Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous. Such diversity has hindered identification of known or expected UDG-inhibitory activities in other genomes. The structural basis for UDG inhibition by ugi is well characterized; yet, paradoxically, the structure of the unbound p56 protein is enigmatically unrevealing of its mechanism. To resolve this conundrum, we determined the structure of a p56 dimer bound to UDG. A helix from one of the subunits of p56 occupies the UDG DNA-binding cleft, whereas the dimer interface forms a hydrophobic box to trap a mechanistically important UDG residue. Surprisingly, these p56 inhibitory elements are unexpectedly analogous to features used by ugi despite profound architectural disparity. Contacts from B-DNA to UDG are mimicked by residues of the p56 helix, echoing the role of ugi’s inhibitory beta strand. Using mutagenesis, we propose that DNA mimicry by p56 is a targeting and specificity mechanism supporting tight inhibition via hydrophobic sequestration.     

Bispecific antibody process development: Assembly and purification of knob and hole bispecific antibodies

Production of knob and hole dual light chain bispecific antibodies poses several unique challenges for development of a feasible industrial scale manufacturing process. We developed an efficient process for the assembly and purification of knob and hole dual light chain bispecific antibodies. Two distinct half‐antibodies targeting two different antigens were expressed separately in Escherichia coli cells and captured independently using Protein A chromatography. When combined, the knob and hole mutations in the CH3 domains promoted heterodimer formation. The hinge region disulfides were reduced and reoxidized to form the disulfide bridge between the two complementary half antibodies. Unreacted half antibodies, noncovalently linked homodimers, covalently linked homodimers, and noncovalently linked heterodimers are impurities closely related to the product of interest and are challenging to remove by standard processes. Characterization of the molecular properties of the half antibodies and high‐throughput screening predicted column chromatography performance and allowed for rapid development of downstream purification steps for removal of unique product‐related and process‐related impurities. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:397–404, 2018     

Growth regulation of human glioblastoma t98g cells by insulin-like growth factor-1 and its receptor

The interaction of insulin-like growth factors (IGFs) with the IGF-1 receptor is an important step in the control of cell proliferation and development. In particular, IGF-1 and IGF-2 are key regulators of central nervous system development, and may modulate the growth of glial tumors. We have investigated the growth factor regulation of the human glioblastoma cell line T98G. These cells growth arrested in serum-free medium at 34°C, despite their secretion of substantial amounts of bioactive IGF-1. To be stimulated to divide, growth-arrested cells required the addition of platelet-derived growth factor (PDGF) or its equivalent, 1% serum. Cell proliferation in serum-free medium could also be obtained by shifting the cells to a temperature of 39.6°C. Treatment of growth-arrested cells with PDGF or temperature shift was accompanied by a transient increase in the expression of the mRNA for the IGF-1 receptor. Transfection with a plasmid constitutively expressing the full cDNA for the human IGF-1 receptor allowed autonomous growth in serum-free medium at 34°C. By contrast, growth induction by growth factors or temperature shift was abrogated by transfection of the cells with a plasmid expressing a 300 bp segment of mRNA antisense to the IGF-1 receptor mRNA. Cloning in soft agar was also inhibited by expression of antisense IGF-1 receptor mRNA. These results demonstrate that the IGF-1 receptor is strictly required for the growth of T98G glioblastoma cells. Moreover, the autocrine interaction of IGF-1 with its receptor regulates both autonomous and anchorage-independent growth of these cells. © 1994 wiley-Liss, Inc.     

The Microtubule-Associated Protein CLASP Sustains Cell Proliferation through a Brassinosteroid Signaling Negative Feedback Loop

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塔里木盆地西北缘核桃坚果生化成分多样性分析

该研究收集新疆塔里木盆地西北缘44份核桃资源,其中树龄超过50 a的实生资源41份、主栽良种3个,并对其主要生化成分蛋白质、糖、脂肪、氨基酸、油酸、亚油酸、亚麻酸、棕榈酸、硬脂酸进行了多样性分析。结果表明:44份资源的生化成分变异幅度大,存在着丰富的多样性。各指标变异幅度由4.93%~30.93%,香农-维纳指数(H')变幅为1.38~2.02。17种氨基酸变异幅度由10.07%~35.71%,香农-维纳指数(H')变幅为1.85~2.20。主要生化成分主成分分析显示蛋白质、糖、脂肪三个主要成分的累计贡献率达81.67%。聚类分析表明,群组间生化成分存在显著差异,群组的聚类与地理分布有明显相关性,流域相同的资源的生化成分构成比例具有相似性。与主栽品种相比,实生资源在糖、蛋白质、脂肪等方面具有更高的变异幅度,因而具有一定的开发潜力。     

Diversity of root-endophytic <Emphasis Type="Italic">Trichoderma</Emphasis> from Malaysian Borneo

Trichoderma species form endophytic associations with plant roots and may provide a range of benefits to their hosts. However, few studies have systematically examined the diversity of Trichoderma species associated with plant roots in tropical regions. During the evaluation of Trichoderma isolates for use as biocontrol agents, root samples were collected from more than 58 genera in 35 plant families from a range of habitats in Malaysian Borneo. Trichoderma species were isolated from surface-sterilised roots and identified following analysis of partial translation elongation factor-1α (tef1) sequences. Species present included Trichoderma afroharzianum, Trichoderma asperelloides, Trichoderma asperellum, Trichoderma guizhouense, Trichoderma reesei, Trichoderma strigosum and Trichoderma virens. Trichoderma asperellum/T. asperelloides, Trichoderma harzianum s.l. and T. virens were the most frequently isolated taxa. tef1 sequence data supported the recognition of undescribed species related to the T. harzianum complex. The results suggest that tropical plants may be a useful source of novel root-associated Trichoderma for biotechnological applications.     

