Sleep pattern and learning in knockdown mice with reduced cholinergic neurotransmission.

Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.

Sleep pattern and learning in knockdown mice with reduced cholinergic neurotransmission

Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.

Identification of Aspergillus flavus isolates as potential biocontrol agents of aflatoxin contamination in crops.

Aspergillus flavus, a haploid organism found worldwide in a variety of crops, including maize, cottonseed, almond, pistachio, and peanut, causes substantial and recurrent worldwide economic liabilities. This filamentous fungus produces aflatoxins (AFLs) B1 and B2, which are among the most carcinogenic compounds from nature, acutely hepatotoxic and immunosuppressive. Recent efforts to reduce AFL contamination in crops have focused on the use of nonaflatoxigenic A. flavus strains as biological control agents. Such agents are applied to soil to competitively exclude native AFL strains from crops and thereby reduce AFL contamination. Because the possibility of genetic recombination in A. flavus could influence the stability of biocontrol strains with the production of novel AFL phenotypes, this article assesses the diversity of vegetative compatibility reactions in isolates of A. flavus to identify heterokaryon self-incompatible (HSI) strains among nonaflatoxigenic isolates, which would be used as biological controls of AFL contamination in crops. Nitrate nonutilizing (nit) mutants were recovered from 25 A. flavus isolates, and based on vegetative complementation between nit mutants and on the microscopic examination of the number of hyphal fusions, five nonaflatoxigenic (6, 7, 9 to 11) and two nontoxigenic (8 and 12) isolates of A. flavus were phenotypically characterized as HSI. Because the number of hyphal fusions is reduced in HSI strains, impairing both heterokaryon formation and the genetic exchanges with aflatoxigenic strains, the HSI isolates characterized here, especially isolates 8 and 12, are potential agents for reducing AFL contamination in crops.

Induced mitotic homologous recombination by the babesicide imidocarb dipropionate in Aspergillus nidulans diploid cells.

Imidocarb dipropionate (IMD) is a chemotherapeutic agent prescribed for the treatment and control of babesiosis; it is known to be a nucleic acid synthesis inhibitor. Although it is an effective babesicide, there are reports of persistent IMD residues retained at high levels in edible tissues of cattle, swine and sheep, raising concerns about potential effects on humans. Since the carcinogenic potential of a chemical compound can be assessed through its effect on the homologous recombination, we investigated whether IMD is recombinogenic in Aspergillus nidulans diploid cells and whether it is capable of inducing homozygosis in genes that were previously heterozygous. This analysis was done with a homozygotization assay applied to a heterozygous diploid strain of A. nidulans. IMD used at non-toxic concentrations (2.5 to 10.0 µM) was recombinogenic, demonstrated by homozygotization indices higher than 2.0 for diploid markers. A diploid homozygous for genetic markers from chromosomes I and II was also produced. Since DNA replication blockers that induce DNA strand breaks have been classified as potent inducers of homologous recombination, the recombinogenic potential of IMD may be due to induction of recombinational repair.

Reduced expression of the vesicular acetylcholine transporter causes learning deficits in mice.

Storage of acetylcholine in synaptic vesicles plays a key role in maintaining cholinergic function. Here we used mice with a targeted mutation in the vesicular acetylcholine transporter (VAChT) gene that reduces transporter expression by 40% to investigate cognitive processing under conditions of VAChT deficiency. Motor skill learning in the rotarod revealed that VAChT mutant mice were slower to learn this task, but once they reached maximum performance they were indistinguishable from wild-type mice. Interestingly, motor skill performance maintenance after 10 days was unaffected in these mutant mice. We also tested whether reduced VAChT levels affected learning in an object recognition memory task. We found that VAChT mutant mice presented a deficit in memory encoding necessary for the temporal order version of the object recognition memory, but showed no alteration in spatial working memory, or spatial memory in general when tested in the Morris water maze test. The memory deficit in object recognition memory observed in VAChT mutant mice could be reversed by cholinesterase inhibitors, suggesting that learning deficits caused by reduced VAChT expression can be ameliorated by restoring ACh levels in the synapse. These data indicate an important role for cholinergic tone in motor learning and object recognition memory.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean.

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Asexual recombination in a uvsH mutant of Aspergillus nidulans.

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

Genotoxicity (mitotic recombination) of the cancer chemotherapeutic agents cisplatin and cytosine arabinoside in Aspergillus nidulans.

