Study of the possible link of 25-hydroxyvitamin D with Epstein-Barr virus and human herpesvirus 6 in patients with multiple sclerosis.

BACKGROUND AND PURPOSE: Although the causes of multiple sclerosis (MS) remain partially unknown, environmental and genetic factors are thought to play a role in its aetiopathogenesis. Hypovitaminosis D, Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6) infections have been described as possible MS triggers. Our aim was to analyse the possible link between 25-hydroxyvitamin D [25(OH)D] and viruses in patients with MS. METHODS: We included 482 patients with MS in a 2-year study. Serum samples were collected to analyse 25(OH)D levels and, according to sample availability, antibody titres against EBV and HHV-6 by enzyme-linked immunosorbent assay. DNA was extracted from blood in order to analyse EBV and HHV-6 viral load by quantitative real-time polymerase chain reaction and to genotype MS-related single nucleotide polymorphisms (rs3135388, rs2248359 and rs12368653) when possible. RESULTS: The 25(OH)D levels were significantly higher in the first semester of the year than in the second. Carriers of the risk allele rs2248359-C showed lower 25(OH)D levels than non-carriers. For EBV, viral load was significantly higher when 25(OH)D levels were low, demonstrating an inverse correlation between 25(OH)D levels and EBV load. CONCLUSIONS: The 25(OH)D levels could be involved in the regulation of EBV replication/reactivation in patients with MS.

Xenopus as a model organism to study heterotrimeric G-protein pathway during collective cell migration of neural crest.

Collective cell migration is essential in many fundamental aspects of normal development, like morphogenesis, organ formation, wound healing, and immune responses, as well as in the etiology of severe pathologies, like cancer metastasis. In spite of the huge amount of data accumulated on cell migration, such a complex process involves many molecular actors, some of which still remain to be functionally characterized. One of these signals is the heterotrimeric G-protein pathway that has been studied mainly in gastrulation movements. Recently we have reported that Ric-8A, a GEF for Gα proteins, plays an important role in neural crest migration in Xenopus development. Xenopus neural crest cells, a highly migratory embryonic cell population induced at the border of the neural plate that migrates extensively in order to differentiate in other tissues during development, have become a good model to understand the dynamics that regulate cell migration. In this review, we aim to provide sufficient evidence supporting how useful Xenopus model with its different tools, such as explants and transplants, paired with improved in vivo imaging techniques, will allow us to tackle the multiple signaling mechanisms involved in neural crest cell migration.

Fat-soluble vitamin deficiencies after bariatric surgery could be misleading if they are not appropriately adjusted.

OBJECTIVE: To evaluate the differences in frequency of fat-soluble vitamin deficiencies if we adjust their levels by its main carriers in plasma in patients undergoing Biliopancreatic diversion (BPD) and Roux-en-Y gastric bypass (RYGB). RESEARCH METHODS & PROCEDURES: We recruited 178 patients who underwent RYGB (n = 116 patients) and BPD (n = 62 patients) in a single centre. Basal data information and one-year after surgery included: anthropometric measurements, fat-soluble vitamins A, E and D, retinol binding protein (RBP) and total cholesterol as carriers of vitamin A and E respectively. Continuous data were compared using T-Student and proportions using chisquare test. RESULTS: There was a vitamin D deficiency of 96% of all patients, 10% vitamin A deficiency and 1.2% vitamin E deficiency prior to surgery. One year after surgery, 33% of patients were vitamin A deficient but the frequency reduced to 19% when we adjusted by RBP. We found a vitamin E deficiency frequency of 0% in RYGB and 4.8% in DBP one year after surgery. However, when we adjusted the serum levels to total cholesterol, we found an increased frequency of 8.7% in RYGB group for vitamin E deficiency and 21.4% in DBP (p = 0.04). CONCLUSION: We have found a different frequency of deficit for fat-soluble vitamin both in BPD and RYGB once we have adjusted for its main carriers. This is clinically relevant to prevent from overexposure and toxicity. We suggest that carrier molecules should be routinely requested when we assess fat-soluble vitamin status in patients who undergo malabsorptive procedures.

