Effects of Aβ1–42 on the Subunits of KATP Expression in Cultured Primary Rat Basal Forebrain Neurons

ATP-sensitive potassium channels (KATP) play a crucial role in coupling metabolic energy to the membrane potential of cells, thereby functioning as cellular "metabolic sensors." Recent evidence has showed a connection between the amyloid neurotoxic cascade and metabolic impairment. With regard to their neuroprotection in other neuronal preparations, KATP channels may mediate a potential neuroprotective role in Alzheimer's disease (AD). To investigate the effects of Abeta1-42 on the subunits of KATP expression in cultured primary rat basal forebrain cholinergic neurons, primary rat basal forebrain neurons were cultured and evaluated. The subunits of KATP: Kir6.1, Kir6.2, SUR1 and SUR2 expressing changes were observed by double immunofluorescence and immunoblotting when the neurons were exposed to Abeta1-42(2 microM) for different time (0, 24, 72 h). We found a significant increase in the expression of Kir6.1 and SUR2 in the cultured neurons being exposed to Abeta1-42 for 24 h, while Kir6.2 and SUR1 showed no significant change. However, after being treated with Abeta1-42 for 72 h, the expression of the four subunits was all increased significantly compared with the control. These findings suggest that being exposed to Abeta1-42 for different time (24 and 72 h) induces differential regulations of KATP subunits expression in cultured primary rat basal forebrain cholinergic neurons. The change in composition of KATP may contribute to resist the toxicity of Abeta1-42.     

On-chip ligation of multiplexing probe-pairs for identifying point mutations out of dense SNP loci

Detailed analyses of dense single nucleotide polymorphism (SNP) loci within rifampin-resistance determining region (RRDR) are very important for the early assessment of drug resistance of Mycobacterium tuberculosis. A strategy was developed here to specifically identify point mutations out of dense SNP loci by on-chip ligation of multiplexing probe-pairs (MPPs). A probe-pair combines a common probe with a discriminating probe which is covalently attached to a DNA chip. The common probe hybridizes to the discriminating probe via a unique "zip-code complement". The allele-specific part on the 3'-end of the discriminating probe becomes covalently ligated to the adjacent part on the 5'-end of the common probe if and only if a mutation is present. Thus upon zip-code recognition, the process of identifying a mutation of interest is entirely located into corresponding well on the chip. As a consequence, cross-reactions and biased competitive attachments to targets, both of which result from the presence of various multiplexing probes, are greatly minimized. Mutation detection was performed by direct visualization using enzyme-linked assay. The method was demonstrated with an initial set of 24 probe-pairs targeting 22 clinically meaningful mutations within an 81-bp RRDR. 130-bp fragments of the rpoB gene from 15 clinical isolates were identified and were in 100% agreement with results from independent sequencing.     

Zfra is an inhibitor of Bcl-2 expression and cytochrome c release from the mitochondria

Zfra is a small size 31-amino-acid C2H2 zinc finger-like protein, which is known to interact with c-Jun N-terminal kinase 1 (JNK1), WW domain-containing oxidoreductase (WWOX, FOR or WOX1), TNF receptor-associated death domain protein (TRADD) and nuclear factor kappaB (NF-kappaB) during stress response. Here, we show that Zfra became phosphorylated at Ser8 (as determined by specific antibody) and translocated to the mitochondria in response to inducers of mitochondrial permeability transition (MPT) (e.g. staurosporine and betulinic acid). Overexpressed Zfra induced cell death. This event is associated, in part, with increased dissipation of mitochondrial membrane potential (MMP) and increased chromosomal DNA fragmentation. Intriguingly, Zfra significantly downregulated Bcl-2 and yet blocked cytochrome c release from the mitochondria. Overexpression of an S8G-Zfra mutant (Ser8 to Gly8 alteration) could not induce cell death, probably due to its failure of translocating to the mitochondria and causing MMP dissipation. Over-expressed proapoptotic WOX1 induced cytochrome c release from the mitochondria. Zfra bound and blocked the effect of WOX1. Taken together, Ser8 is essential for overexpressed Zfra to exert cell death via the mitochondrial pathway. Zfra downregulates Bcl-2 and induces MMP dissipation but causes no cytochrome c release, indicating a novel death pathway from the mitochondria.     

