Modular determinants of antimicrobial activity in platelet factor-4 family kinocidins

Mammalian platelets contain an array of antimicrobial peptides, termed platelet microbicidal proteins (PMPs). Human and rabbit PMPs include known chemokines, such as platelet factor-4 (hPF-4); PMP-1 is the rabbit orthologue of hPF-4. Chemokines that also exert direct antimicrobial activity have been termed kinocidins. A consensus peptide domain library representing mammalian PF-4 family members was analyzed to define structural domains contributing to antimicrobial activity against a panel of human pathogens. Secondary conformations were assessed by circular dichroism spectrometry, and molecular modeling was employed to investigate structural correlates of antimicrobial efficacy. Antimicrobial activity against isogenic peptide-susceptible or -resistant Staphylococcus aureus, Salmonella typhimurium, and Candida albicans strain pairs mapped to the C-terminal hemimer (38-74) and modular domains thereof (49-63 and 60-74). Increasing electrostatic charge and steric bulk were general correlates of efficacy. Structural data corroborated spatial distribution of charge, steric bulk and putative secondary structure with organism-specific efficacy. Microbicidal efficacies of the cPMP antimicrobial hemimer and C-terminal peptide (60-74) were retained in a complex human-blood biomatrix assay. Collectively, these results suggest that modular determinants arising from structural components acting independently and cooperatively govern the antimicrobial functions of PF-4 family kinocidins against specific target pathogens.     

In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates

Background

The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins.

Methods

We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods.

Results

All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)₂ form represents the most favorable architecture for parasite P-proteins.

Conclusion

The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1–P2 heterodimeric intermediate.

General significance

On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.     

Occurrence of Pinus sibirica Du Tour in Da Hinggan Mountains

Based on the morphological and anatomical characteristics of vegetative organs,as well as the ecology and geography, Zhao Guang-yi et al. have reported the occurrence of siberian pine(Pinus sibirica) in the Da Hinggan Mountains continuously since 1981, that arouses disputing, The pine cones of the disputing pine were collected in the Mangui Forestry Bureau in the Da Hinggan Mountains, Northeast China, in autumn of 1989. By comparing it with those of Pinus sibirica and P. koraiensis it is proved that the disputing pine in the Da Hinggan Mountains is P. sibirica, but not P. koraiensis. And the 30-year old wrong name of “Mohe Korean Pine” has been corrected. A map showing the distribution of the P. koraiensis and P. sibirica in the DaHinggan Mountains and its adjacent regionswas drawn according to the 10-year investigation.     

Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer’s Disease

Proteolytic truncation of microtubule associated human (h) Tau protein by caspase-3 at the carboxy (C) terminus has been linked to the pathogenesis of Alzheimer’s Disease (AD). This cleavage likely occurs between Asp⁴²¹↓Ser⁴²² leading to the formation of 421-mer truncated Tau protein which has been found to be present as aggregate in high level after phosphorylation in mortal AD brain tissue compared to normal. At least 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed in hTau and a selected number of them have been implicated in hTau aggregation following latter’s proteolytic truncation. Interestingly, it is further noted that Ser⁴²² residue present in the P1′ position of hTau caspase-3 cleavage region is a potential phosphorylation site. So we became interested to examine in vitro the effect of phospho-Ser⁴²² residue on hTau cleavage by caspase-3 which is a crucial upstream event associated with hTau self-assembly leading to AD pathogenesis. The goal of this project is to study in vitro the caspase-3 cleavage site of hTau protein and to examine the kinetics of this cleavage following Ser⁴²² phosphorylation and treatment with caspase-3 inhibitors. This is achieved by designing peptides from the sequence of hTau protein containing the proposed caspase-3 cleavage region. Peptides were designed from 441-mer major human Tau protein sequence that encompasses the proposed caspase-3 cleavage site [Asp⁴²¹↓Ser⁴²²]. Corresponding phospho-, dextro-Ser⁴²² and dextro-Asp⁴²¹ analogs were also designed. Peptides were synthesized by solid phase chemistry, purified and fully characterized by mass spectrometry. These were then incubated with recombinant caspase-3 enzyme under identical condition for digestion and analyzed for cleavage by mass spectrometry and RP-HPLC chromatograms. Our results indicated that while the control peptide is efficiently cleaved by caspase-3 at Asp⁴²¹↓Ser⁴²² site producing the expected N- and C-terminal fragment peptides, the corresponding phospho-Ser⁴²² peptide remained completely resistant to the cleavage. Substitution of Asp⁴²¹ by its dextro isoform also blocks peptide cleavage by caspase-3. However substitution of Ser⁴²² by its dextro isoform in the peptide did not affect the cleavage significantly. The above results were further confirmed by caspase-3 digestion experiment in the presence of varying amounts of caspase-3 inhibitor (Ac-DQVD-aldehyde) which was found to block this cleavage in a highly effective manner. Our results highlighted the crucial significance of Ser⁴²² phosphorylation and suggest that the kinase associated with this Ser-phosphorylation may protect Tau from aggregation. Thus specific promoters/activators of this kinase may find useful therapeutic benefits in arresting Tau truncation by caspase-3 and the progression of AD. In addition our data demonstrated that Tau-peptides where Ser⁴²² or Asp⁴²¹ are substituted by their respective dextro isomers, exhibit different cleavage kinetics by caspase-3 and this may have important implications in therapeutic intervention of Tau aggregation and associated AD.     

流程再造在达芬奇机器人辅助手术中的应用

目的探讨达芬奇机器人辅助手术中应用流程再造对患者的影响。方法对我院实施达芬奇机器人辅助手术患者108例进行流程再造,观察流程再造前后手术开台时间,医护人员以及患者满意度,术后并发症和住院时间情况。结果流程再造后手术开台时间和并发症均有降低,医护人员满意度和患者满意度均有提高,与流程再造前比较差异均有统计学意义(均P0.05);流程再造前后患者的住院时间比较,差异无统计学意义(P0.05)。结论在达芬奇机器人辅助手术中应用流程再造,能缩短达芬奇机器人辅助手术开台时间,提高医护人员以及患者满意度,减少了患者术后并发症。     

基于脑－肠轴理论探讨重症胰腺炎脑损伤“脑病治肠”的科学内涵

胰性脑病（pancreatic encephalopathy,PE）是重症胰腺炎患者病死的主要原因,目前尚无有效治疗手段。临床运用通腑泻下法进行“脑病治肠”的疗效确切,但其现代医学机制尚未完全阐明。肠道菌群与PE脑损伤的发生发展密切相关,且是脑－肠轴的核心通路,可通过迷走神经直接影响脑功能。通腑泻下代表方剂大承气汤可调节中枢神经系统炎症介质的释放,减轻PE脑损伤。以肠道菌群为切入点,从脑－肠轴调控机制的关键环节入手,全面分析肠道菌群构成、功能丰度及优势菌群,观察到胆碱能抗炎通路（cholinergic anti-inflammatory pathway,CAP）的活化程度影响中枢神经系统炎症因子的释放,最终改变PE脑损伤。本文阐述大承气汤通过肠道菌群调节CAP抗中枢神经系统炎症的分子机制,以期为中医以肠道菌群为靶点治疗PE提供理论依据,也为“脑病治肠”提供有力支持。

     

The Characterization of Rennin Action on κ-Casein Using CM-Cellulose

Rennin action on κ-Casein was studied using the CM-cellulose method which determines the amount of para-κ-casein formed during the enzymatic hydrolysis of κ-casein. The reaction rate was measured as a function of the time and enzyme concentration. A Km value of 8.9 ×10^?4m and a V value of 1.2 × 10^?4 M/min were obtained under the assay condition used in this study. The maximum initial rate of para-κ-casein formation occurred at pH 5.0 and 50°C. The present study also demonstrated that the CM-cellulose method is useful for measuring the rennin activity on κ-casein.     

