Lysophosphatidylcholine triggers cell differentiation in the protozoan parasite Herpetomonas samuelpessoai through the CK2 pathway.

BACKGROUND: Protozoa are distantly related to vertebrates but present some features of higher eukaryotes, making them good model systems for studying the evolution of basic processes such as the cell cycle. Herpetomonas samuelpessoai is a trypanosomatid parasite isolated from the hemipteran insect Zelus leucogrammus. Lysophosphatidylcholine (LPC) is implicated in the transmission and establishment of Chagas disease, whose etiological agent is Trypanosoma cruzi. LPC is synthesized by T. cruzi and its vectors, the hemipteran Rhodnius prolixus and Triatoma infestans. Platelet-activating factor (PAF), a phospholipid with potent and diverse physiological and pathophysiological actions, is a powerful inducer of cell differentiation in Herpetomonas muscarum muscarum and T. cruzi. The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the 2-ester bond of 3-sn-phosphoglyceride, transforming phosphatidylcholine (PC) into LPC. METHODS: In this study, we evaluated cellular differentiation, PLA2 activity and protein kinase CK2 activity of H. samuelpessoai in the absence and in the presence of LPC and PAF. RESULTS: We demonstrate that both PC and LPC promoted a twofold increase in the cellular differentiation of H. samuelpessoai, through CK2, with a concomitant inhibition of its cell growth. Intrinsic PLA2 most likely directs this process by converting PC into LPC. CONCLUSIONS: Our results suggest that the actions of LPC on H. samuelpessoai occur upon binding to a putative PAF receptor and that the protein kinase CK2 plays a major role in this process. Cartoon depicting a model for the synthesis and functions of LPC in Herpetomonas samuelpessoai, based upon our results regarding the role of LPC on the cell biology of Trypanosoma cruzi [28-32]. N nucleus, k kinetoplast, PC phosphatidylcholine, LPC lysophosphatidylcholine, PLA2 phospholipase A2, PAFR putative PAF receptor in trypanosomatids [65], CK2 protein kinase CK2 [16].

Inhibition of transcription and translation in dorsal hippocampus does not interfere with consolidation of memory of intense training.

Findings of several experiments indicate that many treatments that typically interfere with memory consolidation are ineffective in preventing or attenuating memory induced by intense training. As extensive evidence suggests that the consolidation of newly acquired memories requires gene expression and de novo protein synthesis the present study investigated whether intense training prevents consolidation impairment induced by blockers of mRNA and protein synthesis. Rats were given a single inhibitory training trial using a moderate (1.0 mA) or a relatively intense (2.0 mA) foot-shock. Bilateral hippocampal infusions of the mRNA synthesis blocker DRB (10, 40 or 80 ng/0.5 µL/hemisphere) or the protein synthesis inhibitor anisomycin (ANI), an inhibitor de novo protein synthesis (15.62, 31.25, or 62.50 µg/0.5 µL/hemisphere) were administered 15 min prior to training. Retention was measured at 30 min or 48 h following training. DRB and ANI impaired memory of moderate training in a dose-dependent manner without affecting short-term memory. In contrast, memory consolidation was not impaired in the groups trained with 2.0 mA. The findings showed that: (1) inhibitors of transcription and translation in the hippocampus impair the consolidation of memory of inhibitory avoidance learning induced by moderate levels of aversive stimulation and (2) blocking of mRNA and protein synthesis does not prevent the consolidation of memory induced by relatively high levels of aversive stimulation. These findings do not support the hypothesis that gene expression and de novo protein synthesis are necessary steps for long-term memory formation as memory was not impaired if intense foot-shock was used in training.

Behavioral tagging of extinction learning.

Extinction of contextual fear in rats is enhanced by exposure to a novel environment at 1-2 h before or 1 h after extinction training. This effect is antagonized by administration of protein synthesis inhibitors anisomycin and rapamycin into the hippocampus, but not into the amygdala, immediately after either novelty or extinction training, as well as by the gene expression blocker 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole administered after novelty training, but not after extinction training. Thus, this effect can be attributed to a mechanism similar to synaptic tagging, through which long-term potentiation can be enhanced by other long-term potentiations or by exposure to a novel environment in a protein synthesis-dependent fashion. Extinction learning produces a tag at the appropriate synapses, whereas novelty learning causes the synthesis of plasticity-related proteins that are captured by the tag, strengthening the synapses that generated this tag.

