siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2h in medium containing either 1.0mM [(15)NH(4)(+)] or 100μM [(15)N]glutamate. For those exposed to [(15)N]glutamate an additional 100μM was added after 1h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The (15)N incorporation from [(15)NH(4)(+)] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [(15)N]glutamate, the reduced GDH activity led to an increased (15)N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.     

Association of Polymorphisms of the CHI3L1 Gene with Asthma and Atopy: A Populations-Based Study of 6514 Danish Adults

Background

YKL-40 is a chitinase-like glycoprotein encoded by the chitinase 3-like 1 gene, CHI3L1, localized at chromosome 1q32.1. Increased levels of serum YKL-40 have been reported to be a biomarker for asthma and a reduced lung function. Interestingly, the C-allele of the -131 C→G (rs4950928) polymorphism of CHI3L1 has been shown to associate with bronchial hyperresponsiveness and reduced lung function suggesting that variations in CHI3L1 may influence risk of asthma. The objective of the present study was to investigate the association of common variation in the CHI3L1 locus with asthma, atopy and lung function in a large population-based sample of adults.

Methods/Principal Findings

Eleven single nucleotide polymorphisms (SNPs) of CHI3L1 including rs4950928 were genotyped in 6514 individuals. Asthma was defined as self-reported history of physician-diagnosed asthma. Total IgE and specific IgE to inhalant allergens were measured on serum samples. Lung function was measured by spirometry. Homozygosity of the rs4950928 G allele as compared to homozygosity of the C allele was associated with self-reported physician diagnosed asthma (OR 1.5 (95% CI, 1.00–2.26)) and with prevalence of atopic asthma (OR 1.93 (95% CI, 1.21–3.07)) after adjustment for age, sex, smoking status, socio-economic class and BMI. Carriers of rs883125 G allele had a significantly lower prevalence of atopy (OR 0.82 (CI, 0.72; 0.94)) as compared to homozygosity of the C allele. None of the SNPs examined were significantly associated with FEV1. However, two SNPs (rs10399931and rs4950930) appeared to be significantly associated with FEV₁/FVC-ratio. Subgroup analyses of never-smokers did not consistently influence the associations in an either positively og negatively way.

Conclusions

In contrast to previous studies, the rs4950928 G allele, and not the C allele, was found to be associated with asthma. A few other SNPs of the CHI3L1 was found to be significantly associated with atopy and FEV1/FVC ratio, respectively. Thus, more studies seem warranted to establish the role of CHI3L1 gene in asthma and atopy.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

Perinatal transmission of SHIV-SF162P3 in Macaca nemestrina

We developed a SHIV/macaque model of transmission from infected dams to their infants. Ten pregnant dams were infected intravenously with 100 MID(50) of macaque-titered SHIV-SF162P3 during the second trimester. Nine infants were born; the seven surviving beyond day of birth suckled for 6 months. Four of nine infants were infected (transmission rate = 44.4%), with one infection in utero, and three intrapartum and/or immediately post-birth via suckling. Varying levels of binding and neutralizing antibodies were transplacentally transferred to infants. Passive antibodies were detected in plasma on the day of birth and persisted for 5 weeks. Infants infected at or after birth controlled acute and post-acute viremia. Exposure to maternal SHIV-SF162P3 during birth and suckling in the presence of autologous maternal neutralizing antibodies may have affected transmission or pathogenesis in the infants. This transmission model can allow investigation of key parameters involved in perinatal transmission of HIV.     

Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor

Spent sulfite pulping liquor (SSL) is a high-organic content byproduct of acid bisulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15-0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (<1 g/l) were produced, and glycerol yields were approximately 0.12 g glycerol/g sugar consumed. By using 259ST for SSL fermentation up to 130% more ethanol can be produced compared to fermentations using non-xylose fermenting yeast.     

