Adducin Is Required for Desmosomal Cohesion in Keratinocytes

Adducin is a protein organizing the cortical actin cytoskeleton and a target of RhoA and PKC signaling. However, the role for intercellular cohesion is unknown. We found that adducin silencing induced disruption of the actin cytoskeleton, reduced intercellular adhesion of human keratinocytes, and decreased the levels of the desmosomal adhesion molecule desmoglein (Dsg)3 by reducing its membrane incorporation. Because loss of cell cohesion and Dsg3 depletion is observed in the autoantibody-mediated blistering skin disease pemphigus vulgaris (PV), we applied antibody fractions of PV patients. A rapid phosphorylation of adducin at serine 726 was detected in response to these autoantibodies. To mechanistically link autoantibody binding and adducin phosphorylation, we evaluated the role of several disease-relevant signaling molecules. Adducin phosphorylation at serine 726 was dependent on Ca²⁺ influx and PKC but occurred independent of p38 MAPK and PKA. Adducin phosphorylation is protective, because phosphorylation-deficient mutants resulted in loss of cell cohesion and Dsg3 fragmentation. Thus, PKC elicits both positive and negative effects on cell adhesion, since its contribution to cell dissociation in pemphigus is well established. We additionally evaluated the effect of RhoA on adducin phosphorylation because RhoA activation was shown to block pemphigus autoantibody-induced cell dissociation. Our data demonstrate that the protective effect of RhoA activation was dependent on the presence of adducin and its phosphorylation at serine 726. These experiments provide novel mechanisms for regulation of desmosomal adhesion by RhoA- and PKC-mediated adducin phosphorylation in keratinocytes.     

γ辐照对小麦种子延迟发光的影响

应用延迟发光的理论与方法,研究了γ辐照对扬麦13、扬麦15两个品种小麦种子的影响。结果表明:被测样品延迟发光的弛豫过程可以用非线性动力学方程Y=b1+b2e-b3t/1+b4t来进行描述,该方程对测量结果拟合的相关系数达到了99%以上。当t=0时,Y0=b1+b2,即样品的初始发光强度,在光照条件一定时,辐照剂量越高,样品的初始发光强度就越低。F测验结果表明不同辐照剂量对小麦种子初始发光强度的影响达到了极显著的水平,即受照剂量越高,初始发光强度越低,而两种供试小麦间的初始发光强度并无显著差异。结合处理种子苗期的生物学观察,样品(即种子)的初始发光强度与幼苗株高具有一致的变化趋势,即种子的初始发光强度越高,幼苗株高也高。因此,对辐射种子延迟发光过程的分析,可能是了解γ辐照对作物种子影响的一种快速可靠的检测方法。     

利用外源γ-氨基丁酸调控海洋绿藻亚心形四爿藻淀粉积累

微藻被看作第三代生物质能源的来源。微藻淀粉结构与高等植物的高度相似性使其可以作为粮食作物的替代,在生物能源领域有广泛的应用。γ-氨基丁酸(GABA)被认为是一种信号分子,可以调节植物细胞的生长代谢。本研究在缺氮培养条件下添加外源GABA调控海洋绿藻亚心形四爿藻生理代谢和淀粉积累。结果表明,添加外源GABA可以抑制细胞生长,降低光合作用效率;OJIP实验显示,GABA的添加增强了光合器官能量耗散,降低了光能利用效率,阻碍了电子传递,造成额外胁迫,从而促使细胞将碳流更多地分配到淀粉积累,导致藻细胞的淀粉含量、淀粉产量和淀粉产率提高。添加10 mmol/L GABA获得最大淀粉含量39%DW,比未添加GABA的对照组淀粉含量提高39%;同时获得最大淀粉产量和产率为1.72 g·L^-1和0.36 g·L^-1·d^-1,分别比未添加GABA的对照组提高39%和50%。以上结果表明在缺氮条件下添加外源GABA是一种调控亚心形四爿藻细胞代谢并提高其淀粉生产的有效方法。     

Doxorubicin-induced alterations in kidney functioning,oxidative stress,DNA damage,and renal tissue morphology; Improvement by Acacia hydaspica tannin-rich ethyl acetate fraction

Doxorubicin (DOX) is an anthracycline drug used for cancer treatment. However, its treatment is contiguous with toxic effects. We examined the nephroprotective potential of A. hydaspica polyphenol-rich ethyl acetate extract (AHE) against DOX persuaded nephrotoxicity. 36 male Sprague Dawley rats were randomly assorted into 6 groups. Control group received saline; DOX group: 3 mg/kg b.w. dosage of DOX intraperitoneally for 6 weeks (single dose/week). In co-treatment groups, 200 and 400 mg/kg b.w AHE was given orally for 6 weeks in concomitant with DOX (3 mg/kg b.w, i.p. injection per week) respectively. Standard group received silymarin 400 mg/kg b.w daily + DOX (single dose/week). Biochemical kidney function tests, oxidative stress markers, genotoxicity, antioxidant enzyme status, and histopathological changes were examined. DOX caused significant body weight loss and decrease kidney weight. DOX-induced marked deterioration in renal function indicators in both urine and serum, i.e., PH, specific gravity, total protein, albumin, urea, creatinine, uric acid, globulin, blood urea nitrogen, etc. Also, DOX treatment increases renal tissue oxidative stress markers, while lower antioxidant enzymes in tissue along with degenerative alterations in the renal tissue compared to control rats. AHE co-treatment ameliorates DOX-prompted changes in serum and urine chemistry. Likewise, AHE treatment decreases sensitive markers of oxidative stress and prevented DNA damages by enhancing antioxidant enzyme levels. DOX induction in rats also caused DNA fragmentation which was restored by AHE co-treatment. Moreover, the histological observations evidenced that AHE effectively rescued the kidney tissue from DOX interceded oxidative damage. Our results suggest that co-treatment of AHE markedly improve DOX-induced deleterious effects in a dose-dependent manner. The potency of AHE co-treatment at 400 mg/kg dose is similar to silymarin. These outcomes revealed that A. hydaspica AHE extract might serve as a potential adjuvant that avoids DOX-induced nephrotoxicity.     

