Characterization of human phosphodiesterase 12 and identification of a novel 2'-5' oligoadenylate nuclease - The ectonucleotide pyrophosphatase/phosphodiesterase 1

The vertebrate 2-5A system is part of the innate immune response and central to cellular antiviral activities. Upon activation by viral double-stranded RNA, 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2′-5′ oligoadenylate synthetases. The 2-5As bind and activate RNase L, an unspecific endoribonuclease, resulting in viral and cellular RNA decay. Given that most endogenous RNAs are degraded by RNase L, continued enzyme activity will eventually lead to cell growth arrest and cell death. This is averted, when 2-5As and their 5′-dephosphorylated forms, the so-called 2-5A core molecules, are cleaved and thus inactivated by 2′-5′-specific nuclease(s), e.g. phosphodiesterase 12, thereby turning RNase L into its latent form. In this study, we have characterized the human phosphodiesterase 12 in vitro focusing on its ability to degrade 2-5As and 2-5A core molecules. We have found that the enzyme activity is distributive and is influenced by temperature, pH and divalent cations. This allowed us to determine V_max and K_m kinetic parameters for the enzyme. We have also identified a novel 2′-5′-oligoadenylate nuclease; the human plasma membrane-bound ectonucleotide pyrophosphatase/phosphodiesterase 1, suggesting that 2-5A catabolism may be a multienzyme-regulated process.     

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.     

Biophysical characterization of the proton-coupled oligopeptide transporter YjdL

Proton-coupled oligopeptide transporters (POTs) utilize the electrochemical proton gradient to facilitate uptake of di- or tripeptide molecules. YjdL is one of four POTs found in Escherichia coli. It has shown an extraordinary preference for di- rather than tripeptides, and is therefore significantly different from prototypical POTs such as the human hPepT1. Nonetheless YjdL contains several highly conserved POT residues, which include Glu388 that is located in the putative substrate binding cavity. Here we present biophysical characterization of WT-YjdL and Glu388Gln. Isothermal titration calorimetrical studies exhibit a K_d of 14 μM for binding of Ala-Lys to WT-YjdL. Expectedly, no binding could be detected for the tripeptide Ala-Ala-Lys. Surprisingly however, binding could not be detected for Ala-Gln, although earlier studies indicated inhibitory potencies of Ala-Gln to be comparable to Ala-Lys (IC₅₀ values of 0.6 compared to 0.3 mM). Finally, Ala-Lys binding to Glu388Gln was also undetectable which may support a previously suggested role in interaction with the ligand peptide N-terminus.     

Brain glycogen and its role in supporting glutamate and GABA homeostasis in a type 2 diabetes rat model

The number of people suffering from diabetes is hastily increasing and the condition is associated with altered brain glucose homeostasis. Brain glycogen is located in astrocytes and being a carbohydrate reservoir it contributes to glucose homeostasis. Furthermore, glycogen has been indicated to be important for proper neurotransmission under normal conditions. Previous findings from our laboratory suggested that glucose metabolism was reduced in type 2 diabetes, and thus we wanted to investigate more specifically how brain glycogen metabolism contributes to maintain energy status in the type 2 diabetic state. Also, our objective was to elucidate the contribution of glycogen to support neurotransmitter glutamate and GABA homeostasis. A glycogen phosphorylase (GP) inhibitor was administered to Sprague-Dawley (SprD) and Zucker Diabetic Fatty (ZDF) rats in vivo and after one day of treatment [1-13C]glucose was used to monitor metabolism. Brain levels of 13C labeling in glucose, lactate, alanine, glutamate, GABA, glutamine and aspartate were determined. Our results show that inhibition of brain glycogen metabolism reduced the amounts of glutamate in both the control and type 2 diabetes models. The reduction in glutamate was associated with a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in the control but not the type 2 diabetes model. In the type 2 diabetes model GABA levels were increased suggesting that brain glycogen serves a role in maintaining a proper ratio between excitatory and inhibitory neurotransmitters in type 2 diabetes. Both the control and the type 2 diabetic states had a compensatory increase in glucose-derived 13C processed through the TCA cycle following inhibition of glycogen degradation. Finally, it was indicated that the type 2 diabetes model might have an augmented necessity for compensatory upregulation at the glycolytic level.     

