Using light to see and control membrane traffic

Cellular compartmentalization into discrete organelles is maintained by membrane trafficking including vesiculation and tubulation. Recent advances in superresolution imaging have begun to bring these small and dynamic events into focus. Most nanoscopes exploit, and are limited by, switching dyes ON and OFF. Using ground state depletion to switch dyes into long-lived dark states can exploit specific photophysical properties of dyes, such as redox potential or pK(a), and expand the repertoire of nanoscopy probes for multicolor imaging. Seeing is not enough, and new technologies based on homodimerization, heterodimerization and selective release can manipulate membrane trafficking in pulse-chase and light-controlled ways. Herein we highlight the utility and promise of these strategies and discuss their current limitations.     

Protein stability by number: high-throughput and statistical approaches to one of protein science's most difficult problems

Most proteins are only barely stable, which impedes research, complicates therapeutic applications, and makes proteins susceptible to pathologically destabilizing mutations. Our ability to predict the thermodynamic consequences of even single point mutations is still surprisingly limited, and established methods of measuring stability are slow. Recent advances are bringing protein stability studies into the high-throughput realm. Some methods are based on inferential read-outs such as activity, proteolytic resistance or split-protein fragment reassembly. Other methods use miniaturization of direct measurements, such as intrinsic fluorescence, H/D exchange, cysteine reactivity, aggregation and hydrophobic dye binding (DSF). Protein engineering based on statistical analysis (consensus and correlated occurrences of amino acids) is promising, but much work remains to understand and implement these methods.     

Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer

α(1)-Antitrypsin (α1AT) deficiency is a disease with multiple manifestations, including cirrhosis and emphysema, caused by the accumulation of stable polymers of mutant protein in the endoplasmic reticulum of hepatocytes. However, the molecular basis of misfolding and polymerization remain unknown. We produced and crystallized a trimeric form of α1AT that is recognized by an antibody specific for the pathological polymer. Unexpectedly, this structure reveals a polymeric linkage mediated by domain swapping the carboxy-terminal 34 residues. Disulphide-trapping and antibody-binding studies further demonstrate that runaway C-terminal domain swapping, rather than the s4A/s5A domain swap previously proposed, underlies polymerization of the common Z-mutant of α1AT in vivo.     

促卵泡激素结合片段通过P13K／Akt—cyclinD1通路抑制卵巢癌细胞增殖活性

目的：研究促卵泡激素（FSH）结合片段对卵巢上皮性细胞癌细胞株hey增殖活性的影响。方法：应用卵巢上皮性细胞癌细胞株hey作为实验对象,分别加入FSH、FSH结合片段、FSH＋FSH结合片段。用四甲基偶氮唑蓝（MTT）法检测卵巢癌hey细胞的生长状况,Westernblot检测cyclinD1、Akt、pAkt分子的表达。结果：FSH对卵巢癌hey细胞的生长有明显的促进作用,细胞生长活性提高21％。FSH结合片段对卵巢癌细胞的生长有明显抑制作用,对细胞平均抑制率为22％,且能下调cyclinD1的表达,在2．5×10^-9Mol／L达70％。FSH结合片段对FSH有竞争抑制作用,细胞抑制率为18％,能抑制FSH上调cyclinD1的作用,抑制率为61％。FSH能上调pAkt的表达,对Akt的袁达没有明显影响,在40mlU／ml的浓度时pAkt／Akt上调率达224％。FSH结合片段对Akt的表达没有明显影响,但能明显下调pAkt的表达,且呈时间依赖性（在15分钟时达66％）和剂量依赖性（在2．5×10^-9Mol／L达31％）。FSH结合片段能抑制FSH上调pAkt的作用,抑制率为80％。结论：FSH结合片段可通过抑制FSH诱导的P13FdAkt-cycl—inD1信号通路进而抑制上皮性卵巢癌hey细胞的增殖活性,     

乳液法静电纺丝PLGA组织工程缓释纤维的研究

目的：研究Dextran对蛋白药物的释放影响。方法：将模型蛋白BSA溶解于多糖溶液中,通过W/O乳液法静电纺丝制备缓释纤维。采用MicroBCA法测定该纤维体外释放行为,采用SEC-HPLC检测制备前后蛋白的聚集程度,并与不含多糖的BSA纤维做对照。结果：添加Dextran以后蛋白的包封率由52.68%提高到63.92%,第一天突释不大于药物载量的15%,对蛋白单体的保持达到85%以上。结论：Dextran可以改善一般组织工程纤维中蛋白药物的释放,提高蛋白药物在制剂、贮存、释放过程中的稳定性,增加纤维的载药量。     

BMP9 protects septal neurons from axotomy-evoked loss of cholinergic phenotype

Background

Cholinergic projection from the septum to the hippocampus is crucial for normal cognitive function and degeneration of cells and nerve fibers within the septohippocampal pathway contributes to the pathophysiology of Alzheimer''s disease. Bone morphogenetic protein (BMP) 9 is a cholinergic differentiating factor during development both in vivo and in vitro.

