An SRLLR motif downstream of the scissile bond enhances enterokinase cleavage efficiency

In a previous paper, we reported more efficient enterokinase cleavage at a C-terminal non-target LKGDR(201) site compared with an internally sited canonical recognition site, DDDDK(156). When this non-target site was placed internally to replace DDDDK(156) between the thioredoxin moiety and mouse NT-proCNP(1-50), this site was poorly processed leading us to conclude that efficient processing at LKGDR(201) in the first instance was due to its accessibility at the C-terminus of the fusion protein. Subsequently, we reasoned that treatment of thioredoxin-fused NT-proCNP(1-81) would allow us to retrieve full-length NT-proCNP(1-81) without undue processing at the LKGDR(201) site since this non-target site would now be located internally about 36 residues away from the C-terminus and hence not be hydrolyzed efficiently. Surprisingly, ESI-MS data showed that the LKGDR site in thioredoxin-fused human NT-proCNP(1-81) was still very efficiently cleaved and revealed a new but slow hydrolysis site with the sequence RVDTK/SRAAW to yield a peptide consistent with NT-proCNP(58-81). The evidence obtained from these experiments led us to postulate that efficient cleavage at the non-target LKGDR(201) site was not merely influenced by steric constraints but also by the sequence context downstream of the scissile bond. Hence, we constructed variants of thioredoxin-mouse NT-proCNP(1-50) where SRLLR residues (i.e. those immediately downstream from the LKGDR(201) site in NT-proCNP(1-50)) were systematically added one at a time downstream of the internal DDDDK(156) site. To evaluate the relative effects of site accessibility and downstream sequence context on the efficiency of enterokinase cleavage, we have also replaced the native LKGDR(201) sequence with DDDDK(201). Our results showed that incremental addition of SRLLR residues led to a steady increase in the rate of hydrolysis at DDDDK(156). Further variants comprising DDDDK(156)SS, DDDDK(156)SD and DDDDK(156)RR showed that the minimal critical determinants for enhanced enterokinase cleavage are serine in the P1' position followed by a serine or a basic residue, lysine or arginine, in the P2' position. Our data provided conclusive evidence that the influence of downstream sequences on recombinant light chain enterokinase activity was greater than accessibility of the target site at the terminus region of the protein. We further showed that the catalytic efficiency of the native holoenzyme was influenced primarily by residues on the N-terminal side of the scissile bond while being neutral to residues on the C-terminal side. Finally, we found that cleavage of all nine fusion proteins reflects accurate hydrolysis at the DDDDK(156) and DDDDK(201) sites when recombinant light chain enterokinase was used while non-specific processing at secondary sites were observed when these fusion proteins were treated with the native holoenzyme.     

Expression of UNCL during development of periodontal tissue and response of periodontal ligament fibroblasts to mechanical stress in vivo and in vitro

Mutations in two genes, uncoordinated (unc) and uncoordinated-like (uncl), lead to a failure of mechanotransduction in Drosophila. UNCL, the human homolog of unc and uncl, is preferentially expressed in periodontal ligament (PDL) fibroblasts compared with gingival fibroblasts. However, the precise role of UNCL in the PDL remains unclear. The aim of the present study has been to examine whether mechanical stimuli modulate the expression of UNCL in the human PDL in vivo and in vitro and to examine the roles of UNCL in the development, regeneration, and repair of the PDL. We have investigated the expression pattern of UNCL during the development of periodontal tissue and the response of PDL fibroblasts to mechanical stress in vivo and in vitro. The expression of UNCL mRNA and protein increases with PDL fibroblast differentiation from the confluent to multilayer stage but slightly decreases on mineralized nodule formation. UNCL has also been localized in ameloblasts and adjacent cells, differentiating cementoblasts, and osteoblasts of the developing tooth. Strong distinct UNCL expression has further been observed in the differentiating cementoblasts of the tooth periodontium at the site of tension after orthodontic tooth movement. Application of cyclic mechanical stress on PDL fibroblasts increases the expression of UNCL mRNA. These results indicate that UNCL plays important roles in the development, differentiation, and maintenance of periodontal tissues and also suggest a potential role of UNCL in the mechanotransduction of PDL fibroblasts.This work was supported by a grant from the Korea Health 21R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH08-0001).     

A disposable on-chip phosphate sensor with planar cobalt microelectrodes on polymer substrate

Disposable microsensors on polymer substrates consisting of fully integrated on-chip planar cobalt (Co) microelectrodes, Ag/AgCl reference electrodes, and microfluidic channels have been designed, fabricated, and characterized for phosphate concentration measurement in aqueous solution. The planar Co microelectrode shows phosphate-selective potential response over the range from 10(-5) to 10(-2)M in acidic medium (pH 5.0) for both inorganic (KH(2)PO(4)) and organic (adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphates (ADP)) phosphate compounds. This microfabricated sensor also demonstrates significant reproducibility with a small repeated sensing deviation (i.e. relative standard deviation (R.S.D.)<1%) on a single chip and a small chip-to-chip deviation (i.e. R.S.D.<2.5%). Specifically, while keeping the high selectivity, sensitivity, and stability of a conventional bulk Co-wire electrode, the proposed phosphate sensor yields advantages such as ease of use, cost effectiveness, reduced analyte consumption, and ease of integrating into disposable polymer lab-on-a-chip devices. The capability to sense both inorganic and organic phosphate compounds makes this sensor applicable in diverse areas such as environmental monitoring, soil extract analysis, and clinical diagnostics.     

