Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97–118)

Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97–118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1–131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1–131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.     

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.     

N-Glycan-dependent and -independent Quality Control of Human δ Opioid Receptor N-terminal Variants

Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys²⁷ to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys²⁷ reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys²⁷, pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys²⁷ precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys²⁷ variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions.     

Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Mimosine is an effective cell synchronization reagent used for arresting cells in late G₁ phase. However, the mechanism underlying mimosine-induced G₁ cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mm mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G₁-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.     

基因快速检测仪的自动进样控制系统的研制

基因检测是遗传工程的重要技术之一,基因检测技术的自动化对工程的研究具有重要意义,而自动进样控制系统是基因快速检测仪的重要组成部分。该系统的硬件部分主要由液位检测电路及其接口电路组成;软件部分主要由VisualC 6．0编程对硬件实现其自动控制功能。该系统主要包括：控制清洗通道流程、排除废流流程和进样流程等功能,工作模式可根据人机对话方式设定,并能将扩增反应后的试样自动送到基因检测池中进行检测。系统具有快速、操作方便、智能化程度高、准确性高等特点。     

菌根及其在荒漠化土地恢复中的应用

对菌根的作用及其荒漠化对其影响,以及菌根在荒漠化土地恢复中的应用进行了探讨,指出菌根不仅能够促进植物个体养分的吸收,提高植物光合作用,增强植物抗旱、抗盐性,而且能够调节群落内植物间的关系、群落的演替轨道及其生物多样性、耕作、灌溉、过度放牧和开矿等诱发土地荒漠化过程的人类活动直接影响菌根的建立和生存,采用外来菌引入及原来残存菌培育,在恢复区建立丛状菌根植物,菌木化菌根造林及开矿土地管理中表土的存贮等方式都会有力地促进恢复地菌根的建立,并可缩短恢复周期。     

A Proposed Mode of Action of Oil-Based Formulations of a Microbial Herbicide

Formulation of some microbial herbicides in emulsions of vegetable oil can significantly reduce their dependency on long dew periods to allow infection and control of the host weed. Examination of leaves of Viola arvensis , by a range of microscopy techniques, after spraying with an oilin-water emulsion formulation of the potential microbial herbicide Mycocentrospora acerina , suggests that the effect arises as a result of inversion of water into both oil droplets on the leaf surface and oil that penetrates into the leaf tissue. This oil probably mixes with aqueous components from the leaf tissue and, in doing so, forms an invert emulsion. Wesuggest that this supplies the water required by the fungus to initiate and sustain growth so that infection can result.     

Screening Isolates of Hirsutella Species for Biocontrol of Heterodera glycines

The fungi, Hirsutella rhossiliensis and Hirsutella minnesotensis, are two endoparasites of second-stage juveniles (J2) of the soybean cyst nematode (SCN), Heterodera glycines. The objective of this study was to screen for effective fungal isolates to control the nematode in laboratory and greenhouse assays. A total of 93 isolates of H. rhossiliensis and 25 isolates of H. minnesotensis were evaluated for parasitism of SCN J2 on cornmeal agar. Percentage of SCN J2 parasitized by the fungi varied among the fungal isolates. Most H. rhossiliensis isolates parasitized a high percentage of J2. The isolates of H. rhossiliensis obtained from bacteria-feeding nematodes, however, generally did not parasitize J2 on agar. H. minnesotensis parasitized fewer J2 on agar than did H. rhossiliensis . Forty isolates of H. rhossiliensis and four isolates of H. minnesotensis that parasitized a high percentage of J2 on agar were evaluated for their biocontrol potential in soil treated with microwave heating. Most isolates selected from the agar assay also parasitized a high percentage of J2 in the soil but there was variation among isolates. Pathogenicity of 14 isolates of H. rhossiliensis and four isolates of H. minnesotensis to the SCN was also investigated in the greenhouse using untreated field soil. Most isolates of H. rhossiliensis reduced SCN population density and increased plant growth when compared with 1% corn-grits control (culture media). One isolate (OWVT-1) of H. rhossiliensis reduced the SCN egg density by 95% and J2 density by 98% when compared with the control. Isolates of H. minnesotensis, however, neither reduced SCN density nor increased plant growth in the greenhouse.     

Biological Control of Thrips on Ornamental Crops: Interactions Between the Predatory Mite Neoseiulus cucumeris (Acari: Phytoseiidae) and Western Flower Thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), on Cyclamen

Difficulties in controlling outbreaks of Western flower thrips, Frankliniella occidentalis, have obstructed the widespread adoption of biological control in many ornamental crops. The efficacy of the predatory mite, Neoseiulus cucumeris, in controlling F. occidentalis on two cultivars of cyclamen was tested in glasshouse experiments. The establishment and development of F. occidentalis populations was compared in three treatment introductions of N. cucumeris (50, 200 and 350 mites m -2 per week) and an untreated control. F. occidentalis were sampled in the flowers over eight weeks and counted into different life stages. No differences were observed between the two cultivars. All treatments with the predator resulted in a decline in numbers of F. occidentalis compared to the untreated control. Although the proportion of first instar F. occidentalis was similar in all treatments, the level of control varied with the number of N. cucumeris introduced. Lower populations of F. occidentalis, combined with a more rapid decline in their numbers, were observed at the 200 and 350 mites m -2 rates. Numbers of F. occidentalis remained low in the 350 N. cucumeris m -2 rate and the proportion of second instar F. occidentalis in the samples was consistently lower than in the other treatments. Trap counts of adult F. occidentalis were strongly correlated with the numbers of both adult and total F. occidentalis in flower samples. High inoculative releases of N. cucumeris early in the flowering cycle followed by frequent low introductions of predators should provide a strong basis for preventative control of F. occidentalis and other thrips species on cyclamen.