OBJETIVO: Evaluar las diferencias en la frecuencia de las deficiencias de vitaminas liposolubles si ajustamos sus concentraciones mediante sus principales transportadores plasmáticos en pacientes sometidos a derivación biliopancreática (DBP) y derivación gástrica en Y de Roux (DGYR). MÉTODOS DE INVESTIGACIÓN Y PROCEDIMIENTOS: Reclutamos a 178 pacientes sometidos a DGYR (n = 116 pacientes) y DBP (n = 62 pacientes) en un único centro. Los datos de información basal y al año de la cirugía incluyeron: mediciones antropométricas, vitaminas liposolubles A, E y D, proteína de unión al retinol (PUR) y el colesterol total como transportadores de las vitaminas A y E, respectivamente. Los datos continuos se compararon utilizando la t de Student y para las proporciones el test chi cuadrado. RESULTADOS: Hubo una deficiencia de vitamina D en el 96% de todos los pacientes, de vitamina A en el 10% y de vitamina E en el 1,2% antes de la cirugía. Un año después de la cirugía, el 33% de los pacientes tenía deficiencia de vitamina A pero la frecuencia se redujo al 19% cuando ajustamos para la PUR. Encontramos una frecuencia de deficiencia de vitamina E en el 0% de los pacientes con DGYR y en el 4,8% de aquellos con DBP un año después de la cirugía. Sin embargo, cuando ajustamos las concentraciones séricas de colesterol total, encontramos un aumento de la frecuencia de hasta el 8,7% de deficiencia de vitamina E en el grupo con DGYR y del 21,4% en el grupo con DBP (p = 0,04). CONCLUSIÓN: Encontramos una frecuencia diferente de déficit de vitaminas liposolubles tanto en DBP como en DGYR una vez que ajustamos para sus principales transportadores. Esto es clínicamente relevante para evitar la sobreexposición y la toxicidad. Sugerimos que se deberían solicitar de forma rutinaria las moléculas transportadoras a la hora de evaluar el estado de vitaminas liposolubles en pacientes sometidos a procedimientos que entrañan malabsorción.

SCO-spondin from embryonic cerebrospinal fluid is required for neurogenesis during early brain development.

The central nervous system (CNS) develops from the neural tube, a hollow structure filled with embryonic cerebrospinal fluid (eCSF) and surrounded by neuroepithelial cells. Several lines of evidence suggest that the eCSF contains diffusible factors regulating the survival, proliferation, and differentiation of the neuroepithelium, although these factors are only beginning to be uncovered. One possible candidate as eCSF morphogenetic molecule is SCO-spondin, a large glycoprotein whose secretion by the diencephalic roof plate starts at early developmental stages. In vitro, SCO-spondin promotes neuronal survival and differentiation, but its in vivo function still remains to be elucidated. Here we performed in vivo loss of function experiments for SCO-spondin during early brain development by injecting and electroporating a specific shRNA expression vector into the neural tube of chick embryos. We show that SCO-spondin knock down induces an increase in neuroepithelial cells proliferation concomitantly with a decrease in cellular differentiation toward neuronal lineages, leading to hyperplasia in both the diencephalon and the mesencephalon. In addition, SCO-spondin is required for the correct morphogenesis of the posterior commissure and pineal gland. Because SCO-spondin is secreted by the diencephalon, we sought to corroborate the long-range function of this protein in vitro by performing gain and loss of function experiments on mesencephalic explants. We find that culture medium enriched in SCO-spondin causes an increased neurodifferentiation of explanted mesencephalic region. Conversely, inhibitory antibodies against SCO-spondin cause a reduction in neurodifferentiation and an increase of mitosis when such explants are cultured in eCSF. Our results suggest that SCO-spondin is a crucial eCSF diffusible factor regulating the balance between proliferation and differentiation of the brain neuroepithelial cells.

Cyclin-dependent kinase inhibitor p21 controls adult neural stem cell expansion by regulating Sox2 gene expression.

In the adult brain, continual neurogenesis of olfactory neurons is sustained by the existence of neural stem cells (NSCs) in the subependymal niche. Elimination of the cyclin-dependent kinase inhibitor 1A (p21) leads to premature exhaustion of the subependymal NSC pool, suggesting a relationship between cell cycle control and long-term self-renewal, but the molecular mechanisms underlying NSC maintenance by p21 remain unexplored. Here we identify a function of p21 in the direct regulation of the expression of pluripotency factor Sox2, a key regulator of the specification and maintenance of neural progenitors. We observe that p21 directly binds a Sox2 enhancer and negatively regulates Sox2 expression in NSCs. Augmented levels of Sox2 in p21 null cells induce replicative stress and a DNA damage response that leads to cell growth arrest mediated by increased levels of p19(Arf) and p53. Our results show a regulation of NSC expansion driven by a p21/Sox2/p53 axis.

Risk factors for gestational diabetes mellitus in a large population of women living in Spain: implications for preventative strategies.

The aim of this study is to establish a risk appraisal model for GDM by identifying modifiable factors that can help predict the risk of GDM in a large population of 2194 women living in Spain. They were recruited between 2009-2010 when screening for GDM was performed. Participants completed a questionnaire on socio-demographic, anthropomorphic and behavioral characteristics, and reproductive and medical history. A total of 213 (9.7%) women were diagnosed as having GDM. Age, pregestational body weight (BW) and body mass index (BMI), and number of events of medical, obstetric and family history were significantly associated with GDM. After logistic regression model, biscuits and pastries intake <4 times/week, red and processed meats intake <6 servings/week, sugared drinks <4 servings/week, light walking >30 minutes/day, and 30 minutes/day of sports at least 2 days/week, compared with opposite consumption, was associated with less GDM risk. Our study identified several pregestational modifiable lifestyle risk factors associated with an increase in the risk of developing GDM. This may represent a promising approach for the prevention of GDM and subsequent complications. Further intervention studies are needed to evaluate if this appraisal model of risk calculation can be useful for prevention and treatment of GDM.

Ric-8: different cellular roles for a heterotrimeric G-protein GEF.

Signaling via heterotrimeric G-proteins is evoked by agonist-mediated stimulation of seven transmembrane spanning receptors (GPCRs). During the last decade it has become apparent that Gα subunits can be activated by receptor-independent mechanisms. Ric-8 belongs to a highly conserved protein family that regulates heterotrimeric G-protein function, acting as a non-canonical guanine nucleotide exchange factors (GEF) over a subset of Gα subunits. In this review we discuss the roles of Ric-8 in the regulation of diverse cell functions, emphasizing the contribution of its multiple domain protein structure in these diverse functions.

Cloning and spatiotemporal expression of RIC-8 in Xenopus embryogenesis.

RIC-8 is a highly conserved protein that promotes G protein signaling as it acts as a Guanine nucleotide Exchanging Factor (GEF) over a subset of Gα subunits. In invertebrates, RIC-8 plays crucial roles in synaptic transmission as well as in asymmetric cell division. As a first step to address further studies on RIC-8 function in vertebrates, here we have cloned a ric-8 gene from Xenopus tropicalis (xtric-8) and determined its spatiotemporal expression pattern throughout embryogenesis. The xtric-8 transcript is expressed maternally and zygotically and, as development proceeds, it shows a dynamic expression pattern. At early developmental stages, xtric-8 is expressed in the animal hemisphere, whereas its expression is later restricted to neural tissues, such as the neural tube and the brain, as well as in the eye and neural crest-derived structures, including those of the craniofacial region. Together, our findings suggest that RIC-8 functions are related to the development of the nervous system.

Telomere shortening in neural stem cells disrupts neuronal differentiation and neuritogenesis.

Proliferation in the subependymal zone (SEZ) and neurogenesis in the olfactory bulb decline in the forebrain of telomerase-deficient mice. The present work reveals additional effects of telomere shortening on neuronal differentiation, as adult multipotent progenitors with critically short telomeres yield reduced numbers of neurons that, furthermore, exhibit underdeveloped neuritic arbors. Genetic data indicate that the tumor suppressor protein p53 not only mediates the adverse effects of telomere attrition on proliferation and self-renewal but it is also involved in preventing normal neuronal differentiation of adult progenitors with dysfunctional telomeres. Interestingly, progenitor cells with short telomeres obtained from fetal brains do not exhibit any replicative defects but also fail to acquire a fully mature neuritic arbor, demonstrating cell cycle-independent effects of telomeres on neuronal differentiation. The negative effect of p53 on neuritogenesis is mechanistically linked to its cooperation with the Notch pathway in the upregulation of small GTPase RhoA kinases, Rock1 and Rock2, suggesting a potential link between DNA damage and the Notch signaling pathway in the control of neuritogenesis. We also show that telomerase expression is downregulated in the SEZ of aging mice leading to telomere length reductions in neurosphere-forming cells and deficient neurogenesis and neuritogenesis. Our results suggest that age-related deficits could be caused partly by dysfunctional telomeres and demonstrate that p53 is a central modulator of adult neurogenesis, regulating both the production and differentiation of postnatally generated olfactory neurons.

