Evidence for Ca(2+)-permeable AMPA receptors in the olfactory bulb

-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs), a subtype of glutamate receptor, contribute to olfactory processing in the olfactory bulb (OB). These ion channels consist of various combinations of the subunits GluR1–GluR4, which bestow certain properties. For example, AMPARs that lack GluR2 are highly permeable to Ca²⁺ and generate inwardly rectifying currents. Because increased intracellular Ca²⁺ could trigger a host of Ca²⁺-dependent odor-encoding processes, we used whole cell recording as well as histological and immunocytochemical (ICC) techniques to investigate whether AMPARs on rat OB neurons flux Ca²⁺. Application of 1-naphthylacetyl spermine (NAS), a selective antagonist of Ca²⁺-permeable AMPARs (CP-AMPARs), inhibited AMPAR-mediated currents in subsets of interneurons and principal cells in cultures and slices. The addition of spermine to the electrode yielded inwardly rectifying current-voltage plots in some cells. In OB slices, olfactory nerve stimulation elicited excitatory responses in juxtaglomerular and mitral cells. Bath application of NAS with D,L-2-amino-5-phosphonovaleric acid (AP5) to isolate AMPARs suppressed the amplitudes of these synaptic responses compared with responses obtained using AP5 alone. Co²⁺ staining, which involves the kainate-stimulated influx of Co²⁺ through CP-AMPARs, produced diverse patterns of labeling in cultures and slices as did ICC techniques used with a GluR2-selective antibody. These results suggest that subsets of OB neurons express CP-AMPARs, including functional CP-AMPARs at synapses. Ca²⁺ entry into cells via these receptors could influence odor encoding by modulating K⁺ channels, N-methyl-D-aspartate receptors, and Ca²⁺-binding proteins, or it could facilitate synaptic vesicle fusion. GluR2; polyamines; cobalt; glutamate receptor; olfaction     

Biglycan binds to alpha- and gamma-sarcoglycan and regulates their expression during development

The dystrophin-associated protein complex (DAPC), which links the cytoskeleton to the extracellular matrix, is essential for muscle cell survival, and is defective in a wide range of muscular dystrophies. The DAPC contains two transmembrane subcomplexes-the dystroglycans and the sarcoglycans. Although several extracellular binding partners have been identified for the dystroglycans, none have been described for the sarcoglycan subcomplex. Here we show that the small leucine-rich repeat (LRR) proteoglycan biglycan binds to alpha- and gamma-sarcoglycan as judged by ligand blot overlay and co-immunoprecipitation assays. Our studies with biglycan-decorin chimeras show that alpha- and gamma-sarcoglycan bind to distinct sites on the polypeptide core of biglycan. Both biglycan proteoglycan as well as biglycan polypeptide lacking glycosaminoglycan (GAG) side chains are components of the dystrophin glycoprotein complex isolated from adult skeletal muscle membranes. Finally, our immunohistochemical and biochemical studies with biglycan null mice show that the expression of alpha- and gamma-sarcoglycan is selectively reduced in muscle from young (P14-P21) animals, while levels in adult muscle (> or = P35) are unchanged. We conclude that biglycan is a ligand for two members of the sarcoglycan complex and regulates their expression at discrete developmental ages.     

