The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis

Mutations in genes encoding components of the heterotrimeric G-protein complex were previously shown to confer altered sensitivity to increased levels of D-glucose. This suggests that G-protein coupling may be a novel sugar-signaling mechanism in Arabidopsis thaliana. THYLAKOID FORMATION1 (THF1) is here demonstrated in vivo as a Galpha interaction partner that functions downstream of the plasma membrane-delimited heterotrimeric G-protein (GPA1) in a D-glucose signaling pathway. THF1 is a plastid protein localized to both the outer plastid membrane and the stroma. Contact between root plastidic THF1 and GPA1 at the plasma membrane occurs at sites where the plastid membrane abuts the plasma membrane, as demonstrated by F?rster resonance energy transfer (FRET). A probable role for THF1 in sugar signaling is demonstrated by both biochemical and genetic evidence. Root growth in the thf1-1 null mutant is hypersensitive to exogenous D-glucose, and THF1-overexpressing roots are resistant to inhibition of growth rate by high D-glucose. Additionally, THF1 levels are rapidly degraded by D-glucose but not L-glucose. The interaction between THF1 and GPA1 has been confirmed by in vitro and in vivo coimmunoprecipitation, FRET analysis, and genetic epistasis and provides evidence of a sugar-signaling mechanism between plastids and the plasma membrane.     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

Melatonin receptor signaling in pregnant and nonpregnant rat uterine myocytes as probed by large conductance Ca2+-activated K+ channel activity

The mRNAs of MT1 and MT2 melatonin receptors are present in cells from nonpregnant (NPM) and pregnant (PM) rat myometrium. To investigate the coupling of melatonin receptors to Gq- and Gi-type of heterotrimeric G proteins, we analyzed the activity of large-conductance Ca2+-activated K+ (BKCa) channels, the expression of which in the uterus is confined to smooth muscle cells. The melatonin receptor agonist 2-iodomelatonin induced a pertussis toxin (PTX)-insensitive increase in channel open probability that was blocked by the nonselective antagonist luzindole. The 2-iodomelatonin effect on channel open probability was suppressed by overexpression of the Gqalpha-inactivating protein RGS16 and the phospholipase C inhibitor U-73122. The activity of BKCa channels is differentially regulated by protein kinase A (PKA) in NPM and PM cells. Thus, the beta-adrenoceptor agonist isoprenaline inhibited the BKCa channel conducted whole-cell outward current (Iout) in NPM cells and enhanced Iout in PM cells. Additional application of 2-iodomelatonin antagonized the isoprenaline effect on Iout in NPM cells but enhanced Iout in PM cells. All 2-iodomelatonin effects on Iout were sensitive to PTX treatment and the PKA inhibitor H-89. We therefore conclude that melatonin activates both the PTX-insensitive Gq/phospholipase C/Ca2+ and the PTX-sensitive Gi/cAMP/PKA signaling pathway in rat myometrium.     

The autoxidation of tetrahydrobiopterin revisited. Proof of superoxide formation from reaction of tetrahydrobiopterin with molecular oxygen

It has been known for quite some time that tetrahydrobiopterin (H4B) is prone to autoxidation in the presence of molecular oxygen. Evidence has been presented that in this process superoxide radicals may be released, although their intermediacy never has been directly proven. In the present study, the autoxidation of H4B was reinvestigated with the aim to find direct evidence for superoxide formation. By means of two specific assays, namely elicitation of luminescence from lucigenin and ESR-spectrometric detection of the DEPMPO-OOH radical adduct, the release of free superoxide radicals was unequivocally demonstrated. The production of superoxide radicals was further corroborated by interaction with nitric oxide. The kinetics of the autoxidation process was established. Our data fully confirm earlier conclusions that the direct reaction between H4B and oxygen serves as an initiation reaction for the further, rapid reaction of the thus formed superoxide with H4B, thereby very likely establishing a chain reaction process involving reduction of molecular oxygen by the intermediary tetrahydrobiopterin radical. Conclusively, because H4B can per se induce oxidative stress, an in vivo overproduction of this pterin, as is evident in various diseases, may be responsible for the observed acceleration of pathophysiological pathways.     

