Adenosine A2A Receptors Contribute to the Radial Migration of Cortical Projection Neurons through the Regulation of Neuronal Polarization and Axon Formation.

Cortical interneurons born in the subpallium reach the cortex through tangential migration, whereas pyramidal cells reach their final position by radial migration. Purinergic signaling via P2Y1 receptors controls the migration of intermediate precursor cells from the ventricular zone to the subventricular zone. It was also reported that the blockade of A2A receptors (A2AR) controls the tangential migration of somatostatin+ interneurons. Here we found that A2AR control radial migration of cortical projection neurons. In A2AR-knockout (KO) mouse embryos or naïve mouse embryos exposed to an A2AR antagonist, we observed an accumulation of early-born migrating neurons in the lower intermediate zone at late embryogenesis. In utero knockdown of A2AR also caused an accumulation of neurons at the lower intermediate zone before birth. This entails the presently identified ability of A2AR to promote multipolar-bipolar transition and axon formation, critical for the transition of migrating neurons from the intermediate zone to the cortical plate. This effect seems to require extracellular ATP-derived adenosine since a similar accumulation of neurons at the lower intermediate zone was observed in mice lacking ecto-5'-nucleotidase (CD73-KO). These findings frame adenosine as a fine-tune regulator of the wiring of cortical inhibitory and excitatory networks.

Purinergic signalling and brain development.

ATP and adenosine are released from cells as a function of their metabolic activity, being important cell-to-cell communication signals. Both purines are also released from neurons in an activity-dependent manner, with several established roles to fine tune brain function in adults, as best heralded by the effects of caffeine, an antagonist of adenosine receptors. Purines are also dynamically released from early neurogenesis and different purine receptors are dynamically expressed throughout development. Accordingly, emerging evidence supports multiple roles for purinergic signalling in the control of different processes of brain development, such as embryonic neurogenesis, migration of principal neurons and interneurons, guidance for neuronal connectivity, synaptogenesis and synaptic stability/elimination. Although major efforts are still required to unravel the time and space-related engagement of the different components of the purinergic system, the relevance of purines in brain development is heralded by their association with neurodevelopmental disorders, positing novel opportunities to understand and correct brain wiring.

Adenosine A2b receptors control A1 receptor-mediated inhibition of synaptic transmission in the mouse hippocampus.

Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A(2B)R) are G(s/q)--protein-coupled receptors with low expression in the brain. As A(2B)R function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A(2B)R-knockout (A(2B)R-KO) and C57/BL6 mice were submitted to a behavioural test battery (open field, elevated plus-maze, Y-maze). The A(2B)R agonist BAY60-6583 (300 nM) decreased the paired-pulse stimulation ratio, an effect prevented by the A(2B)R antagonist MRS 1754 (200 nM) and abrogated in A(2B)R-KO mice. Accordingly, A(2B)R immunoreactivity was present in 73 ± 5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60-6583 attenuated the A(1)R control of synaptic transmission, both the A(1)R inhibition caused by 2-chloroadenosine (0.1-1 µM) and the disinhibition caused by the A(1)R antagonist DPCPX (100 nM), both effects prevented by MRS 1754 and abrogated in A(2B)R-KO mice. BAY 60-6583 decreased glutamate release in slices and also attenuated the A(1)R inhibition (CPA 100 nM). A(2B)R-KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus-maze and the Y-maze and no alteration of locomotion, anxiety or working memory. We conclude that A(2B)R are present in hippocampal glutamatergic terminals where they counteract the predominant A(1)R-mediated inhibition of synaptic transmission, impacting on exploratory behaviour.

Adenosine A2B receptor activation stimulates glucose uptake in the mouse forebrain.

ATP consumption during intense neuronal activity leads to peaks of both extracellular adenosine levels and increased glucose uptake in the brain. Here, we investigated the hypothesis that the activation of the low-affinity adenosine receptor, the A2B receptor (A(2B)R), promotes glucose uptake in neurons and astrocytes, thereby linking brain activity with energy metabolism. To this end, we mapped the spatiotemporal accumulation of the fluorescent-labelled deoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in superfused acute hippocampal slices of C57Bl/6j mice. Bath application of the A(2B)R agonist BAY606583 (300 nM) triggered an immediate and stable (>10 min) increase of the velocity of 2-NBDG accumulation throughout hippocampal slices. This was abolished with the pretreatment with the selective A(2B)R antagonist, MRS1754 (200 nM), and was also absent in A(2B)R null-mutant mice. In mouse primary astrocytic or neuronal cultures, BAY606583 similarly increased (3)H-deoxyglucose uptake in the following 20 min incubation period, which was again abolished by a pretreatment with MRS1754. Finally, incubation of hippocampal, frontocortical, or striatal slices of C57Bl/6j mice at 37 °C, with either MRS1754 (200 nM) or adenosine deaminase (3 U/mL) significantly reduced glucose uptake. Furthermore, A(2B)R blockade diminished newly synthesized glycogen content and at least in the striatum, increased lactate release. In conclusion, we report here that A(2B)R activation is associated with an instant and tonic increase of glucose transport into neurons and astrocytes in the mouse brain. These prompt further investigations to evaluate the clinical potential of this novel glucoregulator mechanism.

CRMP2 tethers kainate receptor activity to cytoskeleton dynamics during neuronal maturation.

The CRMP2 and CRMP4 proteins are strongly expressed in the developing nervous system, mediating neurite outgrowth, neuronal polarity, and axon guidance. In the present study, we demonstrate the interaction of the CRMP2 and CRMP4 proteins with the GluK5 subunit of the kainate (KA) receptor (KAR) and investigated the role of KARs in modulating the development of cultured mouse DRG neurons. We found that KARs modulate neuronal maturation and neurite outgrowth in a bidirectional manner. Accordingly, low concentrations of KA delayed maturation and enhanced neurite outgrowth, whereas maturation was promoted by higher concentrations of KA that attenuated neuritic elongation. The effects of weak KAR activation were prevented by blocking their noncanonical signaling and involved a differential regulation of CRMP2. Whereas the delay in maturation involves PKC-mediated phosphorylation of CRMP2 at T555 leading to a downregulation of membrane Cav2.2, the promotion of neurite outgrowth is achieved by dephosphorylation at T514 at the growth cones, the latter reflecting PKC-driven enhancement of GSK3ß phosphorylation at S9. Together, these findings indicate that noncanonical KAR signaling influences neuronal development by modulating CRMP2 activity.

Surface-Exposed Protein Moieties of Burkholderia cenocepacia J2315 in Microaerophilic and Aerobic Conditions.

Burkholderia cepacia complex infections remain life-threatening to cystic fibrosis patients, and due to the limited eradication efficiency of current treatments, novel antimicrobial therapies are urgently needed. Surface proteins are among the best targets to develop new therapeutic strategies since they are exposed to the host's immune system. A surface-shaving approach was performed using Burkholderia cenocepacia J2315 to quantitatively compare the relative abundance of surface-exposed proteins (SEPs) expressed by the bacterium when grown under aerobic and microaerophilic conditions. After trypsin incubation of live bacteria and identification of resulting peptides by liquid chromatography coupled with mass spectrometry, a total of 461 proteins with ≥2 unique peptides were identified. Bioinformatics analyses revealed a total of 53 proteins predicted as localized at the outer membrane (OM) or extracellularly (E). Additionally, 37 proteins were predicted as moonlight proteins with OM or E secondary localization. B-cell linear epitope bioinformatics analysis of the proteins predicted to be OM and E-localized revealed 71 SEP moieties with predicted immunogenic epitopes. The protegenicity higher scores of proteins BCAM2761, BCAS0104, BCAL0151, and BCAL0849 point out these proteins as the best antigens for vaccine development. Additionally, 10 of the OM proteins also presented a high probability of playing important roles in adhesion to host cells, making them potential targets for passive immunotherapeutic approaches. The immunoreactivity of three of the OM proteins identified was experimentally demonstrated using serum samples from cystic fibrosis patients, validating our strategy for identifying immunoreactive moieties from surface-exposed proteins of potential interest for future immunotherapies development.

Adenosine A2A receptors control synaptic remodeling in the adult brain.

