Tracing changes in protonation: a prerequisite to factorize thermodynamic data of inhibitor binding to aldose reductase

To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. Here, we study the binding process of inhibitors to aldose reductase (ALR2) with respect to changes of the protonation inventory upon complex formation. Knowledge of such processes is a prerequisite to factorize the binding free energy into enthalpic and entropic contributions on an absolute scale. Our isothermal titration calorimetry (ITC) measurements suggest a proton uptake upon complex formation with carboxylate-type inhibitors. As the protonation event will contribute strongly to the enthalpic signal recorded during ITC experiments, knowledge about the proton-accepting and releasing functional groups of the system is of utmost importance. However, this is intricate to retrieve, if, as in the present case, both, binding site and ligand possess several titratable groups. Here, we present pKa calculations complemented by mutagenesis and thermodynamic measurements suggesting a tyrosine residue located in the catalytic site (Tyr48) as a likely candidate to act as proton acceptor upon inhibitor binding, as it occurs deprotonated to a remarkable extent if only the cofactor NADP+ is bound. We furthermore provide evidence that the protonation state and binding thermodynamics depend strongly on the oxidation state of the cofactor;s nicotinamide moiety. Binding thermodynamics of IDD 388, IDD 393, tolrestat, sorbinil, and fidarestat are discussed in the context of substituent effects.     

Non-regulatory CD8+CD45RO+CD25+ T-lymphocytes may compensate for the loss of antigen-inexperienced CD8+CD45RA+ T-cells in old age

The age-related decline in immune system functions is responsible for the increased prevalence of infectious diseases and the low efficacy of vaccination in elderly individuals. In particular, the number of peripheral naive T-cells declines throughout life and they exhibit severe functional defects at advanced age. However, we have recently identified a non-regulatory CD8+CD45RO+ CD25+ T-cell subset that occurs in a subgroup of healthy elderly individuals, who still exhibit an intact humoral immune response following influenza vaccination. Here, we demonstrate that CD8+CD45RO+CD25+ T-cells share phenotypic and functional characteristics with naive CD8+CD45RA+CD28+ T-cells from young individuals, despite their expression of CD45RO. CD8+CD45RO+ CD25+ T-cells also have long telomeres and upon antigenic challenge, they efficiently expand in vitro and differentiate into functional effector cells. The expanded population also maintains a diverse T-cell receptor repertoire. In conclusion, CD8+CD45RO+CD25+ T-cells from elderly individuals compensate for the loss of functional naive T-cells and may therefore be used as a marker of immunological competence in old age.     

Ammonia-oxidizing Crenarchaeota and nitrification inside the tissue of a colonial ascidian

Marine Crenarchaeota represent an abundant component of the oceanic microbiota that play an important role in the global nitrogen cycle. Here we report the association of the colonial ascidian Cystodytes dellechiajei with putative ammonia-oxidizing Crenarchaeota that could actively be involved in nitrification inside the animal tissue. As shown by 16S rRNA gene analysis, the ascidian-associated Crenarchaeota were phylogenetically related to Nitrosopumilus maritimus, the first marine archaeon isolated in pure culture that grows chemolithoautotrophically oxidizing ammonia to nitrite aerobically. Catalysed reporter deposition (CARD)-FISH revealed that the Crenarchaeota were specifically located inside the tunic tissue of the colony, where moreover the expression of amoA gene was detected. The amoA gene encodes the alpha-subunit of ammonia monooxygenase, which is involved in the first step of nitrification, the oxidation of ammonia to nitrite. Sequencing of amoA gene showed that they were phylogenetically related to amoA genes of N. maritimus and other putative ammonia-oxidizing marine Crenarchaeota. In order to track the suspected nitrification activity inside the ascidian colony under in vivo conditions, microsensor profiles were measured through the tunic tissue. Net NO(x) production was detected in the tunic layer 1200-1800 microm with rates of 58-90 nmol cm(-3) h(-1). Oxygen and pH microsensor profiles showed that the layer of net NO(x) production coincided with O(2) concentrations of 103-116 microM and pH value of 5.2. Together, molecular and microsensor data indicate that Crenarchaeota could oxidize ammonia to nitrite aerobically, and thus be involved in nitrification inside the ascidian tissue.     

Uric Acid Accumulation in an Arabidopsis Urate Oxidase Mutant Impairs Seedling Establishment by Blocking Peroxisome Maintenance

Purine nucleotides can be fully catabolized by plants to recycle nutrients. We have isolated a urate oxidase (uox) mutant of Arabidopsis thaliana that accumulates uric acid in all tissues, especially in the developing embryo. The mutant displays a reduced germination rate and is unable to establish autotrophic growth due to severe inhibition of cotyledon development and nutrient mobilization from the lipid reserves in the cotyledons. The uox mutant phenotype is suppressed in a xanthine dehydrogenase (xdh) uox double mutant, demonstrating that the underlying cause is not the defective purine base catabolism, or the lack of UOX per se, but the elevated uric acid concentration in the embryo. Remarkably, xanthine accumulates to similar levels in the xdh mutant without toxicity. This is paralleled in humans, where hyperuricemia is associated with many diseases whereas xanthinuria is asymptomatic. Searching for the molecular cause of uric acid toxicity, we discovered a local defect of peroxisomes (glyoxysomes) mostly confined to the cotyledons of the mature embryos, which resulted in the accumulation of free fatty acids in dry seeds. The peroxisomal defect explains the developmental phenotypes of the uox mutant, drawing a novel link between uric acid and peroxisome function, which may be relevant beyond plants.     

Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development

Two distinct thioredoxin/thioredoxin reductase systems are present in the cytosol and the mitochondria of mammalian cells. Thioredoxins (Txn), the main substrates of thioredoxin reductases (Txnrd), are involved in numerous physiological processes, including cell-cell communication, redox metabolism, proliferation, and apoptosis. To investigate the individual contribution of mitochondrial (Txnrd2) and cytoplasmic (Txnrd1) thioredoxin reductases in vivo, we generated a mouse strain with a conditionally targeted deletion of Txnrd1. We show here that the ubiquitous Cre-mediated inactivation of Txnrd1 leads to early embryonic lethality. Homozygous mutant embryos display severe growth retardation and fail to turn. In accordance with the observed growth impairment in vivo, Txnrd1-deficient embryonic fibroblasts do not proliferate in vitro. In contrast, ex vivo-cultured embryonic Txnrd1-deficient cardiomyocytes are not affected, and mice with a heart-specific inactivation of Txnrd1 develop normally and appear healthy. Our results indicate that Txnrd1 plays an essential role during embryogenesis in most developing tissues except the heart.     

Helicobacter pylori-induced downregulation of the secretory leukocyte protease inhibitor (SLPI) in gastric epithelial cell lines and its functional relevance for H. pylori-mediated diseases

The secretory leukocyte protease inhibitor (SLPI) exerts antiproteolytic activity towards serine proteases, as well as anti-microbial and anti-inflammatory effects. To investigate its role in H. pylori-mediated diseases, SLPI expression was analyzed by RT-PCR, ELISA and immunohistochemistry in clinical samples and gastric tumor cell lines. Determination of the mucosal SLPI levels in 126 patients confirmed the previously reported downregulation of SLPI in H. pylori-infected patients. The lower SLPI levels in antral biopsies of H. pylori-positive subjects were associated with a 30-fold increase (p<0.01) in neutrophil elastase activity, and a significant negative correlation was demonstrated for both parameters (R=-0.63, p=0.0002). Eradication of the bacterium in a long-term study (5-7 years) led to a recovery of mucosal SLPI expression. In vitro experiments using four gastric tumor cell lines (AGS, MKN-28, MKN-45, NCI-N87) generally confirmed the clinical findings. While the co-incubation of these cell lines with H. pylori resulted in lower or unchanged SLPI protein levels, the corresponding SLPI mRNA amounts were upregulated by up to five-fold (p=0.006) in all cell lines. Taken together, these results indicate that the reduction in antral SLPI levels in H. pylori-infected subjects has a functional relevance for gastric mucosa and the H. pylori-induced decrease in SLPI is primarily regulated at the posttranslational level.     

Internet-Based Brief Intervention to Prevent Unhealthy Alcohol Use among Young Men: A Randomized Controlled Trial

Introduction

Alcohol use is one of the leading modifiable morbidity and mortality risk factors among young adults.

Study Design

2 parallel-group randomized controlled trial with follow-up at 1 and 6 months.

Setting/Participants

Internet based study in a general population sample of young men with low-risk drinking, recruited between June 2012 and February 2013.Intervention: Internet-based brief alcohol primary prevention intervention (IBI). The IBI aims at preventing an increase in alcohol use: it consists of normative feedback, feedback on consequences, calorific value alcohol, computed blood alcohol concentration, indication that the reported alcohol use is associated with no or limited risks for health. Intervention group participants received the IBI. Control group (CG) participants completed only an assessment.

Main Outcome Measures

Alcohol use (number of drinks per week), binge drinking prevalence. Analyses were conducted in 2014–2015.

Results

Of 4365 men invited to participate, 1633 did so; 896 reported low-risk drinking and were randomized (IBI: n = 451; CG: n = 445). At baseline, 1 and 6 months, the mean (SD) number of drinks/week was 2.4(2.2), 2.3(2.6), 2.5(3.0) for IBI, and 2.4(2.3), 2.8(3.7), 2.7(3.9) for CG. Binge drinking, absent at baseline, was reported by 14.4% (IBI) and 19.0% (CG) at 1 month and by 13.3% (IBI) and 13.0% (CG) at 6 months. At 1 month, beneficial intervention effects were observed on the number of drinks/week (p = 0.05). No significant differences were observed at 6 months.

Conclusion

We found protective short term effects of a primary prevention IBI.

Trial Registration

Controlled-Trials.com ISRCTN55991918     

Mikrobiologische Untersuchungen über den Stickstoffhaushalt der Elbe

Ohne Zusammenfassung