营养膜无土栽培技术

前言营养膜技术(Nutrient Film Technique简称NFT)是国外广泛采用的一种特殊的无土栽培方法。它是将植物栽植在一个狭长的不透水的营养槽中,槽中有缓慢流动的营养液。植物根系的一部分浸没在营养液中,一部分露在空气中,植物从这种小环境中吸收水、营养物和氧气。由于这种方法采用很薄一层营养液在根际周围流动,裸露的根     

Effects of tau phosphorylation on proteasome activity

Dysfunction of proteasome contributes to the accumulation of the abnormally hyperphosphorylated tau in Alzheimer's disease. However, whether tau hyperphosphorylation and accumulation affect the activity of proteasome is elusive. Here we found that a moderate tau phosphorylation activated the trypsin-like activity of proteasome, whereas further phosphorylation of tau inhibited the activity of the protease in HEK293 cells stably expressing tau441. Furthermore, tau hyperphosphorylation could partially reverse lactacystin-induced inhibition of proteasome. These results suggest that phosphorylation of tau plays a dual role in modulating the activity of proteasome.     

The Role of the Lipid Bilayer in Tau Aggregation

Tau is a microtubule associated protein whose aggregation is implicated in a number of neurodegenerative diseases. We investigate the mechanism by which anionic lipid vesicles induce aggregation of tau in vitro using K18, a fragment of tau corresponding to the four repeats of the microtubule binding domain. Our results show that aggregation occurs when the amount of K18 bound to the lipid bilayer exceeds a critical surface density. The ratio of protein/lipid at the critical aggregation concentration is pH-dependent, as is the binding affinity. At low pH, where the protein binds with high affinity, the critical surface density is independent both of total lipid concentration as well as the fraction of anionic lipid present in the bilayer. Furthermore, the aggregates consist of both protein and vesicles and bind the β-sheet specific dye, Thioflavin T, in the manner characteristic of pathological aggregates. Our results suggest that the lipid bilayer facilitates protein-protein interactions both by screening charges on the protein and by increasing the local protein concentration, resulting in rapid aggregation. Because anionic lipids are abundant in cellular membranes, these findings contribute to understanding tau-lipid bilayer interactions that may be relevant to disease pathology.     

The Recombinant Amyloid-β Peptide Aβ1-42 Aggregates Faster and Is More Neurotoxic than Synthetic Aβ1-42

Aggregation of the amyloid-β (Aβ) peptide is considered a central event in the pathogenesis of Alzheimer's disease (AD). In order to bypass methodological bias related to a variety of impurities commonly present in typical preparations of synthetic Aβ, we developed a simple, generally applicable method for recombinant production of human Aβ and Aβ variants in Escherichia coli that provides milligram quantities of Aβ in very high purity and yield. Amyloid fibril formation in vitro by human Aβ1-42, the key amyloidogenic Aβ species in AD, was completed threefold faster with recombinant Aβ1-42 compared to synthetic preparations. In addition, recombinant Aβ1-42 was significantly more toxic to cultured rat primary cortical neurons, and it was more toxic in vivo, as shown by strongly increased induction of abnormal phosphorylation of tau and tau aggregation into neurofibrillary tangles in brains of P301L tau transgenic mice. We conclude that even small amounts of impurities in synthetic Aβ—including a significant fraction of racemized peptides that cannot be avoided due to the technical limitations of peptide synthesis—prevent or slow Aβ incorporation into the regular quaternary structure of growing β-amyloid fibrils. The results validate the use of recombinant Aβ1-42 for both in vitro and in vivo studies addressing the mechanisms underlying Aβ aggregation and its related biological consequences for the pathophysiology, therapy, and prevention of AD.     

Extracellular tau is toxic to neuronal cells

The degeneration of neurons in disorders such as Alzheimer's disease has an immediate consequence, the release of intracellular proteins into the extracellular space. One of these proteins, tau, has proven to be toxic when added to cultured neuronal cells. This toxicity varies according to the degree of protein aggregation. The addition of tau to cultured neuroblastoma cells provoked an increase in the levels of intracellular calcium, which is followed by cell death. We suggest that this phenomenon may be mediated by the interaction of tau with muscarinic receptors, which promotes the liberation of calcium from intracellular stores.     

Solution structure and dynamics of human metallothionein-3 (MT-3)

Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the alpha-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (beta-domain) and one in the C-terminus (alpha-domain). The overall fold of the alpha-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the beta-domain exhibits similar internal motion to the alpha-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.     

