Molecular basis for selective uptake and elimination of organic anions in the kidney by OAT1.

In mammals, the kidney plays an essential role in maintaining blood homeostasis through the selective uptake, retention or elimination of toxins, drugs and metabolites. Organic anion transporters (OATs) are responsible for the recognition of metabolites and toxins in the nephron and their eventual urinary excretion. Inhibition of OATs is used therapeutically to improve drug efficacy and reduce nephrotoxicity. The founding member of the renal organic anion transporter family, OAT1 (also known as SLC22A6), uses the export of α-ketoglutarate (α-KG), a key intermediate in the Krebs cycle, to drive selective transport and is allosterically regulated by intracellular chloride. However, the mechanisms linking metabolite cycling, drug transport and intracellular chloride remain obscure. Here, we present cryogenic-electron microscopy structures of OAT1 bound to α-KG, the antiviral tenofovir and clinical inhibitor probenecid, used in the treatment of Gout. Complementary in vivo cellular assays explain the molecular basis for α-KG driven drug elimination and the allosteric regulation of organic anion transport in the kidney by chloride.

Characterization and pharmacokinetic evaluation of microcomposite particles of alpha lipoic acid/hydrogenated colza oil obtained in supercritical carbon dioxide.

The work reported here is an extension of our previous findings in which supercritical composite particles (SCP) of alpha lipoic acid (ALA) masked with hydrogenated colza oil (HCO) named as ALA/HCO/SCP were obtained by the modified particles from gas-saturated solutions (PGSS) process in supercritical carbon dioxide in order to obscure the unpleasant taste and odor of ALA. The masking effect on ALA/HCO/SCP was compared with the widely used mechano-chemically masked formulation of ALA and HCO named as MC-50F. In the present study, ALA/HCO/SCP particles were found to have a significant improvement in regard to bitterness, numbness, and smell compared to ALA bulk powders suggesting they were well coated. The pharmacokinetic parameters for ALA/HCO/SCP and ALA bulk powder gave similar values but were significantly different from those of MC-50F. The amount of ALA absorbed into the body, in the administered ALA/HCO/SCP, was comparable to that absorbed by ALA bulk powder, whereas about half portion of ALA of the MC-50F was not absorbed, because the ALA/HCO/SCP particles were small enough and the particles of MC-50F were relatively large and had smaller specific surface area. Therefore, this study suggested a newly masked candidate may offer functional particles with maintained efficacy.

Crystal structure of plant vacuolar iron transporter VIT1.

The iron ion is an essential cofactor in several vital enzymatic reactions, such as DNA replication, oxygen transport, and respiratory and photosynthetic electron transfer chains, but its excess accumulation induces oxidative stress in cells. Vacuolar iron transporter 1 (VIT1) is important for iron homeostasis in plants, by transporting cytoplasmic ferrous ions into vacuoles. Modification of the VIT1 gene leads to increased iron content in crops, which could be used for the treatment of human iron deficiency diseases. Furthermore, a VIT1 from the malaria-causing parasite Plasmodium is considered as a potential drug target for malaria. Here we report the crystal structure of VIT1 from rose gum Eucalyptus grandis, which probably functions as a H+-dependent antiporter for Fe2+ and other transition metal ions. VIT1 adopts a novel protein fold forming a dimer of five membrane-spanning domains, with an ion-translocating pathway constituted by the conserved methionine and carboxylate residues at the dimer interface. The second transmembrane helix protrudes from the lipid membrane by about 40 Å and connects to a three-helical bundle, triangular cytoplasmic domain, which binds to the substrate metal ions and stabilizes their soluble form, thus playing an essential role in their transport. These mechanistic insights will provide useful information for the further design of genetically modified crops and the development of anti-malaria drugs.

Thalamo-insular pathway conveying orofacial muscle proprioception in the rat.

Little is known about how proprioceptive signals arising from muscles reach to higher brain regions such as the cerebral cortex. We have recently shown that a particular thalamic region, the caudo-ventromedial edge (VPMcvm) of ventral posteromedial thalamic nucleus (VPM), receives the proprioceptive signals from jaw-closing muscle spindles (JCMSs) in rats. In this study, we further addressed how the orofacial thalamic inputs from the JCMSs were transmitted from the thalamus (VPMcvm) to the cerebral cortex in rats. Injections of a retrograde and anterograde neuronal tracer, wheat-germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), into the VPMcvm demonstrated that the thalamic pathway terminated mainly in a rostrocaudally narrow area in the dorsal part of granular insular cortex rostroventrally adjacent to the rostralmost part of the secondary somatosensory cortex (dGIrvs2). We also electrophysiologically confirmed that the dGIrvs2 received the proprioceptive inputs from JCMSs. To support the anatomical evidence of the VPMcvm-dGIrvs2 pathway, injections of a retrograde neuronal tracer Fluorogold into the dGIrvs2 demonstrated that the thalamic neurons projecting to the dGIrvs2 were confined in the VPMcvm and the parvicellular part of ventral posterior nucleus. In contrast, WGA-HRP injections into the lingual nerve area of core VPM demonstrated that axon terminals were mainly labeled in the core regions of the primary and secondary somatosensory cortices, which were far from the dGIrvs2. These results suggest that the dGIrvs2 is a specialized cortical region receiving the orofacial proprioceptive inputs. Functional contribution of the revealed JCMSs-VPMcvm-dGIrvs2 pathway to Tourette syndrome is also discussed.

Gas-saturated solution process to obtain microcomposite particles of alpha lipoic acid/hydrogenated colza oil in supercritical carbon dioxide.

