001), had lower referral rates, and presented more frequently with advanced infection and malnutrition. Distance from the hospital was an important mediator of the association between refugee status and mortality but did not account for all of the increased risk. CONCLUSIONS: Malaria outcomes were more favorable in refugee children referred from a highly outfitted refugee transit center than those referred later from a permanent refugee settlement. Refugee children experienced higher in-hospital malaria mortality due in part to delayed presentation and higher rates of malnutrition. Interventions tailored to the refugee context are required to ensure capacity for rapid diagnosis and referral to reduce malaria mortality.

Whole Blood Transfusion for Severe Malarial Anemia in a High Plasmodium falciparum Transmission Setting.

BACKGROUND: Severe malaria resulting from Plasmodium falciparum infection is the leading parasitic cause of death in children worldwide, and severe malarial anemia (SMA) is the most common clinical presentation. The evidence in support of current blood transfusion guidelines for patients with SMA is limited. METHODS: We conducted a retrospective cohort study of 911 hospitalized children with SMA in a holoendemic region of Zambia to examine the association of whole blood transfusion with in-hospital survival. Data were analyzed in adjusted logistic regression models using multiple imputation for missing data. RESULTS: The median age of patients was 24 months (interquartile range, 16-30) and overall case fatality was 16%. Blood transfusion was associated with 35% reduced odds of death in children with SMA (odds ratio, 0.65; 95% confidence interval, .52-.81; P = .0002) corresponding to a number-needed-to-treat (NNT) of 14 patients. Children with SMA complicated by thrombocytopenia were more likely to benefit from transfusion than those without thrombocytopenia (NNT = 5). Longer storage time of whole blood was negatively associated with survival and with the posttransfusion rise in the platelet count but was not associated with the posttransfusion change in hemoglobin concentration. CONCLUSIONS: Whole blood given to pediatric patients with SMA was associated with improved survival, mainly among those with thrombocytopenia who received whole blood stored for <4 weeks. These findings point to a potential use for incorporating thrombocytopenia into clinical decision making and management of severe malaria, which can be further assessed in prospective studies, and underline the importance of maintaining reliable blood donation networks in areas of high malaria transmission.

Four Artemisinin-Based Treatments in African Pregnant Women with Malaria.

BACKGROUND: Information regarding the safety and efficacy of artemisinin combination treatments for malaria in pregnant women is limited, particularly among women who live in sub-Saharan Africa. METHODS: We conducted a multicenter, randomized, open-label trial of treatments for malaria in pregnant women in four African countries. A total of 3428 pregnant women in the second or third trimester who had falciparum malaria (at any parasite density and regardless of symptoms) were treated with artemether-lumefantrine, amodiaquine-artesunate, mefloquine-artesunate, or dihydroartemisinin-piperaquine. The primary end points were the polymerase-chain-reaction (PCR)-adjusted cure rates (i.e., cure of the original infection; new infections during follow-up were not considered to be treatment failures) at day 63 and safety outcomes. RESULTS: The PCR-adjusted cure rates in the per-protocol analysis were 94.8% in the artemether-lumefantrine group, 98.5% in the amodiaquine-artesunate group, 99.2% in the dihydroartemisinin-piperaquine group, and 96.8% in the mefloquine-artesunate group; the PCR-adjusted cure rates in the intention-to-treat analysis were 94.2%, 96.9%, 98.0%, and 95.5%, respectively. There was no significant difference among the amodiaquine-artesunate group, dihydroartemisinin-piperaquine group, and the mefloquine-artesunate group. The cure rate in the artemether-lumefantrine group was significantly lower than that in the other three groups, although the absolute difference was within the 5-percentage-point margin for equivalence. The unadjusted cure rates, used as a measure of the post-treatment prophylactic effect, were significantly lower in the artemether-lumefantrine group (52.5%) than in groups that received amodiaquine-artesunate (82.3%), dihydroartemisinin-piperaquine (86.9%), or mefloquine-artesunate (73.8%). No significant difference in the rate of serious adverse events and in birth outcomes was found among the treatment groups. Drug-related adverse events such as asthenia, poor appetite, dizziness, nausea, and vomiting occurred significantly more frequently in the mefloquine-artesunate group (50.6%) and the amodiaquine-artesunate group (48.5%) than in the dihydroartemisinin-piperaquine group (20.6%) and the artemether-lumefantrine group (11.5%) (P<0.001 for comparison among the four groups). CONCLUSIONS: Artemether-lumefantrine was associated with the fewest adverse effects and with acceptable cure rates but provided the shortest post-treatment prophylaxis, whereas dihydroartemisinin-piperaquine had the best efficacy and an acceptable safety profile. (Funded by the European and Developing Countries Clinical Trials Partnership and others; ClinicalTrials.gov number, NCT00852423.).