Involvement of glucose-6-phosphate dehydrogenase in reduced glutathione maintenance and hydrogen peroxide signal under salt stress

Cellular redox homeostasis is essential for plant growth, development as well as for the resistance to biotic and abiotic stresses, which is governed by the complex network of prooxidant and antioxidant systems. Recently, new evidence has been published that NADPH, produced by glucose-6-phosephate dehydrogenase enzyme (G6PDH), not only acted as the reducing potential for the output of reduced glutathione (GSH), but was involved in the activity of plasma membrane (PM) NADPH oxidase under salt stress, which resulted in hydrogen peroxide (H₂O₂) accumulation. H₂O₂ acts as a signal in regulating G6PDH activity and expression, and the activities of the enzymes in the glutathione cycle as well, through which the ability of GSH regeneration was increased under salt stress. Thus, G6PDH plays a critical role in maintaining cellular GSH levels under long-term salt stress. In this addendum, a hypothetical model for the roles of G6PDH in modulating the intracellular redox homeostasis under salt stress is presented.Key words: glucose-6-phosphate dehydrogenase, hydrogen peroxide, reduced glutathione, redox homeostasis, salt stressEnvironmental stresses inevitably induce the production of reactive oxygen species (ROS).¹ Reduced glutathione (GSH) is a key substance in the network of antioxidants that include ascorbate, glutathione, α-tocopherol and a serial of antioxidant enzymes,² which metabolizes H₂O₂ mainly via the ascorbate-glutathione cycle, the most important detoxifying system in plants.³ Thus, the regulatory ability to maintain the cellular GSH balance is crucial to confer the resistance to oxidative stress in plants. However, to our knowledge, the regulatory mechanism on the intracellular GSH-pool equilibrium under environmental stresses has been largely unknown in plants.A main source of GSH is regenerated from its oxidative form (GSSG) via glutathione cycling, which uses NADPH as the reductant.⁴ G6PDH is the key enzyme of pentose phosphate pathway that is responsible for the generation of NADPH.⁵ G6PDH has been shown to play a protective role against ROS in human and animal cells,^6,7 and the enhanced expression of G6PDH could enhance the GSH levels and the ability of resistance to oxidative stress.^5,8 In plants, it has been reported that oxidative stress induced by the elicitor stimulated G6PDH activity in tobacco cells,^9,10 and the GSH-biosynthesis inhibitor or GSH precursor could increase or suppressed G6PDH activity, respectively.¹⁰ Interestingly, after G6PDH activity was inhibited, not only GSH levels dramatically decreased, but the elicitor-induced H₂O₂ accumulation was also completely counteracted.^9,10 Thus, the functions of G6PDH under oxidative stress seem to be involved in these two contradictory courses in cells: the regeneration of GSH as well as H₂O₂ accumulation. The role of G6PDH under environmental stresses remained limited to clarify this, so we studied the G6PDH functions with a series of inhibitor or donor of GSH, H₂O₂ and G6PDH in reed calli under salt stress. Our recent studied clearly demonstrated that G6PDH activity was also simultaneously involved in intracellular GSH maintenance and H₂O₂ accumulation in salt stress. Further studies revealed that a plasma membrane (PM) NADPH oxidase, using NADPH as substrate mainly produced by G6PDH, was mainly responsible for the generation of H₂O₂. And H₂O₂, produced under salt stress, induced the increased G6PDH activity and the enzymes of glutathione cycle, which concomitantly resulted in an increased GSH contents. Foyer and Noctor (2005) suggested that the cellular “oxidative signaling” was made possibly by homeostatic regulation by antioxidant redox buffer.¹¹ Based on these, it can be speculated that G6PDH might play an important role in maintaining the cellular redox signals under salt stress in plants.Our recent work provides a new insight into G6PDH functions under environmental stresses in plants. Growing evidences suggest that PM NADPH oxidase is responsible for H₂O₂ accumulation under stresses,^12,13 and H₂O₂ is involved in various signaling pathways in plants, such as defense gene expression, stomatal closure, root growth, programmed cell death (PCD) and so on.¹¹ In addition, GSH, as a key antioxidant, also influences gene expression associated with biotic and abiotic stress responses to maximum defense.² Recent study also reported that G6PDH was involved in NR-dependent NO production, and thus played a pivotal role in establishing tolerance of red kidney bean roots to salt stress.¹⁴ Therefore, the research work is required to further clarify the regulatory mechanism underlying the roles of G6PDH in the cellular redox homeostasis as well as the related signals under environmental stresses in plants.Based on the results obtained so far, a model for G6PDH functions under salt stress is proposed (Fig. 1). In our model, the increased G6PDH activity is tightly correlated with GSH maintenance and H₂O₂ accumulation through PM NADPH oxidase under salt stress in plants. Under salt stress, H₂O₂ activities the activities of G6PDH and the enzymes in glutathione recycle, which finally result in the enhanced glutathione cycling rate and thus the increased GSH levels. This enhanced antioxidant ability can facilitate to maintain a steady-state level of H₂O₂. Eventually, the properly intracellular redox state is established under salt stress and forms a metabolic interface for signals. Thus, we suggest that G6PDH plays a crucial role in establishing this cellular redox homeostasis under salt stress.Open in a separate windowFigure 1Hypothetical model for the roles of G6PDH under salt stress. Under salt stress, G6PDH activity is involved in both GSH maintenance and H₂O₂ accumulation through PM NADPH oxidase. H₂O₂, as a signal, increases the activities of G6PDH, glutathione (GR) and glutathione peroxidase (GPX), which finally enhance glutathione cycle rate and result in the increased GSH levels. This enhanced antioxidant ability could facilitate to keep H₂O₂ in a steady state for signal in salt stress.     

