Temporal pattern of neuronal insulin release during Caenorhabditis elegans aging: Role of redox homeostasis

The insulin‐IGF‐1/DAF‐2 pathway has a central role in the determination of aging and longevity in Caenorhabditis elegans and other organisms. In this paper, we measured neuronal insulin secretion (using INS‐22::Venus) during C. elegans lifespan and monitored how this secretion is modified by redox homeostasis. We showed that INS‐22::Venus secretion fluctuates during the organism lifetime reaching maximum levels in the active reproductive stage. We also demonstrate that long‐lived daf‐2 insulin receptor mutants show remarkable low levels of INS‐22::Venus secretion. In contrast, we found that short‐lived mutant worms that lack the oxidation repair enzyme MSRA‐1 show increased levels of INS‐22::Venus secretion, specifically during the reproductive stage. MSRA‐1 is a target of the insulin‐IGF‐1/DAF‐2 pathway, and the expression of this antioxidant enzyme exclusively in the nervous system rescues the mutant insulin release phenotype and longevity. The msra‐1 mutant phenotype can also be reverted by antioxidant treatment during the active reproductive stage. We showed for the first time that there is a pattern of neuronal insulin release with a noticeable increment during the peak of reproduction. Our results suggest that redox homeostasis can modulate longevity through the regulation of insulin secretion, and that the insulin‐IGF‐1/DAF‐2 pathway could be regulated, at least in part, by a feedback loop. These findings highlight the importance of timing for therapeutic interventions aimed at improving health span.     

棉花Trihelix转录因子GhGT29基因的克隆及功能分析

Trihelix转录因子在植物抵御各种逆境胁迫中扮演重要作用,克隆棉花Trihelix转录因子基因并分析其表达特性和功能,为最终利用转基因手段改良棉花抗逆性奠定基础。本文依据生物信息学分析,采用RT-PCR方法从陆地棉中克隆了一个Trihelix转录因子基因,命名为GhGT29(GenBank登录号：JQ013097)。该基因最大开放阅读框(ORF)为1092 bp,编码363个氨基酸,预测分子量为40.9 kDa,等电点为5.45。SMART蛋白结构预测发现,该蛋白含有1个Trihelix家族典型的SANT结构域。系统进化树分析表明,GhGT29属于Trihelix转录因子SH4亚家族,与拟南芥AtSH4-like1、AtSH4-like2亲缘关系最近。实时荧光定量PCR结果表明,GhGT29受高盐、干旱、低温胁迫和ABA诱导表达;GhGT29在陆地棉的根、茎、叶、花、开花后当天胚珠以及开花后12 d(12 DPA)纤维中均有表达,其中在花中表达量最高,在茎中表达量最低。利用拟南芥原生质体系统进行分析,结果显示GhGT29主要定位于细胞核中,并且具有转录激活活性。以上结果表明GhGT29基因可能参与棉花逆境信号通路中对抗逆功能基因表达的调控。     

Characterization of stress‐responsive lncRNAs in Arabidopsis thaliana by integrating expression,epigenetic and structural features

Agrobacterium genetically transforms plants by transferring and integrating T‐(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus‐tagged VirE2 or Venus‐tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1–Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY‐2 protoplasts, regardless of whether VirE2 was co‐expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium‐mediated transformation or VirE2 subcellular localization.     

Delineating the role of MITF isoforms in pigmentation and tissue homeostasis

MITF, a gene that is mutated in familial melanoma and Waardenburg syndrome, encodes multiple isoforms expressed from alternative promoters that share common coding exons but have unique amino termini. It is not completely understood how these isoforms influence pigmentation in different tissues and how the expression of these independent isoforms of MITF is regulated. Here, we show that melanocytes express two isoforms of MITF, MITF‐A and MITF‐M. The expression of MITF‐A is partially regulated by a newly identified retinoid enhancer element located upstream of the MITF‐A promoter. Mitf‐A knockout mice have only subtle changes in melanin accumulation in the hair and reduced Tyr expression in the eye. In contrast, Mitf‐M‐null mice have enlarged kidneys, lack neural crest‐derived melanocytes in the skin, choroid, and iris stroma, yet maintain pigmentation within the retinal pigment epithelium and iris pigment epithelium of the eye. Taken together, these studies identify a critical role for MITF‐M in melanocytes, a minor role for MITF‐A in regulating pigmentation in the hair and Tyr expression in the eye, and a novel role for MITF‐M in size control of the kidney.     

N-glycosylation and biological activity of recombinant human alpha1-antitrypsin expressed in a novel human neuronal cell line