Cisplatin (cis-diamminedichloroplatinum, cis-DDP) and cytosine arabinoside (ara-C) are anticancer drugs used in the treatment of human cancer. The two chemotherapeutic drugs were tested in current research for their recombinogenic potential in diploid cells of Aspergillus nidulans. Non-cytotoxic concentrations of ara-C (0.4 and 0.8 microM) and cis-DDP (1.5, 3.0 and 6.0 microM) were strong recombinagens in A. nidulans UT448//A757 diploid strain, which induced homozygosis of recessive genetic markers, previously present in heterozygous condition. Drugs significantly increased homozygosity index (HI) values for five nutritional genetic markers when compared with those determined in the absence of anticancer drugs. Since mitotic recombination is a mechanism leading to malignant growth through loss of heterozygosity at tumor-suppressor loci, ara-C and cis-DDP may be characterized as secondary promoters of malignant neoplasia in diagnosed cancer patients, after chemotherapy treatment.

Asexual Recombination in a uvsH Mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Characterization of a Trypanosoma cruzi antigen with homology to intracellular mammalian lectins.

Two cDNAs, isolated from a Trypanosoma cruzi amastigote library immunoscreened with sera from patients with Chagas disease, encode proteins with sequence homology to eukaryotic components of the cellular sorting and recycling machinery. These proteins, denominated TcAGL, present an N-terminal lectin domain and a C-terminal region containing repetitive amino acids and a poly-glutamine tract. They are products of polymorphic alleles of a single copy gene constitutively expressed during the parasite life cycle. Polyclonal antibodies obtained from mice immunized with the recombinant antigen recognize proteins with apparent molecular weight ranging from 95 to 120 kDa in cell lysates from all three life stages and in various strains of the parasite. Sera from Chagas disease patients recognize the recombinant antigen in ELISA and immunoprecipitation assays but not in Western blot assays under denaturing conditions. Consistent with its proposed role in the glycoprotein secreting pathway, immunofluorescence analyses and expression of a green fluorescent protein-tagged TcAGL protein indicate a sub-cellular localization in the vicinity of the flagellar pocket membrane and the Golgi complex of the parasite.

Cockroaches as carriers of fungi of medical importance.

The fungal pathogenic flora of the external surface of 103 cockroaches (Periplaneta americana) collected from the intensive care unit of a hospital were investigated. In this study, a high percentage of test cockroaches (93.2%) were found to carry fungi of medical importance. The main fungi isolated were species of Candida, Aspergillus and Penicillium. Information about the carriage of pathogenic fungi by cockroaches in hospital environment is scanty. The results suggest that cockroaches can play a role in dissemination of fungi, which they can carry on their external surface.

Chlorhexidine digluconate induces mitotic recombination in diploid cells of Aspergillus nidulans.

UNLABELLED: Chlorhexidine digluconate (1,1'-hexamethylene-bis[(5-p-clorophenyl)-biguanide]) is a bisbiguanidine antiseptic, used to decrease plaque formation and to control periodontal diseases. The determination of the frequency of mitotic crossing-over constitutes a very important method for detecting carcinogenic agents. OBJECTIVE: The recombinogenic potential of chlorhexidine digluconate was evaluated on Aspergillus nidulans by the production of cells homozygous for the following nutritional markers: riboA1, pabaA124, biA1, methA17 and pyroA4. METHOD: A. nidulans was exposed to three concentrations of chlorhexidine digluconate (1, 5, and 10 microM). RESULTS: Inhibition of colony development, conidiophore morphological alteration (cytotoxic effect), and the recombinogenic effect, indicated by homozygotization index (HI) values higher than 2.0, were observed for all concentrations of chlorhexidine digluconate. A homozygous pyro+//pyro+ diploid strain and a diploid homozygous for the recessive w gene were isolated from UT448//A757 diploid treated with chlorhexidine digluconate, emphasazing its recombinogenic potential. CONCLUSION: Although, beneficial effects of chlorhexidine, as an antiseptic agent, are reported in the literature, our results revealed that chlorhexidine digluconate, at less levels lowered those used clinically, caused toxic and recombinogenic effects on diploid A. nidulans strain.

Cytotoxicity, antitumoral and antimycobacterial activity of tetrazole and oxadiazole derivatives.

In this study, the antimycobacterial activity of mono and di-substituted tetrazole and oxadiazole derivatives and their precursors was assayed on Mycobacterium tuberculosis H37Rv, and cytotoxicity was evaluated on J774 macrophages and on tumoral cell lines. Structure Activity Relationship (SAR) analysis was performed using Principal Component Analysis (PCA) to determine the relationship between these compounds and their biological activities.