Lessons from the pandemic: new best practices in selecting molecular diagnostics for point-of-care testing of infectious diseases in sub-Saharan Africa.

INTRODUCTION: Point-of-care molecular diagnostics offer solutions to the limited diagnostic availability and accessibility in resource-limited settings. During the COVID-19 pandemic, molecular diagnostics became essential tools for accurate detection and monitoring of SARS-CoV-2. The unprecedented demand for molecular diagnostics presented challenges and catalyzed innovations which may provide lessons for the future selection of point-of-care molecular diagnostics. AREAS COVERED: We searched PubMed from January 2020 to August 2023 to identify lessons learned from the COVID-19 pandemic which may impact the selection of point-of-care molecular diagnostics for future use in sub-Saharan Africa. We evaluated this in the context of REASSURED criteria (Real-time connectivity; Ease of specimen collection; Affordable; Sensitive; Specific; User-friendly; Rapid and robust; Equipment free; and Deliverable to users at the point of need) for point-of-care diagnostics for resource-limited settings. EXPERT OPINION: The diagnostic challenges and successes during the COVID-19 pandemic affirmed the importance of the REASSURED criteria but demonstrated that these are not sufficient to ensure new diagnostics will be appropriate for public health emergencies. Capacity for rapid scale-up of diagnostic testing and transferability of assays, data, and technology are also important, resulting in updated REST-ASSURED criteria. Few diagnostics will meet all criteria, and trade-offs between criteria will need to be context-specific.

Role of mannose-binding lectin in intestinal homeostasis and fungal elimination.

Mannose-binding lectin (MBL) is a soluble lectin of the innate immune system that is produced by the liver and secreted into the circulation where it activates the lectin complement pathway, enhances phagocytosis of microorganisms by leukocytes, and modulates inflammation. MBL can recognize patterns on the surface of different pathogens, including Candida albicans. Our aims were to investigate whether MBL is expressed in the gut epithelium and to examine its effect on the modulation of intestinal inflammation and C. albicans elimination. Using reverse transcriptase-PCR, MBL transcripts were highly expressed in different parts of the mouse gut. MBL expression was also detected by immunoblotting and immunolocalization in response to C. albicans colonization of the gut; the highest expression of MBL was detected in the stomach. Blocking MBL by administering mannans to mice increased C. albicans colonization. MBL-deficient mice had a higher level of colonization than wild-type mice. Dextran sodium sulfate-induced colitis promoted C. albicans dissemination to the kidneys and lungs of MBL-deficient mice. MBL-deficient mice exhibited elevated expression of interleukin (IL)-17, IL-23, dectin-1, and Toll-like receptor-4. This study shows that MBL expression is induced in the gut in response to C. albicans sensing and is required for intestinal homeostasis and host defense against C. albicans.

Four artemisinin-based treatments in African pregnant women with malaria.

