噬菌体φHAU3的cos位点及其与寄主相互作用的功能位点attP和attB的定位

ＨＡＵ３是寄主范围很广的放线菌噬菌体。Ｓｏｕｔｈｅｒｎ杂交实验表明,ＨＡＵ３可以整合到吸水链霉菌应城变种１０－２２和变铅青链霉菌６６的突变体ＺＸ１的染色体中,形成溶原,其溶原菌自发释放ＨＡＵ３,不受热激和紫外线照射的诱导。通过比较ＨＡＵ３衍生噬粒ｐＩＪ８３００的ＤＮＡ酶切片段在加热前、后电泳带谱的区别,将ＨＡＵ３的ｃｏｓ位点在ｐＩＪ８３００的图谱上得到了定位。还利用Ｓｏｕｔｈｅｒｎ杂交的方法定位了ＨＡＵ３与宿主形成溶原时附着位点（ａｔｔＰ）,并利用脉冲电泳技术定位了在变铅青链霉菌ＺＸ７和吸水链霉菌应城变种１０－２２中形成溶原的附着位点（ａｔｔＢ）。这些信息均有利于以ＨＡＵ３为基础的载体的发展和优化。     

Densitometric Quantification and Optimization of Polyphenols in Phyllanthus maderaspatensis by HPTLC

Quantifying and optimizing the polyphenol content of Phyllanthus maderaspatensis was accomplished using a single-solvent HPTLC system. Analyzing hydroalcoholic extracts for kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, we simultaneously quantified and optimized their concentration. In the experiment, the methanol to water ratio (%), temperature (°C), and time of extraction (min) were all optimized using a Box-Behnken statistical design. Kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid were among the dependent variables analyzed. In the HPTLC separation, silica gel 60F254 plates were used, and toluene, ethyl acetate, and formic acid (5:4:1) made up the mobile phase. For kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, densitometric measurements were carried out using the absorbance mode at 254 nm. Hydroalcoholic extract of P. maderaspatensis contains rutin (0.344), catechin (2.62), gallic acid (0.93), ellagic acid (0.172), quercetin (0.0108) and kaempferol (0.06). Further, it may be affected by more than one factor at a time, resulting in a varying degree of reaction. A negative correlation was found between X1 (extraction time (min)) and X2 (temperature), as well as X1 and X3 (solvent ratios). Taking these characteristics into consideration, the method outlined here is a validated HPTLC method for measuring kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid.     

Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden

Background

It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection.

Results

Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember?, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced.

Conclusions

Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

     

Treatment of cyclic vomiting syndrome with co-enzyme Q10 and amitriptyline,a retrospective study

Background  

Cyclic vomiting syndrome (CVS), which is defined by recurrent stereotypical episodes of nausea and vomiting, is a relatively-common disabling condition that is associated with migraine headache and mitochondrial dysfunction. Co-enzyme Q10 (Co-Q) is a nutritional supplement that has demonstrated efficacy in pediatric and adult migraine. It is increasingly used in CVS despite the complete lack of studies to demonstrate its value in treatment     

Differential survival and reproduction in colour forms of Philaenus spumarius give new insights to the study of its balanced polymorphism

1. Colour polymorphisms are common across animals and are often the result of complex selection regimes. Philaenus spumarius (Linnaeus) (Hemiptera, Aphrophoridae) shows a widely studied dorsal colour polymorphism with several described phenotypes whose variation in their occurrence and frequency, as well as their maintenance across time, have been reported. Several selective influences have been suggested to play a role, but the mechanisms underlying the maintenance of this polymorphism are still poorly understood. 2. To explore the adaptive significance of the colour polymorphism of P. spumarius, an experiment was conducted in captivity under semi‐natural conditions to measure survival, reproductive success, and duration of egg maturation. 3. It was found that there was higher longevity, a higher number of oviposition events, and a higher number of eggs laid by trilineatus phenotype females than by typicus and marginellus, supporting previous reports of an increase in trilineatus frequency during the season. The duration of egg maturation did not differ among phenotypes. 4. The higher longevity and fertility of the trilineatus phenotype may compensate, for example, the higher rate of attack by parasitoids and/or higher solar radiation reflectance in this phenotype, which have already been reported in previous studies, constituting a possible mechanism for the maintenance of this polymorphism.     

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.     

Adeno-associated virus-mediated rescue of the cognitive defects in a mouse model for Angelman syndrome

Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII) regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr(286) and Thr(305/306), resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV) vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.     

Facilitating Akt Clearance via Manipulation of Hsp70 Activity and Levels

Members of the 70-kDa heat shock family can control and manipulate a host of oncogenic client proteins. This role of Hsp70 in both the folding and degradation of these client proteins makes it a potential drug target for certain forms of cancer. The phenothiazine family of compounds, as well as the flavonoid myricetin, was recently shown to inhibit Hsp70-ATPase activity, whereas members of the dihydropyrimidine family stimulated ATPase function. Akt, a major survival kinase, was found to be under the regulation of Hsp70, and when the ATPase activity of Hsp70 was increased or decreased by these compounds, Akt levels were also increased or decreased. Also, increasing Hsp70 levels concurrent with inhibition of its ATPase function synergistically reduced Akt levels to a greater extent than either manipulation alone, providing new insights about client fate decisions. Akt reductions mediated by Hsp70 inhibitors were prevented when Hsp70 expression was silenced with small interfering RNA. Inhibiting Hsp70 ATPase function produced cytotoxic events only in breast cancer cell lines where Akt dysfunction was previously shown, suggesting therapeutic specificity depending on the Hsp70 client profile. Thus, increasing Hsp70 levels combined with inhibiting its ATPase function may serve to dramatically reduce Akt levels and facilitate cell death in certain types of cancer.     

Hsc70 Rapidly Engages Tau after Microtubule Destabilization

The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7–9 interact with residues 312–342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.     

Strategies for farmers and policy makers to control nitro-gen losses whilst maintaining crop production

The nitrogen (N) cycle is essentially 'leaky'. The losses of small amounts of nitrate to waters and of ammonia and nitrous oxide to the atmosphere are a part of the global biogeo-chemical N cycle. However, intensive agricultural production, industry and vehicle use have more than doubled the amount of 'reactive' N in the environment, resulting in eutrophication, ecosystem change and health concerns. Research has identified agricultural practices that cause large losses of N and, in some cases, developed solutions. This paper discusses the problems of maintaining productivity while reducing N losses, compares conventional with low input (integrated) and organic farming systems, and discusses wider options. It also looks at the need to integrate studies on N with other environmental impacts, set in the context of the whole farm system, to provide truly sustainable agricultural systems.