Conformation-dependent stability of junctophilin 1 (JP1) and ryanodine receptor type 1 (RyR1) channel complex is mediated by their hyper-reactive thiols

Junctophilin 1 (JP1), a 72-kDa protein localized at the skeletal muscle triad, is essential for stabilizing the close apposition of T-tubule and sarcoplasmic reticulum membranes to form junctions. In this study we report that rapid and selective labeling of hyper-reactive thiols found in both JP1 and ryanodine receptor type 1 (RyR1) with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, a fluorescent thiol-reactive probe, proceeded 12-fold faster under conditions that minimize RyR1 gating (e.g. 10 mM Mg2+) compared with conditions that promote high channel activity (e.g. 100 microM Ca2+, 10 mM caffeine, 5 mM ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H2O2, NO, or O2, all known modulators of the RyR1 channel complex. Using preparative SDS-PAGE, in-gel tryptic digestion, high pressure liquid chromatography, and mass spectrometry-based peptide sequencing, we identified 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin-thioether adducts on three cysteine residues of JP1 (101, 402, and 627); the remaining five cysteines of JP1 were unlabeled. Co-immunoprecipitation experiments demonstrated a physical interaction between JP1 and RyR1 that, like thiol reactivity, was sensitive to RyR1 conformation and chemical status of the hyper-reactive cysteines of JP1 and RyR1. These findings support a model in which JP1 interacts with the RyR1 channel complex in a conformationally sensitive manner and may contribute integral redox-sensing properties through reactive sulfhydryl chemistry.     

Gene regulation by homeobox transcription factor Prox1 in murine hepatoblasts

Protein kinase C (PKC) is a signalling enzyme critically involved in many aspects of synaptic plasticity. In cyprinid retinae, the PKC alpha isoform is localized in a subpopulation of depolarizing bipolar cells that show adaptation-related morphological changes of their axon terminals. We have studied the subcellular localization of phosphorylated PKC alpha (pPKC alpha) in retinae under various conditions by immunohistochemistry with a phosphospecific antibody. In dark-adapted retinae, pPKC alpha immunoreactivity is weak in the cytoplasm of synaptic terminals, labelling being predominantly associated with the membrane compartment. In light-adapted cells, immunoreactivity is diffusely distributed throughout the terminal. Western blot analysis has revealed a reduction of pPKC alpha immunoreactivity in cytosolic fractions of homogenized dark-adapted retinae compared with light-adapted retinae. Pharmacological experiments with the isoform-specific PKC blocker Goe6976 have shown that inhibition of the enzyme influences immunolabelling for pPKC alpha, mimicking the effects of light on the subcellular distribution of immunoreactivity. Our findings suggest that the state of adaptation modifies the subcellular localization of a signalling molecule (PKC alpha) at the ribbon-type synaptic complex. We propose that changes in the subcellular distribution of PKC alpha immunoreactivity might be one component regulating the strength of the signal transfer of the bipolar cell terminal. Uwe D. Behrens and Johannes Borde contributed equally to this work. This research was supported by an SFB-430 grant.     

mTORC1 Is Essential for Early Steps during Schwann Cell Differentiation of Amniotic Fluid Stem Cells and Regulates Lipogenic Gene Expression

Schwann cell development is hallmarked by the induction of a lipogenic profile. Here we used amniotic fluid stem (AFS) cells and focused on the mechanisms occurring during early steps of differentiation along the Schwann cell lineage. Therefore, we initiated Schwann cell differentiation in AFS cells and monitored as well as modulated the activity of the mechanistic target of rapamycin (mTOR) pathway, the major regulator of anabolic processes. Our results show that mTOR complex 1 (mTORC1) activity is essential for glial marker expression and expression of Sterol Regulatory Element-Binding Protein (SREBP) target genes. Moreover, SREBP target gene activation by statin treatment promoted lipogenic gene expression, induced mTORC1 activation and stimulated Schwann cell differentiation. To investigate mTORC1 downstream signaling we expressed a mutant S6K1, which subsequently induced the expression of the Schwann cell marker S100b, but did not affect lipogenic gene expression. This suggests that S6K1 dependent and independent pathways downstream of mTORC1 drive AFS cells to early Schwann cell differentiation and lipogenic gene expression. In conclusion our results propose that future strategies for peripheral nervous system regeneration will depend on ways to efficiently induce the mTORC1 pathway.     

Hormonal and Neuromuscular Responses to Mechanical Vibration Applied to Upper Extremity Muscles

Objective

To investigate the acute residual hormonal and neuromuscular responses exhibited following a single session of mechanical vibration applied to the upper extremities among different acceleration loads.

Methods

Thirty male students were randomly assigned to a high vibration group (HVG), a low vibration group (LVG), or a control group (CG). A randomized double-blind, controlled-parallel study design was employed. The measurements and interventions were performed at the Laboratory of Biomechanics of the University of L''Aquila. The HVG and LVG participants were exposed to a series of 20 trials ×10 s of synchronous whole-body vibration (WBV) with a 10-s pause between each trial and a 4-min pause after the first 10 trials. The CG participants assumed an isometric push-up position without WBV. The outcome measures were growth hormone (GH), testosterone, maximal voluntary isometric contraction during bench-press, maximal voluntary isometric contraction during handgrip, and electromyography root-mean-square (EMG_rms) muscle activity (pectoralis major [PM], triceps brachii [TB], anterior deltoid [DE], and flexor carpi radialis [FCR]).

Results

The GH increased significantly over time only in the HVG (P = 0.003). Additionally, the testosterone levels changed significantly over time in the LVG (P = 0.011) and the HVG (P = 0.001). MVC during bench press decreased significantly in the LVG (P = 0.001) and the HVG (P = 0.002). In the HVG, the EMG_rms decreased significantly in the TB (P = 0.006) muscle. In the LVG, the EMG_rms decreased significantly in the DE (P = 0.009) and FCR (P = 0.006) muscles.

Conclusion

Synchronous WBV acutely increased GH and testosterone serum concentrations and decreased the MVC and their respective maximal EMG_rms activities, which indicated a possible central fatigue effect. Interestingly, only the GH response was dependent on the acceleration with respect to the subjects'' responsiveness.     

Response to "The anglerfish deception"