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation.

DNA damage induces apoptosis and many apoptotic genes are regulated via alternative splicing (AS), but little is known about the control mechanisms. Here we show that ultraviolet irradiation (UV) affects cotranscriptional AS in a p53-independent way, through the hyperphosphorylation of RNA polymerase II carboxy-terminal domain (CTD) and a subsequent inhibition of transcriptional elongation, estimated in vivo and in real time. Phosphomimetic CTD mutants not only display lower elongation but also duplicate the UV effect on AS. Consistently, nonphosphorylatable mutants prevent the UV effect. Apoptosis promoted by UV in cells lacking p53 is prevented when the change in AS of the apoptotic gene bcl-x is reverted, confirming the relevance of this mechanism. Splicing-sensitive microarrays revealed a significant overlap of the subsets of genes that have changed AS with UV and those that have reduced expression, suggesting that transcriptional coupling to AS is a key feature of the DNA-damage response.

Inhibition of mRNA synthesis in the hippocampus impairs consolidation and reconsolidation of spatial memory.

Using two different mRNA synthesis inhibitors, we show that blockade of hippocampal gene expression during restricted posttraining or postretrieval time windows hinders retention of long-term spatial memory for the Morris water maze task, without affecting short-term memory, nonspatial learning, or the functionality of the hippocampus. Our results indicate that spatial memory consolidation induces the activation of the hippocampal transcriptional machinery and suggest the existence of a gene expression-dependent reconsolidation process that operates in the dorsal hippocampus at the moment of retrieval to stabilize the reactivated mnemonic trace.

Activation of cyclic AMP pathway prevents CD34(+) cell apoptosis.

OBJECTIVE: Although cAMP is involved in a number of physiologic functions, its role in hematopoietic cell fate decision remains poorly understood. We have recently demonstrated that in CD34(+)-derived megakaryocytes, cAMP-related agents prevent apoptosis. In this study we addressed the question of whether cAMP also regulates survival of their precursors, CD34(+) cells. METHODS: Apoptosis was evaluated by fluorescence microscopy, and detection of hypodiploid or annexin V(+) cells by flow cytometry. Mitochondrial membrane potential and bcl-xL or caspase-3 expression were assessed by flow cytometry. Colony-forming units were studied by clonogenic assays in methylcellulose. RESULTS: We found that two different cAMP analogs such as Dibutiril-cAMP and sp-5,6-DCl-BIMPS (BIMPS) promoted survival of human umbilical cord-derived CD34(+) cells by suppressing apoptosis induced by either nitric oxide (NO) or serum deprivation. Involvement of PKA and PI3K pathway was demonstrated by the ability of their specific inhibitors Rp-cAMP and Wortmannin or LY294002 respectively to reverse the antiapoptotic effect of BIMPS. Treatment of CD34(+) cell with BIMPS not only restrained the bcl-xL downregulation but also suppressed the loss of mitochondrial membrane potential and caspase-3 activation induced by serum starvation. While thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF) were not able to increase cAMP levels, the antiapoptotic activity exerted by these growth factors was blocked by inhibition of the adenylate cyclase and synergized by BIMPS. Cyclic AMP analogs suppressed the decreased colony formation in cells exposed to NO or serum deprivation. CONCLUSION: Altogether, our results strongly suggest that cAMP appears to be not only a key pathway controlling CD34(+) survival, but also a mediator of the TPO-, G-CSF- and SCF-mediated cytoprotection.

A polar mechanism coordinates different regions of alternative splicing within a single gene.

Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.

Learning twice is different from learning once and from learning more.

The rat hippocampus plays a crucial role in the consolidation of a variety of memories, including that for a one trial inhibitory avoidance learning task in which stepping down from a platform is associated with a footshock. Here we show that this is the case regardless of the intensity of the footshock used and hence, of the strength of the learned response. However, additional learning produced by a second training session in this task does not involve the hippocampus but, instead, the striatum. Memory consolidation of the second trial requires glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, N-methyl-D-aspartate and metabotropic receptors, activation of signaling pathways, gene expression and protein synthesis in the striatum, as are required in the hippocampus during memory consolidation of the first trial.

