Benzodiazepine Receptor Binding in Cerebellar Cortex: Observations in Olivopontocerebellar Atrophy

Benzodiazepine receptor binding was measured in cerebellar cortex of 15 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The majority of these patients had a moderate to marked Purkinje cell loss, as judged by the lowered levels of dentate nucleus gamma-aminobutyric acid (GABA), a marker of Purkinje cells. Despite the reduction in Purkinje cell number cerebellar cortical benzodiazepine receptor density was either normal or slightly elevated in the OPCA patients. These results are in contrast to the findings in a mutant strain of mice deficient in Purkinje cells in which the concentration of benzodiazepine receptors in cerebellum is greatly reduced. Our data indicate that in the human, cerebellar cortical benzodiazepine receptors are either not significantly associated with Purkinje cells or that in OPCA Purkinje cell loss triggers a de novo synthesis of extra benzodiazepine binding sites. It is concluded that, in contrast with the rodent, in the human benzodiazepine receptor binding may not serve as a marker for cerebellar Purkinje cells.     

Down''s Syndrome Individuals Begin Life with Normal Levels of Brain Cholinergic Markers

We measured the activities of the cholinergic marker enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in autopsied brains of seven infants (age range 3 months to 1 year) with Down's syndrome (DS), a disorder in which virtually all individuals will develop by middle age the neuropathological changes of Alzheimer's disease accompanied by a marked brain cholinergic reduction. When compared with age-matched controls cholinergic enzyme activity was normal in all brain regions of the individuals with infant DS with the exception of above-normal activity in the putamen (ChAT) and the occipital cortex (AChE). Our neurochemical observations suggest that DS individuals begin life with a normal complement of brain cholinergic neurons. This opens the possibility of early therapeutic intervention to prevent the development of brain cholinergic changes in patients with DS.     

S-nitrosoglutathione-induced toxicity in Drosophila melanogaster: Delayed pupation and induced mild oxidative/nitrosative stress in eclosed flies

The toxicity of the nitric oxide donor S-nitrosoglutathione (GSNO) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with GSNO at concentrations of 1.0, 1.5 or 4.0 mM. Food supplementation with GSNO caused a developmental delay in the flies. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies that emerged from control larvae and larvae fed on food supplemented with GSNO. Larval exposure to GSNO resulted in lower activities of aconitase in both sexes and also lower activities of catalase and isocitrate dehydrogenase in adult males relative to the control cohort. Larval treatment with GSNO resulted in higher carbonyl protein content and higher activities of glucose-6-phosphate dehydrogenase in males and higher activities of superoxide dismutase and glutathione-S-transferase in both sexes. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by GSNO.     

OXIDIZED LIPIDS DID NOT REDUCE LIFESPAN IN THE FRUIT FLY,Drosophila melanogaster

Aging is often associated with accumulation of oxidative damage in proteins and lipids. However, some studies do not support this view, raising the question of whether high levels of oxidative damage are associated with lifespan. In the current investigation, Drosophila melanogaster flies were kept on diets with 2 or 10% of either glucose or fructose. The lifespan, fecundity, and feeding as well as amounts of protein carbonyls (PC) and lipid peroxides (LOOH), activities of superoxide dismutase (SOD), catalase, glutathione‐S‐transferase (GST), and glutathione reductase activity of thioredoxin reductase (TrxR) were measured in “young” (10‐day old) and “aged” (50‐day old) flies. Flies maintained on diets with 10% carbohydrate lived longer than those on the 2% diets. However, neither lifespan nor fecundity was affected by the type of carbohydrate. The amount of PC was unaffected by diet and age, whereas flies fed on diets with 10% carbohydrate had about fivefold higher amounts of LOOH compared to flies maintained on the 2% carbohydrate diets. Catalase activity was significantly lower in flies fed on diets with 10% carbohydrates compared to flies on 2% carbohydrate diets. The activities of SOD, GST, and TrxR were not affected by the diet or age of the flies. The higher levels of LOOH in flies maintained on 10% carbohydrate did not reduce their lifespan, from which we infer that oxidative damage to only one class of biomolecules, particularly lipids, is not sufficient to influence lifespan.     

Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila

An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20–50%. However, NDX was able only weakly to complement the phenotypes produced by the knockdown of complex I subunits.     

Marked dissociation between high noradrenaline versus low noradrenaline transporter levels in human nucleus accumbens

We recently identified a noradrenaline-rich caudomedial subdivision of the human nucleus accumbens (NACS), implying a special function for noradrenaline in this basal forebrain area involved in motivation and reward. To establish whether the NACS, as would be expected, contains similarly high levels of other noradrenergic markers, we measured dopamine-beta-hydroxylase (DBH) and noradrenaline transporter in the accumbens and, for comparison, in 23 other brain regions in autopsied human brains by immunoblotting. Although the caudomedial NACS had high DBH levels similar to those in other noradrenaline-rich areas, the noradrenaline transporter concentration was low (only 11% of that in hypothalamus). Within the accumbens, transporter concentration in the caudal portion was only slightly (by 30%) higher than that in the rostral subdivisions despite sharply increasing rostrocaudal gradients of noradrenaline (15-fold) and DBH. In contrast, the rostrocaudal gradient in the accumbens for the serotonin transporter and serotonin were similar (2-fold increase). The caudomedial NACS thus appears to represent the only instance in human brain having a striking mismatch in high levels of a monoamine neurotransmitter versus low levels of its uptake transporter. This suggests that noradrenaline signalling is much less spatially and temporally restricted in the caudomedial accumbens than in other noradrenaline-rich brain areas.     

Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking

Hyaluronan (HA) turnover accelerates metastatic progression of prostate cancer in part by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed hyaluronidase 1 (Hyal1) as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of HA and the endocytic recycling marker transferrin. Live imaging of Hyal1, sucrose gradient centrifugation, and specific colocalization of Rab GTPases defined the subcellular distribution of Hyal1 as early and late endosomes, lysosomes, and recycling vesicles. Manipulation of vesicular trafficking by chemical inhibitors or with constitutively active and dominant negative Rab expression constructs caused atypical localization of Hyal1. Using the catalytically inactive point mutant Hyal1-E131Q, we found that enzymatic activity of Hyal1 was necessary for normal localization within the cell as Hyal1-E131Q was mainly detected within the endoplasmic reticulum. Expression of a HA-binding point mutant, Hyal1-Y202F, revealed that secretion of Hyal1 and concurrent reuptake from the extracellular space are critical for rapid HA internalization and cell proliferation. Overall, excess Hyal1 secretion accelerates endocytic vesicle trafficking in a substrate-dependent manner, promoting aggressive tumor cell behavior.     

Forest transitions in Eastern Europe and their effects on carbon budgets

Forests often rebound from deforestation following industrialization and urbanization, but for many regions our understanding of where and when forest transitions happened, and how they affected carbon budgets remains poor. One such region is Eastern Europe, where political and socio‐economic conditions changed drastically over the last three centuries, but forest trends have not yet been analyzed in detail. We present a new assessment of historical forest change in the European part of the former Soviet Union and the legacies of these changes on contemporary carbon stocks. To reconstruct forest area, we homogenized statistics at the provincial level for ad 1700–2010 to identify forest transition years and forest trends. We contrast our reconstruction with the KK11 and HYDE 3.1 land change scenarios, and use all three datasets to drive the LPJ dynamic global vegetation model to calculate carbon stock dynamics. Our results revealed that forest transitions in Eastern Europe occurred predominantly in the early 20th century, substantially later than in Western Europe. We also found marked geographic variation in forest transitions, with some areas characterized by relatively stable or continuously declining forest area. Our data suggest extensive deforestation in European Russia already prior to ad 1700, and even greater deforestation in the 18th and 19th centuries than in the KK11 and HYDE scenarios. Based on our reconstruction, cumulative carbon emissions from deforestation were greater before 1700 (60 Pg C) than thereafter (29 Pg C). Summed over our entire study area, forest transitions led to a modest uptake in carbon over recent decades, with our dataset showing the smallest effect (<5.5 Pg C) and a more heterogeneous pattern of source and sink regions. This suggests substantial sequestration potential in regrowing forests of the region, a trend that may be amplified through ongoing land abandonment, climate change, and CO₂ fertilization.     

Regulation of epithelial Na+ channel activity by conserved serine/threonine switches within sorting signals

The PY and YXXphi motifs are canonical sorting signals involved in trafficking. Nedd4-2 and the mu(2)-subunit of the AP-2 complex target these motifs to facilitate internalization. Epithelial Na(+) channel (ENaC) subunits contain both motifs in their cytosolic COOH termini where they overlap ((S/T)PPPXYX(S/T)phi). Just preceding the PY and embedded within the YXXphi motifs are conserved serine/threonine. We test here whether these conserved Ser/Thr modulate ENaC activity by influencing the function of the internalization domains. We find that co-expression of dominant-negative dynamin (K44A) with ENaC increases channel activity. Conversely, co-expression of Nedd4-2 and epsin with ENaC decrease activity. Alanine substitution of the conserved Thr(628) preceding the PY motif in gamma-mENaC had no effect on basal activity. Channels with this mutation, however, responded to K44A and epsin but not Nedd4-2. Similarly, mutation of the proline repeat in the PY motif of gamma-mENaC disrupted only Nedd4-2 regulation having no effect on regulation by K44A and epsin. Alanine substitution of the conserved Thr within the YXX motif of gamma-mENaC (T635A) increased basal activity. Channels containing this mutation responded to Nedd4-2 but not K44A and epsin. Channels containing the T635(D/E) substitution in gamma-mENaC did not have increased basal activity and responded to Nedd4-2 but not K44A. The double mutant T628A,T635A did not respond to Nedd4-2 or K44A. Mutation of Thr(628) and Thr(635) also disrupted ENaC precipitation with the mu(2)-subunit of the AP-2 complex. Moreover, the YXXphi motif, independent of the PY motif, was sufficient to target degradation with T635A disrupting this effect. These results demonstrate that the overlapping PY and YXXphi motifs in ENaC are, in some instances, capable of independent function and that the Ser/Thr just preceding and within these domains impact this function.