The correspondents argue that “The anglerfish deception” contains omissions, errors, misunderstandings and misinterpretations.EMBO reports (2012) advanced online publication; doi: 10.1038/embor.2012.71EMBO reports (2012) 13 2, 100–105; doi: 10.1038/embor.2011.254The commentary [1] on aspects of genetically modified organism (GMO) regulation, risk assessment and risk management in the EU contains omissions, errors, misunderstandings and misinterpretations. As background, environmental risk assessment (ERA) of genetically modified (GM) plants for cultivation in the EU is conducted by applicants following principles and data requirements described in the Guidance Document (ERA GD) established by the European Food Safety Authority (EFSA) [2], which follows the tenets of Directive 2001/18/EC. The ERA GD was not referenced in [1], which wrongly referred only to EFSA guidance that does not cover ERA. Applications for cultivation of a GM plant containing the ERA, submitted to the European Commission (EC), are checked by the EFSA to ensure they address all the requirements specified in its ERA GD [2]. A lead Member State (MS) is then appointed to conduct the initial evaluation of the application, requesting further information from the applicant if required. The MS evaluation is forwarded to the EC, EFSA and all other MSs. Meanwhile, all other MSs can comment on the application and raise concerns. The EFSA GMO Panel carefully considers the content of the application, the lead MS Opinion, other MSs'' concerns, all relevant data published in the scientific literature, and the applicant''s responses to its own requests for further information. The Panel then delivers its Opinion on the application, which covers all the potential environmental areas of risk listed in 2001/18/EC. This Opinion is sent to the EC, all MSs and the applicant and published in the EFSA journal (efsa.europa.eu). Panel Opinions on GM plants for cultivation consider whether environmental harm might be caused, and, if so, suggest possible management to mitigate these risks, and make recommendations for post-market environmental monitoring (PMEM). The final decision on whether to allow the cultivation of GM plants, and any specific conditions for management and monitoring, rests with the EC and MSs and is not within the remit of the EFSA.Against this background we respond to several comments in [1]. Regarding the Comparative Safety Assessment of GM plants and whether or not further questions are asked following this assessment, the Comparative Safety Assessment, described fully in [2], is not a ‘first step''. It is a general principle that forms a central part of the ERA process, as introduced in section 2.1 of [2]. Each ERA starts with problem formulation and identification, facilitating a structured approach to identifying potential risks and scientific uncertainties; following this critical first step many further questions must be asked and addressed. In [2] it is clearly stated that all nine specific areas of risk listed in 2001/18/EC must be addressed—persistence and invasiveness; vertical gene flow; horizontal gene flow; interactions with target organisms; interactions with non-target organisms; human health; animal health; biogeochemical processes; cultivation, management and harvesting techniques. Under the Comparative Safety Assessment, following problem formulation, each of these areas of risk must be assessed by using a six-step approach, involving hazard identification, hazard characterization, exposure assessment, risk characterization, risk management strategies and an overall risk evaluation and conclusion. Indeed, far from asking “no further questions” [1], the EFSA GMO Panel always sends a sequence of written questions to the applicant as part of the ERA process to achieve a complete set of data to support the ERA evaluation (on average about ten per application).The principle of comparative analysis in ERA—sometimes referred to as substantial equivalence in the risk assessment of food and feed—is not discredited. The comparative approach is supported by all of the world''s leading national science academies [for example, 3]; none has recommended an alternative. The principle is enshrined in risk assessment guidelines issued by all relevant major international bodies, including the World Health Organization, the Food and Agriculture Organization of the United Nations and the Organisation for Economic Co-operation and Development. Critics of this approach have failed to propose any credible alternative baseline to risk assess GMOs. The comparative analysis as described in [2] is not a substitute for a safety assessment, but is a tool within the ERA [4] through which comparisons are made with non-GM counterparts in order to identify hazards associated with the GM trait, the transformation process and the associated management systems, which are additional to those impacts associated with the non-GM plant itself. The severity and frequency of these hazards are then quantified in order to assess the levels of risks associated with the novel features of the GM plant and its cultivation.European Parliament (EP) communications include that “the characteristics of the receiving environments and the geographical areas in which GM plants may be cultivated should be duly taken into account”. We agree, and the ERA GD [2] recognizes explicitly that receiving environments differ across the EU, and that environmental impacts might differ regionally. Therefore, the ERA GD [2] demands that such differences be fully accounted for in cultivation applications and that receiving environments be assessed separately in each of the nine specific areas of risk (see section 2.3.2). Furthermore, [2] states in section 3.5 that the ERA should consider scenarios representative of the diversity of situations that might occur and assess their potential implications. The EP communications state that “the long-term environmental effects of GM crops, as well as their potential effects on non-target organisms, should be rigorously assessed”. This is covered explicitly in section 2.3.4 of [2], and developed in the recent guidance on PMEM [5].The EFSA is committed to openness, transparency and dialogue and meets regularly with a wide variety of stakeholders including non-governmental organizations (NGOs) [6] to discuss GMO topics. That the EFSA is neither a centralized nor a singular voice of science in the EU is clear, because the initial report on the ERA is delivered by a MS, not the EFSA; all MSs can comment on the ERA; and EFSA GMO Panel Opinions respond transparently to every concern raised by each MS. Following publication, the EFSA regularly attends the SCFCAH Committee (comprising MS representatives) to account for its Opinions. The involvement of all MSs in the evaluation process ensures that concerns relating to their environments are addressed in the ERA. Subsequently, MSs can contribute to decisions on the management and monitoring of GM plants in their territories if cultivation is approved.In recent years, several MSs have used the ‘safeguard clause'', Article 23 of 2001/18/EC, to attempt to ban the cultivation of specific GM plants in their territories, despite earlier EFSA Panel Opinions on those plants. But the claim that “the risk science of the EFSA''s GM Panel has been publicly disputed in Member State''s justifications of their Article 23 prohibitions” needs to be placed into context [1]. When a safeguard clause (SC) is issued by a MS, the EFSA GMO Panel is often asked by the EC to deliver an Opinion on the scientific basis of the SC. The criteria on which to judge the documentation accompanying a SC are whether: (i) it represents new scientific evidence—and is not just repetition of information previously assessed—that demonstrates a risk to human and animal health and the environment; and (from the guidance notes to Annex II of 2001/18/EC) (ii) it is proportionate to the level of risk and to the level of uncertainty. It is pertinent that on 8 September 2011, the EU Court of Justice ruled that ‘with a view to the adoption of emergency measures, Article 34 of Regulation (EC) No 1829/2003 requires Member States to establish, in addition to urgency, the existence of a situation which is likely to constitute a clear and serious risk to human health, animal health or the environment''. Scientific literature is monitored continually by the Panel and relevant new work is examined to determine whether it raises any new safety concern. In all cases where the EFSA was consulted by the EC, there has been no new scientific information presented that would invalidate the Panel''s previous assessment.Throughout [1] the text demonstrates a fundamental misunderstanding of the distinction between ERA and risk management. ERA is the responsibility of the EFSA, although it is asked for its opinion on risk management methodology by the EC. Risk management implementation is the responsibility of the EC and MSs. Hence, the setting of protection goals is an issue for risk managers and might vary between MSs. However, the ERA GD [2], through its six-step approach, makes it mandatory for applications to relate the results of any studies directly to limits of environmental concern that reflect protection goals and the level of change deemed acceptable. Indeed, the recent EFSA GMO Panel Opinions on Bt-maize events [for example, 7] have been written specifically to provide MSs and risk managers with the tools to adapt the results of the quantified ERA to their own local protection goals. This enables MSs to implement risk management and PMEM proportional to the risks identified in their territories.The EFSA GMO Panel comprises independent researchers, appointed for their expertise following an open call to the scientific community. The Panel receives able support from staff of the EFSA GMO Unit and numerous ad hoc members of its working groups. It has no agenda and is neither pro- or anti-GMOs; its paramount concern is the quality of the science underpinning its Guidance Documents and Opinions.     

