The early pathophysiology of diabetic retinopathy and the involvement of neural and vascular malfunction are poorly understood. Glial cells provide structural and metabolic support for retinal neurons and blood vessels, and the cells become reactive in certain injury states. We therefore used the streptozotocin rat model of short-term diabetic retinopathy to study glial reactivity and other glial functions in the retina in the first months after onset of diabetes. With a two-site enzyme-linked immunosorbent assay, we measured the expression of the intermediate filament glial fibrillary acidic protein (GFAP). After 1 month, GFAP was largely unchanged, but within 3 months of the beginning of diabetes, it was markedly induced, by fivefold (P < 0.04). Immunohistochemical staining showed that the GFAP induction occurred both in astrocytes and in Müller cells. Consistent with a glial cell malfunction, the ability of retinas to convert glutamate into glutamine, assayed chromatographically with an isotopic method, was reduced in diabetic rats to 65% of controls (P < 0.01). Furthermore, retinal glutamate, as determined by luminometry, increased by 1.6-fold (P < 0.04) after 3 months of diabetes. Taken together, these findings indicate that glial reactivity and altered glial glutamate metabolism are early pathogenic events that may lead to elevated retinal glutamate during diabetes. These data are the first demonstration of a specific defect in glial cell metabolism in the retina during diabetes. These findings suggest a novel understanding of the mechanism of neural degeneration in the retina during diabetes, involving early and possibly persistent glutamate excitotoxicity.

Summary• Niche differentiation for different soil substrates has been proposed as a mechanism contributing to ectomycorrhizal fungal diversity. This hypothesis has been largely untestable because of a lack of techniques to study the in situ distribution of ectomycorrhizal hyphae.• We developed a technique involving soil DNA extraction, PCR and terminal restriction fragment length polymorphism (T-RFLP) analysis for species identification to investigate the vertical distribution of fungal hyphae in four distinct layers of the forest floor (lower litter, F-layer, H-layer, and B-horizon) of a Pinus resinosa plantation.• Fungal communities differed markedly among the four layers. Cluster analysis suggested six different patterns of resource utilization: litter-layer specialists, litterlayer generalists, F-layer, H-layer, and B-horizon species, and multilayer generalists. Known ectomycorrhizal species were found in all six clusters.• This spatial partitioning observed among ectomycorrhizal fungi along a single, relatively simple substrate-resource gradient supports the niche differentiation hypothesis as an important mechanism contributing to ectomycorrhizal fungal diversity.

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of a-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the a-amino group to glutamate. Measurements of synthesis of 14 C-glutamate and of 14 C-glutamine from H 14 CO 3 have shown that 14 C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The speci®c radioactivity of 14 C-glutamine indicated that ,30% of glutamine was derived from 14 CO 2 ®xation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of 14 C-glutamate and 14 C-glutamine from H 14 CO 3 2 was inhibited by 31%. These results suggest that transamination of a-ketoglutarate to glutamate in Mu È ller cells is slow, the supply of branchedchain amino acids may limit¯ux, and that branched-chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/ glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H 14 CO 3 2 . 14 C-metabolites in brain extracts were measured to determine net incorporation of 14 CO 2 and speci®c radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from 14 CO 2 ®xation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for ,60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.

Abstract:Because it is well known that excess branchedchain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with a-ketoglutarate to form the branched-chain a-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BOAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic(BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-14C]glutamate we found that BOKAs, but not BCAAs, stimulate glutamate transamination to a-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an a-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAAJBCKA shuttle, where BOATs and BCAAs provide the amino group for glutamate synthesis from a-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.

Lysine crotonylation (Kcr) is a newly identified histone modification that is associated with active transcription in mammalian cells. Here we report that the chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to β-hydroxybutyryl-CoA. We showed that the negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Significantly, Cdyl transgenic mice manifest dysregulation of histone Kcr and reduction of male fertility with a decreased epididymal sperm count and sperm cell motility. Our study uncovers a biochemical pathway in the regulation of histone Kcr and implicates CDYL-regulated histone Kcr in spermatogenesis, adding to the understanding of the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (Azoospermia Factor c)-deleted infertile men.

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We present a sequence comparison of 12 Sallele-associated proteins from three solanaceous species with gametophytic self-incompatibility: Nicotiana alata, Petunia inflata, and Solanum chacoense. The allelic variants of the S-protein exhibit a very high degree of sequence diversity consistent with their function as recognition molecules. We identify 41 perfectly conserved residues, 18 of which are also conserved in two fungal ribonucleases, RNase T2 and RNase Rh. The residues conserved in both the S-proteins and the ribonucleases include two histidines essential for catalysis, four cysteines involved in disulfide bridges, and hydrophobic residues probably involved in the core structure of the proteins. This conservation between the two ribonucleases and the 12 divergent S-proteins confirms the previously recognized similarity between 3 more closely related N. alata S-proteins and these ribonucleases, and argues strongly for the functional importance of the ribonuclease activity of the S-protein in self-incompatibility. We also identify the 19 most variable residues, which are the prime candidates for the S-allele-specificity determinant. Twelve of these nineteen residues are clustered in two regions of hypervariability, designated HVa and HVb, which are also the most prominent hydrophilic regions of the S-protein. We suggest that these two regions might form parts of the putative pollen recognition site. Identification of these structural features forms a foundation for the study of the molecular basis of selfrecognition and the biochemical mechanism of inhibition of self-pollen tube growth.

Summary• Ectomycorrhizal fungal communities can be structured by abiotic and biotic factors. Here, we present evidence for community structuring by species interactions.• We sampled ectomycorrhizas and forest floor seven times during a 13-month period. The presence of various ectomycorrhizal fungal species was determined for each sample, and species co-occurrence analyses were performed.• For both ectomycorrhizas and forest floor samples there was significantly less cooccurrence among species within the community than expected by chance, mostly because of negative associations involving Cenococcum geophilum or Clavulina cinerea . For some species pairs, there was significantly more co-occurrence than expected by chance. Both nitrogen and tannin additions to the forest floor altered some interactions among species.• The causes of these nonrandom distributions are currently unknown. Future investigations on competition, antibiosis, parasitism and facilitation among ectomycorrhizal fungal species appear to be warranted.