Inhibition of mRNA and protein synthesis in the CA1 region of the dorsal hippocampus blocks reinstallment of an extinguished conditioned fear response.

Memories are extinguished by the repeated presentation of a conditioned stimulus in the absence of an unconditioned stimulus to which it has been associated. It is believed that extinction establishes a new hierarchy of responses rather than an actual forgetting of the original response, which can usually reappear spontaneously after interruption of the extinction process. In this study, our aim was to analyze how profound extinction can be. Rats were trained in a one-trial, step-down inhibitory avoidance paradigm and then were exposed to several extinction sessions in which they were allowed to freely explore the apparatus for 30 sec after having stepped down. Extinction was complete enough so that there was no spontaneous recovery, and test session performance could not be enhanced by pharmacological agents with well known facilitative actions on retrieval. After being submitted to a new training session, control animals reacquired the avoidance response; however, animals failed to do so after receiving bilateral intra-CA1 infusions of either the protein synthesis inhibitor anisomycin or the mRNA synthesis blocker 5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole 15 min before the retraining session. Our results indicate that extinction can be carried to a point at which reinstallment of the conditioned response requires, like the original learning, de novo gene expression and protein synthesis in the CA1 region of the dorsal hippocampus.

Mayaro virus infection cycle relies on casein kinase 2 activity.

Replication of Mayaro virus in Vero cells induces dramatic cytopathic effects and cell death. In this study, we have evaluated the role of casein kinase 2 (CK2) during Mayaro virus infection cycle. We found that CK2 was activated during the initial stages of infection ( approximately 36% after 4h). This activation was further confirmed when the enzyme was partially purified from the cellular lysate either by Mono Q 5/5Hr column or heparin-agarose column. Using this later column, we found that the elution profile of CK2 activity from infected cells was different from that obtained for control cell enzyme, suggesting a structural modification of CK2 after infection. Treatment of infected cells with a cell-permeable inhibitor of CK2, dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), abolished the cytopathic effect in a dose-dependent manner. Together this set of data demonstrates for the first time that CK2 activity in host cells is required in Mayaro virus infection cycle.

Two time periods of hippocampal mRNA synthesis are required for memory consolidation of fear-motivated learning.

Information storage in the brain is a temporally graded process involving different memory types or phases. It has been assumed for over a century that one or more short-term memory (STM) processes are involved in processing new information while long-term memory (LTM) is being formed. It has been repeatedly reported that LTM requires de novo RNA synthesis around the time of training. Here we show that LTM formation of a one-trial inhibitory avoidance training in rats, a hippocampal-dependent form of contextual fear conditioning, depends on two consolidation periods requiring synthesis of new mRNAs. By injecting the RNA polymerase II inhibitors 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole or alpha-amanitin into the CA1 region of the dorsal hippocampus at various times before and after training, we found that hippocampal gene expression is critical in two time windows: around the time of training and 3-6 hr after training. Interestingly, these two periods of sensitivity to transcriptional inhibitors are similar to those observed using the protein synthesis inhibitor anisomycin. These findings underscore the parallel dependence of LTM formation of contextual fear on mRNA and protein synthesis in the hippocampus and suggest that the two time periods of anisomycin-induced amnesia depend at least in part on new mRNA synthesis.

Two critical periods for cAMP-dependent protein kinase activity during long-term memory consolidation in the crab Chasmagnathus.

Activation of the cAMP pathway was found to be implicated in the memory process. In the context-signal learning paradigm of the crab Chasmagnathus, the protein kinase (PKA) activator Sp-5,6-DCl-cBIMPS facilitated long-term memory (LTM) induced by spaced training while the PKA inhibitor 8-chloroadenosine-3', 5'-monophosphorothioate, Rp-isomer (Rp-8-Cl-cAMPS) produced amnesia. In the present report the effect of the PKA inhibitor on long-term retention was assessed when administered (systemic injection of 2 microg/animal) at various times after training. According to previous results obtained with a lower dose, retention is impaired when the drug is administered immediately pretraining. An effect on acquisition was ruled out considering that the drug did not affect the performance during training. On the contrary, no effect of the PKA inhibitor was found with an immediately posttraining injection and amnesia was observed only when training was shortened from 15 to 12 trials (training duration from 45 to 36 min). At 2 and 12 h posttraining Rp-8-Cl-cAMPS injection failed to impair retention, but amnesia was found when the drug was injected at 4 and 8 h after training. In order to assess a possible effect of the drug in retrieval, the PKA inhibitor was administered 15 min before testing, and no amnestic effect was observed. These results suggest that two phases of PKA activity are required during consolidation of LTM, one during training and the other between 4 and 8 h after training. The link between these two periods of PKA activation and the two phases of the transcription factor NF-kappaB activation previously found in this model, as well as the similar time course found in rodents, is discussed. An amnestic effect of the drug was not found when administered immediately before a massed training protocol that yielded an intermediate-term memory, suggesting that in this type of memory PKA activation is not required.

