MULTIPLE ROUTES OF SEXUALITY IN ALEXANDRIUM TAYLORI (DINOPHYCEAE) IN CULTURE1

Alexandrium taylori Balech is a cyst‐forming dinoflagellate species responsible for recurrent blooms in Mediterranean coastal waters. The nuclear development of the cells during the sexual cycle and the effect of different external nitrate and phosphate levels were studied. Nuclear fusion of gametes occurred 6–12 h after the complete cytoplasmic fusion. The U‐shaped nuclei fused through the end of one nucleus and the mid‐area of the other. The mobile and biflagellated zygote had a large, U‐shaped nucleus and may follow three different fates: direct division, short‐term encystment (ecdysal), and long‐term encystment (resting). Ecdysal cysts may divide in >24–96 h into two, four, six, or eight cells before germinating. Meiosis presumably occurred in three locations: in the planozygote, within the ecdysal cyst, and in the planomeiocyte (germling) liberated either from ecdysal or resting cysts. The effects of nutrients on these routes were studied in individually isolated sexual stages. (1) Direct divisions occurred mainly under replete conditions (L1), whereas no direct planozygote divisions were recorded in media with no phosphate added (L‐P). (2) Short‐term encystment was larger in media lacking phosphate (L‐P and L/30) than in medium with no nitrate added (L‐N) or under replete conditions (L1). (3) Long‐term encystment was only observed in medium with no nitrate added (L‐N). The long‐lived resting cyst, not previously described for this species, had a clear double wall, an irregular shape, a flat morphology, and a middle orange spot. No cysts germinated in 1–2 months, whereas 86% of the cysts germinated 2–3 months after being formed. A flow cytometry analysis showed that sexual induction and zygote formation were very fast and highly common processes, zygotes being nearly half of the population at days 3 and 5 after the induction of sexuality in the cultures.     

Fruit infesting tephritids [Dipt.: Tephritidae] and associated parasitoids in Chiapas,Mexico

A total of 1,302 parasitoids representing 8 species and 4 families were recovered from 9,818 fruit fly host fruits sampled. The most common parasitoid species wasDiachasmimorpha longicaudata (Ashmead). Average percent parasitism ranged between 0.44 and 29.23%. Parasitoid emergence data indicate thatAnastrepha ludens (Loew),A. obliqua (Sein),A. serpentina (Wiedeman),A. striata (Schiner) andToxotrypana curvicauda (Gerstaecker) were subject to parasitism. We provide information on the population fluctuation ofAnastrepha ludens, A. obliqua, A. serpentina, A. distincta (Greene),A. striata, A. fraterculus (Wiedeman),A. chiclayae (Greene),A. montei (Costa Lima),A. leptozona (Hendel) andA. tripunctata (Wulp).Anastrepha ludens andA. obliqua were the most common species, representing 95.3% of all fruit fly species caught in McPhail traps.      

Rapid discrimination of sequences flanking and within T‐DNA insertions in the Arabidopsis genome

An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T-DNA insertions is presented that allows for the avoidance of some of the cloning difficulties caused by the concatameric nature of T-DNA inserts. The principle of the procedure is to categorize by size restriction fragments of mutant DNA, produced in separate digestions with NdeI and Bst1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T-DNA construct, the restriction fragments obtained fall into two categories: those showing identical size in both digestions, which correspond to sequences internal to T-DNA concatamers; and those of different sizes, that contain the junctions between plant DNA and the T-DNA insert. Such a criterion makes it possible to easily distinguish the digestion products corresponding to internal T-DNA parts, which do not deserve further attention, and those which presumably include a segment of the locus of interest. Discrimination between restriction fragments of genomic mutant DNA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.     