大兴安岭林区不同植被对冻土地温的影响

植被常常通过反射太阳辐射、遮阳、蒸腾散热、阻风挡雪、保水吸水等来影响下伏多年冻土.但是不同的植被类型,对下伏冻土热状况的影响也不尽相同.为了探讨大兴安岭林区不同植被对冻土的影响,选取大兴安岭森林生态站实验区杜香-真藓-落叶松林、真藓-落叶松林、塔头-落叶松林、柴桦落叶松林和伊图里河镇原冻土观测场塔头湿地5种典型林型,分析不同林型对冻土的温度和冻融作用的影响.研究发现不同林型的不同组分,由于反射率、覆盖度和根系吸水能力的差别,使得各种林型下的地面温度也不相同.在夏季,月平均地面温度从高到低依次为真藓-落叶松林、杜香-真藓-落叶松林、伊图里河塔头湿地、柴桦落叶松林和塔头落叶松林.由于塔头落叶松林存在乔木层和灌木层,与伊图里河塔头湿地相比,8月份平均地面温度差值低10℃以上.柴桦落叶松林两个钻孔的对比实验表明,铲除地表植被会使活动层0.8 m以上部分的地温升高,并且主要发生在8、9、10月份.对冻土而言,林区植被暖季降温的贡献大于冷季增温的贡献.另外,塔头一落叶松林根系吸水能力最强,这种林型下的土壤开始融化和冻结的日期最晚,冻结初期地面降温速率为0.1 ℃/d,而0.2 m以下降温速率几乎为零.同样柴桦落叶松林的塔头根系吸水能力使得其0.5 m和0.8 m的降温速率低于除塔头落叶松林外的其他林型,但是地面上植物的凋零和枯萎会加快地面的冻结速率.真藓-落叶松林的乔灌木层发育不好,地面降温速率大于杜香-真藓-落叶松林和伊图里河塔头湿地,而伊图里河塔头湿地由于没有乔灌木层的庇护,地面以下的降温速率高于其他林型.     

Estimation of Förster's distance between two ends of Dps protein from mycobacteria: distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.     

Responses of radial growth to temperature in Larix gmelinii of the da Hinggan Ling under climate warming北大核心CSCD

Aims The Da Hinggan Ling is amongst the areas in China susceptible to climate warming. The objective of this study is to determine the responses of radial growth to temperature variations in Larix gmelinii growing in different parts of the Da Hinggan Ling in the process of climate warming, by using dendrochronological techniques. Methods We collected tree-ring samples from the southern, the middle and the northern parts of the main Da Hinggan Ling, developed site-specific ring-width chronologies, and synthesized tree-ring indices of the southern, the middle and the northern parts of the study area according to the first principal component loading factors for each chronology. The relationships between radial growth in L. gmelinii and temperature variations were determined with correlation analysis, and the differences in the responses of radial growth to temperature variations among various parts were analyzed and compared with principle component analysis. Important findings There were notable discrepancies in the effects of temperature variations on radial growth in L. gmelinii between the southern and the northern parts of the study area (the middle part > the northern part > the southern part). In the southern part, the mean monthly temperature between the previous November and April of the current year had a significant relationship with tree-ring indices (p < 0.05). In the middle part, the mean monthly temperature during March and October of the current year had a significant relationship with tree-ring indices (p < 0.05), and so did the mean monthly temperature during June and August of the previous year (p < 0.05). The mean monthly temperature during April and May of the current year had a highly significant relationship with tree-ring indices in the northern part (p < 0.01). This study suggests that the warmer and drier regional climate condition caused by elevated temperature has resulted in that soil moisture becomes the main factor limiting the radial growth, and the relationship between tree growth and temperature variations signified with aggravated soil drought under climate warming. The productivity in L. gmelinii as reflected by basal area increment experienced a shift response from cold stress to water stress. In addition, the radial growth in L. gmelinii in the Da Hinggan Ling will likely to show a declining trend in the southern and the middle parts, and an increasing trend in the northern part, in response to rapid warming in the coming decades.