A C-terminal domain in KCC2 confers constitutive K+-Cl- cotransport

The neuron-specific K(+)-Cl(-) cotransporter KCC2 plays a crucial role in determining intracellular chloride activity and thus the neuronal response to gamma-aminobutyric acid and glycine. Of the four KCCs, KCC2 is unique in mediating constitutive K(+)-Cl(-) cotransport under isotonic conditions; the other three KCCs are exclusively swelling-activated, with no isotonic activity. We have utilized a series of chimeric cDNAs to localize the determinant of isotonic transport in KCC2. Two generations of chimeric KCC4-KCC2 cDNAs initially localized this characteristic to within a KCC2-specific expansion of the cytoplasmic C terminus, between residues 929 and 1043. This region of KCC2 is rich in prolines, serines, and charged residues and encompasses two predicted PEST sequences. Substitution of this region in KCC2 with the equivalent sequence of KCC4 resulted in a chimeric KCC that was devoid of isotonic activity, with intact swelling-activated transport. A third generation of chimeras demonstrated that a domain just distal to the PEST sequences confers isotonic transport on KCC4. Mutagenesis of this region revealed that residues 1021-1035 of KCC2 are sufficient for isotonic transport. Swelling-activated K(+)-Cl(-) cotransport is abrogated by calyculin A, whereas isotonic transport mediated by KCC chimeras and KCC2 is completely resistant to this serine-threonine phosphatase inhibitor. In summary, a 15-residue C-terminal domain in KCC2 is both necessary and sufficient for constitutive K(+)-Cl(-) cotransport under isotonic conditions. Furthermore, unlike swelling-activated transport, constitutive K(+)-Cl(-) cotransport mediated by KCC2 is completely independent of serine-threonine phosphatase activity, suggesting that these two modes of transport are activated by distinct mechanisms.     

Modulation of the domain topography of biphasic monolayers of stearic acid and dimyristoyl phosphatidylcholine

The phase diagram of mixed monolayers composed of dimyristoyl phosphatidylcholine (DMPC) and stearic acid (SA) on different subphases was previously reported. It was observed that on acid subphases, liquid-condensed domains with shapes that depend on the SA proportion are formed. For mixtures with 40-45mole% of SA, the domain shape changes from flower-like to circular domains. In this work, we carried out a detailed study of the driving force for the shape change. We find that it is related to the domain density which, in turn, is driven by the domain nucleation process and thus by oversaturation of the system leading to phase segregation. This could be a way of self-regulating the local electrostatics and mechanical properties in membrane surfaces with segregated phase domains.     

Ammonia inactivation of Ascaris ova in ecological compost by using urine and ash

Viable ova of Ascaris lumbricoides, an indicator organism for pathogens, are frequently found in feces-derived compost produced from ecological toilets, demonstrating that threshold levels of time, temperature, pH, and moisture content for pathogen inactivation are not routinely met. Previous studies have determined that NH(3) has ovicidal properties for pathogens, including Ascaris ova. This research attempted to achieve Ascaris inactivation via NH(3) under environmental conditions commonly found in ecological toilets and using materials universally available in an ecological sanitation setting, including compost (feces and sawdust), urine, and ash. Compost mixed with stored urine and ash produced the most rapid inactivation, with significant inactivation observed after 2 weeks and with a time to 99% ovum inactivation (T(99)) of 8 weeks. Compost mixed with fresh urine and ash achieved a T(99) of 15 weeks, after a 4-week lag phase. Both matrices had relatively high total-ammonia concentrations and pH values of >9.24 (pK(a) of ammonia). In compost mixed with ash only, and in compost mixed with fresh urine only, inactivation was observed after an 11-week lag phase. These matrices contained NH(3) concentrations of 164 to 173 and 102 to 277 mg/liter, respectively, when inactivation occurred, which was below the previously hypothesized threshold for inactivation (280 mg/liter), suggesting that a lower threshold NH(3) concentration may be possible with a longer contact time. Other significant results include the hydrolysis of urea to ammonia between pH values of 10.4 and 11.6, above the literature threshold pH of 10.     