The molecular mechanisms underlying circuit re-wiring in the mature brain remains ill-defined. An eloquent example of adult circuit remodelling is the hippocampal mossy fiber (MF) sprouting found in diseases such as temporal lobe epilepsy. The molecular determinants underlying this retrograde re-wiring remain unclear. This may involve signaling system(s) controlling axon specification/growth during neurodevelopment reactivated during epileptogenesis. Since adenosine A2A receptors (A2AR) control axon formation/outgrowth and synapse stabilization during development, we now examined the contribution of A2AR to MF sprouting. A2AR blockade significantly attenuated status epilepticus(SE)-induced MF sprouting in a rat pilocarpine model. This involves A2AR located in dentate granule cells since their knockdown selectively in dentate granule cells reduced MF sprouting, most likely through the ability of A2AR to induce the formation/outgrowth of abnormal secondary axons found in rat hippocampal neurons. These A2AR should be activated by extracellular ATP-derived adenosine since a similar prevention/attenuation of SE-induced hippocampal MF sprouting was observed in CD73 knockout mice. These findings demonstrate that A2AR contribute to epilepsy-related MF sprouting, most likely through the reactivation of the ability of A2AR to control axon formation/outgrowth observed during neurodevelopment. These results frame the CD73-A2AR axis as a regulator of circuit remodeling in the mature brain.

Immunization and Immunotherapy Approaches against Pseudomonas aeruginosa and Burkholderia cepacia Complex Infections.

Human infections caused by the opportunist pathogens Burkholderia cepacia complex and Pseudomonas aeruginosa are of particular concern due to their severity, their multiple antibiotic resistance, and the limited eradication efficiency of the current available treatments. New therapeutic options have been pursued, being vaccination strategies to prevent or limit these infections as a rational approach to tackle these infections. In this review, immunization and immunotherapy approaches currently available and under study against these bacterial pathogens is reviewed. Ongoing active and passive immunization clinical trials against P. aeruginosa infections is also reviewed. Novel identified bacterial targets and their possible exploitation for the development of immunization and immunotherapy strategies against P. aeruginosa and B. cepacia complex and infections are also presented and discussed.

Retrospective analysis of clinical yeast isolates in a hospital in the centre of Portugal: spectrum and revision of the identification procedures.

Abstract We conducted a four-year (2003-2006) retrospective study of yeasts recovered in a hospital laboratory in the centre of Portugal to evaluate the epidemiology of yeast infections. Clinical isolates and data were gathered from 751 patients corresponding to 906 episodes of yeast infection. The isolates were first identified using classical and commercial methods, routinely employed at the hospital laboratory. We then re-identified the same isolates using RFLP of the ITS 5.8S rRNA gene and sequence of the D1/D2 domain of the 26S rRNA gene. Candida parapsilosis isolates were re-identified using the Ban I digestion of the SADH gene. C. albicans was the most frequently isolated of the yeasts found in the analysed specimens, with an overall incidence of 69.6% and then in decreasing order, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei. C. parapsilosis was most frequently recovered from younger patients, decreasing with age, while C. glabrata occurrence increased with age. We found an increased number of cases of fungemia per 100,000 people per year, reaching a maximum of 4.4 during 2006.

The spatial learning phenotype of heterozygous leaner mice is robust to systematic variation of the housing environment.

Providing stimulation and allowing the performance of motivated behaviors through environmental enrichment improves learning and memory in rodents and delays cognitive impairment in neurodegenerative disease models. The leaner mutation affects the Ca(v)2.1 voltage-gated calcium channel alpha(1A)-subunit gene, and homozygous mice show severe phenotypic alterations. Although several authors have described heterozygous mice as normal, recent studies in our laboratory indicate motor and cognitive impairment in tg(la)/+ mice. In the present study, we evaluated whether this impairment is robust to systematic variation of the housing environment from barren to standard and furnished (enriched) cages. Wildtype (n = 55) and tg(la)/+ (n = 79) C57Bl/6J mice were assigned randomly to 1 of the 3 housing systems and tested on the Morris water maze at 6, 12, and 20 mo of age. The results confirmed impaired performance in tg(la)/+ mice, particularly in older mice. At 12 and 20 mo, only wildtype (and not tg(la)/+) mice showed evidence of learning (spending increased time in the target quadrant) during the probe trial. Housing also affected performance: at 12 mo, only mice from furnished cages showed evidence of learning, and in aged mice (20 mo), only those housed in more complex environments showed long-term memory (8 mo after previous testing) of the platform position. In conclusion, a heterozygous mutation in a Ca(2+) channel gene causes cognitive deficits in leaner mice that are robust to environmental variation but attenuated by physical and behavioral stimulation.