Human alpha‐1‐antitrypsin (A1AT) is a protease inhibitor that is involved in the protection of lungs from neutrophil elastase enzyme that drastically modifies tissue functioning. The glycoprotein consists of 394 amino acids and is N‐glycosylated at Asn‐46, Asn‐83, and Asn‐247. A1AT deficiency is currently treated with A1AT that is purified from human serum. In view of therapeutic applications, rA1AT was produced using a novel human neuronal cell line (AGE1.HN®) and we investigated the N‐glycosylation pattern as well as the in vitro anti‐inflammatory activity of the recombinant glycoprotein. rA1AT (300 mg/L) was biologically active as analyzed using elastase assay. The N‐glycan pool, released by PNGase F digestion, was characterized using 2D‐HPLC, MALDI‐TOF mass spectrometry, and by exoglycosidase digestions. A total of 28 N‐glycan structures were identified, ranging from diantennary to tetraantennary complex‐type N‐glycans. Most of the N‐glycans were found to be (α1–6) core‐fucosylated and part of them contain the Lewis X epitope. The two major compounds are a monosialylated diantennary difucosylated glycan and a disialylated diantennary core‐fucosylated glycan, representing 25% and 18% of the total N‐glycan pool, respectively. Analysis of the site‐specificity revealed that Asn‐247 was mainly occupied by diantennary N‐glycans whereas Asn‐46 was occupied by di‐, and triantennary N‐glycans. Asn‐83 was exclusively occupied by sialylated tri‐ and tetraantennary N‐glycans. Next, we evaluated the anti‐inflammatory activity of rA1AT using A1AT purified from human serum as a reference. rA1AT was found to inhibit the production of TNF‐α in neutrophils and monocytes as commercial A1AT does. Biotechnol. Bioeng. 2011;108:2118–2128. © 2011 Wiley Periodicals, Inc.     

Regulation of TrkB receptor tyrosine kinase and its internalization by neuronal activity and Ca2+ influx

Internalization of the neurotrophin-Trk receptor complex is critical for many aspects of neurotrophin functions. The mechanisms governing the internalization process are unknown. Here, we report that neuronal activity facilitates the internalization of the receptor for brain-derived neurotrophic factor, TrkB, by potentiating its tyrosine kinase activity. Using three independent approaches, we show that electric stimulation of hippocampal neurons markedly enhances TrkB internalization. Electric stimulation also potentiates TrkB tyrosine kinase activity. The activity-dependent enhancement of TrkB internalization and its tyrosine kinase requires Ca2+ influx through N-methyl-d-aspartate receptors and Ca2+ channels. Inhibition of internalization had no effect on TrkB kinase, but inhibition of TrkB kinase prevents the modulation of TrkB internalization, suggesting a critical role of the tyrosine kinase in the activity-dependent receptor endocytosis. These results demonstrate an activity- and Ca2+-dependent modulation of TrkB tyrosine kinase and its internalization, and they provide new insights into the cell biology of tyrosine kinase receptors.     

Genetically encoded impairment of neuronal KCC2 cotransporter function in human idiopathic generalized epilepsy

The KCC2 cotransporter establishes the low neuronal Cl⁻ levels required for GABA_A and glycine (Gly) receptor-mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non-synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C-terminus, exhibit significantly impaired Cl⁻-extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (E_Gly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE.     

The occurrence of protein kinase C ϕ and λ isoforms in retina of different species

The localization and immunochemical identification of the novel protein kinase C ϕ (nPKC ϕ) and the atypical protein kinase C λ (aPKC λ) isoforms in retinas of different species were analyzed by immunohistochemistry and SDS-PAGE/Western blotting. nPKC ϕ immunoreactivity is associated with bipolar cells of mammalian (rabbit, rat and guinea pig) retinas but not the non-mammalian goldfish retina which has a lower concentration of nPKC ϕ. However, SDS-PAGE and Western blotting data indicate the antigen recognized by the nPKC ϕ monoclonal antibody in the retina is of a lower molecular weight than that expected for nPKC ϕ. This would suggest nPKC ϕ is more susceptible to degradation/breakdown than other PKC isoforms found in the retina or that the nPKC ϕ antibody may be recognizing an unknown retinal antigen. A comparison of nPKC ϕ and nPKC ϕ is present in the developing retina at an earlier stage than cPKC α. The typical ‘transport’ of cPKC α toward axonal terminals by phorbol-12,13-dibutyrate does not occur for nPKC ϕ yet both are translocated from the cytosolic to membrane compartments. The inner plexiform layer and the inner nuclear layer (putative horizontal cells) of all species examined (rabbit, rat, guinea pig and goldfish) exhibited positive immunoreactivity for aPKC λ as confirmed by SDS-PAGE/Western blotting. Special issue dedicated to Dr. Kinya Kuriyama.     

Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non‐steroidal anti‐inflammatory drugs: role of mitochondrial calcium

Brain damage after insult and cognitive decline are related to excitotoxicity and strongly influenced by aging, yet mechanisms of aging‐dependent susceptibility to excitotoxicity are poorly known. Several non‐steroidal anti‐inflammatory drugs (NSAIDs) may prevent excitotoxicity and cognitive decline in the elderly by an unknown mechanism. Interestingly, after several weeks in vitro, hippocampal neurons display important hallmarks of neuronal aging in vivo. Accordingly, rat hippocampal neurons cultured for several weeks were used to investigate mechanisms of aging‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs. We found that NMDA increased cytosolic Ca²⁺ concentration in young, mature and aged neurons but only promoted apoptosis in aged neurons. Resting Ca²⁺ levels and responses to NMDA increased with time in culture which correlated with changes in expression of NMDA receptor subunits. In addition, NMDA promoted mitochondrial Ca²⁺ uptake only in aged cultures. Consistently, specific inhibition of mitochondrial Ca²⁺ uptake decreased apoptosis. Finally, we found that a series of NSAIDs depolarized mitochondria and inhibited mitochondrial Ca²⁺ overload, thus preventing NMDA‐induced apoptosis in aged cultures. We conclude that mitochondrial Ca²⁺ uptake is critical for age‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs.