Metadoxine, an ion-pair of pyridoxine and l-2-pyrrolidone-5-carboxylate, blocks adipocyte differentiation in association with inhibition of the PKA-CREB pathway

Adipogenesis is orchestrated by the expression of master adipogenic regulators. In particular, phosphorylation of cAMP response element binding protein (CREB) by protein kinase A promotes CREB nuclear translocation, thereby inducing expression of the adipogenic regulators and resulting in adipogenic maturation. Although metadoxine, an ion-pair of pyridoxine and l-2-pyrrolidone-5-carboxylate, has been shown to inhibit lipid accumulation in the liver, its effect on adipocyte differentiation has never been explored. This study investigated the effects of metadoxine on the differentiation of 3T3-L1 preadipocytes and the molecular mechanism. Metadoxine treatment did not inhibit mitotic clonal expansion, but inhibited late-stage cell differentiation, suggesting that metadoxine may block the differentiation step of preadipocytes. Metadoxine inhibited CREB phosphorylation and binding to the cAMP response element, thereby repressing CCAAT/enhancer-binding protein β during hormone-induced adipogenesis. Overall, metadoxine inhibits adipogenic differentiation in association with the inhibition of CREB/cAMP response element-dependent CCAAT/enhancer-binding protein β induction in the protein kinase A-CREB pathway.     

Phosphorylation of the tubulin-binding protein, stathmin, by Cdk5 and MAP kinases in the brain

Regulation of cytoskeletal dynamics is essential to neuronal plasticity during development and adulthood. Dysregulation of these mechanisms may contribute to neuropsychiatric and neurodegenerative diseases. The neuronal protein kinase, cyclin-dependent kinase 5 (Cdk5), is involved in multiple aspects of neuronal function, including regulation of cytoskeleton. A neuroproteomic search identified the tubulin-binding protein, stathmin, as a novel Cdk5 substrate. Stathmin was phosphorylated by Cdk5 in vitro at Ser25 and Ser38, previously identified as mitogen-activated protein kinase (MAPK) and p38 MAPKdelta sites. Cdk5 predominantly phosphorylated Ser38, while MAPK and p38 MAPKdelta predominantly phosphorylated Ser25. Stathmin was phosphorylated at both sites in mouse brain, with higher levels in cortex and striatum. Cdk5 knockout mice exhibited decreased phospho-Ser38 levels. During development, phospho-Ser25 and -Ser38 levels peaked at post-natal day 7, followed by reduction in total stathmin. Inhibition of protein phosphatases in striatal slices caused an increase in phospho-Ser25 and a decrease in total stathmin. Interestingly, the prefrontal cortex of schizophrenic patients had increased phospho-Ser25 levels. In contrast, total and phospho-Ser25 stoichiometries were decreased in the hippocampus of Alzheimer's patients. Thus, microtubule regulatory mechanisms involving the phosphorylation of stathmin may contribute to developmental synaptic pruning and structural plasticity, and may be involved in neuropsychiatric and neurodegenerative disorders.     

Spinach-thylakoid phosphorylation: Studies on the kinetics of changes in photosystem antenna size, spill-over and phosphorylation of light-harvesting chlorophyll a/b protein

The kinetics of LHCP phosphorylation and associated changes in photosystem cross-section and energy ‘spill-over’ from PS II to PS I have been examined in isolated spinach chloroplasts. During an initial phosphorylation period of 3–6 min, in the presence of saturating concentrations of Mg²⁺, the increase in PS I and decrease in PS II cross-section are largely completed, as judged by both measurements of the steady-state redox state of Q and fluorescence yield changes. This corresponds to a period of rapid ³²P incorporation into the low-molecular weight LHCP polypeptide. Subsequent to this initial 3–6-min period there is substantial further phosphorylation of both LHCP polypeptides, which is not accompanied by significant changes in photosystem cross-section, even after the chloroplasts had been unstacked with extensive mixing of PS I and PS II by Mg-removal. It is suggested that there exists a specific ‘mobile’ population of LHCP molecules which is rapidly phosphorylated and which may be enriched in the low-molecular-weight polypeptide. In addition, measurements of the kinetics of the ‘spill-over’ changes upon either Mg²⁺ addition or removal indicate that the continued phosphorylation of LHCP is able to increase the ‘spill-over’ process under favourable ionic conditions.     