Long Withdrawal of Methylphenidate Induces a Differential Response of the Dopaminergic System and Increases Sensitivity to Cocaine in the Prefrontal Cortex of Spontaneously Hypertensive Rats

Methylphenidate (MPD) is one of the most prescribed drugs for alleviating the symptoms of Attention Deficit/Hyperactivity Disorder (ADHD). However, changes in the molecular mechanisms related to MPD withdrawal and susceptibility to consumption of other psychostimulants in normal individuals or individuals with ADHD phenotype are not completely understood. The aims of the present study were: (i) to characterize the molecular differences in the prefrontal dopaminergic system of SHR and Wistar strains, (ii) to establish the neurochemical consequences of short- (24 hours) and long-term (10 days) MPD withdrawal after a subchronic treatment (30 days) with Ritalin® (Methylphenidate Hydrochloride; 2.5 mg/kg orally), (iii) to investigate the dopaminergic synaptic functionality after a cocaine challenge in adult MPD-withdrawn SHR and Wistar rats. Our results indicate that SHR rats present reduced [³H]-Dopamine uptake and cAMP accumulation in the prefrontal cortex (PFC) and are not responsive to dopaminergic stimuli in when compared to Wistar rats. After a 24-hour withdrawal of MPD, SHR did not present any alterations in [³H]-Dopamine Uptake, [³H]-SCH 23390 binding and cAMP production; nonetheless, after a 10-day MPD withdrawal, the results showed a significant increase of [³H]-Dopamine uptake, of the quantity of [³H]-SCH 23390 binding sites and of cAMP levels in these animals. Finally, SHR that underwent a 10-day MPD withdrawal and were challenged with cocaine (10 mg/kg i.p.) presented reduced [³H]-Dopamine uptake and increased cAMP production. Wistar rats were affected by the 10-day withdrawal of MPD in [³H]-dopamine uptake but not in cAMP accumulation; in addition, cocaine was unable to induce significant modifications in [³H]-dopamine uptake and in cAMP levels after the 10-day withdrawal of MPD. These results indicate a mechanism that could explain the high comorbidity between ADHD adolescent patients under methylphenidate treatment and substance abuse in adult life.     

EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP⁺). We investigated the effects of EGb761 pretreatment on MPP⁺ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP⁺, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP⁺ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP⁺ administration. MPP⁺-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP⁺ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP⁺-induced oxidative stress.     

Sympathetic glial cells and macrophages develop different responses to Trypanosoma cruzi infection or lipopolysaccharide stimulation

Nitric oxide (NO) participates in neuronal lesions in the digestive form of Chagasdisease and the proximity of parasitised glial cells and neurons in damaged myentericganglia is a frequent finding. Glial cells have crucial roles in manyneuropathological situations and are potential sources of NO. Here, we investigateperipheral glial cell response to Trypanosoma cruzi infection toclarify the role of these cells in the neuronal lesion pathogenesis of Chagasdisease. We used primary glial cell cultures from superior cervical ganglion toinvestigate cell activation and NO production after T. cruziinfection or lipopolysaccharide (LPS) exposure in comparison to peritonealmacrophages. T. cruzi infection was greater in glial cells, despitesimilar levels of NO production in both cell types. Glial cells responded similarlyto T. cruzi and LPS, but were less responsive to LPS thanmacrophages were. Our observations contribute to the understanding of Chagas diseasepathogenesis, as based on the high susceptibility of autonomic glial cells toT. cruzi infection with subsequent NO production. Moreover, our findingswill facilitate future research into the immune responses and activation mechanismsof peripheral glial cells, which are important for understanding the paradoxicalresponses of this cell type in neuronal lesions and neuroprotection.     

Cultivable fungi present in deep-sea sediments of Antarctica: taxonomy,diversity, and bioprospecting of bioactive compounds

Extremophiles - We accessed the culturable mycobiota present in marine sediments at different depths in Antarctica Ocean. Acremonium fusidioides, Penicillium allii-sativi, Penicillium chrysogenum,...     

Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells

Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O(2)(-)). The superoxide dismutase manganese dependent (SOD2) catalyzes O(2)(-) in H(2)O(2) into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier's different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-α, IFN-γ). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question.