Effects of activation and inhibition of cAMP-dependent protein kinase on long-term habituation in the crab Chasmagnathus.

On sudden presentation of a danger stimulus, the crab Chasmagnathus elicits an escape response that habituates promptly and for a long period. We have previously reported that administration of a cAMP-permeable analog (CPT-cAMP) along with a phosphodiesterase inhibitor (IBMX) improves long-term habituation (LTH). In present experiments we studied the effect of systemic administration of the protein kinase A (PKA) activator Sp-5,6-DCl-cBIMPS and that of the PKA inhibitor Rp-8-Cl-cAMPS on LTH tested 24 h after a weak training protocol (5 trials of danger stimulus presentation) or a strong training protocol (15-30 trials), respectively. A 50 microliters pre-training injection of 75 microM Sp-5,6-DCl-cBIMPS, and to a lesser degree of 25 microM, improved retention of the habituated response but not affect short-term habituation (STH). Like pre-training injection, post-training administration of Sp-5,6-DCl-cBIMPS proved to exert a facilitatory action on retention though with 75 microM dose only. Conversely, both pre- and post-training injection of 25 microM Rp-8-Cl-cAMPS impaired LTH without affecting STH. Thus, the PKA activator Sp-5,6-DCl-cBIMPS enables a weak training to produce LTH while the PKA inhibitor Rp-8-Cl-cAMPS impairs LTH when a strong training is given. Activation of crab PKA by Sp-5,6-DCl-cBIMPS and its inhibition by Rp-8-Cl-cAMPS were assessed using an in vitro PKA activity assay. These results provide independent evidences supporting the view that PKA plays a key role in long-term memory storage in this learning paradigm.

A comparison of apparent mRNA half-life using kinetic labeling techniques vs decay following administration of transcriptional inhibitors.

Several different techniques were used to determine the apparent half-lives of immunoglobulin gamma 2b heavy chain and kappa light chain mRNA's in mouse myeloma 4T001 and a mutant derived from 4T001, i.e., mutant I17. The mutant I17 Ig heavy chain mRNA lacks CH1 and has fused CH2 and CH3 domains resulting in a truncated protein. By all four techniques the Ig heavy chain mRNA from mutant I17 displays a half-life that is approximately 70% the half-life of Ig mRNA in 4T001 cells. However, the absolute values of apparent half-life varied by greater than twofold for both lines among several of the techniques employed. The half-life of Ig gamma 2b mRNA in 4T001 cells was found to be 6.4 h by measuring decay following administration of the adenosine analog DRB to block new mRNA synthesis and 5.7 hr by measuring accumulation in an approach to steady-state labeling protocol. In contrast, the observed Ig mRNA half-lives determined by measuring decay following administration of actinomycin D to block new mRNA synthesis, or in a pulse-chase analysis were 2.9 and 3.8 h, respectively. The apparent half-life for Ig kappa light chain mRNA was the same in the 4T001 and I17 lines using any one technique but the value varied depending on the technique from a high value of 5.9 h following DRB to a low value of 2.4 h with actinomycin decay. Approach to steady-state is theoretically the most accurate method to measure mRNA half-life when that value is less than the doubling time of the cells. Pulse-chase analyses are accurate for measuring mRNA half-life when that value is longer than the effective chase period. Measuring preformed message decay following administration of drugs to block new mRNA synthesis is adaptable over a range of half-lives, but the cells must be shown to retain correct RNA metabolism over the time frame of the experiment. Determining a correct half-life for a particular mRNA may not be feasible using only one method and may, in fact, require several different approaches until a consensus value emerges.