Methodology/Principal Findings

To determine whether BMP9 could protect the adult cholinergic septohippocampal pathway from axotomy-evoked loss of the cholinergic phenotype, we performed unilateral fimbria-fornix transection in mice and treated them with a continuous intracerebroventricular infusion of BMP9 for six days. The number of choline acetyltransferase (CHAT)-positive cells was reduced by 50% in the medial septal nucleus ipsilateral to the lesion as compared to the intact, contralateral side, and BMP9 infusion prevented this loss in a dose-dependent manner. Moreover, BMP9 prevented most of the decline of hippocampal acetylcholine levels ipsilateral to the lesion, and markedly increased CHAT, choline transporter CHT, NGF receptors p75 (NGFR-p75) and TrkA (NTRK1), and NGF protein content in both the lesioned and unlesioned hippocampi. In addition, BMP9 infusion reduced bilaterally hippocampal levels of basic FGF (FGF2) protein.

Conclusions/Significance

These data indicate that BMP9 administration can prevent lesion-evoked impairment of the cholinergic septohippocampal neurons in adult mice and, by inducing NGF, establishes a trophic environment for these cells.     

Asymmetric dispersal and colonization success of Amazonian plant-ants queens

Background

The dispersal ability of queens is central to understanding ant life-history evolution, and plays a fundamental role in ant population and community dynamics, the maintenance of genetic diversity, and the spread of invasive ants. In tropical ecosystems, species from over 40 genera of ants establish colonies in the stems, hollow thorns, or leaf pouches of specialized plants. However, little is known about the relative dispersal ability of queens competing for access to the same host plants.

Methodology/Principal Findings

We used empirical data and inverse modeling—a technique developed by plant ecologists to model seed dispersal—to quantify and compare the dispersal kernels of queens from three Amazonian ant species that compete for access to host-plants. We found that the modal colonization distance of queens varied 8-fold, with the generalist ant species (Crematogaster laevis) having a greater modal distance than two specialists (Pheidole minutula, Azteca sp.) that use the same host-plants. However, our results also suggest that queens of Azteca sp. have maximal distances that are four-sixteen times greater than those of its competitors.

Conclusions/Significance

We found large differences between ant species in both the modal and maximal distance ant queens disperse to find vacant seedlings used to found new colonies. These differences could result from interspecific differences in queen body size, and hence wing musculature, or because queens differ in their ability to identify potential host plants while in flight. Our results provide support for one of the necessary conditions underlying several of the hypothesized mechanisms promoting coexistence in tropical plant-ants. They also suggest that for some ant species limited dispersal capability could pose a significant barrier to the rescue of populations in isolated forest fragments. Finally, we demonstrate that inverse models parameterized with field data are an excellent means of quantifying the dispersal of ant queens.     

Heritable variation in garter snake color patterns in postglacial populations

Global climate change is expected to trigger northward shifts in the ranges of natural populations of plants and animals, with subsequent effects on intraspecific genetic diversity. Investigating how genetic diversity is patterned among populations that arose following the last Ice Age is a promising method for understanding the potential future effects of climate change. Theoretical and empirical work has suggested that overall genetic diversity can decrease in colonial populations following rapid expansion into postglacial landscapes, with potential negative effects on the ability of populations to adapt to new environmental regimes. The crucial measure of this genetic variation and a population''s overall adaptability is the heritable variation in phenotypic traits, as it is this variation that mediates the rate and direction of a population''s multigenerational response to selection. Using two large full-sib quantitative genetic studies (N_Manitoba = 144; N_{South Dakota} = 653) and a smaller phenotypic analysis from Kansas (N_Kansas = 44), we compared mean levels of pigmentation, genetic variation and heritability in three pigmentation traits among populations of the common garter snake, Thamnophis sirtalis, along a north-south gradient, including a postglacial northern population and a putative southern refuge population. Counter to our expectations, we found that genetic variance and heritability for the three pigmentation traits were the same or higher in the postglacial population than in the southern population.     

Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel β-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX(3-9)C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of β2 and β3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.     

E. coli filament formation induced by heterologous S-layer expression

Escherichia coli is a rod-shaped intestinal bacterium which has a size of 1.1-1.5 μm x 2.0-6.0?μm. The fast cell division process and the uncomplicated living conditions have turned E. coli into a widely used host in genetic engineering and into one of the best studied microorganisms of all. We used E. coli BL21(DE3) as host for heterologous expression of S-layer proteins of Lysinibacillus sphaericus JG-A12 in order to enable a fast and high efficient protein production. The S-layer expression induced in E. coli an unusual elongation of the cells, thus producing filaments of > 100 μm in length. In the stationary growth phase, E. coli filaments develop tube-like structures that contain E. coli single cells. Fluorescence microscopic analyses of S-layer expressing E. coli cells that were stained with membrane stain FM (?) 5-95 verify the membrane origin of the tubes. Analyses of DAPI stained GFP-S-layer expressing E. coli support the assumption of a disordered cell division that is induced by the huge amount of recombinant S-layer proteins. However, the underlying mechanism is still not characterized in detail. These results describe the occurrence of a novel stable cell form of E. coli as a result of a disordered cell division process.