Ecological Drivers of Mansonella perstans Infection in Uganda and Patterns of Co-endemicity with Lymphatic Filariasis and Malaria

Background

Mansonella perstans is a widespread, but relatively unknown human filarial parasite transmitted by Culicoides biting midges. Although it is found in many parts of sub-Saharan Africa, only few studies have been carried out to deepen the understanding of its ecology, epidemiology, and health consequences. Hence, knowledge about ecological drivers of the vector and parasite distribution, integral to develop spatially explicit models for disease prevention, control, and elimination strategies, is limited.

Methodology

We analyzed data from a comprehensive nationwide survey of M. perstans infection conducted in 76 schools across Uganda in 2000–2003, to identify environmental drivers. A suite of Bayesian geostatistical regression models was fitted, and the best fitting model based on the deviance information criterion was utilized to predict M. perstans infection risk for all of Uganda. Additionally, we investigated co-infection rates and co-distribution with Wuchereria bancrofti and Plasmodium spp. infections observed at the same survey by mapping geographically overlapping areas.

Principal Findings

Several bioclimatic factors were significantly associated with M. perstans infection levels. A spatial Bayesian regression model showed the best fit, with diurnal temperature range, normalized difference vegetation index, and cattle densities identified as significant covariates. This model was employed to predict M. perstans infection risk at non-sampled locations. The level of co-infection with W. bancrofti was low (0.3%), due to limited geographic overlap. However, where the two infections did overlap geographically, a positive association was found.

Conclusions/Significance

This study presents the first geostatistical risk map for M. perstans in Uganda. We confirmed a widespread distribution of M. perstans, and identified important potential drivers of risk. The results provide new insight about the ecologic preferences of this otherwise poorly known filarial parasite and its Culicoides vector species in Uganda, which might be relevant for other settings in sub-Saharan Africa.     

Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows

We hypothesized that a diet enriched in alpha-linolenic acid would enhance embryonic development relative to diets enriched in linoleic or saturated fatty acids. Twenty-four lactating Holstein cows (86+/-22 d postpartum) were assigned to one of three diets containing saturated fatty acids (SAT; high in palmitic and stearic acids), whole flaxseed (FLX; high in alpha-linolenic acid) or sunflower seed (SUN; high in linoleic acid). Rations were formulated to provide 750 g supplemental fat/cow/d in all dietary groups. Ovulation (Day 0) was synchronized approximately 20 d after diets began. Ultrasound-guided follicular ablation of all follicles >8 mm was performed 5 d after ovulation; super stimulatory treatments began 2 d after follicular ablation, and embryos were collected non-surgically 7 d after AI. Fertilization rate, numbers of follicles and ovulations, and total and transferable embryos did not differ (P>0.05) among dietary groups. Sixty-one transferable embryos were stained and total blastomere number determined. Blastomere number was affected by diet (P<0.01); without regard to stage of development, embryos collected from cows fed SAT had lower (P<0.01) blastomere numbers (mean+/-S.E.M.; 77.1+/-3.9) than those from cows fed FLX (93.4+/-3.3) or SUN (97.2+/-3.5). Differences were most evident in the expanded blastocyst stage; at this stage, embryos of cows fed FLX and SUN diets had more blastomeres (P<0.02) than those of cows fed SAT (115.4+/-6.3, 132.3+/-8.3, and 89.3+/-9.6 cells, respectively). Although our hypothesis was only partially supported, embryonic development was enhanced in Holstein cows fed unsaturated fatty acids compared to those fed saturated fatty acids.     

The kinesin ATK5 functions in early spindle assembly in Arabidopsis

During cell division, the mitotic spindle partitions chromosomes into daughter nuclei. In higher plants, the molecular mechanisms governing spindle assembly and function remain largely unexplored. Here, live cell imaging of mitosis in Arabidopsis thaliana plants lacking a kinesin-14 (ATK5) reveals defects during early spindle formation. Beginning during prophase and lasting until late prometaphase, spindles of atk5-1 plants become abnormally elongated, are frequently bent, and have splayed poles by prometaphase. The period of spindle elongation during prophase and prometaphase is prolonged in atk5-1 cells. Time-lapse imaging of yellow fluorescent protein:ATK5 reveals colocalization with perinuclear microtubules before nuclear envelope breakdown, after which it congresses inward from the poles to the midzone, where it becomes progressively enriched at regions of overlap between antiparallel microtubules. In vitro microtubule motility assays demonstrate that in the presence of ATK5, two microtubules encountering one another at an angle can interact and coalign, forming a linear bundle. These data indicate that ATK5 participates in the search and capture of antiparallel interpolar microtubules, where it aids in generating force to coalign microtubules, thereby affecting spindle length, width, and integrity.     

The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.