Airway inflammation in the elite athlete and type of sport.

BACKGROUND: The prevalence of asthma and bronchial hyper-responsiveness is greater in elite athletes than in the general population, and its association with mild airway inflammation has recently been reported. OBJECTIVE: To study the relationship between the type of sport practised at the highest levels of competition (on land or in water) and sputum induction cell counts in a group of healthy people and people with asthma. MATERIAL AND METHODS: In total, 50 athletes were enrolled. Medical history, results of methacholine challenge tests and sputum induced by hypertonic saline were analysed RESULTS: Full results were available for 43 athletes, who were classified by asthma diagnosis and type of sport (land or water sports). Nineteen were healthy (10 land and 9 water athletes) and 24 had asthma (13 land and 11 water athletes). Although the eosinophil counts of healthy people and people with asthma were significantly different (mean difference 3.1%, 95% CI 0.4 to 6.2, p = 0.008), analysis of variance showed no effect on eosinophil count for either diagnosis of asthma or type of sport. However, an effect was found for neutrophil counts (analysis of variance: F = 2.87, p = 0.04). There was also a significant correlation between neutrophil counts and both duration of training and bronchial hyper-responsiveness among athletes exposed to water (Spearman's rank correlations, 0.36 and 0.47, p = 0.04 and 0.04, respectively). CONCLUSIONS: Elite athletes who practice water sports have mild neutrophilic inflammation, whether or not asthma is present, related to the degree of bronchial hyper-reactivity and the duration of training in pool water.

Anti-inflammatory effects of high-dose inhaled fluticasone versus oral prednisone in asthma exacerbations.

The objective of the present study was to investigate the kinetics of high doses of inhaled steroid fluticasone in comparison with oral steroid prednisone on plasma protein leakage and bronchial eosinophilia in adults with moderate asthma exacerbations. The study design was a randomised, double-blind, placebo-controlled prospective trial. In total, 45 patients treated at the emergency department for moderate asthma exacerbations were recruited and 39 were assigned to receive fluticasone and placebo of prednisone (19 patients), or prednisone and placebo of fluticasone (20 patients). Medication was administered to all patients via a metered-dose inhaler and spacer (16 puffs; 4,000 microg.day(-1) or placebo) plus one pill (prednisone 30 mg.day(-1) or placebo). Spirometry and induced sputum for differential cell counts, albumin and alpha(2)-macroglobulin levels and blood eosinophils, interleukin-5 and granulocyte-macrophage colony-stimulating factor levels were obtained before treatment and at 2, 6 and 24 h after treatment. Symptoms clearly improved after 24 h in both groups. No differences were seen between groups in peak expiratory flow or forced expiratory flow in one second, which improved progressively but then decayed slightly after 24 h. Eosinophil counts in sputum also improved over time in both groups. The effect was faster with fluticasone than with prednisone, but was partially lost at 24 h. However, plasma proteins in sputum and eosinophil count in blood both decreased until 24 h, with no significant differences between groups. There was no correlation between eosinophil counts and plasmatic protein levels. In conclusion, both treatments improved symptoms, airway obstruction and inflammation, and plasma protein leakage at 24 h. Prednisone reduced blood eosinophil counts, while fluticasone reduced airway eosinophil counts, suggesting that the anti-inflammatory performance of fluticasone is exerted locally.

A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells.

