特异性人端粒酶催化亚单位基因小干扰RNA对HeLa细胞生长的抑制作用

通过设计并化学合成人端粒酶催化亚单位(hTERT)特异性siRNA,观察其对hTERT表达水平及肿瘤细胞生长的影响。将hTERT-siRNA以脂质体法转染入HeLa细胞,应用RT-PCR、实时定量TRAP、Western印迹、软琼脂克隆形成实验、荷瘤裸鼠肿瘤内注射等方法检测细胞内hTERTmRNA、蛋白质表达水平及对肿瘤细胞生长的影响。RT-PCR、实时定量TRAP和Western印迹的结果显示hTERT-siRNA明显降低了HeLa细胞内hTERT的mRNA及蛋白质表达水平并伴随有端粒酶活性的下降。克隆形成实验表明hTERT-siRNA组的体外肿瘤形成能力受到抑制。荷瘤裸鼠肿瘤内注射hTERT-siRNA使肿瘤平均体积显著小于对照组。TUNEL凋亡检测表明hTERT-siRNA转染组的凋亡率明显高于对照组。研究表明hTERT特异性siRNA可以明显抑制HeLa细胞内hTERT的表达水平,对其生长有明显抑制作用,是一种有前途的肿瘤治疗新方法。     

Protons @ interfaces: Implications for biological energy conversion

The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from “pulsed” experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the “sources” and “sinks”, the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.     

Developing novel hCT derived cell-penetrating peptides with improved metabolic stability

Many promising therapeutics are currently awaiting their clinical application. Due to their low capability of cell membrane crossing, these compounds do not reach their site of action. One way to overcome this problem might be the fusion of these agents to cell-penetrating peptides (CPP), which are able to shuttle various cargoes across cellular membranes. One disadvantage in using CPP in drug delivery is their low metabolic stability. The aim of our work was to increase the proteolytic resistance of the CPP hCT(9-32), a truncated C-terminal fragment of human calcitonin. Thus, we synthesised six modified N-terminally carboxyfluorescein labelled hCT(9-32) derivatives by replacing positions 12 and/or 16 of hCT(9-32) with either N-methylphenylalanine or d-phenylalanine, respectively. By using confocal laser scanning microscopy we showed that the modifications did neither affect the peptide internalisation efficiency in HeLa nor HEK 293T cells. The metabolic stability of the peptides was investigated in human blood plasma and HEK 293T cell culture supernatant. To analyse the degradation patterns, we used RP-HPLC and MALDI-TOF mass spectrometry. However, we found for all of the new derivatives high metabolic stabilities. In blood plasma, the half-lives for five of the six peptides increased compared to unmodified hCT(9-32). The degradation patterns showed a distinct stabilisation in the N-terminal part of the modified peptides, in the C-terminal part, we found some cleavage to a minor extent. Furthermore, we studied the conformation of the peptides by CD spectroscopy and demonstrated that they possess no cell toxicity. Since our metabolically more stable compounds are still able to pass the cell membrane they provide powerful tools as drug delivery vectors.     

Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-π interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular α-helices and β-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

The contingency of parameters of human encephalograms and Schumann resonance electromagnetic fields revealed in monitoring studies

The contingency of variations in amplitude—frequency parameters of the main modes of extremely-low-frequency resonances of an ionospheric waveguide (Schumann resonances) and changes in human encephalograms in the frequency range of 6–16 Hz has been studied. The results obtained with the use of synchronized monitoring suggest that such contingency, expressed as indices of cross-correlation function, is statistically significant, varying from 0.12 to 0.65 at α = 0.95. It has been established that contingency is largely determined by the current level of solar and geomagnetic activity.     

The Future of Tropical Forest Species1

Deforestation and habitat loss are widely expected to precipitate an extinction crisis among tropical forest species. Humans cause deforestation, and humans living in rural settings have the greatest impact on extant forest area in the tropics. Current human demographic trends, including slowing population growth and intense urbanization, give reason to hope that deforestation will slow, natural forest regeneration through secondary succession will accelerate, and the widely anticipated mass extinction of tropical forest species will be avoided. Here, we show that the proportion of potential forest cover remaining is closely correlated with human population density among countries, in both the tropics and the temperate zone. We use United Nations population projections and continent‐specific relationships between both total and rural population density and forest remaining today to project future tropical forest cover. Our projections suggest that deforestation rates will decrease as population growth slows, and that a much larger area will continue to be forested than previous studies suggest. Tropical forests retracted to smaller areas during repeated Pleistocene glacial events in Africa and more recently in selected areas that supported large prehistoric human populations. Despite many caveats, these projections and observations provide hope that many tropical forest species will be able to survive the current wave of deforestation and human population growth. A strategy to preserve tropical biodiversity might include policies to improve conditions in tropical urban settings to hasten urbanization and preemptive conservation efforts in countries with large areas of extant forest and large projected rates of future human population growth. We hope that this first attempt inspires others to produce better models of future tropical forest cover and associated policy recommendations.     