The EPI bioassay identifies natural compounds with estrogenic activity that are potent inhibitors of androgenic pathways in human prostate stromal and epithelial cells

The reactive stromal phenotype is an important factor for prostate cancer progression and may be a new target for treatment and prevention. A new high efficiency preclinical protocol, the EPI bioassay, reflects the interaction of endocrine, paracrine and immune, (EPI) factors on induced androgen metabolism in human prostate reactive stroma. The bioassay is based on co-culturing human primary prostate stromal cells and LAPC-4 prostatic adenocarcinoma cells in a downscaled format of 96-well-plates for testing multiple doses of multiple target compounds. Metabolism of dehydroepiandrosterone (DHEA) with or without TGFβ1-induced stimulation (D+T) of the reactive stroma phenotype was assessed by increased testosterone in the media and PSA production of the epithelial prostate cancer cells. Using the non-metabolizable androgen R1881, effects from direct androgen action were distinguished from stromal androgen production from DHEA. Stromal cell androgenic bioactivity was confirmed using conditioned media from D+T-treated stromal cell monocultures in an androgen-inducible AR screening assay. We further showed that both agonists to estrogen receptor (ER), DPN (ERβ) and PPT (ERα), as well as estrogenic natural compounds including soy isoflavones attenuated D+T-induced PSA production. Studies with the pure ER agonists showed that activating either ERα or ERβ could inhibit both D+T-mediated and R1881-mediated PSA production with the D+T effect being more pronounced. In conclusion, natural compounds with estrogenic activity and pure ER agonists are very potent inhibitors of stromal conversion of DHEA to androgenic metabolites. More studies are needed to characterize the mechanisms involved in estrogenic modulation of the endocrine-immune-paracrine balance of the prostate microenvironment.     

Impairment of the organization of locomotor and exploratory behaviors in bile duct-ligated rats

Hepatic encephalopathy (HE) arises from acute or chronic liver diseases and leads to several problems, including motor impairment. Animal models of chronic liver disease have extensively investigated the mechanisms of this disease. Impairment of locomotor activity has been described in different rat models. However, these studies are controversial and the majority has primarily analyzed activity parameters. Therefore, the aim of the present study was to evaluate locomotor and exploratory behavior in bile duct-ligated (BDL) rats to explore the spatial and temporal structure of behavior. Adult female Wistar rats underwent common bile duct ligation (BDL rats) or the manipulation of common bile duct without ligation (control rats). Six weeks after surgery, control and BDL rats underwent open-field, plus-maze and foot-fault behavioral tasks. The BDL rats developed chronic liver failure and exhibited a decrease in total distance traveled, increased total immobility time, smaller number of rearings, longer periods in the home base area and decreased percentage of time in the center zone of the arena, when compared to the control rats. Moreover, the performance of the BDL rats was not different from the control rats for the elevated plus-maze and foot-fault tasks. Therefore, the BDL rats demonstrated disturbed spontaneous locomotor and exploratory activities as a consequence of altered spatio-temporal organization of behavior.     

Muscle morphological and strength adaptations to endurance vs. resistance training