Aggregation of the Protein TRIOBP-1 and Its Potential Relevance to Schizophrenia

We have previously proposed that specific proteins may form insoluble aggregates as a response to an illness-specific proteostatic dysbalance in a subset of brains from individuals with mental illness, as is the case for other chronic brain conditions. So far, established risk factors DISC1 and dysbindin were seen to specifically aggregate in a subset of such patients, as was a novel schizophrenia-related protein, CRMP1, identified through a condition-specific epitope discovery approach. In this process, antibodies are raised against the pooled insoluble protein fractions (aggregomes) of post mortem brain samples from schizophrenia patients, followed by epitope identification and confirmation using additional techniques. Pursuing this epitope discovery paradigm further, we reveal TRIO binding protein (TRIOBP) to be a major substrate of a monoclonal antibody with a high specificity to brain aggregomes from patients with chronic mental illness. TRIOBP is a gene previously associated with deafness which encodes for several distinct protein species, each involved in actin cytoskeletal dynamics. The 3′ splice variant TRIOBP-1 is found to be the antibody substrate and has a high aggregation propensity when over-expressed in neuroblastoma cells, while the major 5′ splice variant, TRIOBP-4, does not. Endogenous TRIOBP-1 can also spontaneously aggregate, doing so to a greater extent in cell cultures which are post-mitotic, consistent with aggregated TRIOBP-1 being able to accumulate in the differentiated neurons of the brain. Finally, upon expression in Neuroscreen-1 cells, aggregated TRIOBP-1 affects cell morphology, indicating that TRIOBP-1 aggregates may directly affect cell development, as opposed to simply being a by-product of other processes involved in major mental illness. While further experiments in clinical samples are required to clarify their relevance to chronic mental illness in the general population, TRIOBP-1 aggregates are thus implicated for the first time as a biological element of the neuropathology of a subset of chronic mental illness.     

Symptomatic relief precedes improvement of myocardial blood flow in patients under spinal cord stimulation

Background

Spinal cord electrical stimulation (SCS) has shown to be a treatment option for patients suffering from angina pectoris CCS III-IV although being on optimal medication and not suitable for conventional treatment strategies, e.g. CABG or PTCA. Although many studies demonstrated a clear symptomatic relief under SCS therapy, there are only a few short-term studies that investigated alterations in cardiac ischemia. Therefore doubts remain whether SCS has a direct effect on myocardial perfusion.

Methods

A prospective study to investigate the short- and long-term effect of spinal cord stimulation (SCS) on myocardial ischemia in patients with refractory angina pectoris and coronary multivessel disease was designed. Myocardial ischemia was measured by MIBI-SPECT scintigraphy 3 months and 12 months after the beginning of neurostimulation. To further examine the relation between cardiac perfusion and functional status of the patients we measured exercise capacity (bicycle ergometry and 6-minute walk test), symptoms and quality of life (Seattle Angina Questionnaire [SAQ]), as well.

Results

31 patients (65 ± 11 SEM years; 25 male, 6 female) were included into the study. The average consumption of short acting nitrates (SAN) decreased rapidly from 12 ± 1.6 times to 3 ± 1 times per week. The walking distance and the maximum workload increased from 143 ± 22 to 225 ± 24 meters and 68 ± 7 to 96 ± 12 watt after 3 months. Quality of life increased (SAQ) significantly after 3 month compared to baseline, as well. No further improvement was observed after one year of treament. Despite the symptomatic relief and the improvement in maximal workload computer based analysis (Emory Cardiac Toolbox) of the MIBI-SPECT studies after 3 months of treatment did not show significant alterations of myocardial ischemia compared to baseline (16 patients idem, 7 with increase and 6 with decrease of ischemia, 2 patients dropped out during initial test phase). Interestingly, in the long-term follow up after one year 16 patients (of 27 who completed the one year follow up) showed a clear decrease of myocardial ischemia and only one patient still had an increase of ischemia compared to baseline.

Conclusion

Thus, spinal cord stimulation not only relieves symptoms, but reduces myocardial ischemia as well. However, since improvement in symptoms and exercise capacity starts much earlier, decreased myocardial ischemia might not be a direct effect of neurostimulation but rather be due to a better coronary collateralisation because of an enhanced physical activity of the patients.     

Characterization of a Single-Chain Variable Fragment Recognizing a Linear Epitope of Aβ: A Biotechnical Tool for Studies on Alzheimer’s Disease?

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with devastating effects. Currently, therapeutic options are limited to symptomatic treatment. For more than a decade, research focused on immunotherapy for the causal treatment of AD. However, clinical trials with active immunization using Aβ encountered severe complications, for example meningoencephalitis. Consequently, attention focused on passive immunization using antibodies. As an alternative to large immunoglobulins (IgGs), Aβ binding single-chain variable fragments (scFvs) were used for diagnostic and therapeutic research approaches. scFvs can be expressed in E. coli and may provide improved pharmacokinetic properties like increased blood-brain barrier permeability or reduced side-effects in vivo. In this study, we constructed an scFv from an Aβ binding IgG, designated IC16, which binds the N-terminal region of Aβ (Aβ(1-8)). scFv-IC16 was expressed in E. coli, purified and characterized with respect to its interaction with different Aβ species and its influence on Aβ fibril formation. We were able to show that scFv-IC16 strongly influenced the aggregation behavior of Aβ and could be applied as an Aβ detection probe for plaque staining in the brains of transgenic AD model mice. The results indicate potential for therapy and diagnosis of AD.