Richness and structure of plant communities in temporary pools from western Morocco: influence of human activities

Temporary pools are numerous in coastal plains of Atlantic Morocco and have a rich and diverse flora. These habitats are increasingly under pressure by man impact through grazing by domestic livestock and the development of annual crops in their catchments. The objectives of this work were to evaluate the species richness and the species composition of the vegetation of a sample of 30 pools in this region, in order to assess the structure of the vegetation within pools and to evaluate the role of environmental and anthropogenic factors in their richness and species composition. The results highlighted the species richness of the pools with 300 species found among which annual species were heavily dominant. The structure of the vegetation varied within pools according to a topographical/flooding gradient and between pools in relation with the land use in the surrounding areas. Three vegetation belts were recognized from the centre to the periphery of the pool. The total species richness per pool was not found significantly related to environmental or anthropogenic factors. The species characteristics of the pools were found mostly in the centre and their number affected by hydrological factors and land use in the pool and in the surrounding areas. The species characteristics of woodland habitats and of agriculture crops were found mostly in the peripheral zone. Temporary pools maintain specific communities of both aquatic and amphibious species, probably because of the selection induced by water level variations. The peripheral zone, although often dominated by terrestrial species, is clearly interpreted as part of the pool. It contains amphibious species highly characteristic of the pool for which the irregular flooding is a key environmental factor which decreases competition.     

人为干扰对黄土高原子午岭油松人工林土壤物理性质的影响

研究了放牧、收集枯落物及清灌等人为干扰活动对黄土高原于午岭油松林土壤结构及抗蚀性的影响。结果表明,随人为干扰强度的增加,0～50cm土壤中砂粒含量比无干扰时分别增加了11.83％、37．80％和51．60％;粉粒下降了8．16％、11．83％和15．55％;粘粒下降了8．10％、20．84％和30．72％,土壤表现出粗骨化趋势;〉0.25mm水稳性团聚体含量比无干扰林地分别下降了16．59％、43.12％和61．13％。〉1．0mm的团聚体含量仅为无干扰林地土壤的27．78％和24．34％,1．0—0．25mmm的团聚体下降幅度较小;〉0．05mm微团聚体的比例分别下降了19．39％、32．62％和33．47％。而〈0．05mm微团聚体所占比例随干扰程度的增加而大幅度上升。土壤容重增加了0．11-0．41g／cm^3。土壤总孔隙度分别降低了13．64％、25．47％和39．14％,毛管孔隙下降了7．79％、11．54％和29．32％,非毛管孔隙下降了28．47％、60．79％和64．08％。说明表层土壤非毛管孔隙对人为干扰更为敏感。最大持水量分别下降23．42％、37．15％和52．92％;毛管持水量下降33．79％、43．01％和52．22％;自然含水量下降31．03％、39．34％和46．28％。饱和持水量下降16．14％、28．80％和49．68％;田间持水量下降了12．39％、33．92％和47．47％;土壤有效水含量下降了9．55％、20．55％和58．91％。土壤前3min初渗率下降了38．74％、51．45％和63．23％;稳渗速率下降了54．06％、71．63％和84．10％,相应地受人为干扰林地前30min累计人渗量也较未受人为干扰林地土壤分别低48．15％、65．93％和73．35％。饱和导水率较对照下降了8．73％、33．33％和51．00％。土壤的结构系数。由79．12％下降到27．32％。团聚度由59．48％下降到11．11％,分散率上升了l倍多,分散系数上升了4倍多。土壤枯落物层及有机质的减少是引起土壤物理性质恶化的主要原因,其次是放牧和踏实等活动。     

Evaluation of the phenotypic characteristics of coliform bacteria recovered with two methods: the European Drinking Water Directive reference method and the Colilert 18/Quanti-Tray™ system

Summary The taxonomy of the species belonging to Enterobacteriaceae has undergone a series of revisions. As a consequence, the new classification has caused the analytical detection methods to be updated. According to the European Drinking Water Directive 98/83/CE coliforms/Escherichia coli have to be determined with the ISO 9308-1 reference method. Many technical drawbacks of the procedure as well as limitations regarding the recent taxonomy of coliforms have been pointed out by laboratories working in water quality control. In our investigation, water was analyzed in parallel with the reference method and the rapid Colilert 18/Quanti-Tray™ system. Phenotypic characteristics of isolates were recorded for the verification of the response of the two methods to the new taxonomic approach. Results obtained with the ISO standard confirmed the limitations of the test. In fact, in addition to difficulties linked to the readability of results, it failed to detect a significant proportion of coliforms and E. coli in water. Furthermore, it allowed the growth of microorganisms belonging to other groups. The Colilert 18/Quanti-Tray™ system detected a qualitatively and quantitatively higher number of target microorganisms. It also provided results in a shorter time, allowing the simultaneous detection of E. coli and coliforms with no further confirmation steps.     

Characterization of TonB Interactions with the FepA Cork Domain and FecA N-terminal Signaling Domain

The mechanism of TonB dependent siderophore uptake through outer membrane transporters in Gram-negative bacteria is poorly understood. In an effort to expand our knowledge of the interaction between TonB and the outer membrane transporters, we have cloned and expressed the FepA cork domain (11–154) from Salmonella typhimurium and characterized its interaction with the periplasmic C-terminal domain of TonB (103–239) by isotope assisted FTIR and NMR spectroscopy. For comparison we also performed similar experiments using the FecA N-terminal domain (1–96) from Escherichia coli which includes the conserved TonB box. The FepA cork domain was completely unfolded in solution, as observed for the E. coli cork domain previously [Usher et al. (2001) Proc Natl Acad Sci USA 98, 10676–10681]. The FepA cork domain was found to bind to TonB, eliciting essentially the same chemical shift changes in TonB C-terminal domain as was observed in the presence of TonB box peptides. The FecA construct did not cause this same structural change in TonB. The binding of the FepA cork domain to TonB-CTD was found to decrease the amount of ordered secondary structure in TonB-CTD. It is likely that the FecA N-terminal domain interferes with TonB-CTD binding to the TonB box. Binding of the FepA cork domain induces a loss of secondary structure in TonB, possibly exposing TonB surface area for additional intermolecular interactions such as potential homodimerization or additional interactions with the barrel of the outer membrane transporter.