14-3-3τ Regulates Beclin 1 and Is Required for Autophagy

Background

Beclin 1 plays an essential role in autophagy; however, the regulation of Beclin 1 expression remains largely unexplored. An earlier ChIP-on-chip study suggested Beclin 1 could be an E2F target. Previously, we also reported that 14-3-3τ regulates E2F1 stability, and is required for the expression of several E2F1 target genes. 14-3-3 proteins mediate many cellular signaling processes, but its role in autophagy has not been investigated. We hypothesize that 14-3-3τ could regulate Beclin 1 expression through E2F1 and thus regulate autophagy.

Methods and Findings

Using the RNAi technique we demonstrate a novel role for one of 14-3-3 isoforms, 14-3-3τ, in the regulation of Beclin 1 expression and autophagy. Depletion of 14-3-3τ inhibits the expression of Beclin 1 in many different cell lines; whereas, upregulation of 14-3-3τ induces Beclin 1. The regulation is physiologically relevant as an extracellular matrix protein tenascin-C, a known 14-3-3τ inducer, can induce Beclin 1 through 14-3-3τ. Moreover, rapamycin-induced, serum free-induced and amino acid starvation-induced autophagy depends on 14-3-3τ. We also show the expression of Beclin 1 depends on E2F, and E2F can transactivate the Beclin 1 promoter in a promoter reporter assay. Upregulation of Beclin 1 by 14-3-3τ requires E2F1. Depletion of E2F1, like 14-3-3τ, also inhibits autophagy.

Conclusion

Taken together, this study uncovers a role for 14-3-3τ in Beclin 1 and autophagy regulation probably through regulation of E2F1.     

Comparison of two β-glucosidases for the enzymatic synthesis of β-(1-6)-β-(1-3)-gluco-oligosaccharides

A domain of epiglucan was synthesized by beta-glucosidases. Two beta-glucosidases, an extracellular beta-glucosidase derived from Sclerotinia sclerotiorum grown on xylose, and a commercial lyophilized preparation of beta-glucosidase from Aspergillus niger, were used to synthesize gluco-oligosaccharides from cellobiose and, specially, beta-(1-6) branched beta-(1-3) gluco-oligosaccharides, corresponding to the structure of epiglucan. Gentiobiose, cellotriose, cellotetraose, beta-Glc-(1-3)-beta-Glc-(1-4)-Glc, beta-Glc-(1-6)-beta-Glc-(1-4)-Glc and beta-Glc-(1-6)-beta-Glc-(1-3)-Glc were synthesized from cellobiose by both enzymes. The latter compound was preferentially synthesized by the beta-glycosidase from Sclerotinia sclerotiorum. Under the best conditions, only 7 g l(-1) of beta-Glc-(1-6)-beta-Glc-(1-3)-Glc was synthesized by the beta-glycosidase from Aspergillus niger compared to 20 g l(-1) synthesized with beta-glycosidase from Sclerotinia sclerotiorum.     

Definitive Solution Structures for the 6-Formylated Versions of 1-(βD-Ribofuranosyl)-, 1-(2′-Deoxy-β-D-Ribofuranosyl)-, and 1-β-D-Arabinofuranosyluracil,and of Thymidine

Abstract

ROESY and NOESY NMR spectroscopic analyses of the ribofuranosyl (1a), 2′-deoxyribofuranosyl (1b), and arabinofuranosyl (1c) derivatives of 6-formyluracil in (CD₃)₂SO and D₂O solutions have established that each exclusive 7,05′-cyclic hemiacetal diastereomer of 1a,b and the major 7,O2′-cyclic hemiacetal diastereomer of 1c possess the 7R configuration. In addition, (7R)-1c has been shown to be thermodynamically more stable than (7S)-1c, contrary to our previous indication. A new, higher yielding synthetic route to 1a has been developed, 1b has been obtained for the first time in crystalline form, the route to 1c has been modified to better accommodate large scale preparations, and a new, fourth member of this class, 6-formylthymidine (1d), has been synthesized and its solution structures in (CD₃)₂SO, D₂O, and CD₃OD have been determined. Antitumor and antiviral evaluations of 1a-c have revealed no significant levels of activity.     

A monoclonal antibody specific for non-reducing terminal Galα1-4Gal residues

A mouse monoclonal antibody (87.5) against Gal1-4Gal has been obtained after immunization with the disaccharide glycosidically coupled to a protein. The specificity was determined by studying its binding to a number of glycoconjugates and oligosaccharides.The antibody which was found to be highly specific for terminal Gal1-4Gal residues is a powerful tool for the detection of this structure in glycoproteins and glycolipids by immunochemicalin vitro methods. It is also useful forin vitro quantification of the free disaccharide.A thin layer chromatographic overlay assay using glycolipids and an immunoperoxidase technique is also described. The antibody 87.5 is used in this assay to identify human uroepithelium glycolipids with terminal Gal1-4Gal residues.Abbreviations Lactosylceramide Gal1-4GlcCer - globotriaosylceramide GbOse₃-ceramide, Gal1-4Gal1-4GlcCer - globotetraosylceramide globoside, GbOse₄-ceramide, GalNAc1-3Gal1-4Gal1-4GlcCer     