Fascicle angle (FA) is suggested to increase as a result of fiber hypertrophy and furthermore to serve as the explanatory link in the discrepancy in the relative adaptations in the anatomical cross-sectional area (CSA) and fiber CSA after resistance training (RT). In contrast to RT, the effects of endurance training on FA are unclear. The purpose of this study was therefore to investigate and compare the longitudinal effects of either progressive endurance training (END, n = 7) or RT (n = 7) in young untrained men on FA, anatomical CSA, and fiber CSA. Muscle morphological measures included the assessment of vastus lateralis FA obtained by ultrasonography and anatomical CSA by magnetic resonance imaging of the thigh and fiber CSA deduced from histochemical analyses of biopsy samples from m. vastus lateralis. Functional performance measures included VO2max and maximal voluntary contraction (MVC). The RT produced increases in FA by 23 ± 8% (p < 0.01), anatomical CSA of the knee extensor muscles by 9 ± 3% (p = 0.001), and fiber CSA by 19 ± 7% (p < 0.05). RT increased knee extensor MVC by 20 ± 5% (p < 0.001). END increased VO2max by 10 ± 2% but did not evoke changes in FA, anatomical CSA, or in fiber CSA. In conclusion, the morphological changes induced by 10 weeks of RT support that FA does indeed serve as the explanatory link in the observed discrepancy between the changes in anatomical and fiber CSA. Contrarily, 10 weeks of endurance training did not induce changes in FA, but the lack of morphological changes from END indirectly support the fact that fiber hypertrophy and FA are interrelated.     

Cognitive Functions in Middle Aged Individuals Are Related to Metabolic Disturbances and Aerobic Capacity: A Cross-Sectional Study

Aims

Metabolic disturbances may contribute to cognitive dysfunction in patients with type 2 diabetes. We investigated the relation between cognitive impairment and metabolic deteriorations, low physical fitness, low-grade inflammation and abdominal obesity in middle aged individuals.

Methods

We conducted a cross-sectional study including 40 to 65 year-old patients with type 2 diabetes and limited co morbidity (N = 56), age-matched individuals with impaired glucose tolerance (N = 56) as well as age-matched controls with normal glucose tolerance (N = 72). Specific cognitive functions were assessed with focus on verbal memory, processing speed, executive functions, and a composite overall mean score. Oral glucose tolerance test, VO₂max test, systemic inflammation, DXA scanning and abdominal MRI were measured.

Results

Multiple linear regression analyses adjusting for age, gender and verbal intelligence demonstrated that a low score in processing speed, executive functions and overall cognitive function were related to high fasting C-peptide, as well as low insulin sensitivity, beta-cell function and VO₂max. Measurements of blood glucose, obesity and inflammation were not associated with cognitive function.

Conclusion

Low cognitive scores are seen in middle aged individuals with hyperinsulinemia, low insulin sensitivity, beta-cell function and low aerobic capacity. These findings emphasize the importance of appropriate lifestyle and not only blood glucose control in prevention of cognitive disability.     

Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4(+) T-Cells

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8(+) T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4(+) T-cells, however recently a subset of NKG2D(+) CD4(+) T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D(+) CD4(+) T-cells possesses effector-like functions, thus resembling the subsets of NKG2D(+) CD4(+) T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4(+) T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D(+) CD4(+) T-cells, generated from HCMV-primed CD4(+) T-cells. We show that the HCMV-primed NKG2D(+) CD4(+) T-cells possess a higher differentiated phenotype than the NKG2D(-) CD4(+) T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+) T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4(+) T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4(+) T-cells, whereas it is produced de novo in resting CD4(+) T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+) CD4(+) T-cells, as well as the mechanisms regulating NKG2D cell surface expression.     

Crystal Structure of ATVORF273, a New Fold for a Thermo- and Acido-Stable Protein from the Acidianus Two-Tailed Virus

Acidianus two-tailed virus (ATV) infects crenarchaea of the genus Acidianus living in terrestrial thermal springs at extremely high temperatures and low pH. ATV is a member of the Bicaudaviridae virus family and undergoes extra-cellular development of two tails, a process that is unique in the viral world. To understand this intriguing phenomenon, we have undertaken structural studies of ATV virion proteins and here we present the crystal structure of one of these proteins, ATV. ATV forms tetramers in solution and a molecular envelope is provided for the tetramer, computed from small-angle X-ray scattering (SAXS) data. The crystal structure has properties typical of hyperthermostable proteins, including a relatively high number of salt bridges. However, the protein also exhibits flexible loops and surface pockets. Remarkably, ATV displays a new protein fold, consistent with the absence of homologues of this protein in public sequence databases.