Mechanisms of hydrazine toxicity in rat liver investigated by proteomics and multivariate data analysis

A proteomics approach combined with multivariate data analysis was used to examine the hepatotoxic effect of hydrazine in 30 male Sprague Dawley rats, assigned to four treatment groups and two control groups. Liver samples from the individual animals were resolved by two-dimensional differential gel electrophoresis (2-D DIGE) and protein patterns from the 2-D gels were analyzed by principal component analysis (PCA) and partial least squares regression (PLSR). The PCA plot was able to describe the variation in the protein expression related to dose and time, by separation or clustering of different animal groups. PLSR followed by variable selection (Jack-knifing) was used to select proteins that varied significantly in relation to the dose related response of the hydrazine treatment. The 10 up-regulated and 10 down-regulated proteins with highest rank in the PLSR model were identified by mass spectrometry. Hydrazine treatment induced altered expression of proteins related to lipid metabolism, Ca(2+) homeostasis, thyroid hormone pathways and stress response. Several of the identified proteins have not previously been implicated in hydrazine toxicity and may thus be regarded as new potential biomarkers of induced liver toxicity.     

Antagonistic roles of ESCRT and Vps class C/HOPS complexes in the recycling of yeast membrane proteins

In Saccharomyces cerevisiae, deficiencies in the ESCRT machinery trigger the mistargeting of endocytic and biosynthetic ubiquitinated cargoes to the limiting membrane of the vacuole. Surprisingly, impairment of this machinery also leads to the accumulation of various receptors and transporters at the plasma membrane in both yeast and higher eukaryotes. Using the well-characterized yeast endocytic cargo uracil permease (Fur4p), we show here that the apparent stabilization of the permease at the plasma membrane in ESCRT mutants results from an efficient recycling of the protein. Whereas several proteins as well as internalized dyes are known to be recycled in yeast, little is known about the machinery and molecular mechanisms involved. The SNARE protein Snc1p is the only cargo for which the recycling pathway is well characterized. Unlike Snc1p, endocytosed Fur4p did not pass through the Golgi apparatus en route to the plasma membrane. Although ubiquitination of Fur4p is required for its internalization, deubiquitination is not required for its recycling. In an attempt to identify actors in this new recycling pathway, we found an unexpected phenotype associated with loss of function of the Vps class C complex: cells defective for this complex are impaired for recycling of Fur4p, Snc1p, and the lipophilic dye FM4-64. Genetic analyses indicated that these phenotypes were due to the functioning of the Vps class C complex in trafficking both to and from the late endosomal compartment.     

Mechanisms and rates of bacterial colonization of sinking aggregates

Quantifying the rate at which bacteria colonize aggregates is a key to understanding microbial turnover of aggregates. We used encounter models based on random walk and advection-diffusion considerations to predict colonization rates from the bacteria's motility patterns (swimming speed, tumbling frequency, and turn angles) and the hydrodynamic environment (stationary versus sinking aggregates). We then experimentally tested the models with 10 strains of bacteria isolated from marine particles: two strains were nonmotile; the rest were swimming at 20 to 60 microm s(-1) with different tumbling frequency (0 to 2 s(-1)). The rates at which these bacteria colonized artificial aggregates (stationary and sinking) largely agreed with model predictions. We report several findings. (i) Motile bacteria rapidly colonize aggregates, whereas nonmotile bacteria do not. (ii) Flow enhances colonization rates. (iii) Tumbling strains colonize aggregates enriched with organic substrates faster than unenriched aggregates, while a nontumbling strain did not. (iv) Once on the aggregates, the bacteria may detach and typical residence time is about 3 h. Thus, there is a rapid exchange between attached and free bacteria. (v) With the motility patterns observed, freely swimming bacteria will encounter an aggregate in <1 day at typical upper-ocean aggregate concentrations. This is faster than even starving bacteria burn up their reserves, and bacteria may therefore rely solely on aggregates for food. (vi) The net result of colonization and detachment leads to a predicted equilibrium abundance of attached bacteria as a function of aggregate size, which is markedly different from field observations. This discrepancy suggests that inter- and intraspecific interactions among bacteria and between bacteria and their predators may be more important than colonization in governing the population dynamics of bacteria on natural aggregates.