Quantitative proteomic signature of liver cancer cells: tissue transglutaminase 2 could be a novel protein candidate of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common diseases worldwide, with extremely poor prognosis due to failure in diagnosing it early. Alpha-fetoprotein (AFP) is the only available biomarker for HCC diagnosis; however, its use in the early detection of HCC is limited, especially because about one-third of patients afflicted with HCC have normal levels of serum AFP. Thus, identifying additional biomarkers that may be used in combination with AFP to improve early detection of HCC is greatly needed. A quantitative proteomic analysis approach using stable isotope labeling with amino acids in cell culture (SILAC) combined with LTQ-FT-MS/MS identification was used to explore differentially expressed protein profiles between normal (HL-7702) and cancer (HepG2 and SK-HEP-1) cells. A total of 116 proteins were recognized as potential markers that could distinguish between HCC and normal liver cells. Certain proteins, such as AFP, intercellular adhesion molecule-1 (ICAM-1), IQ motif containing GTPase activating protein 2 (IQGAP2), claudin-1 (CLDN1) and tissue transglutaminase 2 (TGM2), were validated both in multiple cell lines and in 61 specimens of clinical HCC cases. TGM2 was overexpressed in some of the AFP-deficient HCC cells (SK-HEP-1 and Bel-7402) and in about half of the tumor tissues with low levels of serum AFP (17/32, AFP-negative HCC). Trace amounts of TGM2 were found to be expressed in the samples with high serum AFP (26/29, AFP-positive HCC). Moreover, TGM2 expression in liver tissues showed an inverse correlation with the level of serum AFP in HCC patients. Notably, TGM2 existed in the supernatant of the AFP-deficient SK-HEP-1, SMMC-7721 and HLE cells, and it was found to be induced in AFP-producing cells (HepG2) by specific siRNA silence assay. Serum TGM2 levels of 109 HCC patients and 42 healthy controls were further measured by an established ELISA assay; the levels were significantly higher in HCC patients, and they correlated with the histological grade and tumor size. These data suggest that TGM2 may serve as a novel histological/serologic candidate involved in HCC, especially for the individuals with normal serum AFP. These novel findings may provide important clues to identify new biomarkers of HCC and indirectly improve early detection of the disease.     