Inhibition and promotion by light of the accumulation of translatable mRNA of the light-harvesting chlorophyll a/b-binding protein of photosystem II

The amount of in-vitro translatable mRNA of the light-harvesting chlorophyll a/b-binding protein (LHCP) of photosystem II strongly increases in darkness (D) after a 5-min red-light pulse while continuous illumination of mustard seedlings with far-red (FR), red or white light leads only to a slight increase in the amount of translatable LHCP-mRNA. No increase can be observed after a long-wavelength FR (RG9-light) pulse. However, a FR pretreatment prior to the RG9-light pulse strongly increase LHCP-mRNA accumulation in subsequent D. This is not observed in the case of the mRNA for the small subunit of ribulose-1.5-bisphosphate carboxylase. The increase of LHCP-mRNA in D after a FR pretreatment can be inhibited by a reillumination of the seedlings with FR. The inhibition of LHCP-mRNA accumulation during continuous illumination with FR and the strong increase in D following a FR illumination was found to be independent of chlorophyll biosynthesis since no correlation between chlorophyll biosynthesis and translatable LHCP-mRNA levels could be detected. Even strong changes in the amount of intermediates of chlorophyll biosynthesis caused by application of levulinic acid or 5-aminolevulinic acid did not affect LHCP-mRNA levels. Therefore, we conclude that the appearance of LHCP-mRNA is inhibited during continuous illumination, even though illumination leads to a storage of a light singal which promotes accumulation of translatable LHCP-mRNA in D.Abbreviations c continuous - Chl chlorophyll - D darkness - FR far-red light (3.5 W·m^-2) - LHCP light-harvesting chlorophyll a/b-binding protein of photosystem II - NF Norfluration - PChl protochlorophyll(ide) - Pfr far-red absorbing form of phytochrome - Ptot total phytochrome - R red light (6.8 W·m^-2) - RG9-light long-wavelength FR (10 W·m^-2) - SSU small subunit of ribulose-1.5-bisphosphate carboxylase - WL white light - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

Short-term effects of nitrate,nitrite and ammonium assimilation on photosynthesis,carbon partitioning and protein phosphorylation in maize

Maize (Zea mays L. cv. Contessa) was grown with a nitrogen supply that was just sufficient to support maximal biomass production. The third leaves from 14-to 21-d-old plants were harvested and net photosynthesis allowed to attain steady-state rates at an irradiance of either 250 or 700 mol·m^–2·s^–1. Nitrogen in the form of either KNO₃, KNO₂ or NH₄Cl was then supplied to the leaves through the transpiration stream. In all cases the addition of the nitrogen source resulted in an approximate doubling of the total amino-acid content of the leaves within 1 h. The glutamine pool increased to ten times the level found in control leaves in the light in the absence of added nitrogen. Glutamine accounted for about 21–24% of the total amino-acid content in leaves fed with 40 mM NH₄Cl. Nitrate caused a rapid, but transient inhibition of the rate of net CO₂ assimilation, accompanied by an increase in the activity of phosphoenolpyruvate carboxylase and a decrease in the maximum extractable activity of sucrose-phosphate synthase. This demonstrates that the activities of phospho-enolpyruvate carboxylase and sucrose-phosphate synthase are modulated by NO₃^–in the C₄ plant maize, in a similar manner to that observed in C₃ plants. Nitrite or ammonium feeding resulted in decreased rates of CO₂ assimilation for as long as the nitrogen source was supplied. In all cases the degree of inhibition was greatest at high irradiance and least at low irradiance, even though the total amino-acid contents of the leaves were comparable at the time when maximum inhibition of CO₂ assimilation occurred. Measurements of chlorophyll-a fluorescence showed that the quantum efficiency of PSII decreased and non-radiative dissipation of excitation energy increased as CO₂ assimilation was inhibited by nitrate or nitrite. These metabolites had no direct effect on thylakoid PSII-based electron transport. Ammonium ions weakly inhibited O₂ evolution at high concentrations. The addition of nitrogen (KNO₃^–, KNO₂ or NH₄Cl) caused a significant decrease in the phosphorylation state of the light-harvesting chlorophyll-a/b-binding protein of the thylakoid membranes. We conclude that the response of photosynthetic carbon assimilation and electron transport in maize is essentially similar whether nitrogen is supplied in the form of nitrate, nitrite or ammonium, with the noteworthy exception that the nitrogen-induced inhibition of photosynthesis is transient when leaves are supplied with NO₃^–but sustained when NO₂^–or NH₄⁺is provided. We suggest that the observed modulation of phosphoenolpyruvate carboxylase and sucrose-phosphate synthase is mediated by the increase in the endogenous level of glutamine. Furthermore, the transient nature of the inhibition of CO₂ assimilation in the case of NO₃^–, but not NO₂^–or NH₄⁺, may be due to regulation of nitrate reductase.Abbreviations and Symbol Chl chlorophyll - FB-Pase fructose-1,6-bisphosphatase - Gln glutamine - Glu glutamic acid - K_D index of the rate of thermal energy dissipation within the PSII antenna - LHCII light-harvesting chlorophyll-a/b-binding protein - PEPCase phosphoenolpyruvate carboxylase - PFD photon flux density - SPS sucrose-phosphate synthase - _PSII relative quantum efficiency for electron transport by PSIIWe wish to thank Gabriel Cornic (Structure et métabolisme des plantes, Université de Paris-Sud, Orsay, France) for useful discussion. We are grateful to Sylvie Ferrario (Laboratoire du Métabolisme, I.N.R.A., Versailles) for optimising the conditions of assay and extraction of SPS and PEPCase from maize leaves.     

