Dual Role of miR-21 in CD4+ T-Cells: Activation-Induced miR-21 Supports Survival of Memory T-Cells and Regulates CCR7 Expression in Naive T-Cells

Immune cell-type specific miRNA expression patterns have been described but the detailed role of single miRNAs in the function of T-cells remains largely unknown. We investigated the role of miR-21 in the function of primary human CD4+ T-cells. MiR-21 is substantially expressed in T-cells with a memory phenotype, and is robustly upregulated upon αCD3/CD28 activation of both naive and memory T-cells. By inhibiting the endogenous miR-21 function in activated naive and memory T-cells, we showed that miR-21 regulates fundamentally different aspects of T-cell biology, depending on the differentiation status of the T-cell. Stable inhibition of miR-21 function in activated memory T-cells led to growth disadvantage and apoptosis, indicating that the survival of memory T-cells depends on miR-21 function. In contrast, stable inhibition of miR-21 function in activated naive T-cells did not result in growth disadvantage, but led to a significant induction of CCR7 protein expression. Direct interaction between CCR7 and miR-21 was confirmed in a dual luciferase reporter assay. Our data provide evidence for a dual role of miR-21 in CD4+ T cells; Regulation of T-cell survival is confined to activated memory T-cells, while modulation of potential homing properties, through downregulation of CCR7 protein expression, is observed in activated naive T-cells.     

Invader presence disrupts the stabilizing effect of species richness in plant community recovery after drought

Higher biodiversity can stabilize the productivity and functioning of grassland communities when subjected to extreme climatic events. The positive biodiversity–stability relationship emerges via increased resistance and/or recovery to these events. However, invader presence might disrupt this diversity–stability relationship by altering biotic interactions. Investigating such disruptions is important given that invasion by non‐native species and extreme climatic events are expected to increase in the future due to anthropogenic pressure. Here we present one of the first multisite invader × biodiversity × drought manipulation experiment to examine combined effects of biodiversity and invasion on drought resistance and recovery at three semi‐natural grassland sites across Europe. The stability of biomass production to an extreme drought manipulation (100% rainfall reduction; BE: 88 days, BG: 85 days, DE: 76 days) was quantified in field mesocosms with a richness gradient of 1, 3, and 6 species and three invasion treatments (no invader, Lupinus polyphyllus, Senecio inaequidens). Our results suggest that biodiversity stabilized community productivity by increasing the ability of native species to recover from extreme drought events. However, invader presence turned the positive and stabilizing effects of diversity on native species recovery into a neutral relationship. This effect was independent of the two invader's own capacity to recover from an extreme drought event. In summary, we found that invader presence may disrupt how native community interactions lead to stability of ecosystems in response to extreme climatic events. Consequently, the interaction of three global change drivers, climate extremes, diversity decline, and invasive species, may exacerbate their effects on ecosystem functioning.     

The MAPK Pathway Signals Telomerase Modulation in Response to Isothiocyanate-Induced DNA Damage of Human Liver Cancer Cells

4-methylthiobutyl isothiocyanate (MTBITC), an aliphatic, sulphuric compound from Brassica vegetables, possesses in vitro and in vivo antitumor activity. Recently we demonstrated the potent growth inhibitory potential of the DNA damaging agent MTBITC in human liver cancer cells. Here we now show that MTBITC down regulates telomerase which sensitizes cells to apoptosis induction. This is mediated by MAPK activation but independent from production of reactive oxygen species (ROS). Within one hour, MTBITC induced DNA damage in cancer cells correlating to a transient increase in hTERT mRNA expression which then turned into telomerase suppression, evident at mRNA as well as enzyme activity level. To clarify the role of MAPK for telomerase regulation, liver cancer cells were pre-treated with MAPK-specific inhibitors prior to MTBITC exposure. This clearly showed that transient elevation of hTERT mRNA expression was predominantly mediated by the MAPK family member JNK. In contrast, activated ERK1/2 and P38, but not JNK, signalled to telomerase abrogation and consequent apoptosis induction. DNA damage by MTBITC was also strongly abolished by MAPK inhibition. Oxidative stress, as analysed by DCF fluorescence assay, electron spin resonance spectroscopy and formation of 4-hydroxynonenal was found as not relevant for this process. Furthermore, N-acetylcysteine pre-treatment did not impact MTBITC-induced telomerase suppression or depolarization of the mitochondrial membrane potential as marker for apoptosis. Our data therefore imply that upon DNA damage by MTBITC, MAPK are essential for telomerase regulation and consequent growth impairment in liver tumor cells and this detail probably plays an important role in understanding the potential chemotherapeutic efficacy of ITC.     