1-Bromo-3-(1',1'-dimethylalkyl)-1-deoxy-Δ(8)-tetrahydrocannabinols: New selective ligands for the cannabinoid CB(2) receptor

Δ(8)-Tetrahydrocannabinol (26), 3-(1',1'-dimethylbutyl)- (12), 3-(1',1'-dimethylpentyl)- (13), 3-(1',1'-dimethylhexyl)- (14) and 3-(1',1'-dimethylheptyl)-Δ(8)-tetrahydrocannabinol (15) have been converted into the corresponding 1-bromo-1-deoxy-Δ(8)-tetrahydrocannabinols (25, 8-11). This was accomplished using a protocol developed in our laboratory in which the trifluoromethanesulfonate of a phenol undergoes palladium mediated coupling with pinacolborane. Reaction of this dioxaborolane with aqueous-methanolic copper(II) bromide provides the aryl bromide. The affinities of these bromo cannabinoids for the cannabinoid CB(1) and CB(2) receptors were determined. All of these compounds showed selectivity for the CB(2) receptor and one of them, 1-bromo-1-deoxy-3-(1',1'-dimethylhexyl)-Δ(8)-tetrahydrocannabinol (10), exhibits 52-fold selectivity for this receptor with good (28nM) affinity.     

HP1-β is required for development of the cerebral neocortex and neuromuscular junctions

HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-β isotype, and show that the Cbx1^−/−-null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1^−/− mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1^−/− mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization.     

一种异常氨基酸——-1-氨基-环丙烷-1-羧酸的合成

<正> 1 氨基—环丙烷-1-羧酸(ACC)是一种存在于苹果、梨等果汁中的异常氨基酸,是一类新型的植物生长调节剂,广泛应用于植物生理、园艺和农牧业等方面,在实践上也有着重大的经济价值,因而有关ACC的应用文章日益增多。ACC的合成方法有4—5种,我们采用来源方便的丙二酸二乙酯作为基本原料,并对Engeld的方法作了重大改进,成功地合成了ACC。在环化步     

Small animal model for HIV-1 Disease

Development of a viral infection model of the humanimmune systemusingsmall animalsis animportant goal in biomedi-cal research,especiallyinstudiesof HIV-1infection.Thisis particularlyimportant since susceptibilityto HIV-1islimit-edto humans.The C.B-17-scid/scid-mouselacks mature Tand Bcells dueto a defective rearrangement of the Tcell re-ceptor andimmunoglobulin genes.Twotypes of humanlymphoid chimeras have been establishedin scid-mice.The firstsuccess withthe human mouse chimera was achie…     

Setting research priorities for Type 1 diabetes

Diabet. Med. 29, 1321-1326 (2012) ABSTRACT: Aims Research priorities are often set by academic researchers or the pharmaceutical industry. The interests of patients, carers and clinicians may therefore be overlooked and research questions that matter may be neglected. The aims of this study were to collect uncertainties about the treatment of Type?1 diabetes from patients, carers and health professionals, and to collate and prioritize these uncertainties to develop a top 10 list of research priorities, using a structured priority-setting partnership of patients, carers, health professionals and diabetes organizations, as described by the James Lind Alliance. Methods A partnership of interested organizations was set up, and from this a steering committee of 10 individuals was formed. An online and paper survey was used to identify uncertainties. These were collated, and the steering group carried out an interim priority-setting exercise with partner organizations. This group of uncertainties was then voted on to give a smaller list that went forward to the final priority-setting workshop. At this meeting, a final list of the top 10 research priorities was agreed. Results An initial 1141 uncertainties were described. These were reduced to 88 indicative questions, 47 of which went out for voting. Twenty-four were then taken forward to a final priority-setting workshop. This workshop resulted in a list of top 10 research priorities in Type?1 diabetes. Conclusion We have shown that it is possible using the James Lind Alliance process to develop an agreed top 10 list of research priorities for Type?1 diabetes from health professionals, patients and carers.     

Immobilization of the α-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance

The α-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K _m increased, while V _max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized α-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized α-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 α-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.     

3′-(1,2,3-Triazol-1-yl)-3′-deoxythymidine analogs as substrates for human and Ureaplasma parvum thymidine kinase for structure–activity investigations

The pathogenic mycoplasma Ureaplasma parvum (Up) causes opportunistic infections and relies on salvage of nucleosides for DNA synthesis and Up thymidine kinase (UpTK) provides the necessary thymidine nucleotides. The anti-HIV compound 3?-azido-3′-deoxythymidine (AZT) is a good substrate for TK. Methods for a rapid and efficient synthesis of new 3′-α-[1,2,3]triazol-3′-deoxythymidine analogs from AZT under Huisgen conditions are described. Thirteen 3′-analogues were tested with human cytosolic thymidine kinase (hTK1) and UpTK. The new analogs showed higher efficiencies (K_m/V_max values) in all cases with UpTK than with hTK1. Still, hTK1 was preferentially inhibited by 9 out of 10 tested analogs. Structural models of UpTK and hTK1 were constructed and used to explain the kinetic results. Two different binding modes of the nucleosides within the active sites of both enzymes were suggested with one predominating in the bacterial enzyme and the other in hTK1. These results will aid future development of anti-mycoplasma nucleosides.     