BACKGROUND: Information regarding the safety and efficacy of artemisinin combination treatments for malaria in pregnant women is limited, particularly among women who live in sub-Saharan Africa. METHODS: We conducted a multicenter, randomized, open-label trial of treatments for malaria in pregnant women in four African countries. A total of 3428 pregnant women in the second or third trimester who had falciparum malaria (at any parasite density and regardless of symptoms) were treated with artemether-lumefantrine, amodiaquine-artesunate, mefloquine-artesunate, or dihydroartemisinin-piperaquine. The primary end points were the polymerase-chain-reaction (PCR)-adjusted cure rates (i.e., cure of the original infection; new infections during follow-up were not considered to be treatment failures) at day 63 and safety outcomes. RESULTS: The PCR-adjusted cure rates in the per-protocol analysis were 94.8% in the artemether-lumefantrine group, 98.5% in the amodiaquine-artesunate group, 99.2% in the dihydroartemisinin-piperaquine group, and 96.8% in the mefloquine-artesunate group; the PCR-adjusted cure rates in the intention-to-treat analysis were 94.2%, 96.9%, 98.0%, and 95.5%, respectively. There was no significant difference among the amodiaquine-artesunate group, dihydroartemisinin-piperaquine group, and the mefloquine-artesunate group. The cure rate in the artemether-lumefantrine group was significantly lower than that in the other three groups, although the absolute difference was within the 5-percentage-point margin for equivalence. The unadjusted cure rates, used as a measure of the post-treatment prophylactic effect, were significantly lower in the artemether-lumefantrine group (52.5%) than in groups that received amodiaquine-artesunate (82.3%), dihydroartemisinin-piperaquine (86.9%), or mefloquine-artesunate (73.8%). No significant difference in the rate of serious adverse events and in birth outcomes was found among the treatment groups. Drug-related adverse events such as asthenia, poor appetite, dizziness, nausea, and vomiting occurred significantly more frequently in the mefloquine-artesunate group (50.6%) and the amodiaquine-artesunate group (48.5%) than in the dihydroartemisinin-piperaquine group (20.6%) and the artemether-lumefantrine group (11.5%) (P<0.001 for comparison among the four groups). CONCLUSIONS: Artemether-lumefantrine was associated with the fewest adverse effects and with acceptable cure rates but provided the shortest posttreatment prophylaxis, whereas dihydroartemisinin-piperaquine had the best efficacy and an acceptable safety profile. (Funded by the European and Developing Countries Clinical Trials Partnership and others; ClinicalTrials.gov number, NCT00852423.).

A conserved retinoid X receptor (RXR) from the mollusk Biomphalaria glabrata transactivates transcription in the presence of retinoids.

Retinoid X receptors (RXR) are members of the nuclear receptor superfamily of ligand-activated transcription factors that have been characterized in a wide variety of metazoan phyla. They act as heterodimer partners of other nuclear receptors, and in vertebrates also activate transcription as homodimers in the presence of a ligand, 9-cis retinoic acid. In order to test the hypothesis that retinoic acid signaling pathways involving RXRs are present in the Lophotrochozoa, we have sought to isolate conserved members of this family from the platyhelminth parasite Schistosoma mansoni and its intermediate host, the mollusk Biomphalaria glabrata. Here we report that an RXR ortholog from B. glabrata (BgRXR) is better conserved, compared with mouse RXRalpha, both in the DNA-binding domain (89% identity) and in the ligand-binding domain (LBD) (81% identity), than are arthropod homologs. In EMSA, BgRXR binds to the direct repeat response element DR1 as a homodimer or as a heterodimer with mammalian RARalpha, LXR, FXR or PPARalpha. When transfected alone into mammalian cell lines, BgRXR transactivated transcription of a reporter gene from the Apo-A1 promoter in the presence of 9-cis retinoic acid or DHA. Constructs with the Gal4 DNA binding domain fused to the hinge and LBDs of BgRXR were used to show that ligand-dependent activation of transcription by BgRXR required its intact AF-2 activation domain, and that the LBD can form homodimers. Finally, the binding of 9-cis retinoic acid preferentially protected the LBD of BgRXR from degradation by trypsin in a proteolysis protection assay. Our results show that BgRXR binds and is activated by retinoids and suggest that retinoid signaling pathways are conserved in the Lophotrochozoa. The nucleotide sequence reported in this paper has been submitted to the GenBank/EBI Data Bank with accession no. AY048663.

Specific atrial overexpression of G protein coupled human beta 1 adrenoceptors in transgenic mice.

OBJECTIVE: The aim was to develop a transgenic mouse model of atrial beta 1 adrenoceptor overexpression in order to create atrial alteration of the receptor transduction system. METHODS: Transgenic founders were generated after microinjection of the transgene construct into the pronucleus of fertilised mouse eggs. Heterozygous progeny were screened for RNA expression of the human beta 1 adrenoceptor gene under the control of a 0.56 kb proximal promoter of the human atrial natriuretic factor. One line, out of the three obtained, was selected and further characterised for overexpression of the human beta 1 adrenoceptor. Polymerase chain reaction was employed to detect beta 1 adrenoceptor mRNA, and 125I-cyanopindolol (ICYP) binding assays were used to quantify receptors in heart membranes. A quantitative autoradiographic ICYP binding technique was also used to visualise atrial and ventricular beta adrenoceptors in heart sections. RESULTS: The human beta 1 adrenoceptor was overexpressed specifically in the atria of transgenic mice. The level of the beta 1 adrenoceptor was 5-10-fold higher in transgenic mice compared to basal murine beta 1 adrenoceptors in non-transgenic control mice. Left and right atrial receptor overexpression was confirmed by in vitro autoradiography. The human receptors were able to couple to the murine stimulatory G proteins (Gs), as shown by high affinity binding site dosage using the beta adrenoceptor agonist isoprenaline. Isoprenaline displacement studies allowed the determination of two different affinity sites, one of high affinity (KH = 5.8 nM), and one of low affinity (KL = 520 nM). When expressed in terms of protein density (fmol.mg-1), atrial transgenic beta 1 adrenoceptors displayed a threefold increase in high affinity sites (KH) as compared to control mice. Preliminary electrocardiographic data showed supraventricular premature beats in 6/14 transgenic mice v 2/16 control mice. CONCLUSIONS: These transgenic mice may provide a useful pharmacological tool to investigate the pathophysiological consequences of the overactivation of atrial beta 1 adrenoceptor-adenylyl cyclase signalling system.

Agonist-induced signaling and trafficking of the mu-opioid receptor: role of serine and threonine residues in the third cytoplasmic loop and C-terminal domain.

The human mu-opioid receptor and a mutant form, muS/ T[i3+Cter]A, in which all Ser and Thr residues from the third cytoplasmic loop and C-terminal domain were changed to Ala, were studied after expression in CHO-K1 cells. Although the mutant receptors had similar affinities for agonists and EC50 values for inhibition of adenylyl cyclase as compared to wild-type receptors, the Emax were almost 2-fold decreased, suggesting a role of the mutated residues in G-protein coupling. After chronic morphine or etorphine, the EC50 values of the agonists were about 5-fold increased at both receptors but the Emax values were not altered; upon agonist withdrawal forskolin-stimulated cAMP levels were increased to almost 200% of control levels. Sequestration and rapid down-regulation of the mu-opioid receptor were induced by DAGO and etorphine but not morphine. In contrast, the muS/T[i3+Cter]A receptor was not sequestered and was up-regulated (150-380%) after treatment with agonists. The results indicate that the Ser and Thr residues in the third cytoplasmic loop and C-terminus of the mu-opioid receptor are not involved in the limited desensitization or in the adenylyl cyclase superactivation promoted by agonists but that their integrity and/or their phosphorylation is required in the intricate and coordinately regulated pathways involved in receptor signaling and trafficking.

Production of anti-peptide antibodies directed against the first and the second extracellular loop of the human serotonin 5-HT1A receptor.

The second extracellular loop of the beta-adrenergic and muscarinic acetylcholine receptors was shown to be an autoimmune target for antibodies in several autoimmune diseases. These autoantibodies and the antibodies induced against synthetic peptides corresponding to this loop have pharmacological and physiological properties upon receptor recognition which could explain their pathophysiological role. We here describe the immune properties of the first and second extracellular loops of another G protein-coupled receptor, the serotonin 5-HT1A receptor. The injection in rabbits of the free peptides Y16L and G21G corresponding to the first and second extracellular loops respectively induced anti-peptide antibodies with high titer, demonstrating the presence of a T-cell epitope on each peptide. Interestingly, in contrast to the G21G peptide that induced only anti-G21G antibodies (Ab-2 antibodies), the Y16L peptide induced two populations of antibodies. One recognized only the Y16L peptide (Ab-1 antibodies), the other recognized both peptides (Ab-12 antibodies). This reflects the presence on the two peptides of at least two B-cell epitopes. The fact that the G21G peptide induces only one antibody population might indicate that it possesses one immunodominant epitope involved in the Ab-2 antibody production and one cryptic epitope involved in the cross-reaction with the anti-Y16L antibodies. But only Ab-2 antibodies were able to recognize specifically the human protein receptor expressed in E coli in immunoblot.

Synthesis, structure-activity relationships, and pharmacological profile of 9-amino-4-oxo-1-phenyl-3,4,6,7-tetrahydro[1,4]diazepino[6, 7,1-hi]indoles: discovery of potent, selective phosphodiesterase type 4 inhibitors.

The synthesis, structure-activity relationships, and biological properties of a novel series of potent and selective phosphodiesterase type 4 (PDE4) inhibitors are described. These new aminodiazepinoindoles displayed in vitro PDE4 activity with submicromolar IC(50) values and PDE4 selectivity vs PDE1, -3, and -5. Specifically, one compound (CI-1044, 10e) provided efficient in vitro inhibition of TNFalpha release from hPBMC and hWB with IC(50) values of 0.34 and 0.84 microM, respectively. This compound was found to exhibit potent in vivo activity in antigen-induced eosinophil recruitment in Brown-Norway rats (ED(50) = 3.2 mg/kg po) and in production of TNFalpha in Wistar rats (ED(50) = 2.8 mg/kg po). No emetic side effects at therapeutic doses were observed in ferrets.

[Transfusional record and blood grouping methods: essential factors of transfusion safety]. / Dossier transfusionnel et techniques de groupage sanguin: éléments essentiels de la sécurité transfusionnelle.

The clinical and biological control of the whole transfusion process is a major preoccupation for everyone dealing with blood transfusion. Specially when the patient is a female recipient or belongs to a group with a high prevalence of alloimmunisation. This case report points out the outstanding importance of the immune compatibility, which must be strongly maintained to prevent any harmful consequences. The transfusional record transmission and a simple and sensitive blood grouping test are essential to increase transfusion safety.

[Blood transfusion security: one train may conceal another!]. / Sécurité transfusionnelle: un train peut en cacher un autre!

The discovery of antibodies with specificities that are directed toward antigens of high prevalence is a difficult situation to manage in emergency blood transfusion. The reactions they produce interfere with the identification of reactions due to other, clinically significant antibodies. We report a case which illustrates this problem in terms of transfusion safety and time to carry out the tests.

Modulation of oxazolone-induced hypersensitivity in mice by selective PDE inhibitors.

The effects of PDE inhibitors on oxazolone-induced contact hypersensitivity (CS) were studied in mice. Rolipram, Ro 20-1724 and theophylline dose dependently inhibited CS but none caused >53% inhibition. ED(30) values at 24 h before challenge for rolipram, Ro 20-1724 and theophylline were 2.1, 5.4 and 30.4 mg/kg, p.o., respectively. Milrinone and SKF 94836 at 30 mg/kg caused a small, but significant inhibition of 13% and 18%, respectively, although the inhibition (8%) caused by zaprinast was not significant. Betamethasone (10 mg/kg, p.o.) caused a marked inhibition (80%) as did indomethacin (65% at 5 mg/kg, p.o.). Rolipram and Ro 20-1724 inhibited proliferation of mouse lymphoblasts with IC(50) values of 0.08 muM and 0.83 muM, respectively. In contrast, zaprinast caused only a weak inhibition (IC(50) = 119 muM) of lymphocyte proliferation, whereas SKF 94836 and theophylline failed to cause any significant inhibition at 100 muM (26% and 2%, respectively). These findings suggest that PDE IV isozymes play a principal role in mediating CS by inhibiting lymphocyte activation.

Human beta2-adrenergic receptor/GS alpha fusion protein, expressed in 2 ras-dependent murine carcinoma cell lines, prevents tumor growth in syngeneic mice.

We report a strategy of tumor growth inhibition based on the expression of a foreign protein with both potential anti-proliferative and immunogenic properties. To validate our approach, we used 2 ras-mutated murine carcinoma cell lines (carB and C57/PDV) transfected with the gene encoding a fusion protein containing the human beta2-adrenergic receptor and the alpha subunit of the Gs protein (beta2Gs). We previously showed that the sustained activation of the beta2Gs fusion protein expressed in carB cells (carB beta2Gs cells) induced a cAMP-dependent inhibition of cell growth in vitro. Here, we observed inhibition of tumor growth after s.c. inoculation of 2 carB beta2Gs clones (10C2 and 20F4) in syngeneic ICFW mice. We thus selected 3 C57/PDV beta2Gs clones (2D3, 5F3 and 1G1) in which activation of the fusion protein was not efficiently coupled to the cAMP-PKA signaling pathway. Contrasting with carB beta2Gs clones, activation of the fusion protein in these C57/PDV beta2Gs clones did not have any anti-proliferative effect in vitro. Therefore, they were good candidates to assess the immunogenic property of the fusion protein. Accordingly, none of the C57/PDV beta2Gs clones formed tumors in immunocompetent syngeneic C57BL/6 mice, while they were still tumorigenic in nude mice. Most interestingly, all of the beta2Gs clones that did not form tumors, from both cell lines, provided protection against respective wild-type tumor development. Our results show that expression of the beta2Gs fusion protein in cancer cells elicits inhibition of cell proliferation and/or immune rejection of both beta2Gs-modified and wild-type tumor cells.