Catechol-O-Methyltransferase Val158Met Polymorphism Is Associated with Somatosensory Amplification and Nocebo Responses

A large number of unwanted adverse events and symptoms reported by patients in clinical trials are not caused by the drug provided, since most of adverse events also occur in corresponding placebo groups. These nocebo effects also play a major role in drug discontinuation in clinical practice, negatively affecting treatment efficacy as well as patient adherence and compliance. Experimental and clinical data document a large interindividual variability in nocebo responses, however, data on psychological, biological or genetic predictors of nocebo responses are lacking. Thus, with an established paradigm of behaviorally conditioned immunosuppressive effects we analyzed possible genetic predictors for nocebo responses. We focused on the genetic polymorphisms in the catechol-O-methyltransferase (COMT) gene (Val158Met) and analyzed drug specific and general side effects before and after immunosuppressive medication and subsequent placebo intake in 62 healthy male subjects. Significantly more drug-specific as well as general side effects were reported from homozygous carriers of the Val158 variant during medication as well as placebo treatment compared to the other genotype groups. Val158/Val158 carriers also had significantly higher scores in the somatosensory amplification scale (SSAS) and the BMQ (beliefs about medicine questionnaire). Together these data demonstrate potential genetic and psychological variables predicting nocebo responses after drug and placebo intake, which might be utilized to minimize nocebo effects in clinical trials and medical practice.     

Handedness in a nonindustrial society challenges the fighting hypothesis as an evolutionary explanation for left-handedness

Handedness is a heritable trait, and left-handedness is related with increased fitness costs. Left-handedness persists, however, as a minority in every human population investigated. One explanation for this persistence has been put forward in the fighting hypothesis, which postulates that left-handers have a frequency-dependent benefit in fights. Support for this has been found in the finding that left-handedness is relatively frequent in populations with high homicide rates, according to estimates of left-handedness partly based on pictures and films made for a different purpose. We measured handedness based on hand preference in 10 ecologically relevant tasks in 621 subjects in the nonindustrial society of the Eipo (Papua, Indonesia) in which homicide rate was very high. This set of tests was validated in 198 Western students. Contrary to the prediction based on the fighting hypothesis, we did not find a high frequency of left-handedness or a difference between men (who participate in warfare) and women (who do not). These findings challenge the idea that fighting is the driving evolutionary force for the persistence of left-handedness in human populations. Furthermore, we found lower percentages of left- and mixed-handers compared to a Western population who executed the same tasks. Since left-handedness is associated with health problems, we suggest that in a society lacking Western health care, selection pressures against left-handedness may be more intense and therefore its frequency may be reduced.     

Late Signals from the PDGF Receptors Leading to the Activation of the p70-Kinase Are Necessary for the Transition from G1 to S phase in AKR-2B Cells

Platelet-derived growth factor AB (PDGF-AB) has to be permanently present in the culture medium to achieve full proliferation (>90%) of AKR-2B fibroblasts. Upon removal after 1 h incubation time, only a small number of cells (<20%) entered the cell cycle. Concomitantly there was no increase in RNA- and protein-synthesis. The PDGF-receptor autophosphorylation reached a maximum after 30 min incubation with PDGF-AB. Tyrosine phosphorylation was no longer detectable after 2–4 h. The clustering of receptors into coated pits, analyzed by indirect immunofluorescence using a specific antibody against PDGF-β-receptor, showed in contrast to autophosphorylation a biphasic kinetic. A first maximum was reached after 30 min, followed by a complete disappearance of coated pits, which regenerated in a second phase after 3 h and were long lasting. If PDGF-AB was removed after 1 h, the second phase was obliterated.The involvement of two different signalling pathways in these two phases was investigated in detail: (1) The ras-raf-MAP-kinase pathway and (2) the PI-3-kinase/p70^S6-kinase pathway. PDGF-AB addition caused a fast (10 min) activation of MAP-kinase, which returned to background level after 1 h without any further activation later on. In contrast PDGF-AB led to a rapid (15–30 min) activation of the p70^S6-kinase that persisted for 8–12 h just prior to the entry of the cells into S-phase. If PDGF-AB was removed after 1 h, the activation of this kinase ceased 3 h later. PDGF-AA, which is unable to promote division of AKR-2B cells, induced only a shortlasting p70^S6-kinase activation. These observations add further evidence for the involvement of the p70^S6-kinase pathway in the proliferation control of AKR-2B fibroblasts in the late G1 phase (4–8 h after growth factor addition). On the other hand, if the p70^S6-kinase activation was prevented by the addition of 10 nM rapamycin, the cell division was not inhibited but only delayed by 4 h. Similar kinetics were observed when the PI-3-kinase was inhibited by 400 nM wortmannin. It is suggested that a regulatory element exists upstream of the p70^S6-kinase and the PI-3-kinase. This regulatory element should be responsible for the transmission of late signals required for the progression through the cell cycle. This element is not involved in the immediate responses after PDGF-AB addition but must be stimulated within a second later phase of PDGF activation.     

Determinants of substrate specificity and biochemical properties of the sn‐glycerol‐3‐phosphate ATP binding cassette transporter (UgpB–AEC2) of Escherichia coli

Under phosphate starvation conditions, Escherichia coli can utilize sn‐glycerol‐3‐phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68 ± 0.02 μM and 5.1 ± 0.3 μM, respectively, while glycerol‐2‐phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8 Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp‐169 for G3P binding. The purified UgpAEC₂ complex displayed UgpB/G3P‐stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG–UgpC could be functionally reconstituted while a UgpAE–MalK complex was unstable.