Evaluation of exploration and risk assessment in pre-weaning mice using the novel cage test.

Exploration and risk behaviour (risk assessment/risk taking) are critical to enable mice to cope with novel situations and gain control over their environment. Evaluation of those behaviours would therefore be a useful part of early phenotypic characterization of genetically modified mice, allowing early detection of behavioural phenotypes that require special attention and/or are of scientific interest. This study aimed to evaluate exploration and risk behaviour in pre-weaning mice using the novel cage test, which consists in exploration of a novel, clean, Makrolon type III cage. The results of this test were compared with those obtained in more complex and established tests to which the same mice were subjected as adolescents and young adults. Mice of two inbred strains (129S6/Bkl, n=10; C57BL/6Bkl, n=10) and one hybrid (B6CBAF1/Bkl, n=10) were used for validation of the test. The animals were tested in the novel cage (at weaning), the open field test (at 5 weeks), and from 9 weeks of age in three other tests: the elevated plus-maze, the concentric square field and the rat exposure test. The novel cage test effectively detected strain differences in pre-weaning mice as regards exploration and risk behaviour and the results were largely consistent with those obtained in the established tests later in life. In all tests 129S6 displayed a low locomotion and high risk assessment, while C57BL/6 and B6CBAF1 showed high locomotion and exploration. In addition high levels of risk taking were observed in C57BL/6. The novel cage test is rapid, requires no special equipment and is as discriminatory as more complex tests in detecting strain/genotype differences. This suggests that the novel cage test is a valuable tool for evaluation of exploration, risk assessment and risk taking in juvenile mice.

Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation.

Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

Identification and biodegradation potential of a novel strain of Dietzia cinnamea isolated from a petroleum-contaminated tropical soil.

A bacterial strain, named P4, isolated previously from microcosms containing oil-contaminated soil collected from an environmentally protected area of a tropical Atlantic forest (Biological Reserve of Poço das Antas) located in Brazil was identified as Dietzia cinnamea by morphological, biochemical and genotypic tests. Arabian Light and Marlin oils were both degraded when strain P4 was tested for oil degradation ability in microplates. Total Petroleum Hydrocarbons (TPH) analysis, determined by gas chromatography, showed that strain P4 degraded a wide range of n-alkanes, and also pristane and phytane. Furthermore, this strain was also able to grow in mineral liquid media amended with carbazole, quinoline, naphthalene, toluene, gasoline and diesel as the sole carbon sources. The species D. cinnamea has been previously described with only one representative strain isolated from a perianal swab of a patient with a bone marrow transplant. With the results presented here this species is implicated not only as a human pathogen but also as a potential strain for further studies concerning its role for bioremediation of oil contaminated soil.

Presynaptic P2X1-3 and α3-containing nicotinic receptors assemble into functionally interacting ion channels in the rat hippocampus.

Previous studies documented a cross-talk between purinergic P2X (P2XR) and nicotinic acetylcholine receptors (nAChR) in heterologous expression systems and peripheral preparations. We now investigated if this occurred in native brain preparations and probed its physiological function. We found that P2XR and nAChR were enriched in hippocampal terminals, where both P2X1-3R and α3, but not α4, nAChR subunits were located in the active zone and in dopamine-ß-hydroxylase-positive hippocampal terminals. Notably, P2XR ligands displaced nAChR binding and nAChR ligands displaced P2XR binding to hippocampal synaptosomes. In addition, a negative P2XR/nAChR cross-talk was observed in the control of the evoked release of noradrenaline from rat hippocampal synaptosomes, characterized by a less-than-additive facilitatory effect upon co-activation of both receptors. This activity-dependent cross-inhibition was confirmed in Xenopus oocytes transfected with P2X1-3Rs and α3ß2 (but not α4ß2) nAChR. Besides, P2X2 co-immunoprecipitated α3ß2 (but not α4ß2) nAChR, both in HEK cells and rat hippocampal membranes indicating that this functional interaction is supported by a physical association between P2XR and nAChR. Moreover, eliminating extracellular ATP with apyrase in hippocampal slices promoted the inhibitory effect of the nAChR antagonist tubocurarine on noradrenaline release induced by high- but not low-frequency stimulation. Overall, these results provide integrated biochemical, pharmacological and functional evidence showing that P2X1-3R and α3ß2 nAChR are physically and functionally interconnected at the presynaptic level to control excessive noradrenergic terminal activation upon intense synaptic firing in the hippocampus.

Hierarchical glucocorticoid-endocannabinoid interplay regulates the activation of the nucleus accumbens by insulin.

Here we asked if insulin activation of the nucleus accumbens in vitro is reflected by an increase in (3)H-deoxyglucose ([(3)H]DG) uptake, thus subserving a new model to study molecular mechanisms of central insulin actions. Additionally, we investigated the dependence of this insulin effect on endocannabinoids and corticosteroids, two major culprits in insulin resistance. We found that in acute accumbal slices, insulin (3 and 300nM but not at 0.3nM) produced an increase in [(3)H]DG uptake. The synthetic cannabinoid agonist, WIN55212-2 (500nM) and the glucocorticoid dexamethasone (10µM), impaired insulin (300nM) action on [(3)H]DG uptake. The glucocorticoid receptor (GcR) antagonist, mifepristone (10µM) prevented dexamethasone from inhibiting insulin's action. Strikingly, this anti-insulin action of dexamethasone was also blocked by two CB1 cannabinoid receptor (CB1R) antagonists, O-2050 (500nM) and SR141716A (500nM), as well as by tetrahydrolipstatin (10µM), an inhibitor of diacylglycerol lipases-the enzymes responsible for the synthesis of the endocannabinoid, 2-arachidonoyl-glycerol (2-AG). On the other hand, the blockade of the post-synaptic 2-AG metabolizing enzymes, α,ß-serine hydrolase domain 6/12 by WWL70 (1µM) also prevented the action of insulin, probably via increasing endogenous 2-AG tone. Additionally, an anti-insulin receptor (InsR) antibody immunoprecipitated CB1Rs from accumbal homogenates, indicating a physical complexing of CB1Rs with InsRs that supports their functional interaction. Altogether, insulin stimulates glucose uptake in the nucleus accumbens. Accumbal GcR activation triggers the synthesis of 2-AG that in turn binds to the known CB1R-InsR heteromer, thus impeding insulin signaling.

Plant age and genotype affect the bacterial community composition in the tuber rhizosphere of field-grown sweet potato plants.

The hypothesis that sweet potato genotypes containing different starch yields in their tuberous roots can affect the bacterial communities present in the rhizosphere (soil adhering to tubers) was tested in this study. Tuberous roots of field-grown sweet potato of genotypes IPB-149 (commercial genotype), IPB-052, and IPB-137 were sampled three and six months after planting and analyzed by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing analysis of 16S rRNA genes PCR-amplified from total community DNA. The statistical analysis of the DGGE fingerprints showed that both plant age and genotypes influenced the bacterial community structure in the tuber rhizosphere. Pyrosequencing analysis showed that the IPB-149 and IPB-052 (both with high starch content) displayed similar bacterial composition in the tuber rhizosphere, while IPB-137 with the lowest starch content was distinct. In comparison with bulk soil, higher 16S rRNA gene copy numbers (qPCR) and numerous genera with significantly increased abundance in the tuber rhizosphere of IPB-137 (Sphingobium, Pseudomonas, Acinetobacter, Stenotrophomonas, Chryseobacterium) indicated a stronger rhizosphere effect. The genus Bacillus was strongly enriched in the tuber rhizosphere samples of all sweet potato genotypes studied, while other genera showed a plant genotype-dependent abundance. This is the first report on the molecular identification of bacteria being associated with the tuber rhizosphere of different sweet potato genotypes.

Kainate receptors in health and disease.

