Marine diatoms are responsible for up to 20% of global CO 2 fixation. Their photosynthetic efficiency is enhanced by concentrating CO 2 around Rubisco, diminishing photorespiration, but the mechanism is yet to be resolved. Diatoms have been regarded as C 3 photosynthesizers, but recent metabolic labeling and genome sequencing data suggest that they perform C 4 photosynthesis. We studied the pathways of photosynthetic carbon assimilation in two diatoms by short-term metabolic 14 C labeling. In Thalassiosira weissflogii, both C3 (glycerate-P and triose-P) and C4 (mainly malate) compounds were major initial (2-5 s) products, whereas Thalassiosira pseudonana produced mainly C3 and C6 (hexose-P) compounds. The data provide evidence of C 3 -C 4 intermediate photosynthesis in T. weissflogii, but exclusively C 3 photosynthesis in T. pseudonana. The labeling patterns were the same for cells grown at near-ambient (380 mL L 21 ) and low (100 mL L 21 ) CO 2 concentrations. The lack of environmental modulation of carbon assimilatory pathways was supported in T. pseudonana by measurements of gene transcript and protein abundances of C 4 -metabolic enzymes (phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase) and Rubisco. This study suggests that the photosynthetic pathways of diatoms are diverse, and may involve combined CO 2 -concentrating mechanisms. Furthermore, it emphasizes the requirement for metabolic and functional genetic and enzymic analyses before accepting the presence of C 4 -metabolic enzymes as evidence for C 4 photosynthesis.

SUMMARY Mu-Opioid Receptors (MOR) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a novel conditional BAC rescue strategy to show that mice with targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restore opiate reward, opiate-induced striatal dopamine release, and partially restore motivation to self-administer opiates. However, they lack opiate analgesia or withdrawal. Importantly, we used Cre-mediated deletion of the rescued MOR transgene to establish that striatal, rather than a few extrastriatal sites of MOR transgene expression, is needed for the restoration of opiate reward. Together, our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a novel intersectional genetic approach to dissect neurocircuit-specific gene function in vivo.

Diatoms are responsible for up to 40% of primary productivity in the ocean, and complete genome sequences are available for two species. However, there are very significant gaps in our understanding of how diatoms take up and assimilate inorganic C. Diatom plastids originate from secondary endosymbiosis with a red alga and their Form ID Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase) from horizontal gene transfer, which means that embryophyte paradigms can only give general guidance as to their C acquisition mechanisms. Although diatom Rubiscos have relatively high CO(2) affinity and CO(2)/O(2) selectivity, the low diffusion coefficient for CO(2) in water has the potential to restrict the rate of photosynthesis. Diatoms growing in their natural aquatic habitats operate inorganic C concentrating mechanisms (CCMs), which provide a steady-state CO(2) concentration around Rubisco higher than that in the medium. How these CCMs work is still a matter of debate. However, it is known that both CO(2) and HCO (3) (-) are taken up, and an obvious but as yet unproven possibility is that active transport of these species across the plasmalemma and/or the four-membrane plastid envelope is the basis of the CCM. In one marine diatom there is evidence of C(4)-like biochemistry which could act as, or be part of, a CCM. Alternative mechanisms which have not been eliminated include the production of CO(2) from HCO (3) (-) at low pH maintained by a H(+) pump, in a compartment close to that containing Rubisco.

G-protein coupled receptors (GPCRs) are typically present in a basal, inactive state, but when bound to agonist they activate downstream signaling cascades. In studying arrestin regulation of opioid receptors in dorsal root ganglia (DRG) neurons, we find that agonists of delta opioid receptors (δORs) activate cofilin through Rho-associated coiled-coiled containing protein kinase (ROCK), LIM domain kinase (LIMK) and β- arrestin 1 (β-arr1), to regulate actin polymerization. This controls receptor function, as assessed by agonist-induced inhibition of voltage-dependent Ca2+ channels in DRGs. Agonists of opioid-receptor like receptors (ORL1) similarly influence the function of this receptor through ROCK, LIMK and β-arr1. Functional evidence of this cascade was demonstrated in vivo where the behavioral effects of δOR or ORL1 agonists were enhanced in the absence of β-arr1 or prevented by inhibiting ROCK. This pathway allows δOR and ORL1 agonists to rapidly regulate receptor function.

Diel periodicity and effects of inorganic carbon (Ci ) and NO3 (-) on the expression of 11 key genes for primary carbon and nitrogen metabolism, including potential C4 photosynthesis, in the marine diatom Thalassiosira pseudonana Hasle et Heimdal were investigated. Target gene transcripts were measured by quantitative reverse transcriptase-PCR, and some of the gene-encoded proteins were analyzed by Western blotting. The diatom was grown with a 12 h photoperiod at two different Ci concentrations maintained by air-equilibration with either 380 μL · L(-1) (near-ambient) or 100 μL · L(-1) (low) CO2 . Transcripts of the principal Ci and NO3 (-) assimilatory genes RUBISCO LSU (rbcL) and nitrate reductase displayed very strong diel oscillations with peaks at the end of the scotophase. Considerable diel periodicities were also exhibited by the β-carboxylase genes phosphoenolpyruvate carboxylase (PEPC1 and PEPC2) and phosphoenolpyruvate carboxykinase (PEPCK), and the Benson-Calvin cycle gene sedoheptulose-bisphosphatase (SBPase), with peaks during mid- to late scotophase. In accordance with the transcripts, there were substantial diel periodicities in PEPC1, PEPC2, PEPCK, and especially rbcL proteins, although they peaked during early to mid-photophase. Inorganic carbon had some transient effects on the β-carboxylase transcripts, and glycine decarboxylase P subunit was highly up-regulated by low Ci concentration, indicating increased capacity for photorespiration. Nitrogen-starved cells had reduced amounts of carbon metabolic gene transcripts, but the PEPC1, PEPC2, PEPCK, and rbcL transcripts increased rapidly when NO3 (-) was replenished. The results suggest that the β-carboxylases in T. pseudonana play key anaplerotic roles but show no clear support for C4 photosynthesis.

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Conditional responses in rodents such as locomotion have been reported for drugs of abuse and similar to the placebo response in humans, may be associated with the expectation of reward. We examined several conditional opioid-like responses and the influence of drug expectation on conditioned place preference and concomitant conditional locomotion. Male C57BL/6J mice were conditioned with the selective mu opioid receptor agonist fentanyl (0.2 mg/kg, i.p.) in a novel context and subsequently given a vehicle injection. In separate experiments, locomotor activity, Straub tail, hot plate sensitivity, and conditioned place preference (CPP) were measured. Mice exhibited multiple conditional opioid-like responses including conditional hyperlocomotion, a conditional pattern of opioid-like locomotion, Straub tail, analgesia, and place preference. Modulating drug expectation via administration of fentanyl to “demonstrator” mice in the home cage did not affect the expression of conditioned place preference or the concomitant locomotor activity in “observer” mice. In summary, Pavlovian conditioning of an opioid in a novel context induced multiple conditional opioid-like behaviors and provides a model for studying the neurobiological mechanisms of the placebo response in mice.

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