EGCG treatment prevents the cardiac hypertrophy observed in CD mice.

<p>(a) Comparison of the heart/body weight ratios obtained from each genotype showed that cardiac hypertrophy present in CD mice is not observed after EGCG treatment. A two-way ANOVA indicated a significant interaction between genotypes and treatment (F_1,46 = 7.237, <i>p</i> = 0.0099), with a significant effect of genotype (F_1,46 = 6.751, <i>p =</i> 0.0125) but with treatment effect only in CD mice (<i>p</i><0.01, Bonferroni <i>post hoc</i> test). n = 10–15 mice per group. (b) Representative images of hematoxylin-eosin stained transverse sections of heart samples. We can appreciate the thickening of the left ventricular wall of the control CD animals. (c) CD mice cardiomyocytes are significantly larger than in WT animals, while CD animals treated with EGCG show normal cell size. A two-way ANOVA indicated a significant interaction between genotypes and treatment (F_1,12 = 24.05, <i>p</i> = 0.0004), with a significant effect of genotype (F_1,12 = 14.15, <i>p</i> = 0.0027) and treatment effect in CDs (<i>p</i><0.01, Bonferroni <i>post hoc</i> test) but also in WTs (<i>p</i><0.05, Bonferroni <i>post hoc</i> test). n = 4 mice/group. <i>p</i> values are shown with asterisks (genotype) or hashes (treatment) indicating values that are significantly different (Two-way ANOVA with Bonferroni <i>post hoc</i> test). *,^#<i>p</i><0.05; **,^##<i>p</i><0.01; ***,^###<i>p</i><0.001. White, WT; Black, CD. Plain, water; Striped, EGCG. Data are presented as the mean ± SEM.</p

NADPH-oxidase and NO pathways were not affected by EGCG.

<p>(a) DHE staining in cardiac tissue showed a ≈20% increase in the general oxidative stress levels in CD mice (<i>p</i> = 0.04). (b) CD animals showed reduced expression levels of <i>Ncf1</i> (<i>p</i> = 0.0008) as previously described, but expression of other oxidative stress-related genes of the NADPH-oxidase and NO pathways was not affected. n = 3–4 mice per group. <i>p</i> values are shown with asterisks (genotype) indicating values that are significantly different (Unpaired <i>t</i>-test with Welch’s correction). *<i>p</i><0.05; ***<i>p</i><0.001. White, WT; Black, CD. Plain, water. Data are presented as the mean ± SEM.</p

EGCG does not influence brain abnormalities present in CD mice.

<p>(a) Brain weight of water or EGCG fed CD mice was significantly reduced when compared to WT mice. A two-way ANOVA indicated a significant effect of genotype (F_1,37 = 40.72, <i>p</i><0.0001) but no effect of treatment (F_1,37 = 0.3766, <i>p</i> = 0.5432). (b) Dendrite length of CA1 neurons was measured in stratum radiatum (SR). A two-way ANOVA indicated a significant effect of genotype (F_1,37 = 15.98, <i>p</i> = 0.0003) but not effect of treatment (F_1,37 = 1.728, <i>p</i> = 0.1968) (c) Dendrite length of CA1 neurons was measured in stratum oriens (SO). A two-way ANOVA indicated a deleterious effect of treatment in the case of WT mice (F_1,37 = 22.92, <i>p</i><0.0001 with <i>p</i><0.001 Bonferroni <i>post hoc</i> test). (d) EGCG treatment did not increase the number of spines present in apical dendrites of CD neurons. A two-way ANOVA indicated a significant effect of genotype (F_1,37 = 13.13, <i>p</i> = 0.0009) without treatment effect (F_1,37 = 1.722, <i>p</i> = 0.1976). n = 8–13 mice per group. <i>p</i> values are shown with asterisks (genotype) or hashes (treatment) indicating values that are significantly different (two-way ANOVA with Bonferroni <i>post hoc</i> test). *,^#<i>p</i><0.05; **,^##<i>p</i><0.01; ***,^###<i>p</i><0.001. White, WT; Black, CD. Plain, water; Striped, EGCG fed mice. Data are presented as the mean ± SEM.</p

EGCG restores oxidative stress status via NRF2 pathway.

<p>(a) Representative images of cardiomyocytes cultures showed that NRF2 nuclear levels were decreased in CD cardiomyocytes in comparison with WT cells. After EGCG-treatment both genotypes increased the nuclear proportion of NRF2. Blue, DAPI; red, NRF2. (b) Histograms show the quantification of figures showed in (a). A clear interaction effect between genotype and treatment could be observed (F_1,756 = 34,51, <i>p</i><0.0001). n = 145–235 cardiomyocytes per group. (c) <i>Nqo1</i> expression levels were downregulated in CD cardiac tissue (<i>p</i><0.01, Bonferroni post hoc test) and significantly higher after EGCG treatment (<i>p</i><0.05, Bonferroni <i>post hoc</i> test). n = 4 mice/group. <i>p</i> values are shown with asterisks (genotype) or hashes (treatment) indicating values that are significantly different (two-way ANOVA or one-way ANOVA with Bonferroni <i>post hoc</i> test). *^,#<i>p</i><0.05, **^,##<i>p</i><0.01; ***^,###<i>p</i><0.001. White, WT; Black, CD. Plain, water; Striped, EGCG. Data are presented as the mean ± SEM.</p

Impaired cellular calcium homeostasis in CD mice.

<p>(a) The mean frequency of calcium oscillations in CD hippocampal neurons was significantly lower (<i>t</i>₂₀₀ = 5.526, <i>p</i><0.0001) when compared to the mean frequency of WT neurons n = 103–130 cells per genotype. (b) Amplitude (<i>p</i> = 0.0028) and (c) duration (<i>p</i> = 0.0063) of calcium transients were significantly higher in CD neurons when compared to WT neurons. (d) Differences in hippocampal mRNA expression genes related to Ca²⁺ signaling levels of <i>Trpc3</i> were slightly lower in CD mice (<i>p</i> = 0.045), while <i>Ryr2</i> and <i>Ryr3</i> levels were significantly higher in CD mice when compared to WT mice (<i>p</i> = 0.0017 and <i>p</i> = 0.0071, respectively). No significant differences in mRNA expression of other genes related to Ca²⁺ signaling (<i>Cacna1c</i>, <i>Atp2a2</i> and <i>Trpc6</i>) were found. (e) TRPC3 subcellular localization in CD mice primary cultures of hippocampal neurons is not significantly altered compared with WT cells (F_1,77 = 0.3922, <i>p</i> = 0.5331). n = 16–23 cells per genotype. <i>p</i> values are shown with asterisks (genotype) indicating values that are significantly different (two-way ANOVA with Bonferroni <i>post hoc</i> test or unpaired <i>t</i>-test with Welch’s correction). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. White, WT; Black, CD. Plain, water. Data are presented as the mean ± SEM.</p

Effect of EGCG on hippocampal synaptic plasticity markers.

<p>(a) EGCG treatment normalized <i>Bdnf</i> mRNA levels in CD animals. A two-way ANOVA indicated a significant interaction between genotypes and treatment (F_1,38 = 5.512, <i>p</i> = 0.0242), with a significant effect of genotype (F_1,38 = 6.599, <i>p</i> = 0.0143) and treatment (F_1,38 = 30.47, <i>p</i><0.0001). (b) EGCG treatment did not normalize the high <i>Pik3r1</i> mRNA levels observed in the hippocampus of CD mice. A two-way ANOVA indicated a significant effect of treatment (F_1,24 = 7.406, <i>p</i> = 0.0119), since EGCG treatment upregulated <i>Pik3r1</i> mRNA levels in WT animals. n = 6–13 per group. <i>p</i> values are shown with asterisks (genotype) or hashes (treatment) indicating values that are significantly different (two-way ANOVA with Bonferroni <i>post hoc</i> test). *,^#<i>p</i><0.05; **,^##<i>p</i><0.01; ***,^###<i>p</i><0.001. White, WT; Black, CD. Plain, water; Striped, EGCG. Data are presented as the mean ± SEM.</p

EGCG does not influence sociability or anxiety-related behavior in CD mice.