Our understanding of the molecular properties of kainate receptors and their involvement in synaptic physiology has progressed significantly over the last 30 years. A plethora of studies indicate that kainate receptors are important mediators of the pre- and postsynaptic actions of glutamate, although the mechanisms underlying such effects are still often a topic for discussion. Three clear fields related to their behavior have emerged: there are a number of interacting proteins that pace the properties of kainate receptors; their activity is unconventional since they can also signal through G proteins, behaving like metabotropic receptors; they seem to be linked to some devastating brain diseases. Despite the significant progress in their importance in brain function, kainate receptors remain somewhat puzzling. Here we examine discoveries linking these receptors to physiology and their probable implications in disease, in particular mood disorders, and propose some ideas to obtain a deeper understanding of these intriguing proteins.

Performance of juvenile mice in a reach-to-grasp task.

Reach-to-grasp tasks have been used to study rodent models of motor system damage, stroke, and neurodegenerative disorders such as Parkinson's. These tasks are especially useful as they allow evaluation of bilateral or unilateral damage in different regions of the brain. Performing reach-to-grasp tests in juvenile mice may be important to understand motor disorders of early onset. This study evaluated the performance of juvenile and adolescent mice on a reach-to-grasp task. Male and female C57BL/6J mice (n=93) were tested in a forelimb reaching task at postnatal weeks 4, 5, 7, 9 and 12. At all ages mice could learn the task and improved performance with training. Results show that reach-to-grasp tasks can be used to study skill learning in juvenile and adolescent mice. Results are discussed in terms of adapting methodologies (test protocols and arenas) when performing behaviour tests in young mice.

Motor and cognitive deficits in the heterozygous leaner mouse, a Cav2.1 voltage-gated Ca2+ channel mutant.

The leaner mutation in mice affects the Ca(v)2.1 voltage-gated calcium channel alpha(1A)-subunit gene (Cacna1a), causing a reduction in calcium currents predominantly in Purkinje cells. This reduction in calcium currents causes severe progressive cerebellar ataxia, beginning around postnatal day 10, in homozygous leaner mice (tg(la)/tg(la)), while their heterozygous littermates (tg(la)/+) present no obvious behavioral deficits. In humans, heterozygous mutations in the Cacna1a orthologous gene produce a broad range of neurological manifestations. To evaluate the phenotypic status of the tg(la)/+ animals, we assessed motor performance and cognition, at different ages, in these mutant mice. We were able to observe age-dependent impairment in motor and cognitive tasks; balance and motor learning deficits were found in demanding tasks on the rotarod and on the hanging wire test, while spatial learning and memory impairment was observed in the Morris water maze. Progressive dysfunction in escape reflexes, indicative of neurological impairment, was also present in tg(la)/+ animals. Although not presenting major motor alterations, tg(la)/+ mice show age-dependent motor and cognitive deficits.

Saccharomyces cerevisiae Hog1 protein phosphorylation upon exposure to bacterial endotoxin.

The yeast Hog1 protein is both functionally and structurally similar to the mammalian p38, belonging to the same family of mitogen-activated protein (MAP) kinases and responding to extracellular changes in osmolarity. Since p38 mediates lipopolysaccharide (LPS) effects in mammalian cells, we now tested the responsiveness of Hog1 upon exposure of the yeast Saccharomyces cerevisiae to bacterial LPS. In the presence of Escherichia coli LPS (100 ng/ml) and an endotoxically active, hexaacylated, synthetic lipid A (compound 506; 100 ng/ml), Hog1 becomes phosphorylated with a maximum of phosphorylation between 3 and 6 h, whereas a tetraacylated, inactive form of lipid A (compound 406) did not cause any modification in the phosphorylation state of Hog1. A triple labeling immunocytochemical study showed that phosphorylated Hog1 translocates into the nucleus after a 90-min incubation and becomes sparsely located in the cytoplasm. The translocation of the phospho-Hog1 is preceded by an increased expression of the HOG1 gene and concomitant with the expression of the Hog1 target gene, GPD1. We also observed that cells unable to synthesize Hog1 do not resist LPS as efficiently as wild-type cells. We conclude that the yeast S. cerevisiae is able to respond to the presence of Gram-negative bacteria endotoxin and that Hog1 is involved in this response.