2,2′-Dihydroxybenzophenones and their carbonyl N-analogues as inhibitor scaffolds for MDR-involved human glutathione transferase isoenzyme A1-1

The MDR-involved human GSTA1-1, an important isoenzyme overexpressed in several tumors leading to chemotherapeutic-resistant tumour cells, has been targeted by 2,2′-dihydroxybenzophenones and some of their carbonyl N-analogues, as its potential inhibitors. A structure-based library of the latter was built-up by a nucleophilic cleavage of suitably substituted xanthones to 2,2′-dihydroxy-benzophenones (5–9) and subsequent formation of their N-derivatives (oximes 11–13 and N-acyl hydrazones 14–16). Screening against hGSTA1-1 led to benzophenones 6 and 8, and hydrazones 14 and 16, having the highest inhibition potency (IC₅₀ values in the range 0.18 ± 0.02 to 1.77 ± 0.10 μM). Enzyme inhibition kinetics, molecular modeling and docking studies showed that they interact primarily at the CDNB-binding catalytic site of the enzyme. In addition, the results from cytotoxicity studies with human colon adenocarcinoma cells showed low LC₅₀ values for benzophenone 6 and its N-acyl hydrazone analogue 14 (31.4 ± 0.4 μM and 87 ± 1.9 μM, respectively), in addition to the strong enzyme inhibition profile (IC₅₀₍₆₎ = 1,77 ± 0.10 μM; IC₅₀₍₁₄₎ = 0.33 ± 0.05 μM). These structures may serve as leads for the design of new potent mono- and bi-functional inhibitors and pro-drugs against human GTSs.     

Disease modifying therapy for AD?1

Alzheimer's disease (AD) is the most common form of dementia in industrialized nations. If more effective therapies are not developed that either prevent AD or block progression of the disease in its very early stages, the economic and societal cost of caring for AD patients will be devastating. Only two types of drugs are currently approved for the treatment of AD: inhibitors of acetyl cholinesterase, which symptomatically enhance cognitive state to some degree but are not disease modifying; and the adamantane derivative, memantine. Memantine preferentially blocks excessive NMDA receptor activity without disrupting normal receptor activity and is thought to be a neuroprotective agent that blocks excitotoxicty. Memantine therefore may have a potentially disease modifying effect in multiple neurodegenerative conditions. An improved understanding of the pathogeneses of AD has now led to the identification of numerous therapeutic targets designed to alter amyloid beta protein (Abeta) or tau accumulation. Therapies that alter Abeta and tau through these various targets are likely to have significant disease modifying effects. Many of these targets have been validated in proof of concept studies in preclinical animal models, and some potentially disease modifying therapies targeting Abeta or tau are being tested in the clinic. This review will highlight both the promise of and the obstacles to developing such disease modifying AD therapies.     

Evidence for a genetic basis for the class A Thy-1− defect

When Thy-1^– cell lines derived from different Thy-1⁺ murine thymic lymphomas are analyzed by complementation analysis, most fall into the A complementation class. A possible explanation for this result is that the Class A phenotype is due to a mutation in a gene on the X chromosome. To test this idea, selection for 6-thioguanine resistance was carried out on Thy-1⁺ hybrid cell lines between complementary Class A and Class C Thy-1^– mutant cell lines. In some hybrid clones, there was complete concordance between 6-thioguanine resistance and a change of the phenotype of the hybrid from Thy-1⁺ to Thy-1^–. Detailed study of one of these hybrid clones showed that 6-thioguanine resistance was accompanied by loss of hypoxanthine guanine phosphoribosyltransferase activity and that the Thy-1^– phenotype was attributable to loss of the gene complementing the Class A Thy-1^– mutation.Other hybrid clones, however, had some thioguanine resistant lines which remained Thy-1⁺. The degree of concordance was a characteristic of the particular hybrid clone examined and subclones which showed complete concordance could be derived from clones showing incomplete concordance. The variability in the degree of concordance between 6-thioguanine resistance and the Thy-1^– phenotype in different hybrid cell lines was also seen among individual hybrid clones isolated from a fusion between a Class A mutant and normal spleen cell blasts.We conclude from these results that the basis of the Class A Thy-1^– phenotype is genetic, but given the variability in the degree of linkage observed, we cannot determine whether the gene determining the Class A mutant phenotype is X-linked in the normal situation.