Functional Analysis of the C-Terminal Region of γ-Glutamyl Kinase of Saccharomyces cerevisiae

γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ¹-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of Saccharomyces cerevisiae GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in Escherichia coli and S. cerevisiae and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.     

Repeated Batch Production of Theanine by Coupled Fermentation with Energy Transfer Using Membrane-Enclosed γ-Glutamylmethylamide Synthetase and Dried Yeast Cells

γ-Glutamylmethylamide synthetase and dried baker’s yeast cells were enclosed together in a dialysis membrane tube to produce theanine repeatedly by coupled fermentation with energy transfer. The membrane-enclosed enzyme preparation (M-EEP) formed approximately 600 mM theanine from glutamic acid and ethylamine at a 100% conversion rate. M-EEP maintained its productivity of theanine during six consecutive reactions in a mixture containing NAD⁺.     

Syntheses,biological evaluation and SAR of ingenol mebutate analogues for treatment of actinic keratosis and non-melanoma skin cancer

Ingenol mebutate is the active ingredient in Picato® a new drug for the treatment of actinic keratosis. A number of derivatives related to ingenol mebutate were prepared by chemical synthesis from ingenol with the purpose of investigating the SAR and potency in assays relating to pro-inflammatory effects (induction of PMN oxidative burst and keratinocyte cytokine release), the potential of cell death induction, as well as the chemical stability. By modifications of the ingenol scaffold several prerequisites for activity were identified. The chemical stability of the compounds could be linked to an acyl migration mechanism. We were able to find analogues of ingenol mebutate with comparable in vitro properties. Some key features for potent and more stable ingenol derivatives have been identified.     

Fibroblast growth factor 2 and protein kinase C alpha are involved in syndecan-4 cytoplasmic domain modulation of turkey myogenic satellite cell proliferation

Syndecan-4 core protein is composed of extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain functions in transmitting signals into the cell through the protein kinase C alpha (PKCα) pathway. The glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains attached to the extracellular domain influence cell proliferation. The current study investigated the function of syndecan-4 cytoplasmic domain in combination with GAG and N-glycosylated chains in turkey muscle cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Syndecan-4 or syndecan-4 without the cytoplasmic domain and with or without the GAG and N-glycosylated chains were transfected or co-transfected with a small interfering RNA targeting syndecan-4 cytoplasmic domain into turkey muscle satellite cells. The overexpression of syndecan-4 mutants increased cell proliferation but did not change differentiation. Syndecan-4 mutants had increased cellular responsiveness to FGF2 during proliferation. Syndecan-4 increased PKCα cell membrane localization, whereas the syndecan-4 mutants decreased PKCα cell membrane localization compared to syndecan-4. However, compared to the cells without transfection, syndecan-4 mutants increased cell membrane localization of PKCα. These data indicated that the syndecan‐4 cytoplasmic domain and the GAG and N-glycosylated chains are critical in syndecan-4 regulating satellite cell proliferation, responsiveness to FGF2, and PKCα cell membrane localization.     

2-(2-Bromophenyl)-formononetin and 2-heptyl-formononetin are PPARγ partial agonists and reduce lipid accumulation in 3T3-L1 adipocytes

Isoflavones are bioactive compounds that have been shown to decrease lipid accumulation in vitro. However, the knowledge of the isoflavone formononetin is limited. The aim of the study was to assess the effects of formononetin and its two synthetic analogues, 2-(2-bromophenyl)-formononetin and 2-heptyl-formononetin, on lipid accumulation in 3T3-L1 adipocytes and investigate possible mechanisms. Formononetin and the two analogues were added day 0–8 or day 8–10 of the differentiation period, and lipid accumulation, glycerol release and gene expression were measured. Additionally, competitive peroxisome proliferator-activated receptor (PPAR)-γ binding assay, PPARγ transactivation assay and Western blot for phosphorylated AMP-activated protein kinase (AMPK) were performed. Chronic treatment (day 0–8) with formononetin increased lipid accumulation, whereas the two analogues decreased lipid accumulation partly due to decreased differentiation. The two analogues, but not formononetin, also decreased lipid content in mature adipocytes. 2-Heptyl-formononetin increased glycerol release and lipolytic gene expression and decreased lipogenic gene expression. Formononetin did not bind to or activate PPARγ whereas both analogues bound to the receptor and behaved as PPARγ partial agonists in the transactivation assay. Neither of the compounds affected phosphorylation of AMPK. In conclusion, the analogues of formononetin decreased lipid accumulation possibly in part by acting as PPARγ partial agonists.