Activation of a beta 2-adrenergic receptor/Gs alpha fusion protein elicits a desensitization-resistant cAMP signal capable of inhibiting proliferation of two cancer cell lines.

We showed in a previous study that the expression, in Gs-deficient S49 cyc- cells, of a fusion gene encoding the beta 2-adrenergic Receptor (beta 2AR) and the alpha subunit of the Gs protein (Gs alpha) restored beta 2AR-dependent activation of adenylyl cyclase. We report here the extensive characterization of short- and long-term regulation of the beta 2AR/Gs alpha fusion protein activity and its pharmacological effect after expression in two cancer cell lines. In contrast with native beta 2ARs and Gs, the receptor and the alpha s subunit moieties of the beta 2AR/Gs alpha fusion protein did not undergo functional uncoupling. After a sustained incubation with isoproterenol or forskolin, the accumulation of cAMP could still be observed in S49 beta Gs cells, expressing the fusion gene, which showed, in addition, an up-regulation of their beta 2AR binding sites, while in S49 wt cells, the same treatments completely abolished the rise of cAMP and markedly reduced the number of receptors. cAMP-activation of protein kinase A (PKA) is known to modulate proliferation of most cells. We studied the effect of long term beta 2AR/Gs alpha activation on the growth rate of S49 lymphoma cells and carcinoma carB cells, a highly proliferative cancer cell line expressing oncogenic ras protein. The beta 2AR agonist salmeterol blocked the proliferation of both S49 and carB beta 2Gs cells, while this treatment did not change the growth of wild-type cells. In carB beta 2Gs cells, this effect may be reinforced by a significant basal activity of the fusion protein and by agonist-promoted MAP kinase inhibition. In conclusion, the stimulatory overload provided by the beta 2AR/Gs alpha fusion protein led to the inhibition of cAMP-sensitive cancer cell proliferation in vitro.

beta-Adrenergic and muscarinic receptor expression are regulated in opposite ways during senescence in rat left ventricle.

The well-known attenuated sensitivity of senescent heart to isoproterenol is accompanied by a decreased beta1-adrenergic receptors (beta1-AR) density, a down regulation process which may involve several molecular modifications and whose understanding is incomplete. Data concerning the M2-R muscarinic receptors (M2-R) are more contradictory. Both the absolute and relative concentrations of beta1-AR and M2-R as well as the coupling protein G alpha s and G alpha(i2) mRNAs were determined by slot-blot analysis in the left ventricles (LVs) of 6-7 week and 22-month-old male Wistar rats. In addition, the beta-AR and M2-R densities were quantitated by radioactive ligand binding. (1) The M2-R mRNA concentration increases by 92+/-32% in senescent as compared to adult animals; by contrast, the density in M2-R remains unchanged, suggesting that the M2-R expression was not exclusively regulated at a pre-translational level. (2) The beta1-AR mRNA concentration was nearly halved (reduced by 46+/-9.5%) and paralleled the 51+/-5.6% diminution of the beta-AR density which resulted exclusively from the decrease of beta1-AR density without change in the beta2-AR concentration, suggesting a pre-translational regulation of the beta1-AR expression. (3) G alpha(i2) mRNA concentration was unchanged, while G alpha s mRNA concentration was reduced by 26+/-4.6% in senescent compared with adult LVs. To conclude, the different components of the adrenergic and muscarinic systems are differentially regulated during aging.

Cellular signaling by an agonist-activated receptor/Gs alpha fusion protein.

The consequences of agonist-dependent activation of guanine nucleotide-binding protein (G protein)-coupled receptors vary from cell to cell, depending on a complex network of regulations between components of the signaling cascade. Specific interactions between receptors, G proteins, and effectors are difficult to analyze in intact cells. Engineering of receptor-transducer fusion proteins might be an effective strategy to target cellular effectors more efficiently and specifically. As a model, we evaluated the ability of a fusion protein of beta 2-adrenergic receptor bound to the alpha subunit of adenylyl cyclase-stimulatory G protein (Gs alpha) to restore the defective activation of adenylyl cyclase in S49 cyc- cells that lack endogenous Gs alpha. The coupling between the two partners of the fusion protein was functional, and the agonist-dependent activation of the effector was more potent and more productive in transfected than in wild-type S49 cells. The covalent link between receptor and Gs alpha could thus convey an advantage over freely interacting components. Such receptor-G alpha fusion proteins may help to elucidate the complex interactions between members of signaling pathways and may also constitute a useful tool for studying the effects of single effector activation.