Loss of heterozygosity by mitotic recombination in diploid strain of Aspergillus nidulans in response to castor oil plant detergent

A recombinação somática em células diplóides heterozigotas pode atuar como agente promotor de neoplasias por induzir homozigose de genes deletéreos. Por meio desse processo, genes supressores de tumores podem ser completamente suprimidos em células recombinantes. O presente trabalho avaliou a genotoxicidade do detergente derivado do óleo da semente da mamona (Ricinus communis) em células diplóides heterozigotas do fungo filamentoso Aspergillus nidulans. Trabalhos anteriores avaliaram a aplicação dessa solução no tratamento de canais radiculares como líquido irrigador. O potencial recombinagênico desse composto foi estudado pela origem de células homozigotas para os marcadores nutricionais: riboA1, pabaA124, biA1, metA17 e piroA4. A solução, diluída em 1:40, 1:20 e 1:10, induziu alterações morfológicas e atraso no desenvolvimento dos conidióforos da linhagem UT448//UT196 e aumento nas freqüências de recombinação mitótica. Embora trabalhos anteriores relatem a atividade antimicrobiana da solução em estudo, nossos resultados evidenciam a citotoxicidade e o potencial recombinagênico dessa substância.

Loss of heterozygosity by mitotic recombination in diploid strain of Aspergillus nidulans in response to castor oil plant detergent.

Somatic recombination in heterozygous diploid cells may be a promotional agent of neoplasms by inducing homozygosity of defective genes. Tumor suppressor genes may in this way be completely suppressed in recombinant cells. In this work, the genotoxic effects of detergent derived from the castor oil plant (Ricinus communis) in heterozygous diploid cells of Aspergillus nidulans are evaluated. Previous studies have evaluated the application of this substance in endodontic treatments as an irrigating solution. The recombinogenic potential of the compound has been studied through the production of homozygous cells for nutritional markers riboA1, pabaA124, biA1, methA17, and pyroA4. Detergent was diluted to 1:10, 1:20, and 1:40, and morphologic alterations, delay in conidiophore development, and mitotic recombination occurrence were reported for the three dilutions. Although past studies have demonstrated the antimicrobial action of the detergent under analysis, our results revealed its cytotoxic effects and recombinogenic potential.

The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles.

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.

Expression of a functional recombinant Phoneutria nigriventer toxin active on K+ channels.

PnTx3-1 is a peptide isolated from the venom of the spider Phoneutria nigriventer that specifically inhibits A-type K(+) currents (I(A)) in GH(3) cells. Here we used a bacterial expression system to produce an NH(2)-extended mutant of PnTx3-1 (ISEF-PnTx3-1) and tested whether the toxin is functional. The recombinant toxin was purified from bacterial extracts by a combination of affinity and ion-exchange chromatography. The recombinant toxin blocked A-type K(+) currents in GH(3) cells in a fashion similar to that observed with the wild-type toxin purified from the spider venom. These results suggest that recombinant cDNA methods provide a novel source for the production of functional Phoneutria toxins. The recombinant ISEF-PnTx3-1 should be useful for further understanding of the role of A-type K(+) currents in biological processes.

Alternative reproduction pathway in Aspergillus nidulans.

Recombinant haploid segregants were recovered in filamentous fungus Aspergillus nidulans (Eidam) G. Winter directly from the heterokaryons instead of diploid segregants (process described earlier as parameiosis). In spite of the reproductive complexity of A. nidulans, parameiosis has only now been observed in this fungus. Since parameiosis was characterized by the occurrence of genetic recombination inside heterokaryotic hyphae, master strains (uvs+) and uvs mutants with high rate of both mitotic exchanges or chromosome nondisjunction were used to form heterokaryons. Two groups of mitotic segregants were recovered directly from heterokaryons--aneuploids and stable haploids. Heterokaryons formed with uvs mutants produced a higher number of parameiotic segregants compared to the heterokaryons formed with uvs+ strains. Segregants were analyzed by nutritional markers, acriflavine resistance and conidial color. Normal meiotic behavior of haploid recombinants was observed.

Recombinagenic effect of cryptolepine in uvsH+//uvsH+ and uvsH//uvsH diploid strains of Aspergillus nidulans.

Recombinagenic potential of the alkaloid cryptolepine was evaluated in two diploid strains of Aspergillus nidulans--a wild type strain (uvsH+//uvsH+) and a DNA-repair-deficient one (uvsH//uvsH). Treatment of both strains with cryptolepine failed to alter colony growth in culture; its recombinagenic potential was determined by the homozygotization index (in which events of mitotic exchange may cause expression of genes previously masked by the dominant allele). Mitotic crossing-overs were induced by 7 and 14 mg/L doses of cryptolepine in both diploid A. nidulans strains.