<p>(a) In a direct social test, CD mice fed with green tea behaved in the same way that water fed CD mice towards an intruder mice. A two-way ANOVA indicated a significant main effect of genotype (F_1,37 = 19.86, <i>p</i><0.0001), without effect of treatment (F_1,37 = 0.00044, <i>p</i> = 0.9834). n = 7–15 per group. White, WT; Black, CD. Plain, water; Striped, EGCG. Data are presented as the mean ± SEM. (b) Anxiety-like behavior was evaluated in the marble-burying test. A three-way ANOVA revealed no interaction between genotype, treatment and time (F_3,128 = 1.380, <i>p</i> = 0.252), with a significant main effect of genotype (F_1,128 = 306.684, <i>p</i><0.0001) and time (F_3,128 = 17.170, <i>p</i><0.0001) but no effect of treatment (F_1,128 = 0.182, <i>p</i> = 0.670). n = 8–10 per group. Squares, WT; Circles, CD. White and black, water; Green, EGCG fed mice. <i>p</i> values are shown with asterisks (genotype effect) indicating values that are significantly different in a two-way ANOVA with Bonferroni <i>post hoc</i> test (***<i>p</i><0.001).</p

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

<div><p>Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated <i>pgi1-3</i>, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, <i>PGI1</i>. Starch content in <i>pgi1-3</i> source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of <i>pgi1-2</i>, a T-DNA insertion pPGI null mutant. Starch deficiency of <i>pgi1</i> leaves could be reverted by the introduction of a <i>sex1</i> null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of <i>pgi1-2</i> leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of <i>pgi1-2</i> and <i>pgi1-3</i> mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in <i>pgi1</i> leaves, which was accompanied by increased β-amylase activity. Both <i>pgi1-2</i> and <i>pgi1-3</i> mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in <i>pgi1</i> leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in <i>pgi1</i> leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of <i>pgi1</i> leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.</p></div

HSV1 infection activates PKR.

<p>SH-SY5Y cells were infected with HSV1 (1 pfu/cell, 24 h) or were not infected (controls). Immunocytochemistry analysis was carried with the following Abs: anti-p-PKR, anti-p-eIF2-alpha and anti-BACE1 (A). Protein extracts of SH-SY5Y cells infected with HSV1 (3 pfu/cell, 24 h) and uninfected cells (controls) were analysed by Western blotting using the following Abs: anti-BACE1, anti-p-PKR, anti-PKR, anti-p-eIF2-alpha, anti- eIF2-alpha and anti-tubulin. Bands were quantified by densitometric analysis. Results are expressed as the mean ± SEM of 3–4 independent experiments. * p<0.05, ** p<0.01, *** p<0.0005 by Student's <i>t</i> test (B). Sections from mouse dorsal ganglion root (DRG) were obtained from HSV1-infected mice. Contralateral uninfected ganglia were used as controls. Immunohistochemistry analysis was carried out to detect p-PKR (C).</p

<i>pgi1–3</i> leaves lack pPGI activity.

<p>(A) PGI zymogram of proteins extracted from WT (L<i>er</i>) and <i>pgi1–3</i> leaves. (B) Q-sepharose chromatography profile of PGI activity in WT and <i>pgi1–3</i> leaves. In “B”, loaded WT extract contained 850 mU of total PGI activity, whereas <i>pgi1–3</i> extract loaded on the column contained 650 mU of PGI activity.</p