Protein kinase C modulates synaptic vesicle acidification in a ribbon type nerve terminal in the retina

The driving force for neurotransmitter accumulation into synaptic vesicles is provided by the generation of a transmembrane electrochemical gradient (DeltamicroH+) that has two components: a chemical gradient (DeltapH, inside acidic) and an electrical potential across the vesicular membrane (DeltaPsi, inside positive). This gradient is generated in situ by the electrogenic vacuolar H(+)-ATPase, which is responsible for the acidification and positive membrane potential of the vesicle lumen. Here, we investigate the modulation of vesicle acidification by using the acidic-organelle probe LysoTracker and the pH-sensitive probe LysoSensor at goldfish Mb-type bipolar cell terminals. Since phosphorylation can modulate secretory granule acidification in neuroendocrine cells, we investigated if drugs that affect protein kinases modulate LysoTracker staining of bipolar cell terminals. We find that protein kinase C (PKC) activation induces an increase in LysoTracker-fluorescence. By contrast, protein kinase A (PKA) or calcium/calmodulin kinase II (CaMKII) activation or inhibition did not change LysoTracker-fluorescence. Using a pH-dependent fluorescent dye (LysoSensor) we show that the PKC activation with PMA induces an increase in LysoSensor-fluorescence, whereas the inactive analog 4alpha-PMA was unable to cause the same effect. This increase induced by PMA was blocked by PKC inhibitors, calphostin C and staurosporine. These results suggest that phosphorylation by PKC may increase synaptic vesicle acidification in retinal bipolar cells and therefore has the potential to modulate glutamate concentrations inside synaptic vesicles.     

Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition

The molecular basis for the substrate specificity of human caspase-3 has been investigated using peptide analog inhibitors and substrates that vary at the P2, P3, and P5 positions. Crystal structures were determined of caspase-3 complexes with the substrate analogs at resolutions of 1.7 A to 2.3 A. Differences in the interactions of caspase-3 with the analogs are consistent with the Ki values of 1.3 nM, 6.5 nM, and 12.4 nM for Ac-DEVD-Cho, Ac-VDVAD-Cho and Ac-DMQD-Cho, respectively, and relative kcat/Km values of 100%, 37% and 17% for the corresponding peptide substrates. The bound peptide analogs show very similar interactions for the main-chain atoms and the conserved P1 Asp and P4 Asp, while interactions vary for P2 and P3. P2 lies in a hydrophobic S2 groove, consistent with the weaker inhibition of Ac-DMQD-Cho with polar P2 Gln. S3 is a surface hydrophilic site with favorable polar interactions with P3 Glu in Ac-DEVD-Cho. Ac-DMQD-Cho and Ac-VDVAD-Cho have hydrophobic P3 residues that are not optimal in the polar S3 site, consistent with their weaker inhibition. A hydrophobic S5 site was identified for caspase-3, where the side-chains of Phe250 and Phe252 interact with P5 Val of Ac-VDVAD-Cho, and enclose the substrate-binding site by conformational change. The kinetic importance of hydrophobic P5 residues was confirmed by more efficient hydrolysis of caspase-3 substrates Ac-VDVAD-pNA and Ac-LDVAD-pNA compared with Ac-DVAD-pNA. In contrast, caspase-7 showed less efficient hydrolysis of the substrates with P5 Val or Leu compared with Ac-DVAD-pNA. Caspase-3 and caspase-2 share similar hydrophobic S5 sites, while caspases 1, 7, 8 and 9 do not have structurally equivalent hydrophobic residues; these caspases are likely to differ in their selectivity for the P5 position of substrates. The distinct selectivity for P5 will help define the particular substrates and signaling pathways associated with each caspase.     

Effects of tocopherols and 2,2'-carboxyethyl hydroxychromans on phorbol-ester-stimulated neutrophils

Tocopherol vitamers [e.g., alpha-, gamma- and delta-tocopherol (-TOC, γ-TOC and δ-TOC, respectively)] and their water-soluble 2,2′-carboxyethyl hydroxychroman metabolites (e.g., -, γ- and δ-CEHC) all possess antioxidant properties. As a consequence, and similarly to other natural antioxidants, vitamin E compounds may be useful in preventing inflammatory and oxidative-stress-mediated diseases. In this study, we investigated the concentration-dependent effect of tocopherols and water-soluble metabolites on a key event in oxidative stress, for example, the oxidative burst in neutrophils. It was found that not only -TOC but also γ-TOC and δ-TOC as well as -, γ- and δ-CEHC at physiological concentrations inhibit superoxide anion (O₂^•−) production in phorbol-ester-stimulated neutrophils. This effect was mediated by the inhibition of the translocation and activation of protein kinase C (PKC) enzyme, which is the key event in the phorbol-ester signaling. Importantly, CEHCs were stronger inhibitors of PKC as compared with the vitamer precursors, and the gamma forms of both tocopherol and CEHC showed the highest inhibitory activities. Tocopherols, but not CEHCs, directly inhibit the fully activated nicotine–adenine–dinucleotide phosphate (NADPH) oxidase. However, none of the test compounds was able to directly scavenge O₂^•− when tested in a cell-free system. In conclusion, vitamin E compounds can control the neutrophil oxidative burst through the negative modulation of PKC-related signaling and NADPH oxidase activity. As an original finding, we observed that CEHC metabolites might contribute to regulate PKC activity in these cells. These results may have important implications in the anti-inflammatory and antioxidant role of vitamin E compounds.     

Antibacterial colorants: characterization of prodiginines and their applications on textile materials

A strain of Vibrio sp. isolated from marine sediments produced large quantities of bright red pigments that could be used to dye many fibers including wool, nylon, acrylics, and silk. Characterization of the pigments by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) revealed three prodiginine-like structures with nonpolar characteristics and low molecular mass. UV-visible spectra of the major constituent in methanol solution showed absorbance at lambda max 530 nm wavelength. The accurate mass result showed that the main isolated product has a molecular mass of m/z 323.1997. Further analysis using mass fragmentation (MS/MS), 1H NMR, COSY, HMQC NMR and DEPT confirmed the detailed structure of the pigment with an elementary composition of C20H25N3O. Fabrics dyed with the microbial prodiginines demonstrated antibacterial activity.