Intraguild predation and interference by the mealybug predator Cryptolaemus montrouzieri on the parasitoid Leptomastix dactylopii

The compatibility of the encyrtid parasitoid Leptomastix dactylopii with the coccinellid beetle Cryptolaemus montrouzieri against the citrus mealybug, Planococcus citri, is determined by the extent of intraguild predation and interference by the predator. We tested the preference of the adults and fourth-instar larvae of C. montrouzieri for healthy mealybugs and parasitized mealybugs harboring 1-, 4-, 7- and 14-day-old parasitoid larvae. The experiments were conducted in no-choice (only unparasitized mealybugs or parasitized mealybugs of one parasitoid age were offered at one time) and choice (unparasitized and parasitized mealybugs of a specific age were offered simultaneously) tests. Both the adults and larvae of C. montrouzieri fed on unparasitized and parasitized mealybugs but strongly discriminated against the hardened mummies (14 days old). We also investigated the influence of the presence of C. montrouzieri to the foraging effectiveness of L. dactylopii. The level of parasitism by L. dactylopii was reduced from about 13 to 6% when the number of the C. montrouzieri was increased from zero to four. We recommended that the releases of C. montrouzieri should be conducted 14 days after the releases of L. dactylopii to reduce intraguild predation on the parasitized mealybugs and to avoid interference with the foraging parasitoids.     

Dietary antioxidants and primary prevention of age related macular degeneration: systematic review and meta-analysis

Objective To evaluate the effectiveness of dietary antioxidants in the primary prevention of age related macular degeneration (AMD).Design Systematic review and meta-analysis.Data sources Search of seven databases without limits on year or language of publication, and retrieval of references in pertinent reviews and articles.Methods Two reviewers independently searched the databases and selected the studies, using standardised criteria. Randomised clinical trials and prospective cohort studies were included. Of the 4192 abstracts initially identified, 12 studies (nine prospective cohort studies and three randomised clinical trials) met the selection criteria and were included. Data extraction and study quality evaluation were independently reviewed, using standardised criteria. Results were pooled quantitatively using meta-analytic methods.Results The nine prospective cohort studies included 149 203 people, with 1878 incident cases of early AMD. The antioxidants investigated differed across studies, and not all studies contributed to the meta-analysis of each antioxidant. Pooled results from prospective cohort studies indicated that vitamin A, vitamin C, vitamin E, zinc, lutein, zeaxanthin, α carotene, β carotene, β cryptoxanthin, and lycopene have little or no effect in the primary prevention of early AMD. The three randomised clinical trials did not show that antioxidant supplements prevented early AMD.Conclusions There is insufficient evidence to support the role of dietary antioxidants, including the use of dietary antioxidant supplements, for the primary prevention of early AMD.     

Synthesis of the 5-phosphono-pent-2-en-1-yl nucleosides: a new class of antiviral acyclic nucleoside phosphonates

A new class of acyclic nucleoside phosphonates, the 5-phosphono-pent-2-en-1-yl nucleosides and their hexadecyloxypropyl esters, were synthesized from butyn-1-ol. Only the hexadecyloxypropyl esters showed antiviral activity against herpes simplex virus type 1, in vitro. Hexadecyloxypropyl 1-(5-phosphono-pent-2-en-1-yl)-thymine was the most active and selective compound among the synthesized nucleotides with an EC50 value of 0.90 microM.     

植物开花的组蛋白甲基化调控分子机理

开花是指植物从营养生长转变到生殖生长的生理过程,是植物个体发育和后代繁衍的中心环节,既受遗传基础决定,同时又受到温度和光周期等多种环境因素的调控。在拟南芥中,已经分离了大量的与开花相关的基因,从遗传学上已初步形成了一个开花调控的网络。组蛋白甲基化是植物发育过程的重要调节方式,近年来关于其参与开花调控的研究有了重要进展。本文综述了具有代表性的组蛋白H3赖氨酸甲基化修饰参与调控植物开花发育的机制,提出该研究领域的发展方向和前景。     

Effects of chronic heat stress on immune responses of the foot-and-mouth disease DNA vaccination

The main purpose of this study was to assess the effects of chronic heat stress (CHS) on humoral and cellular responses of DNA vaccination. Mice with the CHS were exposed to a temperature set at 38 +/- 1 degrees C, 2h per day, for 35 days, and mice with thermoneutral (TN) temperature were maintained at 24 +/- 1 degrees C for the same period of time. Both groups of mice were immunized with a DNA vaccine-expressed viruscapsid protein 1 (VP1) of foot-and-mouth disease virus (FMDV), and we tested their antigen-specific humoral and cellular responses during the treatments. Compared with the TN group, titers of total Imunoglobulin G (IgG) and IgG1 and expression of interleukin 4 (IL-4) in CD4(+) cells of CHS group were not affected significantly. In contrast, the levels of IgG2a, T cell proliferations, and expression of interferon-gama (IFN-gamma) in both CD4(+) and CD8(+) cells were suppressed significantly, and cytotoxic T-lymphocyte (CTL) responses in vivo were also weakened by the CHS condition. These results indicate that the CHS treatment has negatively affected the immune responses of DNA vaccination and particularly impaired to the cell-mediated responses. It suggests that vaccination in animals is affected by the changes of ambient temperature.