辣椒细胞质雄性不育花药败育及淀粉粒分布的细胞学观察

用PAS反应对辣椒细胞质雄性不育系8214A和保持系8214B花药中的淀粉粒分布进行研究.在减数分裂前,保持系花药与不育系花药的结构和淀粉粒分布相似.保持系花药减数分裂后,药壁绒毡层细胞开始液泡化并体积增大,在药隔薄壁细胞中积累了许多较小的淀粉粒;在小孢子晚期,绒毡层细胞退化,在药隔薄壁细胞中淀粉粒体积增大;在二胞花粉时期,随着花粉大液泡的消失花粉中出现淀粉粒;花粉成熟时,其细胞质中积累了丰富的淀粉粒.不育系花药减数分裂后,由于药室腔的空间不能扩大,四分体被挤压在一起,最终四分体小孢子败育.不育花药的维管组织发育正常,但较多的淀粉粒积累在药隔薄壁细胞中.该种辣椒雄性不育系中.花粉的败育发生在四分体时期.绒毡层细胞结构异常可能影响糖类物质向药室的正常转运.该种辣椒雄性不育系的绒毡层异常与花粉败育有关.     

High genetic differentiation between the M and S molecular forms of Anopheles gambiae in Africa

Background

Anopheles gambiae, a major vector of malaria, is widely distributed throughout sub-Saharan Africa. In an attempt to eliminate infective mosquitoes, researchers are trying to develop transgenic strains that are refractory to the Plasmodium parasite. Before any release of transgenic mosquitoes can be envisaged, we need an accurate picture of the differentiation between the two molecular forms of An. gambiae, termed M and S, which are of uncertain taxonomic status.

Methodology/Principal Findings

Insertion patterns of three transposable elements (TEs) were determined in populations from Benin, Burkina Faso, Cameroon, Ghana, Ivory Coast, Madagascar, Mali, Mozambique, Niger, and Tanzania, using Transposon Display, a TE-anchored strategy based on Amplified Fragment Length Polymorphism. The results reveal a clear differentiation between the M and S forms, whatever their geographical origin, suggesting an incipient speciation process.

Conclusions/Significance

Any attempt to control the transmission of malaria by An. gambiae using either conventional or novel technologies must take the M/S genetic differentiation into account. In addition, we localized three TE insertion sites that were present either in every individual or at a high frequency in the M molecular form. These sites were found to be located outside the chromosomal regions that are suspected of involvement in the speciation event between the two forms. This suggests that these chromosomal regions are either larger than previously thought, or there are additional differentiated genomic regions interspersed with undifferentiated regions.     

History cleans up messes: The impact of time in driving divergence and introgression in a tropical suture zone

Contact zones provide an excellent arena in which to address questions about how genomic divergence evolves during lineage divergence. They allow us to both infer patterns of genomic divergence in allopatric populations isolated from introgression and to characterize patterns of introgression after lineages meet. Thusly motivated, we analyze genome‐wide introgression data from four contact zones in three genera of lizards endemic to the Australian Wet Tropics. These contact zones all formed between morphologically cryptic lineage‐pairs within morphologically defined species, and the lineage‐pairs meeting in the contact zones diverged anywhere from 3.1 to 5.8 million years ago. By characterizing patterns of molecular divergence across an average of 11K genes and fitting geographic clines to an average of 7.5K variants, we characterize how patterns of genomic differentiation and introgression change through time. Across this range of divergences, we find that genome‐wide differentiation increases but becomes no less heterogeneous. In contrast, we find that introgression heterogeneity decreases dramatically, suggesting that time helps isolated genomes “congeal.” Thus, this work emphasizes the pivotal role that history plays in driving lineage divergence.     

Root characteristics of grafted peppers and their resistance to <Emphasis Type="Italic">Fusarium solani</Emphasis>

Root rot caused by Fusarium solani, is one of the most severe diseases in pepper (Capsicum annuum L.). Grafting has been attempted as an effective means to control the disease, but little is known about the disease resistance mechanism in grafted pepper. Therefore, we investigated the changes of biomass, cell structure, and the secondary metabolism in roots of control (non-grafted pepper) and grafted peppers using cvs. Weishi and Buyeding as rootstocks and the cv. Xinfeng 2 as a scion. After a manual inoculation, less F. solani invaded grafted pepper roots and consequently less serious injury to the root cell ultra-structure compared with the control was found. The roots of grafted pepper infected with F. solani exhibited greater biomass production and root activity than the roots of infected controls. Grafting led to an increased content of salicylic acid, benzoic acid, vanillin, lignin, and polyamines, as well as activities of phenylalanine ammonia lyase, polyphenoloxidase, and peroxidase. These results suggest that grafting improved the resistance of peppers to root rot.