We describe a protocol developed/modified by our group for the ex vivo and in vivo assessment of the response to a soluble factor of murine neural stem cells from the adult sub-ventricular zone (SVZ). The procedure includes several experimental options that can be used either independently or in combination. Potential factor effects on self-renewal, survival and proliferation are assayed by means of neurosphere cultures, with the factor administered directly in vitro to the culture plates (Step 1) or infused in vivo immediately before tissue dissociation (Step 3). We also use bromodeoxiuridine (BrdU) retention to label slowly dividing cells in vivo and subsequently perform two different types of experiments. In one set of experiments, the factor is added to primary cultures of stem cells obtained from the BrdU-pulsed animals and effects are tested on label-retaining cells after immunocytochemistry (Step 2). In another set, prolonged intraventricular infusion of the factor in BrdU-pulsed animals is followed by immunohistochemical analysis of BrdU labeling in the intact SVZ (Step 4). The minimum estimated time for the full combined procedure is 45 d.

[Bronchial exudate of serum proteins during asthma attack]. / Exudado bronquial de proteínas séricas en las crisis de asma.

OBJECTIVE: Although altered vascular permeability and edema of the bronchial mucosa are associated with asthma attack, their influence on its severity remains unknown. We address this issue by comparing relative indices for the concentration of albumin (RIAlb) and alpha2-macroglobulin (RIalpha2M) in induced sputum and peripheral blood from patients with exacerbated asthma, patients with stable asthma, and control subjects. PATIENTS AND METHODS: Forty-six volunteers participated in the study: 14 with exacerbated asthma (forced expiratory volume in the first second [FEV1] 74.3% [SD, 20.8%] of reference), 23 with stable asthma (FEV1 93.6% [7.5%]), and 9 controls (FEV1 101.1% [9.9%]). The concentrations of albumin and alpha2-macroglobulin were quantified by immunoturbidimetry and immunonephelometry, respectively. The relative index was then calculated by dividing the concentration in sputum supernatant by the concentration in peripheral blood. RESULTS: The mean RIAlb was 1.2 (1.1) in the control group, 2.9 (3.1) in the stable asthma group, and 6.0 (6.7) in the exacerbated asthma group. The RIalpha2M values were 11.7 (10.9), 11.9 (14.7), and 3.2 (3.8) for the control group and stable and exacerbated asthma groups, respectively. The increases in the RIAlb values between all groups, and the decrease in the RIalpha2M value between the exacerbated asthma and control groups were statistically significant (P<.05). The percentage of neutrophils, but not of eosinophils, in sputum was correlated with the RIAlb (r=0.39; P=.008) but not the RIalpha2M (r=-0.035; P=.82). FEV1 displayed an inverse relationship with the RIAlb (r=-0.43; P=.009) but not with the RIalpha2M (r=-0.206; P=.24). No correlation was found between oxyhemoglobin saturation and either the RIAlb (r=-0.33; P=.19) or the RIalpha2M (r=-0.12; P=.84). CONCLUSIONS: Vascular permeability is altered during asthma exacerbations and appears to be correlated with the presence of neutrophils and the degree of bronchial obstruction.

[Total IgE in the sputum and serum of patients with asthma]. / IgE total en el esputo y suero de pacientes con asma.

BACKGROUND: The correlation between total IgE in induced sputum (IS) and serum is not well defined. The aim of this study was to investigate the relationship between total IgE in IS and total IgE in serum and airway inflammation. METHODS: Twenty-one patients with stable asthma and thirteen healthy controls were studied. Clinical and spirometric data were collected and a skin prick test to the 13 most common aeroallergens in our area was performed in all subjects. Total IgE in IS and serum was determined by the UNICAP immunoanalysis system (Pharmacia Uppsala, Sweden) while albumin concentration in IS and serum was determined using the Cobas Integra turbidimetric method (Roche Diagnostics, Basel, Switzerland). RESULTS: The percentage of eosinophils in EI was 8.7 (11.8) in asthmatic subjects and was 0.5 (1) in healthy controls. Total IgE (KU/L) was 43.2 (23) in asthmatics vs 25.6 (3) in healthy controls in IS, and was 329 (413) in asthmatics vs 57 (78) in controls in serum. Total IgE in IS was significantly correlated with total IgE in serum; r = 0.71 (p = 0.048), but not with the albumin relative index. No correlation was found between IgE and the number of eosinophils in IS. CONCLUSIONS: Total IgE can be measured in IS. Total IgE in IS is mildly correlated with total IgE measured in serum. The lack of correlation between total IgE and albumin in IS suggests that IgE in IS could be locally produced, at least in part.

[Patient preference in the choice of dry powder inhalers]. / Preferencias de los pacientes en la elección de dispositivos de inhalación en polvo.

OBJECTIVE: To investigate a group of patients' preferences among 3 dry powder inhalers--Accuhaler, Easyhaler, and Turbuhaler--and to analyze the features that were most important for motivating choices. MATERIAL AND METHOD: The study enrolled 30 patients with stable asthma with a mean (SD) age of 40 (13) and who habitually used inhaled corticosteroids. The patients were shown in detail how to use each of the devices and were randomized to begin using them in different orders. After using each inhaler for a week, the patients assessed 9 different features on a scale of 0 to 10 with an independent observer. The patients were asked to put the inhalers in order of preference, and finally to demonstrate they could use them correctly. RESULTS: All patients correctly performed the inhalation maneuver at the beginning and the end of the study. The mean final scores out of 90 of the 9 features evaluated were 75 (13) for the Easyhaler, 67 (12) for the Accuhaler, and 65 (14) for the Turbuhaler. Differences were statistically significant between the first and the second device (P=0.02) and the first and the third (P=.001) but not between the Accuhaler and the Turbuhaler (P=.376). Mean rating scores were 8.6 (1.4) for the Easyhaler, 7.3 (1.9) for the Turbuhaler, and 7.1 (1.6) for the Accuhaler. The Easyhaler was the first choice for 53% of patients, the Turbuhaler for 27%, and the Accuhaler for 20%. CONCLUSIONS: The Easyhaler was rated the highest by the patients in the study. The scores were a long way from the maximum score, so research into developing an ideal inhaler must continue.

Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate.

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.

The C2 cytosolic loop of adenylyl cyclase interacts with the activated form of G alpha s.

Using the yeast two-hybrid system, we studied the physical interaction between the complete C1 and C2 cytosolic domains of Xenopus laevis type 9 (xl9C1, xl9C2) and the C2 domain of rat type 6 (r6C2) adenylyl cyclase (AC). Heterodimerization between xl9C1 and xl9C2 and homodimerization between C2 (but not C1) domains was observed. Interaction between C2 and human G alpha s (hG alpha s) was also detected and was dependent on G alpha s activation. In contrast X. laevis G alpha s (xlG alpha s), which is 92% identical to hG alpha s, was unable to interact with any of the three AC cytosolic domains tested, corroborating previous findings that showed no effector activation. Through the construction of chimeras, we demonstrated that the amino-terminal half of xlG alpha s was responsible for the lack of interaction with AC. Chimeras between mouse G alpha i2 and G alpha s (N-mG alpha i2/C-G alpha s), that have previously shown to activate AC to a higher extent than wild-type G alpha s, also interacted with the C2 cytosolic domain and with a higher affinity. Interestingly, N-mG alpha i2/C-xlG alpha s chimera was not only able to interact with C2 but also with the C1 cytosolic domain.

Molecular cloning and expression of an adenylyl cyclase from Xenopus laevis oocytes.

We have cloned a cDNA that encodes a novel Xenopus laevis oocyte adenylyl cyclase (xlAC) using oligonucleotides against conserved mammalian adenylyl cyclase regions. The isolated cDNA is 4372 bp long with an open reading frame of 4065 nucleotides which encodes a protein of 1355 amino acids. Comparison of the deduced amino acid sequence with previously cloned mammalian adenylyl cyclases shows a low identity, 19.7% with type 2 rat adenylyl cyclase and 24.2% with type 4 rat adenylyl cyclase, indicating that this Xenopus isoform represents a new member of this protein family. Gene expression studies of the xlAC by reverse PCR showed that this gene is expressed in all oogenesis stages but not during early embryogenesis. Expression of the xlAC in COS-7 cells resulted in increased basal AC activity, that was stimulated by forskolin, Gpp(NH)p and aluminium fluoride, and was insensitive to calcium and calcium-calmodulin (Ca2(+)-CaM).