PRKCB/protein kinase C,beta and the mitochondrial axis as key regulators of autophagy

Autophagy is the major intracellular system of degradation, and it plays an essential role in various biological events. Recent observations indicate that autophagy is modulated in response to the energy status of the mitochondrial compartment. However, the exact signaling mechanism that controls autophagy under these conditions remains unclear. In this study, we report that the activation of protein kinase C β (PRKCB), a member of the classical PRKCs, negatively modulates the mitochondrial energy status and inhibits autophagy. Furthermore, cells treated with a pharmacological PRKCB inhibitor, and prkcb knockout MEFs showed an increase in autophagy both in vitro and in vivo, as well as an increased mitochondrial membrane potential (Ψ_m), suggesting a strong involvement of mitochondrial energy in the modulation of the autophagy machinery. Finally, we show that factors that increase the Ψ_m oppose the PRKCB-dependent inhibition of autophagy. Altogether, these data underscore the importance of PRKCB in the regulation of autophagy; moreover, the finding that a pharmacological modulation of the Ψ_m modifies autophagy levels may be useful in fighting pathologies (including various types of cancer and neurodegenerative disorders) that are characterized by reduced levels of autophagy.     

The effects of SO2 and O3 on the foliar nutrition of Scots pine, Norway spruce and Sitka spruce in the Liphook open-air fumigation experiment

Foliar elements were analysed in Scots pine, Sitka spruce and Norway spruce over a 6 year period before and during continuous exposure to SO₂ and O₃ in an open-air fumigation experiment. Sulphur dioxide treatment elevated foliar sulphur concentration in all species, and there were increases in foliar nitrogen in the two spruce species but not in pine. The concentrations of cations were frequently increased by SO₂ treatment, but there was no correlation between the sulphur concentration of needles and their total cation charge. SO₂-related elevations of foliar magnesium were correlated with the concentration of this element in soil solution, but the mechanism by which other cations were enhanced remains unclear. The only consistent effects on nutrient ratios were for SO₂ treatments to increase sulphur/cation ratios.     

3′-Untranslated region of doublecortin mRNA is a binding target of the Musashi1 RNA-binding protein

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural stem cells, and is considered to be a stemness factor. A known function of Msi1 is translational repression of specifically bound mRNAs. Although the basic mechanism and some target RNAs have been reported, further survey of interactors is necessary to understand the integrated function of Msi1. By screening using an mRNA display technique, we found that doublecortin (dcx) mRNA is a specific binding target of Msi1 in vitro. We confirmed that Msil repressed translation of a luciferase reporter gene linked to the selected 3′-untranslated region fragment of dcx in Neuro2A cells.     

LncRNA00067110通过靶向Cabyr调控B16-F10细胞增殖、凋亡和黑色素生成

长非编码RNA(lncRNA)是一类转录长度大于200个核苷酸的非编码RNA。现已证明,多个lncRNA是潜在的癌症治疗靶点。LncRNA00067110是从小鼠黑色素瘤B16-F10细胞和正常黑色素细胞转录物组图谱中发现的差异表达基因。为研究lncRNA00067110是否调控B16-F10细胞的增殖、凋亡和黑色素生成,本文通过LncTar预测和双荧光酶活性验证了钙结合酪氨酸磷酸化调节蛋白(Cabyr) 和lncRNA00067110存在靶向关系。通过构建lncRNA00067110的过表达载体,转染B16-F10细胞,经过对B16-F10细胞的转录图谱分析,并对细胞增殖、凋亡和黑色素生成的表型以及相关基因表达变化进行了检测。结果显示,lncRNA00067110靶向Cabyr,在过表达lncRNA00067110的B16细胞中,17个基因呈差异表达。其中,Cabyr的表达被上调,细胞增殖相关基因MEK/ERK/MNK/CREB和黑色素生成相关基因TYR家族成员及CREB的mRNA和蛋白质水平显著被下调,凋亡相关基因AKT和Bcl-2的mRNA水平和蛋白质丰度被上调。进一步通过细胞增殖和凋亡的表型的变化验证了lncRNA00067110的功能。结果提示,lncRNA00067110通过靶向Cabyr,调控相关基因表达,从而抑制B16-F10细胞的增殖和黑色素生成,并诱导黑色素瘤细胞的凋亡,可能成为治疗和抑制黑色素瘤的新的靶点。     

Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein-protein cooperative interactions

The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy. The protein has a dominant tyrosyl 229-nm L(a) CD band that is shown to be approximately the sum of the five individual Tyr contributions. Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer-dimer packing interactions in crystal structures. We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy. Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures. However, there were significant differences in the binding properties of the two mutant proteins. The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly[d(A)] in 2 mM Na(+) much less than did Y41F and the wild-type gene 5 proteins. Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer-dimer interface, was absent in titrations with the Y41H mutant under low salt conditions. In contrast, titrations with the Y41H mutant in 50 mM Na(+) exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band. Thus, protein-protein and g5p-ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.     

Modulation of 1O2-mediated retrograde signaling by the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein, a central integrator of stress and energy signaling

Shortly after the release of singlet oxygen (¹O₂) in chloroplasts, changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Extensive genetic screens aimed at identifying constituents involved in ¹O₂-mediated plastid-to-nucleus signaling have failed to identify extraplastidic signaling components. This finding suggests that ¹O₂-mediated signals are not translocated to the nucleus via a single linear pathway, but rather through a signaling network that is difficult to block by single mutations. The complexity of this signaling network has been tackled by mutagenizing a transgenic flu line expressing the luciferase reporter gene under the control of the promoter of a ¹O₂-responsive AAA-ATPase gene (At3g28580) and isolating second site mutants that constitutively express the reporter gene at a high level. One of the mutants was shown by map-based cloning and sequencing to contain a single amino acid change in the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein. PRL1 suppresses the expression of AAA-ATPase and other ¹O₂-responsive genes. PRL1 seems to play a major role in modulating responses of plants to environmental changes by interconnecting ¹O₂-mediated retrograde signaling with other signaling pathways.     

强心益气方联合瑞舒伐他汀治疗急性心肌梗死的疗效及对患者血清相关指标的影响

目的:探讨强心益气方联合瑞舒伐他汀治疗急性心肌梗死的临床疗效及对患者白细胞计数(WBC)、中性粒细胞比值(NEU)、肌红蛋白及C反应蛋白(CRP)水平的影响。方法:选自2014年6月~2015年12月我院收治的急性心肌梗死患者96例,随机分为观察组与对照组,每组48例。对照组采用瑞舒伐他汀治疗,观察组在对照组基础上给予强心益气方治疗。观察并比较两组患者的治疗疗效以及治疗前后心功能指标、WBC、NEU、肌红蛋白、CRP水平变化,及用药期间不良反应情况。结果:观察组治疗总有效率(89.58%)高于对照组(70.83%)(P0.05);与治疗前比较,两组LVEF、SV治疗后明显升高,而LVEDV明显降低(P0.05);观察组LVEF、SV治疗后高于对照组,而LVEDV低于对照组(P0.05);与治疗前比较,两组WBC、NEU、肌红蛋白、CRP水平治疗后明显降低(P0.05);观察组WBC、NEU、肌红蛋白、CRP水平治疗后低于对照组(P0.05);两组均未见严重不良反应。结论:强心益气方联合瑞舒伐他汀治疗急性心肌梗死患者疗效显著,可降低WBC、NEU、肌红蛋白、CRP含量,安全可靠,值得研究。     

Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues

Constitutive promoters are the most common promoters used to drive the expression of various genes in monocots and dicots. Therefore, it is of intense interest to ascertain their expression patterns in various plant species, organs and during their ontogenic development. In this study, the activity of the CaMV 35S promoter in transgenic tobacco plants was assessed. In contrast to other studies, performed rather on the primary transformants (T₀ generation), here, individuals of T₁ and T₂ generations were used. The expression profiles of the CaMV 35S promoter were tracked within various plant organs and tissues using the GFP marker. Special attention was given to floral tissues for which the original data regarding the CaMV 35S expression were obtained. As expected, distinct developmental and organ/tissue specific expression patterns in a plant body were observed. CaMV 35S activity was detected in most of the plant tissues and during different developmental stages. The GFP signal was not visible in dry seeds only, but it became clearly apparent within 24–48 h after sowing onto the medium, what, among other things, enables the discrimination of transgenic and non-transgenic seeds/seedlings. Afterwards, the most pronounced GFP fluorescence intensity was usually visible in various vascular tissues of both, T₁ and T₂ plants, indicating the high promoter activity. A stable manifestation of the promoter was retained in the next T₂ generation without any evident changes or losses of activity, showing the expression stability of the CaMV 35S.