Structural characterization of microcin E492 amyloid formation: Identification of the precursors

Microcin E492 is a low-molecular weight, channel-forming bacteriotoxin that generates amyloid structures. Using electron microscopy and image processing techniques several structural conformations can be observed. Prior to the conditions that induce amyloid formation and at its initial stage, microcin E492 molecules can be found in two main types of oligomers: a pentameric, pore-like structure consisting of globular monomers of ～25? diameter, and long filaments made up of stacked pentamers. The equilibrium between these structures depends on the properties of the solvent, because samples kept in methanol mainly show the pentameric structure. Amyloid induction in aqueous solvent reveals the presence, together with the above mentioned structures, of several amyloid structures such as flat and helical filaments. In addition, X-ray diffraction analysis demonstrated that the fibrils formed by microcin E492 presented cross-β structure, a distinctive property of amyloid fibrils. Based on the study of the observed structures we propose that microcin E492 has two conformations: a native one that assembles mainly into a pentameric structure, which functions as a pore, and an amyloid conformation which results in the formation of different types of amyloid filaments.     

Global assessment of experimental climate warming on tundra vegetation: heterogeneity over space and time

Understanding the sensitivity of tundra vegetation to climate warming is critical to forecasting future biodiversity and vegetation feedbacks to climate. In situ warming experiments accelerate climate change on a small scale to forecast responses of local plant communities. Limitations of this approach include the apparent site-specificity of results and uncertainty about the power of short-term studies to anticipate longer term change. We address these issues with a synthesis of 61 experimental warming studies, of up to 20 years duration, in tundra sites worldwide. The response of plant groups to warming often differed with ambient summer temperature, soil moisture and experimental duration. Shrubs increased with warming only where ambient temperature was high, whereas graminoids increased primarily in the coldest study sites. Linear increases in effect size over time were frequently observed. There was little indication of saturating or accelerating effects, as would be predicted if negative or positive vegetation feedbacks were common. These results indicate that tundra vegetation exhibits strong regional variation in response to warming, and that in vulnerable regions, cumulative effects of long-term warming on tundra vegetation - and associated ecosystem consequences - have the potential to be much greater than we have observed to date.     

Gamma-aminobutyric acid (GABA) and pentobarbital induce different conformational rearrangements in the GABA A receptor alpha1 and beta2 pre-M1 regions

Gamma-aminobutyric acid (GABA) binding to GABA(A) receptors (GABA(A)Rs) triggers conformational movements in the alpha(1) and beta(2) pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABA(A)R channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABA(A)R pre-M1 regions. Alpha(1)beta(2) GABA(A)Rs containing cysteine substitutions in the pre-M1 alpha(1) (K219C, K221C) and beta(2) (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC(50) values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto alpha(1)K219C and alpha(1)K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For alpha(1)K221Cbeta(2) receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For alpha(1)beta(2)K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.     

The above-ground coarse wood productivity of 104 Neotropical forest plots

The net primary production of tropical forests and its partitioning between long‐lived carbon pools (wood) and shorter‐lived pools (leaves, fine roots) are of considerable importance in the global carbon cycle. However, these terms have only been studied at a handful of field sites, and with no consistent calculation methodology. Here we calculate above‐ground coarse wood carbon productivity for 104 forest plots in lowland New World humid tropical forests, using a consistent calculation methodology that incorporates corrections for spatial variations in tree‐size distributions and wood density, and for census interval length. Mean wood density is found to be lower in more productive forests. We estimate that above‐ground coarse wood productivity varies by more than a factor of three (between 1.5 and 5.5 Mg C ha^?1 a^?1) across the Neotropical plots, with a mean value of 3.1 Mg C ha^?1 a^?1. There appear to be no obvious relationships between wood productivity and rainfall, dry season length or sunshine, but there is some hint of increased productivity at lower temperatures. There is, however, also strong evidence for a positive relationship between wood productivity and soil fertility. Fertile soils tend to become more common towards the Andes and at slightly higher than average elevations, so the apparent temperature/productivity relationship is probably not a direct one. Coarse wood productivity accounts for only a fraction of overall tropical forest net primary productivity, but the available data indicate that it is approximately proportional to total above‐ground productivity. We speculate that the large variation in wood productivity is unlikely to directly imply an equivalent variation in gross primary production. Instead a shifting balance in carbon allocation between respiration, wood carbon and fine root production seems the more likely explanation.