Alpha lipoic acid (ALA), an active substance in anti-aging products and dietary supplements, need to be masked with an edible polymer to obscure its unpleasant taste. However, the high viscosity of the ALA molecules prevents them from forming microcomposites with masking materials even in supercritical carbon dioxide (scCO2). Therefore, the purpose of this study was to investigate and develop a novel production method for microcomposite particles for ALA in hydrogenated colza oil (HCO). Microcomposite particles of ALA/HCO were prepared by using a novel gas-saturated solution (PGSS) process in which the solid-dispersion method is used along with stepwise temperature control (PGSS-STC). Its high viscosity prevents the formation of microcomposites in the conventional PGSS process even under strong agitation. Here, we disperse the solid particles of ALA and HCO in scCO2 at low temperatures and change the temperature stepwise in order to mix the melted ALA and HCO in scCO2. As a result, a homogeneous dispersion of the droplets of ALA in melted HCO saturated with CO2 is obtained at high temperatures. After the rapid expansion of the saturated solution through a nozzle, microcomposite particles of ALA/HCO several micrometers in diameter are obtained.

Thalamic afferent and efferent connectivity to cerebral cortical areas with direct projections to identified subgroups of trigeminal premotoneurons in the rat.

The roles of supramedullary brain mechanisms involved in the control of jaw movements are not fully understood. To address this issue, a series of retrograde (Fluorogold, FG) and anterograde (biotinylated dextran amine, BDA) tract-tracing studies were done in rats. At first, we identified projection patterns from defined sensorimotor cortical areas to subgroups of trigeminal premotoneurons that are located in defined brainstem areas. Focal injections of FG into these brainstem areas revealed that the rostralmost part of lateral agranular cortex (rmost-Agl), the rostralmost part of medial agranular cortex (rmost-Agm), and the rostralmost part of primary somatosensory cortex (rmost-S1) preferentially project to brainstem areas containing jaw-closing premotoneurons, jaw-opening premotoneurons and a mixture of both types of premotoneurons, respectively. The thalamic reciprocal connectivities to rmost-Agl, rmost-Agm, and rmost-S1 were then investigated following cortical injections of FG or BDA. We found many retrogradely FG-labeled neurons and large numbers of axons and terminals labeled anterogradely with BDA in the dorsal thalamus mainly on the side ipsilateral to the injection sites. The rmost-Agl had strong connections with the ventral lateral nucleus (VL), ventromedial nucleus (VM), parafascicular nucleus, and posterior nucleus (Po); the rmost-Agm with the ventral anterior nucleus, VL, VM, central lateral nucleus, paracentral nucleus, central medial nucleus, mediodorsal nucleus and Po; and the rmost-S1 with the ventral posteromedial nucleus and Po. The present results suggest that the descending multiple pathways from the cerebral cortex to jaw-closing and jaw-opening premotoneurons have unique functional roles in jaw movement motor control.

Different corticostriatal projections from two parts of the cortical masticatory area in the rabbit.

The cortical masticatory area (CMA) elicits rhythmic jaw movements in response to repetitive stimulation and is involved in the control of mastication. Based on jaw movement patterns, the CMA is divided into two parts. One is the part of the CMA in which a T-pattern similar to jaw movements during food transport in natural mastication is evoked by electrical stimulation. The other is more dorsomedially located, and during chewing a C-pattern similar to jaw movements can be induced. However, it is still not known which region of the putamen receives projections from the CMA and whether projections originate from both parts of the CMA. In this study, electrophysiological and histological experiments were undertaken in rabbits to investigate projections from the CMA to the putamen. Both experiments showed that the ventral region of the putamen received projections from the CMA. The density of the projections from the CMA area inducing the T-pattern seemed to be higher than that from the area inducing the C-pattern. Furthermore, the peak latency of the evoked potentials from stimulation of the CMA area inducing the T-pattern was shorter than that from stimulation of the area inducing the C-pattern. The data obtained from the present study indicate the functional role of the ventral region of the putamen in the regulation of mastication, and further suggest that the corticostriatal pathway is involved in the transition between behavioral jaw movement patterns.

Experimental pain perception remains equally active over all sleep stages.

The literature on sensory perception during sleep suggests that light sleep (Stage 2) is more responsive to external sensory stimulation (e.g. sound, electrical shock) than deep sleep (Stages 3 and 4) and REM sleep. The main objective of this study was to characterize the specificity of nociceptive stimulation to trigger sleep arousal-awakening over all sleep stages. Thirteen healthy adults (e.g. without pain or sleep problems; six female and seven male of a mean age of 24.2+/-1.3 years) were included in the study. The responses to noxious intramuscular 5% hypertonic infusion were compared to innocuous vibrotactile and to respective control stimulations: isotonic infusion and auditory stimulations. These stimulations were applied during wakefulness and were repeated during sleep. Polygraphic signals (e.g. brain activity, heart rate) signals were recorded to score sleep arousal over all sleep stages. A subjective assessment of sleep quality was made on next morning. No overnight sensitization or habituation occurred with any of the experimental stimulations. The vibratory-auditory stimulations and the noxious hypertonic infusions triggered significantly (P < 0.05) more awakenings in sleep Stage 2 and in REM than their respective control stimulations. In sleep Stage 2, both vibratory + auditory stimulations and the noxious hypertonic infusions has the same awakening response frequency (approximately 30%), however, with the noxious infusions the response frequency were similar in sleep Stages 3 and 4 (P < 0.05) and in REM (trend). Compared to the baseline night, sleep quality was lower following the night with noxious stimulation (90.1+/-2.7 and 73.3+/-7.4 mm, respectively; P < 0.03. These data suggest that pain during sleep could trigger a sleep awaking response over all sleep stages and not only in light sleep.