The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl^?) channels. The EAATs couple the transport of glutamate to the co-transport of three Na⁺ ions and one H⁺ ion into the cell, and the counter-transport of one K⁺ ion out of the cell. The EAAT Cl^? channel is activated by the binding of glutamate and Na⁺, but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, Glt_Ph, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl^? permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl^? permeation and the mechanisms that underlie their split personality.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Quantifying the Impact of Woodpecker Predation on Population Dynamics of the Emerald Ash Borer (Agrilus planipennis)

The emerald ash borer (EAB), Agrilus planipennis, is an invasive beetle that has killed millions of ash trees (Fraxinus spp.) since it was accidentally introduced to North America in the 1990s. Understanding how predators such as woodpeckers (Picidae) affect the population dynamics of EAB should enable us to more effectively manage the spread of this beetle, and toward this end we combined two experimental approaches to elucidate the relative importance of woodpecker predation on EAB populations. First, we examined wild populations of EAB in ash trees in New York, with each tree having a section screened to exclude woodpeckers. Second, we established experimental cohorts of EAB in ash trees in Maryland, and the cohorts on half of these trees were caged to exclude woodpeckers. The following spring these trees were debarked and the fates of the EAB larvae were determined. We found that trees from which woodpeckers were excluded consistently had significantly lower levels of predation, and that woodpecker predation comprised a greater source of mortality at sites with a more established wild infestation of EAB. Additionally, there was a considerable difference between New York and Maryland in the effect that woodpecker predation had on EAB population growth, suggesting that predation alone may not be a substantial factor in controlling EAB. In our experimental cohorts we also observed that trees from which woodpeckers were excluded had a significantly higher level of parasitism. The lower level of parasitism on EAB larvae found when exposed to woodpeckers has implications for EAB biological control, suggesting that it might be prudent to exclude woodpeckers from trees when attempting to establish parasitoid populations. Future studies may include utilizing EAB larval cohorts with a range of densities to explore the functional response of woodpeckers.     

Androgenesis-inducing stress treatments change phytohormone levels in anthers of three legume species (Fabaceae)

Legumes are recalcitrant to androgenesis and induction protocols were only recently developed for pea (Pisum sativum L.) and chickpea (Cicer arietinum L.), albeit with low regeneration frequencies. Androgenesis is thought to be mediated through abscisic acid (ABA) but other phytohormones, such as auxins, cytokinins, and gibberellins, have also been implicated. In view of improving induction protocols, the hormone content of pea, chickpea, and lentil anthers was measured after exposure to cold, centrifugation, electroporation, sonication, osmotic shock, or various combinations thereof using an analytical mass spectrometer. Indole-3-acetic acid (IAA) had a key function during the induction process. In pea, high concentrations of IAA-asparagine (IAA-Asp), a putative IAA metabolite, accumulated during the application of the different stresses. In chickpea, the IAA-Asp concentration increased 30-fold compared to pea but only during the osmotic shock treatment and likely as a result of the presence of exogenous IAA in the medium. In contrast, no treatment in lentil (Lens culinaris) invoked such an increase in IAA-Asp content. Of the various cytokinins monitored, only cis zeatin riboside increased after centrifugation and electroporation in pea and possibly chickpea. No bioactive gibberellins were detected in any species investigated, indicating that this hormone group is likely not linked to androgenesis in legumes. In contrast to the other stresses, osmotic shock treatment caused a reduction in the levels of all hormones analyzed, with the exception of IAA-Asp in chickpea. A short period of low hormone content might be a necessary transition phase for androgenesis induction of legumes. KEY MESSAGE: Five androgenesis-inducing stress treatments changed content of ABA, auxin and cytokinin in anthers of three legumes. Osmotic shock treatment differed because it reduced hormone content to very low levels.     

Multiparametric Characterization of Grade 2 Glioma Subtypes Using Magnetic Resonance Spectroscopic,Perfusion, and Diffusion Imaging

BACKGROUND AND PURPOSE: The purpose of this study was to derive quantitative parameters from magnetic resonance (MR) spectroscopic, perfusion, and diffusion imaging of grade 2 gliomas according to the World Health Organization and to investigate how these multiple imaging modalities can contribute to evaluating their histologic subtypes and spatial characteristics. MATERIALS AND METHODS: MR spectroscopic, perfusion, and diffusion images from 56 patients with newly diagnosed grade 2 glioma (24 oligodendrogliomas, 18 astrocytomas, and 14 oligoastrocytomas) were retrospectively studied. Metabolite intensities, relative cerebral blood volume (rCBV), and apparent diffusion coefficient (ADC) were statistically evaluated. RESULTS: The 75th percentile rCBV and median ADC were significantly different between oligodendrogliomas and astrocytomas (P < .0001) and between oligodendrogliomas and oligoastrocytomas (P < .001). Logistic regression analysis identified both 75th percentile rCBV and median ADC as significant variables in the differentiation of oligodendrogliomas from astrocytomas and oligoastrocytomas. Group differences in metabolite intensities were not significant, but there was a much larger variation in the volumes and maximum values of metabolic abnormalities for patients with oligodendroglioma compared with the other tumor subtypes. CONCLUSIONS: Perfusion and diffusion imaging provide quantitative MR parameters that can help to differentiate grade 2 oligodendrogliomas from grade 2 astrocytomas and oligoastrocytomas. The large variations in the magnitude and spatial extent of the metabolic lesions between patients and the fact that their values are not correlated with the other imaging parameters indicate that MR spectroscopic imaging may provide complementary information that is helpful in targeting therapy, evaluating residual disease, and assessing response to therapy.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

Exon-skipping Splice Variants of Excitatory Amino Acid Transporter-2 (EAAT2) Form Heteromeric Complexes with Full-length EAAT2

The glial transporter excitatory amino acid transporter-2 (EAAT2) is the main mediator of glutamate clearance in brain. The wild-type transporter (EAAT2wt) forms trimeric membrane complexes in which each protomer functions autonomously. Several EAAT2 variants are found in control and Alzheimer-diseased human brains; their expression increases with pathological severity. These variants might alter EAAT2wt-mediated transport by abrogating membrane trafficking, or by changing the configuration or functionality of the assembled transporter complex. HEK293 cells were transfected with EAAT2wt; EAAT2b, a C-terminal variant; or either of two exon-skipping variants: alone or in combination. Surface biotinylation studies showed that only the exon-7 deletion variant was not trafficked to the membrane when transfected alone, and that all variants could reach the membrane when co-transfected with EAAT2wt. Fluorescence resonance energy transfer (FRET) studies showed that co-transfected EAAT2wt and EAAT2 splice variants were expressed in close proximity. Glutamate transporter function was measured using a whole cell patch clamp technique, or by changes in membrane potential indexed by a voltage-sensitive fluorescent dye (FMP assay): the two methods gave comparable results. Cells transfected with EAAT2wt or EAAT2b showed glutamate-dependent membrane potential changes consistent with functional expression. Cells transfected with EAAT2 exon-skipping variants alone gave no response to glutamate. Co-transfection of EAAT2wt (or EAAT2b) and splice variants in various ratios significantly raised glutamate EC₅₀ and decreased Hill coefficients. We conclude that exon-skipping variants form heteromeric complexes with EAAT2wt or EAAT2b that traffic to the membrane but show reduced glutamate-dependent activity. This could allow glutamate to accumulate extracellularly and promote excitotoxicity.