Substituent effect of N-benzylated gramine derivatives that prevent the PP2A inhibition and dissipate the neuronal Ca2+ overload,as a multitarget strategy for the treatment of Alzheimer’s disease

Following the premises of the multitarget-directed ligands approach for the drug R&D against neurodegenerative diseases, where Alzheimer’s disease (AD) outstands, we have synthesized and evaluated analogues of the gramine derivative ITH12657 (1-benzyl-5-methyl-3-(piperidin-1-ylmethyl-1H-indole, 2), which had shown important neuroprotective properties, such as blocking effect of voltage-gated Ca²⁺ channels (VGCC), and prevention of phosphoprotein phosphatase 2A (PP2A) inhibition. The new analogues present different substitutions at the pending phenyl ring, what slightly modified their pharmacological characteristics. The VGCC blockade was enhanced in derivatives possessing nitro groups, while the pro-PP2A feature was ameliorated by the presence of fluorine. Chlorine atoms supplied good activities over the two biological targets aimed; nevertheless that substitution provoked loss of viability at 100-fold higher concentrations (10?μM), what discards them for a deeper pharmacological study. Overall, the para-fluorine derivative of ITH12657 was the most promising candidate for further preclinical assays.     

Three-/four-repeat-dependent aggregation profile of tau microtubule-binding domain clarified by dynamic light scattering analysis

The analysis of the self-assembly mechanism of the tau microtubule-binding domain (MBD) could provide the information needed to develop an effective method for the inhibition of the tau filament formation because of its core region that forms the filament. The MBD domain in the living body consists of similar three or four 31- to 32-residue repeats, namely 3RMBD (R134) and 4RMBD (R1234), respectively. The filament formation of the MBD has been mainly investigated by fluorescence spectroscopy utilizing the β-sheet structure-binding signal sensor thioflavin. This method observes the aggregation indirectly, and provides no information on the time-dependent change in aggregation size or volume. Thus, to determine the structure necessary for initiating MBD self-association, the dynamic light scattering (DLS) method was applied to the analysis of the aggregations of 3RMBD, 4RMBD and their component single repeats and shown to be a powerful tool for directly analyzing filament formation. DLS analysis clearly showed that the building unit for initiating the aggregation is the intermolecular R3-R3 disulfide-bonded dimer for 3RMBD and the intramolecular R2-R3 disulfide-bonded monomer for 4RMBD, and their aggregation processes under physiological condition differ from each other, which has not been clearly revealed by the conventional fluorescence method. The repeat-number-dependent aggregation model of MBD, together with the function of each repeat, reported in this paper should help to devise a method of preventing tau PHF formation.     

An SRp75/hnRNPG complex interacting with hnRNPE2 regulates the 5' splice site of tau exon 10, whose misregulation causes frontotemporal dementia

Tau is a neuronal-specific microtubule-associated protein that plays an important role in establishing neuronal polarity and maintaining the axonal cytoskeleton. Aggregated tau is the major component of neurofibrillary tangles (NFTs), structures present in the brains of people affected by neurodegenerative diseases called tauopathies. Tauopathies include Alzheimer's disease (AD), frontotemporal dementia with Parkinsonism (FTDP-17), the early onset dementia observed in Down syndrome (DS; trisomy 21) and the dementia component of myotonic dystrophy type 1 (DM1). Splicing misregulation of adult-specific exon 10, which codes for a microtubule binding domain, results in expression of abnormal ratios of tau isoforms, leading to FTDP-17. Positions 3 to 19 of the intron downstream of exon 10 define a hotspot of splicing regulation: the region diverges between humans and rodents, and point mutations within it result in tauopathies. In this study, we investigated three regulators of exon 10 splicing: serine/arginine-rich protein SRp75 and heterogeneous nuclear ribonucleoproteins hnRNPG and hnRNPE2. SRp75 and hnRNPG inhibit splicing of exon 10 whereas hnRNPE2 activates it. Using co-transfections, co-immunoprecipitations and RNAi we discovered that SRp75 binds to the proximal downstream intron of tau exon 10 at the FTDP-17 hotspot region; and that hnRNPG and hnRNPE2 interact with SRp75. Thus, increased exon 10 inclusion in FTDP mutants may arise from weakened SRp75 binding. This work provides insights into the splicing regulation of the tau gene and into possible strategies for correcting the imbalance in tauopathies caused by changes in the ratio of exon 10.     

PKA modulates GSK-3beta- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3beta (GSK-3beta), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3beta, but the site-specific modulation of GSK-3beta-catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3beta-catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases.